Practical guide for the application of the Genebank Standards for Plant Genetic Resources for Food and Agriculture Conservation through cryopreservation Food and Agriculture Organization of the United Nations Rome, 2025 Practical guide for the application of the Genebank Standards for Plant Genetic Resources for Food and Agriculture Conservation through cryopreservation Required citation: FAO. 2025. Practical guide for the application of the Genebank Standards for Plant Genetic Resources for Food and Agriculture – Conservation through cryopreservation. Commission on Genetic Resources for Food and Agriculture. Rome. https://doi.org/10.4060/cd6442en The designations employed and the presentation of material in this information product do not imply the expression of any opinion whatsoever on the part of the Food and Agriculture Organization of the United Nations (FAO) concerning the legal or development status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. The mention of specific companies or products of manufacturers, whether or not these have been patented, does not imply that these have been endorsed or recommended by FAO in preference to others of a similar nature that are not mentioned. ISBN  978-92-5-140004-3 © FAO, 2025 Some rights reserved. 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FAO photographs that may appear in this work are not subject to the above-mentioned Creative Commons licence. Queries for the use of any FAO photographs should be submitted to: photo-library@fao.org.  Sales, rights and licensing. FAO information products are available on the FAO website (www.fao.org/ publications) and print copies can be purchased through the distributors listed there. For general enquiries about FAO publications please contact:  publications@fao.org. Queries regarding rights and licensing of publications should be submitted to: copyright@fao.org.  mailto:photo-library@fao.org mailto:publications@fao.org mailto:copyright@fao.org iii Foreword v Acknowledgements vii Preface ix 1. Introduction 1 2. Considerations for cryopreservation 13 3. Acquisition of germplasm 21 4. Cryopreservation of diverse propagule types 29 5. Long-term storage in liquid nitrogen 47 6. Viability/regrowth assessment, monitoring of cryopreserved propagules and post-cryopreservation quality assessment 53 7. Replacement of cryopreserved inventories 65 8. Distribution 71 9. Safety duplication 77 10. Documentation 83 11. Personnel, security and safety 89 12. Infrastructure and equipment 97 13. References 103 14. Further information/reading 111 Annex – Risks and associated mitigation 113 Contents Tables 1. Descriptions of some plant propagule types that can be cryopreserved 8 2. Underlying principles and related genebank operations . for cryopreservation 11 3. Comparison of liquid and vapor phase liquid nitrogen storage 50 4. General infrastructure and equipment recommended for a cryobank 100 Figures 1. Relationship among individuals that comprise a population and those that are preserved as individual genotypes=clones 6 2. Some types of plant propagules that can be cryopreserved 7 3. Relationships among topics presented in the different practical guides for orthodox seeds, field genebanks, in vitro culture, non-orthodox seeds, and cryopreservation 9 4. Major operations for conservation of germplasm through cryopreservation 10 5. Depiction of the process from research to routine implementation of cryopreservation 19 6. Relationships among activities covered in the cryopreservation practical guide 20 7. Summary of the workflow and activities for the acquisition of germplasm 28 8. Summary of the workflow and activities for cryopreservation of diverse propagule types 45 9. Summary of the workflow and activities for long-term storage in liquid nitrogen 51 10. Types of viability assessments for cryopreserved materials 61 11. Summary of the workflow and activities for viability/regrowth assessment, . monitoring of cryopreserved propagules and post-cryopreservation quality assessment 64 12. Summary of the workflow and activities for replacement of cryopreserved inventories 69 13. Summary of the workflow and activities for distribution of germplasm 76 14. Summary of the workflow and activities for safety duplication of germplasm 81 15. Summary of the workflow and activities for documentation 87 16. Summary of the workflow and activities for personnel and security 96 Box 1. Considerations to identify crops or collections to cryopreserve 16 v The international development community and governments are striving to achieve the Sustainable Development Goals (SDGs), including the eradication of hunger, by 2030. The imperative of generating and disseminating the solutions that work for farmers as means to achieve the SDGs provides the backdrop for FAO’s Strategic Framework 2022- 2031. The Strategic Framework aims to transform current suboptimal agricultural and food systems to become more efficient, inclusive, resilient and sustainable as envisaged in its four aspirations: better production, better nutrition, a better environment and a better life. With about 80 percent of food being plant-based, these efforts will benefit greatly from sustainable crop production systems, which generate increased yields of nutritious food with fewer external inputs than are currently the case, even under worsening climate change scenarios. One critical element of such systems is a diverse suite of progressively superior crop varieties that are input use-efficient, nutritious, adapted to target agroecologies, and resilient to biotic and abiotic stresses. Plant breeders need access to the widest possible spectrum of the sources of heritable variations in order to breed such crop varieties. Plant genetic resources for food and agriculture (PGRFA) – that include improved crop varieties, farmers’ varieties/landraces and the wild relatives of crops – are the sources of such variations. The safeguarding of characterized and documented PGRFA in genebanks is a reliable means to ensure their availability to current and future generations – both for direct use and for research and plant breeding. FAO and partners have been cognizant of the critical importance of effective genebank operations to sustainable crop production systems. In addition, in recognition of the global interdependence on PGRFA, facilitated through the exchange of germplasm, the need for the harmonization of genebank procedures has always been at the forefront of FAO’s work on the conservation and sustainable use of PGRFA. This was why FAO, through Foreword vi its Commission on Genetic Resources for Food and Agriculture, published the Genebank Standards for Plant Genetic Resources for Food and Agriculture (Genebank Standards) in 2014. The Genebank Standards provide international standards for ex situ conservation of PGRFA in seed genebanks, field genebanks, in vitro culture and cryopreservation. Deemed a seminal reference material, one feedback provided by genebank practitioners was that the utility of the Genebank Standards would be enhanced through the development of companion volumes that detail the action steps of the genebank workflow in a sequential manner and provide guidance on the complex steps and decisions required. In response to this feedback, FAO has developed this Practical guide for the application of the Genebank Standards for Plant Genetic Resources for Food and Agriculture: Conservation through cryopreservation, along with the Practical guide for the application of the Genebank Standards for Plant Genetic Resources for Food and Agriculture: Conservation of species producing non-orthodox seeds, which was developed as a separate volume. These two companion volumes to the Genebank Standards complement the earlier three volumes published in 2022: the Practical guide for the application of the Genebank Standards for Plant Genetic Resources for Food and Agriculture: Conservation of orthodox seeds in seed genebanks; the Practical guide for the application of the Genebank Standards for Plant Genetic Resources for Food and Agriculture: Conservation in field genebanks; and the Practical guide for the application of the Genebank Standards for Plant Genetic Resources for Food and Agriculture: Conservation via in vitro culture. These companion volumes, prepared in an easy-to-understand format, will be useful for genebank technicians as operational handbooks; for genebank managers as streamlined instructional materials; and, for all interested in genebank operations, a handy reference material. Yurdi Yasmi Director Plant Production and Protection Division vii The Practical guide for the application of the Genebank Standards for Plant Genetic Resources: Conservation through cryopreservation was produced by FAO’s Plant Production and Protection Division under the supervision of Mr Chikelu Mba. Endorsed by FAO’s Commission on Genetic Resources for Food and Agriculture (Commission) at its Twentieth Regular Session, 24 to 28 March 2025, the guidance provided by the body and its Members are gratefully acknowledged. Contributors Bonnie Furman Food and Agriculture Organization of the United Nations Gayle Volk United States Department of Agriculture - Agricultural Research Service Hugh Pritchard deserves special mention for his contributions to the early development of this practical guide. Inputs were also provided by the Members of the Commission, as well as several individuals, in particular Dani Ballesteros, Jean Carlos Bettoni, Luigi Guarino, Sandya Gupta, Niveen Hassan, Fiona Hay, Sarada Krishnan, Charlotte Lusty, Mario Marino, Arshiya Noorani, Bart Panis, Valerie Pence, Esther Uchendu, Rainer Vollmer, Christina Walters and Jinmei Zhang. Special thanks to Alessandro Mannocchi for the design and layout of the publication and to Katheryn Chen for providing illustrations. Thanks also to Suzanne Redfern for her many contributions. The preparation and publication of this practical guide have been made possible by the contribution of many other individuals. FAO thanks them most sincerely for their time, commitment and expertise. Acknowledgements ix The ex situ conservation of plant genetic resources for food and agriculture (PGRFA) in genebanks is aimed at safeguarding them for use by current and future generations – both directly by end users and as materials for research and plant breeding. Genebanks, therefore, ultimately contribute to sustainable crop production systems and hence, food security and nutrition. However, genebanks must be managed effectively in order to conserve these resources in optimal conditions and make,them available for use. Genebanks also play a major role in fostering global collaboration on PGRFA through germplasm exchange, including across national boundaries. The Genebank Standards for Plant Genetic Resources for Food and Agriculture (Genebank Standards), published in 2014, aimed at the harmonization of genebank operations, i.e. the storage of the accessions, their characterization and evaluation and the documentation of associated data, across genebanks and countries. The Genebank Standards set the benchmark for current scientific and technical best practices. Addressing an identified need for the articulation of the stepwise activities of routine genebank operational workflows, the Practical guide for the application of the Genebank Standards for Plant Genetic Resources for Food and Agriculture: Conservation through cryopreservation was developed. Endorsed by FAO’s Commission on Genetic Resources for Food and Agriculture at its Twentieth Regular Session in 2025, this practical guide presents the information contained in the Genebank Standards in a format that presents the action steps of genebank workflow in a sequential manner. The series of interrelated operations presented, are based on the underlying principles of genebank management, namely: identification of accessions; maintenance of viability; maintenance of genetic integrity during storage and regeneration; maintenance of germplasm health; physical security of collections; availability, distribution and use of germplasm; availability of information; and proactive management. Preface x The sections included in this practical guide are: considerations for cryopreservation; acquisition of germplasm, cryopreservation of diverse propagule types, long-term storage in liquid nitrogen, viability/regrowth assessment, monitoring of cryopreserved propagules and post-cryopreservation quality assessment, replacement of cryopreserved inventories, distribution; safety duplication, documentation; and, personnel, security and safety. A summary diagram of the associated workflow and activities supports each of these operations. An additional section considers the suggested infrastructure and equipment for designing or modifying the facilities of a seed genebank. A final section provides a list of references to provide guidance and/or technical background on seed genebank operations and management. An annex identifies the potential risks associated with the different genebank operations and their respective proposed preventive measures. This practical guide is part of a series of publications conceived as companion volumes to the Genebank Standards aimed at facilitating their more widespread application. Genebank managers may use the practical guide as a basis for the development of standard operating procedures, quality management systems or, simply, as a handbook. Conservation through cryopreservation 1 1. Introduction Practical guide for the application of the Genebank Standards for Plant Genetic Resources for Food and Agriculture Conservation through cryopreservation 2 Cryotanks at the USDA-ARS National Laboratory for Genetic Resources Preservation, Fort Collins, United States of America © G . V ol k 3 Introduction to cryopreservation Plant genetic resources that are cryopreserved, stored at liquid nitrogen (LN) temperatures in a living state, have the potential to remain viable for decades at low cost and with minimal maintenance, without requiring regeneration. Cryopreservation technologies have been developed and implemented in numerous genebanks to preserve plant materials, particularly for security back-ups, that would be expensive to replicate in other forms, such as duplicates in field, greenhouse/screenhouse, or in vitro conditions (Panis, Nagel and Van den houwe, 2020; Nagel et al., 2024). Cryopreservation is a complicated, resource-intensive technology that has been successfully applied to secure materials in some genebanks. Successful cryopreservation programmes require long-term funding and institutional support for infrastructure, skilled personnel, and equipment for both research and routine implementation. As a result, cryopreservation is not a required component for all genebanks. Cryopreservation is not a required component for all genebanks Collection types Collections of plant genetic resources conserved ex situ can be maintained in seed genebanks, field genebanks and in vitro/cryopreservation genebanks. Seed genebanks maintain desiccation-tolerant orthodox seeds, where lower moisture and temperature decrease the rate of metabolic processes increasing seed longevity. Field genebank and in vitro conservation activities are aimed at the conservation of plants that are Practical guide for the application of the Genebank Standards for Plant Genetic Resources for Food and Agriculture Conservation through cryopreservation 4 propagated vegetatively or that produce non-orthodox seeds, also known as recalcitrant or intermediate seeds. Field genebanks are also used for plants that produce very few seeds or that have a long life cycle to generate breeding and/or planting materials. It includes the maintenance of plants in the field, greenhouse/screenhouse, or shade houses (FAO 2014). In vitro collections are usually materials that are propagated vegetatively and maintained using standard tissue culture or slow-growth (reduced temperature, low light, modified medium) conditions (Engelmann, 2004). Species producing non-orthodox seed can also be maintained in vitro. Seed bank, field genebank and in vitro collections (as defined above) are generally considered the active collections of the genebank. These are used for regeneration, multiplication, distribution, characterization, and evaluation. The base collection is a backup of the active collection, which can be at –18  oC (for orthodox seeds) or cryostorage, depending on the type of material that is stored. It is recommended that base collections be safety duplicated at a secondary location (FAO, 2014).1 For orthodox seeds, this secondary location is often the Svalbard Global Seed Vault. Genebank components Some genebanks operate from a single facility/location for all activities, while others have a highly dispersed system with genebank units at multiple locations to place crop collections where they can be regenerated or maintained under appropriate field conditions. Single location genebanks have the active and base collections co-located, facilitating the acquisition of materials for cryopreservation. However, this co-location could be a detriment if a natural disaster were to affect both the active and base collection at the same time. In contrast, distributed genebank units within a larger genebank system have the advantage of locating crop collections in diverse conditions that are amenable to the crops, which is key for both seed regeneration and field maintenance of clonally propagated crops. Distributed genebank systems that employ cryopreservation technologies to secure collections may have a single location dedicated for cryopreservation activities, rather than duplicating the unique cryofacility requirements at multiple dispersed locations. To ensure effective long-term storage, this arrangement requires careful coordination among multiple genebank units because materials must be shipped from the active site to the base collection for processing and storage. 1 Standards 4.9.1, 5.10.4, and 6.8.4. 5 1. Introduction Accessions and inventories Within this guide, the term “accession” refers to a unique sample of plant genetic resources in the genebank, which could range from being a specific fruit tree cultivar in the field to a diverse bag of landrace seeds obtained from a market. The term “inventory” refers to one of multiple duplicates of that accession in the genebank. There could be a seed inventory in medium-term storage and another inventory for the same accession in long-term storage. Similarly, there may be multiple inventories (all genetically identical) of a fruit tree cultivar in the field, and additional inventories of the same cultivar stored in LN. In most cases, cryopreservation is used to make a longer-lived, safer inventory of an accession that is maintained elsewhere. Base collections and the role of the cryobank The establishment of secure, safety duplicated base collections at a secondary location, either in cold storage, in the field, in vitro or in a cryobank, is critical to reducing the risk of loss and makes it essential that accession and inventory information is carefully managed. Some genebanks rely on cryopreservation for preserving some of the base collection of an active collection. However, if materials are lost or removed from the active sites, the cryopreserved base collection materials may be the only remaining samples. Some genebanks may strategically use cryostorage to preserve only their inventories for some collections. This is particularly applicable for some recalcitrant and intermediate seed collections for which there may not be corresponding field inventories in the genebank. Cryopreserved collections may be composed of orthodox seeds, materials from a field genebank, materials from in vitro culture or from species producing non-orthodox seeds. Cryopreserved collections may be comprised of materials, such as pollen, that are not included in the other practical guides. Conservation targets The propagule choice to be cryopreserved depends on the plant species, the facilities available, the technical expertise, whether in vitro/cryopreservation protocols have already been developed, and on whether the genes or the specific genetic combination are the programme conservation targets (Bettoni, Bonnart and Volk, 2021; Zhang et al., 2023). Ideally, protocols should be applicable across a wide range of genotypes. One key consideration for plant genetic resource collections is whether the materials that are maintained (as growing plants, seeds or in a cryopreserved state) capture the desired conservation target (Walters et al., 2008). In some seed-propagated cultivars, seed lots are homogeneous, so that some to many of the individuals have the same genotype, Practical guide for the application of the Genebank Standards for Plant Genetic Resources for Food and Agriculture Conservation through cryopreservation 6 according to the level of the self-fertilization (autogamy) of the species. In wild-collected seeds, such as for crop wild relatives, each seed often represents a unique genetic combination, thus there is a wide range of diversity in a single heterogeneous seedlot (Figure 1). Similarly, pollen grains are each unique genetic combinations of male gametes of an individual. In contrast, for clonally propagated crops, such as many fruit, nut and some vegetable cultivars, all the individuals representing a cultivar are genetically identical because they are vegetatively propagated from the same original source plant (Figure 1). For these, each dormant bud or shoot tip collected from an individual (specific genotype) is genetically the same as all the others. Consideration of these factors is key when planning cryopreservation activities. Each of the cryopreserved explants of homogeneous cultivars are genetically identical, so a single individual successfully captures the conservation target. However, a larger quantity of seeds may be necessary to capture the diversity represented in a highly heterogeneous seed lot during storage, regeneration or for distribution. In some cases, seeds of these populations cannot be stored, so multiple genetic lines of clonally propagated cultures can capture the genetic diversity of a wild population (Figure 2). Propagules Cryopreservation technologies can be applied to a wide range of propagule types, including orthodox seeds, intermediate seeds, and recalcitrant seed-derived embryos/ embryonic axes, dormant buds, shoot tips and pollen (Figure 1; Table 1). Figure 1. Some types of plant propagules that can be cryopreserved Recalcitrant seeds e.g. Quercus Orthodox seeds e.g. Phaseolus Pollen e.g. Carya Intermediate seeds e.g. Carica Embryogenic calli e.g. Panax Shoot tips e.g. Vitis Dormant buds e.g. Malus 7 1. Introduction Many of these propagule types have the advantage of cryopreserving organs or regenerable groups of cells, which generally increases the likelihood of multiple cells contributing to the regeneration of a plant after cryo-exposure. Some propagule types, such as recalcitrant seeds (embryos/embryonic axes), have not been extensively implemented as cryopreserved propagules within genebanks, but there are active research programmes with the intent of materials being cryobanked when methods/ resources are available. In contrast, suspension cells and callus are undifferentiated cell types. Although they may be more easily cryopreserved than organized tissues, they also have a greater likelihood of somaclonal variation. Going through dedifferentiation to callus and then regenerating meristems can provide more opportunities for somaclonal variation than the direct outgrowth of cryopreserved meristems. Cryopreservation of suspension cells and callus (i.e. ginseng, Lei et al., 2021), as well as shoots derived from them and somatic embryos, are not covered in this practical guide. This guide includes the cryopreservation of desiccation tolerant pollen. Pollen that is desiccation sensitive can also be cryopreserved using specialized techniques (Nebot et al., 2021), but this not covered herein (i.e. Amphasys AG, 2022; Powerpollen, 2024). Figure 2. Relationship among individuals that comprise a population and those that are preserved as individual genotypes=clones Seed accession = Population Clonal accession = Individual genotype Practical guide for the application of the Genebank Standards for Plant Genetic Resources for Food and Agriculture Conservation through cryopreservation 8 Table 1. Descriptions of some plant propagule types that can be cryopreserved Propagule type Description Orthodox seed Seeds that can be dried to low moisture content and stored at low temperatures to increase seed longevity. Homogeneous or heterogeneous seed lots are preserved. Intermediate seed Seeds that can be at least partially dried, but have shorter longevity at –18 oC, and improved longevity when cryopreserved. Homogeneous or heterogeneous seed lots are preserved. Recalcitrant seed Seeds that are not desiccation-tolerant; they do not dry during the later stages of development and are shed at high water contents (in the range of 0.3–4.0 g g-1). The loss of water rapidly results in decreased vigour and viability, and seed death occurs at relatively high water contents. Homogeneous or heterogeneous seed lots are preserved. Dormant buds Plant buds in a physiologically arrested state during the winter months. Specific genotypes are preserved. Shoot tips Growing point of a plant that contains a meristem and surrounding immature leaves. Specific genotypes are preserved. Pollen Male gametes of plants that are either desiccation tolerant (able to survive in a dry state) or desiccation sensitive (unable to be dried below ~30 percent moisture content, fresh weight basis). Callus Undifferentiated plant cells usually derived from a single individual that can be treated to make somatic embryos or shoots. Specific genotypes are preserved. Purpose of the practical guide The Practical guide for the application of the Genebank Standards for Plant Genetic Resources for Food and Agriculture: Conservation through cryopreservation has been developed to be used as a companion volume to the Genebank Standards for Plant Genetic Resources for Food and Agriculture (Genebank Standards) (FAO, 2014). This practical guide is directly linked to the other practical guides in this series. Practical guides for the application of the Genebank Standards for Plant Genetic Resources for Food and Agriculture include: l Conservation of orthodox seeds in seed genebanks (FAO, 2022a); l Conservation in field genebanks (FAO, 2022b); l Conservation via in vitro culture (FAO, 2022c); l Conservation of species producing non-orthodox seeds (FAO, 2025); and l Conservation through cryopreservation. As a result, it refers to the other practical guides, as appropriate (Figure 3). 9 Figure 3. Relationships among topics presented in the different practical guides for orthodox seeds, field genebanks, in vitro culture, non-orthodox seeds and cryopreservation Conservation in genebanks by means of cryopreservation can be broken down into a series of interrelated operations: acquisition, cryopreservation and storage, viability/regrowth and assessment and monitoring, replacement of cryopreserved inventories, distribution, safety duplication and documentation (Figure 4). This practical guide presents practices and activities critical to each operational area and is based on the underlying principles of genebank management, namely: identification of accessions; maintenance of viability; maintenance of genetic integrity during storage and regeneration; maintenance of germplasm health; physical security of collections; availability, distribution and use of germplasm; availability of information; 1. Introduction Freezer storage Field genebank Orthodox seed Non-orthodox seed In vitro culture Cryopreservation Practical guide for the application of the Genebank Standards for Plant Genetic Resources for Food and Agriculture Conservation through cryopreservation 10 and proactive management (Table 2). It outlines workflows for routine genebank operations for conservation by cryopreservation and supports the application of the Genebank Standards. The purpose of this guide is to present the information contained in the Genebank Standards in a format that details the actions of the genebank workflow in a sequential manner and thereby facilitates more widespread adoption of the Genebank Standards. Genebanks may use the activities outlined in this guide as a basis for the development of standard operating procedures (SOPs) (Harding and Benson, 2015; IITA, 2012), best practices (Benson et al., 2011a), and quality management systems (QMS) (Benson et al., 2011b; CGIAR Genebank Platform, 2021) for conserving these germplasm collections, defining in detail how to carry out each activity. Figure 4. Major operations for conservation of germplasm through cryopreservation Documentation Distribution and Safety duplication Cryopreservation and Storage in liquid nitrogen Viability/regrowth assessment and monitoring Conservation through cryopreservation Acquisition Replacement of cryopreserved inventories 11 Table 2. Underlying principles and related genebank operations for cryopreservation Genebank principle Summarized genebank operations Identity of accessions Passport data collected and recorded. Taxonomic and/or genetic identity verified. Permanent and unique accession number assigned and used in all documentation. Accessions handled carefully to avoid mixing, and all samples labelled and tracked throughout all genebank operations. Maintenance of viability Best practices followed and timing optimized during collecting, processing, introduction into cryopreservation, long-term storage in liquid nitrogen, viability/regrowth assessment and secure backup storage. Cryopreservation conditions optimized and monitored. Replacement undertaken when necessary. Maintenance of genetic integrity Collection and handling of samples conducted in a manner that ensures they represent the original population or clone. Best practices followed and genotypic data collected (when possible) to ensure sample identity and that no somaclonal variation or changes occurred during the cryopreservation, storage, and regrowth processes. Maintenance of germplasm health The stock-plants should preferably be free of fungi, bacteria, and viruses. Quarantine procedures undertaken when needed. Best practices followed during collection, processing, introduction into in vitro culture (if applicable), cryopreservation, and viability/regrowth. Contamination monitored and managed in the laboratory and in the field or greenhouse/screenhouse. Physical security of collections Risk management strategy developed and implemented. Appropriate genebank infrastructure in place and maintained. Accessions safety duplicated. Emergency or contingency plans in place. Availability and use of germplasm Germplasm acquired and distributed according to legal and phytosanitary requirements. Cryopreserved materials usually provided to genebank staff to replace accessions in the active collection or safety duplicates are returned to the original provider. Relevant documentation provided to recipients of genebank material. Availability of information Genebank information management system in place. Passport and accession management data secured by regular data back-ups. Passport and other relevant data available and accessible to external users, as appropriate. Proactive management of genebanks Standard operating procedures developed and available to staff. Data and information generated during genebank activities available to managers and staff. Well-trained staff employed and protected by occupational safety and health measures. Genebank staff capacities kept up to date and training provided as necessary. Cryopreservation and recovery methods are recorded and documented. 1. Introduction Practical guide for the application of the Genebank Standards for Plant Genetic Resources for Food and Agriculture Conservation through cryopreservation 12 Conservation through cryopreservation 13 2. Considerations for cryopreservation Practical guide for the application of the Genebank Standards for Plant Genetic Resources for Food and Agriculture Conservation through cryopreservation 14 In vitro banana cultures at the International Musa Germplasm Transit Centre, Katholieke Universiteit Leuven, Belgium © B . P an is 15 Choosing to cryopreserve The specialized nature of cryobanks – facilities that preserve genetic resources in a cryopreserved state – along with the need for large, regular supplies of LN, can make establishing a cryobank prohibitively expensive for some genebanks. Building and equipping a cryopreservation facility requires reliable, guaranteed resources for technical support, maintenance, cryotanks and LN. This long-term investment and dependence upon LN make some genebanks unable to develop cryobanks. Given the infrastructure required, genebanks should carefully consider their long-term capacity and whether it would be cost-effective to partner with regional or international centres for cryobanking needs. Regional cryobanks may serve designated geographic regions that include multiple national or international genebanks. These regional facilities may also be available as backup locations for collections that are cryopreserved elsewhere. A global back-up cryopreservation facility has also been considered (Acker et al., 2017). Cryopreservation programmes currently have research and implementation components. This is because the technology can sometimes be crop-specific or difficult to implement directly from the literature. At a minimum, cryopreservation methods must be tested on a smaller scale before routine processing begins for large numbers of accessions. Genebanks must also consider the costs to acquire cryotanks, regular supplies of LN, employ skilled technical staff and implement an information management system, as well as a tissue culture facility (for materials that will be derived from or recovered as in vitro plants). This requires long-term institutional support. Storing materials in cryotanks cooled by LN may not be the most cost-effective storage method for standard orthodox seeds that survive for decades or even centuries in freezer Practical guide for the application of the Genebank Standards for Plant Genetic Resources for Food and Agriculture Conservation through cryopreservation 16 storage at –18 oC.2 As a result, genebanks that maintain only collections of orthodox seeds usually do not have associated cryopreservation programmes. Genebanks with small field or in vitro collections may find it more cost-effective to duplicate field or in vitro collections at a secondary site rather than introducing them into LN. In some cases, there may not be effective methods available to cryopreserve the crop of interest, thus a large investment is needed to first develop the method through research and then implement it. Selection of crops and prioritizing materials for cryopreservation If a genebank already has a cryopreservation facility, genebank staff must carefully consider many factors to identify and prioritize which crops and which propagule types will be cryopreserved (Box  1). Cryopreservation may be considered when duplicate collections are prohibitively expensive, methods are available and (ideally) routinely implemented in other genebanks, field collections are vulnerable due to abiotic or biotic stresses, seeds cannot be successfully stored in freezer conditions, in vitro collections do not retain their vigour, funding availability, uniqueness of the collection, or other factors that could prioritize a specific active collection for cryopreservation. In addition, access to adequate quantities and quality of propagules at appropriate physiological states should be considered. Consultations between active and base collection managers as well as genebank administrators and reviews of global crop conservation strategies may reveal collection vulnerabilities and cryopreservation priorities. 2 Conserving orthodox or intermediate seed under cryopreservation is undertaken when seeds are not long-lived under conventional cold storage conditions. Box 1. Considerations to identify crops or collections to cryopreserve l Funding availability for cryopreservation l Duplicate collections are prohibitively expensive l Methods are available and (ideally) routinely implemented in other genebanks l Access to adequate quantities and quality of propagules at appropriate physiological states l Collection has been pathogen-tested and, preferably, free from pathogens (sanitized) l Seeds cannot be successfully stored in freezer conditions l Field collections are vulnerable due to abiotic or biotic stresses l In vitro collections do not retain their vigour l Collection vulnerabilities identified after reviews of global crop conservation strategies l Uniqueness and difficulty to replace the collection l Collection confirmed to be true-to- type l Evaluation and characterization data are available 17 It is critical to select the appropriate propagule type to cryopreserve the desired conservation target. Propagules such as seeds or pollen may be more applicable to capture a larger portion of population genetic diversity than clones. Seed storage behaviour is often described as a continuum between extremely short-lived, desiccation- sensitive recalcitrant seeds and extremely long-lived orthodox seeds. l Most orthodox seeds are stored at –18 to –20 oC for long-term storage. However, there are some shorter-lived orthodox seeds or samples that are very difficult to replace that are candidates for LN storage (Ballesteros et al., 2021; FAO, 2025). For cryopreservation, orthodox seeds should be dried to about 32 percent relative humidity (RH) at 18  oC and then stored in the vapour phase of liquid nitrogen (LNV). l Recalcitrant seeds do not tolerate removal of water that will freeze upon exposure to subzero temperatures, forming ice crystals that will damage the tissues. For recalcitrant seeds, cryopreservation of the whole seed is not usually possible when the seeds are large (>0.5 to 1 cm). For large seeds, the embryonic axes are excised and cryopreserved. The drying, cryoprotection and cooling procedures must be adjusted, and are often species-specific (Ballesteros, Fanega-Sleziiiiak and Daves, 2021). l Intermediate seeds have a storage response that is “intermediate” to recalcitrant or orthodox seed storage behaviour. Intermediate seeds often survive drying at 50–65 percent RH or lower but may be damaged if dried below 25 percent RH. They may lose viability faster in the freezer (–18 °C) than in cold storage (4–8 °C) (FAO, 2025). Examples include, for example, Salix, Populus, Primula, Anemone, and Corylus (Ballesteros, Fanega-Sleziiiiak and Daves, 2021). For clonal conservation of vegetatively propagated plants, dormant buds or shoot tips are preferred over seeds or pollen, as they can be established as whole plants with a high level of genetic fidelity and they also offer the opportunity to preserve selected individuals from the population that display desirable traits, such as disease resistance. Irrespective of the type of the propagule used, successful cryopreservation relies on high-quality source plants, which therefore have a high regrowth capacity. Some genebanks have collections that have been screened and sanitized to eradicate pathogens (fungi, bacteria, viruses and/or viroids). In other cases, genebanks do not have the capacity to screen collections for pathogens, let alone clean them up. The cleanliness of collections should be considered as cryopreservation priorities are determined. Ideally, only sanitized and indexed collections are cryopreserved. The process of sanitization of some collections, may result in in vitro plants that could then be sources of materials for cryopreservation. If unclean collections are cryopreserved, then it may be necessary to re-process those materials for cryopreservation if they are sanitized at a later date. 2. Considerations for cryopreservation Practical guide for the application of the Genebank Standards for Plant Genetic Resources for Food and Agriculture Conservation through cryopreservation 18 In addition, some crop collections in genebanks have been genotyped and/or phenotyped to determine if they are true-to-type with respect to taxonomy and cultivar. These collections that are confirmed to be true-to-type could be considered as priorities for cryopreservation. Method development Cryopreservation method development can range from a straightforward to a long- term endeavour. For orthodox (including short-lived orthodox, which might be referred to as intermediate) seeds, methods are generally available and readily applicable. For recalcitrant seeds, clonal propagules and pollen, methods must be identified through research. This research must identify optimum media for plant in vitro introduction, multiplication and regeneration, as applicable, depending upon the propagule type. In addition, methods and the extent of desiccation, cryoprotectant pre-treatment, cryoprotectant solution treatment, LN exposure, packaging and regrowth must all be optimized. This process of optimization requires many experiments with appropriate controls to determine the necessary parameters. Cryobiology is a state-of-the-art scientific discipline that is constantly evolving. New and improved methods are continuously being developed to preserve an ever-broader range of species and propagule types. One challenge is that methods must often be optimized for the particular species, or even genotype within the same species, and/or crop, and source plant conditions. As a result, methods must be tested in-house to confirm that they can be successfully applied prior to large-scale implementation. Applied research and testing may take months or years. If methods have not been previously developed, it often requires years of experimentation to develop new methods – and genetic resources of some crops have never been successfully cryopreserved despite decades of effort. Numerous reviews and best practices (such as Reed, 2008) have provided information about the key features of plant cryobank facilities, as well as how to select and optimize methods for successful cryopreservation, and these are noted in the text. Some examples are provided from preservation facilities including Royal Botanical Gardens Kew (United Kingdom of Great Britain and Northern Ireland), the USDA-ARS National Laboratory for Genetic Resources Preservation (NLGRP; Fort Collins, United States of America) and the International Potato Center (CIP; Lima, Peru; Vollmer et al., 2016). 19 From research to implementation Generally, methods are developed for four to five accessions that are representative of the desired range of genetic diversity within a crop/species. The process of determining if a method is ready for implementation is called “validation” (Benson et al., 2011b). If plants can be successfully regenerated with viability levels >50 percent (ideally higher) using the identified method, then the method may be ready for larger-scale implementation (Figure 5). In some cases, specific cultivars may not be amenable to the identified procedures and modifications may be needed. Some examples of crops for which cryopreservation has been widely implemented include coffee (intermediate seeds; Dussert et al., 1998), apple dormant buds (Höfer, 1998), and banana, potato, mint, and garlic shoot tips (Nagel et al., 2024). Although it may be labour-intensive and expensive to introduce materials into cryostorage, once cryopreserved the annual cost of their preservation in LN is often much less than the cost of maintaining duplicate field, greenhouse/screenhouse and in vitro plant collections, or regenerating seeds frequently due to their short storage life and is, above all, the safest strategy for the long-term storage of plant genetic resources, complementing traditional field genebanks and in vitro collections. In some cases, genebanks may retain only the cryopreserved accession when the field or in vitro materials are no longer living or when the field or in vitro collections are purposefully downsized. Figure 5. Depiction of the process from research to routine implementation of cryopreservation METHOD READY TO IMPLEMENT • Source plant material healthy and adequate quantity available • Cryopreservation method tested on >3 representative accessions • Regrowth (healthy plant) >50% RESEARCH • Method development • Regrowth assessments • Replicated experiments INSTITUTIONAL CAPACITY IN PLACE ROUTINE IMPLEMENTATION 2. Considerations for cryopreservation Practical guide for the application of the Genebank Standards for Plant Genetic Resources for Food and Agriculture Conservation through cryopreservation 20 When is a collection safely cryopreserved? Cryopreserved collections are usually base collections (backups) of active collections. Genebanks must determine their priority materials to be cryopreserved and the criteria, or standards, to determine if the material is secure. Relationships among conservation activities relating to cryopreservation Activities relating to cryopreservation are not the same as those necessary for orthodox seed, field and in vitro collections. This guide includes sections for many topics that relate cryobank activities (Figure  6), including: acquisition of materials for cryopreservation or that are already in the cryopreserved state; cryopreservation of materials when a method is already available; long-term storage of propagules in either LN or LNV; viability/regrowth assessment, periodic monitoring of viability/regrowth and post-cryopreservation quality assessment; replacement of cryopreserved inventories (when necessary); distribution (usually to replenish materials within the genebank); safety duplication of cryopreserved materials; and documentation. This publication only provides general guidance on the complex steps and decisions required when operating a genebank for cryopreservation. Each genebank will have its own circumstances, and the efficient management of particular collections will require careful consideration and procedural adjustments based on experience. For detailed technical specifications of the steps outlined in this guide, genebank staff will need to consult various sources of information, a few of which are referenced in this document. Figure 6. Relationships among activities covered in the cryopreservation practical guide Acquire Cryopreserve *Data management needed for every step Safety duplicate Assess viabilityReprocess, replace or distribute CryobankActive site 21 3. Acquisition of germplasm Practical guide for the application of the Genebank Standards for Plant Genetic Resources for Food and Agriculture Conservation through cryopreservation 22 Pecan pollen collection at the USDA-ARS National Collection of Genetic Resources for Pecans and Hickories, College Station, United States of America © G . V ol k 23 The genebank is recommended to have documented policies and/or procedures, as applicable, for acquiring germplasm that include abiding by legal, phytosanitary and other regulations and requirements.3 Materials to be cryopreserved are usually sampled from a genebank’s existing collections, where seeds and other propagules received through explorations and donations were originally received by the curators of the main collections.4 In some cases, materials may be received by other cryofacilities in the cryopreserved state. In these cases, propagules must remain cryopreserved throughout the entire transfer process. Detailed information about the acquisition of orthodox seeds, non-orthodox seeds and clonally propagated materials is available in other FAO practical guides (FAO, 2022a,b,c). ✔ Decisions to accept germplasm into a genebank’s collection are guided by the institute’s acquisition policy. The development of an acquisition policy ensures that collections remain manageable and meet users’ needs (Guarino, Rao and Reid, eds, 1995). l Genebank curators may interact with breeders, botanists and other scientists before deciding on new acquisitions. Institutes may also have a crop-specific or general advisory committee in place. l The health and viability status of collected or donated samples, availability of passport information (taxonomic identity, origin of the germplasm, etc.) and sample “uniqueness” (to avoid unnecessary duplicates) should also be considered 3 See Figure 7 at the end of this section for a summary of the workflow and activities for acquisition of germplasm. 4 Specific guidance is available for acquisition of orthodox seeds (FAO, 2022a), cuttings and vegetative materials (FAO, 2022b), materials maintained in vitro (FAO, 2022c), and recalcitrant and intermediate seeds (FAO, 2025). Practical guide for the application of the Genebank Standards for Plant Genetic Resources for Food and Agriculture Conservation through cryopreservation 24 in the decision-making process and are determinants for increasing the efficiency of collection preservation efforts. ✔ Germplasm added to the collection is legally acquired and accompanied by all relevant documentation.5 The process of germplasm acquisition is governed by national and international regulations such as phytosanitary/quarantine regulations and the International Treaty on Plant Genetic Resources for Food and Agriculture (International Treaty) or the Convention on Biological Diversity (CBD) for access to genetic resources (FAO, 2014). l The genebank should communicate with the National Focal Points for the International Treaty or other designated authorities on questions concerning germplasm acquisition. ✔ A permanent and unique accession number is assigned to each sample added to the genebank collection. Once the curator decides to accept a sample into the genebank, a unique accession number must be assigned. l A Digital Object Identifier (DOI) can be requested from the Secretariat of the International Treaty (FAO, 2021a). Both the accession number and the DOI remain with all material derived from the accession during all genebank handling. l If the donated material has an accession number assigned by the donor organization, a DOI, or both, keep these as alternative identifiers in the passport data. This is a critical means of ensuring the unambiguous association of information with the material. ✔ Germplasm added to the genebank collection is accompanied by associated data, as outlined in the FAO/Bioversity Multi-Crop Passport Descriptors.6 It is recommended that all new accessions, whether obtained through collection missions or donation from other institutes, be accompanied by the associated data detailed in the FAO/Bioversity Multi-Crop Passport Descriptors (Alercia, Diulgheroff and Mackay, 2015). l The association of data and associated inventories with the single accession must be clear, for example by using accession numbers and/or DOI. ✔ All acquisition data, including associated metadata, are recorded, validated and uploaded to the genebank’s information management system. Consider the use of electronic devices to avoid transcription errors and for ease of uploading. Otherwise, the use of indelible ink (or pencil) and clear, legible writing are necessary when recording data. The use of barcode labels and barcode readers facilitates accession management, speeds up the process and minimizes human error. 5 Standard 6.1.1. 6 Standard 6.2.2. 25 3.1 Transfer of materials within a genebank Plant materials may need to be transferred between the active site and the cryobank within a genebank system or between one genebank and a regional cryobank. When materials must be transported, specific protocols must be followed. ✔ Protocols for collecting, packaging and shipping seeds and other propagules are followed depending on the target species and through conversations with the cryopreservation programme. l Protocols are available for collecting orthodox seeds (FAO, 2022a) and for collecting propagules from species producing non-orthodox seeds or that are vegetatively propagated (FAO, 2022b,c; FAO, 2025). l Pollen is collected from either populations of plants or from single individuals, depending on the quantity and availability of inflorescences. Pollen preservation is most commonly performed for species with desiccation-tolerant pollen (Ballesteros and Pritchard, 2020). The process of collection is dependent upon species. In some cases, just the male (pollen containing) inflorescence will be collected. In others, entire flowers will be collected. The reproductive stage of flower maturation for successful pollen collection is dependent upon species. Collected materials must be air-dried and sifted as soon as possible after collection to release the pollen (Rajasekharan and Rohini, eds, 2023). l To prevent loss and deterioration of the material, the time between collection, processing and transfer to the genebank should be as short as possible (FAO, 2022b; section 3). ✔ Materials should be shipped overnight if genebank is in a different location, or as quickly as possible, and packaged to ensure safe delivery.7 l Use rigid, insulated packing material. l Shipments should be sent using the fastest means possible, by airfreight or courier, to avoid exposure to adverse environmental conditions and deterioration of quality. l A high degree of coordination between the sender, the shipping company and the receiving genebank is required. The shipping number and tracking information should be shared with the genebank staff; genebank staff should regularly check where packages are and when they are expected to arrive. Genebank staff should be ready to process the samples immediately upon arrival at the genebank. ✔ Visual inspection of the surface of fruits/seeds/buds and under the covering tissues is made, to detect signs of pathogens and insects. l Look for obvious signs of fungi such as discolouration and “furry” fungal colonies. l Check for holes in the seed coat/covering structures, which may indicate the presence of insect larvae and/or eggs and damage of internal tissues. 7 Standard 6.2.4. 3. Acquisition of germplasm Practical guide for the application of the Genebank Standards for Plant Genetic Resources for Food and Agriculture Conservation through cryopreservation 26 l Discard infected/infested seeds/buds according to normal biological waste management procedures; it may be necessary to destroy entire seed lots, if contamination rates are high. ✔ Material arriving at the cryobank is checked for damage/contamination and that it is accompanied by the appropriate documentation. All material arriving at the genebank is visually checked for damage/contamination by an authorized and knowledgeable person in a designated reception area and immediately placed into appropriate environmental conditions. Cross-checking should also be made to ensure the material is as described in the accompanying documentation (which may be sent in advance electronically). l Visual inspection of the surface of fruits/seeds and under the covering tissues is made, to detect signs of pathogens and insects. ¡ Look for obvious signs of fungi such as discolouration and “furry” fungal colonies. ¡ Check for holes in the seed coat/covering structures, which may indicate the presence of insect larvae and/or eggs, and damage of internal tissues. ¡ Discard infected/infested seeds according to normal biological waste management procedures; it may be necessary to destroy entire seed lots, if contamination rates are high. l Decontamination activities, such as treating samples with a surface disinfectant agent, are used (as needed and appropriate) to remove all adherent microorganisms, taking into account any decontamination treatment given prior to packaging and transport.8 l Quarantine measures are applied as necessary. l Inventories are confirmed to ensure that all the expected materials arrived. l All incoming material should be handled as soon as it arrives at the genebank and temporarily stored under conditions appropriate for that propagule type. ¡ Orthodox seed should be cleaned and dried for temporary storage (see FAO, 2022a; section 3). ¡ Recalcitrant seeds should be removed from structures as necessary, cleaned and maintained under short-term humid storage (see FAO, 2025; section 3). For recalcitrant species, intact fruits and seeds should be aerated and perhaps transferred to new, clean loosely-tied polyethylene bags, or sterile plastic buckets/boxes, while still maintaining the moisture content as far as possible: at 6±2 °C unless the seeds/fruits are of a species known to be susceptible to chilling injury, for example, Theobroma cacao, which should be kept at 17–30°C (Hor, Chin and Karim, 1984). ¡ Roots, tubers, cuttings, bulbs and dormant buds should be kept in storage under cool temperatures in a designated storage area free of pests (see FAO, 2022b; section 6). These materials are then used as source materials for explant excision or introduced in vitro for multiplication and cryopreservation. 8 Standard 6.1.5. 27 ¡ In vitro materials should be stored under optimum storage conditions (See FAO, 2022c). n Depending on the condition of the sample, it may be necessary to undergo multiplication prior to storage. ¡ Flowers/pollen are received in a dry state or harvested locally and dried on site. Inflorescences are sifted to remove flower parts and any insects. Moisture content is then adjusted and pollen is prepared for immediate cryopreservation (see section 4). 3.2 Materials transferred to cryobank from another genebank for safety duplication The Genebank Standards recommend that cryopreserved materials are safety duplicated at a secondary location in the cryopreserved state (FAO, 2014; see Chapter 8). As such, genebanks with cryostorage facilities may receive germplasm from another cryobank for the purpose of safety duplication. It is therefore important that the receiving genebank has the long-term capacity to maintain cryopreserved collections. ✔ All necessary agreements and documentation are in place prior to receiving materials. l Agreements should consider duration, best practices and costs. l Ensure that the genebank receives the required documentation (both for the genebank and the host country) and the applicable customs and phytosanitary procedures. This will help ensure timely movement of the germplasm. l The genebank is encouraged to use a Standard Material Transfer Agreement (SMTA) (FAO, 2021b,c). ✔ Material sent to the cryobank is prepared, packaged and sent in a way that ensures material arrives intact and viable.9 l Request that shipments are sent using the fastest means possible, by airfreight or courier, to avoid exposure to adverse environmental conditions and deterioration of quality. l Continuous tracking of the package, if possible, will ensure that cryobank staff are prepared to process the samples upon arrival at the cryobank. ✔ Material arriving at the cryobank is kept at LN or LNV temperatures throughout the transfer process. l Phytosanitary and quarantine regulations are followed, without warming the cryopreserved materials. 9 Standard 6.1.4. 3. Acquisition of germplasm Practical guide for the application of the Genebank Standards for Plant Genetic Resources for Food and Agriculture Conservation through cryopreservation 28 ✔ Received materials are documented. l Inventories are confirmed to ensure that all the expected materials arrived. l Provider receives confirmation that materials arrived successfully and their storage location. Figure 7. Summary of the workflow and activities for the acquisition of germplasm Acquisition of germplasm Germplasm added to the collection is legally acquired and abides by national, regional and international phytosanitary and any other import regulations and requirements. - Follow legal requirements: national regulations, International Treaty on Plant Genetic Resources for Food and Agriculture (Standard Material Transfer Agreement); Convention on Biological Diversity (prior informed consent and mutually agreed terms). - Follow phytosanitary requirements: import permit; phytosanitary certificate. Germplasm is obtained from a genebank’s own collection maintained at a different location from cryopreservation facilities. - Collect germplasm maintained in the field based on breeding system. - Collect from visibly healthy plants. - Ensure correct physiological age/stage for successful processing. - Carefully label and avoiding mixing samples. - Ensure short interval between collecting and transfer to genebank. Germplasm is transported between the active site and the cryobank. - Use rigid, insulated packing material. - Provide packing lists and coordinate shipments between sender and recipient. - Use overnight shipment when possible. Safety duplicates from another cryobank are received. - Have all necessary agreements in place for legal transfer of materials. - Ensure that cryopreserved materials remain at LN temperatures throughout the shipping process. - Inventory and document the materials received. - Provide storage location information to the provider. Record, validate and upload all cryopreservation data, including associated metadata. 29 4. Cryopreservation of diverse propagule types Practical guide for the application of the Genebank Standards for Plant Genetic Resources for Food and Agriculture Conservation through cryopreservation 30 Extracting the banana meristem for cryopreservation © Cr op T ru st /S . L an de rs z 31 The genebank is recommended to have documented policies and/or procedures, as applicable, for the conservation of germplasm through cryopreservation, including guidelines for the preparation of materials for cryostorage.10 Cryopreservation procedures and considerations are dependent upon the species and propagule types to be conserved. Generally, methods of protecting cells focus on generic protocols for seeds and species-specific protocols for clonal propagules. As such, there are no one-size-fits all protocols, and individual protocols go beyond the scope of this guide. It may be necessary to carry out a literature review to investigate whether protocols exist for each step of the cryopreservation process based on the species and type of propagule used and the facility available. If information is not available for a given species, it may be useful to test protocols for a related species. Methods derived from the literature should be thoroughly tested across a wide range of genetic diversity and optimized prior to implementation11 (see Benson et al., 2011a,b; Vollmer et al., 2019; Ballesteros, Fanega-Sleziak and Davies, 2021; Bettoni, Bonnart and Volk , 2021; Nagel et al., 2024;). The text below provides some basic guidance of tested procedures. ✔ The methodology to prepare samples for cryostorage is determined based on the species, propagule type and conditions of the source material. ✔ Only viable and disease-free materials are introduced into cryopreservation.12 ✔ Clean and sterile containers for cryostorage are used. 10 See Figure 8 at the end of this section for a summary of the workflow and activities for cryopreservation of diverse propagule types. 11 Standard 6.5.2. 12 Standard 6.5.1. Practical guide for the application of the Genebank Standards for Plant Genetic Resources for Food and Agriculture Conservation through cryopreservation 32 ✔ In vitro source plants that have not been subcultured for extended lengths of time are used as donors of propagules for cryopreservation, if possible. ✔ Adequate quantities of propagules are cryopreserved to ensure that genetic shifts do not occur if there is a decline in viability during storage.13 l Fewer propagules may be cryopreserved for clonally propagated materials, such as dormant buds and shoot tips, because each of the propagules in the cryopreserved inventories are genetically identical and, as a result, only one successful regrowth event is needed to regenerate an individual identical to the original source plant. ✔ Samples are protected from lethal ice formation by air-drying Air-drying can be achieved through use of the air current of a laminar flow cabinet/ hood or silica gel desiccant, application of cryoprotectants or a combination of both. ✔ Samples are packaged into vessels and materials suitable for storage in LN, such as cryovials, based on the propagule type, size and protocol used. ✔ Secure labels that will remain adhered to the package at LN temperatures are affixed to the packages. ✔ Samples are cooled fast enough to avoid intracellular ice formation during the cooling or warming process. ✔ A temporary holding cryotank is used to store cryopreserved samples prior to viability testing and, if viability reaches the cryobank parameters, the cryopreserved samples are transferred to the permanent cryotanks. ✔ Viability/regrowth assessments are performed on materials immediately after LN exposure to ensure that the cryopreservation procedure was successful.14 ✔ Samples are placed into LN/ LNV for long-term storage. When placing into LN/LNV, it is important to follow the following steps: l Unlock the cryotank and very gently lift the lid. If the lid is opened suddenly excess fogging will occur making it difficult to locate the appropriate rack. l Transfer materials to the cryotank quickly to ensure no warming occurs. ✔ All cryopreservation data, including associated metadata, are recorded, validated and uploaded to the genebank information management. 13 See section 6.7. 14 See section 6. 33 Data to consider include: l inventory identifiers that are associated with source accession; l propagule type; l propagule harvest date and other information related to sample collection (storage time, shipping records, etc.); l cryopreservation date; l storage vessel (cryovial, etc.); l number of propagules per storage vessel; l number of storage vessels in LN; l location of storage vessels in cryotank; l detailed methods for cryopreservation and viability/regrowth; l viability/regrowth level of inventory; l total number of viable propagules in cryostorage for each inventory; and l cryotechnician who processed the material. 4.1 Cryopreservation of orthodox seeds Some orthodox seeds lose viability too quickly in conventional seed genebank storage for freezer storage to be an effective method for long-term ex situ conservation. Cryopreservation of such seeds is recommended. There may be other reasons for conserving orthodox seeds at very low temperatures, for example, if the seed sample represents a particularly valuable species from which it is difficult to collect seeds. Procedures for LN storage of orthodox seeds differ from standard –18  oC storage procedures. In particular, to avoid a potential risk of overdrying when seeds are dried at ambient temperature and then stored at LN temperatures, seeds are not dried to such low moisture contents as used for conventional seedbank storage (Ballesteros, Fanega-Sleziak and Davies, 2021). Optimum conditions (moisture content and cooling rate) that provide high levels of initial survival and seed stability during long-term storage are currently under investigation (C. Walters, personal communication). ✔ Seeds are subjected to initial viability testing following optimized and documented procedures. l Seeds to be cryopreserved should be of high quality and high viability. l It is important to use standard protocols so that viability monitoring tests are comparable, including over time, ideally using replicated testing procedures (section 6; see FAO, 2022a). ✔ Seed samples are equilibrium dried to optimum moisture content for cryostorage. l In general, orthodox seeds dried to 60 percent RH and below survive short- term exposure to LN. The optimal moisture content for storage varies among species, and even within species, (Nits et al., 2024) but generally, adjustment 4. Cryopreservation of diverse propagule types Practical guide for the application of the Genebank Standards for Plant Genetic Resources for Food and Agriculture Conservation through cryopreservation 34 to 30 percent RH should ensure seeds of most species are dried to acceptable moisture content (FAO, 2025; C. Walters, personal communication). l Moisture content can be manipulated by placing seeds in a room with controlled relative humidity and temperature or by placing seeds in chambers with saturated salt solutions. The humidity level within the chamber is dependent upon the salt selected (Pritchard and Nadarajan, 2008). ✔ Seeds are assessed for moisture content to ensure they are adequately dry. l Moisture content of the sample is determined if sufficient quantity of material is available (see FAO, 2025). l The recommended sample size per replicate is 0.5–1.0 g for small seeds and 4.5–5.0 g for larger seeds (cereals, legumes). Moisture content determination should be performed with minimum two independent replicates, and three to four replicates for greater accuracy. ✔ Seeds are packaged into materials suitable for storage in LN, such as cryovials, based on size. l If enough seed and resources are available, it is recommended that seeds be aliquoted into multiple packages to simplify the distribution and/or viability assessment processes. l The quantity of seeds stored is dependent upon seed size, quantity available, storage space available, seed-lot heterogeneity, the number of seeds utilized for viability assessments and the anticipated number of seeds needed for distributions to the active site. ✔ Secure labels that will remain adhered to the package at LN temperatures are affixed to the packages. l Bar codes or labels with indelible ink, suitable for use in LN, with inventory information may be applied for ease in sample identification. ✔ Seeds are cooled to storage temperature.15 l In some cases, a slower cooling rate may be beneficial to prevent damage during introduction into LN. l Slower cooling (<10 oC/min) tends not to lead to mechanical damage. ✔ Seeds are stored in LNV (in most cryobanks). 15 See section 5 on long-term storage in liquid nitrogen. 35 4.2 Cryopreservation of intermediate seeds Intermediate seeds can tolerate drying, but due to their shorter longevity, cryopreservation may be a preferable storage temperature. Intermediate seeds may be cryopreserved as either whole seeds or as excised embryos, most often depending upon seed size. Because the seeds are able to tolerate drying, general procedures may be similar to those of orthodox seeds. A key feature of intermediate seeds is their desiccation tolerance to well below where the cytoplasm naturally vitrifies (C. Walters, personal communication). ✔ Seeds are subjected to initial viability testing following optimized and documented procedures. l Seeds to be cryopreserved should be of high quality and high viability. l It is important to use standard protocols so that viability monitoring tests are comparable, including over time, ideally using replicated testing procedures (see FAO, 2022a; section 4). Preparation of intermediate seeds that are cryopreserved whole ✔ Seed samples are equilibrium dried to optimum moisture content for cryostorage. In general, intermediate seeds dried to 50 percent RH and below survive short-term exposure to LN. The drying levels for LN storage of intermediate seeds are higher than those used for standard storage at –20 oC to avoid drying damage (C. Walters, personal communication). NLGRP adjusts seed moisture levels for cryopreservation by placing seeds at known RHs (50 percent for intermediate seeds). ✔ Seeds are assessed for moisture content to ensure they are adequately dry. l Moisture content of the sample is determined if sufficient quantity of material is available (see FAO, 2025). l The recommended sample size per replicate is 0.5–1.0 g for small seeds and 4.5–5.0 g for larger seeds (cereals, legumes). Moisture content determination should be performed with minimum two independent replicates, and three to four replicates for greater accuracy. Preparation of intermediate seeds that are cryopreserved as excised embryos/ embryonic axes Intermediate seeds that are large (>0.5 to 1 cm) cannot be cryopreserved intact, primarily because the internal tissues would not cool fast enough. 4. Cryopreservation of diverse propagule types Practical guide for the application of the Genebank Standards for Plant Genetic Resources for Food and Agriculture Conservation through cryopreservation 36 ✔ Embryos/embryonic axes are excised from seeds as soon as possible after the seeds arrive at the genebank. Only healthy-looking fruits/seeds with no signs of disease or damage should be used. l Often seed material is disinfected and the axis isolated in a laminar flow hood. Thereafter, all steps in the cryopreservation protocol are sterile. Alternatively, disinfection is applied after the cryopreservation process on entry to tissue culture for regrowth. l Excision of the explant should be optimized, as cutting too close to the apex of an embryonic axis can result in damage and poor subsequent growth. Explant size is dependent upon species and protocol. l Excised embryos/embryonic axes should be immediately placed in antioxidant solution. ¡ An example of antioxidant solution is 50 mg/L ascorbic acid in water or liquid culture medium. ✔ The number of embryos/embryonic axes excised and stored is determined by the number of seeds available, heterogeneity of the seed lot, and the number needed to be placed in long-term storage and for viability assessments. ✔ Propagule water content is reduced through controlled air-drying, encapsulation–dehydration or vitrification or encapsulation–vitrification. l Air-drying of recalcitrant seed embryonic axes is carried out with freshly regenerated silica gel desiccant or in the flow of sterile air in a laminar hood before deep cooling (see Ballesteros, Fanega-Sleziak and Davies, 2021). l Encapsulation-dehydration, vitrification, or encapsulation-vitrification are species-specific procedures developed for cryopreserving recalcitrant seed embryos or embryonic axes (see Ballesteros, Fanega-Sleziak and Davies, 2021). ✔ For either seeds or embryos/embryonic axes, materials are packaged into containers suitable for storage in LN, such as cryovials, based on the propagule type. l If enough seed and resources are available, it is recommended that seeds be aliquoted into multiple packages/cryovials to simplify the distribution and/or viability assessment processes. l Packaging should consider the need to retrieve samples without warming the remaining samples. ✔ Secure labels that will remain adhered to the package at LN temperatures are affixed to the packages. l Bar codes or labels with indelible ink, suitable for use in LN, with inventory information, may be applied for ease in sample identification. 37 ✔ Seeds are cooled to storage temperature.16 l In some cases, a slower cooling rate may be beneficial to prevent damage during introduction into LN. This may help avoid cracking during the cooling process. l Slower cooling (<10 oC/min) tends not to lead to mechanical damage. ✔ Seeds are stored in LNV (in most cryobanks). 4.3 Cryopreservation of recalcitrant and large intermediate seeds Recalcitrant seeds cannot usually withstand sufficient drying for storage at LN temperatures. Recalcitrant seed cryopreservation depends on finding a balance between avoiding desiccation damage and having cooling rates fast enough to prevent ice formation. This usually requires the excision of embryos/embryonic axes. Some information relating to the cryopreservation of recalcitrant seeds for specific taxa may be available in the literature. Taxa-specific methods may take into account the size of the tissue, water relations of embryonic axes and stress-related metabolism (Walters, Volk and Richards, 2008). ✔ Embryos/embryonic axes are excised from seeds as soon as possible after the seeds arrive at the genebank. ✔ Source materials from healthy, mature seeds are used, selecting axes free from mechanical damage and considering the developmental stage of the embryo. ✔ The number of embryos/embryonic axes excised is determined by the number of seeds available, heterogeneity of the seed lot the number needed to be placed in long-term storage and for viability assessments, and is an individual genebank decision. ✔ Healthy-looking fruits/seeds with no signs of disease or damage are used. l In some cases, the fruits/seeds are surface sterilized and excised under sterile conditions. If materials are surface sterilized, it may include treatment with diluted alcohol (ethanol or isopropanol) and/or sodium hypochlorite solutions, followed by sterile water. l Excision of the explant should be optimized, as cutting too close to the apex of an embryonic axis can result in damage and poor subsequent growth. Explant size is dependent upon species and protocol. l Large embryos/embryonic axes, such as those for Persea, may require the use of the plumule. 16 See section 5 on long-term storage in liquid nitrogen. 4. Cryopreservation of diverse propagule types Practical guide for the application of the Genebank Standards for Plant Genetic Resources for Food and Agriculture Conservation through cryopreservation 38 l If materials are not excised under sterile conditions, disinfection is applied after the cryopreservation process on entry to tissue culture for regrowth. l Generally, successful cryopreservation is achievable in small embryonic axes (< 2 mg) that have a water content (WC) <= 1.5 g H2O/g dry weight, equivalent to water potential (WP), where WP = –0.8 MPa, and that do not turn brown too much upon excision (C. Walters, personal communication). ✔ Propagule water content is reduced through controlled air-drying, silica gel, encapsulation–dehydration or vitrification or encapsulation–vitrification. l Drying of recalcitrant seed embryonic axes is carried out with freshly regenerated silica gel desiccant, in the flow of sterile air in a laminar hood or air-drying in a dry climate before deep cooling (see Ballesteros, Fanega-Sleziak and Davies, 2021). l Encapsulation–dehydration, vitrification or encapsulation–vitrification are species-specific procedures that have been reported in the literature for cryopreserving seed embryos or embryonic axes of some recalcitrant seed species (see Ballesteros, Fanega-Sleziak and Davies, 2021). ✔ Seeds are cooled to storage temperature.17 l Fast cooling rates, either with or without cryoprotectant treatment, may achieve successful cryopreservation without ice crystal formation (Walters, Volk and Richards, 2008). One example of seed cooling procedures is provided by Walters, Volk and Richards (2008). ✔ Seeds are stored in LNV (in most cryobanks). 4.4 Cryopreservation of dormant buds Dormant buds of temperate-adapted woody plants may be cryopreserved to provide a backup of specific genotypes (i.e. cultivars) in field collections. Although restricted to a limited number of species, dormant bud cryopreservation is generally much less labour intensive than shoot tip cryopreservation. However, using dormant buds introduces the possibility of cryopreserving virus (if the mother plant was infected), which will require an additional virus eradication procedure to obtain clean plants. Dormant buds are harvested when the plants are fully winter-dormant, prior to the time that dormancy is released. Some growth locations may not result in the dormant buds achieving adequate dormancy for successful cryopreservation of dormant buds. Ideally, twigs containing dormant buds from the previous growing season are harvested after several days of low temperatures. For detailed information, see Volk, Jenderek and Chen (2020b) and Tanner et al. (2021a). 17 See section 5 on long-term storage in liquid nitrogen. 39 ✔ Dormant twigs containing dormant buds are harvested, bundled, placed in sealed plastic bags, and stored in the 0–5 oC cooler until shipment or use. It is important that twigs are processed as soon as possible after harvest and should be kept in insulated boxes or a cooler at around 0 oC until use. l Bud twigs are cut into segments using a saw or pruners. ¡ Each segment is about 3.5 cm long and contains a single bud (alternate arrangement) or two buds (opposite arrangement). ¡ In some cases, dormant buds may be treated with an osmoticum (such as 5 M sucrose) for 96 hours prior to desiccation to increase freeze resistance and cryosurvival of dormant buds (Tanner et al., 2021b). l Bud segments are placed on a rack in a 0 oC cooler at low humidity to dry to a moisture content of 25–30 percent on a fresh weight basis (FWB). ✔ The moisture content of the bud sections is calculated prior to packaging. l The initial moisture content of ten nodal sections is calculated by measuring the fresh weight (FW) and dry weight (DW). l DW is taken after sections, placed in an oven-safe tray, are dried for three days in an oven at 100 oC. l After weights are established, the following equation is used to calculate the initial moisture content: (FW–DW)/FW × 100 percent (on a FWB). l The total FW of the remaining nodal sections is measured, and the desired ending weight is calculated based on a desired moisture content of 25–30 percent (FWB). ✔ After moisture adjustment, bud sections are packaged into polyolefin tubes (ten buds per tube), sealed and cooled to –30  oC at rates ranging from 1  oC per hour to 5  oC per day using a programmable freezer, depending on the procedure. ✔ Tubes containing buds are held at –30  oC overnight and then placed into the LNV. l For dormant bud segments, it is recommended to cryopreserve 17 tubes for each accession, with two tubes to be warmed for viability assessment after LN exposure, and the remaining 15 tubes kept in long-term storage (Volk et al., 2017). 4.5 Cryopreservation of shoot tips Vegetative shoot tips (with meristems) may be cryopreserved for genebank accessions whereby the precise genotype (i.e. cultivar) must be maintained. This method is most often used for species/crops that are vegetatively propagated and are often maintained as plants in the field, greenhouse/screenhouse or in vitro in the genebank. In addition, shoot-tip cryopreservation may be used to cryopreserve wild species that do not have sufficient seeds, cannot be cryopreserved by dormant buds or has recalcitrant seeds for which embryos cannot be successfully cryopreserved. 4. Cryopreservation of diverse propagule types Practical guide for the application of the Genebank Standards for Plant Genetic Resources for Food and Agriculture Conservation through cryopreservation 40 Cryopreservation of shoot tips is more labour-intensive than dormant buds, but it is effective for a wide range of species and can be carried out throughout the year. No single protocol is readily applicable to a wide range of plant species. A benefit of using shoot tips of in vitro material for cryopreservation is that the plants can undergo a virus elimination process before being cryopreserved, allowing for direct usage and dissemination after the plants are recovered. The preferred cryopreservation method will vary with the crop and is dependent upon the equipment available and technical skills. There are numerous steps in the shoot-tip cryopreservation procedure that must be optimized for successful processing. These steps are described in detail by Bettoni, Bonnart and Volk (2021), Bettoni, Chen and Volk (2024), Nagel et al. (2024), and Vollmer et al. (2021) and summarized briefly below (see Volk, ed, 2020). ✔ Ex vitro or in vitro source plant materials are selected for use in shoot-tip cryopreservation. Adequate quantities of shoot tips are needed for cryopreservation. In some cases, sufficient quantities can be acquired directly from plant materials collected from the field or greenhouse/screenhouse. This saves the labour involved in introducing plants into tissue culture and multiplication – which can save months of time. In many cases, it is not possible to successfully cryopreserve directly from ex vitro plants and so in vitro plants must first be established. a) Shoot tips derived from field or greenhouse/screenhouse grown plants Excision of shoot tips from field, greenhouse/screenhouse or growth chamber grown materials may be more efficient than introducing all plant materials into tissue culture prior to use. The use of shoot tips from these sources may be seasonal, depending on the plant physiology, dormancy, etc. of the source material (Ellis et al., 2006; Volk et al., 2015). Examples of protocols whereby shoot tips are derived from field plants: garlic (Ellis et al., 2006); screenhouse plants: citrus (Volk et al., 2015); growth chamber plants: Vitis (Bettoni et al., 2019a,b). l A sufficient quantity of shoot tips within ex vitro plant materials are obtained. Shoots, cloves or other propagative materials (containing shoot tips) are acquired and trimmed to a size that achieves successful surface sterilization. l Explants are surface sterilized to remove all microorganisms prior to cryopreservation. l For citrus shoot-tip cryopreservation directly from screenhouse-grown budwood, nodal sections are surface sterilized with 70 percent isopropanol for three minutes followed by three rinses with sterile water. After transfer to a laminar flow hood, the nodal sections are treated with 10 percent commercial bleach (0.825 percent sodium hypochlorite final concentration) and a drop of Tween 20 (1 drop per 100 mL water) for ten minutes, followed by three rinses with sterile distilled water (Volk et al., 2020). l In some cases, nodal sections taken from ex vitro source plants are cultured in vitro for a few weeks to produce uniform apical shoot tips for excision (Bettoni et al., 2021). 41 b) Shoot tips derived from in vitro cultures l Excision of shoot-tips from in vitro plants requires that the accession is first introduced and multiplied in vitro (see FAO, 2022c). l In vitro culture systems should be optimized to ensure that cultures are clean and that optimal culture media formulation and growth conditions have been established prior to cryopreservation. l Tissue culture plants are multiplied to produce a sufficient number of shoot tips for cryopreservation. l It is important to use cultures that have been introduced into tissue culture relatively recently to reduce the possibility of somaclonal variation. l In vitro plants must be free of endophytes and ideally sanitized so that only pathogen-free plant materials are cryopreserved. Stresses encountered during the cryopreservation process tend to exacerbate the deleterious effects of endophytes in cultured plants (Volk et al., 2022). l In vitro source plants may be pretreated with hormones, reduced temperatures, antioxidants or salicylic acid prior to shoot-tip excision to improve the success of the cryopreservation procedure (Bettoni, Bonnart and Volk, 2021). l In some cases, nodal sections are cultured from the in vitro source plants to produce uniform shoot tips for excision (Vollmer et al., 2021). ✔ Shoot tips are excised from either surface sterilized source material or sterile in vitro cultures in a laminar flow hood that provides sterile conditions, using a binocular microscope. Excised shoot tips are often 0.8–1.5 mm in length. Demonstrations of the process are shown as videos demonstrated in the ebook “Training in plant genetic resources: Cryopreservation of clonal propagules” (Volk, ed, 2020) as well as in a training video published by the International Potato Center (CIP, 2023). l Excised shoot tips may be precultured for hours to days prior to cryoprotection. Shoot tips are usually precultured with elevated sucrose concentration and sometimes with other additives such as salicylic acid, glycerol and/or dimethylsulfoxide (DMSO), on solid, semi-solid or liquid medium, at reduced temperatures. All conditions are optimized to increase the success of the cryopreservation procedure (Bettoni, Bonnart and Volk, 2021). l Excision of propagules can trigger the generation of reactive oxygen species (ROS), which are potentially harmful (Roach et al., 2008). To counteract any negative effect of ROS production,18 freshly isolated shoot tips can be bathed in a solution of vitamin antioxidant (vitamin C) or non-vitamin antioxidant (e.g. glutathione) or elicitors of defense-related proteins in plants (e.g. salicylic acid) or anti-stress compounds (e.g. glycine betaine). The optimum concentration (mM) is determined for each propagule and plant species (Kim and Popova, 2023; Uchendu et al., 2010a,b). 18 Standard 6.5.3. 4. Cryopreservation of diverse propagule types Practical guide for the application of the Genebank Standards for Plant Genetic Resources for Food and Agriculture Conservation through cryopreservation 42 ✔ Excised, precultured shoot tips are exposed to LN Cryoprotection treatments remove freezable water and usually introduce permeable solutes that allow undifferentiated cells in shoot tips to survive LN temperatures. Numerous options are available that use either physical drying or chemical treatments (Bettoni, Bonnart and Volk, 2021; , 2004; Panis, Nagel and Van den houwe, 2020; Reed, 2008). l Encapsulation–dehydration methods place excised shoot tips in calcium alginate beads that are then treated with solutions (often sucrose) of increasing concentration. Beads containing shoot tips are treated with sucrose-enriched medium at either fixed or increasing concentrations for several hours or day and then physically dehydrated by either air-drying in the air current of a laminar flow hood or with silica gel to an optimum moisture content (dependent upon procedure and plant species) prior to placing beads in a vial and plunging the vial into LN (Bettoni, Bonnart and Volk, 2021). l Vitrification methods rely on chemical cryoprotectant treatments, often using plant vitrification solutions (often Plant Vitrification Solution 2 (PVS2; Sakai, Kobayashi and Oiyama, 1990) or Plant Vitrification Solution 3 (PVS3; Nishizawa et al., 1993) for an optimum time and temperature determined for the material to balance osmotic dehydration and chemical toxicity before deep cooling. The duration of cryoprotectant treatment is dependent on the taxa, physiology of the source materials, pretreatments and shoot-tip size, as well as the osmoprotection and cryopreservation protocol. After preculture, shoot tips undergo osmoprotection in loading solution, which usually applies glycerol and sucrose (such as 2 M glycerol + 0.4 M sucrose) to induce tolerance and mitigate the osmotic shock to the subsequent vitrification treatment at either room temperature or 0 oC. Shoot tips are either placed in cryoprotectant solution in vials (vitrification method) or in a droplet of cryoprotectant on a sterile foil strip (droplet-vitrification method) or attached to cryoplates that are submerged in cryoprotectant solution (V-cryoplate) and then plunged into LN. For droplet– vitrification and cryoplate methods, the frozen foil strip or plates, respectively, are then inserted into a pre-cooled cryovial (Bettoni, Bonnart and Volk, 2021). l Encapsulation–vitrification methods combine encapsulation–dehydration and vitrification procedures; shoot tips are encapsulated within calcium alginate beads that are osmoprotected and treated with cryoprotectant solutions prior to LN exposure (Bettoni, Bonnart and Volk, 2021). l Other methods, such as two-step cooling, and D-cryoplate, are also described in the literature (Reed, 2008; Bettoni, Bonnart and Volk, 2021; Niino et al., 2019; Zhang et al., 2023). l In general, vitrification protocols are faster than encapsulation–dehydration and two-step cooling methods. However, shoot tips must be handled carefully and it is critical to have very precise control of exposure time in cryoprotectant solutions. 43 l When conserving a wide range of genotypes that respond differently to a protocol, modifications to the methods or multiple different methods might be needed (Bettoni, Bonnart and Volk, 2021; Zhang et al., 2023). ✔ The quantity of cryopreserved shoot tips per accession varies depending on the desired number of materials that will be placed in cryostorage, infrastructure, availability of technical staff, material availability and preservation goals. The total number of stored propagules should be high enough to be able to produce sufficient quantities of living plants to meet any future needs. For example, the NLGRP, the Indian Council of Agricultural Research-National Bureau of Plant Genetic Resources (ICAR-NBPGR) and the International Potato Center (CIP), place ten cryopreserved shoot tips into 1 mL cryovials). At NLGRP, 17 vials with tenshoot tips each are cryopreserved for each accession, with two warmed immediately for regrowth assessment and 15 remaining in long-term storage. At CIP, 12 vials with ten shoot tips each are cryopreserved for each accession, with two warmed immediately for regrowth assessment and ten remaining in long-term storage. For some crops at ICAR- NBPGR, 20 vials are stored containing 200 explants. For seeds, the number of stored propagules depends on the seed size, space available, seedlot heterogeneity and the expected number needed for viability assessments and for return to the active site. ✔ The number of vials is determined by the number of thaw events deemed necessary for that accession. Protocols at NLGRP aim to cryopreserve 17 cryovials for each accession (a total of 170 shoot tips), with two cryovials warmed for viability assessment after LN exposure, and the remaining 15 vials kept in long-term storage (Volk et al., 2017). At CIP and ICAR-NBPGR, 12 vials with ten shoot tips each are cryopreserved for each accession, with two warmed immediately for regrowth assessment and ten remaining in long- term storage. Other laboratories cryopreserve enough propagules to ensure with high confidence that a desired number of plants can be regenerated (Panis, 2009). l Probabilistic viability calculations are effective tools that have been developed to assist in determining the predicted number of shoot tips that should be cryopreserved, based on the number of explants initially processed and the level of regrowth after LN exposure (Dussert, Engelmann and Noirot, 2003; Volk et al., 2017). 4.6 Cryopreservation of desiccation-tolerant pollen Pollen may be cryopreserved, although this propagule type is not generally considered a backup of genebank accessions that are maintained as plants. Pollen cryopreservation is a vital tool for conservation of dioecious species to conserve male individuals. Preserved pollen may be particularly valuable for breeding programmes for making crosses. Methods should be tested for the species of interest, ideally ensuring that 4. Cryopreservation of diverse propagule types Practical guide for the application of the Genebank Standards for Plant Genetic Resources for Food and Agriculture Conservation through cryopreservation 44 cryopreserved pollen can be employed for successful crosses (see Rajasekharan and Rohini, eds, 2023). Cryopreserved pollen longevity is not fully documented; therefore, periodic viability testing should be performed. In some cases, cryopreserved pollen may be a short- or medium- term storage option, particularly for use in breeding programmes. ✔ Upon receipt, pollen is equilibrated either in a temperature- and humidity- controlled room or ov