Preservation of genetic resources by seed storage Nyat Quat Ng liT A Research Guide 52 Preservation of genetic resources by seed storage Nyat Quat Ng March 1996 International Institute of Tropical Agriculture (IITA) Training Program Fax: (234-2) 241 2221 PMB 5320 Telephone: (234-2) 241 2626 Ibadan Telex: 31417or31159 TROPIBNG Nigeria E-mail (Intemet):IITA@CGNET.COM liT A Research Guides IITA Research Guides provide information and guidance to agricultural researchers, technicians, extension specialists, educators and students involved in research and training. The Research Guides are periodically updated to meet advances in scientific knowledge. liT A permits reproduction of this Research Guide for non- profit purposes. For commercial reproduction, contact the liT A Publications Unit. Editing Ayotunde Oyetunde Kehinde Jaiyeoba Foyinsola Akinrimisi Chiweta Onianwa Nancy Ibikunle Rainer Zachmann Text processing Artwork Layout Coordination Ng, N.Q. 1996. Preservation of genetic resources by seed storage. IITA Research Guide 52. Training Program, International Institute of Tropical Agriculture (IITA), Ibadan, Nigeria. 36 p. Second edrtion. 2 IITA Research Guide 52 Preservation of genetic resources by seed storage Objectives. This guide is intended to enable you to: • differentiate between types of seed; • manage pre-storage conditions; • expl ain the effects of storage conditions; • predict longevity of seed viability; • expla in types of gene banks; • design seed stores; • apply fumigants . Study materials • Samples of orthodox and recalcitrant seed . • Different types of seed dryers. • Diffe rent types of gene banks. • Fumiga nts . Practicals • Harvest and select seed for storage. • Dry seeds using different procedures. • Monitor storage conditions . • Measure moisture content of seeds. • Calculate longevity of seed viability. • Design seed stores. • Apply fumiga nts . 3 Questions 1 Into what two groups can seeds be classified accord- ing to their characteristic seed longevity? 2 What is the effect of harvest on subsequent storabil- ity of seed? 3 When should you harvest seed? 4 What seeds should you select for long-term conser- vation of genetic resources? 5 What is the ideal moisture content for long-term seed conservation? 6 What procedures does IBPGR recommend for seed drying? 7 What are the most important factors affecting seed drying? 8 How does the period of seed viability increase as temperature and seed moisture content decrease? 9 According to the rule of thumb, what effect does a 1% decrease in seed moisture content have on the life of seed? 10 How can you predict seed longevity? 11 What are some factors that may account for differ- ences in seed longevity of genotypes within the same species? 12 What are three broad types of gene banks? 13 What is the purpose of base collections? 14 What are 'preferred' and 'acceptable' standards for long-term seed storage? 15 What is a practical and acceptable standard for the number of seeds in base collections? 16 What safety precautions should you respect when designing a seed store? 17 What common fumigants are used to de-infest seeds? 4 IITA Research Guide 52 Preservation of genetic resources by seed storage I Types of st. .. >d 2 Pre-sto~.gc (."Ondition..~ 3 Storage conditions 4 Predicting longevity of seed viability 5 Types of gene banks 6 Design of seed stores 7 Fumigants 8 Bibliography 9 Suggestions for trainers Abstract. Seve ral ways exist of maintaining the genetic resources of crops. The cheapest and most co n· veni ent way is by storing seeds. However , not a ll crops can be preserved by storing their seeds, beca use they arc eithe r vegeta tively propagated and/or do not produ ce seeds, or the period of their seed viability is short. Ma x· imizing the longevity of seeds requires a good seed store and knowledge of the principles of seed prese rva· tion . This document discusses types of seed, techniques for pre pa ring seeds for storage, factors controll ing longevity of seed, standards of seed stores for geneti c resources conservation, and design of seed s tores . 5 1 Type. 0' seed I Seeds are classified into two groups according to their viability characteristics: • orthodox seeds, • recalcitrant seeds . Orthodox seeds. Orthodox seeds store well for long periods if kept cool and dry. The period of viability of orthodox seeds increases logarithmically, as the stor· age temperature and moisture content of the seeds decrease. Most arable crops belong to this group. Recalcitrant seeds. Recalcitrant seeds generally can· not be dried without damage. Stored under ambient conditions, recalcitrant seeds survive for a period of several days to months. Moist recalcitrant seeds of most species are damaged by sub· zero temperature. To store recalcitrant seeds, special techniques for spe· cific species are required . For example, lemon seeds which are classified as recalcitrant seeds behave like orthodox seeds once the testae have been removed . Presently, it is not possible to give a general recom· mendation on storage of recalcitrant seeds for long periods without the seeds losing viability. This document describes seed preservation and storage techniques for orthodox species. Examples of orthodox and recalcitrant seeds are listed in Table 1. 6 Table 1. Some examples of orthodox and recalcitrant seeds. Orthodox seeds Cereals Food and Oil crops Roots Vege/ables Pastures and grains grain legumes and tubers and others and fodder Ba~ey Asiatic Vigna Brassica Cassava Amarantus Medicago Maize Bambarra- F\ax& Potato Boehmeria Trifolium Millets groundnut Linseed Sweet potato Brassica DaiS Chickpea Groundnul Yam Cotton Rice Cowpea Sesame Kenaf Rye Lablab Sunflower Nicotiana Sorghum Lentil Soybean Pisum Tef! Phaseolus Sugar beet Wheat Pigeon pea Sugarcane Velvel bean Vicia faba Wing bean Recalcitrant seeds Avocado Coffee Oak Ca"",,hor Durian Oil palm Chestnut Filbert Rambutan Cinnamon Jackfruit Rubber Citrus Kolanul Tea Cocoa Mango Walnut Coconut Mangosteen 7 2 Pre-storage . conditions The pre-storage conditions of seeds are just as impor- tant as storage. Pre-storage conditions include: • harvest, • weather, • seed health, • seed drying. Harvest. Time of harvest has a decisive effect on the suhsequent s torability of seed : • seeds harvested before maturity do not survive as long as mature seed ; • over-mature seeds deteriorate quickly; • seeds exposed to unfavora ble environments after maturity have reduced storability . Harvest seed when it is mature. Weather . Take climati c factors into consideration for seed multiplication . Weather conditions during har- vest affect the quality of seeds. If the maturity period coi ncides with the rainy (or wet) season , it is difficult to ha rvest a nd dry the seeds. Some mature seeds may sprout in wet weather, even before harvesting. Try to time planting to produce mature seeds In dry weathe r . Seed h ealth. Unhealthy seeds have poor storability . Causes of unhealthy seeds include: • insect infestation, • diseases, • mechani cal da mage . 8 Chemical control of insect pests and diseases has disadvantages; it can: • result in poisoning, • affect the long-term storage life of the seed. Ideally, for long-term genetic resources conservation, select seeds that are clean and free of chemical treat- ment (unless the chemical does not endanger the longevity and genetic stability of seeds). Do not collect seeds which have been infested by insects or diseases. Seed drying. Mter harvest, dry seeds as quickJy as possible to a moisture content of less than 10 %. Seeds with a high moisture content deteriorate rapidly. The ideal moisture content for long-term seed conservation is: • 4-5 % for legumes, • 5-6 % for cereals. Ultra-dry seeds (2-3 % seed moisture content) may store longer, but require more delicate handling procedures. In most crops, the seed moisture content at the time of harvest is generally high (above 18 %). Sun drying reduces the moisture content to approximately 12 % in most tropical countries. In extremely dry weather with relative humidity of less than 40 %, such as the harmat- tan season in Nigeria, sun/air drying could reduce seed moisture content to less than 9 %. Drying seeds to 5 % seed moisture content requires special techniques. High temperatures (above 40 °C, for instance), can damage the seeds. 9 The following alternative drying procedures are recommended: • forced ventilation, • dehumidified room or cabinet, • sun or air drying. Forced ventilation. Place the seeds in a forced ventila- tion dryer (or air circulated oven) for 2-3 days at 40 °C (for cereal, grasses and less oily types of seeds), or at below 35 °C (for oily seeds such as soybean and other legumes). Clean and fumigate the seeds if necessary. Then ... either: Place the pre-dried seeds in another forced ventilation dryer at a higher temperature of 55-60 °C to bring the seeds down to 5 % moisture content. Note: this alternative is not suitable for oily seeds. or: Place the pre-dried seeds in a dehumidified room or cabinet with relative humidity (Rh) maintained at 10-20 % and at a temperature of less than 25 °C. Dehumidified room or cabinet. Place seeds in a dehu- midified room, cabinet or chamber with a relative humidity (Rh) of about 10 % and temperature of less than 30 DC for 3-5 days. Then clean andlor fumigate the seeds if necessary. Continue drying at a temperature of less than 25 DC until seeds reach the desired moisture content ofless than 6 %. Sun or air drying. Where the facilities described are not available, dry seeds under the sun with partial 10 shading. Do not expose seeds to rain. Spread seeds in a thin layer, under a polythene shelter to protect them from rain. Frequent stirring and turning of seeds promote uniform drying. In air/sun drying, seed moisture content fluctuates according to the Rh of the natural air. In the humid tropics, the daily Rh ranges from 50-90 % or even higher. Under these conditions, seed moisture content (such as rice and cowpea) aver- ages about 13 % ± 1 %. The number of days necessary for seeds to reach the required moisture content varies from species to species. For example, rice seeds reach a moisture con- tent of about 6 % after 7 days of drying in an ait: circu- lated drying room at around 10 % Rh and 25 oC. Most countries in Africa have a long dry season, particularly in the savanna regions. During the dry season, the Rh can be less than 30 %. Under such condi- tions, seeds can be dried to less than 9 % moisture con- tent. You can exploit these conditions for drying, if seeds are harvested during the dry season. Table 2 presents the equilibrium seed moisture content of various types of seeds at specific constant Rh. Use this guide to dry seeds or to estimate the percentage of seed moisture content under a specific Rh. 11 Table 2. Equilibrium moisture content (%) of some common seeds at 25 0c (adapted from IBPGR). Relative humidity (%) 15 30 45 60 75 90 Barley (Hordeum) 6.0 8.4 10.0 12.1 14.4 19.5 Beet (Beta) 5.8 7.6 9.4 11.2 Buckwheat (Fagopyrum) 6.7 9.1 10.8 12.7 15.0 19.1 Cabbage (Brassica) 5.4 6.4 7.6 9.6 Carrot (Daucus) 6.8 7.9 9.2 11.6 Cucumber (Cucumis) 5.6 7.1 8.4 10.1 Egg plant (So/anum) 6.3 8.0 9.8 11.9 Flax (Unum) 4.4 5.6 6.3 7.9 10.0 15.2 Groundnut (Arachis) 2.6 4.2 5.6 9.8 13.0 Lettuce (Lactuca) 5.1 5.9 7.1 9.6 Lima bean (Phaseo/us) 7.7 9.2 11.0 13.8 Maize (Zea) 6.6 8.4 to.2 12.7 14.4 18.8 Mustard (Bmssica) 4.6 6.3 7.8 9.4 Oat (Avena) 5.7 B.O 9.6 11.8 13.8 18.5 Okra (Abe/moschus) B.3 10.0 11.2 13.1 Onion (Allium) B.O 9.5 11.2 13.4 Radish (Raphanus) 5.1 6.8 B.3 10.2 Rice (Oryza) 5.6 7.9 9.8 11.8 14.0 17.6 Rye (Secale) 7.0 8.7 10.5 12.2 14.8 20.6 Sorghum (Sorghum) 6.4 8.6 10.5 12.0 15.2 18.8 Soybean (Glycine) 4.3 6.5 7.4 9.3 13.1 18.8 Tomato (Lycopersicum) 6.3 7.8 9.2 11.1 Turnip (Bmssica) 5.1 6.3 7.4 9.0 Watermelon (Ci/rullus) 5.1 6.3 7.4 9.0 Wheat (Triticum) 6.5 8.5 10.4 12.1 14.6 19.8 Winter squash (Cucurbita) 5.6 7.4 9.0 10.8 12 3 Storege condition. Two most important factors a ffecting the storage condi · tions of seeds are: • storage temperature, • re lative humidity. The period of seed viability increases loga ri t hm ically as the temperature and moistu re content of the seed dec reases. Storage temperature. Temperatu re in the store influ· ences seed viability. High temperatu res promote: • funga l growth , • insect development. High storage temperatures of 50 °C a nd higher injure seeds. The lower the storage temperatu re, the longer the life of the seed . Tem peratures below freezing kill seeds when moisture con tent is high (above 15 'h), but do not harm seeds when moisture content is low (less than 12 'H Remember Harrington's rul e of thumb: for eac h 5 °C ri se in temperatu re, the storage life of seeds is reduced by approx imately ha lf (Harrington 1970 ), Relative humidity. The rela tive humidity in the store determines the seed moisture content, unless seed sam- ples are sealed in moisture proof con tain ers. Seeds germ inate when thei r moisture content is a rou nd 40 'W- or higher , unless they a re subjected to inhibitors . Germinated seeds ca nnot be stored or red ri cd without damage to the seedling. 13 A moisture content between 14 and 20 %, causes seeds to deteriorate rapidly, because of the invasion of microor- ganisms, such as fungi and other seed-borne diseases. At a moisture content below 12 %, fungi and other diseases do not develop. When moisture content is below 9 %, insects (eggs and nymphs) become inactive. Another Harrington's rule of thumb to remember: a 1 % decrease in seed moisture content doubles the life of the seed. 14 .. Predicting longevity of ned viability The two rules of thumb in the previous section give useful approximations of the relationship between seed storage life and storage environment. These rules do not quantify the interactive effects of seed moisture content and storage temperature on seed longevity. The following seed viability model quantifies the combined effects of moisture content and storage temperature on seed longevity (Ellis 1988; Ellis and Roberts 1980). Equation 1: V=K;- PlIO E W H Q [ [K - C logm - C t - C t 2 ) 1 v = probit percentage seed germination rate (viability); P = storage period in days; t = storage temperature in oC; m = percentage seed moisture content (wet basis); Ki = probit value of initial percentage viability; KE. CW. CH and CQ = constant values of individual species . Constant values of several species listed have been esti- mated (Table 3). Use these constants to predict seed viability in those species. The loss of probit viability can be estimated by rear- ranging equation 1 into equation 2. 15 Equation 2: Using equation 2, you can calculate the hypothetical times (in years) for cowpea seed stored under various temperatures and seed moisture contents to decline from an initial viability of 95 % to the lowest acceptable vi abi lity of 85 % (Table 4). According to these esti- ma tes , cowpea seeds with a moisture content of 5 %, stored at ·10 °C or below, could retain a viability of 85 % from their initial viability of 95 % for more than 1 200 y ears . Be cautious a bout the hypothetical times given in Table 4. The equa tion and species constants used in the es timates derive from results obtained from : • limited number of genotypes; • limited conditions of storage; • short period of experiments . Table 3. Constant values for estimation of seed viability. Species KE Cw CH Co Cowpea 9 .102 4 .967 0 .0295 0.000491 Soybea n 7 .292 3.996 0.0295 0 .000491 Chickpea 8 .520 4 .602 0.0295 0.000491 Barley 9 .983 5.896 0.0400 0 .000428 Onion 6 .975 3 .470 0.0400 0 .0004 28 16 Species constants derived from rapid-aging treatments on seeds may not reflect the real relationships between seed survival and storage environments. Table 5 presents the observed percentage of seed germi- nation of 10 randomly selected cowpea samples with a seed moisture content ranging from 6.5-7.5 %, stored for more than 5 years in UTA's base collection seed store, (-20 °C). The obselved percentage of seed germi- nation indicates that: • All accessions, except one (TVu 12393) show germination rates close to the expected values. • In low temperatures and with low seed moisture contents, most cowpea genotypes follow the seed viability model. Table 4. Hypothetical times (in years, to nearest 0.5) for cowpea seed viability to decline from 95 % to 85 % with various seed moisture contents and seed storage tempera- tures . Seed moisture content (% wet bas is) Storage temperature 5 7 8 10 12 -20 1761 .0 331.0 170 .5 56.5 23.0 -10 1253.0 236.0 121.5 40.0 16.0 0 711.5 134 .0 69.0 23.0 9.0 5 493 .0 93.0 47.5 16.0 6.5 10 322.0 60 .0 31.0 10.5 4.0 20 116.0 22 .0 11.0 3.5 1.5 25 64.0 12 .5 6.0 2 .5 1.0 17 • Considerable genotype differences exist among cowpea accessions (TVu 12393 and others) in terms of longevity. Longevity also varies in different genotypes of rice (Figure 1). Gene bank managers need to take account of these genotype differences in seed storability. Results from experiments on cowpea seeds (Table 6) carried out under various storage conditions for a period of about 2 years at IITA can be compared with results in Table 5. Figure 1. Seed viabil ity of two rice varieties stored at 2 0c and 8.5 % moisture content (Chang 1988). seed viability (%) 100 18 /'-...~-_----- Pela 1975 1985 Table 6 shows the predicted viability of the seeds under each storage environment and storage period. The results reveal the following facts: • the observed viability for samples with low moisture contents (7.38-8.54 %) stored at high temperature (30 OC) is generally much lower than the estimated viability; • the observed germination percentage of samples with low moisture content, stored at -20 °C is close to the predicted values; Table 5. Percentage of cowpea seed germination after more than 5 years storage in IITA's base collection (-20 oC); November 1982-1988. Accessions MCl Storage G!;!rmination (%) no. (%) period Initial2 Final2 Estimated3 (Years) TVu 4133 7.5 5.67 94 95 93.8 TVu 4177 7.5 5.67 96 90 95.8 TVu 4337 6.5 5.67 94 98 93.9 TVu 9994 6.5 5.58 94 93 93.9 TVu 11392 6.5 5.33 100 95 99.8 TVu 703 6.5 5.42 97 94 96.9 TVu 4589 6.5 5.33 98 95 97.9 TVu 12393 6.5 5.33 91 80 90.9 TVu 12487 6.5 5.33 90 89 89.8 TVu 4601 6.5 5.25 94 96 93.9 1 Seed moisture content, % wet weight basis. 2 Observed on 200 seeds (TVu 12393 = 400 seeds). 3 Estimated by the seed viability model. 19 • the observed seed germination of samples with moisture content exceeding 12 %, stored at -20 or 5 DC is much lower than the expected viability (this might have been caused by chilling or freezing injury, especially in seeds with a moisture content of over 14 %). Treat the given results with caution. Cowpea acces- sions may not have the expected germination rate over such an extended period as 1 000 years. Some seeds may survive for over 100 years even under below- standard conditions. For example, some barley seeds (12 S0 l, buried underground for over 100 years, were viable. Factors which may account for differences in seed longevity of genotypes within the same species are dif- ferences in: • seed dormancy, • physical structures, • starch and oil content, • seed vigor. Meanwhile, monitoring viability of seeds under stor- age periodically is essential, until more certain data on longevity characteristics of different genotypes is available. 20 Ta bl e II. E xp er im en ta l e nd e xp ec te d se e d vi ab ilit y o f c o w pe a u n de r s to ra ge (N g 19 91 ). St or ag e VI TA 4 VI TA 5 T em pf C ) M el rf2. A B C M C 0 A B C D 30 0 ± 2° 7. 38 98 83 (97 )3 86 (97 ) 84 (96 ) 7. 26 97 86 (96 ) 84 (95 ) 79 (95 ) 90 (94 ) 30 0 ± 20 8. 54 92 70 (88 ) 80 (83 ) 78 (81 ) 8. 46 99 84 (98 ) 90 (97 ) 91 (9 6) 91 (9 6) 30 0 ± 20 12 . 46 96 65 (32 ) 52 (10 ) 47 (3. 6) 14 . 32 4 98 68 (1 ) 12 (0) 25 (0) 0(0 ) 30 0 ± 20 15 .4 6 98 59 (0) 16 (0) 14 (0) 15 .7 1 98 0(0 ) 0( 0) 0(0 ) 0(0 ) 50 ± 2° 7. 38 98 92 (98 ) 90 (98 ) 90 (98 ) 7. 26 97 93 (97 ) 90 (97 ) 89 (97 ) 93 (97 ) SO ±2 0 8. S4 92 86 (92 ) 86 (92 ) 86 (92 ) 8. 46 99 93 (99 ) 94 (99 ) 93 (99 ) 96 (9 7) SO ±2 ° 12 . 46 96 80 (9S ) 74 (9S ) 74 (9S ) 14 . 32 98 80 (97 ) 82 (96 ) 75 (95 ) 78 (95 ) SO ±2 0 IS .4 5 98 68 (96 ) 72 (95 ) 72 (95 ) 15 . 71 98 61 (9S ) 78 (94 ) 70 (93 ) 85 (91 ) · 20 0 ± 1° 7. 38 98 94 (98 ) 94 (98 ) 94 (98 ) 7. 26 97 96 (97 ) 96 (97 ) 95 (97 ) 92 (97 ) - 20 0 ± 1° 8. S4 92 90 (92 ) 91 (92 ) 90 (92 ) 8. 46 99 94 (99 ) 96 (99 ) 94 (99 ) 80 (99 ) - 20 0 ± 1° 12 .4 6 96 85 (96 ) 85 (96 ) 88 (96 ) 14 . 32 98 78 (98 ) 78 (98 ) 80 (98 ) 84 (97 ) - 20 o ± lo 15 .4 54 98 80 (97 ) 80 (97 ) 80 (9 7) 15 . 71 98 52 (97 ) 70 (97 ) 75 (97 ) 82 (9 7) - 1 Se ed m o is tu re c o n te nt , % w et w ei gh t b as is . 2 0 = in iti al s ee d ge rm in at io n. A = 12 m o n th s af te r s to ra ge . B = 16 m o n th s a fte r s to ra ge . C = 19 m o n th s a fte r s to ra ge . D = 23 m o n th s a fte r s to ra ge . 3 Fi gu re in p ar en th es es is th e se e d vi ab ilit y. e st im at ed b y th e se ed v ia bi lity e qu at io n (1 /2 ) a nd c o n st an ts in T ab le 3 . Ki is e st im at ed b y ta ki ng th e pr ab it va lu e of th e in iti al v ia bi lit y. 4 Se ed s w ith a m o is tu re c o n te nt a bo ve 1 4 % m ig ht h av e be en d am ag ed b y fre ez in g. 5 Types of 11_ blinks The term 'gene bank' may refer loosely to any collec- tion of germplasm. The International Board on Plant Genetic Resources (ffiPGR), and FAO distinguish three broad types of gene banks according to the collections they maintain and the conditions under which they are stored: • base collections, • active collections, • working collections. Base collections. Base collections are stored to ensure the long-term viability of material (up to 100 years or longer). Normally, seeds in base collections are not used for routine distribution or interchange of acces- sions . Active collections. Active collections are stored to en- sure medium-term viability (about 30 years). They contain material in the process of evaluation and char- acterization, as well as material represented in base collections . Ideally, all accessions in active collec- tions should be sufficient in quantity for distribution on request . Working collections. Plant breeders and other scien- tists keep working collections in conditions that ensure sufficient longev ity for the purposes of their own research. The working collections of the International Agricultural Research Centers contain much material from active collections, made available through collab- orative networks and national programs. Long-term seed storability aids germplasm conserva- tion. It reduces: 22 • cost of regenerating the materials; • risk of genetic drift; • risk of human error in cultivation. The Panel of Experts on Plant Exploration and Intro- duction of FAO recommended 'preferred' and 'accept- able' standards for long-term seed storage (1975): • Preferred standards specify storage at -18 °c or less in airtight containers at a seed moisture content of 5 ± 1 %. • Acceptable standards specify storage at 5 °C or less in airtight containers at a seed moisture content of 5-7 %, or storage at 5 °C or less in un- sealed containers in a store atmosphere of 20 % Rh. The IBPGR working group accepts, in general, the preferred standards for long-term seed storage: • IBPG R encourages the adoption of preferred standards for base collections. • IBPGR also suggests that the preferred tempera- ture standards could be 'relaxed' to -10 oC, ifthe gene bank is restricted to a few species with good storage characteristics. The most recent recommendations of FAOIIBPGR (1992) on seed storage are: • acceptable: sub-zero temperature « 0 °C with 3-7 % seed moisture content (depending upon species); • preferred: 18 oC or cooler with 3-7 % seed mois- ture content (depending upon species). 23 6 Design of aHd stores J Storage room. The size of a storage room depends on: • number of accessions; • number of seeds in each accession; • sizes of the seeds . An accession in a base collection must contain suffi- cient seeds for: • number and size of sub-samples for routine testing; • number and size of sub-samples for transfer to active collection or multiplication. A practical and acceptable standard for the number of seeds in base collections is: • uniform populations: • variable populations: minimum of 1000 seeds, preferably more than 2 000 seeds . In active collections, the size of the store depends on the number of accessions and the quality of seeds antici- pated for storage and distribution . A suggested stan- dard for active collections is : • uniform populations: • variable populations: 3000 seeds, 5000 seeds. This number can vary greatly depending on demand. Rough estimates of the overall storage space required by a gene bank can be calculated from the total number of accessions anticipated over the next 10 years . Mobile shelving requires much less (roughly halO space than conventional, static shelving. 24 Temperature. For the long-term base collection seed store, maintain temperature at approximately -18 °C. You can relax the temperature to -10 °C or higher depending on the characteristics of seed longevity of the species if necessary. In the active collection seed store, maintain the temperature at less than 10 °C. Use conventional, direct or indirect vapor compression refrigeration to maintain the desirable temperature. If there is a problem of water supply, use air cooled con- densers rather than water cooled condensers. Humidity. No special provision is needed to control Rh in the cold store where accessions are stored in sealed containers. Control humidity to prevent: • evaporators from icing, • insulations from deteriorating, • ferrous materials from rusting. If seeds are not stored in air-tight containers, control Rh at the lowest possible level. When the Rh in the cold store is maintained at 45 %, seeds of most cereals reach an equilibrium moisture content of approximately 10 % (Table 2). Maintaining the seed moisture content of cereal at the 'preferred' standard 5 ± 1 %, requires storage at less than 15 % Rh. In long-term seed stores, it is difficult to maintain the Rh at 15 %. It is cheapest, most convenient and reliable to store seeds in hermetic storage rather than in open containers. On the other hand, in a working collection store at 20 oC, it is relatively easy to maintain Rh at 15-20 %. 25 Under this storage condition, if rice or cowpea seeds are kept in open containers, they can probably remain viable for more than 10 years. This procedure is an efficient way to store breeders' seeds. Several desiccants and dehydrating agents can be used to dry air, such as lithium chloride, silica gel, etc. Commercially made dehumidifiers using these desic- cants are available. Air circulation. Avoid unacceptable temperature gra- dients in the store. Use fans for circulating the air to prevent temperature variations. Insulation. The IBPGR working group recommends that the thickness of insulation for -10 to ·20 °C should be 100-130 mm, with a thermal conductivity of 0.017 w/m °C. Use a reliable insulating material that: • retains its thermal properties, • resists attacks of pests and vermin, • contains a fire retardant. For long-term seed stores, select the following specifi· cations wherever possible: • galvanized steel sandwich panels, containing polyurethane foam mixed with a fire retardant; • heated doors to prevent icing; • seals which do not shrink; • \'entilated or heated floors to prevent frost heave. 26 For the medium-term seed store, some of these specifi- cations can be relaxed. Construction agents will give information and suggestions for insulation. For further information, consult Cromarty et al. (1982). Safety precautions. Respect the following safety pre- cautions: • provide an additional refrigeration unit and a standby generator, in case a refrigeration unit fails or electricity supply is not constant; • install an automatic continuous temperature recording device; • install a warning device to signal any signifi- cant rise in temperature; • construct the store to minimize earthquake damage where necessary; • ensure that the door can be opened from the inside, and place an indicator light outside the store. Site. Select an area with: • reliable electricity supply and voltage stability; • suitable substratum for foundations , adequate drainage and absence of flooding; • a safe distance from dangerous chemicals or fuel storages; • easy access to areas where seeds are threshed and cleaned. 27 Pre-fabricated cold rooms. Pre·fabricated cold rooms for long and medium-term conservation are now available commercially. Though easy to install, the pre-fabricated cold room must be housed in a building. Deep freeze chests operating at approximately -20 oC, such as those used domesticaUy, are suitable for long- term conservation of seeds. They are easy to obtain but have disadvantages: • chests are small and can contain only very limited numbers of samples; • running cost of several units of deep freezers is greater than the cost of a cold room with the same storage capacity. Figure 2. Plan of a gene bank. machinery seed store (5°c) seed store (-20"c) 3 B BOOrd! conforenc. seed drying seed testing seed cleaning ~nd office offico office proceSSH1g 28 Figure 2 shows the plan of a gene bank in~luding the following components: • store rooms, • machinery room, • seed drying room or space for drying oven, • seed cleaning and processing room, • seed testing laboratory, • offices and records room. 29 7· Fumigant. Three common fumigants used to de-infest seeds are: • methyl bromide (MeBd, • phosphine (PH3) gas (trade names: Phostoxin, Detia ), • ethylene dichloride mixed with carbon tetra- chloride (EDC/CT 3: 1 mixture). Methyl bromide. Methyl bromide is a useful fumigant . It is: • commerci ally avai lable; • effective against mites and nematodes; • especia lly useful for short exposure of seeds with moisture content less than 11 % and low oil content . Methyl bromide may retard or impair germination in seeds with ltigh moisture a nd oil content: • after prolonged ex pos u re to methyl bromide (more than 24 hours ), • at ltigh temperatures (above 30 OC), • at high dosage. Methyl bromide is supplied as a liquid under pressure in steel cylinders. Pure methyl bromide is odorless . It is ltighly toxic . Do not a llow untrained personnel to use the fumigant . Inhalation of methyl bromide is fatal and contact with the skin causes severe blistering. 30 The rate of fumigation of seeds (at atmospheric pres- sure) recommended by Monro (1969) are: 16 glm3 for 24 hours 24 glm3 for 24 hours 48 glm3 for 24 hours at at at Phosphine gas. Phosphine gas is: • cheap; • safe with all types of dried seeds; 200C 10-19OC 4-9 0 C • easily administered in small doses by less experienced personnel. Some insect species are resistant to phosphine. and the gas is not effective against dormant nematodes. Phosphine gas is generated when solid aluminium phosphide powder reacts with moist air. Phosphine is very toxic. Release the gas slowly at first. Operators can distribute tablets of phosphide powder safely, with- out wearing respirators. Phosphine kills insects in 3-4 days. The eggs and pupae of Sitophilus spp; the larvae of Trogoderma granavium in diapause, and the hypo- pus resting stage of mites require a 20 % higher dose and a fumigation period of 10-14 days. Low concentrations of phosphine over long periods of exposure (3-14 days), particularly in hot (above 25 OC), humid conditions produce the best results. The gas penetrates seed lots effectively, and passes through thin polythene sheeting (125 microns) and other types of packing when temperatures are not below 5-10 °C . It is scarcely absorbed , and the small residues in the fumi- gated seeds become negligible after 6 hours aeration. 31 Under normal conditions, germination of dry seed is unaffected , but moist seed can be killed. The effect of phosphine on long term seed viability has not been adequately studied. The recommended rates for fumigation of dry seeds of most species are: • 1 gl400 liters for 5 days at 12-15 oC, for 4 days at 16-20 oC, for 3 days at >21 °C. • 1 gllOO liters for 15 hours at 26-28 °C for weevils in kolanuts at 40 % moisture content. Note: One 3g tablet of aluminum phosphide releases 19 phosphine gas. EDC/CT. Ethylene dichloride/carbon tetrachloride mixture is cheap and easy to use, but does not penetrate so well, and is phytotoxic to some seeds. The mixture can damage the human liver if inhaled or splashed on the skin. Pour the liquid into a shallow pan, or sprinkle directly onto sacks. EDC/CT is also safe for use in a metal bin with a well fitted lid or under gas-proof sheeting. Fumigation lasts for 1-3 days, and aeration should last for at least one week after treatment. Germination may be reduced in maize with more than 11 % moisture content and in oil seeds. The recommended rate is 350-500 cm3 per m3 of space. 32 I Bibliography Chang, T.T. 1988. Seed processing, storage conditions and seed viability. Pages 343-352. In: Rice seed health. IRRI, Los Banos, Philippines . Cromarty, A.S.; Ellis, R.H.; Robert, E .H. 1992. The design of seed storage facilities for genetic conserva- tion. IBPGR, Rome, Italy. 96 p. Ellis, R.H . 1988. The viability equation, seed viability nomographs, and practical advice on seed storage. Seed Science and Technology 16: 29-50. Ellis, R.H.; Robert, E.H. 1980. Improved equations for the prediction of seed longevity. Annals of Botany 45: 13-80. Food and Agriculture Organization (FAO). 1975 . Report of the sixth session of the FAO panel of experts on plant exploration and introduction. FAO, Rome, Italy. Gerard, B.M. 1979. Quarantine inspection and control methods for pests in seed lots intended for gene banks. Pages 45-62. In: IBPGR Secretariat. Seed technology for gene banks. International Board for Plant Genetic Resources, (IBPGR), Rome, Italy. 127 p. Harrington, J.F. 1970. Seed and pollen storage for the conservation of plant gene resources . Pages 501-52!. In: Frankel, O.H.; Bennett, E. (eds .J. Genetic resources in plants their exploration and conservation. Backwell , Oxford, England. 554 p. International Board for Plant Genetic Resources (IBPGR). 1979. Seed technology for gene banks. IBPGR, Rome, Italy. 127 p. 33 Mumford, P.M.; Grout, B.W.W. 1979. Desiccation and temperature (-196 OC) tolerance of Citrus limon seed. Seed Science and Technology 7: 407-410. Ng, N.Q. 1991. Long-term seed conservation. Pages 135-148. In: Attere, F.; Zadan, H .; Ng, N.Q.; Perrino, P . (eds .), Crop genetic resources of Africa , Vol. 1. IITA, Ibadan, Nigeria. 305 p. Roberts , E.H. (ed .). 1972. Viability of seeds. Chapman and Hall, London . Roberts, E,H. 1975, Problems of long term storage of seed and pollen for genetic resources conservation , Pages 269-295. In: Frankel, O.H. ; Hawkes, J .G, (eds.). Crop genetic resources for today and tomorrow. Cambridge University Press, Cambridge, 492 p. Villiers, T ,A. 1972. Ageing and the longevity of seeds in field conditions . P ages 265-288. In Heydecker, W. (ed .). Seed Ecology. Proceedings of the Ninet eenth Easter school in Agricultural Science. University of Nottingham, Nottingham Butterworths, London. 34 • SUIIII •• tlon~ tor trelner. If you use this Research Guide in training ... Generally: • Distribute handouts (incl ud ing this Research Guide) to trainees one or several days before your presentation, or distribute them at the end of the presentation. • Do not distribute handouts at the beginning of a pre- sentation, otherwise trainees will read instead of listen to you. • Ask trainees not to take notes, but to pay full atten- tion to the training activity. Assure them that your handouts (and this Research Guide) contain a ll rel- evant information. • Keep your training activities practical. Reduce the- ory to the minimum that is necessary to understand the practical exercises. • Use the questions on page 4 (or a selection of ques- tions) for examinations (quizzes, periodical tests, etc.). Allow consultation of handouts and books during examinations . • Promote interaction of trainees. Allow questions, but do not deviate from the subject. • Respect the time a llotted. 35 BpeCifically: • Discuss with trainees experiences and problems of preservation and maintenance of genetic resources in nature and in gene banks (10 minutes). • Present the content of this Research Guide, using the study materials (or color slides) listed on page 3 (45 minutes). Involve trainees in the calculation of examples. Discuss the tables and illustrations with the help of overhead transparencies. • Conduct the practica1s suggested on page 3 in groups simultaneously (2 hours). Make sure that each trainee has the opportunity to practice. Have resource persons available for each group and practicsl. 36 Internattonal Institute of TropICal Agriculture Instltut International d'agnculture tropc.ale Instltuto Intemaoonal de Agricultuta TropICal (ITA) (ITA) (lIT A) The Intermtlonallnstitute of Tropic. Agriculture (ITA) Is IIIl intern. tion" agricultural research cMter in thlt Consu/Utlve Grot.t? on Intern. tional Agricultural Research (CGIAR), which ;5 an association of about SO count,;"s, intttmational and regional organizations, and private foundations. nT A seeks to increaStt agricultural production In a sustain- able WJlY, in ord", to improve the nutritional starus and well-being of people in tropical sub-Saharan Africa. To achiftw this ~, /ITA c0n- ducts re5elll"Ch and trairnng, provides Informlltion, collects JIf'Id exchanges germplJlsm. lind encOCJ~s transfer of technology, in partnership with African national agncuItUfil/ reSttarch and development programs. L '!nstitur international d'agricu/ture tropiaJe (ITA) est un CMtre in- ttN'nJItionai de recherche agricoIe, membre du Groupe ronsuJtJltif pour la r«herCM agricole intttmationale (GCRAJ), Uf"Ie association regroupant quelque SO PIIYS, organ;satlOi1S intemationales et ~ et fonciations priv~es. L 'Iff A a pour obJectif d 'ac:croItre clurablement la productlOfl agricole, ar", d'amMiorer "a'imentation et Ie bien-Itre des populations de I'Afrique tropicale subsaharienne. Pour IItteindre eet objectif, ,'1fT A ml!ne des IICtM tes de recherche et de formation, divulgcMl des informa- tions, rlunit et «hange du mathlel g~6tiqUt! et encourage Ie transferf de technologies en collaboration avec les programmes nationaux IIfricains de recherche et d6veloppemMt. o Instituto Intemacional de Agricu/rura Tropical (lfTA) e urn centro intemacional de If)vest;g~jo agricola pertencMdo so Grupo Consulrivo para Investig~1o Agricola IntemacionaJ (GCIAJ), uma assoc~1o de cerca de SO paises, organ,z~6es intemadonais e regionais e funda¢es pnvadllS. O/fTA procura aurnentar tiJravelmtYlte II prodllf;1o agrlcolll para meIhorar a a/iment~ e 0 bem-estar das popu/~6es cia Afnca tropk:aI so sui do Sahara. Para alcJIncar esse obj etivo, 0 NT A conduz activid.des de investJg~lo e treinamento, fomece infOfTT1lH;6es, recine e troca material gen~tlCO e favorece a transferlnda de tecnologias en cola- ~Io com os programas nacionais africanos de Invest~lo e desenvoMmiento.