http://cropgenebank.sgrp.cgiar.org/), a website specifically developed and officially launched in ... and monitoring, seed drying, seed storage, and regeneration.
Development of best practices for ex situ conservation of radish germplasm in the context of the Crop Genebank Knowledge Base Imke Thormann, Qiu Yang, Charlotte Allender1, Noortje Bas, George Campbell, Ehsan Dulloo, Andreas W Ebert, Ulrike Lohwasser, Chitra Pandey, Larry D Robertson, Olga Spellman
Abstract Information about crop-specific best practices for ex situ conservation of plant genetic resources has been difficult to find until recently. The CGIAR, together with national and regional partners, started to fill that gap by publishing best practices on the Crop Genebank Knowledge Base (CGKB http://cropgenebank.sgrp.cgiar.org/), a website specifically developed and officially launched in 2010 to provide easy access to knowledge about all aspects of ex situ conservation of specific crops to genebank managers and ex situ conservation researchers. A collaborative study, undertaken by Bioversity International with eight national and international genebanks, utilized the framework provided by the CGKB to develop and publish radish conservation best practices. This paper focuses on two aspects of this study: 1) Differences in procedures and practices in radish conservation currently applied in five key genebank activities, namely, acquisition of germplasm, viability testing and monitoring, seed drying, seed storage, and regeneration. While in a few cases genebanks agreed on a specific best practice to recommend, in others it was not desirable to identify one practice as superior to another, therefore a range of existing practices is described as a variety of equivalent options. The results highlight the importance of proactive genebank management aimed at meeting the standards within the specific context in which a genebank operates. 2) The framework and template provided by the CGKB in guiding the development of genebank best practices, and the CGKB as an excellent resource to widely and freely share best practices with the global community to support the effective management of crop genebanks.
Introduction Radish Radish (Raphanus sativus L.) is an ancient, annual or biennial cultivated vegetable with many uses and is one of the vegetable crops included in Annex I of the International Treaty on Plant Genetic Resources for Food and Agriculture (ITPGRFA). Radish belongs to the Brassicaceae (alt. Cruciferae) family with chromosome numbers 2n = 2x = 18. Cultivated radishes have several wild relatives, R. raphanistrum and its subspecies landra (Moretti ex DC.) Bonnier & Layens, maritimus (Sm.) Thell., microcarpus (Lange) Thell., raphanistrum, rostratus (DC.) Thell.; and R. confusus (Greuter & Burdet) Al-Shehbaz & Warwick (Hanelt and IPK 2001; Zhu et al. 2008). Pistrick (1987) divided cultivated radishes into three groups:
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Authors in alphabetical order from third author onwards.
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convar. oleifera (Raphanus sativus var. oleiformis Pers.), also called R. sativus Leaf Radish Group (Wiersema and León 1999) - oilseed and fodder radishes, which are grown in Southeast Asia and in Europe for leaf fodder, and as green manure. convar. caudatus (Raphanus sativus var. caudatus (L.) L. H. Bailey) also known as R. sativus Rat-Tailed Radish Group (Wiersema and León 1999) – the rat-tailed radishes (also known as mougri, radis serpent) grown for their edible immature green or purple seed pods and leaves. This kind of type is grown in Southeast Asia. convar. sativus (Raphanus sativus var. sativus) - also known as R. sativus Small Radish Group (Wiersema and León 1999) - all forms have edible roots, leaves and germinated radish sprouts, with many different varieties but generally of the small type (radish, small radish, turnip radish, petit rave).
Raphanus sativus L. var. niger J. Kern, also known as R. sativus Chinese Radish Group with the common names Chinese radish, Japanese radish and Oriental radish are recognized by Wiersema and León (1999) as a fourth cultivated group. The small-rooted and short-season types of radishes are cultivated for salads and as fresh vegetables. They are grown in temperate regions of the world and are harvested throughout the year (Crisp 1995). The large-rooted cultivars such as Chinese radish are predominant in East and Southeast Asia (Schippers 2004). They are usually cooked, canned or pickled besides being eaten raw. The leaves and sprouts are used as salad or are cooked, too. The seed pods are cooked for soups in southwest China and Southeast Asia. Wild radish seeds are rich in oil and have an oil content of up to 48 %, which is not suitable for human consumption but has good promise as a source of biofuel (Hoogendoorn and van Kasteren 2010). Farmers also grow oil radishes as fodder and to improve and fertilize the soil. Radishes are used in traditional medicine as non-poisonous materials to treat arthritis, cancer, constipation, coughs, dyspepsia, gallbladder disorders, gallstones and kidney stones, gastric discomfort, whooping cough and liver disorders (Adams 2008). Many radish types are no longer favoured because consumption patterns and preferences have changed. Furthermore, radish needs vernalization for bolting and flowering, making it more expensive for farmers to produce their own seeds as opposed to buying seeds at the market. Old radish landraces and varieties are replaced by modern hybrid varieties and are no longer cultivated. Wild radish species are threatened by ecological, climatic or human factors, and some landraces and varieties are already extinct (Yamaguchi and Okamoto 1997; personal communication Qiu Yang). It is therefore important to maintain and collect the radish diversity still existing in farmers’ fields and in the wild, and to conserve existing radish collections in genebanks in the most appropriate way possible to ensure its continued and sustainable use for both present and future generations. Based on data from WIEWS (http://apps3.fao.org/wiews/wiews.jsp) and personal communications with radish genebank curators, we estimate that there are more than 10,000 radish accessions conserved in over 30 genebanks around the globe. The largest collections are held in China (> 2000 accessions) and Japan (> 800 accessions), accounting together for nearly one third of the total accessions conserved worldwide. Germany, Russian Federation and the United States of America all have collections of over 500 accessions, many of which are cultivated varieties and landraces. Wild radish relatives are stored mainly in genebanks in Australia, Germany and Spain. 2
The Crop Genebank Knowledge Base The demand for novel plant diversity, as a resource for identifying diversity suited to changed ecoclimatic conditions and adaptive traits to be included in breeding programmes, is expected to increase as a consequence of global demographic and climatic change. Germplasm being conserved in genebanks, as well as new germplasm collected from the field, needs to be conserved in the best possible way to warrant the conservation and utilization of its intrinsic genetic variation. Procedures and conditions for conservation are species-specific and therefore require targeted knowledge, but information about crop-specific best practices for ex situ conservation of plant genetic resources has been difficult to find until recently. The System-wide Genetic Resources Program (SGRP) of the Consultative Group on International Agricultural Research (CGIAR), together with national and regional partners, started to fill that gap by compiling and publishing best practices for nine selected crops (banana, barley, cassava, chickpea, forage grasses and legumes, maize, rice and wheat) on the Crop Genebank Knowledge Base (CGKB, http://cropgenebank.sgrp.cgiar.org/) (SGRP 2010; Jorge et al. 2010). The CGKB offers a well structured and globally available knowledge sharing platform for these nine crop-specific best practices, which describe the procedures that, through experience and research, have proven to secure efficient and effective conservation of germplasm of the specific crop. The CGKB website, officially launched in December 2010, aims to contribute to more efficient and effective ex situ conservation of crop genetic resources through facilitating easy access to the knowledge and best practices for genebank management of selected crops and optimization of many other aspects of general genebank management. Scope of the paper The CGKB is as yet limited to major cereals, a legume, forages and two clonally propagated crops. A collaborative study between eight genebanks and Bioversity International used the structure provided by the CGKB to develop and share best practices for radish, a vegetable crop. This paper analyses the observed differences in procedures and practices in radish conservation currently applied in five key genebank activities, namely acquisition of germplasm, viability testing and monitoring, seed drying, seed storage and regeneration. It also describes the experience in using the framework and template provided by the CGKB in guiding the development of genebank best practices, and the CGKB as a resource to widely and freely share best practices with the global community to support the effective management of crop genebanks.
Methodology The CGKB makes available a template for download or online compilation of crop-specific best practices for ex situ germplasm conservation (http://cropgenebank.sgrp.cgiar.org/index.php?option=com_jforms&view=form&id=2&Itemid=551 &lang=english). This template and other best practices already published on the CGKB were used to structure the compilation of radish best practices. A first draft of radish best practices was compiled based on the first-hand experience of the second author, Qiu Yang, who is a radish researcher, and on a literature research. Only very scattered information on radish conservation existed in the literature. This first draft was extended and improved through on-site discussions with radish curators and genebank managers at the Leibniz 3
Institute of Plant Genetics and Crop Plant Research in Germany (IPK) and the Center for Genetic Resources, the Netherlands (CGN), and the collaboration of a further six genebanks who maintain major radish collections and had agreed to collaborate in the development of the best practices (see Table 1). The various steps and procedures applied in the genebanks were compared and discussed within the group and the document went through various stages of review to be finalized. Web pages were added to the CGKB to publish the final version of the radish best practices.
Table 1: Name and location of collaborating genebanks and number of Raphanus accessions conserved as of April 2012 Acronym Genebank Country Raphanus accessions AVRDC The World Vegetable Centre Taiwan 99 CAAS Institute of Vegetables and Flowers of the China 2164 Chinese Academy of Agricultural Sciences CGN Center for Genetic Resources the Netherlands 307 IPK Leibniz Institute of Plant Genetics and Crop Germany 830 Plant Research NGBNational Genebank of the National Bureau of India 253 NBPGR Plant Genetic Resources SASA Science and Advice for Scottish Agriculture United Kingdom 516 USDA Plant Genetic Resources Unit of the United USA 687 States Department of Agriculture in Geneva, NY WARGRU Warwick Genetic Resources Unit, The United Kingdom 398 University of Warwick
Results Best practices for effective and efficient ex situ conservation of radish genetic resources have been compiled, discussed and agreed in a collaborative effort among eight genebanks that conserve radish, based on their experience and expertise. The final document is published on the CGKB (http://cropgenebank.sgrp.cgiar.org/) in the website’s “crops” section, and is freely available to all users. Differences in procedures and practices for radish conservation currently applied in the genebanks were found in five key genebank activities: acquisition of germplasm, viability testing and monitoring, seed drying, seed storage and regeneration. When reference is made to genebanks in the text that follows, the eight collaborating genebanks are intended.
Acquisition Acquisition is the process of collecting or requesting seeds for inclusion in the genebank, together with related information, and based on official documents such as the Standard Material Transfer Agreement (SMTA) required for Annex I crops such as radish. Before accepting a new accession in a 4
genebank, it is a common practice for genebank curators to check the source of the material and ensure that the material is safe to be introduced into the collection. Another key aspect during the acquisition process is the verification of duplicate samples. Genebank curators will take steps to ascertain that the new sample is not already conserved in the genebank, so as to reduce the level of duplication and minimize maintenance costs. There was a general consensus that it is not easy to detect duplicates and no precise and globally accepted definition of duplicates is available. In the case of radish, there are clear differences between the ways in which genebanks treat duplicates (Table 2). While for example IPK validates data of new duplicate accessions and adds the material as a new accession to the collection, the World Vegetable Center (AVRDC) includes the material into the collection assigning a separate seed lot under the original accession number if the collecting site and other relevant passport data are identical with the original accession. CGN either archives the material conserving it at -20°C without further processing or discards it. USDA checks any potential new accession for duplication of existing accessions and will not add a known duplicate accession. The source of accessions is checked and if the new potential accession is from the original source while the existing accession is not, the original accession is inactivated and the new accession is added. The Chinese Academy of Agricultural Sciences (CAAS) sustains that the genebank passport database is not always sufficient to confirm whether a new acquisition is a duplicate of an existing accession. If the germplasm source, name and basic phenotypic traits are the same, they will not introduce it to the long-term collection, but archive and store it under mid-term conditions for further characterization or genetic studies required to confirm or rule out duplication. Very few samples of populations of local varieties and landraces of cross-pollinated crops are really the same, even if they have the same name and come from different families in a same village, except for pure lines. At the National Genebank of the National Bureau of Plant Genetic Resources (NGB-NBPGR) duplicates are identified based on the available passport data and genebank information. This helps to reduce redundancy, minimizes the running costs and makes more space available for conserving diverse accessions. The duplicates can be maintained in an active collection for supply to the users.
Table 2: Summary of practices used in genebanks to verify duplication during the acquisition of new germplasm Genebank Practice for verifying and managing duplicates AVRDC Includes the material into the collection assigning a separate seed lot under the original accession number in case passport data are identical. CAAS Archives and stores under mid-term conditions for further characterization. CGN Either archives materials at -20oC or discards it. IPK After data validation, adds material as a new accession. NGB-NBPGR Check for the duplicates based on available passport and genebank information. Adds it to the active collection for distribution to various users. USDA Does not add duplicates. The source of the material is however checked and if the new potential accession is from the original source, while the existing accession is not, the original accession is inactivated and the new accession is added. WARGRU Uses passport data to determine if new sample is likely to be a duplicate of any 5
existing accessions. Duplicates are usually not entered into collection and are discarded, but in exceptional circumstances they may be accepted and assigned a new and unique accession number.
Another important aspect of acquisition, and an issue of growing concern, is the question of contamination of genebank materials with transgenes. A GMO-free certificate is frequently reported as part of the necessary documentation to be verified upon consignment of the germplasm. In the case of radish, none of the eight genebanks actually requires this certificate, nor do any of the genebanks conserve transgenic radish. So far, very little genetic modification work (Park et al. 2005) has been done on radish and the possibility of acquiring/receiving radish material unintentionally contaminated with GMO germplasm is still very remote. This risk does exist for other crops, for example with maize, cotton and soybean, and avoiding the unintentional presence of transgenes in supposedly non-transgenic germplasm is becoming an increasing concern for many countries. The CGIAR has started to develop general and crop-specific (maize, potatoes, rice) guidelines for maintaining germplasm free from transgenes, which are available online on the CGKB (SGRP 2010). A declaration of the measures taken by the germplasm donor is now considered desirable for GMO risk management by the receiving genebank and should enable the latter to make GMO declarations to subsequent recipients. As GMO-free certificates are not yet compulsory, IPK carries out an internal risk assessment if no certificate is provided on a voluntary basis. This assessment is based on knowledge about the species, the provenance of the material and personal judgment. If no risk is found the material is included in the collection, otherwise it is discarded. The other genebanks considered this a good method and it is included as option in the best practices documentation. The GMO-free certificate is now listed as an optional documentation, in the event that the seed provider issues one or a genebank issues it in the case of safety-duplication and distribution.
Viability testing and monitoring Maintaining seed viability is a critical genebank function which counters the loss of genetic diversity in genebanks and ensures that seeds are of sufficient quality to be successfully used for evaluation and research. Good seed storage conditions maintain germplasm viability, but even under excellent conditions viability declines with storage duration (Walters et al. 2005). Genebanks therefore need to assess viability periodically to detect loss in viability during storage before it falls below the threshold for regeneration. Seed viability is determined through germination tests before seeds are packaged and placed in the genebank storage room and this serves as a reference point for subsequent periodic viability checking during storage. Germination tests The standard germination test recommends the use of a total of 200 seeds, i.e. two replications of 100 seeds for the initial germination test (FAO/IPGRI 1994; ISTA 2011). It is recommended to carry out subsequent tests with 50 – 100 seeds (FAO/IPGRI 1994). In practice the number of seeds that can be used for germination tests often depends on the size of the accession, and curators are aware of the need to save valuable seed. While five of the eight genebanks do apply standard germination tests, three genebanks reported having lowered the routine number of seeds used for germination 6
tests with good results. USDA reports to routinely use only 50 radish seeds for each replication with good results. If the first replicate already yields a sufficient result, the second replication is not always carried out. WARGRU uses a similar protocol but always tests two replicates of 50 seeds. IPK uses 50 radish seeds per germination test without replication if the result is sufficient. This approach is used to reduce costs and to keep the number of seeds used for germination testing to a minimum. Monitoring frequency The monitoring intervals for viability depend on initial germination percentage, specific genebank management aspects and the storage conditions of the base and active collections. The genebanks could be divided into two major groups, those that adopt a fixed interval for monitoring and those that calculate the interval depending on the initial germination rate. The fixed monitoring interval often follows the 1994 genebank standard rules, i.e. viability to be monitored after five years in the active collection and after ten years in the base collection (FAO/IPGRI 1994). But the interval set by the genebank can also be species-specific, such as the case of USDA that monitors both active and base collection of radish – as storage conditions are identical – every 15 years. Other genebanks that keep active and base collections under the same storage conditions usually monitor only one type of collection. CGN only monitors the base collection every 10-20 years depending on initial germination percentage. It does not monitor the active collection, as it is considered too expensive. Their users however often provide feedback on the germination of materials supplied and the follow up to this feedback is imbedded in the Quality Management System of the genebank. IPK monitors the active collection every 10-20 years depending on initial germination percentage, where seeds are stored in glass jars at the same temperature, as in their base collection. They do not monitor the base collection, where samples are stored under vacuum conditions.
Seed drying Prior to storage, seed samples need to be dried to the appropriate moisture content. The critical objective of seed drying and storage is to reduce the frequency of regeneration of the samples by maximizing seed longevity. To obtain the full benefit of refrigerated or freezer storage, it is recommended that genebanks dry seeds to the critical moisture level. The drying conditions and drying time used in genebanks vary, as shown in Table 3.
Table 3. Seed drying environments in genebanks Genebank Temperature (ºC) RH (%) Time AVRDC 18 10 2-3 weeks CAAS 20-25 20-30 7-10 days CGN 15 15 At least 6 weeks IPK 18 10-15 2-3 weeks NGB-NBPGR; 15 15 Up to 4 7
SASA USDA WARGRU
5 15
20 15
weeks 2 months At least 4 weeks
Usually the same drying conditions, in terms of temperature and relative humidity (RH), are used for all crops in a genebank; only drying duration will vary according to the crop and depends mainly upon the initial moisture content of the seed, seed quantity and seed size. NGB-NBPGR also reports that seeds received with high moisture content are pre-dried in a warm, dry environment before being transferred into a walk-in seed drying room or seed drying cabinet.
Storage Appropriate storage conditions and seed quality and quantity are required to maximize seed longevity. This will in turn reduce the frequency of regeneration, thereby reducing genebank maintenance costs and the risks of genetic erosion. For this purpose, all original samples (if enough seeds are available) and their safety-duplicates should be kept under long-term storage in base collections. Where the objective is to store seeds that are required over the medium- or short-term for distribution to users and evaluation of germplasm, seeds are usually conserved in the active or working collection. Variations between seed quantity, quality in terms of germination percentage and conditions for storage of radish seeds in genebanks are described below. Germination percentages for storage Germination percentage is an essential characteristic of an accession. It is assessed during registration monitored during the storage period and its decrease triggers the need for regeneration. Acceptable percentages for storage are found to vary between genebanks. For cultivated radish, CGN, IPK and USDA set the minimum initial germination at 80% for accepting a seed sample into their base collections. This 80% rate is based on acquired long-term experience. They indicate the need to achieve that level of initial germination to maintain the quality and integrity of the base sample. Chances of a rapid drop in germination are considered high when initial germination is less than 80%. In general, the genebanks report that for many species it is too difficult to reach germination of above 85%, the percentage set in the FAO/IPGRI genebank standards (FAO/IPGRI 1994). AVRDC, CAAS and NBPGR follow the 85% germination standard for radish, as recommended in the genebank standards for accessions in base collections (FAO/IPGRI 1994). However, in the case of cultivated radish, the 80% limit as a minimum is found to be agreeable to all genebanks. Compared to cultivated radish, an initial germination of 60% for wild radish species is found to be generally acceptable, although some considered this percentage to be borderline. For the active collections much bigger differences are found between the minimum germination percentages applied for inclusion of accessions and as a trigger for regeneration. Some genebanks like CAAS, CGN and NBPGR use the same germination percentage as for their base collections. IPK and WARGRU use a minimum of 50%. USDA uses a 60% germination trigger at which accessions in 8
the active collection must be regenerated, but continues distributing seeds through to approximately 40-50% germination until the regeneration process is completed. For genetic resources stocks and variety registration collections at the Science and Advice for Scottish Agriculture (SASA), at 50% germination a replacement would be sought, but they do not consider low germination to be a serious problem provided that they are aware of it. Particularly for genetic resources stocks, the percentage of live seed (normal germination % + abnormal germination %) is important, as abnormally germinating seeds often establish into plants which will yield a normal harvest. The genebanks agreed that a minimum of 70% germination for the active collection is an acceptable and recommendable percentage, which also corresponds to the minimum set by the Indian Seed Certification Standards (Tunwar NS and Singh SV 1988). Seed quantity for storage Seed quantity for storage varies considerably between the genebanks. The number of seeds used for an accession in the base collection ranges from about 1000 to 6000. A base sample usually includes as a minimum sufficient seeds for 2 – 3 regeneration cycles, and sometimes also seeds for 2 – 4 germination tests. The number of seeds included per regeneration cycle has been found to be quite variable, from a minimum of 50-100 seeds per regeneration cycle up to 300. One genebank even sets aside 600 seeds per regeneration cycle, of which 300 are used for regeneration and the remaining 300 serve as a back-up, in the event that germination is very low or regeneration fails. Seed numbers included for germination tests vary from 50 to 200 per test. At least one genebank prepares separate seed packages for the various germination tests and regeneration cycles during the seed packaging process. It is agreed that this should be recommended as a good practice as it makes it much easier and quicker to retrieve seeds from storage when necessary. The size of the samples for the active and base collections are the same, except for CGN, where the active sample is smaller, and for IPK and WARGRU, where the active samples are composed of the remaining seeds after the base and safety-duplicate samples have been established. If insufficient seed is available, only an active sample is established and the accession is scheduled for further regeneration. The size of the safety-duplicate sample varies between 600 and 2000. It always includes a minimum of seeds for two regeneration cycles. USDA includes seeds required for as many as ten standard field regeneration attempts. AVRDC prepares two safety-duplicate samples containing 200 seeds per sample. One duplicate sample is stored in Svalbard, the other in an active collection. If access to the safety-duplicate is needed, they can ask the active collection to send one of the packages back, while the other remains as a safety-duplicate.
Table 4: Seed quantities for ex situ conservation in the eight participating genebanks Genebank Base Composition of Active Safety-duplicate sample (No. sample base sample sample (No. of seeds) (No. of of seeds) seeds) 9
AVRDC
1500
CAAS CGN
6000 2600
IPK
1000-2500
NGB-NBPGR
4000
USDA
2000
WARGRU
~900
200 seeds per regeneration cycle, 2x100 for germination testing 3x600 for regeneration, 4x200 for germination testing 2x300 (minimum) for regeneration, 2x50 for germination testing 3x100 for regeneration, 2x100 for germination testing 8x200 for regeneration, 4x100 germination testing Average of ~900 seeds for regeneration
1500
800 (2 samples of 400 seeds each to be stored in two different genebanks)
6000 1800
6000 600
remaining seed
600 (minimum)
4000
No separate safetyduplicates maintained
2000
2000
Remaining seed – includes germination test samples
~900
Storage conditions in active and base collections Although all the genebanks distinguish between active and base collections, the storage conditions are not necessarily distinct as well. All of the genebanks maintain their base collections at temperatures below zero, most of them following the -18°C FAO/IPGRI standard. Storage temperature at SASA and WARGRU is -22°C ± 3°C. Storage conditions for the active collection vary between genebanks depending on the facilities, the environment and resources. Genebanks that are able to store the active collection at the same conditions as the base collection such as CGN, IPK, USDA and WARGRU usually do so to reduce the number of regeneration cycles due to loss of viability. Where this possibility does not exist or is not chosen, active collections are kept under controlled temperature above 0°C, ranging from +2°C at CAAS and +5°C at AVRDC, to +4°- 8°C at NGB-NPBGR. Relative humidity is controlled where seeds are not stored under vacuum conditions and ranges between 30 - 35% RH. AVRDC for example does not control RH, as seed samples are sealed in light vacuum conditions in aluminum foil bags. IPK reports that radish seeds can be stored under ambient conditions for about five years without significant decrease in viability. 10
Regeneration Regeneration is a key operation and an integral responsibility of any genebank. It is the renewal of germplasm accessions by sowing and harvesting the seeds which will possess the same characteristics as the original population. The regeneration process (also called “multiplication”) leads to an increase in the number of seeds stored in the genebank and/or to increased viability of the seeds equal to or above an agreed minimum level. An accession is regenerated when it does not have sufficient seeds for long-term storage or when its germination percentage has dropped below an established minimum threshold. Variations in quantity and germination thresholds have been reported. Seed quantity threshold The threshold for regeneration in terms of seed quantity varies between the genebanks and ranges from 1000 to 1500 seeds or 10g (which can also be less than 1000 seeds, depending on the seed weight). USDA for example chooses 1500 seeds as a trigger for scheduling regeneration of the accession, as this will allow continued distribution while the accession is in the regeneration queue. Germination percentage threshold While seed numbers as a trigger for regeneration vary only slightly between the genebanks, this changes when the germination percentage is considered as the threshold for regeneration. While some genebanks, like CGN and NBPGR, apply the 1994 Genebank Standard of 85% initial germination, others use a considerably lower percentage threshold. AVRDC, SASA and USDA regenerate when germination falls to 60% and WARGRU at 50%. It is reported that at these levels of germination the material is still stable and can easily be regenerated. 50% is a percentage that triggers regeneration particularly of wild species. This low percentage is given by the fact that wild species often have a very low germination percentage.
Discussion The collaborative approach involving genebanks from different countries and continents to elaborate best practices for ex situ conservation of a specific crop has presented the opportunity to compare procedures employed in different realities. The work undertaken has shown that, for a number of steps in the conservation process of radish, different practices exist in the various genebanks. This approach has provided the opportunity to make known and promote to others successful practices which were found to be used in just one of the genebanks such as the internal GMO risk assessment practice used at IPK, through inclusion in the best practices document published on the CGKB. The different approaches to verifying and managing duplicates illustrate a range of options existing in genebanks of which none can be said to be superior to the other, but that depend largely upon the general management approach of the genebank to handle duplicates. The different types of pollinators that can be used for radish, and the timing of their introduction into the cages for artificial pollination during regeneration, are another example in which different equivalent valid practices exist (see 11
http://cropgenebank.sgrp.cgiar.org/index.php?option=com_content&view=article&id=615&Itemid= 822&lang=english ). The drying environments used for radish in the various genebanks differ considerably even although most of them are within the temperature and RH ranges included in the revised genebank standards which recommend drying all seed samples to equilibrium in a controlled environment of 5-20°C and 10-25 % of relative humidity (FAO, pers. communication). Species-specific drying conditions that achieve the critical moisture level at the chosen storage temperature can be determined using water sorption isotherms which show the relationship between the amount of water in the seeds, usually expressed as a percentage of the total seed weight, and their RH. Isotherm relationships, predicted based on seed oil content, are available online at the Kew Seed Information Database (SID) website (http://data.kew.org/sid/viability/index.html). There could be different combinations of relative humidity and drying temperature for a given species to achieve the desired moisture content. However the operational conditions in the genebanks do not provide for the possibility to dry species at those specific combinations of temperature and relative humidity which would be the most appropriate for the specific species, but require having a unique drying environment that is the most appropriate for the whole range of species that are conserved in the collection. For this reason no recommended drying condition is included in the best practices documentation. The critical moisture content was discussed but has not been included in the best practices. Curators indicated that it is not known and is not a value that they take into consideration when drying radish seeds. The size of seed samples included in the active and base collections is another example where the differences are given by the specific context in which a genebank operates, e.g. the expected frequency of requests for radish seeds, the use of samples for characterization and evaluation etc. The reduction in the number of seeds used for germination tests that has been experimented and then implemented as routine practice in IPK provides an example of adjusting standards to improve the management of the collection also rationalizing resources. In genebanks with large collections of different species, such as IPK who test thousands of accessions per year, it allows increasing the number of accessions that can be tested annually and therefore helps to better identify initial decline in viability in accessions. It also helps to avoid sub-sampling of accessions for germination testing, which is sometimes carried out when resources are limited and not all accessions can be tested. As it has been shown that there is intraspecific variation in seed longevity (Nagel et al. 2010; Nagel and Börner 2010), testing of all accessions is a preferred practice to random selection. The differences in practices currently in place in the different genebanks illustrate that although genebanks across the globe share many of the same basic goals, their missions, resources and the systems they operate within often differ. Curators have to proactively apply practical experience and knowledge to germplasm management in a genebank to optimize their own genebank system, and this requires management solutions which may differ substantially across institutions while achieving the same objectives. We have not always been able to identify one single best practice but have found that often the various approaches represent a range of good practices worth describing and including in the radish practices published on the CGKB. The authors therefore suggest to define as best practices for conservation those practices that, within the individual context of resources and operational system of a specific genebank, proactively aim to meet the
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genebank standards required to preserve the genetic integrity of the conserved sample and to make it available to users. The Crop Genebank Knowledge Base provides a very useful structure to guide the development of such crop-specific best practices. Combined with a collaborative and inclusive process such as the one chosen in this study, it represents an ideal opportunity to tap into the breadth of crop-specific experience and expertise available globally, and the website offers an excellent place to make this collective knowledge available to the global conservation community. The CGKB is a free resource that offers the possibility to anybody in the international genebank community to publish and share best practices and other methodologies for any chosen crop, in order to obtain input and comments from peers and users and to support the effective management of crop genebanks.
Acknowledgement We wish to thank CAAS for the financial support of Qiu Yang’s secondment as visiting research fellow for three months to Bioversity International, during which the main compilation work was carried out. We also thank Bioversity International for providing the resources to coordinate the whole process of compilation, development and publication of the best practices.
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References Adams M (2008) Radish. [online]. Available from: http://www.healingfoodreference.com/radish.html. Date accessed: 21 July 2009. Crisp P (1995) Radish, Raphanus sativus (Cruciferae). In: Smartt J, Simmonds NW, editors. Evolution of crop plants. 2nd Edition, Longman Scientific & Technical, UK. pp. 86–89. FAO/IPGRI (1994) Genebank standards. Food and Agriculture Organization of the United Nations and International Plant Genetic Resources Institute, Rome, Italy. Hanelt P, IPK, eds (2001). Mansfeld's Encyclopedia of Agricultural and Horticultural Crops. Springer, Berlin, Germany. Hoogendoorn A, van Kasteren H (2010) Transportation Biofuels. Novel pathways for the production of ethanol, biogas and biodiesel. Royal Society of Chemistry Publishing. UK. 208 pp. ISTA (2011) International rules for seed testing. Edition 2011. International Seed Testing Association. Bassersdorf, Switzerland. Jorge MA, Claessens G, Hanson J, Dulloo ME, Goldberg E, Thormann I, Alemayehu S, Gacheru E, Amri A, Benson E, Dumet D, Roux N, Rudebjer P, Sackville Hamilton R, Sanchez I, Sharma S, Taba S, Upadhyaya HD, van den Houwe I (2010) Knowledge sharing on best practices for managing crop genebanks. Agricultural Information Worldwide 3:2 101-106. Available from: http://journals.sfu.ca/iaald/index.php/aginfo/article/view/179. Date accessed: 14 October 2011. Nagel M, Börner A (2010) The longevity of crop seeds stored under ambient conditions. Seed Science Research 20:1-12. Nagel M, Rehman Arif MA, Rosenhauer M, Börner A (2010) Longevity of seeds - intraspecific differences in the Gatersleben genebank collections. Tagungsband der 60. Jahrestagung der Vereinigung der Pflanzenzüchter und Saatgutkaufleute Österreichs 2009, 179-181. Park B.-J., Liu Z., Kanno A., Kameya T (2005) Transformation of radish (Raphanus sativus L.) via sonication and vacuum infiltration of germinated seeds with Agrobacterium harboring a group of 3 LEA gene from B. napus. Plant Cell Rep. 24: 494-500. Pistrick K (1987) Untersuchungen zur Systematik der Gattung Raphanus. Kulturpflanze 35:224321.Schippers RR (2004) Raphanus sativus L. [online] Record from Protabase. Grubben GJH, Denton OA, editors. PROTA (Plant Resources of Tropical Africa / Ressources végétales de l’Afrique tropicale),Wageningen, Netherlands. Available from: http://database.prota.org/search.htm. Date accessed: 22 March 2012. SGRP (2010) Global Public Goods Project Phase 2 - Final Report. System-wide Genetic Resources Programme (SGRP). Bioversity International, Rome, Italy. Tunwar NS, Singh SV (1988) Indian Minimum Seed Certification Standards CSCB Dept of Agril & Coop Min of Agril GOI, New Delhi pp 388. 14
Walters C, Wheeler LJ, Grotenhuis J (2005) Longevity of seeds stored in a genebank: species characteristics. Seed Science Research 15:1-20. Wiersema JH, León B (1999) World Economic Plants – A Standard Reference. CRC Press, USA. WIEWS [online] World Information and Early Warning System on PGRFA, available from http://apps3.fao.org/wiews/wiews.jsp. Date accessed: 13 September 2011. Yamaguchi H, Okamoto M (1997) Traditional seed production in landraces of daikon (Raphanus sativus) in Kyushu, Japan. Euphytica 95:141-7. Zhu DW, Wang DB, Li XX (2008) Chinese crops and wild relatives, vegetable crops volume (1). Beijing: Chinese Agricultural Press.
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