ISBN 978-958-694-094-8 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Supported by the CGIAR Centro Internacional de Agricultura Tropical (CIAT) Universidad Nacional de Colombia–Sede Palmira Bioversity International Red de Instituciones Vinculadas a la Capacitación en Economía y Políticas Agrícolas en América Latina y el Caribe (REDCAPA) Centre technique de coopération agricole et rurale (CTA) Apartado Aéreo 6713 Cali, Colombia Phone: +57 (2) 4450000 (direct) or +1 (650) 8336625 (via USA) Fax: +57 (2) 4450073 (direct) or +1 (650) 8336626 (via USA) Internet address: www.ciat.cgiar.org Copyright: CIAT 2004 (Spanish); CIAT 2007 (English) All rights reserved. CIAT Publication No. 360 ISBN 978-958-694-095-5 (CD-ROM) Press run: 225 Printed in Colombia November 2007 This course was originally published online, in 2004, in the Spanish language as Curso Multi-Institucional a Distancia sobre Conservación Ex Situ de Recursos Fitogenéticos. The Spanish version was compiled and edited by Benjamín Pineda, Rigoberto Hidalgo, Daniel Debouck, and Mariano Mejía. Scientific editors for the English version: Benjamín Pineda and Rigoberto Hidalgo. Translation into English and style-editing: Elizabeth L. McAdam. Multi-institutional distance learning course on the Ex Situ conservation of plant genetic resources [CD-ROM]. Centro Internacional de Agricultura Tropical (CIAT); Universidad Nacional de Colombia-Sede Palmira; Bioversity International; Red de Instituciones Vinculadas a la Capacitación en Economía y Políticas Agrícolas en América Latina y el Caribe (REDCAPA); Centre Technique de Coopération Agricole et Rurale (CTA), Cali, CO, 2007. 1 CD -- (CIAT publication no. 360) ISBN 978-958-694-095-5 AGROVOC descriptors in English: 1. Germplasm conservation. 2. Genetic resources. 3. Germplasm collections. 4. Gene banks. 5. Plant collections. 6. Biodiversity. 7. Distance education. 8. Training. 9. Internet. 10. Cooperation. Local descriptors in English: 1. Ex-situ conservation. 2. E-learning. AGROVOC descriptors in Spanish: 1. Conservación del germoplasma. 2. Recursos genéticos. 3. Colecciones de material genético. 4. Banco de genes. 5. Colección de plantas. 6. Biodiversidad. 7. Educación a distancia. 8. Capacitación. 9. Internet. 10. Cooperación. Local descriptors in Spanish: 1. Conservación ex-situ. 2. Aprendizaje electrónico. I. Centro Internacional de Agricultura Tropical. II. Universidad Nacional de Colombia-Sede Palmira. III. Bioversity International. IV. Red de Instituciones Vinculadas a la Capacitación en Economía y Políticas Agrícolas en América Latina y el Caribe. V. Centre Technique de Coopération Agricole et Rurale. VI. Tit. VII. Ser. AGRIS subject category: F30 Plant genetics and breeding / Genética vegetal y fitomejoramiento LC classification: SB 123 .3 M8 Project Partners Supported by the CGIAR CIAT Centro Internacional de Agricultura Tropical International Center for Tropical Agriculture www.ciat.cgiar.org The International Center for Tropical Agriculture (CIAT) is a not-forprofit organization that conducts socially and environmentally progressive research aimed at reducing hunger and poverty and preserving natural resources in developing countries. CIAT is one of the 15 centers funded mainly by the 64 countries, private foundations, and international organizations that make up the Consultative Group on International Agricultural Research (CGIAR). Universidad Nacional de Colombia–Sede Palmira National University of Colombia–Palmira www.palmira.unal.edu.co The National University of Colombia is an autonomous university that is attached to the National Ministry of Education, with specific regulations, and defined as a national, public, and state university. Its objective is the development of higher education and research, which shall be promoted by the State by permitting access to it and the simultaneous development of the sciences and the arts thereby attaining excellence. As a Public Institute, it shall be considered as having a pluralistic, pluri-class, and secular character. Moreover, the University does not respond to private interests, allowing it to consider and propose solutions to national problems that go beyond interests related to economic profitability. Bioversity International www.bioversityinternational.org Bioversity International is an independent international scientific organization that seeks to improve the well-being of present and future generations of people by enhancing conservation and the deployment of agricultural biodiversity on farms and in forests. It is one of 15 centres supported by the Consultative Group on International Agricultural Research (CGIAR), an association of public and private members who support efforts to mobilize cutting-edge science to reduce hunger and poverty, improve human nutrition and health, and protect the environment. Bioversity has its headquarters in Maccarese, near Rome, Italy, with offices in more than 20 other countries worldwide. The Institute operates through four programmes: Diversity for Livelihoods, Understanding and Managing Biodiversity, Global Partnerships, and Commodities for Livelihoods. REDCAPA Red de Instituciones Vinculadas a la Capacitación en Economía y Políticas Agrícolas en América Latina y el Caribe Network of Institutions Dedicated to Teaching Agricultural and Rural Development Policies for Latin America and the Caribbean www.redcapa.org.br REDCAPA is a non-profit independent association of universities and research institutions that are dedicated to the study and teaching of topics related to the agricultural and rural sector of Latin America and the Caribbean (LAC). Officially constituted in 1993, it now brings together dozens of institutions of different LAC countries, with European and U.S. universities and institutions as collaborators. REDCAPA is legally registered in Brazil and maintains a complete platform for distance education through the Internet, which is used by its member institutions (specifically their professors) to offer blended learning courses throughout Latin America. CTA Centre technique de coopération agricole et rurale Technical Centre for Agricultural and Rural Cooperation www.cta.int The Technical Centre for Agricultural and Rural Cooperation (CTA) was established in 1983 under the Lomé Convention between the ACP (African, Caribbean and Pacific) Group of States and the European Union Member States. Since 2000, it has operated within the framework of the ACP-EC Cotonou Agreement. CTA’s tasks are to develop and provide services that improve access to information for agricultural and rural development, and to strengthen the capacity of ACP countries to produce, acquire, exchange and utilise information in this area. CTA’s programmes are designed to: provide a wide range of information products and services and enhance awareness of relevant information sources; promote the integrated use of appropriate communication channels and intensify contacts and information exchange (particularly intra-ACP); and develop ACP capacity to generate and manage agricultural information and to formulate ICM strategies, including those relevant to science and technology. CTA’s work incorporates new developments in methodologies and cross-cutting issues such as gender and social capital. CTA is financed by the European Union. Our Collaborators The compilers of the original Spanish version of the Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources, Benjamín Pineda, Rigoberto Hidalgo, Daniel Debouck, and Mariano Mejía, are most grateful to the following people for their collaboration in writing up and improving several sections of the original version: Margarita Baena, María del S Balcázar, Carmen Rosa Bonilla, Carlos Iván Cardozo, Arsenio Ciprián, Norma C Flor, Tito L Franco, Dimary Libreros, Graciela Mafla, César Ocampo, Julio Roa, Manuel Sánchez, Orlando Toro, and Alba Marina Torres. We would furthermore like to thank other collaborators who helped make the English version of this course a publishing reality: Edith Hesse, for her continuous support to this endeavour and active fund raising for the English version of this learning course; Lynn Menéndez, for serving as effective liaison between editors and for coordinating different stages of the production process; Gladys Rodríguez, for her meticulous proofreading; Oscar Idárraga, for the concise layout and work with drawings; and Julio César Martínez, for a meritorious publication and cover design. © Copyright 2007 The Intellectual Property of the Course Materials The intellectual property of the course shall continue, in this English version, in compliance with Clause 6 of the CIAT/IPGRI/Universidad Nacional/REDCAPA Agreement, Final Version, 16 April 2004, which states: • • • • The materials used for this course shall remain in the public domain and shall be freely available to any interested user. Copyright of the materials that had existed before the course was developed shall belong to the institution that generated these materials. Copyright or intellectual rights over the new materials that were developed for the course shall belong to the participating entities that jointly developed them and shall be handled as products for the public domain. Users of these products shall recognize the origin of the materials and, when they use them, shall give due credit to the institutions that developed them. CIAT shall be in charge of placing the course materials in public repositories for distance learning to achieve greater dissemination and use of the same. Furthermore, the English version of the course’s materials shall carry visible and specific acknowledgements of CTA’s support for translation and publication. This information applies to all course materials identified with the logotypes of the participating institutions. Contents Page Preface ix Course Objectives xi Module 1 Basic concepts of conservation for plant genetic resources 1 General comments 1 Lesson 1: Genetic resources, biodiversity, and agrobiodiversity 4 Lesson 2: Conservation: its raison d’être and strategies 12 Lesson 3: Minimum requirements for ex situ conservation 19 2 Germplasm acquisition and introduction (seeds and asexual propagules) 23 General comments 23 Lesson 1: Plant germplasm acquisition: criteria 27 Lesson 2: Plant germplasm acquisition: procedures 32 Lesson 3: Germplasm introduction: transfer regulations and quarantine measures 43 3 Germplasm conservation General comments Submodule A. Multiplication and regeneration Lesson 1: Multiplication Lesson 2: Regeneration Submodule B. Harvesting, conditioning, and quantification Lesson 1: Harvesting Lesson 2: Conditioning and quantification Submodule C. Verifying the biological quality of germplasm 55 55 59 59 68 75 75 88 101 Lesson 1: Basic concepts 101 Lesson 2: Verification procedures 117 Submodule D. Verifying phytosanitary quality 135 Lesson 1: Basic concepts of phytosanitary quality 135 Lesson 2: Procedures for verifying phytosanitary quality 144 Submodule E. Storing germplasm Lesson: Basic concepts of storage, an essential component of the ex situ conservation of germplasm 157 157 vii Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Page Module 4 Germplasm characterization 171 General comments 171 Lesson 1: General concepts of germplasm characterization 174 Lesson 2: Ways of characterizing plant germplasm 184 5 Managing plant germplasm banks General comments 201 Lesson: 205 General aspects of bank management 6 Germplasm documentation Glossary viii 201 217 General comments 217 Lesson: 219 Main aspects of germplasm documentation 235 Preface Many developing countries possess germplasm banks that hold collections of crop species that are significant to humanity’s survival. Yet, these banks are often unable to fulfil even the basic functions of conservation because they lack financial resources and adequately trained staff. Three institutions working in the field of plant genetic resources (PGRs) have recognized this problem and have been collaborating over 10 years on various initiatives; these institutions are the International Center for Tropical Agriculture (CIAT, its Spanish acronym), Bioversity International (formerly the International Plant Genetic Resources Institute or IPGRI), and the National University of Colombia. One initiative was to organize three international training courses on conservation for professionals and technicians working in germplasm banks, botanical gardens, arboreta, and crop diversity projects. Recently, however, obtaining funding for these courses has become increasingly difficult, and distance education was seen as a possible alternative to meet the high demand for training. To embark on this new learning venture, a strategic partnership was established with a provider of computer-supported collaborative learning (CSCL) with over 10 years’ experience, the Network of Institutions Dedicated to Teaching Agricultural and Rural Development Policies for Latin America and the Caribbean (REDCAPA, its Spanish acronym). A coordination committee, made up of representatives from all four partners, defined the objectives of the learning venture and delegated specific staff to take responsibility for given project components. Course objectives and lesson plans were agreed upon jointly by the four partners. Lesson contents were compiled and developed by course tutors and an adult education specialist, with input from university staff and other experts in PGRs and germplasm conservation. The materials were organized into six modules that covered concepts of PGRs, germplasm acquisition, introduction, conservation, characterization, documentation, and germplasm bank management. Dr Daniel Debouck, Head of the Genetic Resources Unit at CIAT, reviewed and endorsed the materials, making valuable suggestions for improvement. The materials were then posted into REDCAPA’s virtual classroom as the course developed. A rich bibliography, containing 234 references and 41 full-text publications, and a glossary of terms related to germplasm conservation were compiled and made accessible to all students. More than 120 professionals applied for the course’s first electronic appearance, but only 30 could be admitted according to clearly defined selection criteria. Those who missed out on the first selection, and another 50 new applicants, are expecting a repetition of the course, thus indicating the usefulness of this kind of training. A detailed evaluation (available at www.ciat.cgiar.org/ccc/ex_situ.htm) of the course resulted in very positive feedback from students (mainly germplasm bank curators and other professionals), the tutors, and the students’ supervisors. The organizers therefore decided to approach the Technical Centre for Agricultural and Rural Cooperation (CTA, its French acronym), based in Wageningen, the Netherlands. The Centre agreed to support the translation of the materials into English so that they can now be made available to a wider audience, both in print and on CD. ix Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources The four institutions who organized the Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources would like to thank CTA for its support in funding the translation and publication of the course materials. We sincerely hope that our publishing the course materials in English and in both print and CD will enable many more people to take advantage of these useful materials, whether for their own studies or for teaching the conservation of PGRs for humanity. x Course Objectives • • • • • To help develop institutional capabilities in the conservation of plant genetic resources (PGRs) by training human resources of the countries participating in the course To improve the efforts of participating countries to conserve their PGRs and thereby increase the social benefits of such an activity To promote social appropriation of knowledge on the conservation of PGRs To strengthen the creative, analytical, and synthesizing capacity of the human talent currently responsible for managing the germplasm banks of Africa To contribute to the education and strengthening of the core of human talent oriented towards understanding such questions as: Why conserve? What should be conserved? For who do we conserve? How do we conserve? • • To provide an environment in which professionals of the participating countries can share their experiences and knowledge on the conservation of PGRs To provide an opportunity for professionals to update their training in the conservation of PGRs xi Module 1 Supported by the CGIAR Basic Concepts of Conservation for Plant Genetic Resources General Comments ‘Biological diversity, or biodiversity, is the term given to the variety of life on Earth. It is the combination of life forms and their interactions with one another, and with the physical environment that has made Earth habitable for humans. Ecosystems provide the basic necessities of life, offer protection from natural disasters and disease, and are the foundation for human culture’ (SCBD 2006). However, over the last two centuries, both biodiversity and agrobiodiversity have entered a stage where they are at high risk of extinction. The main reason is excessive consumption of resources to sustain rapid population growth. Another reason is the considerable degree of ignorance that exists on biodiversity, leading to its consequent destruction. The plants used by humans do not escape this phenomenon, thus awakening global concern. To conserve this precious variability, which represents humanity’s future survival, strategies are being established, two of which are ex situ and in situ conservation. Although, as we shall see, the two types of conservation complement each other, this course focuses on ex situ conservation. Information on the Module This module contains three lessons, each with a rapid evaluation, involving tasks. Objectives When you have completed the module you should be able to: • • • • Justify the raison d’être of conserving plant genetic resources (PGRs) Describe the concepts of biodiversity and agrobiodiversity Describe strategies for in situ and ex situ conservation and state their essential differences Describe the minimum requirements for ex situ conservation Lessons 1. Genetic resources, biodiversity, and agrobiodiversity 2. Conservation: its raison d’être and strategies 3. Minimum requirements for ex situ conservation Bibliography Throughout this module, a bibliography is provided for each section, that is, the General Comments and each Lesson. The bibliographies follow a format of two parts: 1. Literature cited, which includes those references cited in the text itself. Some of these citations were used to develop the original Spanish-language course on ex situ conservation and may therefore appear in Spanish or Portuguese. However, where practical, references to the English versions of the original Spanish-language documents are provided. 1 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources 2. Further reading, which is a list of suggested readings in the English language. Most cover in depth the topics included in this module. A list of Acronyms used in the bibliographies is also given. The idea is to save space by not having to spell out each institution’s full name each time it appears in the references. Acronyms used in the bibliographies CGRFA Commission on Genetic Resources for Food and Agriculture FAO Food and Agriculture Organization of the United Nations IBPGR International Board for Plant Genetic Resources IICA Inter-American Institute for Cooperation in Agriculture IPGRI International Plant Genetic Resources Institute NCBI National Center for Biotechnology Information SCBD Secretariat of the Convention on Biological Diversity Literature cited SCBD. 2006. Global biodiversity outlook 2. Montreal, Canada. 81 p. Also available at http://www.cbd.int/doc/gbo2/cbd-gbo2.pdf Further reading Chang TT. 1985. Principles of genetic conservation. Iowa State J Res 59(4):325–348. Convention on Biological Diversity. 2007. Home page. Available at http://www.cbd.int/ default.shtml FAO. 1996. Report on the state of the world’s plant genetic resources for food and agriculture prepared for the International Technical Conference on Plant Genetic Resources, Leipzig, Germany, 17–23 June 1996. 82 p. Also available at http://www.fao.org/ag/AGP/AGPS/ Pgrfa/pdf/swrshr_e.pdf or http://www.fao.org/iag/AGP/AGPS/Pgrfa/wrlmap_e.htm FAO. 1997. The state of the world’s plant genetic resources for food and agriculture. Rome. 510 p. Also available at http: //www.fao.org/ag/AGP/AGPS/Pgrfa/pdf/swrfull.pdf or http://www.fao.org/iag/AGP/AGPS/Pgrfa/wrlmap_e.htm FAO. (Accessed 28 July 2004) Web site: Biological diversity in food and agriculture. Available at http://www.fao.org/biodiversity/index.asp?lang=en FAO–CGRFA. (Accessed 28 July 2004) Global system on plant genetic resources. Available at http://www.fao.org/ag/cgrfa/PGR.htm Frankel OH; Brown AHD; Burdon JJ. 1995. Conservation of plant biodiversity. Cambridge University Press, UK. 299 p. Hoagland MB. 1978. The roots of life: a layman’s guide to genes, evolution, and the ways of cells. Houghton-Mifflin, Boston, MA. 167 p. IBPGR, comp. 1991. Elsevier’s dictionary of plant genetic resources. Elsevier Science Publishers, Amsterdam, Netherlands. 187 p. 2 Module 1: Basic Concepts of Conservation for Plant Genetic Resources General Comments IPGRI. (Accessed 28 July 2004) In situ conservation. Available at http://www.ipgri.cgiar.org/ themes/in_situ_project/on_farm/espanolpres.htm Keating M. 1993. The earth summit’s agenda for change: a plain language version of Agenda 21 and the other Rio Agreements. Centre for Our Common Future, Geneva, Switzerland. 70 p. Maxted N; Ford-Lloyd BV; Hawkes JG, eds. 1997. Plant genetic conservation: the in situ approach. Chapman and Hall, London. 446 p. Prescott-Allen R; Prescott-Allen C. 1988. Genes from the wild: using wild genetic resources for food and raw materials. Earthscan Publications, London. 112 p. Rao R; Riley KW. 1994. The use of biotechnology for conservation and utilization of plant genetic resources. Plant Genet Resour Newsl 97:3–20. SCBD. 2005. Handbook of the Convention on Biological Diversity, including its Cartagena Protocol on Biosafety, 3rd ed. Montreal, Canada. 1493 p. Also available at http:// www.cbd.int/doc/handbook/cbd-hb-all-en.pdf United Nations. 1993. No. 30619—Multilateral—Convention on Biological Diversity (with annexes): concluded at Rio de Janeiro on 5 June 1992, registered 29 December 1993. Treaty Series, vol 1760, I-30619, pp 142–382. Available at http://www.biodiv.org/doc/ legal/cbd-un-en.pdf Wilson EO. 1989. Current state of biological diversity. In Wilson EO, ed. Biodiversity. National Academy Press, Washington, DC. pp 3–18. Contributors to this Module Rigoberto Hidalgo, Benjamín Pineda, Daniel Debouck, and Mariano Mejía. 3 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Lesson 1 Genetic Resources, Biodiversity, and Agrobiodiversity Objectives • • • To define what is understood by PGRs To describe the basic concepts of biodiversity and agrobiodiversity To analyze the rationale behind conservation and its benefits Introduction Humans depend on plants, which provide food and supply most needs, including clothes and shelter. Plants are also used in industry to make fuels, medicines, fibres, rubber, and other products. However, the number of plants that humans use as food is minimal compared with the number of species existing in nature. Just 30 crops, particularly rice, wheat, and maize, provide 95% of the calories present in the human diet (FAO 1997). Dependency on such a limited number of crops threatens humanity’s food security (Valois 1996). Plant genetic resources are currently of great interest as they are related to satisfying humans’ basic needs and to solving severe problems such as hunger and poverty. Today, almost 800 million people are malnourished. Of these, 200 million are children who no more than 5 years old. In the next 30 years, the world’s population will increase from about 2500 million inhabitants to 8500 million. To supply food for so many people requires an efficient and sustainable improvement of crop yields (FAO 1996). However, even though they help sustain populations and alleviate poverty, PGRs are vulnerable. They can erode and even disappear, thus endangering the continuity of the human species (Jaramillo and Baena 2000). Population increase, industrialization, and the expanding agricultural frontier have contributed towards the loss of germplasm, or genetic erosion. To this we must add the adoption of elite germplasm and the modification and/or destruction of centres of genetic variability. This loss of PGRs demonstrates the urgent need to conserve and sustainably use them (Jaramillo and Baena 2000). What is understood by plant genetic resources? Plant genetic resources are the sum of all combinations of genes resulting from the evolution of plant species. During evolution, the population of any given species is the receptacle of all past changes and of the results of selections made by the environment. Those changes are conserved in the DNA that constitutes the species genome (Hoagland 1985). In other words, genes contain all the information that defines each trait or character of a living being, in this case, plants. An inheritable trait or character is meticulously reproduced in offspring. Consequently, we find in genes information on adaptation, productivity, resistance to adverse conditions such as pests, diseases, stressful climates, and poor soils, and other characteristics of a population’s individuals that are usable by humans to the extent of their knowledge. 4 Module 1, Lesson 1: Genetic Resources, Biodiversity, and Agrobiodiversity In general, PGRs include wild or domesticated plant species that have economic, ecological, or utilitarian potential, whether current or future. The most important of these are those that contribute to food security (IBPGR 1991) and, undoubtedly, are closely related to environmental conservation. Plant genetic resources also include products of classical breeding, biotechnology, and genetic engineering such as transgenic plants, DNA fragments, cloned genes, gene markers, new genetic combinations, silent genes, and chloroplast genomes (FAO 1996; Frankel et al. 1995; Rao and Riley 1994). Why conserve? The conservation of PGRs enables humanity to: • • • • • Broaden the diversity of plant foods and related products it can access. Improve food crops in terms of yield, quality, adaptability to different environmental conditions, and resistance to pests and diseases. Build reserves of breeding materials of native and exotic species that have nutritional or industrial potential. Such potential can be exploited in various ways, for example, in crop improvement programmes that seek higher productivity, resistance to adverse biotic and abiotic conditions, and desired qualities as according to previously established requirements. Economically, help nations to increase the productivity and sustainability of their agriculture and even develop it. Restore and conserve the environment. Who benefits from conservation? Conservation of PGRs directly benefits humanity. As such, investing in conservation generates benefits for society, whether to diversify agriculture or provide environmental services. It is well known that, in the next 30 years, the world’s population may easily exceed 8500 million. As a result, its basic needs must be satisfied, both in food and raw materials for food and agricultural industries. Furthermore, countries that duly conserve their PGRs can better face the challenges of socio-economic development in an increasingly globalized and competitive world. However, programmes must be established to permit the meeting of proposed targets. The spirit of the Convention on Biological Diversity is, currently, of a national character and several countries have PGRs that are not valued within the country as ‘national’ but may be valued from the perspective of a multilateral system. Biodiversity and Agrobiodiversity Biodiversity This generic term was adopted recently to describe the genetic variability of all living things, as represented by micro-organisms, plants, and animals. Current biodiversity is the result of more than 600 million years of evolution. The number of species existing today is uncertain, being more than 5 million but possibly as high as 100 million (Figure 1). 5 6 Period Epoch Time (Ma) Life forms CENOZOIC ERA (Age of Mammals) Quaternary Pleistocene 1.8–0.1 Humans Tertiary Pliocene 5.0–1.8 Mammals, birds Miocene 23–5 Bony fishes Oligocene 38–23 Modern mammals Eocene 54–38 Modern invertebrates Palaeocene 65–54 Primitive mammals MESOZOIC ERA (Age of Reptiles) Cretaceous 146–65 EXTINCTION Ancestral mammals Flowering plants Jurassic 208–146 Dinosaurs Archaeopteryx (primitive birds) Triassic 245–208 EXTINCTION First mammals First dinosaurs Early bony fishes Conifers Figure 1. Geological chart, showing species diversity according to era, together with the “Big Five” mass extinctions (adapted from Bryant 2005). (Continued) Figure 1. (Continued.) Period Epoch Time (Ma) Life forms PALEOZOIC ERA (Ancient Life) Permian 286–245 EXTINCTION Early reptiles Mammal-like reptiles Carboniferous 360–286 Giant insects Large amphibians Primitive plants Devonian 410–360 EXTINCTION Primitive fishes Primitive plants Silurian 440–410 Backboned animals Ordovician 505–440 EXTINCTION Invertebrates Cambrian 544–505 Invertebrates PRECAMBRIAN ERA (Dawn of Life) 4500–544 Unicellular organisms 7 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources A little more than 1.4 million species have been classified. Nearly 1 million correspond to animals (mostly insects) and about 250,000 to plants (Table 1). The least studied have been the micro-organisms but, with the current development of technology, a notable increase in their description is expected in coming years. Table 1. The number of species described. Kingdom Total % Viruses 1,000 Monera 4,760 0.34 Fungi 46,983 3.38 Protista–Algae 26,900 1.93 Protista–Protozoa 0.07 30,800 2.21 Plantae 248,428 17.84 Animalia–others 989,761 71.08 Animalia–Chordata Total 43,853 3.15 1,392,485 100.00 SOURCE: Wilson (1989). Of the groups of living things, plants and animals have been the most exploited by humans to satisfy their basic needs for food, clothes, housing, and health. To this end, our ancestors began, about 15,000 years ago, selecting those species that were useful to them. During that time, nearly 3,000 plant species were tried, of which only a little more than 100 are currently used. Twenty of these 100 answer the basic needs for sustenance. Agrobiodiversity As stated before, of all the species conforming current biodiversity, a special group of plants, animals, and a few micro-organisms was selected by humans for their particular characteristics in answering humans’ needs. Since the dawn of agriculture, such species have been under heavy anthropic selection pressures, a consequence of which was the proliferation of many genetic variants of each species. The sum of those selected species and their thousands of variants was recently designated as agrobiodiversity (FAO 2004). It is the product of natural evolution plus the effect of selection by humans, which process is known as domestication. Over the last 2 centuries, both biodiversity and agrobiodiversity have entered a stage where they are at high risk of extinction. The main reasons are the concentration of agricultural activities that prioritize only a very few crops, excessive consumption of resources to sustain the world’s rapid population growth, and the considerable degree of ignorance about biodiversity. The plants used by humans do not escape this phenomenon, awakening global concern. Strategies are being established to ensure the conservation of that variability, as it represents humanity’s future survival. 8 Module 1, Lesson 1: Genetic Resources, Biodiversity, and Agrobiodiversity Germplasm Banks For conservation strategies to operate, there must be somewhere to identify, store, and maintain the PGRs being conserved. Policies and protocols for their use and distribution must also be put into place. These activities are carried out in germplasm banks, often called gene banks or even germplasm collections. Because of the potential confusion in the use of these terms, we need to clarify them for the context of this course. As discussed previously, germplasm refers to those plant structures that are able to give rise to new generations of a given plant species. By doing so, they carry the total sum of their respective species’ hereditary characteristics. Such structures may be seeds, propagules, or DNA (or gene) fragments. A germplasm collection therefore brings these structures together in a given place; however, in the world of the genetic resources it is understood more as a collection of genotypes, gene libraries, or alleles of a particular species from different locations or sources (geographic and environmental), used as source material in plant breeding and assembled for conservation (IBPGR 1991). Because of the costliness of their maintenance, most collections are specialized, that is, they focus on particular plant species within a certain context such as conservational, agricultural, research and educational, environmental, historic, aesthetic, or economical. The place where the germplasm is gathered is the germplasm bank, which can be seeds stored in cold rooms, living plants in a field, plants conserved as in vitro or cloned DNA fragments from a single genome; by extension, the germplasm banks are also called as gene banks. However, banks are not just physical installations but are also socio-economicpolitical entities that determine the management of the germplasm being held. Germplasm collections or holdings are therefore the “business” of germplasm banks. Because of the inclusive meaning of germplasm, the term gene bank is reserved in the sense of DNA library to describe those infrastructures holding only collections of amplified DNA fragments. Perhaps the best known gene bank is GenBank, which is part of the U.S. National Center for Biotechnology Information (NCBI 2006). Evaluating the Lesson After this lesson, you should understand the raison d’être for conserving PGRs, the concepts of biodiversity and agrobiodiversity, and the role they play in people’s lives. Before going on to the next lesson you should answer, in your own words, each of the following questions. Write a maximum of one page per question. • • • Thinking of the conditions of your own country or region where you live, consider why conservation is necessary. What benefits would the adequate conservation of PGRs bring to your country or region? Briefly explain the differences between biodiversity and agrobiodiversity. 9 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Bibliography Literature cited Bryant PJ. 2005. Biodiversity and conservation: a hypertext book. School of Biological Sciences of the University of California, Irvine, CA, USA. Available at htpp://darwin.bio.uci.edu/ ~sustain/bio65/lec01/b65lec01.htm (accessed 27 Oct 2007). FAO. 1996. Informe sobre el estado de los recursos fitogenéticos en el mundo. Rome. 75 p. (Also available in English at http://www.fao.org/ag/AGP/AGPS/Pgrfa/pdf/swrshr_e.pdf or http://www.fao.org/iag/AGP/AGPS/Pgrfa/wrlmap_e.htm) FAO. 1997. The state of the world’s plant genetic resources for food and agriculture. Rome. 510 p. Also available at http://www.fao.org/ag/AGP/AGPS/Pgrfa/wrlmap_e.htm or http://www.fao.org/iag/AGP/AGPS/Pgrfa/wrlmap_e.htm FAO. (Accessed 28 July 2004) Web site: Diversidad biológica en la alimentación y la agricultura. Available at: http://www.fao.org/biodiversity/doc_es.asp (Also available in English at http://www.fao.org/biodiversity/index.asp?lang=en). Frankel OH; Brown AHD; Burdon JJ. 1995. Conservation of plant biodiversity. Cambridge University Press, UK. 299 p. Hoagland MB. 1985. Las raíces de la vida. Salvat Editores, Barcelona. 167 p. (Translated from the English by Josep Cuello.) (Also published in English as Hoagland MB. 1978. The Roots of Life: A Layman’s Guide to Genes, Evolution, and the Ways of Cells. HoughtonMifflin, Boston, MA, 167 p.) IBPGR, comp. 1991. Elsevier’s dictionary of plant genetic resources. Elsevier Science Publishers, Amsterdam, Netherlands. 187 p. Jaramillo S; Baena M. 2000. Material de apoyo a la capacitación en conservación ex situ de recursos fitogenéticos. IPGRI, Cali, Colombia. pp 9–17 (209 p). Also available at http://www.ipgri.cgiar.org/training/exsitu/web/arr_ppal_modulo.htm NCBI. 2006. GenBank overview. Available at http://www.ncbi.nlm.nih.gov/Genbank/ (accessed 9 Aug 2007). Rao R; Riley KW. 1994. The use of biotechnology for conservation and utilization of plant genetic resources. Plant Genet Resour Newsl 97:3–20. Valois ACC. 1996. Conservação de germoplasma vegetal ex situ. In Diálogo XLV: Conservación de germoplasma vegetal. Proc. Course conducted in Brasília by IICA, 19–30 Sept 1994. IICA, Montevideo, Uruguay. pp 7–11. Wilson EO. 1989. Current state of biological diversity. In Wilson EO, ed. Biodiversity. National Academy Press, Washington, DC. pp 3–18. Further reading Chang TT. 1985. Principles of genetic conservation. Iowa State J Res 59(4):325–348. 10 Module 1, Lesson 1: Genetic Resources, Biodiversity, and Agrobiodiversity Koo B; Pardey PG; Wright BD. 2002. Endowing future harvests: the long-term costs of conserving genetic resources at the CGIAR Centres. IPGRI, Rome. Lovejoy TE. 1997. Biodiversity: what is it? In Reaka-Kudla M; Wilson D; Wilson E, eds. Biodiversity, II: Understanding and protecting our biological resources. Joseph Henry Press, Washington, DC. pp 7–14. Patrick R. 1997. Biodiversity: why is it important? In Reaka-Kudla M; Wilson D; Wilson E, eds. Biodiversity, II: Understanding and protecting our biological resources. Joseph Henry Press, Washington, DC. pp 15–24. SCBD. 2005. Handbook of the Convention on Biological Diversity including its Cartagena Protocol on Biosafety, 3rd ed. Montreal, Canada. 1493 p. Also available at http://www.cbd.int/doc/handbook/cbd-hb-all-en.pdf United Nations. 1993. No. 30619—Multilateral—Convention on biological diversity (with annexes): concluded at Rio de Janeiro on 5 June 1992, registered 29 December 1993. Treaty Series, vol 1760, I-30619, pp 143–382. Available at http://www.biodiv.org/doc/ legal/cbd-un-en.pdf Contributors to this Lesson Rigoberto Hidalgo, Benjamín Pineda, Daniel Debouck, and Mariano Mejía. Next Lesson In the next lesson, you will study the rationale behind conservation and its strategies. 11 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Lesson 2 Conservation: Its Raison d’Être and Strategies Objectives • • • To justify the raison d’être for conserving PGRs To discuss strategies to conserve PGRs To analyze reasons for ex situ conservation and its benefits Introduction As with all living organisms that develop under natural conditions, the population of individuals that form a plant species is in permanent interaction with its surrounding environment. It must adapt and cope with the continuously changing factors that are part of that environment. These factors are biotic (micro-organisms, other plant species, and animals) and abiotic (climate and soils). For a plant species to interact with these factors, it uses information contained in its genome according to its needs for surviving in the environment. The result of this adaptive interaction is an accumulation of genetic information that each species keeps, through variants, among the members of its population and transmits to subsequent generations. The information is carried by genes, which enable the species to adapt to changes that may occur in its environment. In other words, the genetic composition of a population also changes over time or, more exactly, it evolves. As evolutionary processes are dynamic and bring together various forces (according to Theodosius Dobzhansky (1951) mutation, hybridization, selection, and genetic drift), the accumulated changes or information contained in the genes are often lost, and cannot be recovered easily over time. This is when conservation gains raison d’être for its use: to help rescue potentially losable genes, especially those that geneticists and breeders can use. Conservation Strategies Species are believed to originate through the action of forces such as genetic variability, natural selection, and speciation. The pertinent information is recorded in the genomes of the individuals that, in nature, constitute the population of each species. We can detect the natural variability contained in the genomes of a species by collecting samples from different places. The samples would comprise reproductive structures or organs that we can grow, classify, and collect such as seeds, stems, bulbs, stolons, cuttings, rhizomes, tubers, and roots. These collections of live plant parts are called germplasm and are conserved in germplasm banks. They form the basis on which to develop conservation strategies. Because of the heterogeneity of the plants used by humans, no single strategy for conserving the variability of species can be established (Tables 1 and 2). The plants are therefore conserved according to current and future needs and/or usefulness. Plant genetic resources can be conserved in situ in their natural habitats or ex situ under conditions other than those of their natural habitats (IPGRI 2004; Jaramillo and Baena 2000). Or the two methods can be combined into a complementary conservation strategy, where in situ conservation is used to maintain the natural conditions for the creation of new genetic variability and ex situ conservation is used to conserve genetic combinations that 12 Module 1, Lesson 2: Conservation: Its Raison d’Être and Strategies Table 1. Systems of in situ conservation of global biodiversity and agrobiodiversity, according to targeted germplasm and expected conservation period. Class of diversity Conservation system Targeted germplasm Period of conservation Global biodiversity Protected areas • Ecosystems • Wild species (plants and/or animals) Depends on the degree of stability of protected areas Agrobiodiversity Protected areas Wild ancestral Depends on life cycle and number of renewals On farm Traditional varieties Medium or long term Gardens Mixtures of traditional species in communities Depends on life cycle and number of renewals Table 2. Systems of ex situ conservation of agrobiodiversity, according to targeted germplasm and expected conservation period. Conservation systems Targeted germplasm Period of conservation Seed type System Orthodox Seed bank • Cultivated species • Gene pools Medium or long term Botanic gardens • Species for classification • Flora (research) Depends on life cycle and number of renewals Cryopreservation bank Cultivated species Long term DNA bank Special sequences In vitro bank • Cultivated species • Wild species Short term In vivo bank (field) Cultivated species Depends on life cycle and number of renewals Botanic gardens • Classification • Flora (research) Depends on life cycle and number of renewals Pollen bank Cultivated species (male plants) Depends on preservation method Cryopreservation bank Cultivated species Long term DNA bank Special sequences Long term Recalcitrant have been shown to be of value and whose existence in nature may be under threat. Conservation is carried out by using collections of live plant parts in: • • • Germplasm banks, also known as gene banks (e.g., field, seed, in vitro, pollen, or cryopreservation) Botanical gardens Arboreta and herbaria 13 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Plant germplasm banks are centres of resources for live plant parts. These collections of plant parts operate solely to keep them alive and preserve their characteristics for the future benefit of humankind and the environment. Germplasm banks are also called plant genetic resources centres because they give importance to the fact that plants are sources of diversity of genetic characteristics. Conserved plants include economically important food crops (modern and primitive crops, and their wild relatives), horticultural plants, forages, medicinal plants, and trees. In Situ and Ex Situ Conservation Both in situ and ex situ conservation belong to the important set of activities that comprises the management of PGRs. In situ conservation is defined as the conservation of plant or animal species in the habitats in which they had developed naturally (Maxted et al. 1997). In contrast, ex situ conservation is defined as the conservation of a species outside its natural habitat. However, such a definition of ex situ conservation is very broad, belying the complexity of its meaning. For the purposes of this course, the following definition is suggested: Ex situ conservation encompasses all the strategies developed by humans to conserve the germplasm of a plant species outside its natural habitat. It is applied especially to agrobiodiversity. Although in situ conservation is directed mainly at the global biodiversity of wild species through the identification of natural reserves and national parks, some in situ strategies can also be applied to agrobiodiversity. For example, the ancestral wild forms of cultivated species, native landraces, and traditional varieties are conserved in regions that have been centres of traditional agriculture over many years. Some of these strategies include conservation on farms and in household gardens (Tables 1 and 2). When all the variability of a species must be conserved, including its gene pools, then, usually, several ex situ and in situ strategies must be implemented simultaneously and in a complementary fashion. Although both in situ and ex situ conservation have their advantages and disadvantages (Table 3), the most relevant and most discussed disadvantage of ex situ conservation refers to the evolutionary consequences for the species. However, we need to understand that this is precisely what is intended: to stabilize and fix the genetic characteristics of a material so we can take advantage of it as it is. Indeed, it is argued, by taking the germplasm of a species out of its habitat to conserve it in a germplasm bank, its evolutionary development is frozen in terms of the environmental changes that continue to take place in the site from where it was taken. However, other factors exist, such as the disappearance or disturbance of centres of diversity, which encourage us to use an ex situ strategy as the practical solution for saving the still-existing variability. Particularly in recent decades, this type of conservation has become widespread (Hidalgo 1991). When speaking of advantages and disadvantages, we also need to include the relative costs of the two types of conservation. In situ conservation can be very expensive, compared with ex situ conservation, as it requires considerable space to conserve the 14 Module 1, Lesson 2: Conservation: Its Raison d’Être and Strategies Table 3. Relative advantages and disadvantages of ex situ and in situ conservation. Type Advantage Disadvantage Ex situ • Greater diversity of the targeted taxon can be conserved as seed • Easy access for evaluations of resistance to pests and diseases • Easy access for improvement and use • Little maintenance for germplasm conserved over the long term • Freezes evolutionary developments with regard to environmental changes • Genetic diversity is potentially lost with each regeneration cycle In situ • Dynamic conservation with regard to environmental changes • Permits species–pathogen interactions and coevolution • Applicable to many recalcitrant species • Requires active supervision over the long term • Less genetic diversity can be conserved in a single site • Germplasm is not readily available for use • Vulnerable to disasters, natural and/or man-made • Poorly known methodologies or management regimes biological communities of the targeted species. Nevertheless, their different advantages and disadvantages imply that the two types of conservation can complement each other by conserving species that the other cannot. Why ex situ conservation? Applied to cultivated species, ex situ conservation aims to conserve, outside their centre of origin or diversity, both the species and the variability produced during the evolutionary process of domestication. Ex situ conservation can encompass a broad taxonomic spectrum. It is used to protect species, including their wild ancestral species, weedy or regressive forms, and cultivated varieties, particularly those whose original geographic centres of diversity are now under high threat of disturbance or disappearance. What can be conserved ex situ? In theory, all species can be conserved ex situ, provided we can multiply them. We can conserve individual genotypes outside nature, but not the relationships between them and their ecological environment. Traditionally, ex situ conservation has been used for resources important to humans, such as those used for food and agriculture, and whose conservation will provide immediate and future availability, as well as security. FAO’s short Report on the State of the World PGRFA, that is, agrobiodiversity (FAO 1996), mentions 1300 registered collections for plant species, which include about 6.1 million accessions of germplasm conserved ex situ. The FAO WIEWS database indicates that 48% of accessions conserved are cereals, 16% are food legumes, and each of vegetables, roots and tubers, fruits, and forages account for less than 10% of global collections. For Africa, about 50 countries report having 124 collections that conserve more than 350,000 accessions, that is, 6% of the global germplasm. 15 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Among the agricultural species interesting for research and as a basis of human sustenance is a broad range of materials that can be conserved ex situ. These include: • • • • • Wild species, and regressive and weedy forms that belong to cultivated genera and constitute a broad range of important materials for research and crop improvement (Frankel et al. 1995; Prescott-Allen and Prescott-Allen 1988). Wild relatives and regressive forms, which are commonly used as sources of genes to improve traits of interest. They can also provide resistance to diseases and pests. Among the many crops favoured by related wild species, a good example is sugar cane. Modern sugar cane is a complex derived from artificial hybrids, whose pedigree includes the wild species Saccharum spontaneum, which contributed to the crop’s yield, vigour, and resistance to diseases. Other examples are maize, rice, and tomato. Varieties of traditional agriculture, including native landraces, primitive cultivars, and species of cultural importance (e.g., for use in religious ceremonies). Products of scientific improvement programmes, for example, modern and obsolete cultivars, advanced lines, mutants, and synthetic materials. Products of biotechnology and genetic engineering, including transgenic plants, DNA fragments, cloned genes, gene markers, new genetic combinations, silent genes, and chloroplast genomes. Biotechnology and genetic engineering permit the isolation and transfer of genes of plants of agronomic interest, as well as of genes of almost any plant, animal, or bacterium that had not been previously accessed (FAO 1996; Frankel et al. 1995; Rao and Riley 1994). Strategies for Ex Situ Conservation Strategies for ex situ conservation are determined by the biological characteristics of each species, particularly its reproductive system. Indeed, before deciding on any methodology of ex situ conservation, we need to answer the following basic questions in reference to any given species: • • • • • Does it produce sexual seed? If it produces sexual seed, is the seed orthodox or recalcitrant? If it does not produce sexual seed, does it have vegetative reproduction? If it has vegetative reproduction, what is the most suitable propagule for reproducing the species? In addition to producing sexual seed, does it also have vegetative reproduction? The answers to such questions facilitate decision making with regard to the most suitable strategy for the species in question. If the species produces orthodox seeds, then the establishment of a low-temperature seed bank can immediately be considered. However, if the seed is recalcitrant, then it cannot be dried or conserved at low temperatures. Hence, other alternatives need to be sought such as a live field bank or an in vitro bank. If the species does not produce seed, then both live field banks and in vitro banks can be considered. We point out that most cultivated species can be conserved as ex situ collections. 16 Module 1, Lesson 2: Conservation: Its Raison d’Être and Strategies When knowledge on the species is advanced, other additional strategies such as DNA or cryopreservation banks are applied (Table 2). However, these strategies correspond to very different purposes and would be available only to users of advanced technology. Evaluating the Lesson After this lesson, you should understand the reasons for conserving PGRs; conservation strategies, and their advantages and disadvantages; and the PGRs that can be conserved. Before going on to the next lesson you should answer, in your own words, each of the following questions. Write a maximum of one page, per question. • • • • Under the conditions in which you work in your country or region, what materials specifically are being conserved ex situ? What principal benefits does your country or region derive from conserving these materials ex situ? Would the ex situ conservation of this germplasm be advantageous for your country or region? What is your opinion of in situ conservation? If you had to decide on the application of a plan to conserve PGRs, what strategies would you apply? Bibliography Literature cited Dobzhansky T. 1951. Genetics and the origin of species, 3rd ed. Columbia University Press, New York. pp 135–178, 254–275. FAO. 1996. Informe sobre el estado de los recursos fitogenéticos en el mundo. Rome. 75 p. (Also available in English at http://www.fao.org/ag/AGP/AGPS/Pgrfa/pdf/swrshr_e.pdf or http://www.fao.org/iag/AGP/AGPS/Pgrfa/wrlmap_e.htm) Frankel OH; Brown AHD; Burdon JJ. 1995. Conservation of plant biodiversity. Cambridge University Press, UK. 299 p. Hidalgo R. 1991. Conservación ex situ. In Castillo R; Estrella J; Tapia C, eds. Técnicas para el manejo y uso de los recursos genéticos vegetales. Instituto Nacional Autónomo de Investigaciones Agropecuarias, Quito, Ecuador. pp 71–87. IPGRI. (Accessed 28 July 2004) In situ conservation. Available at http://www.ipgri.cgiar.org/ themes/in_situ_project/on_farm/espanolpres.htm Jaramillo S; Baena M. 2000. Material de apoyo a la capacitación en conservación ex situ de recursos fitogenéticos. IPGRI, Cali, Colombia. 209 p. Also available at http:// www.ipgri.cgiar.org/training/exsitu/web/arr_ppal_modulo.htm Maxted N; Ford-Lloyd BV; Hawkes JG, eds. 1997. Plant genetic conservation: the in situ approach. Chapman and Hall, London. 446 p. Prescott-Allen R; Prescott-Allen C. 1988. Genes from the wild: using wild genetic resources for food and raw materials. Earthscan Publications, London. 112 p. 17 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Rao R; Riley KW. 1994. The use of biotechnology for conservation and utilization of plant genetic resources. Plant Genet Resour Newsl 97:3–20. Further reading Chang TT. 1985. Principles of genetic conservation. Iowa State J Res 59(4):325–348. FAO. 1997. The state of the world’s plant genetic resources for food and agriculture. Rome. 510 p. Also available at http://www.fao.org/ag/AGP/AGPS/Pgrfa/pdf/swrfull.pdf or http://www.fao.org/iag/AGP/AGPS/Pgrfa/wrlmap_e.htm FAO. (Accessed 28 July 2004) Web site: Biological diversity in food and agriculture. Available at http://www.fao.org/biodiversity/index.asp?lang=en Ford–Lloyd B; Jackson M. 1986. Plant genetic resources: an introduction to their conservation and use. Edward Arnold Publishers, London. 152 p. Harvis DI; Myer L; Klemick H; Guarino L; Smale M; Brown AHD; Sadiki M; Sthapit B; Hodgkin T. 2000. A training guide for in situ conservation on-farm, version 1. IPGRI, Rome. Also available at http://www.bioversityinternational.org/publications/Pdf/611.pdf Keating M. 1993. The earth summit’s agenda for change: a plain language version of Agenda 21 and the other Rio Agreements. Centre for Our Common Future, Geneva, Switzerland. 70 p. Contributors to this Lesson Rigoberto Hidalgo, Benjamín Pineda, Daniel Debouck, and Mariano Mejía. Next Lesson In the next lesson, you will study the minimum requirements for the ex situ conservation of PGRs. 18 Lesson 3 Minimum Requirements for Ex Situ Conservation Objective To define those minimum requirements for the ex situ conservation of PGRs, taking into account biological, physical, human, and institutional aspects Introduction The conservation of PGRs is a continuous long-term task that implies significant investments of time, personnel, installations, and operation. Such investments should be justified in terms of needs rather than of desire or convenience in conserving a material. The reasons for conserving and targeting specific species should be defined according to logical, scientific, and economic criteria such as need, value, and use of the species, and the feasibility of conserving them (Maxted et al. 1997). Conservation provides maximum benefit when the activities that compose it are closely articulated. The task’s success will be measured in terms of producing the desired result at minimal cost. Requirements for the Ex Situ Conservation of PGRs Conservation should reduce as much as possible the effects of the new environment on the targeted species. Those who conserve germplasm must acquire an in-depth knowledge of the targeted species, that is, their biology, taxonomy, and genetics so that adequate techniques can be developed for representing their genetic variability and ensuring the stability of the original genotypes. Equally important is the documentation of the germplasm because it allows a better understanding and use of the germplasm’s inherent genetic variability, that is, information obtained through, for example, developing passport data, characterization, and evaluation should also be documented. In general, the minimum requirements for the adequate ex situ conservation of PGRs can be grouped as four factors: biological, physical, human, and institutional. Biological requirements • • In-depth knowledge of the species’ biology. This includes mainly the plant’s life cycle and the biology of its reproduction. This information indicates the type of propagule that should be conserved. That is, by knowing if a given species produces sexual seed (true seed), asexual seed (also called vegetative, e.g., stakes, stolons, tubers, and roots), or has the two options, we can determine the type of bank that should be established for it, whether a seed bank, in vivo field bank, or in vitro bank. Whatever type is selected, it should conserve, in the most suitable and practical form possible, the germplasm of that species. Type of pollination. For a species that propagates through sexual seed, knowing its reproduction biology will also help identify the type of pollination of the species, whether it is allogamous or autogamous. This knowledge is essential for correctly managing the germplasm of a species, as it forms the basis on which to establish agronomic protocols for maintaining a field collection of that germplasm. Such 19 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources • • knowledge will also help develop suitable sampling strategies to maintain the original genetic composition of the conserved accessions. Ecological adaptation. Knowing the environmental conditions to which a given species is adapted is also essential. The most relevant data include ranges of altitudes and latitudes, day length, and thermo period. Of lesser importance, although also useful, are the physical conditions of soils and response to excess or deficient water supplies. This information is vital for selecting the most suitable sites for multiplying and/or regenerating the germplasm of the conserved species. Physiology of the reproductive structure. Success in conserving a species depends largely on understanding the physiology of the reproductive structure or organ. These may be botanical seed, meristems, buds, stem pieces, stakes, stolons, tubers, roots, bulbs, or rhizomes. In the case of seeds, principal factors to consider are the determination of orthodox or recalcitrant behaviour, identification of the seed’s cycle of physiological maturity, presence of dormancy, suitable methods for harvesting, and seed health status. Physical requirements The facilities in which materials are to be conserved should guarantee isolation from both environmental factors and pests and diseases. Installations may vary in design and dimensions, depending on the number and size of the samples to be conserved. However, they should have access to a constant supply of electric power and equipment that permit the conditioning, preservation, and regeneration of materials. They should be able to protect the materials from fires, floods, theft, plunder, and other disturbances of public order. The infrastructure to use depends on the type of seed the species has, for example: • • • Species with true sexual seed that can be conserved at low temperatures. Essentially, highly reliable systems are needed to control the temperatures and relative humidity within the rooms where the seeds are conserved. Likewise, suitable seed-drying equipment is needed. To prevent physical damage to the seed, this equipment must be checked before use. Species that propagate through asexual or vegetative seed. These species need suitably selected fields where they will be able to fulfil their normal biological cycles and that the plant part of interest for use will develop normally. To select such a field, the principal variables to consider are altitude, temperatures, day length, rainfall regimes, soil conditions, easy access, and the possibility of continuous use. Species that must be conserved in vitro. The laboratory must be located in a place that will guarantee a stable supply of electric power without the trauma of irregular cuts. The location’s environmental conditions should permit the easy establishment of aseptic conditions, so that pest infestations or disease outbreaks are not sufficiently chronic to endanger the conservation of the germplasm. Requirements for human resources Collections of PGRs should be managed by skilled personnel who are, where possible, from various disciplines (e.g., physiologists, botanists, breeders, and agronomists). They should know the technical aspects of adequately managing the species and the inherent safety procedures of their tasks. Ideally, the collection would depend on a group of 20 Module 1, Lesson 3: Minimum Requirements for Ex Situ Conservation people who are work stable—not exclusively the curator—who can provide continuity to the conservation work, and who are free of political pressures or problems of public order. That is, the personnel usually need to: • • • Be technical personnel with academic training in genetic resources and/or experience in managing germplasm banks or germplasm collections, preferably of cultivated plant species. Be technical personnel who have medium- or long-term continuity, as research on genetic resources usually requires considerable patience and relatively long periods to acquire sound knowledge on the conservation of the species mentioned. Receive cooperation and advice from other professionals such as botanists, taxonomists, geneticists, biologists, physiologists, breeders, agronomists, and refrigeration engineers. Institutional requirements • • • Sustainable institutional, governmental, and political support. Merely creating a germplasm bank does not guarantee the conservation of PGRs of interest to a country, region, province, or given ecosystem. Conservation requires consistent ongoing institutional support in terms of economic, human, and technical resources for maintaining collections and carrying out conservation activities. This aspect is particularly important for the variability of traditional crops in centres of diversity on all continents as, for example, cultivated rice in Africa. According to focus-group interviews with farmers, self-supply of seeds has declined recently, particularly for floating rice. As dependency on local markets for seed supply increases, the farmers are recognizing that a gradual decline of seeds can lead to the total loss of a variety (Synnevåg et al. nd). Policy decisions that distort institutional objectives may be encouraging this situation, as in Latin America, which suffers recurrent difficulties when the institutional mandate differs from that of the conservation unit. A classical example is when the institution is assigned to the Ministry of Agriculture, which, by nature, is a ministry for ‘development’, whereas the conservation unit is, by nature, conservative. Long-term planning. The reasons for conserving targeted species should be defined according to logical criteria. As with any strategic process, the conservation of PGRs implies planning and making decisions based on previous information. Conservation requires the establishment of priorities in terms of types of species to conserve (e.g., species at risk or of interest for food and agriculture), activities to be subsequently carried out with the collected and conserved germplasm, and the resources available to carry them out. Priorities may vary, but the most important objectives are that conservation is for the long term and that the conserved germplasm should be used. Economic resources. Because the nature of ex situ conservation is to ensure perpetuity, it demands the sustained provision over time of economic resources to maintain the physical, human, and technical resources required to upkeep the collections and conduct conservation activities (Koo et al. 2002). Evaluating the Lesson After this lesson, you should know what the minimum requirements are for the ex situ conservation of PGRs, whether these be biological, physical, human, or institutional. 21 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources You have now completed Module 1 of the course but, before going on to the next module, you should answer, in your own words, each of the following questions. Write a maximum of one page per question. • • Under the conditions in which you work in your country or region, what are the minimum requirements for carrying out ex situ conservation? In your opinion and for your case, which of the requirements mentioned in the lesson would need the most attention? Bibliography Literature cited Koo B; Pardey PG; Wright BD. 2002. Endowing future harvests: the long-term costs of conserving genetic resources at the CGIAR Centres. IPGRI, Rome. Maxted N; Ford-Lloyd BV; Hawkes JG, eds. 1997. Plant genetic conservation: the in situ approach. Chapman and Hall, London. 446 p. Synnevåg G; Huvio T; Sidibé Y; Kanouté A. (nd) Farmer indicators for decline and loss of local varieties from traditional farming systems: a case study from northern Mali. Division Landvik of the Norwegian Crop Research Institute, Grimstad, Norway. Further reading Chang TT. 1985. Principles of genetic conservation. Iowa State J Res 59(4):325–348. Harlan JR. 1993. Genetic resources in Africa. In Janick J; Simon JE, eds. New crops. Wiley, New York. Keating M. 1993. The earth summit’s agenda for change: a plain language version of Agenda 21 and the other Rio Agreements. Centre for Our Common Future, Geneva, Switzerland. 70 p. Portères R. 1976. African cereals: eleusine, fonio, black fonio, teff, brachiaria, paspalum, pennisetum, and African rice. In Harlan J, ed. The origins of African plant domestication. Mouton, The Hague, Netherlands. pp 409–452. Contributors to this Lesson Rigoberto Hidalgo, Benjamín Pineda, Daniel Debouck, and Mariano Mejía. Next Module In the lessons of the next module, you will study aspects of procuring and introducing germplasm. 22 Module 2 Supported by the CGIAR Germplasm Acquisition and Introduction (Seeds and Asexual Propagules) General Comments ‘Before collecting, we need to define the targeted species, compile information on them and the sites where they are found, and confirm if financial resources for the expedition are available. We also need to determine a collecting strategy for the samples, envisage how they would be handled in the field so that they survive until they reach the conservation site, and decide how to document them during collection. Furthermore, we also need to request permits from the responsible authorities and respect the regulations established by the country where the collection will be made. Once the permits are obtained, the trip’s logistics can be prepared’ (Jaramillo and Baena 2000). Information on the Module This module contains three lessons, each having its own rapid evaluation and tasks. Objectives When you have completed the module you should be able to: • • • Define the criteria that should be considered when acquiring plant germplasm Describe the procedures for acquiring plant germplasm Describe the legal requirements for access and quarantine, when introducing germplasm Lessons 1. Plant germplasm acquisition: criteria 2. Plant germplasm acquisition: procedures 3. Germplasm introduction: transfer regulations and quarantine measures Bibliography Throughout this module, a bibliography is provided for each section, that is, the General Comments and each Lesson. The bibliographies follow a format of two parts: 1. Literature cited, which includes those references cited in the text itself. Some of these citations were used to develop the original Spanish-language course on ex situ conservation and may therefore appear in Spanish or Portuguese. However, where practical, references to the English versions of the original Spanish-language documents are provided. 2. Further reading, which is a list of suggested readings in the English language, with few exceptions in French. Most of them cover in depth the topics included in this module. A list of Acronyms used in the bibliographies is also given. The idea is to save space by not having to spell out each institution’s full name each time it appears in the references. 23 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Acronyms used in the bibliographies CGRFA COSAVE EPPO EUCARPIA FAO IBPGR ICA ICUC IPGRI IPPC IUCN OIRSA SCBD UNEP Commission on Genetic Resources for Food and Agriculture Comité de Sanidad Vegetal del Cono Sur European and Mediterranean Plant Protection Organization European Association for Research on Plant Breeding Food and Agriculture Organization of the United Nations International Board for Plant Genetic Resources Instituto Colombiano Agropecuario International Centre for Underutilized Crops International Plant Genetic Resources Institute (now Bioversity International) International Plant Protection Convention The World Conservation Union Organismo Internacional Regional de Sanidad Agropecuaria Secretariat of the Convention on Biological Diversity United Nations Environment Programme Literature cited Jaramillo S; Baena M. 2000. Material de apoyo a la capacitación en conservación ex situ de recursos fitogenéticos. IPGRI, Cali, Colombia. pp 9–17 (209 p). Also available at http://www.ipgri.cgiar.org/training/exsitu/web/arr_ppal_module.htm Further reading Allard RW. 1970. Population structure and sampling methods. In Frankel OH; Bennett E, eds. Genetic resources in plants—their exploration and conservation. IBP Handbook No. 11. Blackwell Scientific, Oxford, UK. pp 97–107. Andean Community, General Secretariat. (Spanish version accessed 16 Sept 2004) Treaties and legislation: treaties and protocols; Andean Subregional Integration Agreement, ‘Cartagena Agreement’. Available at http://www.comunidadandina.org/ingles/normativa/ande_ trie1.htm Assy Bah B; Durand-Gasselin T; Engelmann F; Pannetier C. 1989. Culture in vitro d’embryons zygotiques de cocotier (Cocos nucifera L.): Métode, révisée et simplifiée, d’obtention de plants de cocotiers transférables au champ. Oléagineux 44:515–523. Barton JH; Siebeck WE. 1994. Material transfer agreements in genetic resources exchange: the case of the International Agricultural Research Centres. Issues in Genetic Resources No. 1. IPGRI, Rome. Also available at http://www.bioversityinternational.org/ publications/Pdf/109.pdf Brown AHD; Marshall DR. 1995. A basic sampling strategy: theory and practice. In Guarino L; Rao VR; Reid R, eds. Collecting plant genetic diversity: technical guidelines. CAB International, Wallingford, UK. pp 75–91. 24 Module 2: Germplasm Acquisition and Introduction (Seeds and Asexual Propagules) General Comments Engels JMM; Arora RK; Guarino L. 1995. An introduction to plant germplasm exploration and collecting: planning, methods and procedure, follow-up. In Guarino L; Rao VR; Reid R, eds. Collecting plant genetic diversity: technical guidelines. CAB International, Wallingford, UK. pp 31–63. EPPO. 2006. EPPO A1 list of pests recommended for regulation as quarantine pests (version 2006–09). Available at http://www.eppo.org/QUARANTINE/listA1.htm EPPO. 2006. EPPO A2 list of pests recommended for regulation as quarantine pests (version 2006–09). Available at http://www.eppo.org/QUARANTINE/listA2.htm FAO. 1994. The International Code of Conduct for Plant Germplasm Collecting and Transfer. Available at http://www.fao.org/AG/AGp/AGPS/PGR/icc/icce.htm FAO. 1997. International Plant Protection Convention (new revised text approved by the FAO Conference at its 29th Session—November 1997). Available at http://www.fao.org/Legal/ TREATIES/004t2-e.htm Gerard BM. 1984. Improved monitoring test for seed-borne pathogens and pests. In Dickie JB; Linington S; Williams JT, eds. Seed management techniques for genebanks; Proc. Workshop held at the Royal Botanic Gardens, Kew, 6–9 July 1982. IBPGR, Rome. pp 22–42. Glowka L; Burhenne-Guilmin F; Synge H; McNeely JA; Günding L. 1994. A guide to the Convention on Biological Diversity. Environmental Policy and Law Paper No. 30. IUCN, Cambridge, UK. 161 p. Guarino L; Rao VR; Reid R, eds. 1995. Collecting plant genetic diversity: technical guidelines. CAB International, Wallingford, UK. Hawkes JG. 1980. Crop genetic resources field collection manual. IBPGR; EUCARPIA, Rome. 37 p. IPGRI. 1996a. Introduction to collecting; training materials. Rome. Available at http:// www.cgiar.org/ipgri/TRAINING/8-2-1/index.htm (accessed 27 July 2004). IPGRI. 1996b. Planning collecting missions; training materials. Rome. Available at http:// www.ipgri.cgiar.org/training/unit8-1-1/unit8-1-1.htm (accessed 27 July 2004). IPPC, Secretariat. 2006. International standards for phytosanitary measures, 1 to 27 (2006 edition). FAO, Rome. Also available at https://www.ippc.int/servlet BinaryDownloader Servlet124035_ Book_I SPMs_ 2006.pdf? filename=1165395722111_ ISPMs_ 1to27_ 2006_En_with_convention.pdf&refID=124035 Maxted N; Painting K; Guarino L. 1997. Ecogeographic surveys: training materials. IPGRI, Rome. 54 p. Also available at http://www.cgiar.org/ipgri/TRAINING/5-2/index.htm Nath R. 1993. Plant quarantine: principles and concepts. In Rana RS; Nath R; Khetarpal RK; Gokte N; Bisht JS, eds. Plant quarantine and genetic resources management. National Bureau of Plant Genetic Resources of the Indian Council of Agricultural Research, New Delhi, India. pp 19–24. 25 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources SCBD; UNEP. 2003. Convention on Biological Diversity. Available at http://www.biodiv.org/ convention/articles.asp?lg=1 (accessed 6 Sept 2004). Withers LA. 1995. Collecting in vitro for genetic resources conservation. In Guarino L; Rao VR; Reid R, eds. Collecting plant genetic diversity: technical guidelines. CAB International, Wallingford, UK. pp 511–525. Contributors to the Module Rigoberto Hidalgo, Benjamín Pineda, Daniel Debouck, Mariano Mejía, and Graciela Mafla. Next Lesson In the first lesson of the next module, you will study the criteria used for acquiring plant germplasm. 26 Lesson 1 Plant Germplasm Acquisition: Criteria Objectives • • To briefly analyze the process of establishing priorities for acquiring plant germplasm To propose the basic decision-making criteria for acquiring plant germplasm for its conservation Introduction The selection of species for conservation is based on interpretations that, in fact, give rise to subjective valuations. To minimize subjectivity, those who select priority species should sustain their decisions and confirm that the species selected do indeed respond to the proposed objectives. Germplasm may be acquired for many reasons, or combinations of reasons, such as protection, study, improvement, distribution, or completion of an existing collection (Engels et al. 1995). However, an exhaustive analysis should first be done to contribute the elements needed for deciding on what materials to acquire. Establishing priorities is a complex process that includes a range of choices from selecting an analytical method to choose a geographical area to selecting and applying criteria for sampling one population rather than another. However, feasibility—that is, the probability of success of a conservation objective in a given social and political environment—is key to determining priorities and assigning resources. Criteria for Acquisition To acquire germplasm to conserve it, we need to think about how it will be used. This is known as its value of use, that is, its real or potential benefit for food, agriculture, industry, research, or crop improvement (Jaramillo and Baena 2000). A species’ value of use determines the interest, commitment, and priority to conserve it (Jaramillo and Baena 2000; Maxted et al. 1997). Undoubtedly, another important aspect to consider is the level of international commitment, legally binding, of the countries that ratified the Convention on Biological Diversity, which has been international law since 30 December 1993. The Convention governs the conservation of biodiversity, the sustainable use of its components, and the just and equitable participation in the benefits derived from its use (SCBD and UNEP 2003). When germplasm is acquired for its conservation, the criteria related to value of use should be considered. These are listed below: The species’ state of conservation This criterion takes into account that very serious projects have already been established and that excellent germplasm collections are held under ex situ conservation. Acquiring more materials to add to such collections implies making an ex ante evaluation to orient acquisition for such collections. Thus, a species is assessed for the sufficiency of its representation in collections so that conservation activities do not duplicate already existing ones. Furthermore, the quality of available information on the materials should be taken into account. Often, genetic variability or data have not been collected and therefore have never become part of the variability conserved nor of the information kept with the materials. 27 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources For example, maize, rice, and wheat have been collected over decades, whereas other germplasm has not such as Andean roots and tubers like ulluco (Ullucus tuberosus), sweet potato (Ipomoea batatas), isaño (Tropaeolum tuberosum), and arracacha (Arracacia xanthorrhiza), or promising Neotropical fruit trees such as cherimoya (Annona cherimola), papaya (Carica papaya), guava (Psidium guajaba), jaboticaba (Myrciaria cauliflora), cashew (Anacardium occidentale), and borojó (Borojoa patinoi). Even Africa, a very rich region in terms of biodiversity, has a surprisingly low 6% of the world’s total accessions conserved ex situ and only 10% of germplasm banks (FAO 1997). This situation suggests that a lot of germplasm is still to be collected, especially of useful species. For preliminary evaluations, ecogeographic data (Maxted et al. 1997) can be used after careful and duly planned consultations to identify possible collections and assign conservation priorities. Analyzing ecogeographic data is easier when geographic information systems (GIS) are used (IPGRI 2001). A GIS is a system of databases dedicated to the graphic management of geographically referenced spatial data (such as the coordinates of a site or topography), together with logically related non-spatial data (such as the species’ name or its morphological characters). A GIS is also a highly flexible cartographic system that can easily compare a broad range of geographic, ecological, and biological data sets. Once digitized, the cartographic data of maps (often at different scales), aerial photographs, field studies, and remote sensing can be handled and analyzed in various ways. A GIS facilitates understanding of the characteristics of sites where either data had not been recorded during a collection, or data will be used for future collections to locate areas with certain combinations of ecological characteristics. Urgency for conservation The importance of a species for conservation depends on how threatened it is, with priority being given to those in danger of extinction. The level of threat faced by the targeted population can be determined by consulting the IUCN Red List of Threatened Species™, the IUCN’s 2001 list of categories of risk (Glowka et al. 1994; Jaramillo and Baena 2000) (Table 1), or the national entities monitoring at-risk species. Biological importance of the species with respect to other useful species Although some species apparently do not benefit humanity, they interact ecologically with others that do. For example, the interdependence between species of a plant succession of a forest is such that the disappearance of some may endanger the existence of others, including those useful to humans. Contributions in terms of genetic variability The selected species should be genetically different from others already conserved and confirmed to possess a genetic variability that is not being conserved. Although samples should not be acquired of already existing germplasm, it may be appropriate to seek diversity and thereby enrich what is poorly or not represented in the collection. 28 Module 2, Lesson 1: Plant Germplasm Acquisition: Criteria Table 1. Category Categories of plant species in danger of extinction, according to the degree of threat that they face at a given time. Denomination Description 1 Extinct A plant taxon is considered extinct when the individuals composing it are known with certainty to have died. 2 Extinct in the wild A plant taxon is considered extinct in the wild when it is known only as a crop. It is also presumed extinct in the wild when surveys of habitats (exhaustive, at appropriate times, and throughout its historic range) do not record any individuals. 3 Critically threatened When the risk of extinction of a species in the wild and in the immediate future is extremely high. 4 Endangered When the risk of extinction of a species in the wild and in the immediate future is high. 5 Vulnerable, dependent on conservation When removing a species from continuous conservation would expose that species to the category of ‘threatened’ within 5 years. Vulnerable, close to endangered When a species that is not classified as dependent on conservation but is close to being classified as such. Vulnerable, of lesser concern That species which does not fall in either of the previous two subcategories. 6 Species with deficient documentation When the information that exists on a species’ distribution and/or state of its populations does not reliably indicate the risk of extinction to which this species is exposed. A species in this category may be either threatened or at low risk. 7 Not evaluated When a given species has not been evaluated for its level of vulnerability. SOURCES: Glowka et al. (1994); Jaramillo and Baena (2000). Potential usefulness of the species Species that contribute to the satisfaction of basic needs (e.g., food, medicines, and housing) will have greater priority for conservation than others such as ornamentals or those considered as undesirable (e.g., crop weeds). Relative cost of conservation The capacity of the conservation unit for handling materials to be acquired must be considered. ‘Capacity’ refers to the availability and continuity of human, physical, and financial resources to conserve a collection of materials over the medium and long term. Often, ex situ conservation projects start with very ambitious collection activities that do not consider their relatively limited capacity. As a result, within a few years, collections are lost through inadequate and untimely processing. In this sense, collection would not be an end in itself, but would be part of a process. The next stage is to use the acquired variability, which, in fact, depends on the collection’s quality. 29 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources When faced with two equally priority species and a limited budget, cost will determine which will be conserved. The criterion is also applied to the cost of conserving one species versus another or others and to whether the targeted species can be conserved alone or with others of interest. Cultural importance to the community The aesthetic, symbolic, or cultural value of a species for a community (i.e., the role that it fulfils in cultural or religious activities) may determine whether it should be conserved. Examples are plants used as national emblems such as the Quindío wax palm (Ceroxylon quindiuense), Colombia’s national tree (Jaramillo and Baena 2000); the baobab tree (Adansonia digitata), also called Muuyu, emblematic of Africa, a rich reservoir of mythology, folklore and medicines (ICUC 2002) or the forests and jungles that are conserved for their beauty. Evaluating the Lesson After this lesson, you should understand the complexity of prioritizing and establishing criteria for acquiring plant germplasm for conservation. Before beginning the next lesson, complete, in writing, the following tasks: • • Prepare a list of plant species of your country or region that are in danger of extinction according to the categories listed in Table 1 of this lesson. Establish a plan of germplasm acquisition, taking into account the criteria discussed in this lesson. Bibliography Literature cited Engels JMM; Arora RK; Guarino L. 1995. An introduction to plant germplasm exploration and collecting: planning, methods and procedure, follow-up. In Guarino L; Rao VR; Reid R, eds. Collecting plant genetic diversity: technical guidelines. CAB International, Wallingford, UK. pp 31–63. FAO. 1997. The state of the world’s plant genetic resources for food and agriculture. Rome. 510 p. Also available at http://www.fao.org/ag/AGP/AGPS/Pgrfa/pdf/swrfull.pdf or http://www.fao.org/iag/AGP/AGPS/Pgrfa/wrlmap_e.htm Glowka L; Burhenne-Guilmin F; Synge H; McNeely JA; Günding L. 1994. A guide to the Convention on Biological Diversity. Environmental Policy and Law Paper No. 30. IUCN, Cambridge, UK. 161 p. ICUC. 2002. Fruits for the future: baobab. Fact Sheet No. 4. Available at http://www.icuciwmi.org/files/Resources/Factsheets/baobab.pdf IPGRI. 2001. Uso de los SIG en la planificación de colectas de germoplasma. Available at http://www.ipgri.cgiar.org/regions/americas/programas/gisforcollect.htm (accessed 28 July 2004). 30 Module 2, Lesson 1: Plant Germplasm Acquisition: Criteria Jaramillo S; Baena M. 2000. Categorías de especies en peligro de extinción (anexo 3). In Material de apoyo a la capacitación en conservación ex situ de recursos fitogenéticos. IPGRI, Cali, Colombia. Also available at http://www.ipgri.cgiar.org/training/exsitu/web/arr_ppal_ modulo.htm Jaramillo S; Baena M. 2000. Material de apoyo a la capacitación en conservación ex situ de recursos fitogenéticos. IPGRI, Cali, Colombia. pp 9–17 (209 p). Also available at http://www.ipgri.cgiar.org/training/exsitu/web/arr_ppal_modulo.htm (accessed 28 Sept 2004). Maxted N; Painting K; Guarino L. 1997. Ecogeographic surveys: training material. IPGRI, Rome. 54 p. Also available at http://www.cgiar.org/ipgri/TRAINING/5-2/index.htm SCBD; UNEP. 2003. Convention on Biological Diversity. Available at http://www.biodiv.org/ convention/articles.asp?lg=1 (accessed 6 Sept 2004). Further reading Wikipedia. 2007. IUCN Red list. Available at http://en.wikipedia.org/wiki/IUCN_ Red_List Contributors to this Lesson Benjamín Pineda, Rigoberto Hidalgo, Daniel Debouck, and Mariano Mejía. Next Lesson In the next lesson, you will examine methods and procedures for acquiring plant germplasm. 31 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Lesson 2 Plant Germplasm Acquisition: Procedures Objectives • • • To describe the procedures by which germplasm is acquired To indicate how species targeted for conservation are collected To describe the management of acquired germplasm Introduction Once the criteria for germplasm acquisition are defined, the next step is to acquire it, using standard procedures. However, before making the final decision, the curator or collector should remember that if acquisition is done from a another country, international agreements exist that are currently in force such as the Convention on Biological Diversity (CBD), the International Treaty on Plant Genetic Resources for Food and Agriculture (ITPGRFA; FAO–CGRFA 2004), and the FAO and IPGRI’s Technical Guidelines for the Safe Movement of Germplasm (FAO and Bioversity International 1989–2007). These agreements should be considered, especially with respect to the sovereignty of countries over their plant genetic resources (PGRs). The CBD, in accordance with the Charter of the United Nations and the principles of international law, states that nations have the sovereign right to exploit their own resources according to their own environmental policies. However, they also have the obligation to ensure that the activities carried out within their jurisdiction or under their control do not damage the environment of other nations or areas outside their national jurisdiction (United Nations 1993, CBD Article 3: Principle). Furthermore, in recognition of the sovereign rights of nations over their natural resources, the faculty to regulate access to genetic resources corresponds to the national governments and is subject to national legislation (United Nations 1993, CBD Article 15: Access to Genetic Resources). With respect to conservation, exploration, collection, characterization, evaluation, and documentation of PGRs for food and agriculture, Article 5 of the ITPGRFA (FAO–CGRFA 2004) indicates that each contracting party shall follow national legislation in cooperation with the other contracting parties involved. That is, an integrated approach shall be adopted to explore, conserve, and sustainably use PGRs for food and agriculture. In particular, the parties involved shall: • • • • • 32 Conduct studies and inventories of PGRs for food and agriculture, taking into account the situation and degree of variation of existing populations, including those of potential use and, where feasible, evaluate any threat to them. Promote the collection of PGRs for food and agriculture and of relevant information on those that are threatened or are of potential use. Promote or support the efforts of farmers and local communities oriented towards organizing and conserving PGRs for food and agriculture on their farms. Promote the in situ conservation of wild plants related to cultivated ones and of wild plants used for food, including in protected areas; and support, among other things, the efforts of indigenous and local communities. Cooperate in promoting the organization of an effective and sustainable system of ex situ conservation, paying due attention to the need for sufficient documentation, characterization, regeneration, and evaluation; and promote the perfection and transfer Module 2, Lesson 2: Plant Germplasm Acquisition: Procedures • of appropriate technologies to improve the sustainable use of PGRs for food and agriculture. Supervise the maintenance of viability, degree of variability, and genetic integrity of collections of PGRs for food and agriculture. Ways of Acquiring Germplasm Germplasm of interest can be obtained through exploration and collection, exchange, donation, and agreements or conventions. For practical reasons, attempts should be made to obtain the desired materials without resorting to sites of origin. That is, use should be made of donations or exchanges with institutions that hold these materials. When this is not possible, then the materials must be collected from sites where populations of the species of interest exist. Acquisition through exchange and donation The exchange of germplasm is a traditional practice between researchers. Many accessions that today are part of major collections were obtained through exchange or donation. Similarly, materials lost in wars and natural disasters, or through negligence have been recovered by these means. To exchange or receive germplasm by donation, the interested party requests it from the party holding it. Germplasm transfer is made effective through signing an agreement among the parties, in which both the terms of transfer and use of the materials (e.g., conservation, research, or production of commercial varieties) are stipulated. These agreements are known as ‘material transfer agreements in genetic resources exchange’ or MTAs (Barton and Siebeck 1994). Agreements for germplasm transfer should respect the treaties on access to genetic resources held by the countries involved. Because germplasm transfer implies plant health risks, exchange or donation should be made through authorized institutions and according to what is stipulated in the International Plant Protection Convention (FAO 1997). Acquisition through exploration and collection Exploration and collection consist of going to the field to seek and collect the genetic variability of cultivated and wild species that cannot be obtained from germplasm banks, botanic gardens, or other collections (Hawkes 1980; Querol 1988). The reasons for collecting can be various, but the priorities established are based on the species of interest and/or on regions with a broad genetic diversity of the desired material. A collection is justified, for example, when, in a given area, species of interest are endangered, when they are significant for research or use, or when the variability of the targeted species in ex situ collections has been lost or is insufficient. Sometimes, the opportunity for collecting the material can justify collection. Other times, as part of an expedition, germplasm that is not targeted by the mission may be collected, provided that its characteristics will be useful (Engels et al. 1995; IPGRI 1996b; Querol 1988). In any case, the objective for conservation should not be lost sight of. 33 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Before collecting, we need to define the targeted species, compile information on them and the sites where they are found, and confirm if financial resources are available for the expedition. We also need to determine a strategy for collecting samples and handling them in the field so that they will survive until they reach the site of conservation. Finally, we need to know how the samples will be documented during collection. Furthermore, we need to respect international codes and regulations (Box 1) or those established by the country where collection will take place, and request permits from the responsible authorities (Box 2). Box 1 International Code of Conduct for Plant Germplasm Collecting and Transfer (Summary) The International Code of Conduct for Plant Germplasm Collecting and Transfer aims to promote the rational collection and sustainable use of genetic resources, to prevent genetic erosion, and to protect the interests of both donors and collectors of germplasm. The Code, a voluntary one, has been developed by FAO and negotiated by its Member Nations through the Organization’s Commission on Plant Genetic Resources. The Code is based on the principle of national sovereignty over plant genetic resources according with the Convention on Biological Diversity and sets out standards and principles to be observed by those countries and institutions that adhere to it. The Code proposes procedures to request and/or to issue licences for collecting missions, provides guidelines for collectors themselves, and extends responsibilities and obligations to the sponsors of missions, the curators of genebanks, and the users of genetic material. It calls for the participation of farmers and local institutions in collecting missions and proposes that users of germplasm share the benefits derived from the use of plant genetic resources with the host country and its farmers. The primary function of the Code is to serve as a point of reference until such time as individual countries establish their own codes or regulations for germplasm exploration and collection, conservation, exchange and utilization. The Code describes the shared responsibilities of collectors, donors, sponsors, curators and users of germplasm so as to ensure that the collection, transfer and use of plant germplasm is carried out with the maximum benefit to the international community, and with minimal adverse effects on the evolution of crop plant diversity and the environment. While initial responsibility rests with field collectors and their sponsors, obligations should extend to parties who fund or authorize collecting activities, or donate, conserve or use germplasm. The Code emphasizes the need for cooperation and a sense of reciprocity among donors, curators and users of plant genetic resources. Governments should consider taking appropriate action to facilitate and promote observance of this Code by sponsors, collectors, curators and users of germplasm operating under their jurisdiction. The Code recognizes that nations have sovereign rights over their plant genetic resources in their territories and it is based on the principle according to which the conservation and continued availability of plant genetic resources is a common concern of humankind. In executing these rights, access to plant genetic resources should not be unduly restricted. The Code provides a set of general principles which governments may wish to use in developing their national regulations or formulating bilateral agreements on the collection of germplasm. The Code is addressed primarily to governments. All relevant legal and natural persons are also invited to observe its provisions, in particular those dealing with plant exploration and plant collection, agricultural and (Continued) 34 Module 2, Lesson 2: Plant Germplasm Acquisition: Procedures Box 1. (Continued.) botanical activities and research on endangered species or habitat conservation, research institutes, botanical gardens, harvesting of wild plant resources, agroindustry including pharmaceutical plants and the seed trade. The provisions of the Code should be implemented through collaborative action by governments, appropriate organizations and professional societies, field collectors and their sponsors, and curators and users of plant germplasm. FAO and other competent organizations are invited to promote full observance of the Code. The Code should enable national authorities to permit collecting activities within its territories expeditiously. It recognizes that national authorities are entitled to set specific requirements and conditions for collectors and sponsors and that sponsors and collectors are obliged to respect all relevant national laws as well as adhering to the principles of this Code. The Code is to be implemented within the context of the FAO Global System on Plant Genetic Resources, including the International Undertaking and its annexes. In order to promote the continued availability of germplasm for plant improvement programmes on an equitable basis governments and users of germplasm should endeavour to give practical expression to the principles of farmers’ rights. The Code is to be implemented in harmony with: (a) the Convention on Biological Diversity and other legal instruments protecting biological diversity or parts of it; (b) the International Plant Protection Convention (IPPC) and other agreements restricting the spread of pests and diseases; (c) the national laws of the host country; and (d) any agreements between the collector, host country, sponsors and the gene bank storing the germplasm. SOURCE: FAO (1994). Once the permits are obtained, the trip’s logistics are prepared. Exploration and collection are complex activities that put at stake many resources (biological, physical, economic, and human) and require planning (IPGRI 1996a, b; 2001a, b). To understand the objectives of an expedition for collecting PGRs, planning should include the following: • • • • • Regions to visit and crops to collect The human collection team The route to follow The time of the expedition Equipment Acquisition through agreements The germplasm can also be acquired through interinstitutional agreements, where conditions are fixed according to negotiations among the interested parties and which stipulate both the terms of transfer and use of materials. Collecting Targeted Species Once the targeted species are selected, the collector defines the sampling strategy (Brown and Marshall 1995; IPGRI 2000), which will determine how maximum variability will be obtained in the least amount of time. Defining a sampling strategy involves: • • • Locating the collection site or sites Defining the frequency with which samples will be collected, that is, how often will stops be made to collect Defining the methodology by which samples will be collected 35 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Box 2 International Code of Conduct for Plant Germplasm Collecting and Transfer (Chapter III Collectors’ Permits. Articles 6, 7, 8) • Authority for issuing permits. States have the sovereign right, and accept the responsibility, to establish and implement national policies for the conservation and use of their plant genetic resources and, within this framework, should set up a system for the issuance of permits to collectors. Governments should designate the authority competent for issuing permits. This authority should inform proposed collectors, sponsors and the other agencies of the government’s rules and regulations in this matter, and of the approval process to be followed, and of follow-up action to be taken. • Requesting of permits. To enable the permit issuing authority to arrive at a decision to grant or to refuse a permit, prospective collectors and sponsors should address an application to the issuing authority to which they: (a) undertake to respect the relevant national laws; (b) demonstrate knowledge of, and familiarity with, the species to be collected, their distribution and methods of collection; (c) provide indicative plans for the field mission—including provisional route, estimated timing of expedition, the types of material to be collected, species and quantities—and their plans for evaluation, storage and use of the material collected; where possible, the sort of benefits the host country may expect to derive from the collection of the germplasm should be indicated; (d) notify the host country of the kind of assistance, that may be required to facilitate the success of the mission; (e) indicate, if the host country so desires, plans for cooperation with national scholars, scientists, students, non-governmental organizations and others who may assist or benefit from participation in the field mission or its follow-up activities; (f) list, so far as it is known, the national and foreign curators, to whom the germplasm and information is intended to be distributed on the completion of the mission; and (g) supply such personal information as the host country may require. • Granting of permits. The permit issuing authority of the country in which a field mission proposes collecting plant genetic resources should expeditiously: (a) acknowledge the application, indicating the estimated time needed to examine it; (b) communicate to the collectors and sponsors of the proposed collecting mission its decision. In case of a positive decision, conditions of collaboration be established as soon as possible before the mission arrives in the country, or begins fieldwork. If the decision is to prohibit or restrict the mission, whenever possible, the reasons should be given and, where appropriate, an opportunity should be given to modify the application; (c) indicate, when applicable, what categories and quantities of germplasm may or may not be collected or exported, and those which are required for deposit within the country; indicate areas and species which are governed by special regulation; (d) inform the applicant of any restrictions on travel or any modification of plans desired by the host country; (e) state any special arrangement or restriction placed on the distribution or use of the germplasm, or improved materials derived from it; (f) if it so desired, designate a national counterpart for the field mission, and/or for subsequent collaboration; (g) define any financial obligation to be met by the applicant including possible national participation in the collecting team, and other services to be provided; and (h) provide the applicant with the relevant information regarding the country, its genetic resources policy, germplasm management system, quarantine procedures, and all relevant laws and regulations. Particular attention should be drawn to the culture and the society of the areas through which the collectors will be travelling. SOURCE: FAO (1994). 36 Module 2, Lesson 2: Plant Germplasm Acquisition: Procedures • Defining the sample’s optimal size, so that the number of collected seeds and/or propagules will represent the genetic variability available. The collector should not forget to ensure the sample’s representativeness with regard to available genetic variability, as any genetic variability not sampled will never be part of the conserved variability. The sampling strategy is defined according to statistical procedures, requiring that the collector take advice from specialists in this matter. Taking samples during collection To take samples, the collector should bear in mind that collection as such is not separable from other activities. Sampling should consider the biology of reproduction of the targeted material (e.g., allogamous or autogamous plants, plants of intermediate regimes, and plants of asexual reproduction). The collector should take into account the physiology of the conservation organs because, where this is unknown, the collection of seeds or plant parts may not be successful. Regardless of the type of propagule that is collected, the collector must take into account those important aspects that directly influence sampling quality. For instance, the same number of samples should be collected from each plant, preferring those that are in good health and in good physical condition; and the samples’ moisture content and temperatures should be controlled to prevent their drying up or rotting and, thus, affecting their viability (Guarino et al. 1995; Hawkes 1980). If the objective is to collect seeds, then fruits should be harvested because these will keep the seeds viable for longer. Seeds can then be extracted manually. The collected seeds should be fully mature so that they tolerate desiccation without losing viability. For plant parts, fresh propagules and buds should be collected so that they can be regenerated later. Samples can also be collected as complete plants, tubers, rhizomes, or stakes. Plants may be collected in any container, provided that it is safe and easy to transport. Tubers, rhizomes, and stakes should be placed in plastic bags. Another type of sample is to collect in vitro, as discussed below. Sample characteristics The samples acquired should be healthy, represent the diversity targeted and be well documented so that they can enter, without problems, the conservation system of the receiving country and can be later used. The country of origin and, especially, the receiving one, should ensure that the transferred sample is healthy. Accordingly, the germplasm that enters a country must be submitted to sanitary inspection and quarantine (FAO and Bioversity International 1989–2007). In vitro collection In vitro collection consists of taking and transporting in vitro to the laboratory viable plant tissues known as explants (e.g., buds, meristems, and embryos). The explants are extracted, sterilized, and planted onto a culture medium. In vitro collection is practised with species whose samples are difficult to manage such as those of vegetative reproduction or 37 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources non-orthodox (recalcitrant) seed, or when restrictions exist for transporting plant parts. It has been used to collect coconut (Cocos nucifera), cotton (Gossypium spp.), cacao (Theobroma cacao), Prunus, Vitis, grasses, and forages (Withers 1995) and cassava. Handling Acquired Materials according to Germplasm Type Acquired materials are handled according to their germplasm type. Seeds (orthodox or recalcitrant) are identified, conditioned (cleaned and dried), and temporarily stored for later characterization and increase. Plant parts are identified and conditioned, using specific procedures, and processed according to requirements for propagating the materials. The necessary procedures for pre-storage and later temporary storage are also carried out. Care during collection Collections should be carried out carefully, as carelessness or neglect during the activity’s development may damage plant populations and their habitats. This occurs, for example, when large samples are collected from small populations, contaminated germplasm is transported, or species introduced that can displace natives through competition and/or hybridization. Respect of customs, knowledge, and beliefs of the communities dwelling in the collection site will guarantee collaboration during the expedition and in the future. Safety measures should be taken with respect to the personnel who carry out the collection, especially for medical emergencies. Equipment should be handled with care and given due maintenance. Documenting samples during collection Documentation of the samples as they are being collected is fundamental for their identification, characterization, and later use. It should not be forgotten that data that have not been obtained can never be an integral part of either information or of the genetic variability conserved (Painting et al. 1995). Identifying samples in the field is as important as documenting them. In this case, stickers can be placed on them, duly labelling them with sample number, place of origin, collector’s initials, and identification number from the respective recording card. Samples could also be usefully collected for herbaria; photographs taken of the collected materials; and ethnobotanical, ecological, and geographic data (e.g., altitudes, latitudes, heights above sea level, and slopes) also taken (Guarino et al. 1995; Hawkes 1980; Querol 1988). Passport data and collection data are taken during collection and recorded on cards or formats designed for this purpose (see Module 6: Germplasm Documentation). The information includes mainly: • • • • • • 38 The consecutive number of the collection card Genus Species, subspecies, and/or variety of the botanical material Place, province, and country of collection of the sample Name or names of the collector or collectors Collection date Module 2, Lesson 2: Plant Germplasm Acquisition: Procedures Conditioning and storing samples during collection The collected samples should be kept viable until they arrive at the conservation site. They must be conditioned to prevent their damage or contamination. Conditioning includes cleaning the samples, drying them if they are orthodox seeds, or maintaining their moisture content if they are plant parts or recalcitrant or intermediate seeds. Cleaning consists of removing all contaminants from the samples such as stones and soil residues; insects; seeds that are infected, damaged, or are from other species; and plant residues. Drying consists of reducing the moisture content of seeds for storage, using silica gel, equipment for circulating dry air, or spreading them out in thin layers in the shade, in cool and airy places. The conditioned samples should be stored until they are taken to the conservation site. Orthodox seeds are stored in cloth bags, away from light, or in containers that permit the circulation of dry air. Recalcitrant and intermediate seeds and plant parts should be maintained in humidified containers such as newsprint or paper towels, sawdust or sand, or humid inflated plastic bags, changing the air frequently. They can also be stored in polystyrene iceboxes or car refrigerators. To prevent materials losing their viability during collection, partial shipments of samples should be made where possible to the conservation site. The materials must be sent in accordance with the International Code of Conduct for Plant Germplasm Collecting and Transfer (FAO 1994), being clearly identified and accompanied by instructions for handling and documentation. Temporary storage After conditioning, the seeds should be stored at the established conservation site to ensure their availability for increase, characterization, and other procedures characteristic of ex situ conservation. Evaluating the Lesson After this lesson, you should be familiar with the procedures involved in acquiring germplasm, collecting samples of species targeted for conservation, and managing the acquired germplasm. Before going on to the next lesson, prepare a brief, in your own words, on the following themes. Write a maximum of one page per item. • • • If you have had personal experience in applying procedures for acquiring germplasm, then: – Briefly describe your experience, including purposes, achievements, and the difficulties you had. – Prepare a list of suggestions that would be useful for other colleagues who have not had the experience, but are interested in taking it up. If you have not had the experience or have not participated in explorations and collections, briefly describe those procedures that would be the most relevant for germplasm acquisition, including aspects of sample management and documentation. Carefully read Box 3, form an opinion on the subject, and suggest the merits or drawbacks of its application. 39 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Box 3 International Code of Conduct for Plant Germplasm Collecting and Transfer (Chapter IV Responsibilities of Collectors. Articles 9, 10, 11) • Pre-collection. Upon arrival in the host country, collectors should acquaint themselves with all research results, or work in progress in the country, that might have a bearing on the mission. Before fieldwork begins, collectors and their national collaborators should discuss, and to the extent possible, decide on practical arrangements including: (i) collecting priorities, methodologies and strategies, (ii) information to be gathered during collection, (iii) processing and conservation arrangements for germplasm samples, associated soil/symbiont samples, and voucher specimens, and (iv) financial arrangements for the mission • During collection. Collectors should respect local customs, traditions, and values, and property rights and should demonstrate a sense of gratitude towards local communities, especially if use is made of local knowledge on the characteristics and value of germplasm. Collectors should respond to their requests for information, germplasm or assistance, to the extent feasible. In order not to increase the risk of genetic erosion, the acquisition of germplasm should not deplete the populations of the farmers’ planting stocks or wild species, or remove significant genetic variation from the local gene pool. When collecting cultivated or wild genetic resources, it is desirable that the local communities and farmers concerned be informed about the purpose of the mission, and about how and where they could request and obtain samples of the collected germplasm. If requested, duplicate samples should be also left with them. Whenever germplasm is collected, the collector should systematically record the passport data, and describe in detail the plant population, its diversity, habitat and ecology, so as to provide curators and users of germplasm with an understanding of its original context. For this purpose, as much local knowledge as possible about the resources (including observations on environmental adaptation and local methods and technologies of preparing and using the plant) should be also documented; photographs may be of special value. • Post-collection. Upon the completion of the field mission, collectors and their sponsors should: (a) process, in a timely fashion, the plant samples, and any associated microbial symbionts, pests and pathogens that may have been collected for conservation; the relevant passport data should be prepared at the same time; (b) deposit duplicate sets of all collections and associated materials, and records of any pertinent information, with the host country and other agreed curators; (c) make arrangements with quarantine officials, seed storage managers and curators to ensure that the samples are transferred as quickly as possible to conditions which optimize their viability; (d) obtain, in accordance with the importing countries’ requirements, the phytosanitary certificate(s) and other documentation needed for transferring the material collected; (e) alert the host country and the FAO Commission on Plant Genetic Resources about any impending threat to plant populations, or evidence of accelerated genetic erosion, and make recommendations for remedial action; and (f) prepare a consolidated report on the collecting mission, including the localities visited, the confirmed identifications and passport data of plant samples collected, and the intended site(s) of conservation. Copies of the report should be submitted to the host country’s permit issuing authority, to national counterparts arid curators, and to the FAO for the information of its Commission on Plant Genetic Resources and for inclusion in its World Information and Early Warning System on PGR. SOURCE: FAO (1994). 40 Module 2, Lesson 2: Plant Germplasm Acquisition: Procedures Bibliography Literature cited Barton JH; Siebeck WE. 1994. Material transfer agreements in genetic resources exchange: the case of the International Agricultural Research Centres. Issues in Genetic Resources No. 1. IPGRI, Rome. Also available at http://www.bioversityinternational.org/ publications/Pdf/109.pdf (accessed 27 July 2004). Brown AHD; Marshall DR. 1995. A basic sampling strategy: theory and practice. In Guarino L; Rao VR; Reid R, eds. Collecting plant genetic diversity: technical guidelines. CAB International, Wallingford, UK. pp 75–91. Engels JMM; Arora RK; Guarino L. 1995. An introduction to plant germplasm exploration and collecting: planning, methods and procedure, follow-up. In Guarino L; Rao VR; Reid R, eds. Collecting plant genetic diversity: technical guidelines. CAB International, Wallingford, UK. pp 31–63. FAO. 1994. The International Code of Conduct for Plant Germplasm Collecting and Transfer. Available at http://www.fao.org/AG/AGp/AGPS/PGR/icc/icce.htm FAO. 1997. International Plant Protection Convention (new revised text approved by the FAO Conference at its 29th Session–November 1997). Available at http://www.fao.org/Legal/ TREATIES/004t2-e.htm FAO; Bioversity International. 1989–2007. Technical guidelines for the safe movement of germplasm. Rome. Available at http://www.bioversityinternational.org/Themes/ Genebanks/Germplasm_Health/index.asp (with reference to various crops). FAO–CGRFA. 2004. The International Treaty on Plant Genetic Resources for Food and Agriculture. Available at ftp://ftp.fao.org/ag/cgrfa/it/ITPGRe.pdf or http://www.fao. org/ag/cgrfa/itpgr.htm Guarino L; Rao VR; Reid R, eds. 1995. Collecting plant genetic diversity: technical guidelines. CAB International, Wallingford, UK. Hawkes JG. 1980. Crop genetic resources field collection manual. IBPGR; EUCARPIA, Rome. 37 p. IPGRI. 1996a. Introduction to collecting: training materials. Rome. Available at http://www.cgiar.org/ipgri/TRAINING/8-2-1/index.htm (accessed 27 July 2004). IPGRI. 1996b. Planning collecting missions: training materials. Rome. Available at http:// www.ipgri.cgiar.org/training/unit8-1-1/unit8-1-1.htm (accessed 27 July 2004). IPGRI. 2001a. Planificación de una colecta de germoplasma. Available at http:// www.ipgri.cgiar.org/training/unit8-2-1/8-2-1ESDiapositivas.pdf (accessed 28 July 2004). IPGRI. 2001b. Uso de los SIG en la planificación de colectas de germoplasma. Available at http://www.ipgri.cgiar.org/regions/americas/programas/gisforcollect.htm (accessed 28 July 2004). 41 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Jaramillo S; Baena M. 2000. Componentes de una estrategia de muestreo y pasos para definirla (anexo 4). In Jaramillo S; Baena M. 2000. Material de apoyo a la capacitación en conservación ex situ de recursos fitogenéticos. IPGRI, Cali, Colombia. Also available at http://www.ipgri.cgiar.org/training/exsitu/web/arr_ppal_modulo.htm (accessed 28 Sept 2004). Painting KA; Perry MC; Denning RA; Ayad WG. 1995. Guidebook for genetic resources documentation. IPGRI, Rome. Also available at http://www.bioversityinternational.org/ Publications/Pdf/432.pdf Querol D. 1988. Recursos genéticos, nuestro tesoro olvidado: Aproximación técnica y socioeconómica. Industrial Gráfica, Lima, Peru. 218 p. United Nations. 1993. No. 30619–Multilateral–Convention on Biological Diversity (with annexes): concluded at Rio de Janeiro on 5 June 1992, registered 29 December 1993. Treaty Series, vol. 1760, I-30619, pp 143–382. Available at http://www.biodiv.org/doc/legal/ cbd-un-en.pdf Withers LA. 1995. Collecting in vitro for genetic resources conservation. In Guarino L; Rao VR; Reid R, eds. Collecting plant genetic diversity: technical guidelines. CAB International, Wallingford, UK. pp 511–525. Further reading Allard RW. 1970. Population structure and sampling methods. In Frankel OH; Bennett E, eds. Genetic resources in plants—their exploration and conservation. IBP Handbook No. 11. Blackwell Scientific, Oxford, UK. pp 97–107. Assy Bah B; Durand-Gasselin T; Engelmann F; Pannetier C. 1989. Culture in vitro d’embryons zygotiques de cocotier (Cocos nucifera L.): Métode, révisée et simplifiée, d’obtention de plants de cocotiers transférables au champ. Oléagineux 44:515–523. SCBD. 2005. Handbook of the Convention on Biological Diversity, including its Cartagena Protocol on Biosafety, 3rd ed. Montreal, Canada. 1493 p. Also available at http:// www.cbd.int/doc/handbook/cbd-hb-all-en.pdf SCBD; UNEP. 2003. Convention on Biological Diversity. Available at http://www.biodiv.org/ convention/articles.asp?lg=1 (accessed 6 Sept 2004). Contributors to the Module Rigoberto Hidalgo, Benjamín Pineda, Daniel Debouck, and Mariano Mejía. Next Lesson In the next lesson, you will learn about the legal requirements of access and quarantine, with reference to germplasm introduction. 42 Lesson 3 Germplasm Introduction: Transfer Regulations and Quarantine Measures Objectives • • • To indicate the requisites implied in germplasm transfer To describe the procedures for transferring plant germplasm To describe examples of agreements on plant protection and the factors that must be considered when adopting quarantine measures Introduction For national and international crop improvement programmes, which constantly need germplasm, its collection, conservation, use, and global distribution are essential. However, because of its very nature, germplasm can be affected by pests and pathogens which are not globally distributed or reported that threaten their integrity. This resource, which constitutes a treasure, merits conservation and protection against exotic organisms that have high destructive potential. Moving germplasm from one country to another, or from one region to another within a country, involves plant health risks. Such movement is therefore subject to legislation. The parties interested in moving a given germplasm agree on terms of transfer to ensure its legality and that the transported germplasm is healthy. Such agreements must adjust to international regulations that regulate access, safe transfer, and the rights and responsibilities of each party with respect to the use of the transferred germplasm. Before introducing germplasm to a country, requirements must first be met. These include: • • • • Submitting an official application to the entity responsible for the PGRs of the country in which the germplasm is to be acquired Observing requisites according to established regulations Signing agreements on the transfer of PGRs Determining specific procedures for moving or transferring the materials. Once the transactions for acquisition are fulfilled and the germplasm is available, it should be transferred and introduced into the respective bank. When transfer must occur across country borders, then that transfer or movement is achieved through agreements that adjust to current international regulations as expressed in instruments such as the Convention on Biological Diversity (CBD) (Glowka et al. 1994), the International Code of Conduct for Plant Germplasm Collecting and Transfer (ICCPGCT), the International Plant Protection Convention (IPPC), FAO and IPGRI’s Technical Guidelines for the Safe Movement of Germplasm (FAO and Bioversity International 1989–2007). When the germplasm is acquired from within the country, then transfer to the respective banks is subject to regulations established by that country. Germplasm Transfer Germplasm transfer is effected by the interested parties signing an agreement. The agreement should stipulate both the terms of transfer and use of the material (e.g., conservation, research, or production of commercial varieties). An example of these agreements for the transfer of genetic resources (Barton and Siebeck 1994) is that established between the International Center for Tropical Agriculture (CIAT), FAO, and the CGIAR (Figure 1). 43 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources CGIAR MATERIAL TRANSFER AGREEMENT FOR PLANT GENETIC RESOURCES HELD IN TRUST BY CIAT1 The plant genetic resources (hereinafter referred to as the “material”) contained herein are being furnished by the international Centre for Tropical Agriculture (CIAT) under the following conditions: CIAT is making the material described in the attached list available as part of its policy of maximizing the utilization of material for research, breeding and training. The material was either developed by CIAT; or was acquired prior to the entry into force of the Convention on Biological Diversity; or if it was acquired after the entering into force of the Convention on Biological Diversity, it was obtained with the understanding that it could be made available for any agricultural research, breeding and training purposes under the terms and conditions set out in the agreement between CIAT and FAO dated 26 October 1994. The material is held in trust under the terms of this agreement, and the recipient has no rights to obtain Intellectual Property Rights (IPRs) on the material or related information. The recipient may utilize and conserve the material for research, breeding and training and may distribute it to other parties provided such other parties accept the terms and conditions of this agreement.2 The recipient, therefore, hereby agrees not to claim ownership over the material, nor to seek IPRs over that material, or its genetic parts or components, in the form received. The recipient also agrees not to seek IPRs over related information received. The recipient further agrees to ensure that any subsequent person or institution to whom he/she may make samples of the material available, is bound by the same provisions and undertakes to pass on the same obligations to future recipients of the material. CIAT makes no warranties as to the safety or title of the material, nor as to the accuracy or correctness of any passport or other data provided with the material. Neither does it make any warranties as to the quality, viability, or purity (genetic or mechanical) of the material being furnished. The phytosanitary condition of the material is warranted only as described in the attached phytosanitary certificate. The recipient assumes full responsibility for complying with the recipient nation’s quarantine and biosafety regulations and rules as to import or release of genetic material. Upon request, CIAT will furnish information that may be available in addition to whatever is furnished with the material. Recipients are requested to furnish CIAT with related data and information collected during evaluation and utilization. The recipient of material provided under this MTA is encouraged to share the benefits accruing from its use, including commercial use, though the mechanisms of exchange of information, access to and transfer of technology, capacity building and sharing of benefits arising from commercialization. CIAT is prepared to facilitate the sharing of such benefits by directing them to the conservation and sustainable use of the plant genetic resources in question, particularly in national and regional programmes in developing countries and countries with economies in transition, especially in centres of diversity and the least develop-countries. The material is supplied expressly conditional on acceptance of the terms of this Agreement. The recipient’s acceptance of the material constitutes acceptance of the terms of this Agreement. 1. The attention of the recipient is drawn to the fact that the details of the MTA, including the identity of the recipient, will be made publicly available. 2. This does not prevent the recipients from releasing the material for purposes of making it directly available to farmers or consumers for cultivation, provided that the other conditions set out in this MTA are complied with. Figure 1. Material Transfer Agreement between FAO, CIAT, and the CGIAR. 44 Module 2, Lesson 3: Germplasm Introduction: Transfer Regulations and Quarantine Measures Germplasm transfer agreements should respect the treaties on access to genetic resources that the involved countries have. As germplasm transfer implies plant health risks, any exchange or donation should be made through authorized institutions and according to what is stipulated in the International Plant Protection Convention (FAO 1997). In this case, each country commits itself to adopting the legislative, technical, and administrative requisites to act effectively and jointly to prevent the dissemination and introduction of pests of plants and plant products and to promote appropriate measures to combat them. Procedures for Plant Germplasm Transfer Before importing All plant materials and plant products and by-products should meet certain plant health requirements for their importation. An exception would be those products that, by their physical constitution and the processing to which they had been submitted, do not pose plant health risk. In general, before shipping, interested parties should present before each country’s official agency responsible for preventing plant health risks an application, permit, or plant health importation certificate. Figure 2 shows an example. In some cases such as that of Colombia, to import wild flora, an approval issued by the Ministry of the Environment must also be attached to the request. The movement of cassava germplasm from African countries to America is not allowed in the form of vegetative plant parts, unless they are in vitro. Such measures help prevent the introduction of pests or diseases that are, as yet, unreported in the respective countries. Once the interested party obtains the requisite plant health documents for importing plant materials, that party requests its registration of importation from the respective ministry or office. A copy is sent to the exporting country so that the health authority there can issue the plant health certificate (Figure 3) in accordance with the requirements demanded by the receiving country. The requisite plant health document is issued per species and per shipment, and has a determined validity for the respective country. For Colombia, such a document is valid for 90 days. The Colombian Institute of Agriculture and Livestock (ICA) is empowered to suspend it should a quarantine pest be found that would affect national production. Procedures after acquisition According to the regulations of the ICCPGCT, once the field mission is concluded, the collectors and their sponsors should: • • • Submit, on a timely basis, their samples of plants and any associated symbiont, pest, and microbial pathogen that may have been collected to treatment for conservation; the pertinent passport data must be prepared at the same time. Deposit duplicates of all collections and associated materials, as well as records of all corresponding information, in the host country and with agreed-upon persons in charge. Arrange with quarantine officials, directors, and those in charge of seed deposits to ensure that the samples are transferred with the greatest possible speed to a place where conditions for viability are optimal. 45 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Logotype of the Andean Community Country logotype Name and logotype of the official phytosanitary service PHYTOSANITARY CERTIFICATE FOR IMPORT Certificate No. 1. Importer or proprietor of the import --Name or business name: --Commercial or residential address: 2. Name of product (plant/plant product/regulated article) --Scientific name (where original): 3. Quantity, weight, and type of container (where applicable) --In kilos or units: 4. Origin and, where applicable, place of production: 5. Country of origin/reexportation: 6. Point of shipping or departure: 7. Point of entry or entry customs: 8. Means of transport: 9. Use or destiny: 10. Phytosanitary requisites: 11. Observations: 12. Place and date of issue of permit: 13. Name, position, and signature of functionary authorized to issue permit: 14. Seal or security code (optional): ********************************************* Valid for 90 calendar days for the entry of the product, starting from the day of issue and for only one shipment. --Whatever amendment or addition shall invalidate this document --The Competent Authority reserves the right to annul the validity of this Phytosanitary Permit or Document for Import on the appearance of quarantine pests in the exporting Country --This document is not transferable. Figure 2. An example of a plant health certificate for import (taken from the Andean Community 2004). 46 Module 2, Lesson 3: Germplasm Introduction: Transfer Regulations and Quarantine Measures PGT 3–o–BIJ1t1 agriculture ARG13/007 Department: Agriculture REPUBLIC OF SOUTH AFRICA PHYTOSANITARY CERTIFICATE PLANT PROTECTION ORGANISATION OF THE REPUBLIC OF SOUTH AFRICA To: Plant protection organisation(s) of ................................................................................................................................................................. I. DESCRIPTION OF CONSIGNMENT Name and address of exporter ............................................................ TI ............................................................................................................ Declared name and address of consignee ............................................................................................................................................................ ........................................................................................................................................................................................................................... Number and description of packages ................................................................................................................................................................... ........................................................................................................................................................................................................................... E LL D Distinguishing marks .......................................................................................................................................................................................... ........................................................................................................................................................................................................................... C N A E Place of origin ..................................................................................................................................................................................................... C Declared means of conveyance ........................................................... Declared point of entry ............................................................................ Name of produce, quantity declare and purpose .................................................................................................................................................. Botanical name of plants .................................................................................................................................................................................... This is to certify that the plants, plant products or other regulated articles, described herein have been inspected and/or tested according to appropriate official procedures and are considered to be from the quarantine pests specified by the importing contracting party and to conform to the current phytosanitary requirements of the importing contracting party, including those for regulated nonquarantine pests. They are regarded to be practically free from other pests. II. ADDITIONAL DECLARATION ........................................................................................................................................................................................................................... ........................................................................................................................................................................................................................... ........................................................................................................................................................................................................................... ........................................................................................................................................................................................................................... E LL D ........................................................................................................................................................................................................................... E C N ........................................................................................................................................................................................................................... III. DISINFESTATION AND/OR DISINFECTION TREATMENT CA Date ................................................................................................... Treatment ............................................................................................... Chemical (active ingredient) ............................................................... Duration and temperature ...................................................................... Concentration .................................................................................... Additional information ............................................................................ Place of issue ................................................................ Name of authorised officer .................................................. Figure 3. An example of a phytosanitary certificate from the Republic of South Africa. 47 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources • • • Obtain, in accordance with requirements of the importing countries, the plant health certificate or certificates and other documentation needed to transfer the material collected. Warn the host country and the FAO’s Commission on Genetic Resources for Food and Agriculture (CGRFA) of any imminent threat or sign of rapid genetic erosion with regard to plant populations, and to formulate recommendations to remedy the situation. Prepare a joint report on the collection mission, indicating places visited, identifications confirmed, passport data of the samples of collected plants, and the place or places to be used for their conservation. Copies of the report shall be delivered to the authority that grants permits for the host country, to national counterparts and persons in charge, and to FAO. This last shall report to the CGRFA and include the report in the World Information and Early Warning System on PGRFA (WIEWS; FAO 1994). Nationalization On their arrival, the imported plant materials must be accompanied by their respective plant health certificates issued by the health authority of the country of origin. These certificates should be adjusted to the plant health requisites indicated in the plant health documents for importation. The importer should request plant health inspection from the inspection and quarantine service of the plant health organization in the place of entry (sea or river port, airport, or border control). The importer should also present the original plant health certificates from the country of origin and the plant health documents that accompany the materials. Once the documentation has been reviewed and the inspection conducted, the corresponding plant health certificate for nationalization will then be issued, or not, as the case may be. Procedures after nationalization After the material has been introduced into the country, the respective bank determines the risk of transporting plant pathogens during the germplasm’s movement. It establishes flow charts that show where quarantine inspection and plant health control play essential roles in the procedures (Figures 4 and 5). The effectiveness of these measures depends on the seriousness and professionalism that had been applied, logistical support, availability of skilled technical personnel, and availability of specific information on plant pathogens or pests and their potential risk. Political and institutional will to apply the measures is also necessary if regional and international agriculture is to be protected. The simple inspection or visual examination that is frequently practised by quarantine services should be regarded as insufficient for keeping pathogens or pests out of a country. Necessarily, quarantine often consists of officially confining the regulated articles (e.g., plant materials) for observation or research, or for inspection, testing, and/or additional treatment. This is the most effective measure for control and widely applied throughout the world. It encompasses all those activities designed to prevent the introduction and/or dissemination of quarantine pests or ensure their official control. Quarantine is a governmental measure to control the entry of plants, plant parts, or any plant product, soil samples, and live organisms into a given country to prevent the introduction or dissemination of pests, pathogens, and weeds (Nath 1993). It includes inspection to detect pests and pathogens, treatment or cleaning of the samples, and their certification and release if no danger exists, or their destruction if they are highly contaminated or no technology is available to clean them. 48 Module 2, Lesson 3: Germplasm Introduction: Transfer Regulations and Quarantine Measures Collected germplasm Step Cleaning Drying Registration Pre-export Pre-export quarantine Treatment Shipment Post-entry quarantine Post-entry Decisions on quarantine control measures Short-term storage Registration, cleaning, germination, moisture-content determination, drying, seed processing, packaging, storage, monitoring Long-term storage Herbarium samples Shipments Registration, cleaning, germination, moisture-content determination, drying, seed processing, packaging, storage, monitoring Quarantine procedures Figure 4. Flow chart for managing collected germplasm (from Gerard 1984). The principal risk in moving germplasm is the transfer or accidental introduction of pests and pathogens associated with the plant materials. To minimize this risk, effective procedures must be applied to guarantee that the mobilized material is free of pests of quarantine interest. ‘Quarantine pest’ is understood to have economic importance for the area at risk, even if the pest does not exist or, if it does exist, is confined and under official control. A quarantine pest may be of any species, race, or biotype of any harmful animal or plant, or pathogen for plants or plant products (IPPC 1995). A very useful tool in minimizing risk is the Technical Guidelines for the Safe Movement of Germplasm by FAO and Bioversity International (1989–2007), which deal with many species or groups of species. Plant Protection Agreements The reduction of plant health risks in the international movement of plants and plant products is a matter of vital importance, with the responsibility belonging to countries. According to the IPPC, to combat pests of plants and their products, each country must take the steps necessary to establish, in the best possible way, a national organization of plant protection. The responsibilities of such an organization would include: 49 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Seed samples from gene banks Registration and inspection Soil preparation Seed treatments Seeds sown under screenhouse conditions Post-germination inspection Healthy plants Diseased plants Transplant to field plots Transplant to screenhouse Protection against pathogens and pests Curative treatments for plants to prevent pathogen dispersion Periodic crop inspection Propagation from healthy plant tissues Harvest of F1 seed Shipment to gene banks for post-entry registration Protection against infections from other pathogens Periodic crop inspection Harvest of F1 seed Shipment to gene banks for post-entry registration Figure 5. Flow chart for multiplication in germplasm banks (from Gerard 1984). • • • • 50 The issue of certificates based on the plant health regulations of the importing country for shipments of plants, plant products, and other regulated articles. The monitoring of cultivated plants, including in cultivated lands (e.g., fields, plantations, nurseries, gardens, greenhouses, and laboratories), and wild flora; and of plants and plant products in storage or transport. The specific purpose is to report on the presence, outbreak, or dissemination of pests, and combat them. Reports may also have to be presented on request. The inspection of shipments of plants and plant products that circulate in the international traffic. Where appropriate, other regulated articles may be inspected, particularly to prevent the introduction and/or dissemination of pests. The disinfestation or disinfection of shipments of plants, plant products, and other regulated articles that circulate in international traffic to meet plant health requirements. Module 2, Lesson 3: Germplasm Introduction: Transfer Regulations and Quarantine Measures • • • • The protection of at-risk areas and designation, maintenance, and monitoring of areas free of pests and of areas with limited prevalence of pests. The analysis of pest risk. The maintenance of plant health security of shipments after they have been certified in terms of composition, substitution, and re-infestation before export. Personnel training and education. Plant protection organizations and their guidelines At present, following the IPPC guidelines, Latin America has several organizations responsible for plant protection. These include: • • • The Plant Protection Committee of the Southern Cone (COSAVE, its Spanish acronym), which is a regional organization created through agreements among the governments of Argentina, Brazil, Chile, Paraguay, and Uruguay. The Andean Agricultural and Livestock Health System, which forms part of the Andean Subregional Integration Agreement (also known as the ‘Cartagena Agreement’), of the Andean Community, which is constituted by Bolivia, Colombia, Ecuador, Peru, and Venezuela. The International Regional Organization for Plant and Animal Health (OIRSA, its Spanish acronym), formed by Mexico, Guatemala, Belize, El Salvador, Honduras, Nicaragua, Costa Rica, Panama, and the Dominican Republic. It was created to advise, coordinate, and cooperate with national services for agricultural and livestock quarantine of the ministries of agriculture and livestock of the member countries. The goal is to prevent, where possible, the introduction and establishment of new pests in the region. North America and Europe also have plant protection organizations: • • The North American Plant Protection Organization (NAPPO) is a regional organization of plant protection that coordinates efforts between Canada, USA, and Mexico. It aims to protect these countries’ plant resources against the entry, establishment, and dispersion of pests of regulated plants, while facilitating trade among them and with other regions. For Europe, the entity responsible for plant protection is the European and Mediterranean Plant Protection Organization (EPPO). It has 45 members and covers almost all countries in Europe and the Mediterranean Region. EPPO aims to protect plants, develop international strategies against pest introduction and dissemination, and promote effective and safe methods of control. Plant protection organizations in Africa include: • • Inter-African Phytosanitary Council (IAPSC) with 54 members National Plant Protection Organisation (NPPO) of the Republic of South Africa The above-mentioned organizations have established and harmonized plant health standards and procedures that should be taken into account when moving germplasm. Some of these are: • • Decision 515 of the Andean Agricultural and Livestock Health System (Comunidad Andina 2004) Plant Health Requirements Harmonized with Category of Risk for the Entry of Plant Products (COSAVE 2003) 51 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources • • • • Model Manual for the Application of Technical Measures of Agricultural and Livestock Quarantine (OIRSA 2004) General Procedures for Plant Quarantine (Costa Rica) (Servicio Fítosanitario del Estado 2004) Guidelines for the Import and Export of Plants, and Products and By-products of Plant Origin (Colombia) (ICA 2004) List of Pests Recommended for Regulation as Quarantine Pests (EPPO 2006) The IPP has an special web page dedicated to Africa in which there are about 12 documents, mostly handouts very useful to familiarize with the phytosanitary situation on this continent. Furthermore, international organizations such as IPGRI (now Bioversity International) have published technical guidelines for moving germplasm of Acacia spp., Allium spp., edible aroids, sweet potato, cacao, sugar cane, small grains, citrus fruits, coconut, Eucalyptus spp., stone-fruit trees, strawberries, legume grains, Musa spp., yam, potato, Pinus spp., vanilla, grape vine, and cassava (FAO and IPGRI 2004; FAO and Bioversity International 1989– 2007). The guides contain useful information for germplasm transfer. Factors to consider when adopting quarantine measures To guarantee safe germplasm movement in international exchange and adopt quarantine safety measures, the following must be done: • • • • • • Estimate the ‘favourability’ of risk through the risk-to-benefit ratio. The ‘favourability’ of an importation is determined by assessing the associated risk against the benefit of the importation. The benefit should exceed the potential cost of the adverse consequences if a pest or pathogen of quarantine importance enters and becomes established. Estimate the cost-to-benefit ratio to determine if the benefit derived from implementing a quarantine activity or programme exceeds the cost of applying it. Assess the pathogen type of a given pest in terms of its potential for direct destruction or for rapid epidemiological dispersion, even if it is not present in the area targeted for protection or is restricted to areas under effective control. Consider the regions from where the germplasm proceeds, with special reference to centres of origin, experiment stations, or other places generating risk. Consider the susceptibility of materials, including wild ones, to ranges of pathogens. Get acquainted—and this is crucial—with all the existing laws and regulations followed by the importing country. Such knowledge will prevent the destruction of samples through ignorance of simple bureaucratic procedures. Evaluating the Lesson After this lesson, you should be familiar with the legal requirements involved in germplasm transfer, transfer procedures, and plant health agreements. You should also have some understanding of the issues involved in adopting quarantine measures related to the safe transfer of germplasm. This lesson finalizes Module 2 of the course but, before going on to the next module, you should prepare a brief in your own words on the following themes. Write a maximum of one page for each theme. 52 Module 2, Lesson 3: Germplasm Introduction: Transfer Regulations and Quarantine Measures • • If you have had experience in legal transactions for germplasm transfer, then: – Briefly describe the transactions that were carried out and indicate those entities and organizations involved; and – Express your opinion on the effectiveness of the procedures followed for reducing the risk of inadvertently introducing pests (i.e., pathogens, insects, and other agents) of quarantine interest to your country. If you have not had experience with legal transactions for germplasm transfer, then briefly describe what would be the procedures to follow for the safe international transfer of germplasm. Bibliography Literature cited Andean Community, General Secretariat. (Spanish version accessed 16 Sept 2004) Treaties and legislation: treaties and protocols; Andean Subregional Integration Agreement, ‘Cartagena Agreement’. Available at http://www.comunidadandina.org/ingles/normativa/ ande_trie1.htm Barton JH; Siebeck WE. 1994. Material transfer agreements in genetic resources exchange: the case of the International Agricultural Research Centres. Issues in Genetic Resources No. 1. IPGRI, Rome. Also available at http://www.bioversityinternational.org/ publications/Pdf/109.pdf Comunidad Andina, Secretaría General. 2004. Decisión 515 Sistema Andino de Sanidad Agropecuaria. Available at http://www.senasa.gob.pe/sanidad_vegetal defensa_ fitosanitaria/00011.pdf (accessed 16 Sept 2004). COSAVE. 2003. Requisitos fitosanitarios armonizados por categoría de riesgo para el ingreso de productos vegetales. Available at http://www.cosave.org/normas/st3015v020203_ suscCM12.doc (accessed 16 Sept 2004). EPPO. 2006. EPPO standards: EPPO A1 and A2 lists of pests recommended for regulation as quarantine pests; PM 1/2(15) English. Available at http://archives.eppo.org/ EPPOStandards/PM1_GENERAL/pm1-02(15)_A1A2_2006.pdf (accessed 16 Sept 2004). FAO. 1994. The International Code of Conduct for Plant Germplasm Collecting and Transfer. Available at http://www.fao.org/AG/AGp/AGPS/PGR/icc/icce.htm FAO. 1997. International Plant Protection Convention (new revised text approved by the FAO Conference at its 29th Session–November 1997). Available at http://www.fao.org/Legal/ TREATIES/004t2-e.htm FAO; Bioversity International. 1989–2007. Technical guidelines for the safe movement of germplasm. Rome. Available at http://www.bioversityinternational.org/Themes/ Genebanks/Germplasm_Health/index.asp (with reference to various crops). FAO; IPGRI. 2004. Technical guidelines for the safe movement of germplasm. Available at http://www.ipgri.cgiar.org/publications/pubseries.asp?id_serie=11 (accessed 19 Sept 2004). 53 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Gerard BM. 1984. Improved monitoring test for seed-borne pathogens and pests. In Dickie JB; Linington S; Williams JT, eds. Seed management techniques for genebanks; Proc. Workshop held at the Royal Botanic Gardens, Kew, 6–9 July 1982. IBPGR, Rome. pp 22–42. Glowka L; Burhenne-Guilmin F; Synge H; McNeely JA; Günding L. 1994. A guide to the Convention on Biological Diversity. Environmental Policy and Law Paper No. 30. IUCN, Cambridge, UK. 161 p. ICA. 2004. Guía para la importación y exportación de vegetales, productos y subproductos de origen vegetal. Available at http://www.ica.gov.co/CEF/requisitos_ica.htm (accessed 16 Sept 2004). IPPC, Secretariat. 1995. Normas internacionales para medidas fitosanitarias; Principios de cuarentena fitosanitaria en relación con el comercio internacional. Publicación No. 1, Febrero 1995. FAO, Rome. Nath R. 1993. Plant quarantine: principles and concepts. In Rana RS; Nath R; Khetarpal RK; Gokte N; Bisht JS, eds. Plant quarantine and genetic resources management. National Bureau of Plant Genetic Resources of the Indian Council of Agricultural Research, New Delhi, India. pp 19–24. OIRSA. 2004. Manual modelo para la aplicación de las medidas técnicas de la cuarentena agropecuaria. San Salvador, El Salvador. Available at http://www.oirsa.org/DTSV/ Manuales/Manual04/Manual.htm (accessed 16 Sept 2004). Servicio Fitosanitario del Estado. 2004. Procedimientos generales de cuarentena vegetal, 2nd ed. Dirección de Protección Fitosanitaria, Departamento Cuarentena Vegetal, Ministerio de Agricultura y Ganadería, Costa Rica. Available at http://www.protecnet.go.cr/ cuarentena/PROCEDIMIENTOS1.htm (accessed 16 Sept 2004). Further reading IPPC, Secretariat. 2006. International standards for phytosanitary measures, 1 to 27 (2006 edition). FAO, Rome. Also available at https://www.ippc.int/servlet/ BinaryDownloaderServlet124035_Book_ISPMs_2006.pdf?filename=1165395722111_ISPMs_ 1to27_2006_ En_with_convention.pdf&refID=124035 United Nations. 1993. No. 30619–Multilateral–Convention on Biological Diversity (with annexes): concluded at Rio de Janeiro on 5 June 1992, registered 29 December 1993. Treaty Series, vol. 1760, I-30619, pp 143–382. Available at http://www.biodiv.org/doc/legal/ cbd-un-en.pdf Contributors to the Lesson Benjamín Pineda, Daniel Debouck, Rigoberto Hidalgo, and Mariano Mejía. Next Module In the lessons of the next module, you will study germplasm conservation. 54 Module 3 Supported by the CGIAR Germplasm Conservation General Comments Germplasm banks play a crucial role in the conservation and use of biodiversity. They are important institutions, not just for the preservation of germplasm but also for its sustainable use. Germplasm banks are also expected to generate and provide new scientific knowledge and information on ecosystems, species, and genes. The reports presented at the International Technical Conference on Plant Genetic Resources in Leipzig indicated that the number of germplasm banks had grown rapidly since the early 1970s when there were fewer than 10, holding perhaps half a million accessions. At this Conference, 1300 germplasm banks were identified as holding about 6.1 million accessions, of which about 10% were conserved in field collections (FAO 1997). Few African countries have national germplasm banks for agricultural crop species, and the few that do exist are not adequately equipped and organized to attain the continent’s goals (African Ministerial Council on Science and Technology [AMCOST]). Even so, ex situ conservation for forest genetic resources, in the form of field germplasm banks, is practised for most exotic plantation species in Malawi, South Africa, Tanzania, Zambia, and Zimbabwe. In Malawi, field germplasm banks of important seed sources of indigenous species (e.g., Afzelia quanzensis, Khaya anthotheca, and Pterocarpus angolensis) were established. The SADC Regional Gene Bank is also located in Lusaka, Zambia. It stores duplicate samples of germplasm of national institutions (FAO 2003). The information provided by countries of the Andean Region shows that a significant number of germplasm banks house genetic resources of great importance agriculturally, socioeconomically, and in terms of food security. In all, 88 active banks are reported in the Region, of which 60% are managed by public institutions. A further two are national base collection banks (Colombia and Ecuador) and another is being planned (Venezuela). Most of the accessions conserved in the banks correspond to species belonging to Andean flora such as tubers and roots (17,289 accessions), cereals (27,839), vegetables (6415), fruit trees (6331), forest flora (2866), legumes (11,064), forages (426), plants for industry (14,945), and ornamentals (1679) (Comunidad Andina 2002). The conservation of plant genetic resources (PGRs) is not limited to attaining and physically possessing the materials (collection and storage) but also includes ensuring the existence of these under viable conditions and with their original genetic characteristics intact. This is achieved, in the case of seeds or in vitro materials, by controlling storage conditions so that they inhibit or reduce the samples’ metabolism; and, in the case of vegetative planting materials, by maintaining them under optimal agronomic and cropping conditions (Jaramillo and Baena 2000). Information on the Module This module contains five submodules, each of which contains two lessons. Objectives When you have completed the entire module, you should be able to, with regard to plant germplasm: 55 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources • • • • • Describe Describe Describe Describe Describe and long multiplication and regeneration the procedures for harvesting, conditioning, and quantification the procedures for verifying the germplasm’s biological status what constitutes plant health quality and the verification procedures used the alternatives for storing and conserving seeds and propagules in the short term Lessons The lessons for Module 3 on Germplasm Conservation are as follows: Submodule Lessons A. Multiplication and regeneration • • Multiplication Regeneration B. Harvesting, conditioning, and quantification • • Harvesting Conditioning and quantification C. Verifying the biological quality of germplasm • • Basic concepts Verification procedures D. Verifying phytosanitary quality • • Basic concepts of phytosanitary quality Procedures for verifying phytosanitary quality E. Storing germplasm Basic concepts of storage, an essential component of the ex situ conservation of germplasm Bibliography Throughout this module, a bibliography is provided for each section, that is, the General Comments and each Lesson. The bibliographies follow a format of two parts: 1. Literature cited, which includes those references cited in the text itself. Some of these citations were used to develop the original Spanish-language course on ex situ conservation and may therefore appear in Spanish or Portuguese. However, where practical, references to the English versions of the original Spanish-language documents are provided. 2. Further reading, which is a list of suggested readings in the English language, with few exceptions in French. A list of Acronyms used in the bibliographies is also given. The idea is to save space by not having to spell out each institution’s full name each time it appears in the references. Acronyms used in the bibliographies ACIAR AMCOST AOSA 56 Australian Centre for International Agricultural Research African Ministerial Council on Science and Technology Association of Official Seed Analysts Module 3: Germplasm Conservation General Comments APS AVRDC CESAF COSAVE CSSA EPPO FAO IBPGR ICA ICAR ICRISAT IPGRI IPGRI–APO IPPC IRRI ISTA JIRCAS OIRSA OSU SCBD USAID American Phytopathological Society Asian Vegetable Research and Development Center Centro de Semillas y Árboles Forestales Comité de Sanidad Vegetal del Cono Sur Crop Science Society of America European and Mediterranean Plant Protection Organization Food and Agriculture Organization of the United Nations International Board for Plant Genetic Resources Instituto Colombiano Agropecuario Indian Council of Agricultural Research International Crops Research Institute for the Semi-Arid Tropics International Plant Genetic Resources Institute IPGRI Regional Office for Asia, the Pacific and Oceania International Plant Protection Convention International Rice Research Institute International Seed Testing Association Japan International Research Center for Agricultural Sciences Organismo Internacional Regional de Sanidad Agropecuaria Ohio State University Secretariat of the Convention on Biological Diversity United States Agency for International Development Literature cited Comunidad Andina. 2002. Normativa andina: Decisión 523; Estrategia regional de biodiversidad para los países del trópico andino (anexo). Available at http://www.comunidadandina. org/normativa/dec/D523.htm or http://www.observatorioandino.org.co/docs/ ecoandino.pdf FAO. 1997. The state of the world’s plant genetic resources for food and agriculture. Rome. 510 p. Also available at http://www.fao.org/ag/AGP/AGPS/Pgrfa/pdf/swrfull.pdf or http://www.fao.org/iag/AGP/AGPS/Pgrfa/wrlmap_e.htm FAO. 2003. State of forest and tree genetic resources in dry-zone Southern Africa Development Community countries. Prepared by BI Nyoka. Forest Genetic Resources Working Paper FGR/41E. Forest Resources Development Service, Forest Resources Division, FAO, Rome. Available at http://www.fao.org/DOCREP/005/AC850E/ac850e0c.htm Jaramillo S; Baena M. 2000. Material de apoyo a la capacitación en conservación ex situ de recursos fitogenéticos. IPGRI, Cali, Colombia. 209 p. Also available at http://www.ipgri. cgiar.org/training/exsitu/web/arr_ppal_modulo.htm (accessed 28 Sept 2004). Further reading AMCOST. 2007. Conservation and sustainable use of biodiversity. Available at http://www.nepadst.org/platforms/biodiv.shtml Assy Bah B; Durand-Gasselin T; Engelmann F; Pannetier C. 1989. Culture in vitro d’embryons zygotiques de cocotier (Cocos nucifera L.): Métode, révisée et simplifiée, d’obtention de plants de cocotiers transférables au champ. Oléagineux 44:515–523. 57 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Engelmann F, ed. 1999. Management of field and in vitro germplasm collections; Proc. Consultation Meeting, held 15–20 January 1996 at CIAT, Cali, Colombia. IPGRI, Rome. 165 p. FAO. 1997. International Plant Protection Convention (new revised text approved by the FAO Conference at its 29th Session–November 1997). Rome. Available at http://www.fao.org/ Legal/TREATIES/004t2-e.htm FAO; IPGRI. 1994. Genebank standards. Rome. 15 p. Also available at http://www.ipgri.cgiar.org/publications/pdf/424.pdf Frison EA. 1994. Sanitation techniques for cassava. Trop Sci 34(1):146–153. George EF. 1996. Plant propagation by tissue culture: in practice, Part 2. Exegetics, Westbury, UK. 1361 p. George EF; Sherrington PD. 1984. Plant propagation by tissue culture: handbook and directory of commercial laboratories, Part 1. Exegetics, Westbury, UK. 709 p. IPGRI. 2004. Ex situ conservation. Available at http://www.ipgri.cgiar.org/ (accessed 28 July 2004). ISTA. 1993. International rules for seed testing. Seed Sci Technol 21:(Supplement). Ramanatha R. 2001. Principles and concepts in plant genetic resources conservation and use. In Said Saad M; Ramanatha Rao V, eds. Establishment and management of field genebanks: a training manual. IPGRI–APO, Serdang, Indonesia. pp 1–16. Sackville Hamilton, NR; Chorlton KH. 1997. Regeneration of accessions in seed collections: a decision guide. Handbook for Genebanks No. 5. IPGRI, Rome. 75 p. SCBD. 2005. Handbook of the Convention on Biological Diversity, including its Cartagena Protocol on Biosafety, 3rd ed. Montreal, Canada. 1493 p. Also available at http://www.cbd.int/doc/handbook/cbd-hb-all-en.pdf United Nations. 1993. No. 30619–Multilateral–Convention on Biological Diversity (with annexes): concluded at Rio de Janeiro on 5 June 1992, registered 29 December 1993. Treaty Series, vol. 1760, I-30619, pp 143–382. Available at http://www.biodiv.org/doc/legal/ cbd-un-en.pdf Withers LA. 1995. Collecting in vitro for genetic resources conservation. In Guarino L; Rao VR; Reid R, eds. Collecting plant genetic diversity: technical guidelines. CAB International, Wallingford, UK. pp 511–525. Contributors to the Module Benjamín Pineda, Rigoberto Hidalgo, Daniel Debouck, Mariano Mejía, Graciela Mafla, Arsenio Ciprián, Manuel Sánchez, Carmen Rosa Bonilla, and Orlando Toro. Next Lesson In the next lesson, you will learn about germplasm multiplication. 58 Submodule A Lesson 1 Multiplication Multiplication and Regeneration Objectives • • To define germplasm multiplication To review the requirements and procedures for multiplication Introduction Once germplasm has been acquired and introduced, it must be stored (temporarily) to preserve its essential characteristics (the reason for conservation), that is, its physiological and physical qualities, genetic identity, and plant health quality. The temporarily stored germplasm may undergo other important stages as according to the germplasm bank’s goals. These stages would form part of the monitoring needed to conserve the germplasm for the pre-established term. After introducing the germplasm, an essential activity in ex situ conservation of PGRs is preliminary multiplication, or initial increase, of the acquired materials. However, PGRs can be conserved without going through this stage, especially if sufficient conservable material (i.e., highly viable and healthy) had already been acquired. Likewise, instead of conducting a preliminary multiplication, a definitive multiplication programme can be developed according to the germplasm bank’s goals. For in vitro conservation, initial multiplication is probably not necessary. Initial multiplication is key to successful conservation, as acquisition rarely provides security of status of plant health (despite what is expressed in the pertinent certificates) or of the material’s viability. In many cases, the preliminary multiplication of PGRs is carried out as botanical seed because the germplasm bank received very little material and has to increase it to meet a series of requisites for conservation. One requisite is the periodic verification of viability, involving a consumption of seeds from the sample. The idea of requisites leads to an important concept: that the germplasm bank must specify its needs according to its mandate. Because one goal of conservation is to maintain the germplasm’s essential characteristics, five major risks should be avoided during multiplication or regeneration. Best known as the ‘capital sins of conservation’ (Daniel Debouck 2004, personal information), these are: 1. Mechanical mixture with contaminants (other seeds or any material alien to the samples) 2. Infection by pathogens—whether of quarantine importance or not—that can affect the material’s viability 3. Genetic contamination through uncontrolled hybridization; a common event because plants may have received residual cross-pollination, even if they are autogamous 4. Genetic erosion, a common phenomenon in germplasm banks; it occurs when making repeated samplings for different events of increase, permitting the effective multiplication of only a few seeds 59 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources 5. Genetic drift, which is a more subtle effect, where materials produce variable numbers of propagules and, hence, change the gene frequencies that were originally found in the samples on collection Over the time it is conserved, germplasm can decline in quantity (number of seeds) and quality (viability). Sample size shrinks with use and distribution, while viability declines over time, even if the germplasm has been stored under optimal conditions (FAO 1996; Sackville Hamilton and Chorlton 1997). When this happens, the germplasm must be multiplied or regenerated. If the objective is to recover viability, one speaks about regeneration or rejuvenescence; if it is to bring the samples to an optimal size (quantity), then one speaks of multiplication. Increase, multiplication, and regeneration are activities that embrace the same principle: to obtain a given quantity of viable propagules, free of pathogens, and genetically identical to the original. Hence, similar methodologies are applied. As we present this theme we will always refer to the essence of the three activities as multiplication but, for convenience in developing the theme, we will refer to multiplication on the one hand and regeneration on the other. Undoubtedly, when dealing with procedures for germplasm multiplication, we must take into account the two basic strategies of plant reproduction. The first strategy is that of sexual reproduction, which uses the seed as the building block. A seed consists of an embryo, its stored food reserves, and the surrounding protective coats. The other reproductive strategy is asexual or vegetative reproduction, which generates new individuals directly from pieces of mother plants or specialized organs. Thus, in terms of germplasm multiplication, one always refers to the two strategies: reproduction by seed or vegetative reproduction. The Concept of Initial Multiplication Initial multiplication (also known as preliminary multiplication or initial increase) is the increase of introduced germplasm. It is carried out under optimal agronomic conditions to guarantee sufficient viable samples that maintain the original genetic identity. The multiplied material will permit storage, conservation, and distribution of the targeted species, and the establishment of representative populations for characterization and evaluation. Initial multiplication is almost always necessary, as samples obtained through donation, exchange, or field collection have small numbers of seeds and a usually irregular percentage of viability. When a germplasm sample is imported from another country, under normal conditions, before starting initial multiplication, certain plant health transactions should have been fulfilled, as described in Module 2: Germplasm Acquisitions and Introduction (Seeds and Asexual Propagules) (Lesson 3, Figures 4 and 5). The germplasm is then taken to the place of conservation where the samples are verified as being sufficient and viable for conservation. As a safety measure and according to established agreements, initial multiplication is conducted under quarantine and supervised either by the national institutions responsible 60 Module 3, Submodule A: Multiplication and Regeneration Lesson 1: Multiplication for plant health such as the ICA Office for the Prevention of Plant Health Risks for Colombia or according to regional agreements such as the Inter-African Phytosanitary Council (IAPSC) in terms of the stipulations of the International Plant Protection Convention (FAO 1997). These stipulations include inspection to detect pests and pathogens, treating or cleaning samples, and certification and release if no danger exists or the material’s destruction if it is highly contaminated or no technology is available to clean it. Monitoring the need for multiplication As with other conservation activities, multiplication starts with monitoring the samples. It is governed by standards and procedures that specify the quality and quantity of the required material, the number of plants, and the environment (FAO and IPGRI 1994; Sackville Hamilton and Chorlton 1997). A sample is in optimal condition when it is viable and present in sufficient numbers. If, on monitoring, a sample does not fulfil either requirement, it should be multiplied. Size is determined by counting the number of available seeds or propagules per accession. If the sample consists of seeds, the permissible minimum size indicated by the Genebank Standards (FAO and IPGRI 1994) is 1500 to 2000 seeds. No standards exist for the sample size of vegetative propagules conserved in the field or in vitro but between 3 and 20 replications are usually kept per accession, taking into account the number of propagules initially received. Viability is established through observation or testing, depending on sample type. The viability of vegetative material (plants in the field or in in vitro slow growth) is systematically established by observing the health, development, and conditions under which the material is being conserved. If any one of the criteria listed is not met, then the material should be regenerated. If the conserved material is seed, viability is analyzed through germination tests that involve germinating a selection of seeds and evaluating how many (%) germinate. Of those that did not germinate, then observations must be made to determine if they had died or were dormant. Findings are then compared with the initial viability, which had been determined before preliminary multiplication. If viability has declined to 85% or less, then the sample must be regenerated. Germination tests are carried out on a minimum sample of 200 seeds taken at random (FAO and IPGRI 1994). Seeds are placed on paper (towels or rolls) or a substratum (sand or soil) and, depending on the species, incubated at different temperatures until they germinate. The tests should follow the standards indicated in the International Rules for Seed Testing (ISTA 1993). If the germination tests do not give satisfactory results, then complementary tests such as that of tetrazolium and X-rays can be carried out to determine if the embryo is dead, dormant, or non-existent. Once these tests are applied, the material cannot be used later, which means, in practical terms, that sufficient seed must be available to do these tests. Likewise, in practice, even if seed is insufficient, a smaller quantity can be used, for example, two independent replications of 50 seeds each or, if preferred, the necessary minimum quantity for the test can be determined by statistical analysis. 61 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources To decide when to multiply, one should not wait until the sample reaches minimum levels of size and viability, but neither should multiplication be done frequently, as it is expensive and endangers the germplasm’s genetic integrity. Furthermore, viability should take priority over size; thus, multiplying a large sample with low viability is more urgent than multiplying a small sample whose viability is optimal. Requisites for multiplying germplasm Establishing and managing multiplication procedures require prior understanding of propagation techniques and when to use conventional versus biotechnological ones. For vegetative samples, the use of the simplest or most complex techniques as according to the totipotency of cells and/or tissues can strengthen the process. Germplasm multiplication usually requires: • • • • • • • • • • Knowledge of the characteristics of reproduction and biological cycle of the species being multiplied (e.g., whether they reproduce sexually by seed or asexually by stems, cuttings, roots, bulbs, buds, meristems, or stolons; allogamous or autogamous species; and annuals or perennials) To prevent drift, knowledge of the geographical and ecological origins of the material is needed to programme its multiplication under conditions similar to those of the acquisition site Knowledge of the germplasm’s characteristics of adaptation so it can be multiplied in: – The field (site, soils, environmental and biological conditions) – Greenhouses and mesh houses (site, airtightness, temperature, light, humidity) – Growth chambers (place, temperature, light, humidity) – In vitro laboratory (infrastructure, light, temperature, safety) Knowledge of the initial viability and quantity of available material An estimate of yield, for example, g/plant, kg/ha, kg planting material/area, and number of stakes/plant Clarity on multiplication rates, for example, kg planted/kg harvested, area established/ area multiplied, and area established/kg planting material Knowledge of the likely users of the materials produced such as germplasm banks, research institutions, seed companies, and individuals Resources, whether physical, financial, human, or infrastructure Selection of sites, multiplication methods, and area size. Establishment of the size of area needed for multiplication as according to information on yield and requirements of seed or planting materials. How to Increase or Multiply? Procedures • • 62 Adequate preparation of the sites selected for increase (e.g., infrastructure, procedures, machinery, equipment, soil, culture media, and propagation containers) Creation of lists of the species to multiply and preparation of propagules (seeds and/or plant parts) – Seeds (e.g., treatment, scarification, and pregermination) – Plant parts (treatment according to the method to be used, e.g., thermotherapy for in vitro propagation) Module 3, Submodule A: Multiplication and Regeneration Lesson 1: Multiplication • • • • • • • • • Transplanting or sowing of materials in containers with substrata or media suitable for seedling development, previously placed in the sites for increase (e.g., greenhouse, mesh house, or laboratory) Care during growth Plant health inspections, sampling, and analysis to certify health and ‘release’ of the introduced materials Hardening or conditioning of the seedlings for transplanting to the final sites for increase Transplanting to the final sites for increase, according to predefined criteria on location and requisites of the species (e.g., greenhouses, mesh houses, laboratory, growth rooms or chambers, and the field) Agronomic attention and care to ensure successful increase Acquisition of samples for the herbarium, as according to species Harvesting, collecting, conditioning, and temporary storage according to methods as defined by species and material type Documentation of the general multiplication plan Identifying samples In multiplication, as in all germplasm conservation activities, the samples must be precisely identified to prevent mixtures that cause confusion or loss. The location of plots, furrows, and plants undergoing multiplication should be stated on a map and, at the site, with their accession numbers, using weatherproof materials. In cases of doubt, the identification of the plant materials can be confirmed by comparing them with herbarium samples or against available data such as passport data, or from characterization and evaluation. Currently, digital imaging can be used to make photographic records of the materials before their multiplication, so that they may serve as reference to prevent mechanical mixtures or accidental hybridizations. Establishing the germplasm Orthodox seeds can be multiplied in the field, but their multiplication is better done in the greenhouse to prevent genetic recombination and the presence of pests and diseases. Before multiplying the samples, size and viability should be confirmed. The sample’s initial viability will serve as a basis for later monitoring. Plant species that propagate asexually have long growing periods or produce short-lived seeds (i.e., recalcitrant) are normally left in the field. Strategies and procedures for establishing and maintaining collections must be practical, rational, economical, and scientifically well grounded (Engelmann 1999). Planting materials multiplied in the field or greenhouse, using propagules (e.g., stakes or bulbs), are first sterilized or grown in vitro through buds or meristems taken from the original samples. Recalcitrant and intermediate seeds are planted in the field or greenhouse to obtain complete plants, from which buds or meristems are taken to multiply in vitro. The new plants can also be left in the field in the hope that they will produce seeds and thus be multiplied in the field. Once the decision has been made to multiply a sample, plants are established in the multiplication site under optimal conditions of development. The resulting sample of 63 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources germplasm should be viable, healthy, in sufficient quantity for storage, and genetically equal to the original. The type of reproduction a species has will determine those conditions. Species that do not require control over pollination such as those with asexual reproduction or are autogamous are multiplied in the field or mesh houses. If they are multiplied in the field, the germplasm is planted in relatively small plots and in large populations. Those with vegetative reproduction are multiplied through sterilized samples such as stakes, layering, and grafts. Species with sexual reproduction that do need control over pollination (allogamous) are preferably multiplied in greenhouses and mesh houses. They can be multiplied in the field, provided that the area is isolated and pollination is strictly controlled. If the accessions are wild species, they can be multiplied in furrows or plots in the field, mesh houses, or greenhouses, depending on the quantity of available seed and of the species’ requirements. Certain wild species (e.g., Lycopersicon peruvianum) need special environmental conditions to reproduce. Multiplication of germplasm in the field or mesh houses requires space, time, and great quantities of materials and resources. For species multiplied by in vitro tissue culture, not much time or space is needed, permitting work with various sample types and thereby offering the possibility of multiplying a variety of species. It can also ensure healthy samples that are genetically identical to the original. Tissue culture consists of micropropagating apices of axillary buds and meristems until entire plants are obtained. Controlling multiplication conditions in the field Obtaining a sample of good physiological quality (i.e., viable, vigorous, and healthy vegetative propagules or seeds) and identical to the original genotype requires strict control of the environment. The physiological quality of the germplasm depends on its genetic characteristics and on the environment in which it develops. As it can be affected during the growing cycle by adverse environmental factors, any biotic or abiotic stress must therefore be prevented. The selected site should have fertile soil with sufficient water to supply the species’ requirements. Preferably, the site should also be isolated to prevent attacks from pests and pathogens or have facilities for controlling them should they appear. Uniform distances between furrows and between plants should be established, and the agronomic tasks necessary for the given species carried out. Seeds and propagules should be harvested when they are physiologically mature and healthy, taking care to prevent mechanical damage. Controlling the environment to maintain the original genotype consists of preventing accessions from becoming contaminated through pollen exchange (allogamous) or mechanical mixing (autogamous and asexual reproduction). Populations can be isolated in the field or in mesh houses. If they are planted in the field, the area should be isolated, separating the accessions by suitable distances, and submitting them to thinning and pruning to prevent overlapping of plants and mixing of fruits and seeds. Furthermore, allogamous species require strict control over pollination, which is achieved by bagging the reproductive structures and managing populations of insect pollinators. Using individual mesh houses for each accession eliminates risks of contamination but increases costs. 64 Module 3, Submodule A: Multiplication and Regeneration Lesson 1: Multiplication Conditioning and propagating planting materials The plant parts collected are washed and disinfected before propagating and taking them to the conservation site. Disinfection may be carried out with bactericides, fungicides (bulbs and rhizomes), or thermotherapy (stakes). Once they have been disinfected, the planting materials may be propagated in the field, greenhouse, or in vitro. In the field and greenhouse, samples are planted in seedbeds or flower pots and left to grow until plants are obtained from which new samples can be collected. The procedure is repeated until there are enough plants to establish the collection in the definitive site. If propagation is to be in vitro, samples are planted in greenhouses, in soils of optimal nutritional quality. From the resulting plants—preferably the younger ones—explants are extracted and micropropagated in vitro to obtain complete plants that are also taken to the greenhouse. These are planted into sterilized soil and, 2 or 3 weeks later, are transferred to their definitive site in the field. For cassava, micropropagation consists of (a) disinfecting the explants in a solution of sodium or calcium hypochlorite, mercury bichloride, or ethanol; (b) planting them in an in vitro culture medium until new shoots develop; and (c) rooting the shoots until complete plants are obtained (Frison 1994; George 1996; George and Sherrington 1984; Roca and Mroginski 1991). When using seeds, propagation in the field and greenhouse is simple but requires time and space. Nor can it guarantee that the plants obtained will be healthy and genetically identical to the originals. In vitro propagation solves these problems and can be used to propagate many species, even those that reproduce by seed. It is, therefore, more convenient. However, this type of propagation has limitations such as cost, the need for skilled personnel, and the risk of induced somaclonal variation, especially if artificially synthesized hormones are used (e.g., auxins or 2,4-D). Selecting and preparing the site The site selected to conserve the material in the field should be safe and favour plant development. It should be isolated to prevent pest attacks and diseases but easy to access for management tasks. The physical and chemical preparation of the planting site depends on the species’ requirements and on the number of accessions that is expected to be planted in the field. Planting vegetative materials in the field If vigorous plants are taken to the field in a number that represents the genetic variability of the accessions, then the continuity of the conserved materials will be ensured. Plants should be arranged in the field in such a way that no risk of pollen exchange exists, thereby preventing the populations from losing their original genotype. The exact site where each accession is planted should be recorded on a map, with the accessions being identified both in the field and on the plants. Evaluating the Lesson With this lesson, you should have understood the concept of germplasm multiplication; and have generally reviewed the procedures for multiplication, and their requirements and conditions. 65 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Before going on to the next lesson, prepare a well-based plan for germplasm multiplication. Take as an example, the germplasm with which you currently work or, in its absence, with which you are most familiar. Bibliography Literature cited Engelmann F, ed. 1999. Management of field and in vitro germplasm collections. Proc. Consultation Meeting, held 15–20 January 1996 at CIAT, Cali, Colombia. IPGRI, Rome. 165 p. FAO. 1996. Plan de acción mundial para la conservación y utilización sostenible de los recursos fitogenéticos para la alimentación y la agricultura. Rome. 64 p. (Also available in English as Global Plan of Action for the Conservation and Sustainable Utilization of Plant Genetic Resources for Food and Agriculture, and the Leipzig Declaration at http://www.fao.org/ag/ AGP/AGPS/GpaEN/gpatoc.htm). FAO. 1997. International Plant Protection Convention (new revised text approved by the FAO Conference at its 29th Session–November 1997). Rome. Available at http://www.fao.org/ Legal/TREATIES/004t2-e.htm FAO; IPGRI. 1994. Genebank standards. Rome. 15 p. Also available at http://www.ipgri.cgiar. org/publications/pdf/424.pdf Frison EA. 1994. Sanitation techniques for cassava. Trop Sci 34(1):146–153. George EF. 1996. Plant propagation by tissue culture: in practice, Part 2. Exegetics, Westbury, UK. 1361 p. George EF; Sherrington PD. 1984. Plant propagation by tissue culture: handbook and directory of commercial laboratories, Part 1. Exegetics, Westbury, UK. 709 p. ISTA. 1993. International rules for seed testing. Seed Sci Technol 21:(Supplement). Roca WM; Mroginski LA. 1991. Cultivo de tejidos en la agricultura: Fundamentos y aplicaciones. CIAT, Cali, Colombia. 969 p. Sackville Hamilton NR; Chorlton KH. 1997. Regeneration of accessions in seed collections: a decision guide. Handbook for Genebanks No. 5. IPGRI, Rome. 75 p. Further reading Assy Bah B; Durand-Gasselin T; Engelmann F; Pannetier C. 1989. Culture in vitro d’embryons zygotiques de cocotier (Cocos nucifera L.): Métode, révisée et simplifiée, d’obtention de plants de cocotiers transférables au champ. Oléagineux 44:515–523. FAO. 1997. The state of the world’s plant genetic resources for food and agriculture. Rome. 510 p. Also available at http://www.fao.org/ag/AGP/AGPS/Pgrfa/pdf/swrfull.pdf or http://www.fao.org/ag/AGP/AGPS/Pgrfa/wrlmap_e.htm 66 Module 3, Submodule A: Multiplication and Regeneration Lesson 1: Multiplication IPGRI. 2004. Ex situ conservation. Available at http://www.ipgri.cgiar.org/ (accessed 28 July 2004). IPPC. 2005. Inter-African Phytosanitary Council (IAPSC), Phytosanitary Portal (IPP). Available at https://www.ippc.int/servlet/CDSServlet? status= ND1ucHBvemEuMTA2MzkxJjY9ZW 4mMzM9bGVnaXNsYXRpb24mMzc9aW5mbw~~ Ramanatha R. 2001. Principles and concepts in plant genetic resources conservation and use. In Said Saad M; Ramanatha Rao V, eds. Establishment and management of field genebanks: a training manual. IPGRI–APO, Serdang, Indonesia. pp 1–16. Contributors to this Lesson Benjamín Pineda, Rigoberto Hidalgo, Daniel Debouck, Mariano Mejía, Graciela Mafla, Arsenio Ciprián, Manuel Sánchez, Carmen Rosa Bonilla, and Orlando Toro. Next Lesson In the next lesson, you will study major aspects of germplasm regeneration. 67 Submodule A Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Lesson 2 Regeneration Objectives • • • To define what constitutes germplasm regeneration and understand its purposes To describe the criteria and limitations in establishing regeneration protocols To describe the monitoring procedures for decision making on regeneration Introduction Over the time germplasm is kept conserved its viability can diminish, even if the germplasm has been stored under optimal conditions. When this happens, the germplasm must be regenerated or rejuvenated. Regeneration is carried out as a result of information obtained during seed control and usually occurs when the viability of a lot is below an acceptable level, often 85%. This percentage, however, corresponds to the lower limit of acceptability of loss of a sample’s internal variability. Regeneration is also carried out when factors inducing genetic drift or erosion appear, jeopardizing the existence of alleles in the original accession and possibly causing their elimination from the next generation. Regeneration is based on the same principle as multiplication: to obtain a given quantity of viable propagules, free of pathogens, and genetically identical to the original. As a result, similar methodologies are applied. Planning regeneration also requires passport and other data on the accession. Information (when this is adequate) is requested on the number of plants, distances between plots, crop improvement system, any isolation, and pollination method. Most likely, the germplasm bank already has these data recorded as part of its standard practices. Hence, obtaining such data for each accession will not usually be needed before regeneration. If regenerating the accessions is possible in more than one site, then the ‘preferred regeneration site’ should be listed in the inventory file. This will help by eliminating the step of having to consult the passport data file whenever regenerating an accession is planned, thereby simplifying activities. Preferentially, to prevent to the utmost genetic drift, only the most suitable site should be selected. Types of Collections Held in Germplasm Banks To better understand the regeneration protocols, we need to know the types of germplasm collections that are held by germplasm banks around the world. Collections differ according to the purposes of those holding the germplasm. Briefly, four types of collections exist; these are base, active, working, and core. The base collection is for long-term conservation. It comprises that set of accessions, each of which is distinct and, in terms of genetic integrity, as close as possible to the sample provided originally. Active collections comprise accessions that are immediately available for multiplication and distribution. Working collections are those held by breeders, and core collections are a small representation of a larger collection (FAO and IPGRI 1994). 68 Module 3, Submodule A: Multiplication and Regeneration Lesson 2: Regeneration Germplasm Regeneration and Its Purposes ‘Regeneration’ is that process that identifies in time those accessions, already introduced and recorded in the germplasm bank, whose seed (sexual or asexual) needs renewing. It also includes the establishment of protocols or optimal procedures for such renewal. Its essential goal is to maintain optimal quality and the genetic integrity of each accession and minimize the costs of carrying out these protocols. For species with sexual seed, accessions that need to renew their seed are identified basically by monitoring the minimum factors of seed quality (viability and health) or quantity against values or standards previously established for the species. Standards are essential for providing targets that institutes can aim for. However, the problems inherent in setting standards should be considered, including that where some institutes may have difficulties meeting specified standards. In view of these problems, for these cases, two sets of standards are specified: (1) acceptable, that is, minimal but considered adequate, at least for the short term; and (2) preferred, that is, higher and thus safer standards. For most criteria, good scientific reasons exist for meeting the ‘preferred standards’. Efforts should therefore be made to attain them (FAO and IPGRI 1994). For species with asexual or vegetative seed, accessions are conserved continuously as live field collections or in vitro. The factors requiring close attention are those related to quality because, usually, the accessions are clones whose number of individuals remains constant from harvest to harvest. It should be remembered that, with these materials, the biggest risks are confusion of identity (or loss of this) and infection of materials by pathogens, especially those of quarantine interest. When collections are kept in vitro, these risks are not so significant. The ideal goal of regeneration would be to produce seed whose viability is kept at 100% over long periods and whose accessions do not lose their original genetic composition. However, this ideal is difficult to achieve under real-life circumstances. Hence, the following practical goals are suggested for the curator of a collection that is conserved ex situ: • • • • Optimize the quality of seed produced Maximize the quantity of seed produced Wherever possible, maintain the genetic identity of each accession Maximize the cost-to-benefit ratio for each regeneration, that is, minimize costs and efficiently use equipment and resources without sacrificing the three previous objectives Maximizing seed quality ‘Seed quality’ is understood as the maximum quality of an accession that is economically obtainable in terms of its plant health status, viability, and capacity to remain viable over time under storage. This means that, when it is regenerated, an accession should be, as far possible, free of pests or diseases. Moreover, its initial viability after regeneration should be 95% or higher. Viability can be affected by different factors. Hence, to regenerate an accession in a suitable environment, the curator needs to know the optimal physiological state for harvesting, use handling procedures that will not damage the seed, know how to induce germination, and know if some type of dormancy exists (see Module 3, Submodule C). Thus, the curator must 69 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources know the species’ biology, this knowledge being more critical for wild species and forms for which the level of knowledge is not usually as high as for cultivated forms. To maintain the initial viability of a given accession for as long as possible, the seeds of that accession should be harvested when physiologically mature, but as young as possible. The earliest possible processing will ensure that the seed possesses optimal moisture content, which is about 5%, depending on the species, according to Genebank Standards (FAO and IPGRI 1994). These standards also indicate that minimum viability should be 85% for most seeds and 75% for some vegetable and forest species. Optimizing the quantity of seed produced To optimize the quantity of seed produced means that the cost-to-efficiency ratio for regeneration is maximized when the seed produced is sufficient to supply needs for use, before viability falls below the pre-established minimum. If the seed produced is not sufficient, then it must be regenerated more frequently, which is not desirable. In short, the quantities of seed expected from regeneration will depend on four factors: 1. The species’ reproduction system (autogamous or allogamous) 2. Seed size 3. Adequate selection of the regeneration site to prevent problems of fertilization and/or flower abortion and fruit filling 4. Types or categories of conservation that each bank uses With respect to seeds, the ‘acceptable’ standard for base collections, that is, the absolute minimum, is 1000 viable seeds for the accession being conserved; although, of course, any single number is arbitrary. In cases where fewer than 1000 seeds are available, the accession may nevertheless be kept under good storage conditions until such time as further collection or regeneration is possible. For active collections, the ‘preferred’ standard is 1500–2000 viable seeds (FAO and IPGRI 1994). The material’s regenerative capacity versus the time invested in the process should also be taken into account. For example, a tree may take several years before producing major volumes of seed. Thus, the curator must be careful when defining needs for seed for that material (e.g., bank’s mandate), and will verify the germinating quality of the harvested seed before eliminating such a material from the production site. For example, if the interval between one set of seeds to the next is 7 years, then, in reality, the interval could be 14 years if something went wrong in the first cycle. Sackville Hamilton and Chorlton (1997) cited ranges, established by several germplasm banks, of seed quantities for species type (Table 1). Table 1. Ranges of seed quantities for regeneration as per species type. Type of species Number of seeds Autogamous 1,500–6,000 Allogamous 4,000–50,000 Large-seeded species 1,500–4,000 Small-seeded species 2,000–50,000 SOURCE: Sackville Hamilton and Chorlton (1997). 70 Module 3, Submodule A: Multiplication and Regeneration Lesson 2: Regeneration Maintaining genetic identity Ideally, the objective of keeping the original accession’s genetic identity is to maintain jointly the frequency of all alleles of all genes (loci). However, various limitations of a practical order make meeting this objective almost impossible. The two most important are ignorance of the integral genetic composition of the individuals forming the original collected accession, and the physical impossibility implied in regenerating each seed of a conserved accession. Nevertheless, one useful exercise is to minimize the number of regenerations by increasing the number of years between each one, using best practices of conservation, for example, improved drying activities. Another useful exercise is to increase the size of the sample to be regenerated, for example, using 100 plants instead of the 2 or 3, as is normally done, and harvesting the same number of propagules planted per plant planted and harvested. During regeneration or multiplication, certain risks exist that should be avoided as far as possible to maintain the germplasm’s genetic identity. One is the mistaken identification of samples, a situation that, in practical terms, leads to the material’s loss of identity. Another risk is contamination by exotic genes. These genes may come from (1) foreign plants being mixed in during seed preparation, planting, or harvesting; (2) seeds of the same species that come either from previous plantings in the regeneration lot or from nearby plantings; and (3) pollen from other accessions of the same lot or from nearby plantings of the same species or other related species. A third major risk involves genetic drift and selection processes. Changes in gene frequencies occur at random (drift), or are generated by the environment, or are a consequence of management by man (selection). The results are changes in gene frequencies and thus loss of the original genetic composition. Gene frequencies change mainly because: • • • • • The sample being regenerated does not represent the original genetic composition A percentage of the initial seeds planted do not germinate, plants are dead or had not matured The contributions of feminine and masculine gametes are different The genetic composition of pollen and ovules differs from that of the original population Manual pollination had favoured certain phenotypes Criteria and Limitations for Establishing Regeneration Protocols The optimal protocols for regenerating accessions depend on: • • • The characteristics of the species concerned, particularly its reproduction biology, the accession’s physiological condition and original genetic composition, the use it will have, and its value to the collection Documentation on previous multiplications (degree of success or failure) Availability of human resources, infrastructure, and budget Establishing adequate regeneration protocols for most species presents limitations because of poor knowledge of the structure of the original populations. Consequently, these protocols should be flexible so that they can conform with the needs of each bank in terms of categories or types of conservation (short or long term) and research objectives. 71 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources The need to regenerate depends on how and why the accessions concerned are being conserved. The answers relate to each germplasm bank’s conservation objectives. Sexual seed. Regeneration is necessary when the germplasm’s viability has declined to below established limits, or the quantity of sexual seed existing in the bank is less than the minimum number of seeds established by the bank holding the collection. Such a minimum may be established by using the standards recommended by FAO and IPGRI (1994). These standards are designed to maintain the original genetic composition of each accession, and depend on whether the species is autogamous or allogamous. Asexual seed. For species that propagate vegetatively, collections are kept as field collections and the number of plants will depend heavily on the species’ type of propagule, and the availability of land and resources for maintaining the collection in the field. Usually, the number of plants is relatively low because of the high genetic homogeneity of individuals of an accession, as, in most cases, the plants are clones of the same progenitor. Monitoring the Need for Regeneration Determining the need to regenerate or refresh the germplasm starts with monitoring the viability of the samples conserved according to standards and procedures that define the quantity and quality of the material to use, the number of plants, and the environment (Box 1). According to the recommendations offered in paragraph 30 under Viability Monitoring from Genebank Standards (FAO and IPGRI 1994), the purpose of conducting control tests on viability is to determine if regeneration is needed. To save seeds, between 50 and 100 units of the entry can be chosen at random for each control test. The simplest method to ascertain if a substantial loss of viability is occurring (ruling out possible fluctuations in results largely attributable to sampling errors) consists of representing graphically the results of successive control tests against storage period and observing if a gradual loss of viability is occurring. If it is, and seeds are sufficient, another sample of 100 seeds should be extracted at random and tested again for viability. This sampling will guard against unnecessary regeneration. If the decision is made to regenerate, the allotment of seeds for the germination tests will be cancelled, thereby saving seeds, which, under these circumstances, will be more valuable. No standards exist for sample size of plant propagules conserved in the field or in vitro but, usually, 3 to 20 replications are kept per accession. Viability is established through observations or tests, depending on the type of sample. Viability of plant material (plants in the field or in vitro slow growth) is systematically established by observing its health and development, and the conditions under which it is being conserved. If any one of the prior criteria is not being fulfilled, then the material should be regenerated. If the conserved material is seed, then viability is analyzed by practising germination tests, which consist of germinating a sample of seeds to ascertain how many (%) germinate and, of those that do not germinate, determining which have died or are dormant. The findings are then compared with the initial viability—taken during preliminary multiplication—and if the current viability has declined to 85% or less, then the sample should be regenerated. Procedures for determining viability as the key element of monitoring conserved materials are described in more detail in Lesson 2 on verifying the biological status of germplasm (Module 3, Submodule C). 72 Module 3, Submodule A: Multiplication and Regeneration Lesson 2: Regeneration Box 1 Genebank Standards: Regeneration 35. Regeneration standards are needed to ensure that the seeds stored in base collections do not fall below acceptable levels of viability and yet minimize the number of regeneration cycles to ensure that the genetic integrity of accessions is maintained. The regeneration interval will depend on the longevity of the seed in storage and demand for the accession (if seeds are not available from an active collection). 36. Seeds which are produced for storage in base collections should, as far as possible, be of the highest possible viability and free of pests and diseases. Recognizing that the initial germination capacity will depend on the environment during production and processing, maturity and physiological state of the seeds at harvest and genetic differences between species, initial germination values should exceed 85% for most seeds, e.g. cereals, and 75% for some vegetables and even lower for some wild or forest species, which do not normally reach high levels of germination. 37. Regeneration should be undertaken when viability falls to 85% of the initial value. Regeneration methods should follow the standards for the crop, where available, and ensure that sufficient plants are used to maintain the genetic integrity of the accession. As far as possible all sources of selection pressure should be removed, the contribution of seeds from each plant should be equalized and all possible care taken to minimize genetic change. 38. It is desirable to use 100 plants or more for regeneration to avoid the probability of large losses of alleles. However, in wild species this may be limited by the total number of seeds available. Wild species may also vary in breeding system, storage behavior and germination from the related crop species. This should be taken into consideration when deciding when, and how to regenerate an accession. 39. In order to ensure that the genetic integrity is maintained and accessions are distinct, it is recommended that seeds used to plant material for regeneration should be as close as possible genetically to the original germplasm. It is recommended that for active collections, regeneration should be done from original seeds whenever possible or from its offsprings within two or three cycles of regeneration to ensure that genetic integrity is maintained. This implies that, assuming a 15 year storage cycle for the active collection, seeds for regeneration will need to be taken either from the base collection or other original seed in long-term storage once in 45 to 60 years, providing sufficient seeds are regenerated to meet demands on the active collection for distribution. Genebanks carrying out regeneration should also consider what methods they could use to monitor variation during regeneration to measure any changes in genetic constitution in accessions. SOURCE: FAO and IPGRI (1994). Regeneration Protocols Regeneration protocols are essentially the same as those used to multiply germplasm; therefore, see the previous lesson (i.e., Lesson 1, Submodule A). Evaluating the Lesson With this lesson, you should have understood the concept of germplasm regeneration and its purposes, reviewed the criteria for establishing protocols, and learned how to monitor needs for regeneration. 73 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources This lesson finalizes Submodule A of Module 3 but, before going on to the next lesson, prepare a well-based plan for germplasm regeneration. You may use, as an example, the germplasm with which you currently work or, in its absence, with that with which you are familiar. If you are not directly familiar with the process, list and discuss the criteria that, in your opinion, should be taken into account when regenerating a given germplasm. Bibliography Literature cited FAO; IPGRI. 1994. Genebank standards. Rome. 15 p. Also available at http://www.ipgri.cgiar.org/ publications/pdf/424.pdf Sackville Hamilton NR; Chorlton KH. 1997. Regeneration of accessions in seed collections: a decision guide. Handbook for Genebanks No. 5. IPGRI, Rome. 75 p. Further reading Assy Bah B; Durand-Gasselin T; Engelmann F; Pannetier C. 1989. Culture in vitro d’embryons zygotiques de cocotier (Cocos nucifera L.): Métode, révisée et simplifiée, d’obtention de plants de cocotiers transférables au champ. Oléagineux 44:515–523. Frison EA. 1994. Sanitation techniques for cassava. Trop Sci 34(1):146–153. George EF. 1996. Plant propagation by tissue culture: in practice, Part 2. Exegetics, Westbury, UK. 1361 p. George EF; Sherrington PD. 1984. Plant propagation by tissue culture: handbook and directory of commercial laboratories, Part 1. Exegetics, Westbury, UK. 709 p. IPGRI. 2004. Ex situ conservation. Available at http://www.ipgri.cgiar.org/ (accessed 28 July 2004). ISTA. 1993. International rules for seed testing. Seed Sci Technol 21:(Supplement). Ramanatha R. 2001. Principles and concepts in plant genetic resources conservation and use. In Said Saad M; Ramanatha Rao V, eds. Establishment and management of field genebanks: a training manual. IPGRI–APO, Serdang, Indonesia. pp 1–16. Withers LA. 1995. Collecting in vitro for genetic resources conservation. In Guarino L; Rao VR; Reid R, eds. Collecting plant genetic diversity: technical guidelines. CAB International, Wallingford, UK. pp 511–525. Contributors to this Lesson Benjamín Pineda, Rigoberto Hidalgo, Daniel Debouck, Mariano Mejía, Graciela Mafla, Arsenio Ciprián, Manuel Sánchez, Carmen Rosa Bonilla, and Orlando Toro. Next Submodule In the lessons of the next Submodule B, you will study the principal aspects of harvesting, conditioning, and quantifying the germplasm after its multiplication and regeneration. 74 Submodule B Lesson 1 Harvesting Harvesting, Conditioning, and Quantification Objectives • • To describe fruit types, seed parts, and propagules To review the principal aspects of harvesting germplasm and the care needed to guarantee its integrity Introduction Once the germplasm targeted for conservation has been multiplied or regenerated (whether under field conditions or in the greenhouse, mesh house, or laboratory), it is harvested. During multiplication or regeneration, natural biological processes occur that lead to the formation of reproductive structures. For plants that reproduce primarily by seed, flowers form, pollination occurs, and the ovule develops and matures into seed while, simultaneously, the ovary becomes fruit that eventually contains harvestable seeds. Certain types of plants not only produce seeds but also reproduce vegetatively. These plants also form propagules that carry one or more growth buds that, once independent, generate roots to give rise to new plants. New individuals may also result from natural or mechanical fragmentation of any piece of the plant. These are harvestable for conservation purposes. To develop the theme, this lesson will deal with aspects related to harvesting and, in the next lesson, to conditioning and quantification. Before describing what is harvesting, and to help understanding of the process, we will discuss fruit types, principal seed parts, and the propagules associated with plant reproduction. Fruit Types A fruit is the mature ovary that contains the plant’s seed or seeds (Figure 1). To the extent that the ovary develops after fecundation, it changes size, consistency, colour, chemical composition, and shape. Transformations are of two types: (1) dry fruits in which the cells become enveloped in very thick walls that lignify and harden; and (2) fleshy fruits in which the walls gelate and their tissues lose their cohesion, becoming more or less aqueous on ripening. Structures other than the ovary may become part of the fruit such as parts of the floral axis or tissues of foliar origin. Thus, in tomato, for example, the fleshy part is formed by the carpels, which form the ovary; in blackberry, this tissue is formed by petals that have been conserved; and in figs (green or ripe), receptacles of inflorescences form the flesh. During maturation, specific physical and chemical changes occur that lead to fruit senescence and seed dissemination. One very obvious change is the drying of fruit tissues. In certain fruits, this leads to dehiscence and discharge of seeds. The colour of fruits and seed coats may change, and the fruits may soften. Immature fruit is invariably green because of the presence of chlorophyll, but as it ripens, the chlorophyll decomposes and may disappear altogether, exposing other colours, particularly those with certain pigments. Three main criteria are used to classify fruit types: origin, composition, and description. The last is the most useful for harvesting and conditioning purposes. 75 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Fruits A fruit contains the seeds of a plant. True fruits develop exclusively from the ovary, whereas false fruits may also develop from nonovarian tissues such as the receptacle (e.g., strawberry). The fruit’s outside wall is known as the pericarp and is sometimes divided into an outer skin or epicarp, a fleshy part or mesocarp, and an inner layer or endocarp. Main fruit types are listed below. Legume or pod (e.g., pea). The seeds adhere to · the internal face of the fruit wall. To open, the A small dried fruit with only one · Achene. seed. ‘Winged’ achenes (e.g., American pod breaks longitudinally. sycamore or buttonwood) are known as samaras, keys, helicopters, or whirligigs. Seeds Seed Pea pod Sycamore samara (orange, blackcurrant). A fleshy fruit that (plum). Fleshy fruit, in the centre of · Berry · Drupe contains many seeds. which is a hard seed that is often called a ‘stone’. Seed or ‘stone’ Seed Orange Blackcurrant Grain or caryopsis (wheat). The wall of this · small fruit is fused with the seed sheath. Plum (hazelnut, walnut). Dry fruit with a hard · Nut shell that contains only one seed. Shell Seed Wheat grains Hazelnut (apple). This type of fruit has a thick · Pome outer layer, a fleshy layer, and a core. Its seeds are enclosed within a capsule. Pomes are examples of false fruits (see first paragraph). Capsule Seed Apple Figure 1. Examples of different types of fruits and their definitions (from Stockley 1991). 76 Module 3, Submodule B: Harvesting, Conditioning, and Quantification Lesson 1: Harvesting Essentially, a fruit can be classified as dry, fleshy, or originating from an inflorescence. The category depends on whether the ovary concerned had formed hard or fleshy structures, or the flower had one or more pistils, or the flower had been part of an inflorescence. Dry fruits Dry fruits are lignified structures that may or may not open spontaneously. Those that do not open are known as indehiscent and tend to contain a single seed. Such fruits include: • • • • The achene, which is a fruit with a single seed, for example, those of the composite family such as the pappuses of daisies and sunflowers; The caryopsis is similar to the achene, but has the pericarp welded onto the seed, as occurs in grasses; The nut is also similar to the achene but has a hard pericarp, sometimes stony, like acorns and hazelnuts, and The samara, which has winged structures that help its dissemination by wind, as occurs in elms and several other big trees; Dry fruits that open are known as dehiscent. They tend to contain more than one seed. The fruit types falling into this category are: • • • • The follicle, typical of the Ranunculaceae, which opens along the line of suture of its only carpel; The legume or pod, typical of legumes, is similar to the follicle but opens along two sutures; The silicle or silique, common in the Crucifer family, has two halves separated by a partition that persists after dehiscence; and The capsule, which varies considerably in how it opens and the number of compartments it contains; it is typical of the Papaveraceae, Liliaceae, and Primulaceae families. Fleshy fruits Fleshy fruits are aqueous and do not open. Seed is liberated when birds or animals devour the flesh or when this decomposes after falling to the ground after ripening. Principal types of fleshy fruit are: • • • • • Drupe, in which the endocarp tends to be hard and the mesocarp fleshy, as occurs in olives, walnuts, almonds, plums, peaches, or myrobalans; Berry, in which both mesocarp and endocarp are fleshy, as in grapes and tomatoes; Hesperidium, which is a berry that is fleshy between the endocarp and seeds, as in citrus fruits; Pome, which has a coriaceous endocarp and an external part that derives from the floral receptacle, as in apple, pear, or quince; Infructescence, cluster of fruits, derived from an inflorescence or group of inflorescences. Principal types are: – Multiple fruits, in which individual ovaries of many separate flowers cluster together on a common axis. Types include: 77 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources - – Syconium, in which a large number of small drupes from an entire inflorescence are enclosed within a cavity, as in fig; and - Sorosis, which is a group of berries is traversed by a fleshy axis, as in the pineapple and others of the Ananas genus. Aggregate fruit, in which a group of separate fruits develop from the carpels of one flower, as in strawberry or blackberry. Seeds and Their Parts Botanically, an angiosperm seed is a mature ovule that is enclosed within the ovary or fruit. The seeds and fruit of different species vary greatly in the aspect, size, shape, place, and structure of their embryos and presence of food storage tissues. In terms of seed management, the seed cannot always be separated from the fruit, as they sometimes form a unit. In such cases, the fruit itself is treated as ‘seed’, as with maize and wheat (Hartmann and Kester 1971). A seed has three basic parts: embryo, tissues for storing food, and seed coats (Figure 2). Embryo The embryo is a newly formed plant that results from fertilization, that is, from the union of the male and female gametes. Its basic structure consists of an axis with growing points at each extreme, one for the stem and the other for the root, and one or more seminal leaves (cotyledons) set at the embryonic axis. Plants are classified according to their number of cotyledons. Monocotyledonous plants (e.g., grasses and onion) have one cotyledon, whereas dicotyledonous plants (e.g., beans, cowpea, or peach) have two. Gymnosperms (e.g., pine and ginkgo) may have as many as 15. Food storage tissues Food storage tissues in a seed may comprise cotyledons, endosperm, perisperm, or, as in gymnosperms, the haploid female gametophyte. Those seeds in which the endosperm is large and contains most of the stored food are called albuminous seeds. Those that either lack the endosperm or have it reduced to a thin layer surrounding the embryo are called exalbuminous seeds. In the latter, food reserves are found in the cotyledons and the endosperm is digested by the embryo during its development. The perisperm, which originates in the nucellus, occurs in several plant families such as the Chenopodiaceae and Caryophyllaceae. Normally, during seed formation, it is digested by the endosperm as the latter develops. Seed coat or testa One or two seed coats (rarely three) may be formed by sheaths from the seed, from residues of the nucellus, and sometimes by part of the fruit. The coats derive from the integuments of the ovule. During development these coats are modified and at maturity present a characteristic aspect. In general, the outside seed coat dries, hardens, thickens, and takes up a colour that may be coffee coloured or other tone. The inside coat usually remains thin, transparent, and membranous. Within this layer remnants of the nucellus and endosperm may be found, sometimes forming a distinct continuous layer around the embryo. 78 Module 3, Submodule B: Harvesting, Conditioning, and Quantification Lesson 1: Harvesting Seed parts · · Coat or testa. Seed cover, formed from the integuments of the ovule. Coat Orifice (micropyle) by which water enters the ovule Hilum Plumule Radicle Radicle. First or primary root, which forms within the seed and becomes part of the new plant. Radicle Plumule Cotyledon One cotyledon has been removed · Endosperm Cotyledons Upper view of a cross-section of a young bean Cross-section of a mature bean Fleshy outer seed coat Pericarp Stony inner seed coat Scutellum Endosperm Plumule Embryo Plumule. First or primary bud, which forms within the seed and becomes the new plant’s first shoot. Position of the radicle (hidden plumule) Cotyledons have been separated Seed coat has been removed Endosperm. Layer of tissue within the seed, covering the developing plant and contributing food. In some plants such as the pea, the cotyledons absorb and store all the endosperm before the seed matures; in others such as grasses, the endosperm is not completely absorbed until the seed germinates. · Radicle Cotyledon · · Seed (bean) Hilum. Scar on the seed where the ovule had joined the ovary. Cotyledons Seed coat Hypocotylroot axis Coleoptile Endosperm Cotyledon or seed leaf. A simple leaf that forms part of the developing plant. In certain seeds such as those of beans, this leaf absorbs and stores all the endosperm’s food. Monocotyledons (e.g., grasses) are plants with only one cotyledon and dicotyledons (e.g., pea) are plants with two cotyledons. Embryo Perisperm Seed coats Embryo Radicle Endosperm Coleorhiza Magnolia Corn Beet Endocarp Endosperm Seed coats Seed coats Cotyledons Hypocotylroot axis Cotyledons Cotyledons Hypocotylroot axis Hypocotylroot axis Seed coats Fir Endosperm Embryo Olive Embryo Embryo Pear Figure 2. Seeds parts in different plant species (upper drawing from Stockley 1991; lower drawing from Hartmann and Kester 1971). 79 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources In some plants, parts of the fruit adhere to the seed, so that both are regarded as ‘seed’. In certain classes of fruits such as achenes, caryopses, samaras, and schizocarps, the fruit and seed layers are contiguous. In other fruits such as acorns, the fruit and seed coats are separate but the fruit coat is indehiscent. In still others such as the ‘stone’ in many fruit trees (e.g., peach and almond) or the ‘peel’ of the common walnut, the coat is a hardened part of the pericarp but is dehiscent and can be removed without much difficulty. The seed coats provide the embryo with mechanical protection. Hence, the seed can be handled without damage and therefore be transported long distances and stored over long periods. Seed coats significantly influence germination. Vegetative Reproduction: Propagules and Plant Fragments Used for Reproduction Many plants can reproduce vegetatively, that is, through plant parts. Such reproduction is possible because those plants have organs with regeneration capacity. Stem parts can form new roots and root parts can regenerate new stems. Leaves can regenerate new stems and roots. A stem and a root (or two stems), when suitably combined, such as in grafting, form continuous vascular connection to produce a new plant (Hartmann and Kester 1971; Vázquez Y et al. 2004). Vegetative reproduction is one type of asexual reproduction, which typically involves only one progenitor with no fusion of gametes (sexual cells). Plants use diverse mechanisms to reproduce vegetatively. These include: • • • • 80 Specialized storage organs, known as propagules, including: – Rhizomes—horizontal underground stems – Bulbs—bases of swollen leaves – Stem tubers—thickened underground stems – Root tubers—swollen adventitious roots – Corms—solid stem structures, with well-defined nodes and internodes – Stolons—creeping horizontal stems (or runners), which throw out roots that give rise to new plants – Bulbils—small bulbs that grow on the stem or instead of flowers, fall, and grow as new plants – Propagule or adventitious shoots—minute plants that become aligned along leaf margins before these fall to the ground, where they grow into adult plants. Natural or mechanical fragmentation where new individuals originate from any piece or fragment of the plant such as cuttings or stakes. At least one node of the stem or branch is needed to provide a growing point with potential to produce a new plant. Such fragments are almost always vegetative parts of the plant such as stems, modified stems (rhizomes, tubers, corms, and bulbs), leaves, or roots. Use of shoots that are induced naturally or artificially to form roots from a stem that is still joined to the mother plant. Such stems, once rooted, separate to become new plants that grow with their own roots. Today, new plants can be obtained from single cells, tissues, or organs. Any plant part is isolated and cultured in an aseptic, artificial, nutritive environment (in vitro tissue culture). One well-known application is the use of apical meristems or apices, based on the principle that these structures perpetuate themselves and are responsible for the continuous formation of primary tissues and stem appendages (e.g., leaves and stipules). Module 3, Submodule B: Harvesting, Conditioning, and Quantification Lesson 1: Harvesting Harvesting the Germplasm After the plants have grown and borne fruit (in the sense of containing seeds or propagules capable of generating new individuals), harvesting is carried out. The procedures for each case are inherent to the type of germplasm being handled and its predominant reproductive system. Harvesting germplasm that reproduces by seed Harvesting germplasm that reproduces by seed consists of collecting the plant’s fruits, once they are physiologically mature, that is, they are carrying seeds capable of germinating and initiating the development of new plants. When harvesting, the following should be taken into account: • • • • • • • The species being harvested and its type of seed (determines conditioning—drying is critical for species with recalcitrant or short-lived seeds, as they are sensitive to drying, whereas orthodox ones tolerate it better) Stage of maturity of the fruit (physiological maturity according to fruit type is preferred) Procedures for collection (manual or use of special equipment) Selection of fruits during collection (i.e., harvesting only ripe fruits that are not damaged by insects or showing symptoms of pathogen attack) Type of packaging to use (preferably clean cloth or paper bags) and germplasm identification system System of bulk collection and transport to sites for temporary storage Conditions for temporary storage and pre-drying of fruits before final conditioning In general, harvesting should be selective and timely. Fruits that are green, damaged, or diseased should not be harvested. In no way should overripe fruits or those decomposing through saprophytic micro-organisms be included. During harvest, utmost care should be taken to prevent damage or injury likely to degrade the fruits’ physical integrity and their contents. Mechanical injuries produced during harvest may reduce seed viability and lead to the production of abnormal seedlings. Some injuries are internal and cannot be seen at the time but, after storage, manifest themselves as reduced viability. Damage to the seeds is a potential factor in any operation that implies hitting the seeds, especially when machinery is not duly adjusted. Usually, seed suffers less damage if its moisture content is 12%–15% during harvest. As the objective of conservation is to maintain the germplasm’s genetic identity as closely as possible to the lots originally entered, only the offspring of the materials planted originally should be harvested while avoiding atypical materials or other entries or plants that do not correspond to the planted germplasm. However, cross-pollinated species may have natural segregations that may have to be confirmed later. Hence, the reproduction system of the species should be taken into account before ‘atypical’ materials are discarded. A seed reaches maturity when it can be separated from the fruit or plant without endangering its germination. Usually, harvest is facilitated if the fruit is ripe, that is, has acquired the characteristics that lead to natural dissemination. The maturation stages of fruit and seed may not coincide. If the seed is harvested too early or if the embryo has not 81 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources developed sufficiently when the fruit matures, then the seed may be thin, light, shrivelled, and of poor quality. If the harvest is delayed, then the fruits may open, fall, or be eaten by birds or animals. The tendency for fruit drop, that is, a premature fall of fruits and therefore seeds, varies considerably according to plant class. Losses can be reduced by careful management. Usually, harvesting should take place before the fruits dry up too much. Cutting early in the morning when dew is still present may, in some cases, reduce drop (Hartmann and Kester 1971), but the risk that the seeds will be severely affected by fungi is higher. As a result, harvesting should, preferably, take place after the dew has evaporated. Pre-drying fruits Great care should be taken when pre-drying fruits and their contents, as any neglect or error may lead to reduced seed viability and, in extreme cases, to the loss of germplasm. The reason for drying fruits and their seeds is to reduce moisture content to levels that will increase longevity during storage and, therefore, the intervals between regenerations. Several drying methods exist, the most common being the use of a drying chamber or de-humidifier (FAO and IPGRI 1994; Hong and Ellis 1996). When drying fruit, the species seed type must be taken into account. Seed moisture content will determine storage time. Species with short-lived seeds or seeds sensitive to drying (recalcitrant) should be dried out with more care than long-lived ones whose moisture content can be more severely reduced (orthodox seeds). Other seeds can be highly sensitive to moisture loss, being able to tolerate storage for only some days, such as those species with fleshy fruits belonging to the Myrtaceae family (e.g., myrtles, Luma spp., Myrceugenia spp., and Chilean guava). In these cases, seeds should be planted immediately after being extracted from the fruit (Hartmann and Kester 1971; Sandoval 2000). The methods used will depend on available equipment, number and size of samples to be dried, local climatic conditions, and economic cost (Grabe 1989). Preferable ranges for drying are temperatures between 10° and 25°C and relative humidity (RH) between 10% and 35%, whether using a dryer or drying chamber. A suitable drying product is silica gel, which can reduce moisture content to the extremely low levels that characterize ultra-dry seeds. Harvested materials should be dried out as soon as possible after collection to prevent any significant deterioration. The drying period will depend on the size of fruits and seeds, the quantity to be dried, the fruits’ initial moisture content, and the level of relative humidity maintained in the drying chamber. Personnel of germplasm banks must keep in mind that dried seeds, particularly those that are very dry, are often fragile, and therefore susceptible to mechanical injury. Hence, they must always be handled with utmost care (FAO and IPGRI 1994; Hong and Ellis 1996). Some management procedures are described below, according to whether seeds are from woody or herbaceous plants, or from trees and shrubs. Woody plants To harvest the fruits of woody plants, we need to know the characteristics that indicate optimal conditions for harvesting a given class of seed. These include moisture content (dryness), general appearance, and the state of the more-or-less milky colour of the seed. In 82 Module 3, Submodule B: Harvesting, Conditioning, and Quantification Lesson 1: Harvesting some pine species, the specific weight of recently harvested cones is valuable for judging their state of maturity. Some seeds, if they are harvested before the fruit has ripened completely and have not been allowed to dry, germinate better in spring or the season immediately following harvest. Once those seeds dry up and their coats harden, they may not germinate until the second spring or season after they were produced, except by using special handling methods. Examples of those plants for which this practice of early harvest has been found desirable include Cornus, Cotoneaster, Carpinus, Cercis, Hamamelis, Rhodotypos, Viburnum, Juniperus, and Magnolia kobus (Hartmann and Kester 1971). Herbaceous plants Dry fruit seeds. The dehiscent (follicles, capsules, pods, and siliques) and indehiscent fruits (caryopses and achenes) of some materials can be harvested, using special combine harvesters. However, for most plants, fruits or mature infructescences are collected, then cut, and allowed to dry for 1 to 3 weeks before being threshed. Plants may be placed in rows, stacks, or piles to dry. Those plants whose fruits open easily on drying, as for many ornamental species, are cut (frequently by hand) and placed on a canvas or tray. When many plants are dealt with, they may be cut and put out to dry by placing them inverted in a bag and hanging them (Hartmann and Kester 1971). Fleshy fruit seeds. Fleshy fruits (e.g., tomato, pepper, chilli, eggplant, and cucumber) may be harvested ripe or, in exceptional cases, overripe (e.g., cucumber and eggplant). If lots are small, the fruits may be broken and separated, and the seeds cleaned and dried out by hand. Otherwise, seed is separated from the flesh through fermentation, mechanical means, or washing in screens (Hartmann and Kester 1971). Trees and shrubs Both dry and fleshy fruits from trees and shrubs can be harvested by shaking them over a canvas, felling them with poles, using conical hooks fixed on long poles (as for conifers), or picking them by hand. The seeds of some street trees such as elms can be collected with brooms. Seeds of small trees and low shrubs may be harvested by hand, cutting or striking seed-bearing branches. The viability of seed from trees and shrubs varies considerably from year to year, from place to place, and from plant to plant. Before collecting seeds from a specific source, several fruits should be opened and the seeds examined to determine the percentage of welldeveloped embryos. Such an examination is known as the cutting test. Although it is not a reliable test of viability, it helps prevent harvesting seeds from a source that is producing only empty seed. Another test is to examine fruits by X-ray (Hartmann and Kester 1971). Dry dehiscent fruits. Seeds of plants such as certain ligneous legumes (e.g., Acacia triacantha), Caragana spp., Ceanothus spp., poplar, and willow are extracted from capsules and pods. The fruit of these plants are dried by spreading them out in thin layers on canvases; cloths; on the floor; or on shelving in open sheds, using trays with wire mesh bottoms. Air-drying takes 1 to 3 weeks. Fleshy fruits. Fleshy fruits include berries (grape), drupes (peach, plum), pomes (apple, pear), aggregate fruits (raspberry, strawberry), and multiple fruits (blackberry). With these 83 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources kinds of fruits, the flesh should be removed as soon as possible to prevent decomposition and before the seed is damaged. Methods that are suitable for small lots of seeds are cleaning by hand, trampling in vats, and scraping through screens. Relatively large fruits can be conveniently cleaned by placing them in a wire basket and washing them with water at high pressure. For larger lots, a hammer mill or macerator can be used. The macerator is constructed with a hermetic feeder, the water is passed through it, together with the fleshy fruits, and the crumbled mass passes to a tank where both flesh and seed are separated by flotation. Germplasm that reproduces vegetatively Harvesting vegetative planting material depends on the type of propagule of the species (Figure 3), and the procedures to apply depend on the management established for the case (Vázquez Y et al. 2004). The procedures established for managing parts of stems, roots, leaves, or specialized structures (e.g., tubers, bulbs, corms, stolons, rhizomes, tuberous roots, and buds) should be revised so that identical, whole, and healthy plants are regenerated. With respect to health, acquisition should necessarily guarantee, where possible, during harvest and conditioning, planting materials that are free of pathogens. One way of guaranteeing this is to pay attention to three basic aspects: isolation of the production site, adopting health and inspection measures, and periodic testing (Hartmann and Kester 1971). Depending on species and type, propagules are usually short-lived and should be planted within a very short period after harvest. In general, when harvesting vegetative planting materials, the following should be taken into account: • • • • • • • The species being harvested The propagules’ stage of maturity Procedures for collection (manual or use of special equipment) Selection of the propagules during collection (i.e., harvesting only mature propagules that are not damaged by insects or nematodes, or showing symptoms of pathogen attack) Type of packaging to use (e.g., baskets or pita-fibre sacks) and germplasm identification system System of bulk collection and transport to sites for temporary storage Conditions for storing and conserving the materials before new plantings begin Evaluating the Lesson After this lesson, you should be familiar with the most important aspects of harvesting germplasm to help guarantee its integrity such as fruit types, seed parts, and propagules. Before going on to the next lesson, comment on your experiences with harvesting and managing fruits or propagules to obtain seeds or planting materials for germplasm conservation. Emphasize the procedures and care needed to be successful. If you are not directly familiar with these processes, list and discuss the criteria that, in your opinion, should be taken into account when harvesting and managing fruits and propagules destined for conservation. 84 Module 3, Submodule B: Harvesting, Conditioning, and Quantification Lesson 1: Harvesting Vegetative reproduction Besides producing seeds, some plants possess a special type of asexual reproduction known as vegetative reproduction or propagation. That is, a part of the plant can give rise to a new plant by itself. · Corm (e.g., saffron). A short thick stem, similar to the bulb, except that it stores food within the stem itself. · Rhizome (grasses, ferns, lilies). Thick stem with layered leaves and growing horizontally underground. It produces roots along its length and buds that give rise to new shoots. Rhizome sectioned in half Adventitious roots Roots Saffron corm · Rhizome of mint Bulb (daffodil). Short thick stem surrounded by layered leaves and containing food reserves. It forms in the soil from an old and dying plant, and represents the first latent stage of a new plant that will emerge as a shoot at the beginning of the following season. · New bud Stolon or runner (strawberry). A stem grows horizontally from a point close to the plant base. It then produces roots at intervals along the stem and new plants grow from these points. Stolon The shoot will come from this point Bulb sectioned in half Layered leaves Adventitious roots Daffodil bulb · New plant Microphotograph of a longitudinal section of a stem apex from Coleus sp. Short and thick stem Tuber (potato). Short, thickened, subterranean stem that stores food and produces buds that give rise to new plants. Potato tubers Old strawberry plant Potato plant Apical meristem (region where active cell division takes place) Procambial strand (cells that produce vascular tissue) Bud in embryo Leaf primordium (embryonic) Cortex (layer between epidermis and vascular tissue) Vascular tissue Epidermis (outer layer of cells) Pith Figure 3. Different types of propagules (corm, rhizome, bulb, stolon, and tuber) for vegetative reproduction (from Stockely 1991). The microphotograph shows a longitudinal section of a stem apex from Coleus sp. (from Kindersely 1994). 85 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Bibliography Literature cited FAO; IPGRI. 1994. Genebank standards. Rome. 15 p. Also available at http://www.ipgri.cgiar. org/publications/pdf/424.pdf Grabe DF. 1989. Measurement of seed moisture. In Stanwood PC; Miller MB, eds. Seed moisture: Proc. Symposium, held 30 November 1987. Special Publication No. 14. CSSA, Madison, WI. pp 69–92. Hartmann HT; Kester DE. 1971. Propagación de plantas: Principios y prácticas. (Translated from the English by Antonio Marino Ambrosio.) Editorial Continental, Mexico, DF. pp 141–223. (Available in English as Hartmann HT; Kester DE; Davies FT, eds. 1990. Plant Propagation: Principles and Practices, 5th ed. Englewood Cliffs, NJ. 647 p.) Hong TD; Ellis RH. 1996. A protocol to determine seed storage behavior. Technical Bulletin No. 1. IPGRI, Rome. 64 p. Also available at http://www.ipgri.cgiar.org/publications/pdf/ 137.pdf Kindersley D. 1994. Enciclopedia visual seres vivos. Santillana; Casa Editorial El Tiempo, Bogotá, Colombia. p 150. Sandoval S, A. 2000. Almacenamiento de semillas. CESAF-Chile No. 14. CESAF of the Faculty of Forest Sciences, Universidad de Chile, Santiago. Available at http://www.uchile.cl/ facultades/cs_forestales/publicaciones/cesaf/n14/1.html (accessed 24 Oct 2004?). Stockley C. 1991. Diccionario de biología. (Translated from the English by Icíar Lázaro Trueba.) Editorial Norma, Bogotá, Colombia. pp 32–35. (Available in English as Illustrated Dictionary of Biology [Practical Guides]. Usborne Publishing, London.) Vázquez Y, C; Orozco A; Rojas M; Sánchez ME; Cervantes V. 2004. La reproducción de las plantas: Semillas y meristemas. Available at http://omega.ilce.edu.mx:3000/sites/ ciencia/volumen3/ciencia3/157/htm/lcpt157.htm (accessed 24 Oct 2004). Further reading Baskin CC; Baskin JM. 1998. Ecologically meaningful germination studies. In Seeds: ecology, biogeography, and evolution of dormancy and germination. Academic Press, San Diego, CA. pp 5–26. Chin HF. 1994. Seed banks: conserving the past for the future. Seed Sci Technol 22:385–400. Ellis RH; Hong TD; Roberts EH. 1985. Seed technology for genebanks. Handbook for Genebanks No. 2, vol. 1. IBPGR, Rome. 210 p. Hong TD; Linington S; Ellis RH. 1998. Compendium of information on seed storage behaviour, vol. 1: Families A–H. Royal Botanic Gardens, Kew, London. 400 p. ISTA. 1999. International rules for seed testing. Seed Sci Technol 27:1-333 (Supplement 21). 86 Module 3, Submodule B: Harvesting, Conditioning, and Quantification Lesson 1: Harvesting Rao NK; Hanson J; Dulloo ME; Ghosh K; Nowell D; Larinde M. 2006. Manual of seed handling in genebanks. Handbooks for Genebanks No. 8. IPGRI, Rome. Contributors to this Lesson Benjamín Pineda, Daniel Debouck, Alba Marina Torres, Rigoberto Hidalgo, Mariano Mejía, Graciela Mafla, Arsenio Ciprián, Manuel Sánchez, Carmen Rosa Bonilla, and Orlando Toro. Next Lesson In the next lesson, you will study the principal aspects of conditioning and quantifying germplasm after its multiplication and regeneration. 87 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Lesson 2 Conditioning and Quantification Objectives • • • To discuss the concept of quality for germplasm To describe the process of conditioning germplasm To describe the generalities of quantifying the harvest Introduction If the multiplication and harvesting tasks have been successful and the germplasm’s identity is successfully maintained, then the tasks of conditioning and preparing for storage for conservation become essential. Conditioning is perhaps the most delicate process, requiring special attention because the long-term viability of materials depends on it. An error in drying, for example, may lead to an inexorable reduction of viability and thus to loss of germplasm in the short term. Once the germplasm is harvested, then obtaining the seed or propagules becomes essential. Acquisition is based on fructifications or on collected plant parts. A series of processes and controls is applied to ensure that germplasm with the requisite quality for conservation is acquired. Given that conditioning is a critical stage in managing germplasm for conservation and that its successful conservation is a function of its quality, this theme will first be discussed. The Concept of Quality The total quality of a given germplasm refers to the degree of adequacy that its genetic, physiological, physical, and health attributes have for that material’s conservation. Genetic quality This attribute refers to the degree to which the germplasm conserves its original genotypical characteristics, that is, the degree to which it carries the genes that are to be conserved and were present in the material when it was first introduced into the germplasm bank or collection. Genetic quality can be ensured by planting authentic and pure seeds, and maintaining this authenticity and purity during multiplication through preventive methodologies such as isolation, selection of appropriate fields, verification inspections, and rigorous management to prevent undesirable mixtures. Physiological quality The tangible result of physiological quality lies in the seed’s faculty to germinate, emerge, and give rise to uniform and vigorous plants. Good physiological quality implies integrity of structures and physiological processes that permit the seeds to be kept not only alive, but also with high vitality index. Physical quality For seeds, this refers to such attributes as size, shape, brilliance, colour, and weight that were characteristic of the accession or entry. It also includes the seed’s own integrity, that is, 88 Module 3, Submodule B: Harvesting, Conditioning, and Quantification Lesson 2: Conditioning and Quantification it is not fractured, damaged by insects, or stained by the action of micro-organisms, and is free of any contaminant. For vegetative planting materials, physical quality refers to the organs or plant fragments containing functional generative parts (e.g., buds, meristems, apices, roots, and primordia) showing no physical or mechanical deterioration. Seed health quality This quality includes that set of characteristics that the germplasm must possess to ensure absence of pathogens transmittable by plant parts and/or micro-organisms that cause deterioration during conservation. Conditioning Conditioning consists of appropriately preparing the germplasm after harvest to achieve conservation goals by treating seeds or propagules accordingly. Those procedures most used for treating seeds and vegetative planting materials are reviewed below. Dry fruit seeds Seeds are conditioned by applying procedures that take into account the type of fruit from which they come. For dry fruit seeds, procedures include threshing or shelling fruits, cleaning by blowing or sieving, drying (20°C; 22% relative humidity or RH), temporary storage in cold rooms (5°C and 22% RH), final selection of seeds, final drying with cool air (20°C; 22% RH), and final packaging in hermetic containers or vacuum-packing in aluminium bags for conservation in cold rooms (-20°C), according to goals. When managing and packaging seeds during different stages of the process, they should be placed in cloth bags (muslin), especially during drying, and then in hermetic containers to prevent the seeds from rehydrating. Threshing or shelling. Shelling or threshing can be carried out manually or be mechanized. The procedures used depend on the species and fruit type. Any threshing operation basically implies a process whereby the harvested fruits are beaten or passed through rollers to separate the seeds from the rest of the plant. A heavily used machine is the combine thresher, the central part of which is a revolving cylinder that works as a beater. It also has couplings with other devices that separate the threshed seed from husks and straw. This type of machine is used to harvest large seed lots. With small lots, seeds can be separated by threshing and cleaning them by hand in a screen (Hartmann and Kester 1971). For legumes, seeds are extracted by striking or trampling the pods and sieving them through a screen, shelling them by hand, or rubbing them with a special implement (Figure 1). However, care must be taken to verify seed performance as according to species, because striking them is sometimes counterproductive, cracking and therefore spoiling them. The extraction of conifer seeds requires special procedures. The cones of some species will open if they are dried in the open air for 2 to 12 weeks. Others must be forcibly dried at higher temperatures in special ovens. On drying, the cone scales open, exposing the seeds. 89 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Threshing pods with a hand-held tamper Shelling Sieves or screens Winnowing machine Pre-drying fruits in a cool-air drier (20°C; 35% RH) Pneumatic separator cleaning grass seeds Light seed Heavy seed Schematic cross-section of a pneumatic separator Hopper Pneumatic separators Light seed Intermediate seed Heavy seed Seed Platform Ventilator Final classification of seeds, using a magnifying glass Schematic cross-section of a seed separator using air and gravity Figure 1. 90 Conditioning seeds. Procedures and equipment used (photographs by B Pineda, GRU, CIAT; diagrams from CIAT 1989). Module 3, Submodule B: Harvesting, Conditioning, and Quantification Lesson 2: Conditioning and Quantification They must then be shaken or raked to separate the seeds, which should then be immediately removed, as the cones may close again (Hartmann and Kester 1971). The seeds of some grasses (Poaceae) and cereals have aristas, beards, or glumes, which cannot be completely separated during threshing, thus impeding their effective classification. To remove them, the seeds must be either rubbed manually or placed into a specialized machine that rubs the seeds against revolving hammer arms that remove the coats, thresh the spikes, and, generally, polish the seeds (CIAT 1989). Conifer seeds have appendages or wings, which are removed, except in species where the seed coats damage easily, as in incense cedar (Libocedrus sp.). Fir (Abies spp.) seeds also damage easily, but they can be separated from the wings if care is taken. Seeds from Sequoia spp. have wings that cannot be separated from the seed. In small lots, the wings can be removed by rubbing the seeds between wet hands, or else trampling or striking seeds in partly filled sacks. For larger lots, special dewinging machines are used. After dewinging, the seeds are cleaned to remove residues of wings and other light materials. The final step is to separate the filled and heavier seeds from the lighter ones, using pneumatic separators or gravity (Hartmann and Kester 1971). Cleaning and selection. Once threshed, the seeds must be cleaned to remove rubbish, twigs and other unwanted plant parts, parts of foreign plants, and seeds of other crop or weed species. Small lots can be cleaned, using a screen or passing them through a container to another and allowing air to drag away the lightest materials. Removing the rubbish is a pre-cleaning operation by which materials that are larger or smaller than the seeds are separated from them. The operation is manual, using sieves or screens (Figure 1) and a seed blower, or with cleaners that combine hoppers, sieves, and ventilators to eliminate the light materials (CIAT 1989; Hartmann and Kester 1971). Seeds can be separated mechanically from undesirable materials during cleaning only if they differ from them in one or more physical properties. The properties most used correspond to weight or density, colour, texture, size, width or thickness, length, and electrostatic properties. These permit the design of specialized devices that take advantage of the differences between seeds and contaminants to clean. The devices usually combine air currents (Figure 1), different-sized screens, gravity (Figure 1) or texture separators, slopes, vibrators, magnetic cylinders, and photoelectric cells (CIAT 1989; Hartmann and Kester 1971). When cleaning equipment is used, care must be taken to remove harvest residues or seeds remaining in its interior before processing another accession to prevent contamination and undesirable mixing. Also, the equipment should be cleaned of dust and other residues to prevent contaminating the seed with the reproductive structures of micro-organisms such as fungi, bacteria, and nematodes that usually associate with plant materials during the plants’ growth and fructification in the field. After carrying out the basic cleaning processes and having obtained the seeds, the procedures for finishing or final selection are conducted (Figure 1). Seeds are examined under low-powered magnifying glasses to discard those with otherwise invisible spots, fissures, wounds, or deformities. Special equipment such as pneumatic or gravity separators is also used to eliminate empty or low-density seeds. 91 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Drying. Drying consists of reducing the moisture content of seeds to a minimum level for metabolic activity, without their losing viability. To dry or reduce the seed’s moisture content, its original moisture content must first be determined by quantifying, either directly or indirectly, the water they contain. Direct determinations are made through gravimetric methods, chromatography, or spectrophotometry. Indirect methods include hygrometric methods, infrared spectroscopy, nuclear magnetic resonance, and chemical reactions of seeds (Grabe 1989). Currently on the market are electronic analysers (moisture meters) or special balances with infrared heating chambers that permit rapid and precise quantification of moisture content of small samples of seeds (Figure 2). If such technology is not available, then the other methods mentioned Figure 2. 92 Drying seeds. Left, balances to determine seed moisture content and, right, cool-air drying room (20°C; 22% RH) (photographs by B Pineda, GRU, CIAT). Module 3, Submodule B: Harvesting, Conditioning, and Quantification Lesson 2: Conditioning and Quantification above can be used. All these methodologies are described in the following publications: Seed Technology for Genebanks (Ellis et al. 1985), A Protocol to Determine Seed Storage Behavior (Hong and Ellis 1996), and Manual of Seed Handling in Genebanks (Rao et al. 2006). To precisely determine moisture content, the gravimetric method (ISTA 1999) is recommended with some modifications to sample size, given that this method is destructive. Many pre-postharvesting operations require rapid determinations (which are less precise) that can be carried out with portable equipment. Such determinations are based on the electrical properties of water in the seeds such as conductivity and capacitance (ability of an electric conductor to carry a charge at a given potential; ability to store electrical charge). The physical relationships between the moisture content (MC) of a seed, temperature, and relative humidity form the basis for drying. The maximum quantity of water that air can contain depends on the temperature. An indirect measure of air humidity is relative humidity (RH). The concept of RH can be expressed as follows: if air at 10°C contains 5 g of water/kg of dry air, but its capacity for saturation is 20 g of water/kg, then its RH is 5/20 × 100 = 25%. For the same water vapour content of the air, if the air’s temperature rises, then its RH drops and vice versa. Water in the seed (i.e., MC) tends to balance (moisture content balance or MCB) out with the humidity of the surrounding air. Hence, dry air, that is, with a low RH (20%–25%), can rapidly dry the seed to reach an MCB. The time taken to reach the MCB depends on the species (anatomy and food reserve tissues) and temperature. Likewise, humid air increases a seed’s MC. Thus, the air used for drying should be recycled and dried out. Certain chemicals are able to absorb moisture from the air; perhaps the most common is silica gel. A relationship exists between seed longevity, storage temperature, and seed MC, thus demanding that a suitable combination of temperature and moisture be taken into account. The first requisite to consider is the low MC of seeds, which can be as low as 5%. A calculation made with sesame (Sesamum indicum L.) found that reducing seed MC from 5% to 2% will increase seed longevity by 40 times. However, a lower limit of tolerable MC exists, depending on the species. Hence, a limit of 5% is usually established (FAO and IPGRI 1994). Before proceeding with drying, the procedures and degrees of desiccation that the materials require should be well understood. In terms of maintaining the viability of the germplasm, deficient or excessive drying without sufficient basis is a very high risk, to which seeds should not be exposed. Hence, experiments should be carried out to determine the type of drying that can be applied with minimal risk. Most seeds should be dried after harvest. Seeds with more than 20% MC heat up if they are piled up for several hours, thus reducing their viability. Drying must begin in the field, immediately after collection and/or extraction of seeds (see Module 3, Submodule B, Lesson 1). Drying can occur naturally in the open air or artificially by heat or other methods. Drying temperatures should not be higher than 43°C (110°F) and, if seeds have high moisture content, the ideal temperature is 32°C (90°F). Too rapid a drying can cause shrivelling and fracture of the seeds and sometimes harden the coats. The MC at which seeds can be conserved without risk is between 8% and 15%, although some seeds should 93 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources be conserved moist. Nevertheless, drying at temperatures at more than 40°C can be disastrous for germplasm conservation. In Latin America, there have been cases where seed longevity has been no longer than 5 years and the percentage of germination no more than 25% (Daniel Debouck 2004, personal communication). Methods of natural drying (e.g., drying in the open air under shade) do not reduce MC below 8%–10%, which is suitable for short-term conservation, that is, 2 to 3 years. Drying in the direct sun is not recommended because, in many cases, the germplasm can be exposed to high temperatures for too much time, thus causing irreversible damage to seed viability. Artificial drying can, with the help of equipment that permits air circulation at different temperatures or silica gel, be an easy and effective method (Hong and Ellis 1996). Electronic driers permit programming of drying cycles, temperatures, flows, and speeds of the drying air. Drying should be carried out in rooms especially designed for the purpose (Figure 2). In such rooms, combinations of dry (20%–22% RH) and cool (temp. 15°–25°C) air can be managed to reduce the percentage of MC in the seeds to 4%–6%, suitable for long-term conservation. However, the species should be taken into account because, in some cases, they would be overdried. The type of substances in the seed’s reserves also influence the MCB in the drying room. Sugars have the most affinity for water, followed by proteins, starches, and oils. This means that, for a given RH, oleaginous seeds may contain less moisture than proteinous or starchy seeds. Once the drying is finished, the MC is measured again to confirm that the required level (5%–12%) has been reached and to determine if the samples need to be submitted to a new cycle of drying or rehydration. The temperatures and times of drying must be established accurately so not to endanger the samples, as repetitive procedures can reduce viability. Fluctuations in MC reduce the seeds’ longevity, as they increase the seeds’ respiratory rate. The increase causes the seeds’ reserves, which are designed to feed the embryo during germination, to be consumed through respiration as metabolism is increased, thereby reducing the seeds’ quality (Hartmann and Kester 1971). Most long-lived or intermediate seeds can tolerate drying (orthodox) to 4%–6% for storage over prolonged periods at low temperatures. Moisture content can be increased, but only if the temperature is reduced. The seed’s MC will determine the duration of storage. In general, short-lived seeds are sensitive to drying (recalcitrant). These seeds have a high MC and lose their viability when this is reduced (Hartmann and Kester 1971). Seeds of this type are found in species such as oaks (Quercus spp.), walnuts (Juglans spp.), araucaria pines (Araucaria spp.), Chilean hazel (Gevuina avellano), and Beilschmiedia spp. (Hartmann and Kester 1971; Sandoval S 2000). Fleshy fruit seeds These seeds must be separated from the flesh that surrounds them. For tomato, macerated fruits are placed in barrels or large vats and left to ferment for about 4 days at about 21°C (70°F), stirring occasionally. If the fermentation is left for too long, the seeds may germinate. At higher temperatures, fermentation time will be shorter. As seeds separate from the pulp, the healthier and heavier ones sink to the bottom of the vat. The pulp remains at the surface, together with the empty seeds and other foreign materials. After extraction, the 94 Module 3, Submodule B: Harvesting, Conditioning, and Quantification Lesson 2: Conditioning and Quantification seeds are washed and dried, either in the sun or in a drier. Additional cleaning is sometimes needed to remove dried pulp and other materials. For cucumbers and similar fruits, special machines are used to extract and clean the seed from the pulp. After separation, the seeds are washed and dried as is done for fermentation (Hartmann and Kester 1971). The small berries of some species of Juniperus and Viburnum are difficult to process because of their size and the difficulty of separating the seeds from pulp. One way of managing such seeds is to pound them with a kitchen roller, soak them in water for several days, and remove the pulp by flotation. A better method for extracting seeds from small fleshy fruits is to use an electric mixer of the type used in soda fountains or a blender. To prevent damage to the seeds, the blender’s metal blades can be replaced with a piece of rubber that is cut from a tyre and fixed horizontally to the machine’s revolving axis. A fruit and water mixture is then placed into the blender’s glass and agitated for 2 min. When the flesh has separated from the seed, it can then be removed by flotation. Some fruits such as those of juniper (Juniperus spp.) must be pounded before extracting the seed (Hartmann and Kester 1971). Plant parts Plant parts are conditioned according to the type of propagule of the species, and to the management established for its case. Where no information is available, then the requisite research must be conducted. The procedures for conditioning stem parts, root parts, leaves, or specialized structures (e.g., tubers, bulbs, corms, stolons, rhizomes, tuberous roots, buds, meristems, and apices) targeted for conservation are specific to each species. For example, for cassava (Manihot esculenta), the factors to take into account when conditioning stakes include the plant’s age, its health status, stem parts to use, stem diameter and length, number of buds, type of cut to make, and the treatment, if any, before temporary storage (Lozano et al. 1977). Generally, because they concern specialized organs, parts, or fragments of live plants, plant parts cannot have their MC reduced. Nor can they be exposed to long-term storage. As a result, they must be handled with great care. If materials are to be conditioned for in vitro conservation, explants are extracted, preferably from the youngest plants, for micropropagation. This procedure consists of (a) disinfecting the explants in a solution of sodium or calcium hypochlorite, or ethanol; (b) planting them in an in vitro culture medium until new shoots develop; and (c) rooting the shoots to obtain whole plants (Frison 1994; George 1996; George and Sherrington 1984; Roca and Mroginski 1991). Packaging Once conditioning is finished and the verifications of MC are carried out, the material, in the case of the seeds, is ready for packing and transporting to the storage site. Both container and storage site should respond to the requirements of the species and guarantee survival of the samples. To pack seeds, diverse types of containers exist, with varied shapes and materials and ranging from paper and aluminium envelopes to plastic or glass bottles (Figure 3) and tins of different metals. More than its shape or material, the container must be airtight, that is, it 95 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Figure 3. Types of containers and holders for seeds and plant parts. Upper part—plastic bottles and aluminium bags for seeds; lower part—plastic crates for transporting planting materials (photographs by B Pineda, GRU, CIAT). isolates the germplasm sufficiently to prevent it from absorbing moisture and/or becoming contaminated. Selection of the container depends on seed characteristics and on the period for which they are expected to be conserved. In practice, it is also determined by the bank’s resources, as containers not only vary in shape and materials, but also in costs and availability on the market. Aluminium bags are the most recommended as they can be hermetically closed, using a heated stamp (Figure 3). Airtight containers, for example, are optimal but expensive. The investment involved depends on what the material is destined for. Jaramillo and Baena (2000) describe a series of containers commonly used in germplasm banks. 96 Module 3, Submodule B: Harvesting, Conditioning, and Quantification Lesson 2: Conditioning and Quantification For plant parts, given their relative perishability and short storage periods, when required or when the material permits it, the containers or packaging used should maintain the germplasm fresh. It is also essential that the containers protect the material from damage to its buds or other generative areas of the material, whether from mechanical injury or deterioration caused by other agents during transport to the planting site. Currently, the market provides numerous options of plastic crates (Figure 3) that are especially designed to transport and manage perishable products that can be useful for germplasm management. Quantifying Germplasm Seeds or propagules are usually counted by hand. For seeds, however, automated solutions are available such as counters, counter heads connected to a vacuum system (Figure 4) and other commercial equipment. Indirect estimates can also be made such as by weight, which are less precise. The technique consists of determining the unitary weight of the seed by taking at random four replications of 100 seeds (g/100 seeds) (ISTA 1999) and making the respective calculations based on the weight of materials ready for storage. Although the count is indeed based on apparently simple operations, it is highly significant because it forms the basis by which the germplasm bank knows what it has and for what ends. If the bank assumes the responsibility to conserve, it has the obligation to test viability on a periodic basis, check samples for plant health quality, to conserve and distribute. For these activities, it must have a record of how many propagation units will be needed to fulfil pre-established plans. Evaluating the Lesson After this lesson you should be familiar with the most important aspects of conditioning and quantifying germplasm, as well as with the concept of total quality. Before going on to the next lesson, describe your experiences in conditioning seeds or propagules for storage for conservation, emphasizing the procedures and care needed to be successful. If you are not directly familiar with these processes, list and discuss the criteria that, in your opinion, should be taken into account to condition seeds and propagules destined for conservation. Bibliography Literature cited CIAT. 1989. Principios del acondicionamiento de semillas; Guía de estudio para ser usada como complemento de la unidad audiotutorial sobre el mismo tema. Scientific consultant: Albert H Boyd. Serie: 04SSe-03.01. Cali, Colombia. 34 p. Ellis RH; Hong TD; Roberts EH. 1985. Seed technology for genebanks. Handbook for Genebanks No. 2, vol. 1. IBPGR, Rome. 210 p. FAO; IPGRI. 1994. Genebank standards. Rome. 15 p. Also available at http://www.ipgri.cgiar. org/publications/pdf/424.pdf 97 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Valve Filter Vacuum pump Pipes connected to the vacuum pump Solenoid Hose Nozzle for large seeds Hose Nozzle for small seeds Figure 4. Vacuum system and nozzles used to quantify seed according to size (photographs by B Pineda, GRU, CIAT, and a vacuum pump catalogue). 98 Module 3, Submodule B: Harvesting, Conditioning, and Quantification Lesson 2: Conditioning and Quantification Frison EA. 1994. Sanitation techniques for cassava. Trop Sci 34(1):146–153. George EF. 1996. Plant propagation by tissue culture: in practice, Part 2. Exegetics, Westbury, UK. 1361 p. George EF; Sherrington PD. 1984. Plant propagation by tissue culture: handbook and directory of commercial laboratories, Part 1. Exegetics, Westbury, UK. 709 p. Grabe DF. 1989. Measurement of seed moisture. In Stanwood PC; Miller MB, eds. Seed moisture: Proc. Symposium held 30 Nov 1987. Special Publication No. 14. CSSA, Madison, WI. pp 69–92. Hartmann HT; Kester DE. 1971. Propagación de plantas: Principios y prácticas. (Translated from the English by Antonio Marino Ambrosio.) Editorial Continental, Mexico, DF. pp 141–223. (Available in English as Hartmann HT; Kester DE; Davies FT, eds. 1990. Plant Propagation: Principles and Practices, 5th ed. Englewood Cliffs, NJ. 647 p.) Hong TD; Ellis RH. 1996. A protocol to determine seed storage behavior. Technical Bulletin No. 1. IPGRI, Rome. 64 p. Also available at http://www.ipgri.cgiar.org/publications/pdf/ 137.pdf ISTA. 1999. International rules for seed testing. Seed Sci Technol 27:1–333 (Supplement 21). Jaramillo S; Baena M. 2000. Material de apoyo a la capacitación en conservación ex situ de recursos fitogenéticos. IPGRI, Cali, Colombia. 209 p. Also available at http://www.ipgri. cgiar.org/training/exsitu/web/arr_ppal_modulo.htm (accessed 27 July 2004). Lozano JC; Toro JC; Castro A; Bellotti AC. 1977. Production of cassava planting material. Series GE-17. CIAT, Cali, Colombia. 28 p. Rao NK; Hanson J; Dulloo ME; Ghosh K; Nowell D; Larinde M. 2006. Manual of seed handling in genebanks. Handbooks for Genebanks No. 8. IPGRI, Rome. Roca WM; Mroginski LA, eds. 1991. Cultivo de tejidos en la agricultura: Fundamentos y aplicaciones. CIAT, Cali, Colombia. 969 p. Sandoval S, A. 2000. Almacenamiento de semillas. CESAF–Chile No. 14. CESAF of the Faculty of Forest Sciences, Universidad de Chile, Santiago. Available at http://www.uchile.cl/ facultades/cs_forestales/publicaciones/cesaf/n14/1.html (accessed 24 Oct 2004). Further reading Baskin CC; Baskin JM. 1998. Ecologically meaningful germination studies. In Seeds: ecology, biogeography, and evolution of dormancy and germination. Academic Press, San Diego, CA. pp 5–26. Chin, HF. 1994. Seed banks: conserving the past for the future. Seed Sci Technol 22:385–400. Hong TD; Linington S; Ellis RH. 1998. Compendium of information on seed storage behaviour, vol. 1: Families A–H. Royal Botanic Gardens, Kew, London. 400 p. 99 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Contributors to this Lesson Benjamín Pineda, Alba Marina Torres, Daniel Debouck, Carlos Iván Cardozo, Rigoberto Hidalgo, Mariano Mejía, Graciela Mafla, Arsenio Ciprián, Manuel Sánchez, Carmen Rosa Bonilla, and Orlando Toro. Next Lesson In Lesson 1 of the next Submodule C, you will study the principal aspects of monitoring the biological status (physiological quality) of germplasm. 100 Submodule C Lesson 1 Basic Concepts Verifying the Biological Quality of Germplasm Objectives • • • To discuss the basic concepts of viability, vigour, dormancy, and germination in seeds To mention the essential structures of seedlings used to evaluate germplasm viability To mention general aspects of viability, vigour, germination, and essential components of plant parts for vegetative propagation Introduction To conserve germplasm, an essential condition is that it must be viable, that is, it must be alive and able to regenerate new plants capable of independent existence. Within this order of ideas, the verification of the biological or physiological status of germplasm targeted for conservation takes on special importance. Once conditioning has been carried out, the material should be verified to check that it was correctly prepared to guarantee its successful conservation in terms of the bank’s plans and goals. Also, following the germplasm bank’s norms, the viability of conserved germplasm should be monitored at least every 5 years according to species and storage conditions; or, in their absence (as for vegetative planting materials), adjustments made as required per species. These periodic checks should be compared with each species’ initial viability and conservation conditions (FAO and IPGRI 1994). The verification of viability requires the understanding of concepts of the germplasm’s physiological, physical, and health quality, as defined in Lesson 2 on conditioning (Module 3, Submodule B). Moreover, as a minimum, general knowledge is needed on viability and vigour as applied to seeds and, indeed, to vegetative planting materials. Also important is being able to identify those essential structures that are evaluated to determine physiological status and interpret results. This lesson deals with these themes. Viability and Vigour The viability of germplasm refers to that property of being alive, that is, possessing the ability to regenerate new plants capable of independent existence. In other words, the germplasm is able to germinate and produce normal and vigorous seedlings that can complete again the species’ life cycle. In terms of quantification, seed viability measures the number of seeds in a lot that are alive and could develop into plants that will reproduce under appropriate field conditions (Rao et al. 2006). In the case of seeds, the viability of germplasm must be understood as being variable over time (i.e., longevity is variable) and, depending on the species, a function of its adaptation to the habitat where, ecologically, the plants had originally developed. The seeds of each plant species present typical germination mechanisms that respond to the effect of natural selection induced by predominant environmental conditions on the nature and physiology of seeds (Vázquez Y et al. 2004). Knowledge of this behaviour is useful for making the adjustments needed to establish viability tests to confirm physiological status. 101 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Usually, the half-life of a seed is between 5 and 25 years. Seeds lose their viability for highly diverse reasons. One is that their food reserves are exhausted and, hence, the germinative capacity is lost. The less active a seed’s metabolism is, the more long-lived it will be (García B 2004). The vigour of a species’ seed is its capacity for the rapid and uniform emergence and normal development of seedlings under a broad range of field conditions (AOSA 1983; OSU 2004). As ISTA defined it during its 1977 congress, vigour is the total sum of those properties of a seed or lot of seeds that determine the level of activity and capability of this during germination and seedling emergence (Perry 1981). The definition considers specific aspects of performance that had been regarded as evident variations, associated with differences in seed vigour such as: • • • • Biochemical processes and reactions during germination such as respiratory activity and enzymatic reactions Speed and uniformity of seed germination and seedling growth Speed and uniformity of seedling emergence and growth in the field The capacity of seeds to emerge under unfavourable conditions Factors that influence seed vigour The causes of variations in vigour are many and diverse. To clarify the concept, ISTA has established a list of the most commonly known factors that affect vigour, including: • • • • • • • Genetic constitution Environmental conditions and nutrition of the parent plant State of maturity at harvest Seed size, weight, and density Physical integrity State of ageing and deterioration Pathogens Generally, seed development involves a series of ontogenetic states such as fertilization, nutrient accumulation, seed drying, and dormancy. Any disturbance to development may alter the seed’s potential performance (Delouche and Baskin 1973). The challenge for tests, then, is to determine vigour by identifying one or more quantifiable parameters commonly found in association with seed deterioration. A hypothetical model has been developed that will eventually be used to better estimate seed vigour, even including the process of deterioration (Delouche and Baskin 1973). The model graphically visualizes the extent to which deterioration may increase and the seed’s vitality diminish (Figure 1). In other words, in real terms, vigour can be seriously affected. The beginning of cell membrane degradation precedes loss of germination. Hence, a highly sensitive test for estimating vigour would be that which monitors membrane integrity. There are sufficient experimental arguments to support this assertion. Membranes are essential for many metabolic events that occur in a seed, including respiration, which provides the seed with the energy needed for subsequent plant development (OSU 2004). 102 Module 3, Submodule C: Verifying the Biological Quality of Germplasm Lesson 1: Basic Concepts Degradation of cellular membranes Diminished respiration, energy production, and biosynthesis Diminished capacity to store Lack of uniformity and heightened sensitivity to environmental factors Less productivity Abnormalities and extreme weakness Deterioration Vigour Death Vitality Less capacity for synthesis Figure 1. Slower germination speed Less growth and development Diminished resistance Lack of uniformity and failures in emergence Less germination Hypothetical model that shows the relationship between vigour, vitality, and deterioration, and the indicators proposed (model adapted and redrawn by B Pineda from Delouche and Baskin 1973). The endoplasmic reticulum is an organelle made up of membranes where many enzymes are produced that bring about the translation of RNA (ribonucleic acid). Thus, any disturbance in the function of such membranes can reduce the ATPs associated with the supply of energy to cells and retard the synthesis of specific enzymes essential to growth. With loss of respiration and biosynthetic capacity, the germination rate is reduced, as manifested in a lack of uniformity in seed lots. Other events associated with deterioration are loss of storability and of the capacity to resist disease. When degraded seeds are subjected to biological and environmental pressures, they show a poor rate of emergence under field conditions and poor yields. Finally, these subtle manifestations of loss in seed quality find expression in an increased incidence of abnormal seedlings—a component of germination tests (OSU 2004). Dormancy Higher plant forms may show dormancy or interruption of growth in meristematic tissue, for example, in growth buds of branches, as well as in seeds. In seeds, dormancy, also known as latency, resting, or quiescence, is the last phase of its ontogeny or formation and development. It is an essential stage for seed survival as it maintains them in waiting until environmental conditions are propitious for germination and plant production. In practical terms, dormancy refers to the state in which viable seeds fail to germinate, even under conditions normally favourable for germination (Rao et al. 2006). 103 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources The development of seed dormancy involves water loss, differentiation of the seed’s integuments or coats, interruption of genetic transcription and protein synthesis, and reduced respiration and other activities of intermediary metabolism (Vázquez Y et al. 2004). During the final stages of ontogeny, particularly that of storing food reserves and maturation, and according to species, carbohydrates (starches and sugars), proteins, and lipids are accumulated. When this process ceases and the seed has completed its development, drying begins. The synthesis of oligosaccharides increases. These substances are involved in the tolerance of drying and may, as does raffinose, prevent the cytoplasm from crystallizing in the desiccated mature seed. In some species, during maturation, the plant hormone abscisic acid (ABA) plays an important role in controlling dormancy until conditions are such that the seed can germinate and initiate the formation of a new seedling (Bolaños 2004; Vázquez Y et al. 2004). When the seed separates from the plant that produced it, it enters quiescence or reduced metabolism, that is, it does not show external signs of activity within it. This resting is called quiescence when the reason for the lack of germination is basically a lack of water, as when seeds are stored under artificial conditions or remain in the fruits united to the parent plant for long periods (Gooding et al. 2000; Vázquez Y et al. 2004). However, resting is called dormancy when the seed does not germinate, despite conditions being optimal in terms of temperature, air, and humidity for radicle emergence and seedling growth (Gooding et al. 2000; Vázquez Y et al. 2004). The lack of germination can be attributed to the existence of a chronologically regulated period of interrupted growth and reduced metabolism during the plant’s life cycle. This is an adaptive strategy to survive unfavourable environmental conditions (Vázquez Y et al. 2004). Dormancy allows seeds to distinguish a good site for germinating. For example, those that require light to germinate will not do so if they are buried in soil or shaded by other plants. Thus, through dormancy, seeds perceive information on external environmental conditions, including the season in the year! For example, the seeds of some species must undergo a period of low temperatures, close to zero degrees, indicating that winter has arrived. Then, as temperatures increase and the first rains fall, thus indicating the beginning of spring, germination occurs and the seedling is established during a time of the year when survival is most likely. This limits those species to certain geographical regions where temperatures drop in winter (Moreno C 2004). The establishment of dormancy is regulated by hereditary factors that determine a plant’s endogenous physiological mechanisms, which interact with factors of the environment in which it grows. In the long run and over millions of individuals, this gives rise to evolutionary changes in the plants. Among the most important environmental conditions are (1) climatic variations in temperature and relative humidity, (2) microclimatic variations derived from physiographic and biotic aspects such as the spectral quality of light and thermoperiod, and (3) the specific characteristics of the place to which the plants are adapted for establishment and growth. Micro- and macroclimatic variations, and the hormonal and nutritional conditions of the parent plant greatly influence the establishment of dormancy in its seeds during their development. This means that variations may exist between harvests of seeds from a given species, depending on the time and place of production (Vázquez Y et al. 2004). 104 Module 3, Submodule C: Verifying the Biological Quality of Germplasm Lesson 1: Basic Concepts The permanence of dormancy depends on the plant’s species and on its adaptation to the conditions of the habitat where it originally grew or evolved. The duration of dormancy may vary considerably, from very short periods to several years. Types of dormancy Several types of dormancy have been defined. Rao et al. (2006) group all types into either embryo dormancy or seed-coat dormancy. However, for purposes of our theme of ex situ conservation, we will briefly describe those most mentioned: Innate or endogenous dormancy becomes manifest when the embryo stops growing (while the seed is still in the parent plant) and continues until the endogenous impediment ceases. From that moment, the seeds are ready to germinate when suitable environmental conditions occur. The presence of chemicals in the embryo inhibiting germination or the embryo’s immaturity is probably the main cause of such dormancy. The duration of innate dormancy is highly variable according to species and, in some cases, may even differ between seeds from one individual. Some experiments indicate that, certain tropical seeds possess processes comparable with the postmaturation that is characteristic of many temperate-climate trees, whose seeds germinate only after winter has elapsed. That is, in the laboratory, when such tropical seeds are exposed or stored under low temperatures, or given applications of plant hormones such as gibberellic acid, they show dormancy (Vázquez Y et al. 2004). Physiological dormancy involves mechanisms that physiologically inhibit the embryo’s radicle from emerging. Such dormancy can be attributed to the low permeability of the integuments around the embryo to oxygen; or to the embryo’s lack of sufficient growth potential to break the seed coats, as the endosperm cells restrict the radicle’s growth. Physiological dormancy may range from non-deep through intermediate to deep dormancy, differing according to species (Baskin and Baskin 1998b). Induced or secondary dormancy occurs when seeds are in a physiological position to germinate but are in an environment that is highly unfavourable such as having little oxygen, CO2 concentrations that are higher than those of the atmosphere, or high temperatures. These unfavourable factors induce reversible physiological alterations in the seeds, whereby they fall into a state of secondary dormancy and are no longer able to germinate, even though they remain alive. In some cases, this type of dormancy can be broken, using hormonal stimulation. Induced dormancy can sometimes compound other types of dormancy or replace them (Vázquez Y et al. 2004). Secondary dormancy can be induced in mature seeds once they are separated from the parent plant or, if dormancy was already present, induced into a deeper level. This process occurs when the seeds are subjected to unfavourable conditions for germination, for example, anaerobiosis, which occurs when there is excess water or low oxygen levels in the atmosphere, or the seed coats are not readily permeable. High temperatures may also have the same effect. Sometimes the lack of a single requirement for germination such as light will induce dormancy. However, it should be remembered that each species responds in particular ways (Moreno C 2004). Imposed or exogenous dormancy. In nature, this dormancy occurs in seeds that can germinate under suitable conditions of humidity and mean temperature, that is, suitable for 105 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources the habitat that they occupy. However, they remain dormant because of one factor or another such as lack of light or particular requirements of temperature or oxygen. This dormancy is controlled by the physical conditions of the environment surrounding the seed. It occurs in seeds found in the soil and which germinate only after a disturbance that modifies, for example, the light regime or oxygen content (Vázquez Y et al. 2004). Physical dormancy. A major reason for a seed not germinating is the impermeability of its seed coats to water. This impermeability is associated with the presence of one or more walls of impermeable palisade cells (Baskin and Bask in 1998b). Many plants produce seeds whose external coat is hard and impermeable to water or gases and even their micropyle is provided with a barrier that impedes the penetration of water to the embryo. This characteristic appears frequently in several plant families, particularly the Fabaceae or legumes, Malvaceae, and Bombacaceae (Vázquez Y et al. 2004). In forest soil, the seed coat gradually becomes permeable through weathering, microbial degradation, soil factors such as saponins, or temperature fluctuations, with germination occurring slowly. This mechanism of passive dormancy is particularly frequent in dry tropical forests, and may have originated as a mechanism for seed persistence in the soil over the season that is unfavourable to growth (Vázquez Y et al. 2004). Chemical dormancy occurs when inhibitors of germination are deposited in the pericarp or seed coats. These substances include compounds that are produced in the seed or are transmitted to it, thus blocking the embryo’s growth (Baskin and Baskin 1998b). Embryo dormancy occurs when an embryo that has been extracted from its seed is incapable of germinating under suitable conditions. This type of dormancy is controlled, on the one hand, by the cotyledons that inhibit growth of the embryonic axis and, on the other, by substances that inhibit germination such as abscisic acid (Moreno C 2004). Coat dormancy. The structures responsible for this mechanism are the seed coats, but frequently include other seed parts such as the endosperm, pericarp, and, in grasses, the glume, palea, and lemma. These structures impose dormancy in several ways by interfering with the entry of water and gas exchange, containing chemical inhibitors, impeding the escape of inhibitors present in the embryo, modifying the light arriving at the embryo, and mechanical constriction. Interference with the entry of water is a major cause of dormancy in seeds with hard coats, particularly in legumes, but also in species of Convolvulaceae, Cannabaceae, Chenopodiaceae, Gramineae, Malvaceae, and Solanaceae. Many seeds have mucilaginous coats and, in some cases, this mucilage is believed to produce impermeability as in, for example, Sinapis arvensis and Blepharis persica. Some species, mainly from the Mimosoideae (legumes) group, present a small aperture in the coat, which has a plug known as the strophiole that must be broken for water to enter. In some cases, such as for Iris or Rosa spp., the coat contains substances that inhibit germination. Sometimes these inhibitors are inside the seed and the coat’s impermeability prevents their escaping. For example, in oats, dormancy is maintained by the lemma and palea. When these are removed and the naked seeds placed in a moist environment, the seeds germinate. In the vast majority of seeds that require light to germinate, the coat imposes dormancy. It acts as a filter through which light must pass. It should be remembered that seeds 106 Module 3, Submodule C: Verifying the Biological Quality of Germplasm Lesson 1: Basic Concepts requiring light to germinate are stimulated when a certain ratio of active to inactive phytochromes is established—a product of the combined action of red and far red light. Seed coats have different thicknesses and different pigmentations, thus modifying the quantity and quality of light that reaches the embryo. For example, in Chenopodium album, germination rates of seeds exposed to 15 min of light are, for thick coats at 49–53 µm, only 27%; 44–99 µm thick, 47%; and 24–28 µm, 62% (Moreno C 2004). Influence of the parent plant’s environment on seed dormancy The seed’s germinative characteristics have a strong genetic component that manifests itself during the seed’s development. At the same time, the environmental conditions to which the parent plant is subjected affect this process and influence the type and degree of dormancy of the seeds. Seeds of a given species (e.g., Lactuca sativa or Stellaria media) produced in a period of low temperatures presented a deeper dormancy than seeds produced in warmer periods. The daylength that the parent plant experiences (especially during the last days of seed maturation) affects the dormancy of some species. Some desert species show a correlation between daylength and coat permeability. The environmental humidity that the parent plant experiences during seed maturation can also determine the seed coat’s degree of impermeability (Moreno C 2004). Classifying seeds by their dormancy Hartmann and Kester (1971) suggest that seeds can be classified according to their response to specific environmental conditions and management methods. The groups are: Group I. Seeds that have a hard and impermeable coat and, accordingly, the embryo cannot absorb water. This group includes seeds with such hard coats that they resist the growing embryo. Group II. Seeds with dormant embryos that respond to pre-germinative chilling. A. Seeds that need a single period of chilling. B. Seeds that require a warm period for the root or embryo to develop before the chilling period. C. Seeds that need two consecutive chilling periods separated by a warm one. Group III. Seeds that combine an impermeable coat with a dormant embryo. Group IV. Seeds that contain chemical inhibitors that can be removed by percolation. Group V. Recently harvested dormant seeds that become germinable after dry storage (postharvest dormancy). A. Seeds in which germination is promoted by light. B. Seeds in which germination is inhibited by light. C. Seeds in which germination is inhibited by high temperatures. The Association of Official Seed Analysts, in its Rules for Seed Testing (AOSA 1983), differentiates between hard and dormant seeds. ‘Hard seeds’ are those that cannot absorb moisture as they possess an impermeable coat (groups I and III above). ‘Dormant seeds’ are those that can absorb moisture, but do not germinate because of restrictive influences within the seed that block some physiological reaction in the embryo, thus impeding the initiation of germination (groups II, IV, and V above) (Hartmann and Kester 1971). 107 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Germination Germination is known as the resumption of active growth of the embryo, which results in the rupture of the seed coats and the emergence of a new seedling capable of independent existence. When the seed separates from the plant that produced it, it is quiescent, that is, it does not show external signs of activity. For germination to take place, three conditions should be met: • • • The seed should be viable, that is, the embryo should be alive and able to germinate. The seed’s internal conditions should be favourable for germination, that is, any physical or chemical barrier to germination should have disappeared. The seed must be exposed to favourable environmental conditions. The essential factors are availability of water, appropriate temperature, provision of oxygen, and, sometimes, light. Although each of these conditions may have different effects on any given seed, more frequently, the interaction among them determines the beginning of germination (Hartmann and Kester 1971). Phases of germination Germination encompasses a complex sequence of processes (Figure 2) that imply complex changes of a biochemical, morphological, and physiological nature. These processes are described by authors such as García B (2004), Grierson and Covey (1984), Hartmann and Kester (1971), and Moreno C (2004). In general, the processes are seen as occurring in five stages, as described on the next page. 1 Activation of the embryo Release of gibberellins 2 Gene induction by gibberellins in the aleurone layer 3 Production and release of hydrolytic enzymes 4 Enzyme action on the endosperm reserve materials 5 Release of nutrients (monomers) 6 Absorption of nutrients by the embryo Seminal coat Aleurone layer 4 3 Endosperm Enzymes 5 2 Nutrients Gibberellins 6 Cotyledon Coleoptile 1 Cauline apex Hypocotyl/ radicle axis Embryo Radical apex Figure 2. 108 Sequence of germination processes in cereals such as wheat and barley (from García B 2004). Module 3, Submodule C: Verifying the Biological Quality of Germplasm Lesson 1: Basic Concepts The first stage starts with the dry seed imbibing water, its coats softening, and the protoplasm hydrating. This process is largely physical and occurs even in nonviable seeds. As a result of absorbing water the seed swells and its coats may break. In the second stage, cellular activity in the seed begins. It is characterized by the appearance of specific enzymes and the elevation of the respiration rate, particularly in cereal grains such as barley and wheat. The process is controlled by the embryo, which uses gibberellic acid, previously synthesized by the embryonic axis and/or scutellum (cotyledon in grasses) to send signals to the aleurone layer. At the same time, the coleoptile of the growing embryo begins to synthesize indoleacetic acid, which initiates the differentiation of vascular tissue through which gibberellic acid is transported towards the aleurone layer. In response to the gibberellic acid, the aleurone synthesizes the enzymes required for germination, except β-1,3-glucanase, the synthesis of which is still not completely understood. The third stage involves the enzymatic digestion of the insoluble complex reserve materials (mostly carbohydrates and fats, with some proteins) into soluble forms that are translocated to the areas of active growth. This whole process is carried out by enzymes secreted by the aleurone layer that surrounds the endosperm. The enzymes are α-amylase, protease, ribonuclease, and β-1,3-glucanase, the last attacking the hemicellulose in the walls. All are secreted towards the endosperm and, in only 3 or 4 days, they totally liquefy it. The endosperm begins dissolving; the cell walls degrade; the protein reserves are hydrolyzed to form amino acids; and the starch is also hydrolyzed to form reduced sugars that then become sucrose to be transported to the embryo. Other enzymes appear in the aleurone, weakening the seed coats and permitting the root end to pass through them. Cell elongation and root emergence are events that are associated with the beginning of germination. Cellular division can also occur in early stages but is, apparently, independent of cell elongation. Fourth stage. Those substances in meristematic regions are assimilated, providing energy for cellular activities and growth, and the formation of new cellular components. Fifth stage. The seedling grows by the usual processes of division, growth, and division of new cells in the growing points. The seedling depends on the seed reserves until such time as the leaves can adequately handle photosynthesis. To summarize, germination occurs in the following stages: imbibition or hydration; germination as such (including enzymatic and respiratory activity, digestion, translocation, and assimilation) when water absorption is reduced; growth, characterized by a renewed increase in water absorption and respiratory activity; and the associated emergence of the radicle. Essential Structures of the Seed and Seedling To evaluate their physiological status and interpret the viability indicators in the respective tests, the essential structures of seed and seedlings must be identified. The following description also complements what was already discussed in Module 3, Submodule B, Lessons 1 and 2. 109 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources A seed constitutes three basic parts: the embryo, food storage tissues, and seed coats. The embryo is a new plant that results from fertilization, that is, from the union of a male and female gamete. Its basic structure consists of an axis with growing points at each extreme—one for the stem and one for the root—and one or more seminal leaves (cotyledons) fixed at the embryonic axis. In dicotyledonous plants, the embryo consists of three components: radicle, which will form the root; plumule, which will give rise to the stem; and two cotyledons, which are part of the seed’s reserve tissues and which will ultimately be used as photosynthetic organs (Figure 3A). In monocotyledonous plants, only one cotyledon is found. It does not have photosynthetic characteristics during germination and constitutes a protective tissue called the scutellum. It separates the embryo from the endosperm, which is the principal reserve tissue in all monocotyledons (Figure 3B). Development of seedlings and essential structures The seed’s absorption of water, increased respiration, and the biochemical changes that it undergoes (as described above) awakens the embryo into growing and subsequently developing into a growing seedling. Normally, ‘seedlings’ refers to tender young plants that emerge from the soil, having developed directly from seed embryos. Usually, the first sign of germination is the appearance of the radicle, from which originates the primary root. In monocotyledons, this part of the seedling lasts only a very short period, as it promptly develops secondary roots. In gymnosperms and dicotyledons, the radicle develops into the primary or principal root that lasts the plant’s entire life. In other species, hypocotyl growth is the first visible manifestation of germination. This pattern is found in families such as the bromeliads, palms, Chenopodiaceae, Onagraceae, Saxifragaceae, and Typhaceae. The growth of this structure is important in epigeal germination, as it lifts the cotyledons out of the soil (Figure 4A). As the episperm tears, the cotyledons become exposed to sunlight, converting them into photosynthesizing organs during the seedlings’ first stages of growth. The epicotyl develops late. Epigeal germination is common in seeds with endosperm, for example, in castor bean (Ricinus communis), onion (Allium cepa), and Rumex spp. It is also found in seeds without endosperm such as those of bean (Phaseolus vulgaris), squash (Cucurbita pepo), cucumber (Cucumis sativus), mustard (Sinapis alba), groundnut (Arachis hypogaea), and lettuce (Lactuca sativa). In hypogeal germination, the hypocotyl grows very little and the cotyledon usually remains within the soil. The epicotyl starts elongating early so that the first two leaves are in the air and receiving sunlight (Figure 4B). Hypogeal germination appears in seeds with endosperm such as those of wheat (Triticum aestivum), maize (Zea mays), barley (Hordeum vulgare), date palm (Phoenix dactylifera), and rubber (Hevea spp.). It also appears in seeds without endosperm, for example, pea (Pisum sativum) and broad bean (Vicia faba). 110 Module 3, Submodule C: Verifying the Biological Quality of Germplasm Lesson 1: Basic Concepts (A) Trifoliate leaves Radicle Plumule Primary leaf Seed integument Bud Epicotyl Petiole Cotyledons Epicotyl Seed integument Hypocotyl Wilted cotyledon Hypocotyl Cotyledon Secondary roots Primary root Secondary roots Primary root Primary root Primary leaf (B) Plumule Seed coat Hairs Coleoptile Coleoptile Aleurone layer Endosperm Scutellum Coleorhiza Coleoptile Radicle Plumule Coleorhiza Radicle Secondary roots Figure 3. Primary root Essential structures of seeds and seedlings. (A) Dicotyledons (Magnoliopsida), for example, beans (Phaseolus vulgaris L.). (B) Monocotyledons (Liliopsida), for example, wheat (Triticum aestivum L.) (from Robbins et al. 1966). 111 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources (A) Onion Bean Seed coat Epicotyl Cotyledon Hypocotyl Cotyledon First leaf Seed coat Bulb Seed coat Primary root Secondary roots Primary root Pea (B) Epicotyl Maize Coleoptile Cotyledon Coleorrhiza Seed coat Primary root Figure 4. Secondary roots Primary root Adventitious roots Types of germination. (A) Examples of seedlings with epigeal germination: Allium cepa (onion) and Phaseolus vulgaris (bean). (B) Examples of seedlings with hypogeal germination: Pisum sativum (pea) and Zea mays (maize) (from Moreno C 2004). To evaluate the parameters of viability such as percentage and speed of germination, and seedling morphology, commercial-seed experts, as represented by the International Seed Testing Association (ISTA) define certain terms and apply criteria that should be taken into account (ISTA 1999). Nevertheless, because germplasm of cultivated species is per se diverse, these rules are a guide only. More worthwhile is to make careful observations of the performance of each species being tested for the parameters being evaluated. During laboratory tests, seedling performance should also be correlated with field performance. 112 Module 3, Submodule C: Verifying the Biological Quality of Germplasm Lesson 1: Basic Concepts ‘Essential structures of seedlings’ refers to those structures that are essential for continuous and satisfactory plant development such as the radicular system, embryonic axis, cotyledons, terminal buds, and, for Poaceae, coleoptile (Figure 3). Depending on the species, ISTA experts (1999) define a seedling as consisting of a specific combination of the following structures, essential for subsequent development: • • • • Radicular system, made up of the primary root and, in certain cases, secondary or seminal roots Embryonic axis, made up of the hypocotyl, epicotyl, and terminal bud; and, for Poaceae, the mesocotyl One or more cotyledons In all Poaceae, the coleoptile Viability, Vigour, Dormancy, Germination, and Essential Structures of Vegetative Planting Materials The availability of information on viability, vigour, dormancy, and germination of vegetative planting materials (plant fragments or specialized organs) is not as abundant as for seeds. However, except for the logical differences that exist, understanding and applying these concepts to this type of material in an analogous manner as for seeds are essential. The concepts of viability and vigour, which refer to the properties of being alive and able to generate new plants that emerge quickly and uniformly, are applied generally. Dormancy or the interruption of growth in meristematic tissues can appear in higher plant forms. Deciduous and subdeciduous trees are best known for exhibiting this type of behaviour. They are characterized by a phenological phase of leaf fall and meristematic dormancy at the end of the growing season. Where stakes are used for propagation, serious difficulties in rooting can be caused by the presence of chemical inhibitors, as in grape vine (Hartmann and Kester 1971). When speaking of the germination of vegetative planting materials, we cannot talk about resuming active growth in the embryo, because this type of structure does not exist in such materials. Instead, we must refer to meristematic buds or regions that can be activated to generate shoots that will develop into new plants. With respect to the essential structures for vegetative reproduction, propagules (see Module 3, Submodule B, Lesson 1) or plant fragments should have buds or meristematic tissues able to generate shoots, and have accumulated nutritive reserves to feed them until they develop photosynthesizing organs able to sustain an independent plant (Figure 5). Evaluating the Lesson After this lesson, you should be familiar with the most important concepts of germplasm viability and vigour, and how it relates to deterioration, and the phenomena of dormancy and germination. Before going on to the next lesson, answer the following question: When planning and executing methods to verify a germplasm’s viability, which of the concepts revised in this lesson do you consider as the most important? Briefly explain why. 113 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Tuberous root Sweet potato (Ipomoea batatas) Petiole (leaf stem) Leaf Terminal shoot Aerial stem Secondary branch Secondary shoot Root Root tuber (thickened adventitious root) Corm Gladiolus (Gladiolus sp.) Remains of flower shoot Bulb with bud Hippeastrum (Hippeastrum sp.) Bud New foliage leaf New corm forming at the base of the bud Bud Apical shoot (flower bud) Rudimentary protective leaf Rudimentary protective leaf Thickened stem containing food reserves Adventitious root Vascular tissue Fleshy rudimentary leaf containing food reserves Figure 5. Stem Adventitious root Tuberous root and longitudinal sections of a corm and bulb, showing principal components (from Kindersley 1994). Bibliography Literature cited AOSA, Seed Vigor Test Committee. 1983. Seed vigor testing handbook. Contribution No. 32 to the Handbook on Seed Testing. Stillwater, OK, USA. 93 p. 114 Module 3, Submodule C: Verifying the Biological Quality of Germplasm Lesson 1: Basic Concepts Baskin CC; Baskin JM. 1998. Types of seed dormancy. In Seeds: ecology, biogeography, and evolution of dormancy and germination. Academic Press, San Diego, CA. pp 27–47. Bolaños L. 2004. Fisiología vegetal: Resúmenes, Tema 13: Desarrollo de las semillas—Dormición y germinación, 3rd ed. Centro de Ciencias Biológicas of the Universidad Autónoma de Madrid. Available at http://www.uam.es/personal_pdi/ciencias/bolarios/FisioVegetal/ FisioVegetal200405/resumenes200405.htm (accessed 11 Nov 2004). Delouche JC; Baskin CC. 1973. Accelerated aging techniques for predicting the relative storability of seed lots. Seed Sci Technol 1:427–452. FAO; IPGRI. 1994. Genebank standards. Rome. 15 p. Also available at http://www.ipgri.cgiar. org/publications/pdf/424.pdf García B, FJ. 2004. Biología y botánica, Parte III: Tema—Germinación de semillas. Universidad Politécnica de Valencia, Spain. Available at http://www.euita.upv.es/varios/biologia/ Temas/tema_17.htm (accessed 11 Nov 2004). Gooding MJ; Murdoch AJ; Ellis RH. 2000. The value of seeds. In Black M; Bewley JD, eds. Seed technology and its biological basis. Sheffield Academic Press, UK. pp 3–41. Grierson D; Covey S. 1984. Plant molecular biology. Blackie & Son; Chapman & Hall, New York. pp 86–89. Hartmann HT; Kester DE. 1971. Propagación de plantas: Principios y prácticas. (Translated from the English by Antonio Marino Ambrosio.) Editorial Continental, Mexico, DF. pp 141–223. (Available in English as Hartmann HT; Kester DE; Davies FT, eds. 1990. Plant Propagation: Principles and Practices, 5th ed. Englewood Cliffs, NJ. 647 p.) ISTA. 1999. International rules for seed testing. Seed Sci Technol 27:1–333. (Supplement 21). Kindersley D. 1994. Enciclopedia visual seres vivos. Santillana; Casa Editorial El Tiempo, Bogotá, Colombia. 150 p. Moreno C, P. 2004. Vida y obra de granos y semillas. Available at http://omega.ilce.edu.mx: 3000/sites/ciencia/volumen3/ciencia3/146/htm/vidayob.htm (accessed 10 Nov 2004). OSU, Department of Horticulture and Crop Science. 2004. Seed vigor and vigor tests. Available at http://www.ag.ohio-state.edu/~seedsci/svvt01.html (accessed 10 Nov 2004). Perry DA, ed. 1981. Manual de métodos de ensayos de vigor. (Translated from the English by L Martínez V and Francisco Gonzáles T.) Instituto Nacional de Semillas y Plantas de Vivero ‘José Abascal’, Madrid, Spain. 56 p. (Available in English as ‘Manual on methods for testing vigor’ in Handbook of Vigor Test Methods. ISTA, Bassersdorf, Switzerland.) Rao NK; Hanson J; Dulloo ME; Ghosh K; Nowell D; Larinde M. 2006. Manual of seed handling in genebanks. Handbooks for Genebanks No. 8. IPGRI, Rome. Robbins WW; Weier TE; Stocking CR. 1966. Botánica. (Translation from the English by Alonso Blackaller V.) Editorial Limusa–Wiley, Madrid. pp 266–277. (Available in English as Botany: an introduction to plant science. Wiley, New York.) 115 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Vázquez Y, C; Orozco A; Rojas M; Sánchez ME; Cervantes V. 2004. Reproducción de las plantas: Semillas y meristemas. Available at http://omega.ilce.edu.mx:3000/sites/ciencia/ volumen3/ciencia3/157/htm/sec_5.htm (accessed 10 Nov 2004). Further reading Baskin CC; Baskin JM. 1998. Ecologically meaningful germination studies. In Seeds: ecology, biogeography, and evolution of dormancy and germination. Academic Press, San Diego, CA. pp 5–26. Ellis RH; Hong TD; Roberts EH. 1985. Seed technology for genebanks. Handbook for Genebanks No. 2, vol. 1. IBPGR, Rome. 210 p. Hong TD; Ellis RH. 1996. A protocol to determine seed storage behavior. Technical Bulletin No. 1. IPGRI, Rome. 64 p. Also available at http://www.ipgri.cgiar.org/publications/pdf/137.pdf Hong TD; Linington S; Ellis RH. 1998. Compendium of information on seed storage behaviour, vol. 1: Families A–H. Royal Botanic Gardens, Kew, London. 400 p. ISTA. 1987. Cold test. In Handbook of vigour test methods. Bassersdorf, Switzerland. pp 28–37. Lozano JC; Toro JC; Castro A; Bellotti AC. 1977. Production of cassava planting material. Series GE-17. CIAT, Cali, Colombia. 28 p. Contributors to this Lesson Benjamín Pineda, Alba Marina Torres, Daniel Debouck, Carlos Iván Cardozo, Rigoberto Hidalgo, Mariano Mejía, Graciela Mafla, Arsenio Ciprián, Manuel Sánchez, Carmen Rosa Bonilla, and Orlando Toro. Next Lesson In the next lesson, you will become familiar with the principal procedures for verifying the biological status of germplasm, both seeds and plant parts. 116 Lesson 2 Verification Procedures Objectives • • To describe the procedures most used to evaluate indicators for germplasm viability and vigour To review some concepts used to measure germplasm viability and vigour Introduction The germplasm’s biological or physiological status is verified either after conditioning or through periodic samplings during conservation under storage. It is a major activity for quality control. As mentioned in the previous lesson, applying it demands knowledge of the species and their peculiarities with regard to germination and the presence or absence of dormancy; the environmental and endogenous conditions that influence the behaviour of seeds or propagules; and the evaluation procedures and methods to conduct and their periodicity. Some of these requirements have already been discussed in the previous lesson. In this lesson, we consider procedures and methods for evaluating indicators of germplasm viability and vigour. First, we point out that periodic evaluation of germplasm viability is indispensable for ascertaining that the materials conserved ex situ are remaining in good condition. These evaluation procedures are regulated and standardized by institutions such as FAO and IPGRI (now Bioversity International) to help germplasm bank managers guarantee, through specific tests, the biological integrity of the PGRs under their responsibility (Box 1). Viability Indicators and their Evaluation The indicators most used to determine viability are germination and vigour. For their assessment, different methods have been designed, whose application depends on the type of seeds to evaluate, and the availability of resources, facilities, and installations. Before implementing tests to evaluate the viability and vigour of a given species, information should be collected on the characteristics of that species. We must have data on the habitat where the germplasm was originally collected, and likewise on seed type (orthodox or recalcitrant), age, and the conditioning and storage conditions to which the materials had been submitted. A sufficient supply of seeds is also advisable to permit additional experiments, should not enough information be available on the material. Many different methods are available to test seed viability, of which the most accurate and reliable is the germination test. Others include biochemical tests, which have the advantage of being quicker, but are not as accurate as the germination test (Rao et al. 2006). Evaluation of viability involves a series of stages from preliminary testing to the application of highly elaborate procedures. The first stage is to establish a preliminary germination test in a suitable environment and under recommended conditions of light and temperature, preferably those of the habitat where the germplasm had originally been collected. The results obtained from this test will orient the next procedures. 117 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Box 1 FAO and IPGRI guidelines for gene banks Viability monitoring 26. Genebank managers have the responsibility to provide conditions which will maintain the viability of each accession held within the genebank above a minimum value. Hence accession viability must be monitored. The preferred standard is that this obligation extends not just to the genebank, which can be considered the originator of the accession, but also to those genebanks holding a duplicate of the accession. 27. Viability will usually be assessed by means of a germination test, although other test procedures (such as the topographical tetrazolium test) may be required in order to clarify whether the non-germinating seeds in these tests are non-viable or whether their dormancy has not been broken during the test. Empty seeds not already removed before storage should be removed before beginning the germination test. An IBPGR handbook (Appendix II, IBPGR, 1985) is available which provides both general and specific advice on the conduct of germination tests and appropriate dormancy-breaking procedures. 28. The minimum standard is that accession viability monitoring tests be carried out at, or soon after, receipt and subsequently at intervals during storage. The initial germination test should be carried out on a minimum of 200 seeds drawn at random from the accession. 29. The period between viability monitoring tests will vary among species and will also depend upon the seed storage conditions. Genebanks should regularly conduct monitoring tests. Under the preferred storage conditions for base collections, the first monitoring test should normally be conducted after 10 ye ars for seeds with high initial germination percentage. Species known to have poor storage life or accessions of poor initial quality should be tested after 5 years. The interval between later tests should be based on experience, but in many cases may well be greater than 10 years. Note that where the preferred conditions of storage are not being met, then monitoring may need to be more frequent. Where a genebank has been operating for some years under the preferred conditions and has obtained sufficient information from their own monitoring tests on the range of material they work with to justify more extended monitoring intervals then this should be done. 30. The objective of the viability monitoring test is to decide whether regeneration is required. It is recommended that, in order to save seeds, 50 - 100 seeds be drawn at random from the accession for each monitoring test. The simplest method of determining whether substantial loss in viability is occurring, and distinguishing between this and the fluctuation in test results which is largely a consequence of sampling error, is to plot the results of successive monitoring tests against the period of storage and to see whether a progressive trend of loss in viability can be detected. Where such an indication is obtained, it is recommended that, provided sufficient seeds are available, a further sample of 100 seeds are drawn at random for a further viability monitoring test to reduce the probability that regeneration is initiated prematurely. Once it has been decided that an accession should be regenerated, further germination tests should be suspended to save valuable seeds. 31. It is essential that genebanks have, or have access to, sufficient laboratory equipment to enable viability monitoring tests to be carried out in a regulated, uniform and timely manner. In some cases the particular problems of the species maintained will require the provision of more specialized equipment, e.g. X -ray equipment to test for empty seeds and/or insect-damaged seeds. (Continued) 118 Module 3, Submodule C: Verifying the Biological Quality of Germplasm Lesson 2: Verification Procedures Box 1. (Continued.) 32. Initial germination testing and viability monitoring during storage requires adequate facilities to carry out these tests according to the conditions described in paragraphs 27 to 31. It is acceptable that a base collection should have access to suitable seed testing facilities and it is preferred that these should be at the same site as the base collection. 33. In the case of active collections, it is suggested that monitoring every 5 years will normally be satisfactory. However, this should be adjusted up or down depending upon the species stored, initial viability, and the storage environment. Where base and active collections are maintained side -by-side within the National Agricultural Research System under the preferred conditions for base collections then the advice for base collections should be followed for the active sample and in most cases it will not be necessary to sample from the base collection until the results for the active collection sample suggest this is necessary, or the latter becomes depleted. Note that this comment only applies in situations where the base and the active collections represent the same original seed sample which has simply been divided at random into the base and active samples. 34. There is no non-destructive viability monitoring test currently available. It is recommended that where the number of seeds within an accession is limited, and regeneration is feasible, the seedlings produced during accession viability monitoring tests should be grown out to provide a fresh stock of seeds (e.g. for distribution) providing, of course, that the number of seedlings available is sufficient for regeneration. SOURCE: FAO and IPGRI (1994). Should the seed not germinate, then tests must be carried out to discover the reason why (see Module 3, Submodule C, Lesson 1) and the recommended treatments then carried out (Table 1). In such a case, respirometric or biochemical activity tests must be conducted (Vázquez Y et al. 2004) before carrying out further procedures that may be unsuccessful. An example of such a test is that of soaking seeds in a solution of 2,3,5triphenyltetrazolium chloride (TTC). Viable seed, that is, seeds with biochemically active embryos become red on soaking. The test is based on the activity of the dehydrogenase enzyme systems linked to respiration in living things. For seeds, these systems are associated with the viability of the embryo and its consequent loss when no enzymatic activity exists. As the tissues of viable embryos respire, through oxidation and reduction, they liberate hydrogen ions. The hydrogen then combines with the TTC (Figure 1), which is normally colourless, producing, through reduction, a formazane, which is an insoluble nondiffusive pigment that colours tissues (Baskin and Baskin 1998a; Delouche et al. 1971). N C6H 5 N C6H 5 N +2e+2H C N N+ C 6H 5 Cl – 2,3,5-triphenyltetrazolium chloride Figure 1. C 6H 5 NH C6H5 +H +Cl – C N N C6H5 Formazane Reaction of 2,3,5-triphenyltetrazolium chloride with hydrogen that is released enzymatically by viable embryos to obtain formazane (from Delouche et al. 1971). 119 120 Table 1. Testing and treating nongerminating seeds: possible reasons why seed may not germinate, the corresponding treatments for correction, and the natural conditions that the treatments simulate. Reason Observed symptom Applicable treatment Observations Natural conditions simulated by treatment Hard seed coat Impermeability to water; Impermeability to gases; Physical barrier to embryo expansion Mechanical scarification by incision or scraping Locate the embryo in the seed so not to damage it Abrasion and wearing away of the seed coat by the action of soil particles, or partial rupture by other agents or microbial attacks Scarification with sulphuric and hydrochloric acids Test with different concentrations (from 10% to 100%) and exposure times to the acids Passage through the digestive tracts of animals, with different digestion times Thermal scarification by immersion in hot water at different temperatures up to 100°C Maintain constant temperatures or cool gradually Thermal effect of fires over damp soil and the time taken to cool Thermal scarification by dry heating to as high as 100°C Base temperatures on those reported for different soil depths during fires; take great care with times of exposure Effect of high temperatures on vegetation during fires over dry soil Variable temperature periods (thermoperiods) Thermoperiods can be achieved in incubators or germination chambers, or with any of the previous methods. To determine the amplitude of thermal fluctuation, consider what would occur in the species’ habitat Temperature variations that occur during the day, with the highest temperatures corresponding to those registered in the soil at the highest insolation (Continued) Table 1. (Continued.) Reason Observed symptom Applicable treatment Observations Natural conditions simulated by treatment Embryo’s anatomical or physiological immaturity Germination does not start Postmaturation time; Cold temperatures; Hormones (i.e., physiological) The embryo may mature over time; Apply stratification (chilling under moisture); Use exogenous gibberellins (gibberellic acid) in concentrations from 500 to 1500 ppm or more, according to the depth of dormancy Environmental conditions of temperate or similar zones Inhibited embryonic growth Germination does not start because of inhibitors present in the seed or lack of external stimuli Time, washing The inhibitors may degrade over time or be eliminated with abundant washing Environmental conditions of tropical, subtropical, or similar areas Light, temperature, changes in humidity Different spectral colours (red, blue, far-red, white light). Mineral salts (potassium nitrate in concentrations of 0.1%–1%) alter cellular permeability, replacing, for example, the role of light in germination. Hyperosmotic solutions (polyethylene glycol or Carbowax 6000 in solutions with osmotic potentials of 10 to 15 bars) modify the seed’s internal permeability. Use gibberellins in concentrations from 250 to 1000 ppm; auxins (IAA, 2,4-D); cytokinins (kinetins); or combinations (e.g., kinetin + Ethephon, i.e., ethylene + gibberellic acid) (Continued) 121 122 Table 1. (Continued.) Reason Observed symptom Applicable treatment Quiescent seed Does not start germination, because resting Water and adequate environment Reversible physiological change Does not start germination Time, hormones, other stimuli, light, temperature, changes in hydration Irreversible physiological change: damaged embryo, partial death, total death SOURCE: Vázquez Y et al. (2004). The change is of such magnitude that no treatment would give results Observations Natural conditions simulated by treatment Conditions that expose the seed to damp soil Physiological activity can recover over time. Gibberellins in concentrations of 250 to 1000 ppm. Auxins (IAA, 2,4-D); cytokinins (kinetins); or combinations (e.g., kinetin + Ethephon (ethylene + gibberellic acid). Mineral salts (potassium nitrate in concentrations from 0.1% to 1%) alter cellular permeability, replacing, for example, the role of light in germination. Different spectral colours (red, blue, far-red, white light). Hyperosmotic solutions (polyethylene glycol or Carbowax 6000 in solutions with osmotic potential of 10 to 15 bars) modify the seed’s internal permeability Micro-environment of seeds under given conditions Module 3, Submodule C: Verifying the Biological Quality of Germplasm Lesson 2: Verification Procedures The reaction occurs within the cells and, as the pigment is not diffusive, a clearly distinguishable delineation appears between breathing (viable) and nonbreathing tissue (dead). Breathing tissue acquires a red or pink colour while the second retains its natural colour (Figure 2). The position and size of the necrotic areas in the embryo, endosperm, and/or gametophytic tissue determine if the seed can be classified as viable or nonviable. I II III IIIB IV 1 2 3 4 5 Figure 2. Guide for evaluating the reaction of seed tissue to 2,3,5-triphenyltetrazolium chloride (TTC) in cereals. The stylistic figures in column I show viable and therefore entirely coloured embryos. The other columns show the maximum area of flaccid or necrotic (and therefore uncoloured) tissue permitted for viable seeds. The exception is column IIIB, Row 1, which shows a nonviable seed. It has necrotic (uncoloured) tissue in the centre of the scutellum, indicating heat damage. Row 1 corresponds to wheat (I), common rye (II), barley (III and IIIs), and oats (IV) when bisected for evaluation; Row 2, cross-sections of oat seeds; Row 3, sections of barley seeds prepared by the excised embryo method; Row 4, sections of common rye seeds prepared by the excised embryo method; Row 5, sections of wheat seeds prepared by the excised embryo method (from ISTA 1999). 123 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources According to the ISTA guidelines (1999), in the TTC test or topographical tetrazole test, viable seeds become completely stained, whereas the dead seeds do not. However, partial staining can occur because of the variable proportions of necrotic tissue in different areas of the embryo, indicating that not all seed tissues are dead. The recommended sample size for this test is four replications of 100 seeds each. Should seed availability be low, the International Standards for Genebanks (FAO and IPGRI 1994) recommend using a minimum of two replicates with 100 seeds per replicate. If the test results show that germination is below 90%, an additional 200 seeds should be tested, using the same method. Overall seed viability is then taken as the mean of the two tests (Rao et al. 2006). However, this test can be carried out only on those firm seeds that did not germinate in the standard germination test. The TTC test is usually applied to dormant seeds, although it can be used with any seed. Determining Germination and Vigour To implement viability tests, the following activities should be conducted: • • • • • Preconditioning procedures established Test types defined: – Germination in sand, soil, or other substrate – Germination on germination paper Sites determined for conducting the tests: – Germinators or other infrastructure – Laboratory, growth rooms, incubators Procedures established for executing the tests: – Reception and preparation of samples, and verification of identification – Preparation of substrates and tools needed for the tests - Germination paper, sand, soil, other substrates, identifiers, trays, containers - Reagents - Adequate in-depth planting and spacing in trays; placement on germination paper and/or in trays - Placement of trays, containers, or germination paper in the site or equipment suitable for germination - Agronomic attention and care to ensure the test’s success (e.g., humidity, temperature, and light) Type of data to collect determined and their evaluation for decision-making Preconditioning Often, to make seeds germinate and succeed in the viability test, the seeds’ internal conditions must be favourable for the process. This means that barriers of a physical, chemical, physiological, or other nature must disappear to permit germination (see Module 3, Submodule C, Lesson 1). If this has not happened, then preconditioning procedures must be carried out, like those described below or in guidelines for testing germination of the most common crop species, as suggested by Rao et al. (2006). Mechanical scarification. The aim is to modify hard or impermeable seed coats. The seed coat is ruptured, scratched, or mechanically altered to make them permeable to water 124 Module 3, Submodule C: Verifying the Biological Quality of Germplasm Lesson 2: Verification Procedures or gases. Although seed extraction and cleaning during harvest probably does a certain amount of scarification, most seeds with hard coats show improved germination with additional artificial treatment. Rubbing seeds with sandpaper, scratching them with a file, or incising them with blades, as according to case, would be sufficient. Manual scarification on any part of the seed coat is effective, but the micropylar region should be avoided, as it is the most sensitive part of the seed and is where the radicle is located (Rao et al. 2006). Scarification should not be so extensive that it damages the seeds. To determine the optimal time for germination, a few seeds may be sown in a test plot, soaked to observe swelling, or examined under a magnifying glass. Under the last, the seeds should appear dull in colour, but not so cut up that the seed’s internal parts are exposed (Hartmann and Kester 1971). Soaking in water. Reasons for soaking seeds in water include modifying the hard coats, removing inhibitors, softening the seeds, and reducing germination time. Sometimes, this treatment overcomes seed-coat dormancy or, in other cases, stimulates germination. For some seeds, their impermeable coats can be softened by placing them into hot water (170°–212°F or 77°–100°C) at four to five times their volume. The fire is removed immediately and the seeds left to soak for 12 to 24 h in the gradually cooling water. Then, using suitable screens, the swollen seeds are separated from those that did not swell. The latter are once again subjected to the same treatment or to another method. In some cases, seeds can be boiled for a few minutes but this procedure is too hazardous, as exposure to such high temperatures can damage the seeds. In certain cases, the inhibitors present in the seeds can be lixiviated by washing or soaking them in water. Seeds that ordinarily germinate slowly can be soaked before being put out to germinate, thereby shortening emergence time (Hartmann and Kester 1971). Scarification with acid. Scarification with acid helps modify hard or impermeable seed coats. Soaking in concentrated sulphuric acid is an effective method but the acid must be used with care as it is very corrosive. It reacts violently with water, considerably elevating temperatures and producing splatters. Protective clothing should be worn to protect the operator’s skin and eyes. Dry seeds are placed in glass or earthen containers and covered with concentrated sulphuric acid (specific weight 1.84) at a ratio of one part seed to two parts acid. To achieve uniform results and prevent the accumulation of dark and resinous material that is sometimes present in seeds, the mixture can be gently stirred at suitable intervals. Because stirring the seeds may elevate temperatures, vigorous stirring of the mixture should be avoided to prevent damage to the seed and minimize splattering of the acid. The most desirable range of temperatures is 60° to 80°F (15°–27°C). With higher temperatures, the contact period is shortened and lengthened with lower temperatures. The duration of the treatment should be carefully standardized. At the end of the treatment, the acid is drained away and the seeds washed. The acid used should be thrown away onto ground that is not in use, never into drain pipes. All possible speed should be used when washing the seeds. Abundant water is needed to dilute the acid, reduce temperatures, and prevent splattering. Washing for 10 min under running water is considered sufficient. The wet seeds can be planted immediately or dried and stored for later planting (Hartmann and Kester 1971). 125 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Chilling under moisture (cold stratification). The main goal of this treatment is to expose seeds to the low temperatures that are frequently needed to obtain prompt and uniform germination. This treatment is necessary for the seeds of many tree and shrub species to encourage physiological changes in the embryo (postmaturation). Seeds are put in refrigerators or, during winter, outside in covered boxes or in holes, 15 to 30 cm deep in the earth. Seeds are placed in containers on a moistened germination substrate and kept at 3° to 5°C in a refrigerator for a minimum of 7 days (Rao et al. 2006). The time needed to complete postmaturation depends on the class of seeds and sometimes on the individual plots. For most seeds, the necessary stratification period ranges between 1 and 4 months. During this period, the seeds should be examined periodically. If they are dry, they need to be moistened again. At the end of the postmaturation period, some seeds may germinate in storage. To sow them, the seeds are removed from their containers and separated from the medium, with care being taken not to damage the moist seeds (Hartmann and Kester 1971). Combining two or more pregermination treatments. Two or more treatments are combined to either overcome the effects of an impermeable seed coat and dormant embryo (double dormancy) or encourage the germination of seeds with complex embryo dormancy. The combination of mechanical or acid scarification or soaking in hot water with chilling under moisture is effective for seeds that have both hard impermeable coats and embryo dormancy. Any of the three treatments can be used to modify seed coats (Hartmann and Kester 1971). Planting time. Planting in a given time of the year can be used to encourage the postmaturation of dormant seeds and to comply with special requirements for germination. This procedure can help save a certain amount of time and use of special equipment that otherwise would have been needed. Seed is planted outside directly in the seedbed or cold bed at a time in the year when the natural environment provides the necessary conditions for postmaturation. If seeds are left in the seedbed for a long time, they must be protected from desiccation, adverse environmental conditions, animals, birds, diseases, and competition with weeds (Hartmann and Kester 1971). Dry storage. Recently harvested seeds of many annual or perennial herbaceous plants do not germinate if they have not gone through a period of dry storage. This postharvest dormancy can last a few days or several months, depending on the plant species. Because dry storage is the usual method for handling and keeping most seeds of cereals, vegetables, and flowers, this dormant period is usually over by the time seeds arrive for viability tests. Otherwise, drying the seeds will facilitate germination (Hartmann and Kester 1971). Chemical stimuli. Many recently harvested but dormant seeds respond to soaking in chemical stimuli such as potassium nitrate, gibberellic acid, thiourea, and sodium hypochlorite. • • 126 Potassium nitrate is used at 0.2%, with the seeds placed in germination trays or in petri dishes, and the substrate is moistened with solution. Gibberellic acid (GA), a plant hormone, increases the germination rate of certain classes of dormant seeds, increases the speed of germination, encourages plant growth, and Module 3, Submodule C: Verifying the Biological Quality of Germplasm Lesson 2: Verification Procedures • • overcomes stunting or dwarfism in dormant epicotyls. Seeds are soaked in an aqueous solution of gibberellic acid at variable concentrations (100 and 500 mg/litre), according to the species’ response (Hartmann and Kester 1971). Thiourea [CS (NH2)2] has been used experimentally to promote germination in some dormant seeds, particularly those that do not germinate in darkness or at high temperatures, or require cold-moisture treatment. Aqueous solutions, ranging from 0.5% to 3%, are used. As thiourea somewhat inhibits growth, seeds should not be soaked for more than 24 h. They should then be rinsed with water (Hartmann and Kester 1971). Sodium hypochlorite is used to encourage the germination of seeds like those of rice. It blocks the effect of inhibitors dissolved in the water found in the husk. A commercial concentrate of sodium hypochlorite is used at a ratio of 1 part of the chemical to 100 parts of water (Hartmann and Kester 1971). Germination tests A germination test consists of exposing seeds to favourable environmental conditions such as moisture, temperature, oxygen, and, in certain cases, light, to ensure the embryo resumes active growth. Hence, different media or substrates are used (Table 2), as well as installations and equipment to obtain the expected results (Figure 3). Standards and norms have been established for germination tests in terms of duration, number of seeds, drying levels, incubation temperatures, and assessment (ISTA 1999). However, these standards should be applied with care, making adjustments as according to germplasm type. Table 2. Culture media and substrates used in germination tests. Medium or substrate Observations Agar-agar 1% A medium with stable moisture content and low contamination. It can be used in the field or laboratory. Very useful under shady conditions, as it conserves moisture for longer. Care must be taken when working under direct sunlight, as it dehydrates and condensed water accumulates in the petri plate. The medium facilitates radicle emergence and seedling transplant. Filter paper Provides good support, but care must be taken to prevent excessive drying. Paper towels More economical to use, but needing the same care as filter paper to prevent excessive drying. Vermiculite, perlite, and similar substrates Useful for large seeds only. Conserve moisture for more time than does paper, but moisture levels still need watching. Depth is easy to control. Soil As useful as perlite and similar substrates. Has the advantage of contributing nitrogenous compounds that stimulate germination. Sand This medium is very popular but must be washed thoroughly to eliminate salts before use. Drains more easily than soil and so needs a controlled water supply. 127 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources (B) (C) (A) Figure 3. (A) Planting in trays containing sterilized sand, (B) germination chambers, and (C) shelving carrying trays of growing seedlings (photos by B Pineda, GRU, CIAT). Detailed information on the various methods for determining seed viability can be found in the following publications: ISTA’s International Rules for Seed Testing (1999) and its Handbook on Seedling Evaluation (2003), the manuals Seed Technology for Genebanks by Ellis et al. (1985) and Seed Vigor Testing Handbook by AOSA (1983), and Hong and Ellis’ Protocol to Determine Seed Storage Behavior (1996). ISTA’s Rules suggest a sample size of four replications of 100 seeds for the viability test. However, sample size will depend on the quantity of seed available and, in some cases, may be reduced. What is important is being able to make several replications rather than just one large sample. A frequently used number is three replications of 50 seeds each (Baskin and Baskin 1998b). Relationships between vigour and germination In some cases, tests for vigour are different from those for germination. However, during evaluations of germination, data are taken that also serve to estimate vigour, for example, germinative strength, germination speed, seedling emergence and development, uniformity of germination, and sensitivity to factors of environmental stress during germination and emergence. A close relationship between the two concepts can therefore be established (Figure 4). Standard germination tests are carried out under conditions optimal for activating the embryo in seeds. During seedling development, observations pertinent to vigour are made. Some of these assess and classify seedling growth (Figure 5) and the percentage and speed of emergence. Others consider response to stress factors such as cold temperatures (10°C) and chilling (18°C), rapid aging, and osmotic pressure (AOSA 1983). 128 Module 3, Submodule C: Verifying the Biological Quality of Germplasm Lesson 2: Verification Procedures Maximum degree of vigor Greater vigor Less vigor Seedling capable of emergence and continued growth under favorable conditions; the higher the seedling on the vigor scale, the greater its chance of success under unfavorable conditions Normal sprouts of germination tests “Vigor” tests applicable in this area Seedling capable of emergence, but incapable of continued growth; abnormal sprouts of seed analysis tests Seed not dead in all its parts, but incapable of emergence Dead seeds of seed analysis tests Seed completely dead Figure 4. A graphic representation of the relationship between germination and vigour (from AOSA 1983). The ISTA experts, to validate germination tests, make counts of and categorize seedlings as complete normal seedlings, normal seedlings with mild defects, and abnormal seedlings. In each case, parameters are defined according to the presence, absence, or level of defects in essential structures such as the radicular system (primary root, secondary or seminal roots), embryonic axis (hypocotyl, epicotyl, terminal bud, and the mesocotyl in Poaceae) (ISTA 1999). 129 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources (A) Figure 5. (B) (C) Classification of cotton seedlings in the final count in the vigour test. (A) Vigorous normal seedlings. (B) Normal seedlings, but less vigorous and with some defects: a. necrotic cotyledon; b. atrophied primary root; c. lack of one cotyledon; d. necrotic hypocotyl; e. absent primary root. (C) Abnormal seedlings: f. deteriorated cotyledons and epicotyl; g. lack of both cotyledons; h. weak secondary roots and absent primary root; i. damaged hypocotyl; j. shortened hypocotyl (from AOSA 1983). Measuring Germination and Vigour Germination percentage and germination rate Measuring germination implies assessing two parameters: germination percentage and germination rate. Germination percentage should be related to time, indicating the number of seedlings produced over a given period. Germination rate can be measured, using several methods. One determines the number of days required to obtain a specified germination percentage. Another calculates the average number of days (MDG) needed for the plumule or radicle to emerge, as follows: Mean days = (N1T1 + N2T2 …. NxTx)/Total number of germinating seeds where, N is the number of seeds that germinated within consecutive intervals of time; and T is the time elapsed between the beginning of the test and the end of the given interval of measurement. 130 Module 3, Submodule C: Verifying the Biological Quality of Germplasm Lesson 2: Verification Procedures Viability is represented by the germination percentage, which expresses the percentage of seedlings that were actually produced of the number that could have been produced by a given number of seeds (i.e., (Nseedlings/Npotential) × 100). The germination should be rapid and seedling growth vigorous. This is seed vitality, or germinative capacity, and can be represented by germination speed. If the sequence of time is measured for the germination of a seed lot or for the emergence of seedlings in a seedbed, a typical pattern is usually found for the germination curve (Figure 6). An initial delay occurs as germination begins, then the number of seeds that germinate rapidly increases, followed by a reduced rate of appearance. When viability is less than 100%, determining the exact final point becomes difficult (Hartmann and Kester 1971). 100 C G Germination (%) 80 T PV 60 40 20 0 0 4 8 12 B 16 20 24 28 32 X 36 Days Figure 6. Typical germination curve of a seed sample. After an initial delay, the number of seeds that germinate increases and then diminishes. This type of curve can be used to measure germination. See text for explanation (redrawn from Czabator, cited in Hartmann and Kester 1971). According to Hartman and Kester (1971), Kotowski has used the inverse of this formula multiplied by 100 to determine a coefficient of speed, whereas Czabator suggested another measurement for seeds of woody perennials where germination maybe slow; that measure includes both speed and percentage of germination, that is, the germination value (GV). To calculate GV, the germination curve (Figure 6) should be obtained by periodically counting radicle or plumule emergence. The important values of the curve are the T value, where the speed of germination begins to diminish, and the G value, which is the final percentage of germination at the end of an X interval of measurement. (Several T values are, in fact, taken to indicate the times between the beginning of the test and the end of given intervals of measurement.) These points divide the curve into a fast and slow phase. Peak value (PV) is 131 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources the percentage of germination at T, divided by the number of days needed to arrive at T. The mean daily germination (MDG) is the final percentage of germination divided by the number of days the test is held. Therefore (Figure 6): GV = PV × MDG GV = (68/13) × (85/34) GV = 5.2 × 2.5 = 13 A test usually takes 10 days to 4 weeks, but may last as long as 3 months for seeds that are slow to germinate. A normal seedling usually has a well-developed root and stem, although the criterion ‘normal seedling’ varies among the different seed classes. Moreover, abnormal seedlings, and hard, firm, dead, or rotten seeds may occur. Abnormal seeds may result from: • • • • • • • Diminished vitality because of age Poor storage conditions Damage, whether mechanical, or by insects or disease Poisoning from overdosage of fungicides Frost damage Nutrient deficiencies such as minerals (e.g., Mn and B in peas and beans) Poisoning from toxic materials sometimes found in metallic germination trays, substrates, or piped water In general, firm seeds can be distinguished from the nonviable; firm seeds are solid, swollen, and free of moulds, or they show erratic germination. Any seed that does not germinate should be examined to determine possible reasons (Hartmann and Kester 1971). Viability and vigour of vegetative planting materials Although the concepts of viability and vigour have been developed and applied to seeds, they can be extended, with the necessary adjustments, by analogy to vegetative planting materials (propagules and plant parts). Basically, for the respective evaluations, plant parts (e.g., roots, stems, and leaves) or specialized plant organs (e.g., bulbs, corms, tuberous roots, rhizomes, and pseudobulbs) that have the capacity for regeneration would be examined for their viability, that is, for their capacity to regenerate healthy and vigorous plants. Parameters would include germination percentage, expressed as the proportion by number of propagules or reproduction units used that have produced seedlings. The vitality of the reproduction units, or germinative capacity, could be represented, as for seeds, by germination speed or germination rate. Because little information is available on evaluating the biological or physiological status of vegetative planting materials, pertinent research is advisable. When working with ex situ conservation, the knowledge needed for the successful management of this type of PGRs should be generated and disseminated. 132 Module 3, Submodule C: Verifying the Biological Quality of Germplasm Lesson 2: Verification Procedures Evaluating the Lesson After this lesson you should be familiar with the procedures most used to evaluate indicators of germplasm viability and to assess the respective tests. Before going on to the next lesson, consider the following problems: • • If you have worked in the verification of germplasm viability, comment on the difficulties you have encountered during tests and indicate how you resolved them. If you have not had experience or have not carried out viability tests, indicate how you would conduct those procedures with the materials in your bank. If you prefer, carry out a small practice experiment, describing what you did and what results you obtained. Bibliography Literature cited AOSA, Seed Vigor Test Committee. 1983. Seed vigor testing handbook. Contribution No. 32 to the Handbook on Seed Testing. Stillwater, OK, USA. 93 p. Baskin CC; Baskin JM. 1998a. Ecologically meaningful germination studies. In Seeds: ecology, biogeography, and evolution of dormancy and germination. Academic Press, San Diego, CA. pp 5–26. Baskin CC; Baskin JM. 1998b. Types of seed dormancy. In Seeds: ecology, biogeography, and evolution of dormancy and germination. Academic Press, San Diego, CA. pp 27–47. Delouche JC; Still TW; Raspet M; Liehard M. 1971. Prueba de viabilidad de la semilla con tetrazol. (Translated from the English by USAID.) Centro Regional de Ayuda Técnica of USAID, Mexico. 71 p. (Available in English as Delouche JC; Still TW; Raspet M; Lienhard M. 1962. The Tetrazolium Test for Seed Viability. Miss Agric Exp Stn Tech Bull 51.) Don R, ed. 2003. ISTA Handbook on Seedling Evaluation, 3rd ed. Bassersdorf, Switzerland. Ellis RH; Hong TD; Roberts EH. 1985. Seed technology for genebanks. Handbook for Genebanks No. 2, vol. 1. IBPGR, Rome. 210 p. FAO; IPGRI. 1994. Genebank standards. Rome. 15 p. Also available at http:// www.ipgri.cgiar.org/publications/pdf/424.pdf Hartmann HT; Kester DE. 1971. Propagación de plantas: Principios y prácticas. (Translated from the English by Antonio Marino Ambrosio.) Editorial Continental, Mexico, DF. pp 141–223. (Available in English as Hartmann HT; Kester DE; Davies FT, eds. 1990. Plant Propagation: Principles and Practices, 5th ed. Englewood Cliffs, NJ, 647 p.) Hong TD; Ellis RH. 1996. A protocol to determine seed storage behavior. Technical Bulletin No. 1. IPGRI, Rome. 64 p. Also available at http://www.cgiar.org/ipgri/doc/ download.htm ISTA. 1999. International rules for seed testing. Seed Sci Technol 27:1–333. (Supplement 21). Rao NK; Hanson J; Dulloo ME; Ghosh K; Nowell D; Larinde M. 2006. Manual of seed handling in genebanks. Handbooks for Genebanks No. 8. IPGRI, Rome. 133 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Vázquez Y, C; Orozco A; Rojas M; Sánchez ME; Cervantes V. 2004. Reproducción de las plantas: Semillas y meristemas. Available at http://omega.ilce.edu.mx:3000/sites/ciencia/ volumen3/ciencia3/157/htm/sec_5.htm (accessed 10 Nov 2004). Further reading Delouche JC; Baskin CC. 1973. Accelerated aging techniques for predicting the relative storability of seed lots. Seed Sci Technol 1:427–452. Gooding MJ; Murdoch AJ; Ellis RH. 2000. The value of seeds. In Black M; Bewley JD, eds. Seed technology and its biological basis. Sheffield Academic Press, UK. pp 3–41. Grierson D; Covey S. 1984. Plant molecular biology. Blackie & Son; Chapman & Hall, New York. pp 86–89. Hong TD; Linington S; Ellis RH. 1998. Compendium of information on seed storage behaviour, vol. 1: Families A–H. Royal Botanic Gardens, Kew, London. 400 p. ISTA. 1987. Cold test. In Handbook of vigour test methods. Bassersdorf, Switzerland. pp 28–37. OSU, Department of Horticulture and Crop Science. 2004. Seed vigor and vigor tests. Available at http://www.ag.ohio-state.edu/~seedsci/svvt01.html (accessed 10 Nov 2004). Perry DA, ed. 1981. Handbook of vigor test methods. ISTA, Bassersdorf, Switzerland. Contributors to this Lesson Benjamín Pineda, Alba Marina Torres, Daniel Debouck, Carlos Iván Cardozo, Rigoberto Hidalgo, Mariano Mejía, Graciela Mafla, Arsenio Ciprián, Manuel Sánchez, Carmen Rosa Bonilla, and Orlando Toro. Next Lesson In Lesson 1 of the next Submodule D, you will study aspects of plant health quality and its verification. 134 Submodule D Lesson 1 Basic Concepts of Phytosanitary Quality Verifying Phytosanitary Quality Objectives To review: • • • The importance of evaluating the phytosanitary quality of germplasm The basic criteria for such evaluation The relationship between the phytosanitary quality control and different stages of ex situ conservation Introduction One essential component of the ex situ conservation of germplasm is its phytosanitary quality. This component, as previously defined in Module 3, Submodule B, Lesson 2, refers to the absence of pathogens associated with planting materials and/or micro-organisms that cause deterioration during multiplication and storage. To obtain germplasm that meets this requirement, it is essential that, throughout the different stages of the ex situ management cycle, control measures are applied that ensure the materials are subjected to as little risk as possible. Consequently, plant health quality control attempts to: • • • • • Reduce the risks involved in transferring germplasm from one country or region to another. Contribute to maintaining the material free of pathogens of quarantine interest and/or of those that imply risk for conservation in terms of causing total or partial losses in the production of planting materials (seeds or propagules), genetic erosion, poor quality, or deterioration during storage. Facilitate the availability of germplasm without plant health restrictions for the users. Facilitate decision-making for saving materials affected by damaging micro-organisms or of quarantine interest. Contribute to compliance with international standards. The application of control measures involves the verification of results. To this end, different procedures and methodologies are used, some of which will be mentioned as we develop the theme. First, we will consider some concepts to better understand the theme ‘verification of plant health quality’. Basic Concepts and Criteria for Evaluating the Phytosanitary Quality Phytosanitary quality refers to the concept whereby germplasm undergoing ex situ conservation is found free of pathogens of quarantine interest and/or those associated with the planting materials (whether seeds or propagules) and which may cause deterioration or contribute to genetic erosion. When considering pathogens of quarantine interest, the definitions of the International Plant Protection Convention (IPPC) should be taken into account. Under the rubric ‘Pest’ (Box 1), pathogens (Figure 1) include micro-organisms (bacteria, spiroplasms, phytoplasmas, Phytomonas spp., fungi, and nematodes) and biotic agents (viruses and viroids) that can cause disease in plants (Agarwal and Sinclair 1987; González 1976; Neergaard 1977). 135 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Box 1 IPPC definitions for pests Pest: any species, strain or biotype of plant, animal or pathogenic agent injurious to plants or plant products. Regulated non-quarantine pest: a non-quarantine pest whose presence in plants for planting affects the intended use of those plants with an economically unacceptable impact and which is therefore regulated within the territory of the importing contracting party. Regulated pest: a quarantine pest or a regulated non-quarantine pest. Quarantine pest: a pest of potential economic importance to the area endangered thereby and not yet present there, or present but not widely distributed and being officially controlled. SOURCE: FAO (1997). Diseases caused by pathogens can sometimes lead, ultimately, to a plant’s death, without its being able to produce the necessary reproductive structures and thereby causing loss of genotype (or genetic erosion). In other plants, pathogens cause the loss or reduction of the conservable product or reduce its quality by infecting and remaining within the tissues, so that when the germplasm is transported, so are the pathogens. Similarly, when infected materials are used for improvement programmes or multiplication, results are severely affected. To evaluate the plant health quality of germplasm and plan suitable management measures, several aspects should be taken into account, as discussed below. Origin of materials This must be considered as each collection site (country or region where the material was collected) has a level of risk associated with the availability of information on its pathogens and the activities carried out for their control (Table 1). Pathogen inventory and quarantine classification, according to region or country With this information, suitable measures for control can be implemented to guarantee the safe transfer and management of germplasm. For example, for the purposes of management, pathogens are classified in three groups: • • • Group A: Dangerous pathogens that have a high epidemic potential but are not found in the region of introduction (exotic pathogens). Group B: Pathogens possessing moderate epidemic potential but not found in the regions of introduction or are occurring in restricted areas under effective control. Group C: Pathogens that are not considered to be of quarantine importance, but which affect the quality of planting materials. When germplasm comes from high-risk sites (i.e., categories 1 to 7, Table 1), it may carry pathogens of category A, B, or C. Given the characteristics of the pathogens included in groups A and B, the material should be filtered through post-quarantine control (closed quarantine). The procedure includes the production of seeds or propagules from the introduced germplasm and release of the same, if their health is duly verified. When information is available for 136 Module 3, Submodule D: Verifying Phytosanitary Quality Lesson 1: Basic Concepts of Phytosanitary Quality Types of pathogens • • • • • • • • Bacteria Spiroplasms Phytoplasmas Phytomonas spp. Fungi Nematodes Viruses Viroids Fungi Bacteria Cytoplasm Capsule Pathogens possess races Cell wall Pilus Flagellum Ribosomes DNA Plasmatic membrane Prokaryotic organisms that lack chlorophyll and multiply by binary fission. Their colonies usually have a gelatinous aspect. More than 200 species cause diseases in plants. Viruses Obligate molecular parasites that possess RNA or DNA and have similar structures to the normal macromolecules found in cells. They depend absolutely on the mechanisms of the host’s live cells to synthesize proteins and generate energy. Figure 1. Non-vascular, heterotrophic organisms that lack chlorophyll and other photosynthesizing pigments. Usually filamentous, they reproduce by means of sexual or asexual spores. More than 100,000 species exist, 8000 of which are pathogenic on plants. Nematodes Cylindrical elongated worms that may be macro- or microscopic in size. They live in the soil or water and can parasitize animals or plants. In the case of the latter, they attack underground or aerial organs, using stylets to extract sap. Most of the species that attack plants belong to the phylum Nematoda, class Secernentea, order Tylenchida. Pathogens associated with planting materials (diagrams adapted by B Pineda from different sources used in a personal lecture, 2004). 137 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Table 1. Germplasm collection sites and associated risk. Collection site Risk levela Collected from any site but plant identity at least to genus was unknown at the time of collection (e.g., grasses collected vegetatively) 1 Collected in a centre of plant and/or pest diversity 2 Collected in the wild, far removed from agricultural areas; wilderness 3 Tubers, roots, seeds, etc., collected in the market place 4 Farms 5 Orchards, plantations 6 Experimental fields (except those where plants are screened for resistance) 7 Experimental plots isolated from commercial plantings or located where certain pests are not present 8 Commercial greenhouses with floor beds 9 Commercial greenhouses with raised benches 10 Research greenhouses with raised benches 11 Approved certification 12 Plant tissue cultures, aseptic plantlet cultures, etc., particularly when derived from pathogen-tested mother plants 13 Collected from other containment facilities/quarantine stations provided the plants are pathogen-tested and grown under a high level of phytosanitation 14 a. ‘1’ denotes the highest hazard/risk site; ‘14’, the lowest. SOURCE: Kahn (1999). pathogens of groups B and C, then the planting materials can be analyzed through suitable sampling and released if found free of infection or contamination. Levels of risk that germplasm movement implies The movement of germplasm inevitably implies a level of significant risk. Planting materials (seeds, propagules, or plant parts) that are transported or transferred may well harbour pathogens within their tissues and thus carry them without their being noticed (Brunt et al. 1990; Frison and Feliu 1991; FAO and IPGRI 2004). Risk may be greater or lower, depending on: • • • • • 138 The plant genus or species; Presence of pathogens of quarantine importance transmitted through seed or planting material, or in association with the planting material in the exporting country or region of origin; Geographical distribution of such pathogens, and their life cycle and type of parasitism; The volume and frequency of international exchange of plants and planting materials; and The favourability of environmental factors. Module 3, Submodule D: Verifying Phytosanitary Quality Lesson 1: Basic Concepts of Phytosanitary Quality Not all agents found in germplasm necessarily take on economic or quarantine importance. Many are ubiquitous and are established in the importing country; others are economically important and may be of quarantine significance. Even so, despite their ubiquity, pathogens have races that vary in aggressiveness according to the regions or countries where they are registered. Hence, race may become a risk factor in germplasm transfer. Plant Health and its Relationship with Different Stages of Germplasm Management Plant introduction This stage—the transfer of germplasm from one country or region to another—should be understood as involving plant health risks and that, therefore, the procedure is subject to legislation (IPPC). Each party interested in moving the germplasm should agree on the terms of transfer, assuring the other parties of the legality of transport and of compliance with established plant health requirements (Barton and Siebeck 1994; COSAVE 2003; EPPO 2004; FAO 1997; OIRSA 2004). The principal risk in moving germplasm is the transfer of pests and pathogens, which must be detected during quarantine, a procedure that includes inspection to detect pests and pathogens, treatment or cleaning of samples, and certification and release of material where no danger is presented, or its destruction if it is heavily contaminated or no technology is available to clean it (Nath 1993). Germplasm that propagates through seed is planted under greenhouse conditions (closed quarantine) so that it germinates and develops seedlings (Figure 2). During establishment, the germplasm is inspected by plant health authorities, and, where necessary, samples are made and analyzed in the laboratory to intercept possible pathogens of quarantine interest. When materials of vegetative reproduction (propagules or plant fragments) are introduced, procedures require more care, as each material taken from the mother plant may contain pathogens that are usually found in association with it. In this case, the initial selection of an adequate source of propagation is essential. Individual plants should be examined with all care to discover possible genetic disorders, bud variations, and symptoms of viruses or other disease pathogens. If a ‘clean’ plant is not found, then the pathogen must be eliminated by sanitizing a small part of the plant such as a stake, bulb layer, growth bud, or meristem that can constitute adequate initial material for propagation. To obtain ‘clean’ planting material, various techniques are used, as not all have necessarily the same effectiveness for all plants or for all pathogens. An example of a cleaning technique is the system applied by CIAT to sanitize cassava (Manihot esculenta), a species that propagates vegetatively and is conserved in vitro (CIAT 1980, 1982; Mafla et al. 1992; Roca et al. 1991). The procedures are: • • Select mother plants, from which stakes, 15–20 cm long with vigorous buds, are taken. They are then surface-disinfected by submersion for 5 min in a solution of the insecticide dimethoate (Sistemin® at 0.3%), left to dry for 1–2 h under shade, and planted in pots that contain a sterilized substrate (e.g., soil to sand at 1:2). Apply thermotherapy for 3 weeks. That is, the pots with the seedlings are placed in a thermotherapy chamber, with a temperature range of 40°C (day) to 35°C (night), 139 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources • • • • • illumination at 3000–4000 lux, and high relative humidity. Three or 4 weeks after thermotherapy, cut the buds to be used to extract meristems. The blade must be disinfected with detergent before each cut. Sterilize the tissue by placing the cut buds in a beaker with a mesh that facilitates their management in the laboratory. In a laminar flow chamber, they are disinfected by rapid submersion in 70% alcohol, rinsed with sterilized water, placed into a solution of sodium hypochlorite at 0.5% for 5 min, and finally rinsed three times with sterilized bi-distilled water. Isolate and plant the meristems (tissue structures of 0.3–0.5 mm, which include the meristematic dome with one or two leaf primordia). Under aseptic conditions in a laminar flow chamber and under the visual field of a stereomicroscope (10X–40X), the apical bud is caught with forceps and the appendages (leaves and stipules) covering the apex removed with a scalpel until a brilliant structure, Figure 2. Quarantine. Entrance to the quarantine greenhouse for 0.3–0.5 mm long with 1 or 2 leaf primordia, introductions, seeds being appears. Then a fine cut is made. This operation planted, and developing should be carried out very quickly and carefully seedlings (photos by B Pineda, to prevent excessive dehydration and possible GRU, CIAT). death of the meristem. The explant is placed in a culture medium suitable for growth and development, ensuring that the basal part remains on the medium’s surface. Incubate the culture. That is, the planted meristems are placed in test-tube racks and taken to a growth chamber with temperatures at 26°–28°C; illumination at 1000 lux, using fluorescent lamps, type daylight; and a 12-h day length. Leave seedlings to develop. This stage usually lasts 3 to 4 weeks, after which the explants must be transferred to a medium suitable for growth and development, with a prior cut in their bases to prevent any possible callus from forming. Three weeks after having been transferred to the new medium, the explants begin growing. Roots emerge, and a completely developed plant becomes available for continuing with the indexing tests (Module 3, Submodule D, Lesson 2). Multiplication or regeneration In this stage, germplasm produced in the open field runs the inherent risk that material may be lost or may deteriorate on being exposed to various adverse environmental or biological agents. Disease from pathogen attack can sometimes cause the eventual death of plants without their succeeding in producing the reproductive structures needed for conservation, thus resulting in the loss of genotype (i.e., genetic erosion). In other plants, conservable products may be lost or reduced in number, or their quality lost as pathogens infect tissues 140 Module 3, Submodule D: Verifying Phytosanitary Quality Lesson 1: Basic Concepts of Phytosanitary Quality and remain within them in such a way that transporting the germplasm would also mean transporting the pathogens. During production, agronomic principles are applied and, hence, include all the practices needed to obtain the quantity and quality of propagules required for the different purposes of conservation. A major agronomic practice is the control of diseases caused by different types of pathogens. To apply control methods to achieve success in multiplication or increase, we must not only know the type of agronomic requirements of a given species, but also have information on the diseases that can affect the targeted germplasm, including causal agents, life cycle, mechanisms of dissemination, and factors favouring their development. Harvest Practices of plant health control also play an important role in this stage. Timely harvesting and good management of the germplasm obtained are fundamental to maintaining the germplasm’s health, provided that, during production, adequate control measures had been applied. Seeds should also be harvested, preferably during the dry season, as harvesting in rainy or humid periods increases the risk of deterioration by micro-organisms that normally associate with the seeds. It is essential that harvested materials are placed in paper or cloth bags and duly identified before being transported to the sites for predrying and later conditioning. Conditioning During this stage, rigorous measures should be applied when cleaning equipment, tables, and places where activities are carried out, as seeds can be easily contaminated with microorganisms normally present in residues from fruits or harvesting. Contaminating microorganisms can contribute to the germplasm’s deterioration during the subsequent steps of conservation if these precautions are not taken. Testing the biological status Essentially, test conditions must be guaranteed to permit the germination of materials without risk of contamination. In particular, the plant health quality of soil, water, trays or pots, and other implements used in evaluations must be checked to minimize the risk of contaminating seedlings and obtaining erroneous data on the germplasm’s biological status. Evaluating the Lesson After this lesson, you should be familiar with the most important aspects of plant health quality control, the criteria to consider for the respective evaluations, and the relationship of plant health control with different stages of ex situ conservation. Before going on to the next lesson, briefly discuss the importance of plant health quality control in the context of ex situ conservation of materials with which you are familiar. Bibliography Literature cited Agarwal VK; Sinclair JB. 1987. Principles of seed pathology, vols I and II. CRC Academic Press, Boca Raton, FL. 141 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Barton JH; Siebeck WE. 1994. Material transfer agreements in genetics resources exchange: the case of the International Agricultural Research Centres. Issues in Genetic Resources No. 1. IPGRI, Rome. Also available at http://www.bioversityinternational.org/ publications/Pdf/109.pdf Brunt AA; Crabtree K; Gibbs AJ. 1990. Viruses of tropical plants. CAB International; ACIAR, Wallingford, UK. 707 p. CIAT. 1980. El cultivo de meristemas de yuca; Guía de estudio para ser usada como complemento de la unidad audiotutorial sobre el mismo tema. Scientific contents: William M. Roca. Serie 04SC-02.02. Cali, Colombia. 40 p. CIAT. 1982. El cultivo de meristemas para el saneamiento de clones de yuca; Guía de estudio para ser usada como complemento de la unidad audiotutorial sobre el mismo tema. Scientific contents: William M Roca and Upali Jayasinghe. Serie 04SC-02.05. Cali, Colombia. 45 p. COSAVE. 2003. Requisitos fitosanitarios armonizados por categoría de riesgo para el ingreso de productos vegetales. Available at http://www.cosave.org/normas/ st3015v020203_suscCM12.doc (accessed 16 Sept 2004). EPPO. 2006. EPPO A1 list of pests recommended for regulation as quarantine pests (version 2006-09). Available at http://www.eppo.org/QUARANTINE/listA1.htm EPPO. 2006. EPPO A2 list of pests recommended for regulation as quarantine pests (version 2006-09). Available at http://www.eppo.org/QUARANTINE/listA2.htm FAO. 1997. International Plant Protection Convention (new revised text approved by the FAO Conference at its 29th Session—Nov 1997). Available at http://www.fao.org/Legal/ TREATIES/004t2-e.htm FAO; IPGRI. 2004. Technical guidelines for the safe movement of germplasm. Available at http:/ /www.ipgri.cgiar.org/publications/pubseries.asp?id_serie=11 (accessed 19 Sept 2004]. Frison EA; Feliu E, eds. 1991. FAO/IBPGR technical guidelines for the safe movement of cassava germplasm. FAO; IBPGR, Rome. 48 p. Also available at http://www.ipgri.cgiar.org/ publications/pdf/349.pdf González LC. 1976. Introducción a la fitopatología. IICA, San José, Costa Rica. 148 p. Kahn R. 1999. Biological concepts. In Kahn RP; Mathur SB, eds. Containment facilities and safeguards for exotic plant pathogens and pests. APS, St. Paul, MN. p 11. Mafla B, G; Roca WM; Reyes R; Roa E, JC; Muñoz M, L; Baca G, AE; Iwanaga M. 1992. In vitro management of cassava germplasm at CIAT. In Roca WM; Thro AM, eds. Proc. International Scientific Meeting Cassava Biotechnology Network, held in 1992 at Cartagena de Indias, Colombia. Working document no. 123. CIAT, Cali, Colombia. pp 168–174. Nath R. 1993. Plant quarantine: principles and concepts. In Rana RS; Nath R; Khetarpal RK; Gokte N; Bisht JS, eds. Plant quarantine and genetic resources management. National Bureau of Plant Genetic Resources of the ICAR, New Delhi, India. pp 19–24. 142 Module 3, Submodule D: Verifying Phytosanitary Quality Lesson 1: Basic Concepts of Phytosanitary Quality Neergaard P. 1977. Seed pathology, vol. I. Halsted Books, New York. 839 p. OIRSA. 2004. Manual modelo para la aplicación de las medidas técnicas de la cuarentena agropecuaria. San Salvador, El Salvador. Also available at http://www.oirsa.org/DTSV/ Manuales/Manual04/Manual.htm (accessed 16 Sept 2004). Roca WM; Nolt B; Mafla G; Roa J; Reyes R. 1991. Eliminación de virus y propagación de clones en la yuca (Manihot esculenta Crantz). In Roca WM; Mroginski LA, eds. Cultivo de tejidos en la agricultura: Fundamentos y aplicaciones. CIAT, Cali, Colombia. pp 403–420. Further reading Andean Community, General Secretariat. (Spanish version accessed 16 Sept 2004) Treaties and legislation: treaties and protocols; Andean Subregional Integration Agreement, ‘Cartagena Agreement’. Available at http://www.comunidadandina.org/normativa/ande_trie1.htm Ayabe M; Sumi S. 2001. A novel and efficient tissue culture method—‘stem-disc dome culture’—for producing virus-free garlic (Allium sativum L.). Plant Cell Rep 20(6):503–507. Christensen CM. 1972. Microflora and seed deterioration. In Roberts EH, ed. Viability of seeds. Syracuse University Press, New York. pp 60–93. Gerard BM. 1984. Improved monitoring test for seed-borne pathogens and pests. In Dickie JB; Linington S; Williams JT, eds. Seed management techniques for genebanks; Proc. Workshop held at the Royal Botanic Gardens, Kew, 6–9 July 1982. IBPGR, Rome. pp 22–42. Leonhardt W; Wawrosch Ch; Auer A; Kopp B. 1997. Monitoring of virus diseases in Austrian grapevine varieties and virus elimination using in vitro thermotherapy. Plant Cell Tissue Organ Cult 52:71–74. Spiegel S; Frison EA; Converse RH. 1993. Recent developments in therapy and virus-detection procedures for international movement of clonal plant germplasm. Plant Dis 77(12): 1176–1180. Available at http://www.protecnet.go.cr/cuarentena/PROCEDIMIENTOS1.htm Contributors to this Lesson Benjamín Pineda, Norma C Flor, Graciela Mafla, Daniel Debouck, Mariano Mejía, María del S Balcázar, and Julio Roa. Next Lesson In the next lesson, you will become familiar with the principal procedures for controlling the plant health quality of germplasm. 143 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Lesson 2 Procedures for Verifying Phytosanitary Quality Objective To describe the general procedures for verifying phytosanitary quality Introduction To speak about phytosanitary quality without referring to health seems senseless. As human beings, we pay attention to our health every day, to maintain us active, vigorous, and with high levels of productivity. With plants, however, we do not check their health in the way they deserve. If we are to conserve germplasm (whether as propagules or seeds), then maintaining its health in the broad sense of the word is as essential for it as it is for us. Ex situ conservation, especially those activities dealing with introduction, multiplication, and regeneration, requires considerable care in maintaining the germplasm’s health for the length of its biological cycle. Being attentive during a plant’s cycle so that it is not affected by pests and diseases (pathogens) should translate into a healthy plant whose products (seeds and propagules) are equally healthy, that is, they also possess the desired attributes for phytosanitary quality. The verification of germplasm health, which results from the above-mentioned activities, should be carefully carried out. Likewise, information obtained for decision-making on germplasm management should be generated and collected according to the germplasm bank’s objectives. Hence, procedures and techniques for diagnosing plant diseases should be used. Some of these will be mentioned in this lesson. General Procedures To verify the plant health status (i.e., quality) of a given germplasm, whether during plant establishment and development or in its products, we use procedures commonly employed in phytopathology to identify diseases and apply control measures (Agarwal and Sinclair 1987; Gerard 1984; FAO and IPGRI 2004; ISTA 1999; Neergaard 1977). These procedures can be conducted, either (1) directly at the site of confinement (quarantine) or production (field, greenhouses, and laboratories) through periodic inspections, or (2) through specialized procedures, some of which are described below. Plant health inspection As materials develop in the sites of quarantine, multiplication, regeneration, and determination of the biological quality, periodic inspections and observations should be practised to determine the presence of symptoms that would permit identification of diseases. Severity and incidence of the observed diseases should then be evaluated and the causal agents identified. Where direct identification is not possible during inspection, then representative samples for specialized diagnosis should be collected. The samples can initially be examined in the laboratory under a stereomicroscope to find signs that may indicate the pathogen’s nature. If such inspection proves the presumed presence of pathogens, whether of a fungal, bacterial, 144 Module 3, Submodule D: Verifying Phytosanitary Quality Lesson 2: Procedures for Verifying Phytosanitary Quality viral, or other nature, then methodologies suitable for isolating and identifying them must be applied. These methodologies include the use of humidity chambers; isolation on specialized culture media; observation under light or electron microscope; use of identification keys; and verification, using Koch’s postulates (Box 1), in healthy materials of the same species. Box 1 Koch’s Postulates* 1. The micro-organism should be always associated with the disease, and the disease, in its turn, should not appear without the micro-organism being or having been present. 2. The micro-organism should be isolated under pure culture and its specific characters should be studied. 3. When a healthy host is inoculated with the pure culture under favourable conditions, it should produce symptoms of the disease. 4. The micro-organism should be re-isolated from the inoculated host and should show the same characteristics under culture as that previously isolated. * Adaptation from those originally recommended by Koch in 1881 for similar studies with humans and animals. They apply only to facultative parasites (in the saprophytic phase). The postulates must be reformulated for obligate parasites. SOURCE: González (1976). We point out that, in diseased tissue, not only is the casual micro-organism found but, often, other micro-organisms are also found, living as saprophytes and even growing in culture. Pure cultures must therefore be established for each organism separately and its pathogenicity determined, as according to Koch’s postulates (Box 1). Seed analysis. Seed analysis should be carried out in the laboratory on representative samples. This activity is usually executed by expert personnel, who use equipment and verification procedures already established for the type of pathogen being examined (Langerak et al. 1988). Before conducting seed analysis, personnel should clearly understand that obligate (those requiring live tissue for survival) and facultative parasites (having a saprophytic phase) exist in association with seeds and that their detection requires specific procedures. Obligate parasites do not grow on culture media, whereas facultative parasites do. Also important to consider are the types of association that tend to occur between pathogens and seeds (Figure 1). Sometimes, they associate internally; other times, externally (Figure 1). When they are found active and embedded in seed tissues, then they are parasitic and cause infections. However, no relationship of parasitism exists if they are external and are being passively carried on seed coats, harvest residues, or particles of contaminated soil (infestation); or are mixed with seed (concomitant contamination) (Neergaard 1977). 145 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Embryonic axis (radicle, hypocotyl, plumule, and primary leaves) FF FF FF FF FF VVV VVV VVV V FF VVVVVVVVVVVV VVVVVVVV FF Hypocotyl Primary leaves BB BBBBBBBBBB BBBBBBBB BBB BBB BBB B Micropyle Radicle BB FF Hilum Raphe FF FF FF FF FF Seed coat FF FF FF FF FF FF FF FF FF FF Figure 1. VVV VVV VVV V Plumule VV VVV VVV VVV V FF BB Cotyledon FF FFFFFFF FFF Radicle VV VVVVVVVVVVVV VVVVVVVV BBB BBB BBB B BBBBBBBB BB VVV VVV VVV V FF FF FF FF FF Association between pathogens and seeds. Fungi (F), bacteria (B), and viruses (V) may be found internally, causing infection in the tissues, or externally as contaminants, being either mixed or in external contact with tissues (graphic design by B Pineda, GRU, CIAT). The basic procedures for verifying the plant health quality of seeds include preliminary activities (sampling, reception, registration, and storage), preparatory work, and analysis, duly organized to fulfil the entrusted function (Figure 2). Overall, these procedures include: • • • • • • • • • 146 Sampling, identification, packaging, and dispatch of materials to the laboratory. Reception of samples, and verification of identity and size. Division of samples for diagnosis according to pathogen type (samples separated for fungi, bacteria, viruses, and other pathogens, as according to requirements). Preparation of elements needed to verify pathogens according to type (i.e., separate elements for fungi, bacteria, viruses, and other pathogens, as according to requirements). Visual inspection of samples to detect mixtures and abnormalities in seeds. Processing and planting of samples according to pathogen type (procedures for fungi, bacteria, viruses, and other pathogens, as according to requirements). Incubation. Observation and analysis. Reports and documentation. Module 3, Submodule D: Verifying Phytosanitary Quality Lesson 2: Procedures for Verifying Phytosanitary Quality (A) (B) Preparation of samples for analysis Pre-storage Reception and registration Final storage Sampling Inspection for admixtures Praparatory work Washing and extraction of fungi Washing and sedimentation for nematodes Extraction of viruss Extraction of bacteria Serological, biochemical Preparation of substrates Sterile work Nonsterile plating and sowing Incubation Cleaning and washing up Fungi Nematodes Use of incubators, growth chambers, greenhouse Viruses Bacteria Sclerotia Cysts or nematode galls Weed seeds Insects Final analysis • Arrows show the functional relationships among various activities and the required facilities in a multifunctional germplasm-health laboratory. Figure 2. A multifunctional germplasm-health laboratory. (A) The area for the microscopic analysis of seeds. Example of the Germplasm Health Laboratory of the Genetic Resources Unit at CIAT. (B) Flow chart of activities. (Chart is adapted from Langerak et al. 1988.) 147 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources The methodology to determine the seed health status uses different techniques to detect pathogens, depending on the class and type of micro-organism (e.g., obligate or facultative parasite, or saprophytes), the characteristic relationship it has with seeds (infection, infestation, or concomitance), or level of precision required. A given technique is selected according to the test’s purpose and the objectives for analysing seed health (Tables 1, 2, and 3). Once the results of the analyses are obtained with respect to the germplasm’s quality (Figure 3), decisions can be made and management carried out accordingly. When standards for the quality demanded are met, the germplasm can be conserved according to the bank’s goals. Any affected materials found may be subjected to cleaning procedures that will help rescue, reintroduce, multiply, or regenerate them. Analysing materials for vegetative propagation The procedures and methodology for analysing the phytosanitary quality of propagules or plant fragments used for vegetative propagation are based on the same principles as for seeds. Normally, in the field, germplasm can be infected by pathogens to a greater or lesser extent and, hence, needs its plant health to be monitored according to circumstances. Evaluations and analyses must be conducted with special care, as any plant part (e.g., stem, root, leaf, cutting, root cutting, stake or stem cutting, meristem, or callus) or organ (e.g., bulb, corm, rhizome, cutting, or tuber) used for propagation contains pathogens such as fungi, bacteria, viruses, viroids, or nematodes. Materials for vegetative propagation are at greater risk and require stricter plant health procedures. In fact, for analysis, observations can be carried out directly on the organs or plant parts to be used or the material can be planted and germinated and the results assessed in the seedlings with the naked eye, as described for seeds. Plant health inspection is especially relevant for selecting materials for use in propagation. Usually, the selected clone comes from a single plant from which several propagative units (which should be as disease-free as possible) are taken. Selection starts with observing and evaluating in detail the material that is to be multiplied for conservation. All aerial (e.g., leaves, branches, stems, flowers, and fruits) and subterranean organs (e.g., roots, tubers, and bulbs) should be inspected to ensure they are in suitable condition. That is, they do not present spots, mosaics, or malformations in leaves and stems; stems do not present fissures, spots, or aqueous wounds, but do present a healthy and brilliant epidermis; and, overall, the material does not present necrosis, cankers, or rot in any organ; or wilt, dwarfism, or abnormal growth. Even if the planting material is at very high risk with respect to plant health, the current tendency is to use in vitro culture techniques and thermotherapy as an alternative to safely maintain the germplasm free of pests and pathogens. That is, to guarantee the acquisition of materials of high plant health quality, the above-mentioned techniques are combined and verifications are carried out, applying several diagnostic techniques (indexing), according to case (Figure 4). 148 Table 1. Methods used to detect fungi and bacteria in analyses of seed health. Method Type of observation Verifications Fungi Bacteria 149 Examination of dry seeds With the naked eye, lenses of low magnification, and/or stereomicroscope Presence of discoloration, morphological abnormalities, and pathogenic fructifications mixed with the seeds Discoloration, presence of morphological abnormalities, and bacterial growth Examination of seeds immersed in water With stereomicroscope, light close to ultraviolet Presence of fungal fructifications or characteristic fluorescence Bacterial growth, presence of characteristic fluorescent pigments Rinse from seeds With microscope, of sediments obtained after centrifuging Presence of spores, remains of fructifications, chlamydospores, and microsclerotia – Incubation of seeds (blotter test) With stereomicroscope and microscope, after incubation, under conditions of light, temperature, and humidity suitable for seeds to germinate and fungi to grow and sporulate Presence of fructifications, spores, mycelia, sclerotia, and microsclerotia Bacterial growth, presence of characteristic fluorescent pigments Incubation of seeds (agar-plate test) With stereomicroscope and microscope, after incubation, under conditions of light and temperature suitable for seeds to germinate and fungi to grow and sporulate; Visual appraisal of bacterial colonies Presence of fungal colonies with fructifications, spores, mycelia, sclerotia, and microsclerotia Presence of colonies typical of the bacterial species being followed up Biological tests (seedling-symptom test) Visual appraisal, after seeds germinate (whether in soil, or on paper towelling or filter paper; or grown in test tubes on culture medium) under controlled conditions, i.e., suitable confinement, light, humidity, and temperature Symptoms in seedlings (absence of symptoms does not necessarily indicate that seedlings are free of fungi, as latent infections can occur) Symptoms in seedlings and presence of the bacterium when isolated (Koch’s postulates must be applied) Biological tests (pathogenicity tests) Visual appraisal, of symptoms on adapted hosts, induced by inoculation with colonies of fungi or bacterial associated with the seeds Symptoms in seedlings as a consequence of the inoculations; presence of fructifications, spores, and mycelia (Koch’s postulates) Symptoms in seedlings as a consequence of the inoculations; presence of the bacterium for re-isolation (Koch’s postulates) (Continued) 150 Table 1. (Continued.) Method Type of observation Verifications Fungi Bacteria Biological tests (using bacteriophages) Visual appraisal, of colonies affected by specific bacteriophages – Presence of lytic zones around the bacterial colonies Observations of fluorescence Observations under light close to ultraviolet (340 nm) Pale blue, yellow, or greenish fluorescence, according to pathogen or organism present in the seeds Pale blue, yellow, or greenish fluorescence, according to pathogen or organism present in the seeds Histopathological methods With microscope, of seed tissues duly processed and stained Presence of hyphae and, in some cases, fructifications within tissues Presence of bacteria within the tissues Embryo counts With microscope, of embryos duly processed and stained Presence of fungal structures (e.g., Ustilago spp. and Sclerospora graminicola) – Immersion in sodium hydroxide method With stereomicroscope, of seeds treated with NaOH at 0.2% for 24 h at 18°–25°C Presence of fungal structures (e.g., Tilletia barclayana or Neovossia horrida) – Biochemical tests (serology, ELISA, microprecipitin, and immunofluorescence) Observation of precipitation reactions, and coloration or fluorescence, according to the type of test used Presence of precipitates, and coloration or fluorescence, according to the fungus intercepted Presence of precipitates, and coloration or fluorescence, according to the bacterium intercepted Molecular and biochemical methods Observations of gels or colour reactions, according to the pathogen or type of test used Presence of coloured bands or colorimetric reactions, according to the pathogen intercepted Presence of coloured bands or colorimetric reactions, according to the pathogen intercepted Table 2. Methods used to detect viruses and viroids by analysing seed health. Method Type of observation Verifications Examination of dry seeds With naked eye, lenses of low magnification, and/or stereomicroscope Discoloration, reduced size, presence of morphological abnormalities Biological tests (seedling-symptom test) Visual appraisal, after seeds germinate (whether in soil, or on paper towelling or filter paper; or grown in test tubes on culture medium) under controlled conditions, i.e., suitable confinement, light, humidity, and temperature Observation of symptoms in seedlings Biological tests (pathogenicity tests) Visual appraisal, of symptoms on suitable hosts, induced by inoculation of seed extracts or suspect seedlings Symptoms in seedlings or in indicator plants as a consequence of inoculations Histopathological methods With electron or optic microscope, of seed tissues duly processed and stained Presence of viruses or viral inclusions within the tissues Observations with electron microscope: negative staining, immunosorbent electron microscopy (ISEM and ornamentation) With electron microscope, of seed tissues duly processed and stained Presence of viruses or viral inclusions within the tissues Biochemical tests (serology, ELISA, agglutination [chloroplast and latex], tagged antibodies, and immunofluorescence) Observation of precipitation reactions, and coloration or fluorescence, according to the type of test used Presence of precipitates, and coloration or fluorescence, according to the virus intercepted Molecular techniques Observation of gels or colour reactions according to the pathogen or type of test used Presence of coloured bands or colorimetric reactions, according to the virus intercepted 151 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Table 3. Methods used to detect nematodes by analysing seed health. Method Type of observation Verifications Examination of dry seeds With naked eye, lenses of low magnification, and/or stereomicroscope Discoloration, presence of morphological abnormalities, or nematode cysts mixed with seeds Examination of seeds immersed in water With stereomicroscope Presence of nematodes, eggs, larvae, and cysts Extraction of nematodes from affected tissues or soil mixed with seeds With stereomicroscope, after extraction Presence of nematodes, eggs, larvae and cysts One example is the procedure employed by the Genetic Resources Unit at CIAT for cassava (Manihot esculenta), a plant that propagates vegetatively. The process begins with selecting mother plants, from which stakes are taken and subjected to thermotherapy. Meristems are then cut, cultured, and micropropagated in vitro (see Module 3, Submodule D, Lesson 1). They are then propagated in specialized culture medium for 4–5 weeks in a growth chamber until they are large enough to transplant to the greenhouse. Once transplanted to pots, they are given appropriate care to obtain stems and leaf tissue for applying screening tests, which are applied according to available facilities and to the viruses being targeted for detection (e.g., common cassava mosaic virus or CCMV, cassava X virus or CsXV, and frogskin disease or FSD). The techniques applied may include diagnoses based on visible symptoms, grafts, serological methods of diffusion, precipitation, enzyme immunoassays (ELISA), and PCR (Figure 3). The materials that show negative results for the different indexing tests should be maintained or propagated under conditions that prevent re-infection. If materials show positive results to the indexing tests, then thermotherapy is re-initiated, followed by meristem culture, and subjected again to indexing tests (CIAT 1982; Roca and Mroginski 1991; Roca et al. 1991a, b). Evaluating the Lesson After this lesson, you should be familiar with the general procedures for verifying plant health quality. Before going on to Submodule E, briefly comment on the importance of verifying plant health quality. If you have had some experience in this regard, indicate what procedures were applied. 152 Module 3, Submodule D: Verifying Phytosanitary Quality Lesson 2: Procedures for Verifying Phytosanitary Quality (B) (A) (C) (D) (E) (F) (G) (H) Figure 3. Results of applying diagnostic techniques in the control of plant health quality in seeds. (A) Peanut seeds examined under ultraviolet light: left, healthy seeds; right, seeds infected by Fusarium oxysporum. (B) Bean seeds infected by various pathogens, mixed with healthy seeds. (C) Beans seeds infected by anthracnose. (D) Bean seeds infected by Macrophomina phaseoli, mixed with healthy seeds. (E) Seeds of beans infected by Curtobacterium flaccumfaciens pv. flaccumfaciens. (F) Bacterial colonies of Xanthomonas campestris pv. phaseoli in YDCA and MXP. (G) Bean seedling infected by bean common mosaic virus (BCMV). (H) ELISA plate: coloured wells correspond to samples with viruses and colourless wells to healthy seeds. (From Ahmed and Ravindener Reddy 1993 [A]; Schwartz and Pastor-Corrales 1989 [C and E]; photographs by B Pineda, GRU, CIAT [B, D, F, G, and H].) 153 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources (A) PATHOGEN ERADICATION Stakes MOTHER PLANT Cryopreservation In vitro Meristem tips 0.2–0.3 mm Thermotherapy 40°C to 35°C Germplasm bank -196°C CONSERVATION Negative Positive MICROPROPAGATION PCR Grafting (FSD) CLONE INDEXATION ELISA (CVMV) (CCMV, CsXV, ACMV) PROPAGATION Indicator clone ‘MCol 2063’ (Secundina) (B) THERMOTHERAPY Positive Stake production, using clones and indicator plant grown in greenhouse Inserting and Preparation securing graft Indicator clone Stake acquisition Clone for evaluation Debudding stock Indicator clone Negative Clone for evaluation Graft Stock Water Evaluation of materials Growth room with suitable light and temperature Figure 4. 154 System for eradicating pathogens (A) and indexing cassava germplasm (B). (Diagrams redesigned by B Pineda from the Flow Chart for the In vitro Management of Manihot Germplasm at CIAT by Flor et al. nd.) Module 3, Submodule D: Verifying Phytosanitary Quality Lesson 2: Procedures for Verifying Phytosanitary Quality Bibliography Literature cited Agarwal VK; Sinclair JB. 1987. Principles of seed pathology, vols I and II. CRC Academic Press, Boca Raton, FL. Ahmed KM; Ravindener Reddy Ch. 1993. A pictorial guide to the identification of seed-borne fungi of sorghum, pearl millet, finger millet, chickpea, and groundnut. Information Bulletin No. 34. ICRISAT, Patnacheru, Andhra Pradesh, India. p 139. CIAT. 1982. El cultivo de meristemas para el saneamiento de clones de yuca; Guía de estudio para ser usada como complemento de la unidad audiotutorial sobre el mismo tema. Scientific contents: William M Roca and Upali Jayasinghe. Serie 04SC-02.05. CIAT, Cali, Colombia. 45 p. FAO; IPGRI. 2004. Technical guidelines for the safe movement of germplasm. Available at http://www.ipgri.cgiar.org/publications/pubseries.asp?id_serie=11 (accessed 19 Sept 2004]. Flor NC; Pineda B; Mafla G. (nd) Cassava in vitro collection cleaned against seedborne diseases of quarantine importance. Genetic Resources Unit of CIAT, Cali, Colombia. (Poster.) Gerard BM. 1984. Improved monitoring test for seed-borne pathogens and pests. In Dickie JB; Linington S; Williams JT, eds. Seed management techniques for genebanks; Proc. Workshop held at the Royal Botanic Gardens, Kew, 6–9 July 1982. IBPGR, Rome. pp 22–42. González LC. 1976. Introducción a la fitopatología. IICA, San José, Costa Rica. 148 p. ISTA. 1999. International rules for seed testing. Seed Sci Technol 27:1–333. (Supplement 21). Langerak CJ; Merca SD; Mew TW. 1988. Facilities for seed health testing and research. In IRRI. Rice seed health; Proc. International Workshop on Rice Seed Health, held 16–20 March 1987. Los Baños, Laguna, Philippines. pp 235–246. Neergaard P. 1977. Seed pathology, vol I. Halsted Books, New York. 839 p. Roca WM; Mroginski LA, eds. 1991. Cultivo de tejidos en la agricultura: Fundamentos y aplicaciones. CIAT, Cali, Colombia. 969 p. Roca WM; Nolt B; Mafla G; Roa J; Reyes R. 1991a. Eliminación de virus y propagación de clones en la yuca (Manihot esculenta Crantz). In Roca WM; Mroginski LA, eds. Cultivo de tejidos en la agricultura: Fundamentos y aplicaciones. CIAT, Cali, Colombia. pp 403–420. Schwartz, HF; Pastor-Corrales MA, eds. 1989. Bean production problems in the tropics, 2nd ed. CIAT, Cali, Colombia. 726 p. Further reading Ayabe M; Sumi S. 2001. A novel and efficient tissue culture method—‘stem-disc dome culture’— for producing virus-free garlic (Allium sativum L.). Plant Cell Rep 20(6):503–507. 155 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Christensen CM. 1972. Microflora and seed deterioration. In Roberts EH, ed. Viability of seeds. Syracuse University Press, New York. pp 60–93. EPPO. 2006. EPPO A1 list of pests recommended for regulation as quarantine pests (version 2006-09). Available at http://www.eppo.org/QUARANTINE/listA1.htm EPPO. 2006. EPPO A2 list of pests recommended for regulation as quarantine pests (version 2006-09). Available at http://www.eppo.org/QUARANTINE/listA2.htm FAO. 1997. International Plant Protection Convention (new revised text approved by the FAO Conference at its 29th Session—Nov 1997). Available at http://www.fao.org/Legal/ TREATIES/004t2-e.htm Frison EA; Feliu E, eds. 1991. FAO/IBPGR technical guidelines for the safe movement of cassava germplasm. FAO; IBPGR, Rome. 48 p. Also available at http://www.ipgri.cgiar.org/ publications/pdf/349.pdf Leonhardt W; Wawrosch Ch; Auer A; Kopp B. 1997. Monitoring of virus diseases in Austrian grapevine varieties and virus elimination using in vitro thermotherapy. Plant Cell Tissue Organ Cult 52:71–74. Mafla B, G; Roca WM; Reyes R; Roa E, JC; Muñoz M, L; Baca G, AE; Iwanaga M. 1992. In vitro management of cassava germplasm at CIAT. In Roca WM; Thro AM, eds. Proc. International Scientific Meeting Cassava Biotechnology Network, held in 1992 at Cartagena de Indias, Colombia. Working document no. 123. CIAT, Cali, Colombia. pp 168–174. Nath R. 1993. Plant quarantine: principles and concepts. In Rana RS; Nath R; Khetarpal RK; Gokte N; Bisht JS, eds. Plant quarantine and genetic resources management. National Bureau of Plant Genetic Resources of the ICAR, New Delhi, India. pp 19–24. Spiegel S; Frison EA; Converse RH. 1993. Recent developments in therapy and virus-detection procedures for international movement of clonal plant germplasm. Plant Dis 77(12):1176–1180. Available at http://www.protecnet.go.cr/cuarentena/ PROCEDIMIENTOS1.htm Contributors to this Lesson Benjamín Pineda, Norma C Flor, Graciela Mafla, Daniel Debouck, Mariano Mejía, María del S Balcázar, and Julio Roa. Next Lesson In the next Submodule E, you will study the principal aspects of storing (conserving) germplasm. 156 Submodule E Lesson Basic Concepts of Storage, an Essential Component of the Ex Situ Conservation of Germplasm Storing Germplasm Objective To review the basic requirements and principal alternatives for storing germplasm Introduction The conservation of PGRs is not limited to the mere attainment and physical possession of materials (collection and storage) but must also ensure their existence under viable conditions and with their original genetic characteristics intact. For seeds or materials conserved in vitro, this is achieved by controlling storage conditions so that they inhibit or reduce the samples’ metabolism; for planting materials, by maintaining them under optimal agronomic conditions. After germplasm has been multiplied or regenerated, following all precautions to maintain its genetic identity, it is harvested and conditioned to conserve its physical and physiological integrity. Its viability and plant health status are then verified according to established procedures. The next step is to keep the germplasm under stable storage conditions so that it retains its viability over the longest period possible. To achieve this end, the required conditions must be established, including the determination of infrastructure and provision of necessary equipment and resources, as according to the magnitude of the collection and the bank’s objectives. As we develop this theme, some major topics will be described on storing germplasm as seed or maintaining it as planting material. Alternatives for Storing Germplasm Germplasm can be stored as seed, or maintained in the field or in vitro, or under cryopreservation, depending on how the species reproduces and reacts to storage. These characteristics, in their turn, determine the conditions under which it will remain viable. Planting materials may be conserved as complete plants in the field or as tissue cultured in vitro. If a species reproduces by seed, its reaction to drying needs to be determined before storage to find out if it is orthodox, recalcitrant, or intermediate. This reaction will determine the form, time, and conditions under which the samples must be stored. If a species should possess orthodox seed, then it would be best conserved as seeds. If its seed is recalcitrant or intermediate, then it should be conserved in the field or in vitro, as these types of seeds can be conserved as seeds for only very short periods and under special conditions. Seeds For the storage of seeds in the collections conserved in germplasm banks, well-founded standards have been established (FAO and IPGRI 1994) (Box 1). These norms, in essence, consider storage conditions to be those that maintain seed viability by reducing respiration and other metabolic processes without damaging the embryo. The most important 157 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Box 1 Genebank standards: seed storage conditions Seed storage conditions for base collections 17. Acceptable: Sub-zero temperatures (<0ºC) with 3-7% seed moisture content (depending upon species). Preferred: -18ºC or cooler with 3–7% seed moisture content (depending upon species). The above seed moisture content standard may need to be raised in exceptional cases where there is strong evidence that problems can arise at this moisture content (e.g. seed breakage during seed handling). 18. The preferred standards for storage of -18ºC or less with about 5% moisture content should not be relaxed. However, it should be emphasized that the choice of seed storage conditions by an individual genebank depends upon the species stored and the length of storage period envisaged before regeneration is likely to be required. Hence some flexibility is required with regard to what should be considered acceptable, particularly for those circumstances in which refrigeration to the extent required by the above preferred standard cannot be provided. Owing to the nature of the relation between seed longevity, storage temperature and seed moisture content, the same storage life can be achieved by different combinations of temperature and moisture. 19. The tendency to overemphasize the benefits of reduction in temperature compared to those in moisture content should be avoided. With regard to the effect of temperature, the relative response of longevity to reduction in seed storage temperature is very similar among diverse orthodox species, but the relative benefit of a given reduction in temperature becomes less as temperature is reduced (at least, that is, within the ranges usually investigated down to -20ºC). Thus, longevity is increased by a factor of almost 3 if storage temperature is reduced from 20ºC to 10ºC; by 2.4 from 10ºC to 0ºC; by 1.9 from 0ºC to -10ºC; but by only 1.5 from -10ºC to -20ºC. 20. In contrast, the relative benefit to longevity of reduction in moisture content: (i) varies among species; and (ii) becomes greater for each successive reduction in moisture content. This variation among species appears to be largely a function of difference in seed composition (which influences the equilibrium relation between seed moisture content and relative humidity). 21. A calculation which was made some years ago (but which, like many calculations involving extended periods of longevity, is to some extent based on extrapolation) to put the relative benefits of reduction in each of storage temperature and moisture content in context concerns the crop sesame (Sesamum indicum L.). The effect of a reduction from 5% to 2% seed moisture content provides about a forty-fold increase in longevity. This is about the same relative benefit as a reduction in temperature from +20ºC to -20ºC. However, in most crops the benefit of desiccation to longevity does not extend to such low moisture content values. 22. There is a low-moisture-limit to the increase in longevity observed to occur with reduction in seed storage moisture content. The value of this limit varies among species, but it is thought that this variation is also related to differences in seed composition such that equilibrium relative humidities at the critical moisture content are similar for different species. One estimate of this value is moisture contents in equilibrium with about 10–12% r.h. at 20ºC. It is reasonable to maximize the benefit of desiccation to subsequent longevity by drying seeds to 10–12% r.h. at 20ºC and then storing hermetically at ambient, but preferably cooler temperatures, if the storage temperature could not be controlled, or where the reduction in temperature provided by refrigeration is not adequate to meet the preferred standard for temperature. This approach has been previously described as “ultra-dry storage”. However, in some species this standard is actually slightly greater than the original 5% standard (e.g. 6–6.5% moisture content in pea). (Continued) 158 Module 3, Submodule E: Storing Germplasm Lesson: Basic Concepts of Storage … Box 1. (Continued.) 23. Whether seeds are stored dry or ultra-dry, it is essential that all seeds be “conditioned” or “humidified” (by placing in a very moist atmosphere, usually overnight but occasionally slightly longer in the case of very large seeds) prior to testing for germination or growing out. Seed storage conditions for active collections 24. Active collections should be kept in conditions which would ensure that accession viability remain above at least 65% for 10 to 20 years, being the only standard which should be provided. The precise storage regimes used to fulfil this objective will vary depending upon the species stored, the prevailing ambient environment and the relative local costs of (principally) electricity and labour. As indicated in the preceding section, different combinations of storage temperature and moisture can provide the same longevity. However, it could be emphasized that, in most locations, the reduction and control of seed storage moisture content will be a more cost-effective approach than controlling temperature. SOURCE: FAO and IPGRI (1994). conditions for achieving these results are seed moisture content reduced to appropriate levels, low temperatures, and modified storage atmosphere. Usually seeds are stored for variable times after harvesting. For orthodox seeds to remain viable under conservation, they must maintain low and constant moisture content. To ensure this, as described in Module 3, Submodule B, Lessons 1 and 2, on conditioning, the seeds are subjected to drying, and then kept in hermetically sealed containers to prevent their coming into contact with atmospheric humidity and becoming rehydrated (FAO and IPGRI 1994). The reaction of seeds to drying for storage has been studied for many species, and information exists for more than 2000 genera of about 250 families (Hong et al. 1996). When insufficient information is available on the species of interest, research is needed to classify its seeds (Figure 1) and determine their characteristics (Hong and Ellis 1996). Recently collected seeds Viability test Drying All viable seeds germinated Viability test Viable Orthodox and intermediate seeds Figure 1. Storage at low temperatures Nonviable Viable Nonviable Recalcitrant seeds Orthodox seeds Intermediate seeds Procedures for determining the behaviour of seeds in terms of their tolerance of drying (from Vázquez Y et al. 2004). 159 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Orthodox seeds can be dried to very low moisture content without being harmed—at least, to a level of constant moisture that is kept in balance with an environmental relative humidity of 10%. Longevity increases when moisture content is reduced and storage is kept at low temperatures in a quantifiable and predictable way (Sandoval S 2000; Vázquez Y et al. 2004). Recalcitrant seeds cannot be dried below a relatively high level of moisture content without being damaged. Although species vary greatly in the critical moisture content below which viability is reduced, some species will die rapidly, even if in balance with an environmental relative humidity of 98%–99%. Most recalcitrant seeds die when their moisture content is in balance with an environmental humidity of 60%–70% (corresponding to a seed moisture content of 16%–30%, fresh weight). Despite considerable research, no good method has been found for maintaining the viability of such seeds in storage, particularly those of tropical origin (Sandoval S 2000; Vázquez Y et al. 2004). Intermediate seeds are sensitive to drying to a relatively low level of moisture content (7% to 10%, in balance with an environmental relative humidity of 30%–50%). The conditions regarded as ideal for long-term storage of orthodox seeds (5% moisture content and -18°C) are potentially harmful for intermediate seeds and should not be used, as they will be killed within few months. Even so, intermediate seeds can be stored for as long as 10 years if they are dried to 7%–10% of their moisture content and maintained at laboratory temperatures (Sandoval S 2000; Vázquez Y et al. 2004). Another requirement for maintaining seed viability is storage at low temperatures in environments that are poor in oxygen. Low temperatures invariably extend the life of seeds in storage and can generally counteract the adverse effects of high moisture content. Modifying the atmosphere in which seeds are stored by vacuum packaging, increasing levels of carbon dioxide, or replacing oxygen with nitrogen or other gases can, according to various studies, benefit the very short-lived seeds of some tropical plants. For example, rubber (Hevea brasiliensis) seeds can be kept in sealed containers filled with 40%–45% of carbon dioxide. Sugar-cane seeds can also be dried in the open air and packaged in sealed tins in which the air is displaced by carbon dioxide, and 9 g of calcium chloride per litre of capacity are added. The whole is then stored at temperatures close to freezing point (Hartmann and Kester 1971). The combination of low moisture content, sealed containers, low temperatures, and, in some cases, modified storage atmosphere help prevent the ageing and degeneration of cellular tissues, phenomena that occur over time as substances accumulate from the metabolism the organism uses to stay alive. These substances are believed to inactivate enzymes and nucleic acids, preventing cellular membranes from fulfilling their function as selectively permeable barriers in the exchange of compounds and thus resulting in the accumulation of not only metabolically inert materials but also mutagenic substances. The organelles within cells, the cells themselves, and organs may possibly become ineffective through constant use. In other cases, mutations may increase with age. Over time, an organism becomes more inefficient as mutations increase in number, leading to the production of defective proteins that affect the organism’s deoxyribonucleic acid (DNA). During storage, seeds accumulate genetic damage, which manifests as chromosomal 160 Module 3, Submodule E: Storing Germplasm Lesson: Basic Concepts of Storage … aberrations occurring during the first phases of cellular division in germination. Thus, many effects of ageing will appear, not so much during germination, but during the cellular differentiation and formation of the seedling (Moreno C 2004; Roos 1982). Types of storage Open storage (no control of temperature or humidity) is not recommended for germplasm conservation. Seed longevity depends largely on the relative humidity and temperature of storage atmosphere. It also depends on the class of seeds and their condition at the beginning of storage. Maintaining the viability of stored seeds therefore depends on the region’s climatic conditions, with the most adverse occurring in warm humid regions and the best in cold dry regions. In the latter areas, the most preferred seeds are those with hard coats, provided they have been dried appropriately. Fumigation or applications of insecticides may be needed to control insect infestations (Hartmann and Kester 1971; Sandoval S 2000). Warm storage with humidity control is a better technique than the previous one, provided seeds are stored in sealed bags that ensure the minimization of fluctuations in humidity and remain in rooms with controlled temperatures. For vegetable seeds, the following recommendations (Hartmann and Kester 1971) serve as a general guide: • • • The environmental air of seeds exposed to 27°C (80°F) for more than a few days must have a relative humidity of no more than 45%; Seeds exposed to 21°C (70°F) should be kept at a humidity of no more than 60%; and Very short-lived seeds (e.g., onion and peanut) should be conserved at even lower humidity levels. Dried seeds may also be stored in sealed containers made of materials resistant to moisture. Many types of containers are used that vary in duration, resistance, cost, protective capacity against rodents and insects, and ability to hold or transmit humidity (Hartmann and Kester 1971; Sandoval S 2000). Cold storage (with or without humidity control) is the best method as seeds are kept with low moisture content, in sealed containers, and at low temperatures, thus prolonging their longevity to the utmost. After conditioning and packaging, seeds can be stored in rooms for the long, medium, or short term, according to goals (Figure 2). Storage conditions for maintaining the samples viable are determined according to species, the reason for conserving it, and the planned storage time. Conservation temperatures depend on the storage period envisaged. Most species with orthodox seed can be conserved for indefinite periods at temperatures between -10°C and -20°C, with a moisture content of 3%–7% and a viability value of no less than 85%. Seeds conserved under these conditions can be kept for 70 to 100 years. It should be remembered that the benefit of reducing temperatures is less than the benefit of reducing seed moisture content. Studies have suggested that greater benefit is obtained when temperatures are reduced considerably. That is, longevity triples when the temperature drops from 20° to 10ºC; multiplies by (×) 2.4 when it drops from 10° to 0°C; × 1.9 from 0° to 10°C; 161 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources 5°C 35% RH Short-term cold room Figure 2. -20°C Long-term cold room Short-term (left) and long-term (right) storage rooms for seeds kept at low temperatures (photographs by B Pineda, GRU, CIAT). and × 1.5 from -10° to -20°C. Hence, standards suggest that acceptable temperatures for conservation are those that are below 0ºC and preferably below -18ºC (FAO and IPGRI 1994). Toole (cited by Hartmann and Kester 1971) recommends that, for vegetable seeds stored at 40°–50°F (4.5°–10°C), relative humidity should be no more than 70% but preferably no more than 50%. When taken out of storage with a humidity of more than 50%, seeds should be dried to a safe moisture content unless they are planted immediately. Storage temperatures kept as low as freezing point may be desirable if the need justifies the additional cost. Temperatures below freezing point may be used for prolonged storage and for conifers such as the silver fir (Afoles) or spruce (Picea sp.). If seeds are to be conserved for the medium term (10–20 years, maximum 30), they can be maintained at temperatures between 0° and 15°C (usually 1°–4°C), with a moisture content between 3% and 7% and a viability value of no less than 65%. For short-term conservation, seed can be stored in airconditioned rooms (Cromarty et al. 1985; Engle 1992; Towil and Roos 1989). The equipment used for cold storage usually consists of appropriately designed cold rooms, which should be hermetic and able to maintain as constant temperatures, relative 162 Module 3, Submodule E: Storing Germplasm Lesson: Basic Concepts of Storage … humidity, and light intensity. Machines for refrigeration, dehumidification, and control of light hours are used. In particular, the rooms should be designed for the samples they will store, the period over which they will remain in the rooms, and the area’s climate where the rooms are established. Usually, cold rooms should be built with prefabricated panels of galvanized steel, joined by polyurethane foam, and insulated to protect the germplasm from outside conditions. Each room must have two independent refrigeration systems, a constant and stable energy supply, and verification instruments such as mercury and wet- and drybulb thermometers. Information on the infrastructure and equipment required can be found in the manual by Cromarty et al. (1985) for designing seed storage installations. The cold rooms for seed storage can be modified considerably in terms of size and complexity, depending on the needs for each storage installation. Spacious underground chambers, constructed with thick concrete walls, are ideal for very large installations. They would be isolated from atmospheric changes in temperature, require less energy to function, and, especially, can resist environmental catastrophes or wars (Vázquez Y et al. 2004). The simplest system, when operating with few resources and personnel, is to store orthodox seeds in a domestic freezer, whether horizontal or vertical. Care must be taken to prevent prolonged changes of temperature during electrical faults by using a generator that turns on automatically when a fault occurs. Desiccator Seeds can also be stored in glass vials sealed under heat. This method consists of first placing predried seeds in the vials and plugging with cotton wool. The air is then replaced with carbon dioxide and the vials sealed under heat. A desiccator can also be added, placing Seal it between the cotton wool plug and the vial opening before sealing (Figure 3). This method facilitates the management of seeds when no Cotton wool special cold room is available in which to open the containers. That is, the vials can be left to gradually take up the room temperature before opening them. Thus, only a small sample of seeds is exposed, whenever seeds must be extracted from the bank. Cold and humid storage involves placing seeds in containers that maintain humidity, Cotton wool Glass vial Seeds In all cases, whether dealing with a small seed bank or one of national importance, a stable conservation policy for the germplasm is indispensable, together with an administrator and personnel, who are well prepared and interested in the project. Also necessary is continuous economic support to ensure the perpetuation of installations and their effective operation (Vázquez Y et al. 2004). Figure 3. Glass vials are used to store seeds (left and centre, photographs by B Pineda, GRU, CIAT; right, sketch from Vázquez Y et al. [2004] and redrawn by B Pineda). 163 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources or mixing them with materials that hold moisture such as damp sand. This method is applied to some recalcitrant seeds for a short period, together with an oxygen supply for respiration. This procedure is similar to that described for seed stratification (see Module 3, Submodule C, Lesson 2). Examples of plants whose seeds require this treatment are Acer spp. (maples, especially A. saccharinum), Aesculus spp. (buckeyes or horse-chestnuts), Carpinus caroliniana (American hornbeam), Carya spp. (hickories), Castanea spp. (chestnuts), Corylus spp. (hazelnuts), Citrus spp. (citrics), Fagus spp. (beeches), Juglans spp. (walnuts), Nyssa sylvatica (tupelo), and Quercus spp. (oaks) (Hartmann and Kester 1971). Many tropical tree species produce seeds with high moisture content and fast metabolic rates, and which behave as recalcitrant. They are characterized by their inability to reorder the structure of their cellular components as water exits from the cells during dehydration. That is, the protoplasm loses its functional structure and does not recover it on rehydrating. The presence of free water in the cells eliminates the protective effect of freezing because ice crystals form that damage the cells. However, recent studies show that some dehydration treatments (Table 1) carried out on these seeds (e.g., papaya) permit storing them for longer (Vázquez Y et al. 2004). Table 1. Treatments that can be carried out with recalcitrant seeds for their storage. Treatment Observations Effect Gradual and careful dehydration under controlled conditions of temperature, aeration, and air humidity The degree of dehydration that seeds can tolerate at different temperatures without losing their viability must be determined Increases longevity briefly Dehydration in the presence of substances that protect the cellular ultrastructure The level of dehydration at which the cellular ultrastructure is protected when conducting treatments with proline, betaine, saccharose, and other products must be determined Increases the possibility of dehydrating seeds to a greater degree Dehydration at very low temperatures in the presence of cryoprotectants or through the use of respiratory inhibitors The levels of dehydration tolerated, and the effects of temperature, cryoprotectants, and inhibitors used must be determined Duration of seed reserves increases and, as a result, viability is lasts longer when the seeds are stored under suitable conditions SOURCE: Vázquez Y et al. (2004). Quantity of seeds to store The first concern to resolve is how many seeds would be used within a period, averaging 15 years. Norms for germplasm banks recommend that a base collection should be represented by a minimum of 1000 viable seeds (FAO and IPGRI 1994). An active collection can be represented by 1500 (presuming 100 seeds per year are distributed for use), and 500 seeds destined for testing viability—one initial and at least four periodic tests—and plant health, to total 3000 as a minimum for each entry. 164 Module 3, Submodule E: Storing Germplasm Lesson: Basic Concepts of Storage … This quantity of seeds should be taken as a suggestion for the minimum number of seeds to conserve rather than as a magic number. This is because variations should be taken into account, such as the species’ reproductive mode. Allogamous or cross-pollinating species should be represented by a larger number of seeds, as they represent a wider genetic heterogeneity of individuals. Similarly, species with very long life cycles such as woody species must be represented by the largest possible number of seeds, because the plants’ regeneration cycle is reached only after a growing period of several years. Likewise, species that have an abundant and easy production can be conserved, using a larger number of seeds. Those international germplasm banks that are responsible for conserving the germplasm of several countries must keep a larger number of seeds, as they must repatriate seeds to the country of origin and maintain one or two duplicates for safety reasons to conserve in other institutions. Plant materials Conservation in the field. This method is applied to species that are perennial, arboreal, wild, semi-domesticated, or heterozygous, and to those with vegetative reproduction, short-lived seeds, or seeds that are sensitive to drying. Many important varieties of field, horticultural, and forestry species are either difficult or impossible to conserve as seeds (i.e., seeds do not form or, if they do, they are recalcitrant). Hence, they are conserved in field germplasm banks (Rao 2001). Field conservation thus involves conditioning the material where needed, multiplying it, selecting and preparing a site, planting the materials, and recording information on the accessions’ location. As mentioned before, vegetative propagation implies the use of parts taken directly from plants. As a result, during multiplication, there is a risk of propagating pathogens and other pests that associate with such plant material. Consequently, any programme aiming to maintain propagative disease-free clones that are genetically identical to the original materials must undertake the following three steps: 1. Initial selection from sources of planting materials that are characteristic of the species and free of serious pathogens; 2. Maintenance of such materials in sites with adequate protection against re-infection or genetic change; and 3. Establishment of a propagation and distribution system by which such materials are disseminated without becoming infected before reaching the user. To conserve the material in the field, it must first be conditioned. The collected plant material is washed and disinfected before propagating it and taking it to the conservation site. Disinfection may be carried out with bactericides, fungicides (bulbs and rhizomes), or thermotherapy (stakes). The planting material, once disinfected, is propagated in the field, greenhouse, or in vitro. In the field and greenhouse, samples are planted on seedbeds or in pots, and left to grow until plants are obtained from which new samples can be collected. The procedure is repeated until the number of plants needed to establish the collection in the definitive site is reached. If propagation is to be in vitro, then samples are planted in the greenhouse, in soils of optimal nutritional quality. From the resulting plants—preferably the youngest—explants 165 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources are extracted and micropropagated in vitro until complete plants are obtained. These are taken again to the greenhouse, planted in sterilized soil, and, after 2 or 3 weeks, transferred to the definitive site in the field. Micropropagation consists of (a) disinfecting the explants in a solution of sodium or calcium hypochlorite, mercuric bichloride (HgCl2), or ethanol, (b) planting them in an in vitro culture medium until new shoots develop, and (c) rooting the shoots until entire plants are obtained (George 1996; George and Sherrington 1984; IPGRI and CIAT 1994; Jaramillo and Baena 2000; Roca and Mroginski 1991). Propagation in the field and greenhouse is simple but requires time and space. It does not guarantee that the plants obtained are healthy and genetically identical to the originals. In vitro propagation solves these problems, making the propagation of many species possible and more convenient, even for those that reproduce by seed. The site selected to conserve materials in the field should be safe and favour the plants’ development. It should also be isolated to prevent attacks from pests and diseases but easy to access for management. The physical and chemical preparation of the planting site depends on the species’ requirements and the number of accessions expected to be planted in the field. Taking vigorous plants to the field in a number that represents the accessions’ genetic variability will ensure the continuity of the conserved materials. The plants are so arranged in the field that they do not exchange pollen and the populations do not lose their original genotype. The exact site where each accession was planted should remain recorded on a map; and the accessions identified both in the field and on the plants. In vitro conservation and storage. This system is gaining ever-growing importance as a tool of conservation and germplasm exchange because it permits the maintenance of a wide range of species, with a diversity of healthy samples, in a small space, and permits their easy exchange. However, such a system requires technology and knowledge that is still developing, protocols for each species, and considerable resources. This means that alternative conservation options should be evaluated before deciding on in vitro conservation. It is best applied for those species that are difficult to conserve as seed or in the field. Tissue culture permits the in vitro conservation of a broad range of species in various types of samples such as complete plants, seeds, sprouts, buds, cauline apexes, meristems, ovules, embryos, cells in suspension, protoplasts, anthers, pollen, and DNA. The in vitro conservation of germplasm focuses on controlling the normal growth of viable explants— either reducing it or stopping it—by managing the constitution of the culture medium and/ or storage conditions. As with conservation in the field, materials are conditioned, planted—in vitro in this case—and taken to the conservation site (Figure 4). Conditioning consists of disinfecting samples and washing them in distilled water to eliminate excess disinfectant. The solutions most used are sodium hypochlorite (NaOCl) at 1%–3%, calcium hypochlorite (Ca(OCl)2) at 6%–12%, and ethanol at 70%. Explants (the smaller, the better) are extracted from the cleaned sample and planted in culture media placed in glass containers. They are subjected to one of two ways of in vitro conservation: slow growth or cryopreservation. In both cases, the medium and conservation environment must be sterilized and storage conditions controlled (George 1996; Jaramillo and Baena 2000; Roca and Mroginski 1991). 166 Module 3, Submodule E: Storing Germplasm Lesson: Basic Concepts of Storage … Slow growth. Slow growth consists of reducing the explants’ development by modifying the culture medium and/or conditions under which they are maintained. Through the culture medium, growth can be reduced by increasing the osmotic potential (adding mannitol, proline, glycerol, or sucrose), adding growth inhibitors (abscisic acid), and reducing or suppressing nutrients that the explants need for growing (carbon and nitrogen). Growth is also limited by controlling the conditions under which the samples are stored, either using small containers or reducing the temperature, light, and partial oxygen pressure. Reducing the temperature is the most effective way of controlling the explants’ growth by reducing metabolic activity. However, it is equally important to ensure and maintain a slow growth rate to keep the explants viable for the longest possible time. Hence, a combination of methods should be used. Samples in slow growth are kept in rooms with low temperatures for periods that may vary from some months to usually two years. The temperature will Figure 4. In vitro germplasm bank for cassava depend on the species and variety, although (Manihot esculenta) germplasm, GRU, CIAT. most in vitro crops are kept at temperatures between 20° and 30°C. Lower temperatures can reduce even further the growth of some species but will negatively affect others. Some Prunus species, for example, conserve well at -3°C, whereas temperatures below 15°C will quickly destroy explants of Musa spp. (Pérez-Ruíz 1997), and those below 18°C will damage many cassava varieties (Roca and Mroginski 1991). Materials conserved in slow growth need to be renewed every so often, even though they have continued growing, albeit slowly. Samples are micropropagated and transferred to a fresh medium for recovery and strengthening. When new explants have been established, they are propagated again, and taken again to the conservation medium. An example of the successful application of this methodology is cassava at CIAT, Colombia, where about 6017 accessions are conserved (IPGRI and CIAT 1994; Jaramillo and Baena 2000; Roca and Mroginski 1991). Cryopreservation consists of placing explants in liquid nitrogen (-196°C) to stop growth while conserving viability and genetic and physiological stability. This technique is recent, with good prospects, as it allows the storage for indefinite periods any species that can tolerate and outlive freezing. Hence, it is particularly useful for conserving species with 167 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources non-orthodox seed or vegetative reproduction, and difficult to conserve in rooms or in the field (Ashmore 1997; Benson 1999; Engelmann and Takagi 2000; Jaramillo and Baena 2000). Cryopreservation consists of (a) growing the explants in vitro (pregrowth), (b) drying them to the permissible minimum according to species, (c) treating them with cryoprotectants (glycerol, sucrose, mannitol, proline, polyethylene glycol) to prevent crystallization of intracellular liquids, (d) freezing in liquid nitrogen, (e) storing, (f) thawing, and (g) treating to recover viable plants (Jaramillo and Baena 2000; Pérez-Ruíz 1997; Rao and Riley 1994; Wang et al. 1993). The success of cryopreservation depends on the species’ reaction to freezing, which means that each species requires specific protocols. Various techniques exist such as encapsulation/dehydration, vitrification, encapsulation, desiccation, pregrowth, pregrowthdesiccation, and droplet-freezing (Ashmore 1997). But studies in this field, such as those carried out by CIP (Peru) with potatoes that tolerate freezing and CIAT with cassava, are still based on trial and error (Rao and Riley 1994). The methodology has limitations, the principal ones being the difficulty and time required to regenerate entire plants from the conserved structures. Evaluating the Lesson After this lesson, you be familiar with the basic requirements and main alternatives and procedures for storing plant germplasm. Before going on to the next Module 4, briefly describe the type or types of storage used in your bank. If your work does not involve storing germplasm, describe that mode of storage that would be the most suitable according to the resources available to the bank of your institution. Bibliography Literature cited Ashmore SE. 1997. Status report on the development and application of in vitro techniques for the conservation and use of plant genetic resources. IPGRI, Rome. 67 p. Benson EE. 1999. Plant conservation biotechnology. Plant Conservation Biotechnology Group, School of Science and Engineering, University of Abertay Dundee, Dundee, Scotland. 309 p. Cromarty AS; Ellis RH; Roberts EH. 1985. The design of seed storage facilities for genetic conservation. Handbook for Genebanks No. 1. IBPGR, Rome. 100 p. Engelmann F; Takagi H, eds. 2000. Cryopreservation of tropical plant germplasm: current research progress and application. JIRCAS; IPGRI, Rome. 496 p. Engle LM. 1992. Introduction to concepts of germplasm conservation. In Chadna ML; Anzad Hossain AMK; Monowar Hossain SM, comps. Germplasm collection, evaluation, documentation, and conservation; Proc. Course offered by AVRDC, Bangladesh Agricultural Research Council, and Bangladesh Agricultural Research Institute, 4–6 May 1992, Bangladesh. AVRDC, Taiwan. pp 11–17. 168 Module 3, Submodule E: Storing Germplasm Lesson: Basic Concepts of Storage … FAO; IPGRI. 1994. Normas para bancos de genes. Rome. 15 p. Also available at http:// www.fucema.org.ar/fucema/legislacion/otros/normasgenesfao.htm (Also available in English as Genebank Standards at http://www.ipgri.cgiar.org/publications/pdf/424.pdf) George EF. 1996. Plant propagation by tissue culture: in practice, Part 2. Exegetics, Westbury, UK. 1361 p. George EF; Sherrington PD. 1984. Plant propagation by tissue culture: handbook and directory of commercial laboratories, Part 1. Exegetics, Westbury, UK. 709 p. Hartmann HT; Kester DE. 1971. Propagación de plantas: Principios y prácticas. (Translated from the English by Antonio Marino Ambrosio.) Editorial Continental, Mexico, DF. pp 119–223. (Available in English as Hartmann HT; Kester DE; Davies FT, eds. 1990. Plant Propagation: Principles and Practices, 5th ed. Englewood Cliffs, NJ. 647 p.) Hong TD; Ellis RH. 1996. A protocol to determine seed storage behavior. Technical Bulletin No. 1. IPGRI, Rome. 64 p. Also available at http://www.ipgri.cgiar.org/publications/pdf/ 137.pdf Hong TD; Linington S; Ellis RH. 1996. Seed storage behavior: a compendium. Handbooks for Genebanks No. 4. IPGRI, Rome. 656 p. Also available at http://www.cgiar.org/ipgri/doc/ download.htm IPGRI; CIAT. 1994. Establishment and operation of a pilot in vitro active genebank: report of a CIAT–IBPGR collaborative project, using cassava (Manihot esculenta Crantz) as a model. IPGRI, Rome. 59 p. Jaramillo S; Baena M. 2000. Material de apoyo a la capacitación en conservación ex situ de recursos fitogenéticos. IPGRI, Cali, Colombia. Also available at http://www.ipgri.cgiar. org/training/exsitu/web/arr_ppal_modulo.htm Moreno C, P. 2004. Vida y obra de granos y semillas. Available at http://omega.ilce.edu. mx:3000/sites/ciencia/volumen3/ciencia3/146/htm/vidayob.htm Pérez-Ruíz C. 1997. Conservación in vitro de recursos genéticos. In VI international course on the ‘Conservation and use of plant genetic resources for food and agriculture’. Conducted in Spanish at San Fernando de Henares by the Ministry of Food, Agriculture, and Fishery, the National Institute of Agricultural and Food Research and Technology, the Spanish Agency for International Cooperation, and the Inter-American Development Bank, 3–28 November 1997. Escuela Central de Capacitación Agraria, San Fernando de Henares, Spain. 4 p. Rao R. 2001. Principle and concepts in plant genetic resources conservation and use. In Said Saad M; Ramanatha Rao V, eds. Establishment and management of field genebanks: a training manual. IPGRI–APO, Serdang, Indonesia. pp 1–16. Rao R; Riley KW. 1994. The use of biotechnology for conservation and utilization of plant genetic resources. Plant Genet Resour Newsl 97:3–20. Roca WM; Mroginski LA, eds. 1991. Cultivo de tejidos en la agricultura: Fundamentos y aplicaciones. CIAT, Cali, Colombia. 969 p. 169 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Roos EE. 1982. Induced genetic changes in seed germplasm during storage. In Khan AA, ed. 1982. The physiology and biochemistry of seed development, dormancy and germination. Elsevier Biomedical Press, Amsterdam, the Netherlands. pp 409–434. Sandoval S, A. 2000. Almacenamiento de semillas. CESAF–Chile No. 14. CESAF of the Faculty of Forest Sciences, Universidad de Chile, Santiago. Available at http://www.uchile.cl/ facultades/cs_forestales/publicaciones/cesaf/n14/1.html Towil LE; Roos EE. 1989. Techniques for preserving of plant germplasm. In Knutson L; Stoner AK, eds. Biotic diversity and germplasm preservation, global imperatives. Kluwer Academic Publishers, Dordrecht, the Netherlands. pp 379–403. Vázquez Y, C; Orozco A; Rojas M; Sánchez ME; Cervantes V. 2004. Reproducción de las plantas: Semillas y meristemas. Available at http://omega.ilce.edu.mx:3000/sites/ciencia/ volumen3/ciencia3/157/htm/sec_5.htm Wang BSP; Charest P; Downie B. 1993. Ex situ storage of seeds, pollen, and in vitro perennial woody plant species. Forestry Paper 113. FAO, Rome. 85 p. Further reading Baskin CC; Baskin JM. 1998. Ecologically meaningful germination studies. In Seeds: ecology, biogeography, and evolution of dormancy and germination. Academic Press, San Diego, CA. pp 5–26. Chin HF. 1994. Seed banks: conserving the past for the future. Seed Sci Technol 22:385–400. Ellis RH; Hong TD; Roberts EH. 1985. Seed technology for genebanks. Handbook for Genebanks No. 2, vol. 1. IBPGR, Rome. 210 p. Grabe DF. 1989. Measurement of seed moisture. In Stanwood PC; Miller MB, eds. Seed moisture: Proc. Symposium, held 30 Nov 1987. Special Publication No. 14. CSSA, Madison, WI. Hong TD; Linington S; Ellis RH. 1998. Compendium of information on seed storage behaviour, vol. 1: Families A–H. Royal Botanic Gardens, Kew, London. 400 p. Ramanatha R. 2001. Principles and concepts in plant genetic resources conservation and use. In Said Saad M; Ramanatha Rao V, eds. Establishment and management of field genebanks: a training manual. IPGRI–APO, Serdang, Indonesia. pp 1–16. Sackville Hamilton NR; Chorlton KH. 1997. Regeneration of accessions in seed collections: a decision guide. Handbook for Genebanks No. 5. IPGRI, Rome. 75 p. Contributors to this Lesson Benjamín Pineda, Alba Marina Torres, Daniel Debouck, Carlos Iván Cardozo, Rigoberto Hidalgo, Mariano Mejía, Graciela Mafla, Arsenio Ciprián, Manuel Sánchez, Carmen Rosa Bonilla, and Orlando Toro. Next Lesson In the next Module 4, you will study aspects of germplasm characterization. 170 Module 4 Supported by the CGIAR Germplasm Characterization General Comments Plants are living things that have morphological, structural, and functional characteristics that enable them to adapt to the habitat where they are established, interacting with changing environmental conditions. Furthermore, they have internal information systems that coordinate and control all the processes pertaining to life maintenance, so that they succeed in sustaining a certain degree of permanence across space and time. Under natural conditions, over time, and as a function of their evolution and needs for adaptation, plants have accumulated, in coded form in their genome, the results obtained. Knowledge of those coded and therefore usable plant attributes and properties converts them into valuable resources (i.e., plant genetic resources or PGRs), worthy of conservation. As described previously (Module 1, Lesson 1, page 2): Plant genetic resources are the sum of all combinations of genes and their variants, resulting from the evolution of plant species. During evolution, a plant population is the receptacle of all past changes and of the results of selections made by the environment, which are expressed as DNA that is exactingly organized and conserved in genomes (Hoagland 1985). In other words, genes contain all the information that defines each trait or character of a living being, in this case, plants. An inheritable trait or character is meticulously reproduced in offspring. Consequently, we find in genes information on adaptation, productivity, resistance to adverse conditions such as pests, diseases, stressful climates, and poor soils, and other characteristic of a population’s individuals that are usable by humans to the extent of their knowledge. In general and according to previous statements, important information is believed to exist in plant genomes and to express itself as morphological, structural, or functional attributes. It is contained in germplasm, which therefore becomes the holder of a species’ entire sum of hereditary characteristics. However, it should be emphasized that, to use it, germplasm should be understood in detail, that is, the type of attributes it possesses should be determined. The process of gaining such understanding is known as germplasm characterization. Information on the Module Module 4 contains two lessons and a brief evaluation exercise for each lesson. Objectives When you have completed this module, you should be able to: • • Justify and conceptualize plant germplasm characterization Describe types of germplasm characterization 171 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Lessons 1. General concepts of germplasm characterization 2. Ways of characterizing plant germplasm Bibliography Throughout this module, a bibliography is provided for each section, that is, the General Comments and each Lesson. The bibliographies follow a format of two parts: 1. Literature cited, which includes those references cited in the text itself. Some of these citations were used to develop the original Spanish-language course on ex situ conservation and may therefore appear in Spanish or Portuguese. However, where practical, references to the English versions of the original Spanish-language documents are provided. 2. Further reading, which is a list of suggested readings in the English language. Most cover in depth the topics included in this module. A list of Acronyms used in the bibliographies is also given. The idea is to save space by not having to spell out each institution’s full name each time it appears in the references. Acronyms used in the bibliographies AVRDC CGN CIP FAO IBPGR IITA INIA IPGRI RHS Asian Vegetable Research and Development Center Centre for Genetic Resources—Netherlands Centro Internacional de la Papa Food and Agriculture Organization of the United Nations International Board for Plant Genetic Resources International Institute of Tropical Agriculture Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (Spain) International Plant Genetic Resources Institute Royal Horticultural Society Literature cited Hoagland MB. 1985. Las raíces de la vida. (Translated from English by Josep Cuello.) Salvat Editores, Barcelona. 167 p. (Also published in English as Hoagland MB. 1978. The roots of life: a layman’s guide to genes, evolution, and the ways of cells. Houghton-Mifflin, Boston, MA, 167 p.) Further reading Bioversity International. (Accessed 17 Aug 2007) Descriptors lists. Available at http:// www.bioversityinternational.org/Themes/Germplasm_Documentation/Crop_Descriptors/ index.asp CGN. 2000. About CGN molecular markers. Available at http://www.cgn.wageningen-ur.nl/pgr/ research/molgen/right.htm#top (accessed 14 Dec 2004). FAO. 1997. The state of the world’s plant genetic resources for food and agriculture. Rome. 510 p. Also available at http://www.fao.org/ag/AGP/AGPS/Pgrfa/pdf/swrfull.pdf or http://www.fao.org/iag/AGP/AGPS/Pgrfa/wrlmap_e.htm 172 Module 4: Germplasm Characterization General Comments Hickey M; King C. 2000. The Cambridge illustrated glossary of botany terms. Cambridge University Press, UK. 208 p. IPGRI. 1996. Descriptors for tomato (Lycopersicon spp.). Rome. Simpson MJA; Withers LA. 1986. Characterization using isozyme electrophoresis: a guide to the literature. IBPGR, Rome. 102 p. Stalker HT; Chapman C. 1989. Scientific management of germplasm: characterization, evaluation and enhancement. IBPGR, Rome. 194 p. Stockley C. 1991. Illustrated dictionary of biology [practical guides]. Usborne Publishing, London. Van Hintum TJL; Van Treuren R. 2002. Molecular markers: tools to improve genebank efficiency. Cell Mol Biol Lett 7(2B):737–744. Available at http://www.cmbl.org.pl/072B/ 72B13.PDF (accessed 14 Dec 2004). Westman AL; Kresovich S. 1997. Use of molecular techniques for description of plant genetic variation. In Callow JA; Ford-Lloyd BV; Newbury HJ, eds. Biotechnology and plant genetic resources, conservation and use. Biotechnology in Agriculture Series, No. 19. CAB International, New York. Contributors to the Module Benjamín Pineda, César Ocampo, Rigoberto Hidalgo, Alba Marina Torres, Daniel Debouck, Mariano Mejía, Arsenio Ciprián, and Orlando Toro. Next Lesson In the next lesson, you will study the general concepts of germplasm characterization. 173 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Lesson 1 General Concepts of Germplasm Characterization Objectives • • To justify the raison d’être of characterizing plant germplasm To conceptualize this process Introduction Plant genetic resources are conserved to use them. Using them is only possible if their characteristics or attributes are known in detail and their possible uses visualized (Jaramillo and Baena 2000). Such knowledge and visualization can be achieved only through the study of the morphological, structural, and functional attributes of germplasm as the carrier of all the hereditary characteristics of any given species. Heritable traits or characters of plant germplasm are studied precisely during characterization, where those aspects already mentioned and others of a population’s individuals that can be used by humans are studied such as adaptation, productivity, and resistance to adverse conditions (e.g., pests, diseases, and climatic and soil stresses). When germplasm is already physically held in a bank or collection, it must be duly accompanied by passport data (i.e., origin, geographical location, and characteristics of the habitat where it was collected and of the environment such as climate and soils). Characterization can then proceed, taking into account that it is a fundamental stage of ex situ conservation and that important reasons exist to justify it. That is, characterization: • • • Permits estimation of the true genetic diversity that is being conserved—the principal raison d’être of a germplasm bank; Is valuable for providing germplasm banks with complete information on the characteristics of a given germplasm, thereby contributing to an optimal ex situ management of collections. Otherwise, its absence would convert such banks into simple depositories of materials of no significant usefulness. Facilitates the use of germplasm collections by improvement programmes and crop research. To characterize germplasm, basic skills in botany (i.e., plant biology or phytology) are essential, particularly in the three principal divisions (taxonomy, morphology, and plant physiology). An understanding of systematics and genetics, among others, is also important. Botanical knowledge of the germplasm of a given species conserved ex situ provides key information for its optimal characterization, especially to better select both materials to characterize and methodologies to use. Taxonomy should be understood as the science that uses, as criteria for classification, properties of organisms such as morphology. The criterion that is currently accepted as the basis for taxonomy is that which reflects the phylogeny of living things and compares characters of whatever nature, whether morphological, anatomical, or cytogenetic. Consequently, taxonomy is a prime tool for germplasm characterization. 174 Module 4, Lesson 1: General Concepts of Germplasm Characterization Systematics is the science of diversity. That is, it is the organization of the total set of knowledge on organisms. It includes phylogenetic, taxonomic, ecological, and palaeontological information. It permits seeing a global vision of the diversity of conserved materials, and has a predictive character, which permits the better selection of germplasm to characterize and methodologies to use, and improves analysis of results. Genetics is the study of the nature, organization, function, expression, transmission, and evolution of the coded genetic information found in organisms. It is fundamental for a maximum evaluation of the data obtained from characterization. Characterization Definition Through characterization, we can estimate the variation that exists in a germplasm collection in terms of morphological and phenological characteristics of high heritability. Such variation may also include the variability expressed by biochemical and molecular markers, that is, by characteristics whose expression is little influenced by the environment (Hidalgo 2003; Jaramillo and Baena 2000; Ligarreto 2003). In the characterization of plants, the expression of constant qualitative characters is recorded throughout a given plant’s various physiological stages (phenotype). Data are taken according to specific descriptors, for example: • • • For the seedling stage: hypocotyl colour and pubescence, length of the primary leaf, and petiole thickness; For the stages before and during flowering: plant height and growth habit, leaf position, flower colour, and days to flowering; and During the stage of production: number, size, and shape of fruits and yield. These data are added to the passport data previously recorded during the collection or procurement of materials (Jaramillo and Baena 2000). Variability as a major element in characterization When characterizing a species, the variability existing in the genome of the population of individuals forming it is estimated. Thus, the genome of a given species of animals or plants contains all the information coded in the form of genes and their variants, which are needed both to establish the morphological identity of the members of that species and to develop all the processes and functions vital for their survival. For higher plants, any given species is estimated to have more than 400,000 genes with particular functions. As a result of evolutionary and environmental effects, many of these genes also have variants, which are accumulated among the different members composing the species. The sum of all effects of the genes and their variants is designated as the genetic variability of that species (Hidalgo 2003). All variability produced during evolution and/or domestication is stored in the genome, that is, among the members of the populations forming the species. It may, or may not, find expression in characteristics that identify those members. Accordingly, with respect to its expression, the variability contained in the genome of a species can be separated into that which (1) finds expression as visible characteristics that form the phenotype, and (2) does not find expression as visible characteristics but generally deals with the plant’s internal processes or products (Hidalgo 2003). 175 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources The first category, which refers to characterizing visually detectable variability, includes a plant’s: • • • Morphology and structure, used primarily for its botanical and taxonomic classification; Characteristics that affect its agronomic management and production and are therefore of interest to breeders and agronomists; and Reaction to environmental stimuli, whether biotic such as pests and diseases or abiotic such as droughts, mineral deficiencies, and temperature changes. This type of characterization is called evaluation and, for its correct quantification, usually requires experimental designs that are separate from morpho-agronomic characterization trials (Hidalgo 2003). The second category, which characterizes variability that is not detectable by simple visual observation, is called molecular because it refers to the identification of cellular products and/or internal functions (Hidalgo 2003). Evaluation Evaluation consists of recording those characteristics that depend on environmental differences (e.g., disease resistance or susceptibility to drought). Hence, an accession may be evaluated in many sites, with perhaps significantly differing results for several descriptors. However, another accession characterized in many different sites may well yield similar results across sites. Once evaluated, an accession is unlikely to be characterized again, except to control its integrity and to check if it still represents the genetic composition of the original entry. Evaluation may also describe the variation existing in a specific collection for attributes of agronomic importance that are strongly influenced by the environment such as yield. It is carried out in different sites, results varying according to environment and to genotype-byenvironment interaction (Jaramillo and Baena 2000). Characterization per se and evaluation are complementary activities that describe the qualitative and quantitative attributes of the accessions of a given species to differentiate them; determine their usefulness, structure, genetic variability, and relationships among them; and identify the genes that promote their use in crop production or improvement. The two activities require precision, care, and constancy, and include a significant datarecording component. These two activities have in common the use of descriptors, which are characters that are considered to be important and/or useful for describing a sample population of species. A descriptor may assume different values—it can be expressed as a numerical value, scale, code, or descriptive quality (Jaramillo and Baena 2000). Objectives of characterization In characterizing a collection, regardless of size, the following objectives can be established (Hidalgo 2003): • 176 Identify the accessions of a germplasm collection so that they can be clearly distinguished or individualized. Module 4, Lesson 1: General Concepts of Germplasm Characterization • • • • • Measure the genetic variability of the group under study; for which one, several, or all possible categories of variability can be included, that is, phenotypic, evaluative, and molecular, using previously defined descriptors. Establish the collection’s representativeness and its relationship with the species’ variability in a region or with its entire range of variability. Study the genetic structure, that is, the way the collection under study is composed in relation to variants or their combinations, forming groups or identifiable populations. The foregoing is influenced by in situ demographic factors such as population size, reproduction biology, and migration. Identify the percentages of duplication of accessions that can exist within a single collection or compared with other collections of the species. Identify special genes or particular alleles that may be of individual character or found in unique combinations, and may find expression in visible characters (morphological or of evaluation) in different stages or combinations of stages. These genes are usually called ‘genetic stocks’ and are used for research of immediate practical application, as in the case of resistance to biotic factors. Stages of characterization Characterization, together with its methodologies, is a comprehensive tool that can be used for both germplasm acquisition and the adequate management of the different stages of ex situ conservation. It includes: • • • • Comprehensive knowledge of one or more species. Presentation of questions that help improve understanding of the conserved germplasm. Use of improved and suitable methodology for characterization. Data analysis, using the best statistical techniques available, or where the data obtained are descriptive only, their presentation according to good logic. Descriptors For the characterization and evaluation of accessions, descriptors are used. These generally correspond to characteristics or attributes whose expression is easy to measure, record, or evaluate and which refer to an accession’s form, structure, or behaviour. Descriptors help differentiate accessions by expressing their attributes precisely and uniformly, thereby simplifying the accessions’ classification, storage, and recovery, and the use of their data. In other words, descriptors are the characteristics through which germplasm can be known and its potential usefulness determined. They should be specific to each species, differentiating genotypes and expressing each attribute precisely and uniformly. Many attributes can be used to describe a material but the really useful ones are those that can be detected by the naked eye; be easily recorded; have high heritability, high taxonomic and agronomic value; are readily applicable to small samples; and can differentiate one accession from another (Hidalgo 2003). Types of descriptors To identify germplasm entries (accessions), lists of descriptors have been established for use in accordance with the management stage in which information must be collected. These include (Hidalgo 2003; IPGRI 2004): 177 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources • • • • Passport data, which provide the basic information for the accession’s general management, including registration in the germplasm bank and other information for identification. They also describe the parameters that must be observed when making the original collection. Management descriptors, which provide the bases for managing the accessions in the germplasm bank and help during multiplication and regeneration. Descriptors of site and environment, which describe the specific parameters of the site and environment. They also help in the interpretation of results when characterization and evaluation trials are carried out. Descriptors for characterization and evaluation. This lesson will emphasize descriptors for characterization and evaluation, although all are important when analyzing a germplasm collection in an integrated manner. Descriptors for characterization Characterization descriptors permit relatively easy discrimination between phenotypes. They are usually highly inheritable characters that are easily detected by the naked eye and find expression in all environments. Descriptors related to phenotypic characters mostly correspond to the morphological description of the plant and its architecture. These characters are called morphological descriptors and can be grouped into two types: botanical-taxonomic and morpho-agronomic. Botanical-taxonomic descriptors correspond to morphological characters such as the shape of the root, stem, leaf, flowers, fruit, and seeds (Figure 1) that describe and identify the species and are common to all individuals. Most of these characters have high heritability and present little variability. Morpho-agronomic descriptors include those morphological characters that are relevant in the use of cultivated species. They can be qualitative or quantitative, and may include some botanical-taxonomic characters and others that do not necessarily identify the species, but are important in terms of agronomic needs, genetic improvement, marketing, and consumption. Examples of these characters include leaf shape; pigmentation of roots, stems, leaves, and flowers; colour, shape, and brilliance in seeds; size, shape, and colour of fruits; and plant architecture as expressed in growth habit and branching types. Some germplasm bank curators include descriptors related to yield components to indicate the potential of the conserved germplasm for this character. Most of these descriptors have acceptable local heritability but are affected by environmental changes. These latter are also called evaluation descriptors. Evaluation descriptors Characters for this type of descriptors include yield, agronomic productivity, and susceptibility to stress. They also include biochemical and cytological characters, which are usually of greater interest for crop improvement. Not all plant characteristics are expressed with the same intensity. Some, especially the quantitative, can present different degrees of expression, and are recorded in terms of scales of value (usually between 1 and 9), known as descriptor states (IPGRI 1996). Such descriptors are found for resistance or susceptibility to different types of biotic (pests and diseases) and abiotic stress (drought, salinity, acidity, or 178 Module 4, Lesson 1: General Concepts of Germplasm Characterization (A) Mangrove Fibrous roots Ivy Lateral root Tap root (carrot) Stilt roots Adventitious roots Aerial roots Tap root. First or primary root, which gives rise to smaller roots known as lateral or secondary roots. Many vegetables are, in fact, enlarged primary roots. (B) Adventitious roots are born directly from stems, bulbs (specialized stems), or cuttings. Aerial roots are born from stems and do not grow into soil. They may be used for climbing, as in ivy. In many plants, aerial roots also absorb moisture from the air. (D) (C) Oval Round Grooved Acicular Fibrous roots. A fibrous root system is formed by numerous roots of equal size that all produce smaller lateral roots. In contrast to the tap root system, the primary root is not prominent. Stilt roots are specialized aerial roots. They grow from stems and later into soil that may be located under water. They support heavy plants such as mangroves. Filiform segments Ligulate Lingulate Linear Ensiform Falcate Lorate Eliptic Oblong Cruciform Papilionaceous Liliaceous Campanulate Funnel-shaped Rotate Orchidaceous Tubular Winged Urceolate Lanceolate Salver-shaped Saccate Personate with spur Oblanceolate Galea Triangular Labiate Furrowed Deltoid Obdeltoid Trullate Ligulate Galeate (Consolida sp.) Trumpetshaped Obovate Female Male Square with strengthened corners Corky Orbicular Oval Ovate Cordate Gibbous (Nematanthus gregarius) Dissimilar segments (Iris sp.) Sepals petaloid (Anemone sp.) Achlamydeous (Salix sp.) (E) Lomentum (Ornithopus sp.) Legume (Vicia sp.) Reticulate pod of Arachis hypogaea 4-winged pod of Tetragonolobus purpureus Inflated many-seeded pod with papery walls of Colutea arborescens Figure 1. (F) Spirally coiled pod of Medicago sativa 1- or 2-seeded pod with beak of Trigonella caerulea Examples of morphological characters that are used as descriptors to characterize germplasm accessions. (A) Root types; (B) stem forms; (C) leaf shapes; (D) flower types; (E) fruit types; (F) seed types. (From Stockley 1991 [A]; Hickey and King 2000 [B, C, D, and E]; D Debouck 2004, GRU, CIAT [F].) 179 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources low soil fertility). Most of the descriptors of this category depend on the environment for their expression and, accordingly, require special experimental designs for their evaluation. Evaluation may also involve complex methods of molecular or biochemical characterization. Other descriptors Sometimes, characterization data and morpho-agronomic evaluation are insufficient for establishing differences between species or between accessions. In these cases, genome characteristics may be studied such as the karyotype, chromosome number, and ploidy level. The genome itself can be studied directly, using biochemical (isoenzymes; Simpson and Withers 1986) and molecular markers (microsatellites, restriction fragment length polymorphisms or RFLPs, randomly amplified polymorphic DNA or RAPD, and quantitative trait loci or QTLs). These methodologies help locate genes of interest with greater accuracy but do not evaluate the effect of the environment on the expression of those genes (Westman and Kresovich 1997). Accordingly, they do not replace—but complement—characterization and morpho-agronomic evaluation (IPGRI 2004; IPGRI and CIP 2003). Descriptor attributes Depending on whether they involve characterizing or evaluating germplasm, the descriptors used may have various attributes. The principal ones are summarized in Table 1. Practical Recommendations for Characterization The characterization of genetic variability has several limitations that are common to almost all germplasm banks at national and international institutions. These should be taken into account when planning procedures. First is the limited quantity and poor quality of available seeds, which does not permit flexibility in multiplication and characterization tasks. Second is the poor documentation of collections, mainly because a high percentage of germplasm existing in banks was the product of opportunistic collections that were made, using criteria based on agronomic characteristics rather than on genetic resources. Finally, resources are scarce for the sustainable maintenance of germplasm banks. This is reflected Table 1. Attributes of characterization and evaluation descriptors for plant germplasm. 180 Activity Attributes of characters or descriptors Examples Characterization Qualitative Environmentally stable Mendelian heredity Mono- and oligogenic Easily manipulable in genetic improvement Flower and seed colours Proteins Isoenzymes Marker-based PCR Evaluation Quantitative Influenced by the environment Additive heredity Oligo- and polygenic Difficult to handle in genetic improvement Yield Plant height Protein contents Flowering Maturation time Module 4, Lesson 1: General Concepts of Germplasm Characterization by low numbers of accessions and reduced quantities of seeds per accession (Hidalgo 2003). Accordingly, when characterizing a germplasm collection, the following recommendations should be considered: • • • • • • • • • • A complete knowledge of the species’ biology is necessary, especially on reproduction— sexual, asexual, autogamous, and allogamous—as well as on the centres of origin and domestication. Adequate documentation provides useful elements for establishing a preliminary idea of the reference collection. With that idea, the variability to be found in the materials can be inferred, even before initiating characterization. Also, by clearly defining the objectives for characterization, unnecessary steps can be saved. Objectives should be clearly established, taking into account the goals being sought, whether these be ascertaining variability in the group or representativeness of the collection, studying the structure, identifying duplicates, or detecting special genes. Regardless of established objectives, prior experimental planting should be carried out to discover, in general, the overall variability of the collection, the facility in recording descriptors, and the usefulness of descriptors for seed characterization and multiplication. Before attempting the definitive characterization, accessions should be homogenized according to their morphotypes. This is especially important for wild forms and native landraces, which, in their original state, are frequently mixtures of morphotypes in terms of, for example, seed types, growth habits, flower colours, and fruit types. Even if the germplasm bank conserves a complete sample of the original, the characterization of an accession that has a mixture of morphotypes enormously hinders data analysis. If standardization cannot be made when preparing seeds, then, where possible, prior experimental planting should be attempted to achieve this purpose. To obtain better and more information for the statistical analysis and reliability of differences among materials and variables, 3 to 5 plants per accession and a minimum of two replications should be established. When the availability of seeds or planting materials is low, thereby making the establishment of replicated plots of each accession impossible, then the most homogeneous plot possible should be selected to prevent the effects of variable soil conditions. In these cases, the correct acquisition of data will facilitate comparative analysis between accessions and even between variables. If the principal objective is to measure group variability, then descriptors should be selected that are as discriminatory as possible. This will help save time by avoiding repetitive data collection and will simplify analysis. Accordingly, the descriptor lists published by IPGRI for the species under study should be consulted. When designing characterization tasks, a statistician or related professional should be consulted on field design, suitable ways of recording and analysing data, and interpretation of results (IPGRI 2001). The use of currently available automated programs helps in understanding the procedures related to advanced statistical methods for data analysis for characterization, especially multivariate ones. The key is to know how to interpret results at the point where biological knowledge of the species is important in explaining the results of data analysis (IPGRI 2001). 181 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Evaluating this Lesson After this lesson, you should be familiar with the basic concepts of plant germplasm characterization and with the types of descriptors used in this process. Before going on to the next lesson, answer the following questions: 1. If you have had personal experience in germplasm characterization, comment briefly on the type of descriptors you used and why? 2. If you do not have personal experience in germplasm characterization, give your opinion on the process and its importance for ex situ conservation, based on the contents of the lesson and the Recommended Reading list. Bibliography Literature cited Bioversity International. (Accessed 17 Aug 2007) Descriptors lists. Available at http:// www.bioversityinternational.org/Themes/Germplasm_Documentation/Crop_Descriptors/ index.asp Hickey M; King C. 2000. The Cambridge illustrated glossary of botany terms. Cambridge University Press, UK. 208 p. Hidalgo R. 2003. Variabilidad genética y caracterización de especies vegetales. In Franco TL; Hidalgo R, eds. 2003. Análisis estadístico de datos de caracterización morfológica de recursos fitogenéticos. Boletín Técnico No. 8. IPGRI, Cali, Colombia. pp 2–26. Also available at http://www.ipgri.cgiar.org/publications/pdf/894.pdf (accessed 14 Dec 2004). IPGRI. 1996. Descriptors for tomato (Lycopersicon spp.). Rome. IPGRI. 2001. The design and analysis of evaluation trials of genetic resources collections: a guide for genebank managers. Technical Bulletin No. 4. Rome. IPGRI; CIP. 2003. Descriptores del ulluco (Ullucus tuberosus). Rome. Available at http:// www.ipgri.cgiar.org/publications/pubseries.asp?ID_SERIE=13 (accessed 14 Dec 2004). Jaramillo S; Baena M. 2000. Material de apoyo a la capacitación en conservación ex situ de recursos fitogenéticos. IPGRI, Cali, Colombia. 209 p. Also available at http:// www.ipgri.cgiar.org/training/exsitu/web/arr_ppal_modulo.htm (accessed 14 Dec 2004). Ligarreto G. 2003. Caracterización de germoplasma. In Franco TL; Hidalgo R, eds. 2003. Análisis estadístico de datos de caracterización morfológica de recursos fitogenéticos. Boletín Técnico No. 8. IPGRI, Cali, Colombia. pp 77–79. Also available at http:// www.ipgri.cgiar.org/publications/pdf/894.pdf (accessed 14 Dec 2004). Simpson MJA; Withers LA. 1986. Characterization using isozyme electrophoresis: a guide to the literature. IBPGR, Rome. 102 p. Stockley C. 1991. Illustrated dictionary of biology [practical guides]. Usborne Publishing, London. 182 Module 4, Lesson 1: General Concepts of Germplasm Characterization Westman AL; Kresovich S. 1997. Use of molecular techniques for description of plant genetic variation. In Callow JA; Ford-Lloyd BV; Newbury HJ, eds. Biotechnology and plant genetic resources, conservation and use. Biotechnology in Agriculture Series, No. 19. CAB International, New York. Further reading FAO. 1997. The state of the world’s plant genetic resources for food and agriculture. Rome. 510 p. Also available at http://www.fao.org/ag/AGP/AGPS/Pgrfa/pdf/swrfull.pdf or http://www.fao.org/iag/AGP/AGPS/Pgrfa/wrlmap_e.htm Hoagland MB. 1978. The roots of life: a layman’s guide to genes, evolution, and the ways of cells. Houghton-Mifflin, Boston, MA, 167 p. Stalker HT; Chapman C. 1989. Scientific management of germplasm: characterization, evaluation and enhancement. IBPGR, Rome. 194 p. Contributors to this Lesson Benjamín Pineda, César Ocampo, Rigoberto Hidalgo, Alba Marina Torres, Daniel Debouck, Mariano Mejía, Arsenio Ciprián, and Orlando Toro. Next Lesson In the next lesson, you will study ways of characterizing plant germplasm. 183 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Lesson 2 Ways of Characterizing Plant Germplasm Objective To describe types of plant germplasm characterization Introduction Under natural conditions, or during domestication, any population of individuals that is part of a plant species is found in permanent dynamic interaction with its environment. It constantly adapts to the biotic and abiotic factors of that environment by adapting the information contained in its genome to the needs for survival in the environment (Hidalgo 2003). The result of such adaptive interaction is an accumulation of genetic information that is stored in the genome, leading to variability among the members of the population. Such variability may or may not find expression in characters, which should be appropriately identified through an activity known as ‘characterization’. During characterization, the existing variability in the genome of the population of individuals is estimated, and methodologies designed for this purpose are used to encompass all aspects related to biodiversity. However, this lesson will focus mainly on morphological, biochemical, and molecular characterization, which have the most applicability to plant germplasm. What Is Involved in Characterization? During characterization, each entry or accession is systematically described. Descriptors are selected and used according to the category of activity (see Module 4, Lesson 1). The best characterization is that where all the characteristics of a germplasm can be observed and recorded. Hence, different methodologies should encompass the categories of diversity that is characteristic of plant germplasm, including those that are biological (i.e., morphological, physiological, and anatomical), taxonomic, ecological, geographical, biochemical, molecular, genetic, and cytogenetic in nature. Each category of characterization offers a series of opportunities for acquiring information that could be very useful for understanding the germplasm. Nevertheless, the categories most used by germplasm banks are those that deal with morphological, biochemical, and molecular characterization. For each of these categories, different methodologies or techniques have been developed, which can be applied individually or complementarily to characterize germplasm. None of the available techniques is superior to the others, even over a wide range of applications, as each permits the observation and recording of different parts of the total diversity available for characterization. The three categories of characterization should be used in a complementary way to estimate the genetic diversity of collections and, accordingly, help establish criteria for improving their representativeness. None of the three is replaced or excluded by another, as each has a different history and can show different facets of the diversity being examined. When a technique must be selected, the following activities should be carried out: 184 Module 4, Lesson 2: Ways of Characterizing Plant Germplasm • • • • • • • • • • Define the type of information needed in terms of the desired results. Define the level of discrimination, that is, the taxonomic level (e.g., within and/or between populations, between species, or between genera) at which genetic variation will be estimated. Estimate reproducibility, as this parameter will genetically identify a collection’s accessions and estimate its genetic variation. Define the genomic coverage or number of loci that the technique is likely to include. Define the number of alleles required in individual loci—this is needed when hypervariability is required. Know the mode of inheritance. Verify the availability of samples for each technique considered. Thus, germplasm will not be sacrificed to develop a process. Normally, recording morphological descriptors will not consume samples, as the multiplication carried out in the greenhouse and field can be used at the same time to characterize germplasm. Estimate the costs of the respective techniques. Estimate the speed each technique would take. Trained personnel must be available. Morphological characterization Characterization is carried out on a representative population of an accession, using a list of descriptors for the species. The representative population of the species is that which represents the accession’s total genetic variability so that all the characteristics that it possesses can be observed and recorded. With regard to variability, a representative population is expected to contain at least 95% of the accession’s alleles. Population size will be determined by the species’ type of reproduction. For example, if it is allogamous (i.e., highly variable), the population should be larger than if it were autogamous (i.e., variability is low). Descriptors are those characteristics by which germplasm can be known and its potential usefulness determined (see Module 4, Lesson 1). For characterization, lists of very useful descriptors have been prepared by IPGRI for more than 110 plants species (IPGRI 1996, 2004), including crops of African importance such as sweet potato (Ipomoea batata; Huamán 1991), taro (Colocasia esculenta; IPGRI 1999), yam (Dioscorea spp.; IPGRI and IITA 1997), and shea tree (Vitellaria paradoxa; IPGRI and INIA 2006). However, if little studied crops are being characterized and a list of descriptors is unavailable, then the most relevant characters for the case must be identified. Material to be characterized is planted in the field or greenhouse, in duly identified plots and under uniform management conditions. Once the targeted populations are established, the characteristics of the species are observed throughout various developmental stages and their expression recorded in terms of a selected set of descriptors. A case of characterization can be seen in Box 1, for ulluco (Ullucus tuberosus). Data are systematically and consistently taken and recorded in an orderly way to facilitate their later statistical analysis and to ensure that the information, based on the same descriptors but obtained from different regions, is comparable and compatible. 185 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Box 1 Characterizing ulluco (Ullucus tuberosus) 7. Plant descriptors The entries to be characterized should be maintained in the same environment, receive the same agronomic and conservation management, and be planted at the same density and in the period most appropriate for their growth and development. Plant characters should be recorded during full flowering (130–150 days after planting), whereas tuber characters should be recorded immediately after harvest. The characters of both plant and tubers should be recorded for a representative number of the population for each entry. The recording of data on plant colour and especially tubers is complex and difficult because of the variation existing among most of them. Hence, attempts have been made to simplify the variation of each colour and indicate the most representative. These should be recorded, using the Colour Chart of the Royal Society of Horticulture (RHS Colour Chart). The characters indicated below are stable and appropriate for identifying morphotypes and/or duplicates. The numbers and letters in parentheses correspond to the colour or colours listed in the RHS Colour Chart. 7.1. Plant data 7.1.1. Plant growth habits 1 Erect 2 Creeping 7.1.2. Elongated stems Where elongated stems are present during full flowering, three to seven stems per plant stand out from the foliage, with a tendency to be decumbent to creeping, and covering more than 50% of the furrow towards the end of the plant’s vegetative cycle. 0 Absence of elongated stalks 1 Erect elongated stems 2 Decumbent elongated stems 3 Creeping elongated stems 7.1.3. Stem colour 1 Pale yellowish green (145A–D) 2 Pale yellowish green (145A–D) predominant, with pale red (pink) (51C, D) irregularly distributed along the length of the stem 3 Greyish red (178B) predominant, with yellowish green (146C, D) irregularly distributed along the length of the stem 4 Greyish red (178A, B; 182A, B) 7.1.4. Pigmentation of aristas/stem angles 0 Absent 1 Present 7.1.5. Leaf blade shape See Figure 3 1 Ovate 2 Cordate 3 Deltoid 4 Semi-reniform (Continued) 186 Module 4, Lesson 2: Ways of Characterizing Plant Germplasm Box 1. (Continued.) 7.1.6. Foliage colour 1 Pale yellowish green (145A, 146D) 2 Yellowish green (146A) 3 Dark yellowish green (147A) 7.1.7. Colour of lower leaf surface 1 Pale yellowish green (146B–D) 2 Pale yellowish green (146B–D), with reddish purple (59A–D) 3 Reddish purple* (59A-D) 7.1.8. Petiole colour 1 Pale yellowish green (144A–D) 2 Yellowish green (144A, B; 146A–C), with arista/angle pigmented 3 Greyish red (178A–D) predominant, with yellowish green (146B) 4 Greyish purple (183D) predominant, with yellowish green (146B) 7.1.9. Flowering habit 0 Absent 3 Scarce 5 Moderate 7 Abundant 7.1.10. Shape of inflorescence axis (rachis) 1 Predominantly straight 2 Predominantly zigzag 7.1.11. Colour of inflorescence axis (rachis) 1 Pale yellowish green (144A–D) 2 Yellowish green (144A, B; 146B–D), with reddish purple (58A, B) irregularly distributed 3 Reddish purple (58A; 59A, B) predominant, with green 7.1.12. Sepal colour 1 Yellowish green (150D; 154C, D) 2 Pale reddish purple (58C, D) 3 Reddish purple (58A; 59A, B) 7.1.13. Petal colour 1 Yellowish green (151C, D) 2 Yellowish green (151C, D), with reddish purple (59B–D) apex 3 Yellowish green (151C, D) with reddish purple (59A–C) apex and margins 4 Reddish purple (59A, B), with yellow orange (14C; 15C, D) base 7.1.14. Tendency to form flowers with more than five petals 0 Absent 1 Present 7.2. Data on tubers 7.2.1. Predominant colour of tuber surfaces 1 Yellowish green (145B–D, 147D, 148D) 2 Yellowish white (8D) * The dish purple becomes progressively more intense towards the end of the cropping cycle. (Continued) 187 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Box 1. (Continued.) 3 4 5 6 7 8 9 10 11 12 Yellow (10A) Dark yellow (13B) Greyish yellow (162C) Yellow orange (19A) Pale orange (22A; 24B) Orange (26A, B) Reddish orange (33A) Pale red (pink) (51C, D) Red (46D) Reddish purple (61A) 7.2.2. Secondary colour of tuber surfaces 0 Absent 1 Yellowish white (4D) 2 Pale red (pink) (54C) 3 Reddish purple (61A) 7.2.3. Distribution of secondary colour on tuber surfaces 0 Absent 1 Eyes 2 Irregularly distributed 3 Eyes and irregularly distributed 7.2.4. Tendency to produce chimeras 0 Absent 1 Present 7.2.5. General tuber shape See Figure 4 1 Round 2 Cylindrical 3 Semi-falcate 4 Twisted 7.2.6. Colour of cortical area 1 Yellowish green (145B, C) 2 Yellowish white (4D) 3 Yellow (12C; 13A) 4 Orange (26A) 5 Reddish orange (33A–D) 6 Pale red (pink) (50C; 51C, D) 7 Red (46D, 53B) 8 Reddish purple (61A) 7.2.7. Colour of central cylinder 1 Yellowish green (145B, C) 2 White (155A–D) 3 Yellowish white (4D) 4 Yellow (12C; 13A) 5 Yellowish orange (20B) 7.3. Notes Add any further information here SOURCE: IPGRI and CIP (2003). 188 Module 4, Lesson 2: Ways of Characterizing Plant Germplasm Information is sometimes taken from observations that record the presence or absence of a characteristic (e.g., thorns or trichomes) or sometimes taken as quantities, for example, number of fruits, plant height, or number of stamens. Structures should then be counted and/or measured, using tape measures, rules of several sizes, and graduators. Highly precise recording of data will need tools such as: • • • • • • • • • Colour charts, for example, the RHS Colour Chart (1982), the Methuen Handbook of Colour (Kornerup and Wanscher 1984), or the Munsell Plant Tissue Charts (Munsell Color 1975, 1977) Vernier calibrators Microscopes or stereomicroscopes Balances pH meters Durometers (to measure the resistance or hardness of peel and pulp) Stoves (to calculate quantities of water and dry matter) Chemical reagents Laboratory instruments (for enzymatic and molecular characterization and evaluation) Biochemical characterization In this type of characterization biochemical markers are used that are principally isoenzymes (metabolism enzymes) and total proteins (e.g., seed storage proteins). These markers have two very useful characteristics as tools for characterizing plant germplasm: they occur naturally, and their expression is not influenced by epistatic effects (Simpson and Withers 1986). Isoenzymes. These molecular forms of a single enzyme have affinity for a given substrate found in the tissue of an organism and are coded by different loci. These differentiate among themselves according to size (weight), shape, and electrical charge. When they are coded by different alleles from a simple locus they are called alloenzymes. Isoenzymes can be found in the same subcellular compartment, in different compartments of the cell, or in different cells or tissues of an organism; and can be produced in any developmental stage of the plant. The principal characteristics of isoenzymes include simplicity, a minimum quantity of material for study, low cost, a genome coverage of 10 to 20 loci per species, and the absence of epistatic and environmental influences. Allelic expression is codominant, permitting comparisons between species or between populations of a single species, and the detection of hybrids and gene introgression. Generally, applications of isoenzymes in the characterization of plant germplasm conserved ex situ can be summarized as follows: germplasm identification (fingerprinting), detection of redundant germplasm (genetic duplicates), analysis of the genetic structure of plant populations, plant systematics, evolution of domesticated plants, and molecular studies (Simpson and Withers 1986). 189 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Total proteins are components of plants and, as genetic markers, they are characterized by: • • • • • A high level of polymorphism; Limited environmental influence on their electrophoretic patterns; Simple genetic control; A complex molecular base for genetic diversity; and Homologues among protein types of different taxons, which enables them to be identified (taxonomy) or have their relationships established (phylogeny). Seed proteins have been heavily used in the analysis of genetic diversity within and between populations, and in the study of relationships between genomes or species, especially serial polyploidy. Table 1 provides a comparison of attributes of isoenzymes and seed proteins as biochemical markers. Table 1. Attributes of total seed proteins and metabolic enzymes (isoenzymes or alloenzymes) that can be used in studies on characterization, evolution, genetics, and germplasm improvement. Attributes Seed proteins Enzymes (isoenzymes or alloenzymes) Polymorphism High Low Environmental stability High Moderate to high Stability when domesticated Unaffected by selection pressures Unaffected by selection pressures Genome coverage Moderate (<10 loci) Low (<50 loci) Inheritance Biparental and codominant Biparental, maternal, and codominant Molecular base Complex Simple Comparability of studies High Good Sample types for analysis Dry seeds (conservation time is not important) Multiple conserved tissues Practicability Quick, simple, inexpensive, and easy to transfer Quick, simple, inexpensive, and easy to transfer Molecular characterization In the last 2 decades, molecular characterization of plant germplasm has gained great importance for both the quantity and quality of results obtained (Mendoza-Herrera and Simpson 1997; Westman and Kresovich 1997). Previously, these results could not be obtained, as they were based on characterizing the phenotype, especially morphologically, and, to a lesser extent, biochemically. Now, a large variety of molecular methodologies, based on DNA, is now available, making the direct characterization of the genotype possible (Westman and Kresovich 1997). Hence, these modern methodologies provide the means of 190 Module 4, Lesson 2: Ways of Characterizing Plant Germplasm knowing the genetic diversity of a germplasm collection, in terms of its measurement and distribution. Furthermore, conclusions can now be drawn on the phylogenetic relationships among the accessions of a collection and even among taxons. However, the use of this type of germplasm characterization carries with it the same philosophy that is applied to morphological and biochemical characterization: that it be used to complement the other types. Hence, together, they provide the best technique for characterizing the genetic diversity of a germplasm collection, meaning that phenotypic characterization can never be ignored in the study of the targeted biological material. Currently, thanks to advances in molecular biology, markers are now being used. These markers have a DNA molecular nature and are highly sensitive to changes in the genotype of individuals. This situation has permitted major advances in studies on the genetic characterization of plant germplasm. The selection of the marker to use depends on the study objectives, the availability of the germplasm to characterize, cost, and the marker’s inherent characteristics. Research on DNA-based technologies has been favoured with the availability of numerous markers such as those based on restriction fragment length polymorphisms (RFLPs) and the polymerase chain reaction (PCR). From these two techniques multiple techniques have derived, for example, random amplified polymorphic DNA (RAPD); amplified fragment length polymorphism (AFLP); and variable number tandem repeats (VNTR), that is, both minisatellites and microsatellites (or simple sequence repeats or SSR). A monomorphic molecular marker is invariable in all organisms studied, but when a marker presents differences in molecular weight, enzymatic activity, structure, or restriction sites, it is polymorphic. The degree of variation is sometimes such that these markers are called hypervariable. To characterize PGRs, the following markers are used: RFLPs (restriction fragment length polymorphisms). The technique that uses these markers was developed at the end of the 1970s. It is based on detecting, through digestion with the same restriction enzyme, DNA fragments of different molecular weights in different organisms. The use of RFLPs in plants represents a good alternative for conducting various studies related to the three genomes that exist in plants: nuclear (nDNA), mitochondrial (mtDNA), and chloroplast (cpDNA). This technique has proved very useful in studies on plant phylogeny and genetic diversity, and for identifying cultivars for varietal protection. RAPDs (random amplified polymorphic DNA). This technique has proved to be one of the most versatile since its development in 1990. It is very convenient and quick, requiring little DNA that, moreover, does not need to be very pure. It does not presuppose previous knowledge of the sequence, and can quickly and simultaneously distinguish many organisms. However, one drawback is that the amplified fragments tend to correspond to redundant DNA rather than to DNA that is linked to some trait. Nor does the technique give information on the number of copies of the amplified sequence in the genomic DNA. This technology has been used to catalogue fruit, select varieties, and differentiate among clonal lines. It is also used to analyse varieties of celery, grape, lemon, and olive, and to study the genetic diversity of crops and their relationships with wild ancestors. 191 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources AFLPs (amplified fragment length polymorphisms). The technique was developed in 1995. It combines the use of restriction enzymes and oligonucleotides for PCR, so that very specific molecular markers are obtained without needing to know the sequence beforehand. One special advantage of this technique is its capacity to generate many molecular markers in a single reaction. Minisatellites (VNTRs). These are repeated sequences that occur in eukaryotes. They are found repeated in tandem and are scattered throughout the genome, representing many loci. Each locus has a distinct number of variable repeats, thus associating itself with specific alleles of high variability. These sequences are used as probes of SSRs. Minisatellites have been used to study genetic diversity and to identify (‘fingerprint’) individuals in various species, both for accession description and detection of genetically duplicated accessions (i.e., redundant germplasm). Microsatellites or SSRs (simple sequence repeats). This technique was described in 1989. Genomes carry a ubiquitous and abundant DNA known as ‘microsatellites’ that comprises mono-, di-, tri-, and tetra-nucleotides repeated in tandem. This DNA, which is highly polymorphic, has been used as molecular markers when the sequence of the repeated motif is cloned and sequenced for use in population analysis. In this way, numerous trees have been successfully studied, even though some trees showed a narrower variability of microsatellites than expected. Other variations of this technique are inter-simple sequence repeats (ISSR), inter-sequence microsatellite amplified (IMA), inter-sequence amplified (ISA), inter-sequence repeat amplified (IRA), and random amplified microsatellite polymorphisms (RAMP). Microsatellites have also been used to measure the genetic diversity of various annual crops such as soybean (Glycine max), rice (Oryza sativa), maize (Zea mays), barley (Hordeum vulgare), wheat (Triticum aestivum), and rape (Brassica sp.). Although microsatellites have been developed for perennial species, their numbers have been few. For Eucalyptus spp., microsatellites have been transferred between populations and species of the same genus. Additionally, these markers help in estimating heterozygosity, thus presenting a great advantage in mapping quantitative characteristics, comparing maps that include QTLs, and studying genetic flow in trees. Germplasm Evaluation Once the morphological and anatomical characteristics of the germplasm are known through characterization, the information for determining its potential for use is broadened through evaluation. This process describes the agronomic characteristics of accessions in the maximum number of environments possible. These characteristics are usually quantitative variables, which are influenced by the environment and low heritability as, for example, yield or resistance to biotic or abiotic stress. The goal is to identify adaptable materials that have useful genes for food production and/or crop improvement. Most cases of evaluation are carried out by breeders (Jaramillo and Baena 2000). Evaluation complements characterization and is also carried out on a representative population of the species, using descriptors (Box 2). It can be carried out in the field, greenhouse, or laboratory, depending on the characteristic being evaluated, and following the same procedures. Unlike characterization, where plants are planted only once, the 192 Module 4, Lesson 2: Ways of Characterizing Plant Germplasm Box 2 Evaluating ulluco (Ullucus tuberosus) 8. Plant descriptors 8.1. Plant emergence in the field (sprouting) (days) Determine from the day of planting until at least 50% of plants for each entry has emerged or sprouts 1 Early (<40 days) 2 Intermediate (40–60 days) 3 Late (>60 days) 8.2. Days to flowering Count from the day of planting until at least 50% of plants for each entry has flowered 0 No flowering 1 Early (<130 days) 2 Intermediate (130–150 days) 3 Late (>150 days) 8.3. Duration of flowering Record from the appearance of the first flowers in at least 50% of plants for each entry until senescence appears in more than 50% of plants 0 No flowering 1 Short (<30 days) 2 Intermediate (30–60 days) 3 Long (>60 days) 8.4. Days to harvest Record from the day of planting until more than 50% of plants for each entry has become senescent 1 Early (<7 months) 2 Intermediate (7–8 months) 3 Late (>8 months) 8.5. Plant height (cm) Measure during full flowering from the base of the main stem to the apical buds (it should be understood that the main stem or stems are measured, not the elongated ones) 1 Short (<25 cm) 2 Intermediate (25–35 cm) 3 Tall (>35 cm) 8.6. Leaf length (cm) 8.7. Leaf width (cm) 8.8. Length of petiole (cm) Measure from the base of the petiole to the base of the central nervure 1 Short (<3 cm) 2 Intermediate (3–6 cm) 3 Long (>6 cm) (Continued) 193 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Box 2. (Continued.) 8.9. Weight of tubers per plant (kg) 1 Low (<0.7 kg) 2 Intermediate (0.7–2.0 kg) 3 High (>2.0 kg) 8.10. Notes Add any further information here 9. Susceptibility to abiotic stress Record under clearly specified artificial and/or natural conditions; code the observations on a numerical susceptibility scale from 1 to 9, where: 1 Very low or no visible signs of susceptibility 3 Low 5 Intermediate 7 High 9 Very high 9.1. Low temperatures Record under natural conditions during days of frosts 9.2. High temperatures Record under natural conditions during the hot season 9.3. Drought Record daily under natural conditions for at least 4 weeks 9.4. Days of hail Record during days of hail 9.5. High soil moisture Record under flood conditions for more than 4 weeks 9.6. Soil salinity 9.7. High soil acidity 9.8. Alkalinity 9.9. Shade 9.10. Notes Add any further information here 10. Susceptibility to biotic stress For each case, specify the origin of infestation or infection, whether natural or inoculation in the field or laboratory. Record such information under descriptor 10.4. Notes. Code plant susceptibility according to a numerical scale of 1 to 9, where: 1 Very low or no visible signs of susceptibility (0%) 3 Low (1%–25%) 5 Intermediate (26%–50%) 7 High (51%–75%) 9 Very high (76%–100%) (Continued) 194 Module 4, Lesson 2: Ways of Characterizing Plant Germplasm Box 2. (Continued.) 10.1. Pests Causal organism Common names in English, Spanish, and Quechua Symptom in: 10.1.1. Cylydrorhinus sp. Ulluco weevil; gorgojo del ulluco; ulluku kuru Tuber (Tu) 10.1.2. Copitarsia turbata White grub; gusano de tierra; silwi kuru Stem 10.1.3. Agrotis sp. Cutworms 10.1.4. Ludius sp. Wireworm; gusano alambre; k’aspi kuru Tu 10.1.5. Epitrix sp. Flea beetle; pulguilla saltadora; piki k’uti Foliage (Fo) 10.1.6. Frankliniella tuberosi Black thrips; trips negro; yawa; k’ello kuru Fo 10.1.7. Bothynus sp. White grub; gusano blanco; gusano arador; lakato Tu 10.1.8. Scarabaeidae White grubs; gusanos blancos; gusanos aradores; wali kuru Tu Potato rosary nematode; nematodo rosario de la papa Roots 10.1.9. Nacobbus aberrans 10.2. Diseases 10.2.1. Alternaria sp. Leaf spot; mancha anillada Fo 10.2.2. Alternaria alternata Alternaria blotch; mancha anillada Fo 10.2.3. Alternaria solani Early blight; mancha anillada Fo 10.2.4. Cladosporium sp. Black mould; mancha Fo 10.2.5. Ascochyta sp. Ascochyta blight; mancha oval Fo 10.2.6. Pleospora sp. Foliar blight; mancha zonada Fo 10.2.7. Aecidium ulluci Ulluco rust; roya de la papalisa Fo 10.2.8. Botrytis cinerea Grey rot; pudrición gris Fo/Tu 10.2.9. Pythium ultimum Black rot; leak; gotero Fo/Tu 10.2.10. Rhizoctonia solani Rhizoctonia blight; rizoctoniasis Fo/Tu 10.2.11. Phoma exigua Stem rot; gangrena Fo/Tu 10.2.12. Hypochnus sp. Rot; pudrición Tu 10.2.13. Rhizopus oryzae Soft rot; pudrición Tu 10.2.14. Fusarium oxysporum Wilt; pudrición Tu 10.2.15. Dematophora sp. White root rot; lanosa Tu 10.2.16. Thielaviopsis basicola Black tuber rot; manchado del tubérculo Tu 10.2.17. Verticillium dahliae Verticillium wilt; marchitez Fo 10.2.18. Erwinia carotovora subsp. carotovora Bacterial soft rot; pudrición suave Tu 10.3. Viruses Viruses are best coded according to the following scale: 1 Susceptible 2 Resistant 3 Immune (Continued) 195 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Box 2. (Continued.) Causal organism Common names in English, Spanish, and Quechua Symptom in: 10.3.1. Ullucus virus C (UVC) Virus C del ulluco Fo 10.3.2. Potato leafroll virus (PLRV) Virus del enrollamiento de la papa Fo 10.3.3. Arracacha virus A (AVA) Virus A de la arracacha Fo 10.3.4. Papaya mosaic virus (PapMV) Virus del mosaico de la papaya Fo 10.3.5. Ullucus mosaic virus (UMV) Virus del mosaico del ulluco Fo 10.3.6. Tobacco mosaic virus (TMV) Virus del mosaico del tabaco Fo 10.3.7. Potato virus T (PVT) Virus T de la papa Fo 10.3.8. Andean-potato latent virus (APLV) Virus latente de la papa andina Fo 10.4. Notes Add any further information here 11. Biochemical markers 11.1. Isoenzymes Indicate, for each enzyme, the tissue analysed and zymogram type. Each enzyme can be specifically recorded as 11.1.1; 11.1.2, etc. Examples: phosphatase acid (ACPH); esterases α and β (EST A and B); isocitrate dehydrogenase (IDH); malate dehydrogenase (MDH); phosphogluconate dehydrogenase (PGD); phosphoglucose isomerase (PGI); phosphoglucose mutase (PGM); and peroxidases. 11.2. Other biochemical markers (e.g., polyphenol profiles) 12. Molecular markers Describe any specific, useful, or distinctive feature for this accession, and indicate the probe-enzyme combination analysed. Some of the more commonly used basic methods are described below. 12.1. Restriction fragment length polymorphism (RFLP) Indicate the probe-enzyme combination (this criterion can be used for nuclear, chloroplast, or mitochondrial genomes). 12.2. Amplified fragment length polymorphism (AFLP) Indicate the combinations of initiating pairs and the exact molecular size of the products (used for nuclear, mitochondrial, or chloroplast genomes). 12.3. DNA amplification fingerprinting (DAF); random amplified polymorphic DNA (RAPD); arbitrarily primed polymerase chain reaction (AP-PCR) Indicate accurately the experimental conditions (initiators, etc.) and the molecular size of the products (used for nuclear, mitochondrial, or chloroplast genomes). (Continued) 196 Module 4, Lesson 2: Ways of Characterizing Plant Germplasm Box 2. (Continued.) 12.4. Sequence-tagged microsatellite site (STMS) Indicate initiating sequences and the exact size of the products (can be used for nuclear or chloroplast genomes). 12.5. Other molecular markers 13. Cytological characteristics 13.1. Number of mitotic chromosomes 13.2. Ploidy level (2x, 3x, 4x, etc.) 13.3. Chromosome pairing during cellular division Make observations of chromosome pairing during meiosis and mitosis. Descriptions should be based on the mean of observations across several cells. 13.4. Other cytological characteristics 14. Identified genes SOURCE: IPGRI and CIP (2003). germplasm must be planted at the same time in different environments and over several years. Hence, evaluating all accessions is not economically feasible. Instead, a preliminary evaluation must be conducted to observe how accessions adapt to the new environment. Those that perform well against a check are further evaluated in terms of a specific objective. The planning of evaluation tests should take into account the species, evaluation objective, sites, and follow an experimental design with several sites and replications. Germplasm evaluation also requires a uniform management of plots and a systematic collection and recording of the data observed to facilitate statistical analysis and allow conclusions to be made on the material’s usefulness (IPGRI 2001). Statistical Tools for Characterization During characterization, visible characteristics of a species may well be more or less uniform, with not all expressed at the same level of intensity. Some members of the population may present sufficiently different degrees of expression that they translate into different types of data or categories of variables. Different categories of data therefore exist according to the expression of the descriptor, being either qualitative or quantitative. If expression is qualitative, then binary data (i.e., double state data), sequential data (ordinal), and nonsequential data (nominal) can be generated. If expression is quantitative, then the generated data may be discontinuous (or discreet) and continuous. These are usually organized into a basic data matrix (Hidalgo 2003). Germplasm characterization generates a considerable quantity of data that must be analysed. One analytical tool comprises statistics, through which scientific methods compile, organize, summarize, present, and analyse the data to obtain valid conclusions and make decisions. 197 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources From a practical viewpoint and considering the impossibility of studying an entire population or universe, representative samples of the population under study are examined. Because deductions at a given time may not be absolutely certain, the language of probability is used to formulate conclusions. Moreover, statistics may be inferential when referring to conditions under which a deduction is valid, or descriptive or deductive, when statistics are used only to describe and analyse a given group without drawing conclusions or inferences to a greater group (Spiegel and Lindstrom 2000). Data can be analysed by using simple or complex methods, which range from the use of figures and statistics for main tendencies and dispersions to multivariates. Analysis aims to reduce the volume of information typical in studies of this nature. By applying these methods on the basic matrix of data, conclusions can be made on the variability and usefulness of germplasm. Hence, data should faithfully represent the characteristics and behaviour of the accessions studied (Hidalgo 2003). Evaluating this Lesson After this lesson, you should be familiar with the ways used to characterize plant germplasm. You have now finished Module 4: Germplasm characterization. Before going on to the next Module 5, comment briefly on the type of germplasm characterization you have used in your work. If you do not have experience with these processes, consider the importance of characterization for the ex situ conservation of plant germplasm. Bibliography Literature cited Hidalgo R. 2003. Variabilidad genética y caracterización de especies vegetales. In Franco TL; Hidalgo R, eds. 2003. Análisis estadístico de datos de caracterización morfológica de recursos fitogenéticos. Boletín Técnico No. 8. IPGRI, Cali, Colombia. pp 2–26. Available at http://www.ipgri.cgiar.org/publications/pdf/894.pdf (accessed 14 Dec 2004). Huamán Z, ed. 1991. Descriptors for sweet potato. CIP; AVRDC; IBPGR, Rome. IPGRI. 1996. Descriptores para el tomate (Lycopersicon spp.). Rome. 44 p. (Also available in English as Descriptors for tomato (Lycopersicon spp.). Rome.) IPGRI. 1999. Descriptors for taro (Colocasia esculenta). Rome. IPGRI. 2001. The design and analysis of evaluation trials of genetic resources collections: a guide for genebank managers. Technical Bulletin No. 4. Rome. IPGRI. (Accessed 14 Dec 2004) Descriptors lists. Available at http://www.ipgri.cgiar.org/ publications/pubseries.asp?ID_SERIE=13 (now available at http:// www.bioversityinternational.org/Themes/Germplasm_Documentation/Crop_Descriptors/ index.asp). IPGRI; CIP. 2003. Descriptores del ulluco (Ullucus tuberosus). Rome. Available at http:// www.ipgri.cgiar.org/publications/pubseries.asp?ID_SERIE=13 (accessed 14 Dec 2004). 198 Module 4, Lesson 2: Ways of Characterizing Plant Germplasm IPGRI; IITA. 1997. Descriptors for yam (Dioscorea spp.). Rome. IPGRI; INIA. 2006. Descriptors for shea tree (Vitellaria paradoxa). Rome. Kornerup A; Wanscher JH. 1984. Methuen handbook of colour, 3rd ed. Methuen, London. Mendoza-Herrera A; Simpson J. 1997. Uso de marcadores moleculares en la agronomía. In Avance y perspectiva. Available at http://www.hemerodigital.unam.mx/ANUIES/ipn/ avanpers/ene97/vol1608/vol608.html (accessed 14 Dec 2004). Munsell Color. 1975. Munsell soil color chart. Baltimore, MD, USA. Munsell Color. 1977. Munsell color charts for plant tissues, 2nd rev. ed. Macbeth Division of Kollmorgen Corporation, Baltimore, MD, USA. RHS. 1982. RHS colour chart. London. Simpson MJA; Withers LA. 1986. Characterization using isozyme electrophoresis: a guide to the literature. IBPGR, Rome. 102 p. Spiegel MR; Lindstrom DP. 2000. Estadística (Schaum, serie fácil). McGraw-Hill, Mexico. 138 p. (Also available in English as Spiegel MR; Lindstrom DP. 1999. Statistics. Schaum’s Easy Outlines series. McGraw Hill, New York.) Westman AL; Kresovich S. 1997. Use of molecular techniques for description of plant genetic variation. In Callow JA; Ford-Lloyd BV; Newbury HJ, eds. Biotechnology and plant genetic resources, conservation and use. Biotechnology in Agriculture Series, No. 19. CAB International, New York. Further reading CGN. 2000. About CGN molecular markers. Available at http://www.cgn.wageningen-ur.nl/pgr/ research/molgen/right.htm#top (accessed 14 Dec 2004). FAO. 1996. The state of the world’s plant genetic resources for food and agriculture. Rome. 510 p. Also available at http://www.fao.org/ag/AGP/AGPS/Pgrfa/pdf/swrfull.pdf or http://www.fao.org/iag/AGP/AGPS/Pgrfa/wrlmap_e.htm Hickey M; King C. 2000. The Cambridge illustrated glossary of botany terms. Cambridge University Press, UK. 208 p. Stalker HT; Chapman C. 1989. Scientific management of germplasm: characterization, evaluation and enhancement. IBPGR, Rome. 194 p. Stockley C. 1991. Illustrated dictionary of biology [practical guides]. Usborne Publishing, London. Van Hintum TJL; Van Treuren R. 2002. Molecular markers: tools to improve genebank efficiency. Cell Mol Biol Lett 7(2B):737–744. Available at http://www.cmbl.org.pl/072B/ 72B13.PDF (accessed 14 Dec 2004). 199 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Contributors to this Lesson Benjamín Pineda, César Ocampo, Rigoberto Hidalgo, Alba Marina Torres, Daniel Debouck, Mariano Mejía, Arsenio Ciprián, and Orlando Toro. Next Lesson In the next Module 5, you will study aspects of germplasm bank management. 200 Module 5 Supported by the CGIAR Managing Plant Germplasm Banks General Comments ‘Companies do not work on the basis of improvisation. Almost everything is planned in advance. Planning figures as the primary function of administration, precisely because it serves as the basis for other functions. Planning is the administrative function that determines in advance those objectives that should be reached and what must be done to reach them. It therefore concerns a theoretical model for future action. It begins by determining objectives and detailing the plans necessary for reaching them in the best way possible. Planning is to define objectives and choose in advance the best course of action to reach them. Planning defines where the company wants to arrive, what must be done, when, how, and in what sequence’ (Chiavenatto 1997). The management of germplasm banks for ex situ conservation includes a sequential development of stages, that is, collection → multiplication → regeneration → documentation → characterization → evaluation → and, lastly, distribution. After 2 decades of intense concern to create germplasm banks, interest is shifting towards developing strategies to improve the composition and management of collections. The increasing global emphasis on short-term solutions has further increased the need to justify and streamline long-term conservation and, consequently, the need to ensure that decisions are optimal for the long term (Sackville Hamilton et al. 2002). Information on the Module This module deals with the management of plant germplasm banks according to administrative principles. It contains one lesson and a brief evaluation. Objective When you have completed this module, you should be able to identify the most important aspects of managing plant germplasm banks. Next Lesson The next lesson deals with general aspects of managing germplasm banks. Bibliography Throughout this module, a bibliography is provided for each section, that is, the General Comments and the Lesson. The bibliographies follow a format of two parts: 1. Literature cited, which includes those references cited in the text itself. Some of these citations were used to develop the original Spanish-language course on ex situ conservation and may therefore appear in Spanish or Portuguese. However, where practical, references to the English versions of the original Spanish-language documents are provided. 2. Further reading, which is a list of suggested readings in the English language, with few exceptions in Spanish. Most cover in depth the topics included in this module. 201 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources A list of Acronyms used in the bibliographies is also given. The idea is to save space by not having to spell out each institution’s full name each time it appears in the references. Acronyms used in the bibliographies AECI AVRDC FAO IBD IBPGR IICA INIA INIAP IPGRI JABG JIRCAS NRC Agencia Española de Cooperación Internacional Asian Vegetable Research and Development Center Food and Agriculture Organization of the United Nations Inter-American Development Bank International Board for Plant Genetic Resources Inter-American Institute for Cooperation in Agriculture Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria Instituto Nacional Autónomo de Investigaciones Agropecuarias International Plant Genetic Resources Institute Japan Association of Botanical Gardens Japan International Research Center for Agricultural Sciences National Research Council Literature cited Chiavenatto I. 1997. Introducción a la teoría general de la administración, 4th ed. McGrawHill, Bogotá, Colombia. pp 249-263. Sackville Hamilton NR; Engels JMM; van Hintum TJL; Koo B; Smale M. 2002. Accession management: combining or splitting accessions as a tool to improve germplasm management efficiency. Technical Bulletin No. 5. IPGRI, Rome. Further reading Brown ADH. 1988. The case for core collections. In Brown AHD; Frankel OH; Marshall DR; Williams JT, eds. The use of plant genetic resources. Cambridge University Press, UK. pp 136-156. Brown AHD; Frankel OH; Marshall DR; Williams JT, eds. 1988. The use of plant genetic resources. Cambridge University Press, UK. 382 p. Chang TT. 1988. The case for large collections. In Brown AHD; Frankel OH; Marshall DR; Williams JT, eds. The use of plant genetic resources. Cambridge University Press, UK. pp 123-135. Chang TT; Dietz SM; Westwood MN. 1989. Management and use of plant germplasm collections. In Knutson L; Stoner AK, eds. Biotic diversity and germplasm preservation: global imperatives. Kluwer Academic Publishers, Dordrecht, Netherlands. pp 127-159. Ellis RH; Roberts EH. 1991. Seed moisture content, storage, viability and vigour. Seed Sci Res 1:275-279. Ellis RH; Hong TD; Roberts EH. 1985. Seed technology for genebanks. Handbook for Genebanks No. 2, vol. 1. IBPGR, Rome. 210 p. Engelmann F; Takagi H, eds. 2000. Cryopreservation of tropical plant germplasm: current research progress and application. JIRCAS; IPGRI, Rome. 496 p. 202 Module 5: Managing Plant Germplasm Banks General Comments Engle LM. 1992. Introduction to concepts of germplasm conservation. In Chadna ML; Anzad Hossain AMK; Monowar Hossain SM, comps. Germplasm collection, evaluation, documentation, and conservation; Proc. Course offered by AVRDC, Bangladesh Agricultural Research Council, and Bangladesh Agricultural Research Institute, 4–6 May 1992, Bangladesh. AVRDC, Taiwan. pp 11-17. FAO. 1996. Global plan of action for the conservation and sustainable utilization of plant genetic resources for food and agriculture, and the Leipzig Declaration. Available at http://www.fao.org/ag/AGP/AGPS/GpaEN/gpatoc.htm FAO; IPGRI. 1994. Genebank standards. Rome. 15 p. Also available at http://www.ipgri.cgiar. org/publications/pdf/424.pdf (accessed 30 Nov 2004). Frankel OH; Brown AHD; Burdon JJ. 1995. Conservation of plant biodiversity. Cambridge University Press, UK. 299 p. Glowka L; Burhenne-Guilmin F; Synge H; McNeely JA; Gündling L. 1994. A guide to the Convention on Biological Diversity. Environmental Policy and Law Paper No. 30. IUCN, Cambridge, UK. 161 p. Also available at http://www.iucn.org/themes/law/ elp_publications_guide-s.html Heywood VH. 1991. The changing role of the botanic garden. In Bramwell D; Hamann O; Heywood V; Singe H, eds. Botanic gardens and the world conservation strategy. Academic Press, London. pp 3-18. Heywood VH. 1992. Efforts to conserve tropical plants: a global perspective. In Adams RP; Adams JE, eds. Conservation of plant genes, DNA banking and in vitro biotechnology. Academic Press, London. pp 1-14. Hodgkin T; Brown AHD; van Hintum TJL; Vilela-Morales EA, eds. 1995. Core collections of plant genetic resources. John Wiley and Sons, Chichester, UK. 269 p. IPGRI. 1998. Directory of germplasm collections. Rome. Available at http://www.cgiar.org/ipgri/ doc/dbintro.htm (accessed 24 Dec 2004). IPGRI. 1998. Germplasm documentation: databases. Rome. Available at http://www.cgiar.org/ ipgri/doc/dbases.htm IPGRI; CIAT. 1994. Establishment and operation of a pilot in vitro active genebank: report of a CIAT–IBPGR collaborative project using cassava (Manihot esculenta Crantz) as a model. Rome. 59 p. NRC. 1993. Crop diversity: institutional responses in managing global genetic resources; agricultural crop issues and policies. National Academies Press, Washington, DC. 171 p. Painting KA; Perry MC; Denning RA; Ayad WG. 1993. Guidebook for genetic resources documentation. 295 p. Also available at http://www.bioversityinternational.org/ publications/pdf/432.pdf Paroda RS; Arora RK. 1991. Plant genetic resources—Conservation and management: concepts and approaches. Regional Office for South and Southeast Asia, IBPGR, India. 392 p. 203 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Plucknett DL; Williams TJ; Smith NJH; Anishetty NM. 1987. Gene banks and the world’s food. Princeton University Press, NJ, USA. 1987. 264 p. Puzone L; Hazekamp T, comps. 1998. Characterization and documentation of genetic resources utilizing multimedia databases. Proc. Workshop held by IPGRI, 19–20 Dec 1996, Naples, Italy. IPGRI, Rome. 67 p. Shan-An H. 1991. Features and functions of botanical gardens in China. In Proc. First International Conference of Botanic Gardens, held in Tokyo by the JABG, Asia Division, 20–22 May 1991. JABG, Japan. pp 63-75. Sharma BD. 1991. Botanic gardens and their role in present day context of the Indian subcontinent. In Proc. First International Conference of Botanic Gardens, held in Tokyo by the JABG, Asia Division, 20-22 May 1991. JABG, Japan. pp 30-44. Toll J. 1995. IPGRI’s concerns for field genebank management; CGIAR System-wide Genetic Resources Programme consultation exercises. In Field genebank management: problems and potential solutions; Proc. Workshop held in Mayagüez by IPGRI, 12–18 Nov 1995. IPGRI, Rome. 2 p. Toll J; Tao KL; Frison E. 1994. Genebank management. In Frison E; Bolton M, eds. Ex situ germplasm conservation; Proc. Workshop held in Prague, 7–9 Oct 1993. FAO; IPGRI, Rome. pp 10-16. Towil LE; Roos EE. 1989. Techniques for preserving of plant germplasm. In Knutson L; Stoner AK, eds. Biotic diversity and germplasm preservation: global imperatives. Kluwer Academic Publishers, Dordrecht, Netherlands. pp 379-403. Wilkes H. 1989. Germplasm preservation: objectives and needs. In Knutson L; Stoner AK, eds. Biotic diversity and germplasm preservation: global imperatives. Kluwer Academic Publishers, Dordrecht, Netherlands. pp 13-41. Williams T. 1989. Germplasm preservation: a global perspective. In Knutson L; Stoner AK, eds. Biotic diversity and germplasm preservation: global imperatives. Kluwer Academic Publishers, Dordrecht, Netherlands. pp 81-115. Withers LA. 1995. Collecting in vitro for genetic resources conservation. In Guarino L; Rao VR; Reid R, eds. Collecting plant genetic diversity: technical guidelines. CAB International, Wallingford, UK. pp 511-525. Zhiming Z. 1991. Ex situ conservation of wild plants in Beijing Botanical Garden. In Proc. First International Conference of Botanic Gardens, held in Tokyo by the JABG, Asia Division, 20-22 May 1991. JABG, Japan. pp 75-80. Contributors to the Module Benjamín Pineda, Daniel Debouck, Rigoberto Hidalgo, and Mariano Mejía. Next Lesson In the next lesson, you will study general aspects of managing plant germplasm banks. 204 Lesson Module 5 General Aspects of Bank Management Objective To identify the most important aspects of managing plant germplasm banks Introduction Activities for the ex situ conservation of PGRs are usually concentrated within germplasm banks, which handle collections of plant materials to maintain them alive and preserve their characteristics for appropriate use. Conservation is carried out, using collections of live plant materials in the field (botanical gardens and arboreta), seeds, or in vitro plants. However, the mere existence of a bank does not secure the conservation of PGRs of interest to a country, region, province, or given ecosystem. To achieve the goals of conservation, germplasm banks must be planned, structured, established, and well managed according to their objectives and to the requirements typical of ex situ conservation. These requirements may include significant biological, physical, human, and institutional aspects (see Module 1, Lesson 3). The management of germplasm banks, as for any business enterprise, is administrative and, as such, must take into account the principles that deal with the four basic functions of administration: planning, organization, direction, and control. This lesson briefly refers to each of these principles and other aspects of germplasm bank management. Administrative Principles Management is a widespread activity and essential to every collective human effort. It consists of orienting, directing, and controlling the efforts of a group of individuals to achieve a common objective (Chiavenatto 1997). For PGRs, clearly, management should lead to the conservation of targeted species. Planning During planning, objectives are defined and the best course of action for reaching them selected in advance. That is, planning determines the objectives and details the plans needed to reach them in the best possible way. It therefore determines and describes the goals to be achieved, and what must be done, when, how, and in what sequence (Chiavenatto 1997). Establishing objectives. Objectives are the future results that are hoped to be attained. They are the chosen targets that must be reached within a certain period, applying given available or possible resources. In reality, objectives exist for the entity (e.g., germplasm bank) as a whole, for each of its divisions or departments separately, and for each of its specialists (e.g., different sections established within a germplasm bank and specialists such as geneticists, biologists, and phytopathologists). 205 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources The organization’s objectives can be visualized as a hierarchy that ranges from global or organizational at the top down to operative or operational, involving simple instructions for daily routines. Thus, planning comprises as much of long-term strategies and policies for reaching the organization’s global objectives as of sets of plans that detail daily activities for achieving immediate objectives within each division or organ of the organization. Based on its organizational objectives, the entity (germplasm bank) can set its policies, directives, goals, programmes, procedures, methods, and standards. Policies refer to the establishment of the organization’s objectives or intentions to orient administrative action. Directives are principles established to permit the attainment of the intended objectives. As objectives are ends, directives serve to establish adequate means for reaching them and for channelling decisions. Goals are short-term targets. They are often confused with immediate objectives or with departmental or section objectives. Programmes comprise the necessary activities for meeting each goal. Attainment of goals is planned through programmes, which are specific plans. They are highly variable and may include an integrated set of minor plans. Procedures or routines are the modes by which the programmes must be carried out or organized. Procedures are plans that prescribe the chronological sequence of specific tasks needed to carry out determined jobs. Methods are plans prescribed for the performance of a specific task. Usually, the method is attributed to each person who occupies a position or carries out a task, and details exactly how that task is done. Method is more limited in scope than procedure. Procedures and methods generally use flow charts to represent the flow of tasks or operations (Figure 1). Norms are rules that delimit and safeguard procedures. They are direct and objective orders for the course of action to be followed. They are specific guides for action, and usually define what should or should not be done. In the case of a national germplasm bank, for example, its objectives would be: • • • • • • • Long-term conservation of PGRs at the national level Germplasm regeneration Characterization and evaluation of specific germplasm Organization of germplasm exploration and collection at the national level Germplasm introduction National and international exchange of germplasm and information Training, education, and organization of technical meetings and workshops Planning scope. In addition to the hierarchy of objectives, a planning hierarchy also exists, comprising three levels: strategic, tactical, and operational. • 206 Strategic planning is the broadest planning for the organization. It projects for the long term, with effects and consequences foreseen over several years. It covers the entity as a whole, encompassing all its resources and areas of activity. It is concerned with drafting objectives at the organizational level, being defined at the peak of the organization. It corresponds to the greater plan, to which all other plans are subordinated. Module 5, Lesson: General Aspects of Bank Management Germplasm introduction (Seeds and/or planting materials) Prior requisites, procedures, documentation, management Repatriation, Distribution, Duplication for Security Initial increase (Greenhouse, mesh house, tissue culture laboratory) Plant quarantine, inspection, release of germplasm Documentation (Procedures, documentation) Long-term conservation Seeds: preparation for conservation, final drying, packing, cold storage Planting materials: planting in culture medium for conservation, preparations for cryopreservation, planting in field or greenhouse Short-term conservation Multiplication Seeds: preparation for conservation, final drying, packing, cold storage Planting materials: planting in culture medium for conservation, preparations for cryopreservation, planting in field (Procedures, documentation) Regeneration Periodic monitoring (Procedures, documentation) Seeds: field/ greenhouse, mesh house, etc. Planting materials: in vitro, growth rooms in greenhouse, field Deficient health Evaluation of plant health quality Deficient viability (Procedures, documentation) Deficient quantity Evaluation of biological status (viability, vigour, vitality) (Procedures, documentation) Characterization/ Evaluation Descriptors Field/greenhouse, mesh house, laboratory, in vitro (Procedures, documentation) Harvesting/Conditioning and quantification (Procedures, documentation) Seeds: pre-drying, processing (cleaning, selection, drying, counting, etc.) Planting materials: processing, treatments Figure 1. Proposed flow chart for the ex situ management of germplasm, in which different stages of the process are visualized and certain tasks and operations are mentioned. Thick arrows indicate normal flow of operations; thin arrows indicate conditioning. (From Flow Chart for Germplasm Management at GRU, CIAT, redrawn by B Pineda.) 207 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources • • Tactical planning is done at the departmental level. It projects for the medium term, usually the annual fiscal period. It covers each department, encompassing its specific resources, and is concerned with reaching the department’s objectives. It is defined within each department of the entity. Operational planning is that carried out for each task or activity. It projects for the short term, for what is immediate. It covers each task or activity separately and is concerned with meeting specific targets. It is defined for each task or activity. Operational planning usually constitutes goals, programmes, procedures, methods, and norms. Types of plans. Plans are the product of planning and constitute the intermediate event between planning and implementation. They have common purposes: prediction, programming, and coordination of a logical sequence of events, which, if they are successfully applied, must lead to the attainment of the objectives orienting them. They are usually strategic, tactical, or operational in character, and tend to be of several types: • • • • Procedures, related to methods Budgets, related to money Programmes or programming, related to time Norms or regulations, related to conduct Organization Organization, as a function and integrated part of administration, is the act of organizing, structuring, and integrating resources and pertinent organs of its administration; and of establishing relationships among them and the functions of each. It depends on planning, direction, and control to form the administrative process. So that objectives can be attained, plans executed, and people performing efficiently, activities must be suitably grouped, in a logical manner. Authority should be distributed so that it prevents conflicts and confusion. Organization consists of: • • • Determining specific activities needed to attain planned objectives (specialization) Grouping activities into a logical structure (departmentalization) Allotting activities to specific positions and people (occupations and tasks) Direction Direction constitutes the third administrative function, following planning and organization. Once planning is defined and organization established, things must happen. This is the role of direction: to activate the entity, giving it the dynamics to function. Direction is related to action, such as implementation, and has much to do with people. It is directly related to actions taken with the entity’s human resources. People need to apply themselves to their positions and functions, to be trained, to be guided and motivated to obtain the results expected from them. The function of direction is related directly to the way by which the objective or objectives must be reached, by means of the activity of the people comprising the entity. Thus, direction is the administrative function that refers to the interpersonal relationships between administrators at all levels of the entity and their respective subordinates. 208 Module 5, Lesson: General Aspects of Bank Management So that planning and organization are effective, they need to be made dynamic and to be complemented by the orientation given to people, through adequate communication, skilful leadership, and motivation. To direct subordinates, the administrator—at whatever level of the entity—must communicate, lead, and motivate. As no entity exists without people, direction constitutes one of the most complex administrative functions in that it implies orientation, assistance with execution, communication, motivation; in short, with all the processes that administrators use to influence their subordinates so that they behave according to expectations and thus achieve the entity’s objectives. Control As an administrative function, control aims to ensure that the results of that which was planned, organized, and directed shall adjust, as much as possible, to the previously established objectives. The essence of control resides in verifying if the controlled activity is attaining the objectives with the desired results. Control consists, basically, of guiding every activity towards a given end. As a process, control presents phases that require explanation. Other Aspects of Germplasm Banks Germplasm banks are established to meet a research institution, country, or region’s objective to conserve plant materials. A bank carries out different activities that range from acquiring germplasm, discovering its characteristics and potential profit, and ensuring its survival to maintaining it available for users and disseminating information that promotes its use. Banks are usually assigned to an institution or group of people (curators) with the capacity and resources to maintain germplasm under optimal conditions for the required period (IPGRI 1998; Jaramillo and Baena 2000). Types of banks Germplasm banks are classified according to (Jaramillo and Baena 2000; Painting et al. 1993): • • • Sample type, that is, seed, field (including botanical gardens and arboreta), or in vitro Number of species conserved (mono-, oligo-, and poly-specific) Mandate of the institutions to which they are assigned (institutional, national, regional, or international) Banks according to sample type conserve orthodox seeds over the short, medium, and long term under controlled conditions of humidity and temperature (Ellis and Roberts 1991; Withers 1995). Examples of seed banks are abundant, and include those of beans at CIAT (Colombia), maize and wheat at CIMMYT (Mexico), Capsicum, Cucurbita, and Solanum spp. at CATIE (Costa Rica), rice at IITA (Nigeria) and IRRI (Philippines), and sorghum at ICRISAT (India). 209 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Field banks conserve species whose storage in the form of seed is problematic or unlikely. They can include botanical gardens and arboreta, which, traditionally, were established to classify and study interspecies variation (mainly medicinal species) and whose current objective is to conserve species that are rare, in danger of extinction, and/ or useful for restoring ecosystems (Frankel et al. 1995; Heywood 1991; Querol 1988). Examples of field banks include those of cassava at CIAT (Colombia), forages at INTA (Argentina), potatoes and Andean roots and tubers at CIP (Peru), cassava and citruses at CENARGEN (Brazil), and yam at IITA (Nigeria). Examples of botanical gardens include the José Celestino Mutis and the Faculty of Agronomy at the University of Caldas (Colombia), the Arenal and the Lankester (Costa Rica), and the Lancetilla (Honduras). In vitro banks are collections of germplasm maintained under laboratory conditions that reduce or suspend growth in the samples. In vitro banks conserve species that cannot be conserved as seed, but as different sample types such as entire plants, tissues (apices, meristems, and calluses), and DNA fragments (Frankel et al. 1995). Three examples are the cassava banks located at CIAT (Colombia), and at IITA (Nigeria), and potato in CIP (Peru). Banks according to number of species conserved can be mono- and oligo-specific, conserving, respectively, one or a few species on a short- or medium-term basis. Examples of this category are banks for research programmes in national and international centres such as those of the soybean germplasm bank for the oleaginous crops programme at CORPOICA (Colombia), and maize for the tropical acid soils improvement programme at CIMMYT (Colombia). Poly-specific banks are established as national centres of PGRs for given countries, and are used for research and improvement. Conservation is long term, and a broad range of species of current or potential interest is distributed. One example of this type of bank is that of INTA in Argentina, which maintains, among others, collections of Arachis spp., Linum usitatissimum, Triticum spp., Zea mays, Sorghum spp., Gossypium hirsutum, Glycine max, Solanum spp., and Helianthus spp. (Jaramillo and Baena 2000). IITA in Nigeria maintains species of legumes and root and tubers. Banks according to institutional mandate are normally assigned to an institution whose mandate, nature, or geographical scope is reflected in its objectives. Such banks are therefore called institutional, national, regional, or international banks. Institutional banks conserve only germplasm used for research by the institute to which they are assigned, for example, that of the Federal University of Viçosa (Brazil) conserves germplasm only from the Lycopersicon and Solanum genera. Regional banks are established as collaborative entities between several countries to conserve germplasm and support research of a given region. In Latin America, one example is the bank at CATIE (Costa Rica), which holds collections of several genera such as Capsicum, Cucurbita, and Solanum. The banks attached to international agricultural research centres were initially established to support improvement programmes, conserve germplasm of crops under their respective mandates and of other crops. Two examples are the germplasm banks of Phaseolus and Manihot spp., and tropical forages at CIAT (Colombia) and of Zea, Triticum, Hordeum, and Secale spp. at CIMMYT (Mexico). 210 Module 5, Lesson: General Aspects of Bank Management Organizing the germplasm To manage germplasm, banks organize their materials as germplasm collections or groupings of accessions that represent a genetic variation that is targeted for conservation and/or use. Such collections may contain from tens to thousands of samples, maintained under appropriate environments and conditions. Germplasm collections are classified as base, active, core, or working. Base collection. It groups the possible genetic variability of the species of interest, including wild relatives, intermediate forms, cultivars, landraces, and elite germplasm (Vilela-Morales and Valois 1996a, b). It is established to conserve long-term germplasm and recover missing accessions. It is not used to distribute or for exchange (NRC 1993; Plucknett et al. 1992; Towil and Roos 1989; Vilela-Morales and Valois 1996a). It may contain seed samples (orthodox only) or planting materials. If it contains seeds, these are conditioned to a moisture content of 3%-7%, packed in sealed containers, and stored in chambers at temperatures between -10° and -20°C (FAO and IPGRI 1994; Paroda and Arora 1991; Towil and Roos 1989; Vilela-Morales and Valois 1996a). If vegetative materials are conserved, they are either maintained in the field or cryopreserved. For the variability it contains and the function it fulfils, a base collection is strategic for a country. It should be duplicated and under the charge of an institution that can answer for the germplasm’s survival. It is normally the responsibility of a national programme or international agricultural research centre. Examples of base collections include those of Arachis spp. at CENARGEN (Brazil), Phaseolus and Manihot spp. at CIAT (Colombia), Zea and Triticum spp. at CIMMYT (Mexico), and Andean roots and tubers at CIP (Peru), and African legumes at IITA. Active collection. It is a duplicate of the base collection, established on a short- and medium-term basis for management and distribution. It may conserve germplasm as seed, in the field, or in vitro. If it conserves seeds, these are stored at moisture content of 3%-7% and at temperatures between 0°C and 15°C (Engle 1992; NRC 1993). If the active collection is established in vitro, the material is conserved in slow growth. Active collections may be the responsibility of a variety of institutions, both public and private, including international research centres; national, regional, provincial, and municipal programmes; universities; and nongovernmental organizations. Two examples of active collections are those of maize at CIMMYT (Mexico) and cassava at CIAT (Colombia). Core collection. The core collection aims to represent the genetic variability of a large collection by bringing together the broadest genetic variability of a species in the smallest number of samples possible (Brown 1988). It is formed by duplicating a base collection, separating the accessions that will constitute the core collection (70%-80% of variability represented in 10%-15% of the accessions), and taking the rest to a reserve collection. The core collection is established to facilitate management and promote use of the germplasm. It permits the detection of duplicates in the base collection and helps set priorities for characterizing and evaluating the samples. It also offers easy access to the conserved materials (Frankel et al. 1995; Hodgkin et al. 1995; Pérez-Ruíz 1997). 211 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources The core collection conserves seed or planting materials under the same conditions as an active collection. As with the other two types mentioned above, a core collection is the responsibility of international centres, national programmes, or collaborative programmes for specific crops. Examples of core collections are those of potato at INTA (Argentina) and IBTA (Bolivia), cassava and potato at CENARGEN (Brazil), potato and sweet potato at CNPH (Brazil), and cassava at CIAT (Colombia). Information systems permit the creation of a virtual core collection. If a germplasm material is well documented and the documentation system permits specific searches, the virtual core collection is obtained by seeking and marking the accessions that have the characteristics of interest (Jaramillo and Baena 2000). Working collection or breeder’s collection is established to provide germplasm to researchers, institutions, or research and/or improvement programmes. It contains accessions with characteristics of interest for crop improvement, although it is not representative of the species’ genetic variability. It conserves seeds or plants over the short term. Seeds are kept at room temperature but, if the climate is hot and humid, then the rooms have air conditioning and dehumidifiers. Plants are also conserved in the field or greenhouse. Working collections are normally the responsibility of crop improvement programmes (Jaramillo and Baena 2000). Evaluating the Lesson After this lesson, you should be familiar with the general aspects of managing plant germplasm banks. Before going on to the next Module 6, do the following exercise: • • Describe the administrative structure of your bank and, if possible, the various functions, together with the names of the people in charge of them. If you do not work in a bank, indicate the basic functions that a germplasm bank must have, as outlined in this lesson. Bibliography Literature cited Brown ADH. 1988. The case for core collections. In Brown AHD; Frankel OH; Marshall DR; Williams JT, eds. The use of plant genetic resources. Cambridge University Press, UK. pp 136-156. Chiavenatto I. 1997. Introducción a la teoría general de la administración, 4th ed. McGrawHill, Bogotá, Colombia. pp 249-263. Ellis RH; Roberts EH. 1991. Seed moisture content, storage, viability and vigour. Seed Sci Res 1:275-279. 212 Module 5, Lesson: General Aspects of Bank Management Engle LM. 1992. Introduction to concepts of germplasm conservation. In Chadna ML; Anzad Hossain AMK; Monowar Hossain SM, comps. Germplasm collection, evaluation, documentation, and conservation; Proc. Course offered by AVRDC, Bangladesh Agricultural Research Council, and Bangladesh Agricultural Research Institute, 4-6 May 1992, Bangladesh. AVRDC, Taiwan. pp 11-17. FAO; IPGRI. 1994. Genebank standards. Rome. 15 p. Also available at http:// www.ipgri.cgiar.org/publications/pdf/424.pdf Frankel OH; Brown AHD; Burdon JJ. 1995. Conservation of plant biodiversity. Cambridge University Press, UK. 299 p. Heywood VH. 1991. The changing role of the botanic garden. In Bramwell D; Hamann O; Heywood V; Singe H, eds. Botanic gardens and the world conservation strategy. Academic Press, London. pp 3-18. Hodgkin T; Brown AHD; van Hintum TJL; Vilela-Morales EA, eds. 1995. Core collections of plant genetic resources. John Wiley and Sons, Chichester, UK. 269 p. IPGRI. 1998. Directory of germplasm collections. Rome. Available at http://www.cgiar.org/ ipgri/doc/dbintro.htm (accessed 24 Dec 2004). Jaramillo S; Baena M. 2000. Material de apoyo a la capacitación en conservación ex situ de recursos fitogenéticos. IPGRI, Cali, Colombia. 209 p. Available at http:// www.ipgri.cgiar.org/training/exsitu/web/arr_ppal_modulo.htm (accessed 14 Dec 2004). NRC. 1993. Crop diversity: institutional responses in managing global genetic resources; agricultural crop issues and policies. National Academies Press, Washington, DC. 171 p. Painting KA; Perry MC; Denning RA; Ayad WG. 1993. Guía para la documentación de recursos genéticos. IPGRI, Rome. 310 p. Also available at http://www.cgiar.org/ipgri/ doc/download.htm [Also available in English as Guidebook for Genetic Resources Documentation (295 p) and at http://www.bioversityinternational.org/publications/ pdf/432.pdf] Paroda RS; Arora RK. 1991. Plant genetic resources—Conservation and management: concepts and approaches. Regional Office for South and Southeast Asia, IBPGR, India. 392 p. Pérez-Ruíz C. 1997. Conservación in vitro de recursos genéticos. In VI Curso Internacional sobre Conservación y Utilización de Recursos Fitogenéticos para la Agricultura y la Alimentación. Proc. Course held by the Ministry of Agriculture, Fishing, and Food, INIA, AECI, and IDB, 3-28 Nov 1997, San Fernando de Henares. Escuela Central de Capacitación Agraria, San Fernando de Henares, Spain. 4 p. Plucknett DL; Williams TJ; Smith NJH; Anishetty NM. 1992. Los bancos genéticos y la alimentación mundial. Colección Investigación y Desarrollo No. 21. IICA; CIAT, San José, Costa Rica. 257 p. [Also available in English as Gene Banks and the World’s Food. Princeton University Press, NJ, USA (1987; 264 p)] 213 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Querol D. 1988. Recursos genéticos, nuestro tesoro olvidado: Aproximación técnica y socioeconómica. Industrial Gráfica, Lima, Peru. 218 p. Towil LE; Roos EE. 1989. Techniques for preserving of plant germplasm. In Knutson L; Stoner AK, eds. Biotic diversity and germplasm preservation: global imperatives. Kluwer Academic Publishers, Dordrecht, Netherlands. pp 379-403. Vilela-Morales EA; Valois ACC. 1996a. Principios genéticos para recursos genéticos. In Diálogo XLV: Conservación de germoplasma vegetal. Proc. Course held by IICA, 19–30 Sept 1994, Brasília. IICA, Montevideo, Uruguay. pp 35-48. Vilela-Morales EA; Valois ACC. 1996b. Principios para la conservação de uso de recursos genéticos. In Diálogo XLV: Conservación de germoplasma vegetal. Proc. Course held by IICA, 19-30 Sept 1994, Brasília. IICA, Montevideo, Uruguay. pp 13-34. Withers LA. 1995. Collecting in vitro for genetic resources conservation. In Guarino L; Rao VR; Reid R, eds. Collecting plant genetic diversity: technical guidelines. CAB International, Wallingford, UK. pp 511-525. Further reading Brown AHD; Frankel OH; Marshall DR; Williams JT, eds. 1988. The use of plant genetic resources. Cambridge University Press, UK. 382 p. Castillo R; Estrella Tapia J, eds. 1991. Técnicas para el manejo y uso de los recursos genéticos vegetales. INIAP, Quito, Ecuador. 248 p. Chang TT. 1988. The case for large collections. In Brown AHD; Frankel OH; Marshall DR; Williams JT, eds. The use of plant genetic resources. Cambridge University Press, UK. pp 123-135. Chang TT; Dietz SM; Westwood MN. 1989. Management and use of plant germplasm collections. In Knutson L; Stoner AK, eds. Biotic diversity and germplasm preservation: global imperatives. Kluwer Academic Publishers, Dordrecht, Netherlands. pp 127-159. Ellis RH; Hong TD; Roberts EH. 1985. Seed technology for genebanks. Handbook for Genebanks No. 2, vol. 1. IBPGR, Rome. 210 p. Engelmann F; Takagi H, eds. 2000. Cryopreservation of tropical plant germplasm: current research progress and application. JIRCAS; IPGRI, Rome. 496 p. FAO. 1996. Global plan of action for the conservation and sustainable utilization of plant genetic resources for food and agriculture, and the Leipzig Declaration. Available at http://www.fao.org/ag/AGP/AGPS/GpaEN/gpatoc.htm FAO. 1997. The state of the world’s plant genetic resources for food and agriculture. Rome. 510 p. Also available at http://www.fao.org/ag/AGP/AGPS/Pgrfa/pdf/swrfull.pdf or http://www.fao.org/iag/AGP/AGPS/Pgrfa/wrlmap_e.htm 214 Module 5, Lesson: General Aspects of Bank Management Glowka L; Burhenne-Guilmin F; Synge H; McNeely JA; Gündling L. 1994. A guide to the Convention on Biological Diversity. Environmental Policy and Law Paper No. 30. IUCN, Cambridge, UK. 161 p. Also available at http://www.iucn.org/themes/law/ elp_publications_guide-s.html Heywood VH. 1992. Efforts to conserve tropical plants: a global perspective. In Adams RP; Adams JE, eds. Conservation of plant genes, DNA banking and in vitro biotechnology. Academic Press, London. pp 1-14. IPGRI. 1998. Germplasm documentation: databases. Rome. Available at http://www.cgiar.org/ ipgri/doc/dbases.htm IPGRI; CIAT. 1994. Establishment and operation of a pilot in vitro active genebank: report of a CIAT–IBPGR collaborative project using cassava (Manihot esculenta Crantz) as a model. Rome. 59 p. Puzone L; Hazekamp T, comps. 1998. Characterization and documentation of genetic resources utilizing multimedia databases. Proc. Workshop held by IPGRI, 19-20 Dec 1996, Naples, Italy. IPGRI, Rome. 67 p. Shan-An H. 1991. Features and functions of botanical gardens in China. In Proc. First International Conference of Botanic Gardens, held in Tokyo by the JABG, Asia Division, 20-22 May 1991. JABG, Japan. pp 63-75. Sharma BD. 1991. Botanic gardens and their role in present day context of the Indian subcontinent. In Proc. First International Conference of Botanic Gardens, held in Tokyo by the JABG, Asia Division, 20-22 May 1991. JABG, Japan. pp 30-44. Toll J. 1995. IPGRI’s concerns for field genebank management; CGIAR System-wide Genetic Resources Programme consultation exercises. In Field genebank management: problems and potential solutions; Proc. Workshop held in Mayagüez by IPGRI, 12-18 Nov 1995. IPGRI, Rome. 2 p. Toll J; Tao KL; Frison E. 1994. Genebank management. In Frison E; Bolton M, eds. Ex situ germplasm conservation; Proc. Workshop held in Prague, 7-9 Oct 1993. FAO; IPGRI, Rome. pp 10-16. Wilkes H. 1989. Germplasm preservation: objectives and needs. In Knutson L; Stoner AK, eds. Biotic diversity and germplasm preservation: global imperatives. Kluwer Academic Publishers, Dordrecht, Netherlands. pp 13-41. Williams T. 1989. Germplasm preservation: a global perspective. In Knutson L; Stoner AK, eds. Biotic diversity and germplasm preservation: global imperatives. Kluwer Academic Publishers, Dordrecht, Netherlands. pp 81-115. Zhiming Z. 1991. Ex situ conservation of wild plants in Beijing Botanical Garden. In Proc. First International Conference of Botanic Gardens, held in Tokyo by the JABG, Asia Division, 20-22 May 1991. JABG, Japan. pp 75-80. 215 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Contributors to this Lesson Benjamín Pineda, Daniel Debouck, César Ocampo, Rigoberto Hidalgo, and Mariano Mejía. Next Module In the next Module 6, you will study principles of germplasm documentation. 216 Module 6 Supported by the CGIAR Germplasm Documentation General Comments ‘Germplasm conservation, in its various stages, includes a range of activities for which information is required or from which information is derived. This may refer to species, their sites of origin, or activities or stages of conservation. The action of recording, organizing, and analysing conservation data is known as documentation. It is fundamental for understanding germplasm and making decisions on its management. Germplasm increases in value as more is known about it; hence, the importance of its being well documented’ (Jaramillo and Baena 2000). Information on this Module This module contains one lesson, and includes its respective evaluation. Objectives When you have completed this module you should be able to: • • • Understand what documentation of plant genetic resources (PGRs) signifies, and its importance in the routine management and scientific use of a germplasm bank Define the stages of constructing a documentation system Document the most common operational procedures of a germplasm bank This Module’s Lesson The lesson for this module examines some main aspects of documenting germplasm during procedures for ex situ conservation. Bibliography Throughout this module, a bibliography is provided for each section, that is, the General Comments and the Lesson. The bibliographies follow a format of two parts: 1. Literature cited, which includes those references cited in the text itself. Some of these citations were used to develop the original Spanish-language course on ex situ conservation and may therefore appear in Spanish or Portuguese. However, where practical, references to the English versions of the original Spanish-language documents are provided. 2. Further reading, which is a list of suggested readings in the English language. Most cover in depth the topics included in this module. A list of Acronyms used in the bibliographies is also given. The idea is to save space by not having to spell out each institution’s full name each time it appears in the references. Acronyms used in the bibliographies FAO Food and Agriculture Organization of the United Nations IBPGR International Board for Plant Genetic Resources IPGRI International Plant Genetic Resources Institute 217 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Literature cited Jaramillo S; Baena M. 2000. Material de apoyo a la capacitación en conservación ex situ de recursos fitogenéticos. IPGRI, Cali, Colombia. 209 p. Available at http://www.ipgri.cgiar. org/training/exsitu/web/arr_ppal_modulo.htm (accessed 14 Dec 2004). Further reading IPGRI. 1996. Descriptors for tomato (Lycopersicon spp.). Rome. IPGRI. 2004. Descriptors lists. Available at http://www.ipgri.cgiar.org/publications/pubseries. asp?ID_SERIE=13 (accessed 14 Dec 2004). Konopka J. 1988. Workshop on exchange of information. Plant Genet Resour Newsl 61:38. Konopka J; Hanson J. 1985. Information handling systems for genebank management. In Konopka J; Hanson J, eds. Proc. Workshop held at the Nordic Genebank, Alnarp, Sweden, 21-23 Nov 1984. IBPGR, Rome. pp 21-28. Painting KA; Perry MC; Denning RA; Ayad WG. 1993. Guidebook for genetic resources documentation. 295 p. Also available at http://www.bioversityinternational.org/ publications/pdf/432.pdf Stalker HT; Chapman C. 1989. Scientific management of germplasm: characterization, evaluation and enhancement. IBPGR, Rome. 194 p. Contributors to the Module Benjamín Pineda, Tito L Franco, Margarita Baena, Dimary Libreros, Mariano Mejía, Rigoberto Hidalgo, and Daniel Debouck. Next Lesson In the next lesson, you will study the main aspects of germplasm documentation. 218 Lesson Main Aspects of Germplasm Documentation Objective To describe the main aspects of documentation as applied to procedures for ex situ conservation in a plant germplasm bank Introduction At present, activities with PGRs are being coordinated globally through computer networks (e.g., SINGER), making possible the most efficient use of existing PGRs. However, they require accurate data that are easy to recover and use. Such data are generated within the germplasm banks where the PGRs are conserved. In many germplasm banks, data on collections are currently found scattered among many sources such as electronic files, paper files, and field notebooks. Such a dispersion of data represents problems for standardizing data within the same bank and for exchanging information and germplasm between banks and institutions. A documentation system is therefore needed to support the bank as a source of information to help in its planning and operation, and in its interactions with other banks and entities. Usually, a germplasm bank has limited time and few human and financial resources available. Hence, priorities must be set and decisions made as to which activities are more important than others. In any decision made, information will play a very important role, making it proper organization essential. The ex situ conservation of germplasm involves many stages, that is, acquisition, multiplication, regeneration, characterization, evaluation, conservation, and distribution. Each stage includes a wide range of activities (see Modules 2, 3, 4, and 5) that, in their turn, require or generate information. This information should be recorded, organized, and analysed to better understand the germplasm, make decisions on its management, and provide the added value that it deserves. This complex of activities to record, organize, and analyse information is known as documentation. This lesson briefly explores this theme, taking into account its importance as an essential component of conservation activities. Documentation and Its Implications ‘Documentation’, in terms of work with PGRs, is understood to be the process of identifying, acquiring, classifying, storing, managing, and disseminating information on germplasm. Documentation implies the organization of a documentation system that will store and conserve data. Manual or computerized methods can be used, or a combination of these (Painting et al. 1993). Germplasm banks also need a documentation system as a tool for setting priorities, planning activities, and managing resources. It helps counteract the dispersion of information, thereby facilitating better access to collections and, thus, more efficient use of germplasm. Usually, a documentation system is used to store, maintain process, analyse, and exchange data that are typical of conservation activities (Painting et al. 1993). 219 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Germplasm banks differ in their activities and in the way these are organized, because of the different species they conserve as well as the human resources, budget and facilities available. Because documentation systems support all these activities, different banks will also have different systems. Although many documentation systems of germplasm banks show similarities in design and operation, each will differ to the extent that they are developed according to the bank’s needs for documentation and information (Painting et al. 1993). Documentation System Characteristics For a documentation system to be usable according to the bank’s goals, it must possess particular characteristics (Painting et al. 1993), such as those mentioned below: • • • • • Veracity and/or validity of information. For information retrieved from a documentation system to be useful, it should contain accurate, reliable, and up-to-date data. Otherwise, the documentation system would be useless. Ease of data retrieval. The system should facilitate the simple and rapid recovery of information. If certain information is needed regularly and several hours must be invested to locate it each time, then valuable time is being lost in recovery. It should be remembered that the documentation system should work for the user, not vice versa. Easy operation. When the documentation system is user-friendly, fewer errors will be found, making the system more accessible for other people. An easy-to-use system requires minimum training. Flexible operation. The documentation system should not be rigid. It should be able to cope with different information requirements and be adaptable to procedural changes in the germplasm bank. Organized data. Data should be organized into groups that are practical for recording, storing, maintaining, and recovering information, taking into account users’ needs. Stages for the establishment of a documentation system Constructing a documentation system, whether manual and/or computerized, requires planning and detailed analysis before it is designed. Indeed, the process should tend towards a detailed analysis if a flexible and user-friendly system is to be constructed. Six stages are involved: • • • 220 Obtaining information on the bank’s needs. In-depth understanding of the germplasm bank’s needs, establishment, and available resources is needed. This will provide essential information that will help develop documentation objectives. It will also help in decisions on the better use and management of available resources. Defining documentation objectives. These objectives may include the documentation of passport data, inventorying, procedures for seed management, data distribution, characterization tests, and evaluation. They may also include information dissemination. To define areas of documentation, the germplasm bank’s fields of work and their needs and priorities for documentation must be clearly understood. Analysing procedures. This activity explores the bank’s most relevant procedures, and determines each procedure’s needs for resources and the distinct types of data it Module 6, Lesson: Main Aspects of Germplasm Documentation • • • generates and uses. The foregoing will greatly assist decisions on handling data in the best way possible, for example, the desirability of using computerized versus manual forms. Analysis will also indicate how one procedure is related to another. This information will help construct a flow chart that shows the relationships among procedures and information flow within the bank. It will also help in later decisions on the best way of handling data and defining documentation procedures. Identifying significant descriptors. The most important descriptors of accessions in the bank should be identified and organized into groups that facilitate the documentation system’s operation and maintenance. These groups can be thought of as separate books, files, or forms in a manual system, or separate files in a computerized system (e.g., ‘characterization of wild Arachis’ or ‘viability tests’). These groups are practical in terms of recording and using data, and recovering information. Developing data formats and recording forms. A major task is to simplify data recording, either manually or in screen formatting, for each stage of documentation. Developing documentation procedures and implementing the system. This is the final phase of constructing the documentation system. It involves establishing documentation procedures to facilitate the system’s operation, their implementation, and the training of personnel in the system’s use. Documenting Operational Procedures in a Germplasm Bank A germplasm bank follows a sequence of steps or stages in managing germplasm from its entry to the bank to its storage or distribution. These steps or stages are called operational procedures and constitute the essence of data generation and processing. Usually, all the bank’s procedures generate information that should be recorded and stored for later recovery or consultation (Painting et al. 1993). In general, there are two main large operational procedures, one related to the bank’s management per se (acquisition, inventories, regeneration, distribution and conservation), and other related to the germplasm per se (characterization and evaluation); each one of them will have, in turn, nested suboperational procedures. Usually, procedures for banks that handle seeds, vegetative planting materials in the field, or in vitro materials can be diagrammatically outlined, whereby priority for documentation can be observed for each procedure (Figure 1). It should be remembered that the preparation of these and the setting of priorities for documentation are a function of the bank’s type and objectives. In some cases, few activities will be diagrammed (e.g., banks that conserve only one species) and in others, multiple activities (e.g., international germplasm banks). Documenting common procedures The most common procedures in germplasm banks include activities such as registration of samples (data of accessions), collection, cleaning, drying, viability testing, inventorying, distribution, and regeneration. These require, for their documentation, the use of descriptors (Figure 2), which, on being recorded, constitute conservation data. For the purposes of this lesson, only some are mentioned. Others can be consulted in IPGRI’s lists of specific descriptors (IPGRI 2004; IPGRI and CIP 2003) and yet others are mentioned in the topic on germplasm characterization and evaluation (Module 4, Lessons 1 and 2). 221 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Germplasm introduction (Seeds and/or planting materials) H Prior requisites, procedures, documentation, management H Repatriation, Distribution, Duplication for Security Initial increase (Greenhouse, mesh house, tissue culture laboratory) Plant quarantine, inspection, release of germplasm Documentation (Procedures, documentation) H Long-term conservation Seeds: preparation for conservation, final drying, packing, cold storage Planting materials: planting in culture medium for conservation, preparations for cryopreservation, planting in field or greenhouse Short-term conservation Multiplication Seeds: preparation for conservation, final drying, packing, cold storage H Planting materials: planting in culture medium for conservation, preparations for cryopreservation, planting in field (Procedures, documentation) H Regeneration Periodic monitoring (Procedures, documentation) Seeds: field/ greenhouse, mesh house, etc. Planting materials: in vitro, growth rooms in greenhouse, field H Deficient health H H Deficient viability Evaluation of plant health quality Characterization/ Evaluation (Procedures, documentation) Descriptors Field/greenhouse, mesh house, laboratory, in vitro (Procedures, documentation) Deficient quantity H H Evaluation of biological status (viability, vigour, vitality) H Harvesting/Conditioning and quantification (Procedures, documentation) (Procedures, documentation) Seeds: pre-drying, processing (cleaning, selection, drying, counting, etc.) M Figure 1. 222 H M Planting materials: processing, treatments General operational procedures of a germplasm bank and its documentation priorities. H = high priority; M = medium priority; thick arrows indicate normal flow of operations; thin arrows indicate conditioning. (From Flow Chart for Germplasm Management at GRU, CIAT, redrawn by B Pineda.) No. Descriptor 1 Number of accession Accession Collection ! ! 2 Reference for lot 3 Collection type 4 Scientific name ! 5 Cultivar name/pedigree ! 6 Donor’s name ! 7 Donor’s identification number ! 8 Other accession numbers ! 9 Acquisition date ! 10 Date of last regeneration or multiplication Drying Viability Inventory Distribution Regeneration ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 11 Collector’s number ! 12 Collector’s organization ! 13 Trip identifier ! 14 Collection date ! 15 Country of collection ! 16 Province/state ! 17 Location of collection site ! 18 Latitude of collection site ! 19 Altitude of collection site ! 20 Origin (if different to collection site) ! 21 Source of collection ! 22 State of sample ! 23 Sample type ! 24 Common or local name ! 25 Number of sampled plants ! Figure 2. Cleaning Significant descriptors used to record data during the general documentation of plant genetic resources (from Painting et al. [1993] and adapted by B Pineda). (Continued) 223 224 Figure 2. (Continued.) No. Descriptor Accession Collection Cleaning Drying 26 Land use ! 27 Vegetation type ! 28 Genetic erosion ! 29 Soil type ! 30 Crop use ! 31 Cultural practices ! 32 Identification number(s) of photography ! 33 Identification number of herbarium ! 34 Topography ! 35 Slope ! 36 Aspect ! 37 Annual rainfall ! 38 Season of monthly rainfall ! 39 Soil pH ! 40 Soil texture ! 41 Date of cleaning seed ! 42 Reference for cleaning method ! 43 Total seed estimate ! 44 Proportion of empty seed ! 45 Seed treatment ! 46 Operator (cleaning) ! 47 Reference for drying method ! 48 Determination of final moisture content (%) ! 49 Date of determination of final moisture content ! 50 Total dry weight of seeds ! Viability Inventory Distribution Regeneration ! ! (Continued) Figure 2. (Continued.) No. Descriptor 51 Accession Collection Cleaning Drying 1000-seed weight for small seeds ! 100-seed weight (for large seeds) ! Viability Inventory Distribution Regeneration ! ! 52 Reference for viability method ! 53 Date of viability test ! 54 Viability (%) ! 55 Operator (viability) ! 56 Location of seeds in storage ! 57 Total quantity of seeds ! 58 Minimum quantity of seed permitted ! 59 Number of packages/containers ! 60 Supply date ! 61 Quantity of seeds sent ! 62 Reference for receiver’s address ! 63 Plant health certificate number ! 64 Exports permit number ! 65 Receiver’s imports permit number ! 66 Postal registration number ! 225 67 Regeneration site ! 68 Collaborator ! 69 Reference for plot ! 70 Planting date ! 71 Transplanting date ! 72 Planting density ! 73 Germination in field (%) ! 74 Number of plants established ! 75 Days from planting to flowering (no.) ! 76 Reproduction system ! 77 Harvest date ! Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Registering samples. The registration of samples consists of assigning each sample (or accession) a unique identification number and recording data received with the samples, including those known to be descriptors of the accession. The data recorded would include: • • • • • • • Accession number (a unique number assigned to each accession) Other numbers associated with the accession (e.g., code numbers for collectors and donors) Scientific name (genus, species, subtaxa, and authorities) Common name(s) of the cultivated species Cultivar name(s) or pedigree Date of acquisition of sample (incorporation into the germplasm bank) Date of last regeneration Collection data are also known as passport data and refer to the data reported when the sample was first collected. They form an essential part of the information on the conserved germplasm. These collection data or descriptors can be numerous, depending on the degree of detail in which information is needed. FAO and IPGRI have jointly prepared a list of passport descriptors of many crops (Box 1) to provide uniform coding systems for common passport descriptors of various crops (FAO and IPGRI 2001). This list should not be regarded as a basic list of descriptors because, to fully describe the germplasm, other passport descriptors must necessarily be recorded. The passport data most commonly documented on registering samples include (Figure 2): • • • • • • • Collection date Collector’s name, number, and institute Country and province or state of collection Locality, latitude, longitude, and altitude of collection site Origin of sample (e.g., household garden, market, or farm) State of sample (e.g., wild, landrace, or advanced cultivar) Number of sampled plants We point out that these collection descriptors are considered as ‘essential’ for registering samples. However, many more may be used, depending on the level of detail at which information is to be recorded at the germplasm bank. For example, some banks may wish to record ethnobotanical data, and others further information on the collection site and environment (e.g., topography, soils, and vegetation). Seed cleaning. The seeds to be conserved in a germplasm bank should be, as far as possible, clean and free of broken seeds, residues, or infested or infected seeds. To save time, some banks do not document this procedure; others consider that such data have little practical or scientific value. Nevertheless, information could be collected on seed management during harvest and conditioning to permit corrections where necessary. Some descriptors suggested for this procedure are: • • • • • • 226 Accession number Date of procedure Method used Total number of seeds Empty seeds (%) Operator (name of person who carried out the test) Module 6, Lesson: Main Aspects of Germplasm Documentation Box 1 Multi-Crop passport descriptors 1. Institute code (INSTCODE) Code of the institute where the accession is maintained. The codes consist of the 3-letter ISO 3166 country code of the country where the institute is located plus a number. The current set of Institute Codes is available from the FAO website (http://apps3.fao.org/wiews/). 2. Accession number (ACCENUMB) This number serves as a unique identifier for accessions within a genebank collection, and is assigned when a sample is entered into the genebank collection. 3. Collecting number (COLLNUMB) Original number assigned by the collector(s) of the sample, normally composed of the name or initials of the collector(s) followed by a number. This number is essential for identifying duplicates held in different collections. 4. Collecting institute code (COLLCODE) Code of the Institute collecting the sample. If the holding institute has collected the material, the collecting institute code (COLLCODE) should be the same as the holding institute code (INSTCODE). Follows INSTCODE standard. 5. Genus (GENUS) Genus name for taxon. Initial uppercase letter required. 6. Species (SPECIES) Specific epithet portion of the scientific name in lowercase letters. Following abbreviation is allowed: ‘sp.’. 7. Species authority (SPAUTHOR) Provide the authority for the species name. 8. Subtaxa (SUBTAXA) Subtaxa can be used to store any additional taxonomic identifier. Following abbreviations are allowed: ‘subsp.’ (for subspecies); ‘convar.’ (for convariety); ‘var.’ (for variety); ‘f.’ (for form). 9. Subtaxa authority (SUBTAUTHOR) Provide the subtaxa authority at the most detailed taxonomic level. 10. Common crop name (CROPNAME) Name of the crop in colloquial language, preferably English (i.e. ‘malting barley’, ‘cauliflower’, or ‘white cabbage’). 11. Accession name (ACCENAME) Either a registered or other formal designation given to the accession. First letter uppercase. Multiple names separated with semicolon without space. For example: Rheinische Vorgebirgstrauben;Emma;Avlon 12. Acquisition date [YYYYMMDD] (ACQDATE) Date on which the accession entered the collection where YYYY is the year, MM is the month and DD is the day. Missing date (MM or DD) should be indicated with hyphens. Leading zeros are required. 13. Country of origin (ORIGCTY) Code of the country in which the sample was originally collected. Use the 3-letter ISO 3166-1 extended country codes. (Continued) 227 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Box 1. (Continued.) 14. Location of collecting site (COLLSITE) Location information below the country level that describes where the accession was collected. This might include the distance in kilometres and direction from the nearest town, village or map grid reference point (e.g. 7 km south of Curitiba in the state of Parana). 15. Latitude of collecting site1 (LATITUDE) Degree (2 digits), minutes (2 digits), and seconds (2 digits) followed by N (North) or S (South) (e.g. 103020S). Every missing digit (minutes or seconds) should be indicated with a hyphen. Leading zeros are required (e.g. 10----S; 011530N, 4531--S). 16. Longitude of collecting site1 (LONGITUDE) Degree (3 digits), minutes (2 digits), and seconds (2 digits) followed by E (East) or W (West) (e.g. 0762510W). Every missing digit (minutes or seconds) should be indicated with a hyphen. Leading zeros are required (e.g. 076----W). 17. Elevation of collecting site (masl) (ELEVATION) Elevation of collecting site expressed in metres above sea level. Negative values are allowed. 18. Collecting date of sample [YYYYMMDD] (COLLDATE) Collecting date of the sample, where YYYY is the year, MM is the month, and DD is the day. Missing data (MM or DD) should be indicated with hyphens. Leading zeros are required. 19. Breeding institute code (BREDCODE) Institute code of the institute that has bred the material. If the holding institute has bred the material, the breeding institute code (BREDCODE) should be the same as the holding institute code (INSTCODE). Follows INSTCODE standard. 20. Biological status of accession (SAMPSTAT) The coding scheme proposed can be used at 3 different levels of detail: either by using the general codes (in boldface) such as 100, 200, 300, 400 or by using the more specific codes such as 110, 120 etc. 100) Wild 110) Natural 120) Semi-natural/wild 200) Weedy 300) Traditional cultivar/landrace 400) Breeding/research material 410) Breeder’s line 411) Synthetic population 412) Hybrid 413) Founder stock/base population 414) Inbred line (parent of hybrid cultivar) 415) Segregating population 420) Mutant/genetic stock 500) Advanced/improved cultivar 999) Other (Elaborate in REMARKS field) 1. To convert from longitude and latitude in degrees (°), minutes ('), seconds (''), and a hemisphere (North or South and East or West) to decimal degrees, the following formula should be used: d° m' s''=h *(d+m/60+s/3600) where h=1 for the Northern and Eastern hemispheres and –1 for the Southern and Western hemispheres i.e. 30°30'0'' S=–30.5 and 30°15'55' N=30.265. (Continued) 228 Module 6, Lesson: Main Aspects of Germplasm Documentation Box 1. (Continued.) 21. Ancestral data (ANCEST) Information about either pedigree or other description of ancestral information (i.e parent variety in case of mutant or selection). For example a pedigree ‘Hanna/7*Atlas/Turk/8*Atlas’ or a description ‘mutation found in Hanna’, ‘selection from Irene’ or ‘cross involving amongst others Hanna and Irene’. 22. Collecting/acquisition source (COLLSRC) The coding scheme proposed can be used at 2 different levels of detail: either by using the general codes (in boldface) such as 10, 20, 30, 40 or by using the more specific codes such as 11, 12 etc. 10) Wild habitat 11) Forest/woodland 12) Shrubland 13) Grassland 14) Desert/tundra 15) Aquatic habitat 20) Farm or cultivated habitat 21) Field 22) Orchard 23) Backyard, kitchen or home garden (urban, peri-urban or rural) 24) Fallow land 25) Pasture 26) Farm store 27) Threshing floor 28) Park 30) Market or shop 40) Institute, Experiment station, Research organization, Genebank 50) Seed company 60) Weedy, disturbed or ruderal habitat 61) Roadside 62) Field margin 99) Other (Elaborate in REMARKS field) 23. Donor institute code (DONORCODE) Code for the donor institute. Follows INSTCODE standard. 24. Donor accession number (DONORNUMB) Number assigned to an accession by the donor. Follows ACCENUMB standard. 25. Other identification (numbers) associated with the accession (OTHERNUMB) Any other identification (numbers) known to exist in other collections for this accession. Use the following system: INSTCODE:ACCENUMB;INSTCODE:ACCENUMB;… INSTCODE and ACCENUMB follow the standard described above and are separated by a colon. Pairs of INSTCODE and ACCENUMB are separated by a semicolon without space. When the institute is not known, the number should be preceded by a colon. 26. Location of safety duplicates (DUPLSITE) Code of the institute where a safety duplicate of the accession is maintained. Follows INSTCODE standard. (Continued) 229 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Box 1. (Continued.) 27. Type of germplasm storage (STORAGE) If germplasm is maintained under different types of storage, multiple choices are allowed, separated by a semicolon (e.g. 20;30). (Refer to FAO/IPGRI Genebank Standards 1994 for details on storage type.) 10) Seed collection 11) Short term 12) Medium term 13) Long term 20) Field collection 30) In vitro collection (Slow growth) 40) Cryopreserved collection 99) Other (Elaborate in REMARKS field) 28. Remarks (REMARKS) The Remarks field is used to add notes or to elaborate on descriptors with value 99 or 999 (=Other). Prefix remarks with the field name they refer to and a colon (e.g. COLLSRC:riverside). Separate remarks referring to different fields are separated by semicolons without space. SOURCE: FAO and IPRGI (2001). Seed drying. In a germplasm bank, orthodox or intermediate seeds are dried to reduce their moisture content to acceptable levels without affecting their viability. This procedure is applicable only to seed germplasm collections. Usually, on receiving the sample, the initial moisture content is first determined. If this is very high, then the seeds are dried, using a suitable method, to reduce moisture content to the desired level. Once seeds are dried, some banks determine the total weight of the dried seeds and the 100- or 1000-seed weight, depending on their size. The most commonly used descriptors for seed drying are: • • • • • • • • Accession number Initial moisture content Drying method Date of measurement Final moisture content Total dry weight of seeds 1000-seed weight 100-seed weight (for large seeds) Seed viability. Germination under laboratory conditions is defined as the emergence and development of those essential structures that indicate, for the class of seed being analysed, the seed’s ability to become a normal plant under favourable conditions. The results of this test indicate the percentage of live seeds of an accession that can produce plants under appropriate conditions (Module 3, Submodule C, Lessons 1 and 2). In terms of bank management, the viability of seeds must be known, as it indicates when a sample should be regenerated. Otherwise, the accession could be lost if its viability drops to very low levels. Typical descriptors for a seed viability test are: • • 230 Accession number Lot reference (any date, code, or number that uniquely identifies the accession’s regeneration or multiplication cycle) Module 6, Lesson: Main Aspects of Germplasm Documentation • • • • Collection type (e.g., whether base or active collection) Reference for method used (e.g., absorbent tissue or tetrazolium test) Viability (%) Operator (name of person who carried out the test) Storage. Once the seeds have been dried and cleaned, and their percentage of viability recorded, they are stored in cold rooms (or under normal conditions, according to case). The following data or descriptors are recorded: • • • • • • • • Accession number Lot reference Collection type Location in cold room Total quantity of seeds stored per accession 1000-seed weight 100-seed weight (for large seeds) Minimum quantity permitted for seeds (this parameter helps determine when more seeds should be produced or multiplied) Germplasm distribution. Linked to the information mentioned above on storage, information on the distribution of germplasm that the bank carries out should also be recorded. For example, some banks continually distribute germplasm for improvement programmes or for exchange with other banks. In these cases, to maintain efficient control over the bank’s holdings of materials, a record must be kept of the materials being distributed. Typical descriptors that should be considered are: • • • • • • Accession number Lot reference Date of exit of material Quantity of seed sent Data on receiver Plant health certificate number (if applicable) Small banks, which have a very limited distribution of materials, would probably not need a sophisticated documentation system. Recording distribution information in a book or other means would be sufficient. Duplication for security. The maintenance of germplasm duplicates for security is a major conservation activity in banks. For its documentation, the descriptors used are those of FAO’s World Information and Early Warning System on Plant Genetic Resources for Food and Agriculture (WIEWS) (Box 2). They provide codes for locating the institution where a given germplasm is kept, in addition to other pertinent data. Regeneration or multiplication. Regeneration or multiplication is carried out in response to data obtained through the seed monitoring control or during the growth cycle of the vegetative propagated species, which is conducted at given intervals of time to test the viability of each accession in storage and ascertain the quantity of seeds it has. The principal data or descriptors used to record regeneration or multiplication are: • Accession number 231 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Box 2 Descriptors used by FAO’s World Information and Early Warning System on Plant Genetic Resources for Food and Agriculture (WIEWS) 1. Location of safety duplicates (DUPLSITE) Code of the institute where a safety duplicate of the accession is maintained. The codes consist of 3-letter ISO 3166 country code for the country where the institute is located plus number or an acronym as specified in the Institute database that will be made available by FAO. Preliminary codes (i.e. codes not yet incorporated into the FAO Institute database) start with an asterisk followed by a 3-letter ISO 3166 country code and an acronym. Multiple numbers can be added and should be separated with a semicolon. 2. Availability of passport data (i.e. in addition to what has been provided) (PASSAVAIL) 0 Not available 1 Available 3. Availability of characterization data (CHARAVAIL) 0 Not available 1 Available 4. Availability of evaluation data (EVALAVAIL) 0 Not available 1 Available 5. Acquisition type of the accession (ACQTYPE) 1 Collected/bred originally by the institute 2 Collected/bred originally by joint mission/institution 3 Received as a secondary repository 6. Type of storage (STORTYPE) Maintenance type of germplasm. If germplasm is maintained under different types of storage, multiple choices are allowed, separated by a semicolon (e.g. 2;3). (Refer to FAO/IPGRI Genebank Standards 1994 for details on storage type.) 1 Short-term 2 Medium-term 3 Long-term 4 In vitro collection 5 Field genebank collection 6 Cryopreserved 99 Other (elaborate in REMARKS field) SOURCE: Quek et al. (1999). • • • • • • • • • • • 232 Lot reference Collection type Regeneration site Plot reference (of field, furrow, and plot number) Planting date Planting density Germination in the field (%) Established plants (no.) Days from planting to flowering (no.) Harvest date Cultural practices Module 6, Lesson: Main Aspects of Germplasm Documentation Germplasm characterization and evaluation. Germplasm characterization refers to the recording of highly inheritable descriptors (or data) that are readily seen and are expressed in all environments (Module 4, Lesson 2). They mostly include: • • • • • • • Accession number Plant descriptors (morphological characterization) Susceptibility to abiotic stress (evaluation) Susceptibility to biotic stress (evaluation) Biochemical markers (molecular characterization) Molecular markers (molecular characterization) Cytological characteristics Data analysis The data generated in different processes may be analysed according to requirements (Franco and Hidalgo 2003). Depending on the level of requested analyses and reports required by the germplasm bank, statistical analysis tools can be used (see Module 4, Lesson 2). Evaluating the Lesson After this lesson, which is the last of this course, you should be familiar with the main aspects of plant germplasm documentation, particularly those procedures that are common to germplasm banks dedicated to ex situ conservation. Before finishing the course, do one of the following exercises: 1. If you have personal experience in germplasm documentation, select one of the operational procedures used in your bank and briefly illustrate it with the descriptors used by the bank. 2. If you do not have personal experience with germplasm documentation, give your opinion on the process and its importance for ex situ conservation. Base your answer on the contents of the lesson and list of recommended reading. Bibliography Literature cited FAO; IPGRI. 2001. FAO/IPGRI multi-crop passport descriptors. December 2001. Rome. Available at http://www.bioversityinternational.org/publications/Pdf/124.pdf FAO; IPGRI. 1994. Normas para bancos de genes. Rome. 15 p. Also available at http:// www.fucema.org.ar/fucema/legislacion/otros/normasgenesfao.htm (accessed 30 Nov 2004). (Also available in English as Genebanks Standards at http://www.ipgri.cgiar.org/ publications/pdf/424.pdf) Franco TL; Hidalgo R, eds. 2003. Análisis estadístico de datos de caracterización morfológica de recursos fitogenéticos. Boletín Técnico No. 8. IPGRI, Cali, Colombia. 89 p. Also available at http://www.ipgri.cgiar.org/publications/pdf/894.pdf (accessed 14 Dec 2004). 233 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources IPGRI. 1996. Descriptores para el tomate (Lycopersicon spp). Rome. 44 p. (Also available in English as Descriptors for tomato (Lycopersicon spp.) and at http:// www.bioversityinternational.org/publications/pdf/286.pdf) IPGRI. 2004. Descriptors lists. Available at http://apoweb/Attachments/dip-manual.PDF (accessed 25 October 2007). IPGRI; CIP. 2003. Descriptores del Ulluco (Ullucus tuberosus). Rome. Also available at http://www.ipgri.cgiar.org/publications/pubseries.asp?ID_SERIE=13 (accessed 14 Dec 2004). Jaramillo S; Baena M. 2000. Lista de descriptores de pasaporte de cultivos múltiples (anexo 9). In Jaramillo S; Baena M. Material de apoyo a la capacitación en conservación ex situ de recursos fitogenéticos. IPGRI, Cali, Colombia. 209 p. Available at http://www.ipgri.cgiar. org/training/exsitu/web/arr_ppal_modulo.htm (accessed 14 Dec 2004). Quek P; Zhang Z; Mathur PN. 1999. Data interchange protocol manual: Appendix 1, p 11. Available at www.ipgri.cgiar.org/regions/apo/apoweb/Attachments/dip-manual.PDF (accessed 25 October 2007). Painting KA; Perry MC; Denning RA; Ayad WG. 1993. Guía para la documentación de recursos genéticos. IPGRI, Rome. Also available at http://www.ipgri.cgiar.org/publications/pdf/ 507.pdf (accessed 29 Dec 2004). [Also available in English as Guidebook for Genetic Resources Documentation (295 p) and at http://www.bioversityinternational.org/ publications/pdf/432.pdf] Further reading FAO; IPGRI. 1994. Genebank standards. Rome. 15 p. Also available at http://www.ipgri. cgiar.org/publications/pdf/424.pdf Konopka J. 1988. Workshop on exchange of information. Plant Genet Resour Newsl 61:38. Konopka J; Hanson J. 1985. Information handling systems for genebank management. In Konopka J; Hanson J, eds. Proc. Workshop held at the Nordic Genebank, Alnarp, Sweden, 21-23 Nov 1984. IBPGR, Rome. pp 21-28. Stalker HT; Chapman C. 1989. Scientific management of germplasm: characterization, evaluation and enhancement. IBPGR, Rome. 194 p. Contributors to this Lesson Benjamín Pineda, Tito L Franco, Margarita Baena, Dimary Libreros, Mariano Mejía, Rigoberto Hidalgo, and Daniel Debouck. Next Activity Review the lessons and prepare for the final evaluation as required by the course teachers. Thank you for your dedication and interest in the matter of conserving germplasm for the benefit of humanity 234 Glossar y Supported by the CGIAR Glossary As was discussed in several lessons, the conservation of a given species involves not only the conservation of its physical seed (sexual or vegetative) but also of its genetic resources. The field of genetic resources is relatively new, and includes several disciplines, each with its own, frequently unfamiliar, jargon. Because no specialized glossaries are readily available on the subject of plant genetic resources, we compiled this glossary for the convenience of the course students. We added other terms related to genetic resources but not used in the course because they may appear in the reading of the bibliographies provided. A Abiotic (adj.) related to physical and chemical factors of the environment such as water, temperature, and soil. Accession or entry (a) A sample of a plant, line, or population maintained in a germplasm bank or breeding programme for conservation and use. (b) A sample of germplasm that represents the genetic variation of a population. Acclimatization (a) The adaptation of an individual to a different climate. (b) In a broader sense, the adjustment of a species or population to a new environment, that is, a new habitat, after several generations. Active collection a group of germplasm samples or accessions stored for the short to medium term and maintained for the purposes of study, distribution, or use. (See also Base collection; Core collection; Working collection.) Adventitious (adj.) (a) accidental, for example, said of a plant that is not native to the targeted site, but is introduced accidentally by humans. (b) In plants, said of structures that develop in unusual places, for example, roots growing on stems, leaves, or old roots. AFLP or amplified fragment length polymorphism the polymorphism of a length of an amplified fragment of DNA. Afrotropic a biogeographical region, which spans several habitat types, but has strong biogeographic affinities, particularly at taxonomic levels higher than the species level. The region includes Africa south of the Sahara Desert, part of the Arabian Peninsula, and includes neighbouring islands such as Madagascar in the western Indian Ocean. (See also Neotropics.) Agamic reproduction see Vegetative propagation. 235 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Agriculture (adj. agricultural) the science or practice of cultivating the soil to raise crops and/or rearing animals. Agrobacterium a genus of soil bacteria that introduces genes into certain plants through their plasmids. Albumen see Endosperm. Allele (a) Each of two forms of a gene present in the same place (locus) in a pair of homologous chromosomes. (b) One of the alternative states of the same gene. Allelomorph (a) The characteristic specified by an allele. (b) Formerly, also used for allele. Alloenzymes isoenzymes whose variants are coded by different alleles at the same locus. Alloenzymes are also allelic and, as a result, are ideal for studying populations. Allogamy cross-fertilization in plants. Allogamous plants are those plants that preferentially cross-pollinate. Alloploidy (adj. alloploid) a condition whereby an organism has more than two sets of chromosomes in their cells, with each set coming from a different species. Altitudinal transect an imaginary line that is traced from a point in a mountain range to sea level. It is used to study altitudinal variations among plant communities. Amino acids organic molecules that contain amino and carboxyl groups. They are protein monomers. An enormous variety of proteins exist because of the large number of combinations and lengths. Amplified fragment length polymorphism see AFLP. Aneuploid an individual that has a chromosome number that is not an exact multiple of the haploid chromosome complement. (See also Ploidy level.) Anfiploid or anfidiploid an individual that originates from hybridization between species and possesses the total chromosome complement of the parental species. It is usually produced by the duplication of the chromosome number of the F1 plant hybrid. Angiosperms or Angiospermae a taxon of plants, whose principal characteristic is that they present true flowers. Angiosperms are divided into monocotyledons and dicotyledons. 236 Glossary Annual a plant that takes one year or less to complete its life cycle and produce seeds. (See also Biennial; Perennial.) Anther in flowers, that part of the stamen that contains pollen. Anthesis in flowers, the dehiscence of anthers, when pollen is dispersed. Anthropic (adj.) having an origin or resulting from human activity. Antibiotic: literally, life destroyer. This term includes all antimicrobial substances, regardless of origin, whether derived from micro-organisms (e.g., bacteria and fungi), synthetic chemical products, or genetic engineering. Antibody a defence substance (protein) synthesized by the immune system of an animal organism in response to the presence of a foreign protein (antigen), which it then neutralizes. Plants are a significant source of substances with which to manufacture drugs having antibody characteristics. Apetalous flower a flower with no petals. Apomixis a phenomenon whereby asexual reproduction occurs instead of normal reproduction through reduction division and fertilization. It is common in some plant species where embryos do not result from meiosis and fertilization, but from certain asexual processes. Seedlings produced in such a way are called apomictic. Those plants that reproduce only through apomictic embryos are known as obligate apomitics, and those whose embryos are either sexual or apomictic are designated as facultative apomictics. (See also Vegetative propagation.) Arboretum (pl. arboreta) a garden where trees and shrubs are cultivated for study and display. Asexual reproduction see Vegetative propagation. At risk see Endangered. Autochthonous see Endemism. Autogamy in plants, self-fertilization. Autogamous plants are preferentially self-pollinated. 237 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Autopolyploid or autoploid an organism whose somatic cells carry more than two sets of chromosomes, with all sets coming from parents of the same species. Autotrophs see Primary producers. B Bank (a) The place where a collection or collections are held. (b) The socio-economic-political entity responsible for the safe-keeping of a collection or series of collections. (See also DNA library; Germplasm bank.) Base collection the widest and most complete collection of germplasm accessions. It is stored over long periods, for conservation purposes. It is used only to fill gaps in the active collection. (See also Core collection; Working collection.) Basic seed produced from seed developed by the plant breeder and so managed that the original genetic identity and purity of a given variety is faithfully conserved. The production of basic seed is carefully supervised or used by the representatives of an agricultural experiment station. Basic seed is the starting point for obtaining certified seed, either directly or through registered seed. Biennial a plant living for two years and fruiting only in the second. (See also Annual; Perennial.) Biochemical markers various isoenzymes that catalyze the substrate itself or other enzymes and which are used to evaluate the enzymatic heterogeneity of plants, that is, the genetic variability between individuals at the level of enzymes and proteins. They indirectly evaluate the genome, based on their enzymatic products, and are susceptible to the environment. Biodiversity or biological diversity the set of all plant and animal species in a given region, including their genetic materials and the ecosystems of which they are part. Biological containers containers designed as protection mechanisms in the use of organisms in genetic engineering applications. Their purpose is to minimize the ‘ability’ of organisms used to survive, persist, and self-replicate. The process is also known as ‘genetic weakening’ and leads to ‘engineeringly’ diminished organisms. Biological diversity see Biodiversity. 238 Glossary Biological heritage that group of living things belonging to a given geographical area, and which are or could be of economic, biological, or social value for the human communities that live in that area. Commonly spoken of as the ‘national patrimony of biological resources’, it implies those living things that belong and would have potential value for a given country. Biology the science that deals with the study of living things and vital phenomena in all their aspects. Biomass and ecosystem productivity (a) The biomass of an ecosystem is the mass of all organisms that constitute the biocoenosis (i.e., community of organisms inhabiting a particular biotope). (b) It can also be defined as the chemical energy stored in such a mass. It may be expressed in grams per fresh weight, grams per dry weight, grams of carbon, or calories per unit volume or surface. (See also Productivity.) Biome see Ecological region. Biometry the science that deals with the application of statistical methods to biological problems. Biomolecules formerly called immediate principles, these are the basic architectural elements of living things. Inorganic biomolecules are, above all, water, mineral salts, and gases such as oxygen and carbon dioxide. The four groups of organic compounds, exclusive to living things, are carbohydrates, lipids, proteins, and nucleic acids. Biotechnology that set of technological applications that use biological systems and live organisms or their derivatives to create or modify products or processes for particular uses. Biotic (adj.) (a) Related to live organisms and organic components of the biosphere. (b) In agriculture, a biotic factor or agent is frequently associated with three important groups that affect crop yield: pests (including nematodes), diseases, and weeds. Biotype a population in which all individuals have an identical genotype. Bofedal a Spanish term referring to a type of wet meadowlands made swampy from underground water seepage and found in Andean high plains. The botanical communities are composed mainly of plants from the Cyperaceae and Juncaceae families, often of compact or cushiony growth. (See also Bog.) 239 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Bog a plant community that develops an organic substrate, with hydric saturation, in environments of high precipitation and low temperatures. Two principal types of bogs are recognized: those that constitute mainly mosses, and those that are dominated by Cyperaceae and Juncaceae. (See also Bofedal.) Breeder’s collection see Working collection. Breeding seed seed (or vegetative planting material) produced by the plant breeder or sponsoring institution from the original form and used to produce basic seed. Bryophytes a taxonomic category of plants that corresponds to mosses. The principal characteristic of these ancient plants is the absence of the corm or true stem. They do not have roots and reproduce by spores. Bulb a specialized underground organ that consists of an axial stem that is short, fleshy, usually vertical. It carries in its apex a meristem or floral primordium that is covered by thick and fleshy scales. C Callus the initial tissue formed by the cellular division of explants. It is usually uniform, not having been differentiated into organized tissues. Carbohydrates organic biomolecules formed by polyalcohols with an aldehyde or ketone group. They owe their name to their empirical formula, which is Cx(H2O)y, although some compounds may differ slightly from this general formula. They include glucosides (yielding sugars), glycides, glycols, and sugars. They carry out the energy, plastic, or structural functions of cellular structures, and store information that determine cellular identity. Categories of species conservation international experience suggests that we cannot set a population number or a minimum surface of habitat to delimit each category for determining the degree to which a taxon is in danger of extinction. Consensus and the criteria of specialists in flora and fauna should be used to set the conservation status of each taxon. (See also Endangered; Extinct in the wild; Indeterminate threat; Insufficiently known; Out of danger; Rare; Vulnerable.) Cell the structural and functional unit of plants and animals that typically consists of a mass of cytoplasm that encloses a nucleus (except in prokaryotes) and is bound by a membrane that is differentially permeable. It is the simplest unit of life that reproduces by division. Normally, each cell contains genetic material in the form of DNA incorporated into a cellular nucleus, which splits as the cell divides. Higher organisms contain large quantities of interdependent cells. Even so, these can be treated as independently as free cells in appropriate culture media. 240 Glossary Certified seed the progeny of basic seed, which is produced and used in such a way that it maintains a satisfactory level of purity and genetic identity. It has been approved and certified by an official agency for certification. Cespitose plants grass species or perennial graminoids that form mats or tufts, or grow very closely together to cover the ground as lawns. Character or characteristic see Trait. Characterization the measurement or evaluation of the presence, absence, or degree of specificity of traits whose expression is little modified by the environment. Chimaera or mosaic (a) An interspecific hybrid. (b) An organism whose tissues are of two or more genetically distinct classes. (c) An individual that presents two or more genetically different cell lines as a consequence of an anomaly in the first mitosis of the zygote. Chromatid one of two filamentous structures that form in the duplication of a chromosome to form other chromosomes. Chromosome an elongated intracellular organelle found in the nucleus and consisting of DNA associated with proteins. It constitutes a linear series of functional units known as genes, which conserve their individuality from one cell generation to the next. The chromosome number is typically constant in any given species. Chromosome number the usual constant number of chromosomes in a somatic cell that is characteristic of a particular species. CITES Convention on International Trade in Endangered Species of Wild Fauna and Flora. Cleistogamy pollination and fertilization within a closed flower. Clonal multiplication see Vegetative propagation. Clone (a) A group of plants that originate from the vegetative propagation of a single plant. (b) A population of cells or organisms of identical genotype. (c) A group of cells or organisms with identical genetic constitution and background and which are derived through binary division or asexual reproduction. (d) A population of recombinant DNA molecules with the same sequence. 241 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Cloned gene a gene copied from an initial gene. It is inserted into a molecular vector through in vitro recombination techniques. Co-evolution (a) The joint evolution of two or more organisms that are interrelated either positively or negatively. (b) Any situation in which two organisms act as selective agents on each other, for example, the Mexican acacia and the ants that inhabit it or Opuntia acanthocarpa (a cactus) and ants. Coironal a Spanish term referring to a particular type of grassland plant formation dominated by cespitose plants and typical of Andean highlands. Collection the action of gathering together, assembling, or grouping similar things into one place, usually with a particular focus. A plant germplasm collection therefore brings together the germplasm, whether as seeds, propagules, or other genetic material, of particular plants. The focus may be conservational, agricultural, research and educational, environmental, historic, aesthetic, or economical. (See also Active collection; Base collection; Core collection; DNA library; Germplasm bank; Working collection.) “Comisión Nacional de Bioseguridad” National Biosafety Commission, which is a central and autonomous advisory organ of Spanish managers on all issues related to GMOs. Commercialization of GMOs, GMO marketing, or ‘placing on the market’ of GMOs (a) The act of making available to third parties, of free or onerous character, those products that are totally or partially composed of genetically modified organisms. (b) Any act that implies a delivery of genetically modified organisms, or of products containing them, to third parties. Community type (a) A plant or animal association. (b) A given characteristic plant or animal community that is distinguishable by the habitual presence of a dominant species or group of species. It usually finds expression in the existence of a plant community with a given floral composition. Complete flower a flower that has all four essential organs (sepals, petals, stamens, and pistils). (See also Incomplete flower.) Conifers ancient plants of the Gymnosperm order and whose principal characteristics are that they do not present true flowers but wooden structures, and their leaves are usually needle-like or aciculate. Consanguinity the pairing of organisms that are closely related; in plants, this is usually achieved through self-pollination. 242 Glossary Conservation the conservation of plant genetic resources refers to the maintenance of populations in their natural habitat (in situ conservation) or to samples of these populations in germplasm banks (ex situ conservation). Conservation presumes that the materials are useful or potentially useful and seeks to maintain and manage them for both current and future benefits. Continuous variation see Quantitative trait. Core collection a collection that groups, into a minimum number of accessions, the greatest variability existing in a base collection. (See also Active collection; Working collection.) Corm (a) The swollen base of a stem shoot, wrapped in dry leaves that look like scales. (b) A solid stem structure, with nodes and well-defined internodes. Corolla petals, considered collectively. Correlation the mutual relationship between two things in such a way that an increase or reduction in one is usually associated with an increase or reduction in the other. Linear correlation is determined by the coefficient of correlation, the value of which may vary from -1 to +1. Cotyledons one or a pair of primary leaves of the embryo within the seed and, commonly, the first to emerge in germination. Cretaceous a geological period that is famous for a mass extinction of species, known as the ‘K-T extinction event’, after which modern-day species of both plants and animals evolved. Cross (a) An organism produced by mating parents of different genotypes. (b) To hybridize. Cross-fertilization the fusion of an ovule with a sperm cell from two individuals that have different genotypes. (See also Allogamy.) Cross-pollination the transfer of pollen from the anther of one plant to the stigma of a flower of another genotypically distinct plant. (See also Self-pollination.) Crossing-over the exchange of segments between the chromatids of two homologous chromosomes during meiosis. Crown in horticulture, that part of a plant’s stem that is located in the soil or below its surface, from which new shoots originate. 243 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Cryopreservation the conservation of materials at very low temperatures (-196°C), usually in containers with liquid nitrogen. Cultivar see Variety. Cultivated species or domesticated species a species whose evolution has been influenced by humans seeking to meet their own needs. Curator-in-charge that personality, natural or legal, who conserves and administers plant genetic resources. Cytogenetic map the configuration of coloured bands of chromosomes observed under the optic microscope after staining. Cytology the science that deals with the structure, function, and life cycle of cells. Cytoplasm (adj. cytoplasmic) the protoplasm of a cell, except the nucleus. Cytoplasmic inheritance heredity that depends on hereditary units of the cytoplasm. D Darwinism the theory of evolution by means of natural selection put forward by Charles Darwin in his book Origin of Species published in 1859 and by Alfred Russel Wallace. The theory was based on their observations of the genetic variability that exists within any given species. Dehiscence the rupture or opening of a fruiting structure or anther. Deoxyribonucleic acid see DNA. Descriptors the quantitative or qualitative characteristics, whether morphological, agronomic, or ecogeographic, that permit the identification of a plant at different taxonomic levels. Desert originally, a geographical expression that encompasses climatic, botanical, and edaphic concepts. The vegetation growing in such areas is sparse and highly adapted, including cacti, succulents, and spiny shrubs. 244 Glossary Detasselling the elimination of immature tassels or ears. This practice is followed in the seed production of hybrid maize. Determinate (adj.) said of an inflorescence in which the terminal flowers open first, thus impeding the extension of the floral axis. An example is the corymb. (See also Indeterminate.) Dichogamy in plants, the maturing of male and female organs at different times, thereby ensuring cross-fertilization. (See also Protandry; Protogyny.) Dicotyledons (a) Those plants or plant species that has two cotyledons or the first pair of leaves forming in the embryo within the seed. (b) Any plant whose outstanding characteristic is the presence of two embryonic leaves at germination. (See also Monocotyledons.) Dihybrid the result of a cross between parents that differ in two specified genes. Dioecious (adj.) said of a plant species that has male and female flowers on different individuals. (See also Monoecious.) Diploid (a) An organism that has two sets (genomes) of chromosomes, that is, a chromosome number of 2n, as in a zygote. The somatic tissue is normally diploid, in contrast to the gametes, which are haploid. (b) (adj.) Having two sets of chromosomes. (See also Ploidy level.) Discontinuous variation see Qualitative trait. Disease the alteration or deviation from the normal physiological state in one or more parts of the plant for generally known causes, and which manifests according to characteristic symptoms and signs, and whose development is more or less foreseeable. (See also Pathogen.) Dissemination of GMOs the release to the environment of genetically modified organisms. DNA or deoxyribonucleic acid nucleic acid formed by nucleotides in which the sugar is deoxyribose and the nitrogenous bases are adenine, thymine, cytosine, and guanine. Except in retroviruses, which have RNA, DNA codifies the information for cell reproduction and operation and for the replication of the DNA molecule itself. It represents the security copy or deposit of primary genetic information that, in eukaryotic cells, is confined to the nucleus. DNA bank see DNA library. 245 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources DNA cloning or gene cloning a technique in genetic engineering that consists of isolating and multiplying a given gene or fragment of recombinant DNA by incorporating it into a host cell (usually a bacterium or yeast) and then isolating copies of DNA thus obtained. (See also Genetic engineering; Molecular cloning.) DNA fingerprint (a) The pattern of DNA fragments obtained in restriction analysis of certain highly variable repeated DNA sequences within the genome. Their number and arrangement are virtually unique to each individual and can be used to identify that individual. (b) The graphic representation of that pattern. DNA library, DNA bank, gene bank, or genomic library (a) A bank whose holdings comprise genes or fragments of genes. (b) A collection of recombinant DNA molecules that carry insertions that represent an organism’s entire genome. (c) A collection of DNA fragments amplified in cloning vectors. The cloned fragments may come from genomic (chromosomal) DNA or from complementary DNA (cDNA). DNA sequence the order of sequence of the nitrogenous bases of the nucleotides that constitute DNA and which codes for all genetic information. When it is a codifier (exon), it defines the order of the amino acids that form the corresponding protein. Documentation with reference to plant genetic resources, the procedure by which information (data) on germplasm is identified, acquired, classified, stored, handled, and disseminated. Domesticated species see Cultivated species. Dominant (adj.) (a) Said of a trait that manifests in the phenotype of a hybrid to the exclusion of the counterpart (recessive) trait. (b) Said of a plant that, by extension of its foliage or root system, modifies and controls the local environment. (c) Constituting the hegemony and biological maximum of one or more species in a community type or of a biological form in a community or plant formation. Dominance is manifested in the biological form’s relative contribution to the community’s biomass, or as a combination of characters that enables that form to manifest greater participation in a community’s physiognomy. Dominant gene (a) A gene that needs only one dose to be expressed, thereby masking the presence of its recessive allele. Most dominant alleles represent the evolved and completely functional state of the gene. (b) A gene that manifests itself exclusively in a hybrid, that is, to the exclusion of its counterpart (recessive) allele. (See also Recessive gene.) Donor parent see Recurrent parent. 246 Glossary Dormancy (a) The state of metabolic rest during which the seed is incapable of germinating because of its structural characteristics (embryo or seed coats) or the effect of external conditions (e.g., light, temperature, aeration, and moisture). (b) The quality of being latent. (c) The state in which a seed, bud, or reproductive structure of a plant is found at rest, inactive, quiescent, or dormant but which can initiate activity when the necessary conditions for activation occur. Duplicate a germplasm sample that was mistakenly introduced into a collection as a different accession but which is genetically identical to others already in the collection. Duplicate genes two or more pairs of genes that produce identical effects, whether together or separately. E Ear see Spike. Ecogeographic study the collection and synthesis of information that is ecological, geographical, and taxonomic in nature, the results of which can be used to establish priorities and strategies for germplasm collection and conservation. Ecological region, biome, or ecozone a large geographical region with distinctive plant and animal groups. These groups form a whole that has a characteristic composition resulting from the groups’ adaptation to the region’s climate and geography. Examples include tropical rainforest, grassland, desert, and tundra. Ecological system a system comprising living things and the physical environment where they live. The system is characterized by interdependent relationships based on a recursive interaction that extends for over 5000 million years on our planet. Ecology the science that studies living things at their different levels of organization and their interrelationships among themselves and with the environment. Ecosystem a dynamic complex of communities of plants, animals, and micro-organisms and their non-living environment; which complex acts as a functional unit. Ecosystemic harvest with reference to natural forest products, the harvest that enormously surpasses the ecosystem’s natural productivity. In this case, not only is the annual productivity harvested, but also the biomass and soil developed over centuries or millennia. Ecosystemic harvest implies the reduction of the natural resource base and ecosystem productivity. (See also Harvest.) 247 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Ecozone see Ecological region. Egg in plants, the female gamete. (See also Sperm cell.) Emasculation the elimination of anthers from a flower, either closed or open, before pollen is released. Embryo the rudimentary plant within a seed. The embryo originates from the zygote. Embryo sac see Megasporangium. Endangered or at risk a category of conservation status, describing taxa that are in danger of extinction and whose survival is unlikely if the causal factors of danger continue operating. It refers to those species (i) of which only a few specimens exist in nature and whose existence is seriously threatened if the causal factors of danger are not removed; (ii) whose populations have been reduced to a critical level; (iii) whose habitat has been so drastically reduced as to be considered in immediate danger of disappearance, thereby leading to their extinction; or (iv) that are possibly already extinct but have been seen in the wild within the last 50 years. (See also Categories of species conservation.) Endemism the condition of being endemic, autochthonous, or indigenous. Said of a plant or associated animal that originates in a given country or region and is restricted to that region, that is, aboriginal or native to a given geographical area. Endosperm or albumen a triploid tissue that comes from the triple fusion of a spermatic nucleus with the two polar nuclei in the megagametophyte. In seeds of certain species, the endosperm persists as storage tissue for food reserves, which are used during the development of both the embryo and seedling during germination. Entry see Accession. Environment the sum total of external influences acting on the life, development, and survival of an organism or group of organisms. Enzyme a biological catalyst, normally a protein, that mediates and promotes a chemical process without itself being altered or destroyed. Enzymes are extremely efficient catalysts and specifically linked to particular reactions. Epiphytotic the unexpected development and usually general distribution of a destructive disease of plants. 248 Glossary Ethnobotany the study of folklore and history of use, with particular reference to plants. Evaluation the measurement, observation, and analysis of a germplasm collection with a view to detecting its potential use. It generally uses descriptors of quantitative traits that are affected by the environment. Evolution the history of changes that are at first molecular, then cellular, and finally organic as a result of mutations in DNA; their reproduction; and selection processes, and which are heritable. (See also Co-evolution; Darwinism.) Ex situ conservation ex situ literally means out of the original place; hence, the conservation of plant genetic resources outside the areas where they had developed naturally (i.e., outside their natural habitats). Exons DNA sequences, specific to genes, and which codify for amino-acid sequences in proteins. Explant a segment of tissue or an organ obtained from a plant (e.g., leaf, root, anther, shoot, bud, embryo, and meristem) and used to initiate an in vitro culture. Extinct in the wild a category of conservation status, where a species is considered extinct in its natural distribution when it has not been located or sighted in the wild state for the last 50 years (a criterion used by CITES). (See also Categories of species conservation.) F Farmers’ rights those rights attributed to farmers for their contribution (past, current, or future) to the conservation, improvement, and availability of plant genetic resources. Fats see Lipids. Fertilization the fusion of an ovule and sperm cell (male gamete), forming a zygote. Filament in flowers, the column of the stamen that sustains the anther. Flora (a) A group of plants with characteristics in common. (b) A set of plant species that is found in a given place. It is usually described in terms of a systematic or alphabetical list of all the plant taxa recorded in that place. Floret a small flower of an inflorescence, as in the case of a grass panicle or compound spike. 249 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Food chain a line that can be established in an ecosystem among organisms that feed, one from another. An example of a food chain is plant → butterfly → house wren → barn owl. The food chains are interconnected through common links, creating a food or trophic network. Food security the capacity and facility of access by all people, over time, to a sufficient quantity of food that permits them to live active and healthy lives. Forest a plant community dominated by tall trees or woody plants with few or no branches at the base. Frost heave alternating freezing and thawing that causes the soil and the plants it carries to lift. The plants become separated from the soil or their roots are destroyed. G Gametes or sexual cells cells that, when fused, form the zygote. In plants, the gametes are the male sperm cell and the female egg. Gene (a) The physical and functional unit of hereditary material that determines a trait or characteristic of an individual and is transmitted from generation to generation. Its material base is constituted by a part of a chromosome known as locus and which codifies information through DNA sequences. By interacting with other genes, cytoplasm, and the environment, it affects or controls the development of a trait. (b) The receptacle of genetic material that is particular to a given species. Gene bank see DNA library; Germplasm bank. Gene cloning see DNA cloning. Gene expression the protein product resulting from the set of mechanisms that decode the information contained within a gene, processing it through transcription and translation. Gene flow the exchange of genetic material between populations through the dispersion of gametes and zygotes. Gene interaction the modification of gene action through non-allelic genes. 250 Glossary Genetic code a code written according to the distribution of nucleotides in the polynucleotide chain of a chromosome. It governs the expression of genetic information in proteins, that is, the succession of amino acids in the polypeptide chain. Information on all genetically determined characteristics of living things is stored in DNA and deciphered through four nitrogen bases. Each succession, adjacent to three bases (or codon), governs the insertion of a particular amino acid, of which there are four: adenine, guanine, thymine, cystosine. In RNA, thymine is replaced by uracil. This information is transmitted from one generation to the next through the production of exact replicates of the code. Genetic drift a random fluctuation of genetic frequencies of a population from generation to generation, caused by factors such as natural selection. It is more evident in small isolated populations, and may lead to the fixation of an allele and to the extinction of the other. Genetic engineering, genetic manipulation, or recombinant DNA technology (a) The process of forming new combinations of hereditary material by inserting nucleic acid molecules, obtained from outside the cell, into any virus, bacterial plasmid, or other vector system outside the cell. Thus, the host organism incorporates the new hereditary material in a way that does not appear natural, but where such molecules are able to reproduce continuously. (b) That set of techniques used to introduce a foreign heterologous gene into an organism to modify its genetic material and products of expression. (See also DNA cloning; Molecular cloning.) Genetic erosion the loss of genetic diversity, that is, of genetic materials, including individual genes or combinations of genes (genetic complexes), genotypes, and species. Genetic identity the characteristic that should be maintained during conservation. This refers to the maintenance, as a set, of all the alleles of all the accession’s genes. Genetic instability susceptibility of stored seeds to cumulative genetic changes (with age), resulting in the alteration of the initial genetic structure of the conserved sample. Genetic integration the insertion of a DNA sequence into another through recombination. Genetic manipulation see Genetic engineering. Genetic map a descriptive diagram of the genes in each chromosome. Genetic material all material, whether of plant, animal, microbial, or other origin, that contains functional units of heredity. 251 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Genetic recombination (a) A combination of alleles from different parents that produce a recombinant individual. Such an organism or progeny may result from a crossing event or from an independent reorganization of different chromosomes during meiosis. (b) In genetics, the term refers to new combinations of sequences that result from the physical interaction of two DNA molecules. In vitro, the term refers to genetic re-arrangement among DNA fragments from different or noncontiguous origins. In vivo, this occurs between homologous copies of a single gene (chromosomal manipulation) or as a result of integrating a genetic element (transposon, prophage, or transgene) into the genome. Genetic resources (a) That set of population samples, whether from plants, animals, or micro organisms, that is acquired to provide useful genetic traits with present or potential value. (b) The good or medium potential found in genes. (c) The genetic variability stored in chromosomes and other structures containing DNA. (See also Plant genetic resources.) Genetic stability the maintenance of a certain degree of genetic balance in each individual of a population. Genetic uniformity the condition in which individuals of a population present identical or very similar genetic structures, so that one may deduce that they will behave similarly and will have the same susceptibility in terms of biotic and abiotic stresses. This condition potentially endangers the persistence of such a population, a situation that is known as genetic vulnerability. Both situations are more likely to occur when the population has been genetically improved, and whose tendency is to give rise to genetically uniform populations, whether homozygous or heterozygous. Genetic variability the degree of genetic variation existing in a population or species, as a consequence of the evolutionary processes to which it has been subjected. It is that set of differences present among individuals of a single species. Genetic variability is the basis on which plant breeders develop new varieties. Genetic variation the heritable variation, derived from changes in genes, usually because of environmental factors. Genetic vulnerability the condition where the risk of exposure is high for plants that are susceptible to certain pathogens, pests, and environmental stress as a result of genetic uniformity, induced by breeding. (See also Genetic uniformity.) Genetically modified organism or GMO any organism whose genetic material has been modified in a way that would not happen naturally in mating (or multiplication) or in natural recombination. GMOs are classified as high or low risk, according to the nature of the receiving or parental organism, and the characteristics of both vector and insert used in the operation. 252 Glossary Genetics the science that deals with reproduction, inheritance, variation, and the set of phenomena and problems related to descendancy, that is, the science that deals with heredity. Genome (a) The set of all genes of an organism, of all the genetic patrimony stored in the set of its DNA or chromosomes. (b) Also a set of chromosomes, as appears within a gamete, corresponding to the haploid number of chromosomes of a given species. Genomic library see DNA library. Genotype (a) The genetic composition of an organism, that is, the total sum of its genes, both dominant and recessive. (b) A group of organisms with the same genetic composition. (c) The genetic constitution of one or more genes of an organism with respect to a particular hereditary trait or set of traits. (d) In plants, that set of hereditary factors that regulate the organism’s way of reacting to external stimuli. Genotypic ratio the proportion of different genotypes of a given progeny. (See also Phenotypic ratio.) Germ cell one of two cells found in the pollen grain and which divides by mitosis into sperm cells. This division may occur before or after pollination. Germination in plants, the resumption of the embryo’s growth under favourable conditions after the seed has matured and dispersed, and the emergence of the young root and shoot from the seed. Germination is taken as completed when photosynthesis begins and the plant no longer relies on the food stored in the seed. Germplasm (a) The base material of heredity, that is, the structure that carries the total sum of hereditary characteristics of a species. The word ‘germplasm’ supposes that the structure is able to give rise to a new generation, transmitting its genetic characteristics. (b) The total genetic variability, represented by germ cells, available for a given population of organisms. (c) The potential hereditary materials of a species, considered collectively. Germplasm bank or gene bank an entity constituted to conserve genetic resources. For plants, it is the most practical method of safeguarding genetic material, storing samples of landraces, breeding products, varieties not in use, and wild species. Glumes bracts or leaves found on the outside of each spikelet in a grass inflorescence or spike. GMO see Genetically modified organism. 253 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources GMO marketing see Commercialization of GMOs. Graft a procedure by which two parts of living plant tissue are brought together so that they continue living and later behave as one plant. Grassland that type of vegetation or plant formation dominated by grasses and herbaceous plants. Various forms are found such as the Sahel; savannah; steppe; and veldt. Gymnosperms plants of ancient origins that present the characteristic of not possessing true flowers. In their place are reproductive structures known as cones. H Habitat (a) A particular place in the environment occupied by organisms or communities of organisms and with which they interact. The habitat is described in relation to those interactions. (b) A place where a plant or animal grows. Haploid (a) The cell or individual that has one set of chromosomes. (b) The reduced number (n), as of a gamete. (c) (adj.) Having one set of chromosomes. (See also Ploidy level.) Harvest (a) The collection of fruits to obtain the seeds. (b) Ecologically, it is understood as the removal, at a given moment, by humans of part of the biomass from the ecosystem. (See also Ecosystemic harvest.) Hereditary disease a disease that has, as its cause, the alteration of genetic material and which is transmitted from generation to generation. Heredity (adj. hereditary) or inheritance the transmission of genetically based characteristics from parents to progeny or from generation to generation. Heritability (a) The capacity of being inherited. (b) That part of the variation observed in a progeny that is due to heredity. Heterosis or hybrid vigour the increase in vigour, growth, size, yield, or functional activity of a hybrid progeny in terms of its parents, resulting from the crossing of genetically different organisms. Heterotrophs see Primary consumers; Secondary consumers. 254 Glossary Heterozygote (a) The genetic condition whereby the individual possesses two different alleles in a locus. (b) Also an organism with one or more heterozygous gene pairs. (c) An organism that does not reproduce exactly the same as itself. (See also Hybrid.) Heterozygous (adj.) said of an organism that has different alleles in the locus corresponding to homologous chromosomes. An organism may be heterozygous for one or more genes. (See also Homozygous.) Hexaploid an organism that has six sets of chromosomes, that is, with a chromosome number of 6n. (See also Ploidy level.) Homologue exchange the exchange of segments between chromatids of two homologous chromosomes during meiosis. Homologous chromosomes or homologues chromosomes that pair up during the first division in meiosis. Each member of the pair comes from a different parent and has a sequence corresponding to the locus of genes. Homozygous (adj.) said of an organism or homozygote that has similar genes in the corresponding loci of homologous chromosomes. An organism may be homozygous for one, several, or all genes. (See also Heterozygous.) Hormone a chemical substance of specialized action that acts as messengers to those cells that respond to its stimulus, thereby controlling tissues and organs in any part of the organism. The difference between animal and plant hormones is that animal hormones are created in particular organs and regulate almost all organic functions. Host (a) An animal or plant that harbours or nourishes another organism (e.g., parasite). (b) In genetic engineering, that organism, whether microbial, animal, or plant, whose metabolism is used to reproduce a virus, plasmid, or other form of DNA foreign to that organism and which incorporates elements of recombinant DNA. Hybrid (a) The first generation of offspring of a cross between two individuals that differ in one or more genes. (b) The progeny of a cross between species of the same genus or distinct genera. (See also Heterozygote.) Hybrid vigour see Heterosis. 255 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Hybridization (a) The crossing of genetically different individuals, thereby generating new genetic combinations and variability. (b) A method for creating new varieties, using crosses to obtain genetic recombinations. (c) The generation of a molecule, cell, or organism combined with genetic material from different organisms. Traditionally, hybrids were produced by crossing distinct varieties of animals or plants by aligning or pairing the bases of two simple-stranded DNA molecules that are homologous or complementary. The technology of cellular fusion and transgenic manipulation are new hybridization modalities introduced by genetic engineering. Hybridize to produce hybrids by crossing individuals with different genotypes. I Immediate principles see Biomolecules. Immune (adj.) (a) Free of attack from a given pathogen. (b) Not subject to a given disease. Imperfect flower a flower that does not have stamens or pistils. (See also Perfect flower.) In situ conservation in situ literally means ‘in the original place’. The conservation of plant genetic resources in the areas where they had developed naturally and, in the case of cultivated species or varieties, in the surroundings of the area where they had acquired their distinctive properties. In vitro (adj.) literally, in the glass. Said of anything studied and manipulated in laboratory test tubes, that is, outside the live organism. Incompatibility in plant reproduction, (a) the absence of fertilization and later seed formation. (b) The condition in which viable gametes cannot fuse because, for example, the stigma reduces or restricts the growth of the pollen tube; the formation of reproductive organs is not synchronized; or structural and/or functional barriers exist such as dichogamy, protandry, and protogyny. (c) The impossibility of achieving fertilization and seed formation through self-pollination, usually because of sluggish growth of the pollen tube in the style tissue. Incomplete dominance the production of an effect by two different alleles. This effect is intermediate between those produced by the same alleles under homozygous conditions. (See also Partial dominance.) Incomplete flower a flower that lacks one or more of the four essential organs (sepals, petals, stamens, and pistils). (See also Complete flower.) 256 Glossary Independent association a random association of two or more pairs of segregating genes in gametes. Indeterminate (adj.) said of an inflorescence in which the terminal flower is the last to open. The flowers are formed in axillary buds and the floral axis may elongate indefinitely by means of a terminal bud. An example is the raceme. (See also Determinate.) Indeterminate threat a category of conservation status, describing taxa that are known to be either endangered, vulnerable, or rare, but not which one. (See also Categories of species conservation.) Indigenous see Endemism. Infection the invasion of a living being by a pathogen, thereby triggering disease. Inflorescence a group of flowers growing on a floral axis, and having a characteristic arrangement and form of development. Inflorescence arrangements may be determinate or indeterminate. (See also Panicle; Spike.) Inherit to receive from predecessors. In organisms, the chromosomes and genes are transmitted or inherited from one generation to the next. Inheritance see Heredity. Inoculate (a) To place an inoculum where it will produce an infectious disease. (b) To introduce bacterial nitrogen fixers into the soil, usually by treating seeds before planting. Inoculum the spores, bacteria, or mycelium fragments of pathogens that can infect plants or soil. Insufficiently known (adj.) (a) A category of conservation status, describing taxa that are thought to belong to a given category related to risk of extinction but for which insufficient information is available. (b) Also said of species or other taxa thought to belong to a given category, but whose status is to be defined through future research. (See also Categories of species conservation.) Introns DNA sequences that do not code for genes and whose function is unknown. Inverse transcription the synthesis of complementary DNA from genomic RNA of retroviruses done by the enzyme known as inverse transcriptase. 257 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Isoenzymes multiple molecular forms of an enzyme that occur within an organism. They have the same catalytic function (catalyzing the same substrate) but possess different kinetic properties (e.g., reaction speed). (See also Alloenzymes.) K Kilobase or kb the unit used to measure the length of a DNA fragment, itself made up of a series of bases. 1 kb = 1000 bases. L Latency (adj. latent) see Dormancy. Leaf (pl. leaves) in plants, an expanded outgrowth of a stem, usually green, and the main photosynthetic organ of most plants. Legislation sui generis a particular form of protection of intellectual property, especially designed to cover certain criteria and needs. Lemma in a grass spikelet, the lower bract of two that protect the floret. (See also Palea.) Life form (a) The characteristic morphology of a mature organism. (b) According to the Raunkiaer system, the mechanisms by which plants survive the unfavourable season. Raunkiaer originally listed five types of life forms: phanerophytes, chamaephytes, hemicryptophytes, cryptophytes, and therophytes. His classification has since been broadened to include other mechanisms for plant survival under unfavourable conditions such as those used by, for example, epiphytes, succulents, halophytes, climbers, and hydrophytes. Line a group of individuals that descend from a common ancestor. Members of such a group are usually more closely related to each other than those of a variety. Lineage (a) A group of individuals whose descent can be traced back to a single ancestor. (b) In evolution, a sequence of species, each of which is considered to have evolved from its predecessor. Linkage the relationship that exists between two or more genes that tend to be inherited together because they are located on the same chromosome. This determines that combinations of these genes, like those of the parents in the gametes, are more frequent than their recombinations. Linkage group a group of genes distributed linearly in a chromosome. 258 Glossary Linkage map a diagram of a chromosome, indicating the position of genes. Lipids or fats a group of chemically very diverse organic biomolecules with the common characteristics of insolubility in water, solubility in polar organic dissolvents, and with little density. Liposomes artificially constructed spherical vesicles made up of two or more layers of lipids. Liposomes are used as gene vectors. Llaretal a Spanish term referring to a particular type of plant community in which pulvinate plants predominate. It is characteristic of the high plains and highlands of South America and is often made up of Umbelliferae species, and the Azorella and Laretia genera, called commonly llaretas. Locus (pl. loci) (a) A position on a chromosome where the gene controlling a given trait is located. (b) In genetics, the point on a chromosome occupied by a gene. Lodicule one of two structures, similar to scales, at the base of an ovary of a grass flower. Longevity the length of life. In seeds, it refers to the time that these remain alive. Longevity depends on the species and on the seeds’ storage conditions. Loricifera a new small phylum of marine sediment-dwelling animals. So far, 22 species from 8 genera have been described. About another 100 species have been collected but are not yet described. The phylum was discovered in 1983 by Reinhardt Kristensen, in Roscoff, France. M Male sterility in flowering plants, a condition whereby pollen is not produced or is sterile, or that part of the male organ that produces it does not function. Marker gene that gene whose function and location are known and which expresses certain characteristics or very notable phenotypic differences that permit the analysis of its heredity, establish its presence in the genome, and detect recombination events. Mass selection that system of plant improvement in which the seed of individual plants is selected on the basis of phenotype and then mixed and used to produce the next generation. Megagametophyte in plants, the female gametophyte. Typically, a female gamete of seven cells with eight nuclei. It originates from the megaspore. 259 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Megasporangium or embryo sac the structure in which the megaspores are produced and the megagametophyte later develops. It eventually forms the nucellus. Megaspore (a) One of four haploid spores that originate from meiotic divisions of the stem cell. (b) In plants, also the diploid megaspore found in the ovule and which undergoes three successive meiotic divisions to give rise to the megagametophyte. It is formed in the megasporangium from a stem cell when it undergoes meiosis. Megaspore stem cell a diploid cell of the ovary, which gives rise, through meiosis, to four haploid megaspores. Meiosis the two successive nuclear divisions of a cell. In the first (or reduction) nuclear division, the diploid chromosome number is reduced to a haploid number. The second nuclear division is mitotic. (See also Mitosis.) Meristem (a) A region of rapid cellular division (mitosis). (b) Undifferentiated tissue from which cells tend to form differentiated and specialized tissues. Meristems found in growing areas such as buds and apexes. Messenger RNA see mRNA. Microbe see Micro-organism. Micro-injection a technique, carried out under the microscope, that introduces a gene in solution into a cell, using a micropipette. Micro-organism or microbe a microscopic organism, usually a bacterium, alga, fungus, or protozoan. Microsatellites, simple sequence repeats, or SSRs short DNA sequences made up of 1 to 6 nucleotides that repeat themselves consecutively 10 or more times. These simple DNA sequences are highly variable and can be studied, using a fast and relatively simple methodology. Microspore in plants, one of four haploid spores that originate from the meiotic division of the microspore stem cell in the anther and gives rise to a pollen grain. Microspore stem cell or pollen stem cell a diploid cell of the anther, which gives rise, through meiosis, to four haploid microspores. 260 Glossary Mitosis the nuclear division of a cell, whereby chromosomes divide longitudinally, forming two daughter nuclei, each of which has a chromosome complex like that of the original nucleus. (See also Meiosis.) Molecular biology (a) That part of biology which deals with biological phenomena at the molecular level. (b) In a restricted sense, it includes the interpretation of these phenomena on the basis of participation of proteins and nucleic acids. Molecular cloning (a) A technique of genetic engineering that consists of inserting a segment of foreign DNA of a given length into a vector that replicates itself in a specific host. (b) The formation of heritable material that can propagate or grow through culturing from a line of genetically identical organisms. (See also DNA cloning; Genetic engineering.) Molecular markers gene markers that are used to directly evaluate the genome (DNA), or part thereof without their being affected by the environment, thus conferring greater accuracy. Monocotyledons modern plants, whose principal characteristic is to develop only one embryonic leaf (cotyledon) on germinating. (See also Dicotyledons.) Monoecious (adj.) said of a plant species that has both staminate and pistillate flowers on the same individual. (See also Dioecious.) Monosomic (adj.) (a) Refers to a chromosome that lacks its homologous partner. (b) A haploid chromosome in an individual that otherwise would be a normal diploid. Morphometry the study of anatomical measures. Mosaic (a) In plant pathology, a viral disease characterized by the presence of irregular patches of different colours or colour intensities. (b) See Chimaera. mRNA or messenger RNA a molecule of RNA that represents a negative copy of amino acid sequences in a gene. The non-coding sequences (introns) have already been extracted. With few exceptions, mRNA has a sequence close to 200 adenines (polyA tail), united to its 3' extreme, which is not coded by DNA. Multiple alleles a series of alleles or alternative forms of a gene. A normal heterozygous diploid would have only two genes of an allelic series. Multiple alleles originate by repeated mutations of a gene, in which each mutant produces different effects. 261 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Multiple genes two or more pairs of independent genes that produce complementary or cumulative effects on a single trait of the phenotype. Mutation (a) A change in the breeding material. It may arise from changes in a pair of DNA bases, particular gene, or chromosomal structure. (b) An unexpected variation in the hereditary material of a cell. (c) A sudden variation or alteration in an organism, which is then said to be a mutant, especially when such alteration is heritable by following generations. It may involve changes in genes (genic mutation) or chromosomes (chromosomal mutation). A genic mutation consists of a change in one allele or another of a gene. A chromosomal change may consist of, for example, a duplication, inversion, or exchange. N Naked DNA (a) DNA that is deprived of its proteinic or lipidic coat. (b) In gene transfer, the term refers to DNA made up of a bacterial plasmid that contains the gene to be transferred. It is injected directly in the targeted tissue where it is usually expressed without being integrated in the genome of the host cells. Native race a population of usually heterozygous plants that were commonly developed in traditional agricultural systems through direct selection by farmers and which, characteristically, are adapted to local conditions. Natural cross in plants, a result of cross-fertilization, usually under natural conditions, where one parent of a plant’s genetic constitution is different to the other. Natural selection (a) The elimination of random alleles, without intervention from humans. (b) The process Darwin called the ‘struggle for survival’, whereby those organisms least adapted to their environment tend to die and the better adapted to survive. According to Darwinism, natural selection acts on a varied population, causing its evolution. Natural selection appears as the inevitable result of three basic facts of life: overpopulation, variability, and heredity. Naturalized (adj.) said of a plant that is not native to a country or region but lives there, surviving as if it were indigenous. Neotropics a biogeographical region, which spans several habitat types, but has strong biogeographic affinities, particularly at taxonomic levels higher than the species level. It extends from southern Mexico, through Central America and the West Indies, and includes the South American continent. (See also Afrotropic.) Nucellus the megasporangium, after it eventually forms the inner layer of the ovule wall. 262 Glossary Nucleic acids biomolecules formed by nucleotide macropolymers or polynucleotides. Present in all cells, they constitute the basic material of heredity that is transmitted from one generation to another. Two types exist: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Nucleoside a combination of a pentose sugar with a purine or pyrimidine nitrogenous base. Nucleotide a monomer of nucleic acids, made up of a combination of a nitrogenous base (purine or pyrimidine), sugar (ribose or deoxyribose), and a phosphate group. It is the product of hydrolysis of nucleic acids through the action of nucleases. Nullisomic a plant that, if it were not for the lack of a pair of particular chromosomes, would be a normal diploid. O Obsolete variety those plant varieties that are no longer cultivated commercially but may be kept in collections for use in improvement programmes. Offshoot or tiller (a) A characteristic type of lateral shoot or branch that develops from the base of the principal stem in certain plants. (b) The term ‘tiller’ is applied to several lateral shoots that emerge from the crowns of monocotyledons such as grasses. Operator a special segment of DNA, adjacent to the promoter, that is part of the controlling region for operon transcription. The operator interacts with the repressor protein, thus regulating the synchronized transcription of the corresponding operon. Operator gene the gene that stimulates the structural gene into functioning. Its activities may be modified by the regulatory gene. Operon a set of genes, comprising an operator gene and the structural genes that it controls. Organism a biological entity able to reproduce itself or transfer genetic material. Microbiological entities are included within this concept, whether or not they are cellular. Almost all organisms are formed of cells, which may then be grouped into organs, and these into systems, each of which carries out particular functions. Orthodox seed seed that can be dried to low levels of moisture content and stored at low temperatures over long periods without losing viability. (See also Recalcitrant seed.) 263 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Out of danger a category of conservation status, describing a species or other taxon that had been included in a higher category on the continuum to extinction, but is now considered to be in a relatively safe state of conservation due to the adoption of effective conservation measures or elimination of previously existing threats. (See also Categories of species conservation.) Ovary in flowers, the swollen base of the pistil, in which seeds are formed. Ovule in plants, (a) the female gamete or germ cell. (b) The structure that contains the female gamete or megagametophyte and becomes seed after fertilization. P Palea in a grass spikelet, the upper of two bracts that protect the floret. (See also Lemma.) Panicle an open and branched inflorescence, with flowers possessing stalks or pedicels. Parasite (adj. parasitic) a living being that, for all or part of its life, derives its food from another living being (the host). The host is usually harmed to some extent by the association. Parthenocarpy in plants, the production of fruits without fertilization and normally without seeds. Parthenogenesis (a) The development of an individual from a gamete without fertilization. (b) Unisexual reproduction, where females give rise to offspring without being fertilized by males, for example, rotifers and certain crop pests such as aphids. Partial dominance (a) The lack of complete dominance. (b) The production of an intermediate hybrid among reproducing types. (See also Incomplete dominance.) Patent the exclusive right granted to the ownership of an invention as a social counterpart to the innovation. Pathogen (adj. pathogenic) (a) The producer or causal agent of a disease. (b) An organism able to incite disease. Pathogenicity the capacity of an organism to cause or incite disease. PCR or polymerase chain reaction a technique for analysing the genome by an unlimited amplification of minuscule but particular parts of DNA. It is a revolutionary method of exponential amplification of DNA that uses the intervention of a heat-stable enzyme, the Taq polymerase. 264 Glossary Pentaploid an organism that has five sets of chromosomes, that is, with a chromosome number of 5n. (See also Ploidy level.) Peptide a polymer or amino acid chain. Perennial a plant that lives for 3 years or more. They may be woody such as shrubs and trees, or herbaceous. Herbaceous perennials may be evergreen; deciduous (i.e., the aerial organs are annual but the underground organs such as rhizomes and bulbs are persistent); or monocarpic, that is, living for many years until flowering and fruiting, after which they die, for example, Agave spp. (See also Annual; Biennial.) Perfect flower a flower that has stamens and pistils. (See also Imperfect flower.) Persistent (adj.) said of a plant organ that remains inserted or does not fall at maturity once it has fulfilled its physiological function. Pests in agriculture, organisms such as insects, nematodes, and other plants that attack crops and livestock. (See also Disease; Pathogen; Weeds.) Phenotype (a) The final appearance of an individual that results from the interaction of its genotype with a given environment. (b) The observable characteristics of an organism. (c) The physical or external appearance of an organism, in contrast to its genetic constitution (genotype). (d) A group of organisms of similar external physical constitution. (e) The set of all apparent characters expressed by an organism, whether these be hereditary or not. Phenotypic ratio the proportion of different phenotypes of a given progeny. (See also Genotypic ratio.) Physiognomy that aspect of a plant community or species that is subject to visual appraisal. It depends on the set of special structures and characteristic forms of its biological constituents. Physiological race those pathogens of the same species and variety that are similar structurally, but differ in their physiological and pathological characteristics and, especially, in their ability to parasitize different varieties of a given host. Phytosanitary quality or plant health quality the set of characteristics that plant germplasm should have with respect to the presence or absence of pathogens transmissible in planting materials and/or micro-organisms that cause deterioration during conservation. 265 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Pistil in flowers, the female organ where the seed originates. It comprises the ovary, style, and stigma. Pistillate flower a flower that bears pistils but has no stamens. (See also Staminate flower.) ‘Placing on the market’ of GMOs see Commercialization of GMOs. Plant community a more or less complex group of plants that occupy a certain area, regardless of the character, composition, and structure that the plants present. Plant formation that group of plant communities, delimited in nature by particular physiognomic characters, depending on the dominant forms of life and the way in which space is occupied. A plant formation represents the expression of given living conditions and has, as its base, a particular type of environment. Plant genetic resources these are the total of all the gene combinations produced during plant evolution. They range from wild species with agricultural potential to cloned genes. The term implies that the material has or may have economic or utilitarian value, whether current or future, perhaps the most important being that which contributes to food security. (See also Genetic resources.) Plant health quality see Phytosanitary quality. Plasmagene the cytoplasmic unit of heredity. Plasmid (a) A non-cellular form of life. (b) A circular fragment of double-stranded DNA that contains some genes and is found within certain bacteria. It acts and replicates independently of bacterial DNA and may pass from one bacterium to another. As with proviruses, they do not produce diseases but induce small mutations in cells. They are used as vectors in genetic engineering. Ploidy level the complete number of complements or basic sets of chromosomes that a cell or organism has. The living unit may be haploid, diploid, triploid, tetraploid, pentaploid, or hexaploid if it possesses 1, 2, 3, 4, 5, or 6 basic sets of chromosomes, respectively. A polyploid is that which has more than two sets of chromosomes, and an aneuploid does not have an exact set. Point mutation a type of mutation that causes the replacement of a single-base nucleotide with another nucleotide. Often includes insertions or deletions of a single base pair. 266 Glossary Polar nuclei the two central nuclei found within the megasporangium and which join with the second sperm cell in triple fusion. In certain seeds, the product of this triple fusion gives rise to the endosperm. Pollen a fine powder produced by anthers and male cones of seed plants, composed of pollen grains. Each grain encloses a developing male gamete, itself having originated from a microspore. Pollen stem cell see Microspore stem cell. Pollen tube a tube that, under favourable circumstances, develops from the pollen grain after being placed on the stigma of a flowering plant. It grows down the style to the ovary and eventually to an ovule. The sperm cell is carried to its destination in the tip of the pollen tube. Pollination the transfer of pollen from the anther to the stigma in flowering plants or from the male to the female cone in gymnosperms. (See also Cross-pollination; Self-pollination.) Polycross an isolated group of plants or clones distributed so that random cross-pollination can occur. Polymer a chemical compound formed by the combination of repeated structural units (monomers) or linear chains of the same molecule. Polymerase chain reaction see PCR. Polyploid an organism with more than two sets of chromosomes in its cells. (See also Ploidy level.) Population a group of individuals of a species living in the wild in a given area. It is the most significant level of organization of a species and is also of evolutionary and conservational significance. Precaution a basic criterion that governs, a priori, any environmental action. The criterion is incorporated into the Maastricht Treaty on the European Union, by which any substance, organism, or technology must demonstrate its compatibility with the environment and public health before its production and use are authorized. Prevention a basic criterion that governs, a posteriori, any environmental action. The criterion is incorporated into the Maastricht Treaty on the European Union, by which the original cause of existing environmental damage is avoided to prevent it recurring. 267 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Primary consumers or heterotrophs these organisms take advantage of the chemical energy stored in the organic matter of primary producers. This level is composed of herbivores. Primary producers or autotrophs these take advantage of the energy from light, using photosynthesis. They are able to synthesize organic matter from inorganic matter. This level corresponds to that of green plants. (See also Primary consumers; Secondary consumers.) Primary production this represents the increase in biomass of the primary or photosynthetic producer organisms. Gross primary production refers to the biomass synthesized through the photosynthetic activity of primary producers. Prion a protein of infectious character that is able to reproduce. It originates from a natural and innocuous protein that is transformed into a harmful form, able to resist proteases, ionization, and ultraviolet radiation. Although it is found mostly in animals, being responsible for diseases such as bovine spongiform encephalopathy, Creutzfeldt-Jakob disease, and kuru, it is also found in certain fungi and plants. Production that process which increases biomass per unit of time. It may be measured in mg cm–3 day–1, kg ha–1 year–1, or kcal ha–1 year–1. It expresses the idea of biomass available per unit of time for use by the next trophic level without endangering the ecosystem’s stability. Production = Biomass/Time. Productivity this is the relationship between production and biomass. In algae, for example, which reproduce daily, that is, they double their mass every 24 hours, productivity is 100%. In contrast, the average productivity in land plants does not reach 0.3%. For example, an almond tree forms almonds only once a year. Productivity = Production/Biomass. (See also Biomass and ecosystem productivity.) Prokaryotes organisms whose cells possess a single chromosome and no membrane to isolate it from the cytoplasm. This means that such an organism lacks a true cellular nucleus. The most representative examples are blue-green algae and bacteria. Promoter a region of DNA that is involved in and necessary for the initiation of transcription. It includes the RNA polymerase binding site (the starting point of transcription) and various other sites at which gene regulatory proteins may bind. Propagule any structure that serves to vegetatively propagate or multiply a plant, for example, cuttings, tubers, differentiated tissues, and cells. 268 Glossary Prophylaxis or preventive treatment in phytosanitary procedures, a measure or set of measures taken to prevent the occurrence of disease. This may include the use of protectants, which are usually chemical agents, to prevent a given disease or diseases among plants. Protandry the condition of hermaphrodite plants where male gametophytes mature and are shed before female gametophytes are mature. (See also Dichogamy; Protogyny.) Proteins biomolecules formed by amino-acid macropolymers or macropolypeptides. They function as enzymes, hormones, and contractile structures that endow organisms their characteristic size, metabolic potential, colour, and physical capacities. Protocol a document of standardization that establishes the rationale, objectives, design, methodology, and foreseen analysis of results, and the conditions under which such activities are to be carried out and developed. Protogyny in hermaphrodite plants, the condition where female gametes mature and are shed before male gametes mature. (See also Dichogamy; Protandry.) Protoplast a cell that is isolated and deprived of its cell wall. Pteridophyta a taxonomic class of plants of ancient origins. The plants are principally characterized by the absence of true roots (they have rhizomes) and reproduce by spores. Pulvinate (adj.) said of vegetation that develops in the form of pads or cushions. Pure line (a) A genetically pure line where all its members are homozygous, having originated from the self-fertilization of a simple homozygous individual. (b) Genetically pure individuals (homozygotes) who originated from self-fertilization and whose offspring are equally homozygous and homogeneous. Q Qualitative trait or discontinuous variation a trait whose observed variation is discontinuous, or which presents several states. It is usually controlled by one or a few genes, with little or no influence from the environment (e.g., yellow flower versus white flower). Quantitative trait (a) A trait that is determined by a series of independent genes that have cumulative effects. (b) Also continuous variation where a trait whose observed variation is continuous and is usually controlled by many genes, with strong influence from the environment. 269 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Quarantine a procedure of legal character that consists of confining or isolating plants or other materials introduced from other countries. They are then subjected to inspection to detect plant health problems that could threaten the agriculture of the country which they are entering. R RAPD or random amplified polymorphic DNA polymorphic DNA amplified at random. Rare (adj.) (a) A category of conservation status given to taxa whose world populations are small, but are not currently at risk of extinction or vulnerable, even though they are subject to a certain degree of risk. These taxa are normally located in restricted geographical areas or habitats, or have extremely low density over a more-or-less broad distribution. (b) With respect to species, the term refers to intraspecific taxa that apparently have always been scarce and are in their last stages of natural extinction. (c) Or the term refers to species with very restricted distribution, few defences, and few powers of adaptation. (See also Categories of species conservation.) rDNA or recombinant DNA a DNA molecule formed by recombining DNA fragments from different origins. The protein that codes is a recombinant protein. Recalcitrant seed seed that cannot be dehydrated nor conserved at low temperatures without suffering damage. It can be stored for only few days or weeks under special treatment. Species that have recalcitrant seeds or do not produce seeds are usually conserved in field germplasm banks. In these areas, collections of live plants are kept, that is, the germplasm is conserved as a permanent live collection. (See also Orthodox seed.) Recessive gene (a) That gene which needs a double ‘dose’ to be expressed. (b) A gene that does not manifest itself in the presence of a counterpart or dominant allele. (See also Dominant gene.) Recombinant DNA see rDNA. Recombinant DNA technology see Genetic engineering. Recombination the formation of new gene combinations as a result of cross-fertilization between individuals that differ in their genotype. Recurrent parent or donor parent in plant improvement, in a back cross, that parent with which the hybrid material is again crossed. 270 Glossary Recurrent selection that system of genetic improvement designed to increase the frequency of genes favourable for yield or other characteristics through repeated selection cycles. Reduction division see Meiosis. Regeneration or rejuvenation within the context of germplasm banks, the cultivation of a sample of an accession (e.g., seed, clone, in vitro plant, or other propagule) to produce fresh, viable, and sufficient samples of plants from which sexual or asexual seeds with similar genetic constitution can be harvested, and which permits the preservation, in a better state, of the seed or propagule when stored. Registered seed progeny of basic seed or certified seed that is produced and used in such a way that it satisfactorily maintains its identity and genetic purity. It is approved and certified by an official certification agency. Regressive form with reference to crops, a species related to the cultivated form, growing in the wild, but not used in agriculture. It usually shows characteristics of both the cultivated species and its wild relatives. Regulatory gene an ancient gene whose mutations can influence evolution. It also modifies the action of the operator gene. Rejuvenation see Regeneration Relicts (adj. relict) in the sense of relics, those plants that had been dominant in other times, but which are now scarce. By extension, a country’s original vegetation that remains or persists. Replication (a) That process by which a DNA or RNA molecule originates from another identical one. (b) Generally, the duplication of nucleic acid. Replicon a nucleic acid structure that can replicate. Replicons include chromosomes of eukaryote cells, prokaryotic nuclear DNA, plasmids, and viral nucleic acids. Representative sample a sample that contains at least 95% of alleles (genetic variability) of the sampled population. Repressor gene that gene which represses the operator gene. 271 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Resilience the capacity of the ecosystem to fluctuate between given limits and thereby restore itself to its original state after disturbance. Such capacity operates within certain limits, beyond which the system is not able to return to the condition of pre-disturbance and, hence, is degraded towards pioneering successional states. The limits of resilience differ for different ecosystems, as does the speed of recovery. (See also Stability.) Resistance that characteristic of a host plant that enables it to prevent or delay the development of a pathogen or other harmful factor. (See also Susceptibility.) Restriction enzymes enzymes that bacteria synthesize in defence against the invasion of foreign DNA from, for example, bacteriophages, thereby degrading that DNA while remaining themselves protected through particular methylations. A restriction enzyme always divides the DNA in the same site, specific loci, or targeted sequences. Their scissor-like behaviour opened the doors to genetic engineering. RFLP or restriction fragment length polymorphism polymorphism along the length of restriction fragments. Rhizome an underground stem that is usually horizontal and elongated. It differs from a root by the presence of nodes and internodes, sometimes scale-like leaves, and shoots in the nodes themselves. Ribonucleic acid see RNA. Ribosomes small cellular organelles found in all living beings where protein synthesis is carried out. Risk the possibility or probability that a future harm will happen. RNA or ribonucleic acid nucleic acid formed by nucleotides in which the sugar is ribose, and the nitrogen bases are adenine, uracil, cytosine, and guanine. They act as intermediaries and complement the genetic instructions coded in the DNA. Several different types of RNA exist, related to protein synthesis; these are messenger RNA (mRNA, ribosomal RNA (rRNA), transfer RNA (tRNA), and heterogeneous nuclear RNA (hnRNA). RNA is normally the product of the transcription of DNA template, although, in retroviruses, RNA acts as the template and DNA is the copy. Root the descending portion of the plant, fixing it in the soil. It also absorbs water and minerals and has a characteristic arrangement of vascular tissues. Ruderal (adj.) said of environments and plant and animal species that are linked to human activities, either directly or indirectly. 272 Glossary Runner see Stolon. S SO the symbol used to designate the original self-fertilized plant. S1, S2, etc. symbols used to designate the first self-fertilization (progeny of plant S0), second self-fertilization (progeny of plant S1), etc. Sahel a transitional zone lying immediately south of the Sahara Desert that is transitional between the northern desert and the southern savannah. It is a semi-arid zone of short grasslands with acacia. (See also Grassland.) Savannah a large region of Africa that lies between the Sahel and the belt of tropical moist broadleaf forests near the equator. Two forms are recognized: the Sudanian savannah (also called Sudan—unrelated to the nation of the same name), which is a belt of tall grasslands, and the forest-savannah, which is a transitional zone between the grasslands and the belt of tropical rainforests. (See also Grassland.) Scarification any process to rupture, scratch, or mechanically alter seed coats to make them permeable to water or gases. Scrub see Shrublands. Secondary consumers, also heterotrophs these feed on the primary consumers. This level is composed of carnivores. (See also Primary producers.) Secondary production this represents the speed of storing energy at the levels of consumers and decomposers. It is therefore the increase in biomass per unit of time and space at these levels. Seed a mature ovule with its embryo and all the normal seed coats. In some plants, the seed also has an endosperm. Seed tubers or seed pieces small tubers or cut pieces of tubers used for planting new-season crops. They are stored during winter and later transplanted for seed production. Seed tubers are obtained from plants that have not been thinned. Seed pieces are obtained by cutting up tubers so that each piece carries an eye or set of eyes that, on planting, will grow into a new plant, as in potato. 273 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Segregation the separation of homologous chromosomes (and, consequently, their genes) of the different parents during meiosis. Selection (a) Any natural or artificial process that permits an increase in the proportion of certain genotypes or groups of genotypes in successive generations. (b) A plant or line that originates from a selection process. Types of selection include mass selection, natural selection, progeny selection, reciprocal recurrent selection, and recurrent selection. Self-fertile (adj.) in plants, able to become fertilized and produce seeds after self-pollination. (See also Self-sterility.) Self-fertilization in plants, the fusion of an ovule with the sperm cell of the same flower, another flower of the same plant, or clone. (See also Autogamy.) Self-fertilized line (a) A pure line that usually originates from self-pollination and selection. (b) The product of self-fertilization. Self-incompatibility see Self-sterility. Self-pollination in plants, the process whereby pollen is transferred from the anther to the stigma of the same flower, another flower of the same plant, or clone. (See also Cross-pollination.) Self-sterility or self-incompatibility in plants, the incapacity, usually physiological, to incur fertilization and produce seeds after self-pollination. (See also Self-fertile.) Sexual cells see Gametes. Sexual reproduction reproduction on the basis of germ cells and the fusion of gametes. (See also Vegetative propagation.) Shoot (a) In vascular plants, that part derived from the plumule (being the stem) and usually leaves. (b) A sprouted part, branch, or offshoot of a plant. (See also Crown; Sucker.) Shrublands or scrub a type of plant community in which dominate shrubs that are usually highly branched at the base. It is typical of semi-arid and arid areas. Simple sequence repeats see Microsatellites. 274 Glossary Somaclonal variation the variation observed in somatic cells, which divide mitotically in tissue culture. Depending on the species, this variation may be genetic, phenotypic, or in habitat. Many of these modifications are transferred to progenies of regenerated plants. Somatic (adj.) refers to diploid cells, normally with one set of chromosomes from the male parent and another set from the female parent. Somatic embryos those embryos that originate from the fusion of somatic cells, that is, not from gametes. Speciation the formation of one or more new species from one already existing. It occurs when an isolated population develops certain distinctive characteristics as a result of natural selection and loses the possibility of reproducing with the rest of the population, even when no geographical or physical reasons are apparent to prevent it. Species (a) A taxonomic class formed by the set of natural populations that can cross among themselves, whether in fact or potentially. That is, determined empirically, two individuals belong to the same species if they can generate reproducible offspring; otherwise, they are of different species. (b) A unit of classification that is a subdivision of a genus. (c) A group of closely related individuals and descendants of common origin. (See also Speciation; Taxon.) Specific (adj.) (a) Related to species. (b) Also said of the characteristic effect on the cells or tissues of members of a given species or which interacts with them, for example, of infectious agents. Sperm cell in plants, part of the male gametophyte. The germ cell in the pollen grain undergoes mitosis to produce two sperm cells, one of which will fuse with the egg in the ovule to form the zygote. The other sperm cell will fuse with the two polar cells in the ovule to form the endosperm that later feeds the embryo. (See also Egg.) Spike also known as ear or tassel: an inflorescence with a more-or-less elongated axis, throughout which the flowers are almost sessile. Spikelet a unit of a spike, that is, of an inflorescence typical of grasses. It is formed by glumes, the rachis, and florets. SSRs see Microsatellites. Stability the ability of ecological systems to persist over time despite external disturbances, whether of natural or anthropic origin. (See also Resilience.) 275 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Stamen in plants, the male reproductive organ of the flower that comprises the anther and filament. Staminate flower a flower that bears stamens but has no pistils. (See also Pistillate flower.) Static conservation the type of conservation that stops the natural processes of evolution and co-evolution of genetic resources, hence conserving them in isolation and outside their natural habitats. The term is applied specifically to ex situ conservation. Stem the main axis of a vascular plant. It bears buds, leaves or scale leaves, and reproductive structures such as flowers. It is usually borne above ground and has a characteristic arrangement of vascular tissue. (See also Bulb; Corm; Crown; Rhizome; Stolon; Tuber.) Steppe (a) A term that, in its etymology, has a strong geographical sense, usually including the concept of desert with very cold winters. (b) In its strictly botanical meaning, it corresponds to grassland plant formations found in cold high areas of Asia where cespitose plants dominate, forming patches and leaving exposed areas. It includes other short plants that are perennial, herbaceous, or woody in nature. The exposed areas may be temporarily occupied by annual plants. (See also Grassland.) Sterility the impossibility of completing fertilization and acquiring seed because of defective pollen or ovules, other aberrations, or unusual or seasonal plant activity. Stigma in flowering plants, that part of the pistil that receives pollen. Stolon or runner (a) A specialized creeping stem, capable of forming roots and shoots at its nodes. (b) Specifically, those prostrated or scattered stems that develop in leaf axils in the crown of the mother plant, grow horizontally over the ground, and form new plants from its nodes. Strain in microbiology, that set of viruses, bacteria, or fungi that have the same gene pool. Structural gene (a) That gene which regulates the formation of an enzyme or other protein required for a cell’s structure or metabolism. (b) In evolutionary terms, that gene of ancient origin that contributes to the basic structure of a life form (a). (See also Evolution.) Style in flowering plants, the column that connects the ovary to the stigma. Subculture the aseptic transfer of part of a plant in a collection to a fresh medium for renewal and strengthening. 276 Glossary Sucker (a) A shoot that originates in a plant but from under the soil surface. This term is more precisely used to designate a shoot that originates from an adventitious bud on a root. (b) Loosely, the term also refers to shoots originating close to the crown, even though they arise from stem tissue. Susceptibility the extent to which a plant, vegetation complex, or ecological community would suffer if it were exposed to a pathogen or other harmful factor (regardless of whether it receives such exposure). A host plant is said to be susceptible if it cannot prevent or retard the harmful effect of a pathogen or other noxious factor. (See also Resistance.) Sustainability the concept of using resources, while renewing them in a given period. Sustainable development ‘Sustainable development is that development that serves the needs of the present generations without undermining the needs of future generations’ (Brundtland Report 1986). ‘It is a process of sustained and equitable improvement of the quality of life, based on the conservation and protection of the environment in such a way that it does not compromise the expectations of future generations’ (Law 19,300, Colombian Congress). Sustainable use the use of components of biodiversity in such a way and at such a rate that it does not cause long-term reduction of biodiversity, thereby maintaining the possibilities of meeting the needs and aspirations of current and future generations. Synthetic variety advanced generations of seed mixtures of free pollination from a group of lines, clones, or self-fertilized lines, or of hybrids among them. System a coherent set of interacting elements that can be isolated from the rest of the universe according to appropriate criteria. Systematics the study of systems, from both the abstract and applied viewpoints. T Tassel see Spike. Taxon (pl. taxa) a taxonomic group (e.g., species, genus, or family) at any level within a classification system of living beings that are thought to have certain similarities and a given degree of evolutionary relatedness. Taxa are typically organized hierarchically from the largest categories (e.g., kingdom, division, or class) where the members are less related to the smallest (e.g., species, subspecies, or variety), where the members are closely related. 277 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Test cross that process whereby a hybrid is crossed with one of its parents, or with a genetically equivalent recessive homozygote. The test is used to prove homozygosis or linkage. Tetraploid an organism that has four sets of chromosomes, that is, a chromosome number of 4n. (See also Ploidy level.) Thermotherapy in plants, a treatment to disinfect plant material, using heat. For cassava, for example, the treatment consists of taking selected stem cuttings to the greenhouse or growth room and subjecting them to 3 weeks of temperatures at 40°C during the day and 35°C during the night, and a daylength of 12 hours. To increase effectiveness, the treatment is combined with in vitro tissue culture. Tiller see Offshoot. Totipotent (adj.) capable of anything. Said of cells that can give rise to cells of any kind. Toxins (a) Substances, usually albuminoid, made by living beings, especially microbes, that act as poisons, even in tiny quantities. (b) Proteins responsible for particular functions in certain bacteria and which are poisonous to certain other organisms. Trait, character, or characteristic (a) A plant’s structural or functional attribute that results from interaction among genes and with the environment in which the plant develops. (b) A distinctive trait that is the expression of a gene. (c) The expression of a gene as manifested in a phenotype. Transcription the biosynthesis of an RNA molecule by polymerizing complementary nucleotides to a DNA sequence. This RNA molecule is a precursor of messenger RNA (mRNA) and represents a true copy of the complementary DNA sequence from which it has been transcribed. A specific sequence placed in front of the gene (promoter) acts by identifying the initiation site for transcription. In RNA, uracil (U) occupies the positions that thymine (T) has in DNA. It is the working copy of given DNA segments. Transformation (a) In bacteria, one of the natural processes, together with conjugation and transduction, for transferring genetic material from one bacterium to another, involving the direct integration of DNA. (b) Experimentally, it consists of introducing a DNA fragment into a bacterium to stimulate genetic recombination. (c) By extension (and loosely), the term sometimes designates an identical process that affects eukaryotic cells (yeasts, animal, and plant cells). Transgene a gene has been introduced from another species such that it can usually be transmitted to that organism’s offspring. 278 Glossary Transgenesis the artificial introduction of new genetic material into the genome of a plant through genetic engineering such that this new material can be inherited by progeny. This technique permits associations of genes that do not exist in nature, as they have been made to jump barriers between species and even higher taxa such as kingdoms. Transgenic plant (a) A plant carrying a transgene. (b) That plant whose genome has been altered by in vitro manipulation. Translation an exchange of information contained in the sequence of the four nucleotides of mRNA due to the arrangement of the 20 amino acids in the structure of polypeptide chains. Each amino acid unites with a small specific RNA molecule, designated as transfer RNA (tRNA), and which serves to identify it. This molecule transfers the free amino acids from the solution to the point of formation of the polypeptide chains when so indicated by instructions contained within the messenger RNA (mRNA) molecule. Translocation the structural modification of chromosomes by which a chromosomal segment changes its relative position within the chromosome itself (intrachromosomal translocation) or between chromosomes (interchromosomal translocation). (See also Transposition.) Transposition: (a) The change of position of given pairs of bases in the DNA sequence. (b) The translocation of a chromosomal segment to another position within the same chromosome. (See also Translocation.) Transposon a mobile genetic element with a defined DNA sequence and which can be transferred to new positions in the cell’s chromosome without losing the copy in its original position. Moreover, it behaves as a true intracellular parasite. Transposable elements of eukaryotes are grouped into two categories, according to their mechanism of transposition: class 1 (retrotransposons), which jump to the genome through an intermediate step, that is, through RNA and with the intervention of the enzyme known as inverse transcriptase; and class 2, which transpose directly from one chromosomal site to another by means of a different enzyme (transposase). Trihybrid the result of one cross between parents that differ in three specific genes. Triploid (a) An organism which has three sets of chromosomes, that is, a chromosome number of 3n. (b) (adj.) Having three sets of chromosomes. (See also Ploidy level.) Tuber a modified stem structure that develops underground as a consequence of swelling in the subapical part of a stolon and the subsequent accumulation of reserve materials. 279 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources V Variety or cultivar among cultivated plant species, (a) that plant which differs by one or more traits. When it reproduces by seed or asexually, these traits are conserved. (b) Taxonomically, a subdivision of a species. (c) Agronomically, a group of similar plants that, by structure and performance, may differ from other varieties within the same species. (See also Obsolete variety; Synthetic variety.) Vascular bundles structures that are present in higher plants and whose function is to transfer liquids through modified canals. Vector a carrier, that is, that which transfers an agent from one host to another. (a) A system that permits the transfer, expression, and replication of foreign DNA in host cells for later cloning or transgenesis. It involves a DNA molecule (bacterial plasmid, artificial microsome of yeast, or bacterium) or a defective virus. (b) By extension, a vector is the entire system of gene transfer, for example, a synthetic system like that of liposomes. Vegetation that spatial structure or mode of organization of the set of plant species found in a given place. It is usually described by examining stratification and coverage, alluding to the species present and the dominant forms of life. Vegetative propagation, clonal multiplication, asexual reproduction, or agamic reproduction a type of reproduction that does not involve the formation and fusion of gametes, leading to the constitution of homogeneous clones. (See also Apomixis; Sexual reproduction.) Veldt or veld open temperate grasslands found in southern Africa. (See also Grassland.) Vernalization that treatment of plants with heat or cold to modify successive stages leading to maturity. For some species, vernalization can be achieved by exposing germinating seed at temperatures slightly above freezing point. Viability (a) The capacity of an organism to continue living after birth. (b) In seeds, the capacity to germinate when they possess all they need to do so. The fact that a seed is alive does not guarantee that it will germinate, even under optimal conditions, as phenomena such as dormancy or inactive states can occur. Virion the extracellular form of the virus, that is, before it has entered the host cell to replicate. Viroids causal agents of certain plant diseases. Their name derives from their similarity to viruses, from which they are differentiated by a lack of a capsid. Virulence the relative capacity of a pathogen to incite disease. 280 Glossary Virus an infectious cell-free entity that, even though it can survive extracellularly as a virion, is an obligate parasite because it can replicate only within specific live cells, generating no energy or metabolic activity. The permanent components of a virus are nucleic acid (DNA or RNA, single or double stranded) and a protein coat known as capsid. In some cases, these basic structures have an outer lipid membrane or envelope (also called peplos), which sometimes also carries glycoprotein spikes. Voluntary release of GMOs the deliberate introduction of a GMO or combination of GMOs into the environment without measures of containment such as physical barriers or a combination of these with chemical or biological barriers having been adopted to limit the GMOs’ contact with the human population and the environment. Vulnerable a category of conservation status, describing taxa that are believed to become at risk of extinction should the causal factors of threat continue operating. Such taxa include those for which most or all their populations are diminishing due to overexploitation, widespread habitat destruction, or other environmental alterations; or whose populations have been seriously exhausted and whose definitive protection is still not ensured; or whose populations are still abundant, but are under threat from severe adverse factors throughout their area of distribution. Vulnerable taxa are those whose populations have been reduced to such critical levels or whose habitats have been so drastically reduced that they are at imminent risk of extinction. (See also Categories of species conservation.) W Weeds (a) In agriculture, plants or species that grow where farmers do not want them. (b) In ecology, plants that have adapted to disturbed environments or open habitats. Wild species those groups of organisms that are regularly found in nature and have not been domesticated. Wild type in genetics, a species or organism that carries the normal form of a gene or genes, as opposed to a mutant. Working collection or breeder’s collection that collection of germplasm accessions used for crop research and improvement. (See also Active collection; Base collection; Core collection.) X Xenia the immediate effect of pollen on the characteristics of endosperm. Z Zygote the cell that results from the fusion of gametes. 281 Multi-Institutional Distance Learning Course on the Ex Situ Conservation of Plant Genetic Resources Bibliography Principal sources consulted Brundtland Report. 1986. CIED (Centro de Investigación, Educación y Desarrollo). (Accessed 27 Aug 2004) Glosario de términos: Biodiversidad y recursos genéticos. Lima, Peru. Also available at http://www.ciedperu.org/cendoc/biodiver/biodiv.htm Huiña-pukios Limitada. 2002. Glosario ecológico. In Difusión y conservación de la biodiversidad (Web site). Rengo, Chile. Available at http://www.geocities.com/biodiversidadchile/ glosario1.htm (accessed 1 Sept 2004]. Instituto de Biotecnología, Universidad Nacional Agraria ‘La Molina’. (Accessed 1 Sept 2004) Glosario biotecnología. Lima, Peru. Available at http://www.lamolina.edu.pe/institutos/ ibt/manualbiotec/glosario.html Jaramillo S; Baena M. 2000. Glosario. In Material de apoyo a la capacitación en conservación ex situ de recursos fitogenéticos. IPGRI, Cali, Colombia. 209 p. Also available at http://www.ipgri.cgiar.org/training/exsitu/web/arr_ppal_modulo.htm Law 19,300 of the Colombian Congress. Lawrence E, ed. 1995. Henderson’s dictionary of biological terms, 11th ed. Longman Group, Harlow, Essex, UK. 693 p. Poehlman JM. 1965. Mejoramiento genético de las cosechas. (Translated from the English by Nicolás Sánchez.) Editorial Limusa-Wiley, Mexico. pp 435–441. (Also available in English as Poehlman JM. 1986. Breeding Field Crops, 3rd ed. AVI Publishing, Westport, CT, USA.) Wikipedia Web site at http://en.wikipedia.org/wiki/Main_Page (accessed July–Aug 2007). Zaid A; Hughes HG; Porceddu E; Nicholas F. 2001. Glossary of biotechnology for food and agriculture: a revised and augmented edition of the Glossary of Biotechnology and Genetic Engineering. Research and Techology Paper 9. FAO, Rome. Available at http://www.fao.org/biotech/index_glossary.asp?lang=en (accessed 27 Aug 2004). Further reading (from Jaramillo and Baena 2000) Committee on Managing Global Genetic Resources: Agricultural Imperatives, National Research Council. 1993. Managing global genetic resources: agricultural crop issues and policies. National Academies Press, Washington, DC. 449 p. Frankel OH; Bennett E. 1970. Genetic resources in plants: their exploration and conservation. FAO; International Biological Programme, Oxford, UK. 554 p. Frankel OH; Brown AHD; Burdon JJ. 1995. Conservation of plant biodiversity. Cambridge University Press, UK. 299 p. 282 Glossary Glowka L; Burhenne-Guilmin F; Synge H; McNeely JA; Gündling L. 1994. A guide to the Convention on Biological Diversity. Environmental Policy and Law Paper No. 30. IUCN, Cambridge, UK. 161 p. Hartmann HT; Kester DE; Davies FT, eds. 1990. Plant propagation: principles and practices, 5th ed. Englewood Cliffs, NJ. 647 p. Hickey M; King C. 2000. The Cambridge illustrated glossary of botanical terms. Cambridge University Press, UK. 208 p. Hong TD; Ellis RH. 1996. A protocol to determine seed storage behavior. Technical Bulletin No. 1. IPGRI, Rome. 64 p. Also available at http://www.ipgri.cgiar.org/publications/pdf/ 137.pdf Hong TD; Linington S; Ellis RH. 1996. Seed storage behavior: a compendium. Handbooks for Genebanks No. 4. IPGRI, Rome. 656 p. Also available at http://www.cgiar.org/ipgri/doc/ download.htm IBPGR, comp. 1991. Elsevier’s dictionary of plant genetic resources. Elsevier Science Publishers, Amsterdam, Netherlands. 187 p. Jones SB; Luchsinger AE. 1986. Plant systematics, 2nd ed. McGraw-Hill, New York. Winburne, JN. 1962. A dictionary of agricultural and allied terminology. Michigan State University Press, East Lansing, MI. 905 p. Compilers of this Glossary Benjamín Pineda and Mariano Mejía. 283 CIAT Publication No. 360 Corporate Communications and Capacity Strengthening Unit Production editing: Gladys Rodríguez Production: Oscar Idárraga (layout) Julio César Martínez (cover design) Printing: Imágenes Gráficas S.A., Cali, Colombia