Regeneration

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Page compiled by: Bioversity International/ILRI, Addis Ababa, Ethiopia (Alexandra Jorge); ILRI, Addis Ababa, Ethiopia (Jean Hanson) including information extracted from: Rao NK, Hanson J, Dulloo ME, Ghosh K, Nowel D and Larinde M. 2006. Manual of seed handling in genebanks. Handbooks for Genebanks No. 8. Bioversity International, Rome, Italy. 147pp.

What is regeneration

Regeneration is the renewal of germplasm accessions by sowing seeds or planting vegetative materials and harvesting the seeds or plant materials which will posses the same characteristics as the original population.

Germplasm regeneration is the most critical operation in genebank management, because it involves risks to the genetic integrity of germplasm accessions due to selection pressures, out-crossing, mechanical mixtures and other factors. The risk of genetic integrity loss is usually high when regenerating genetically heterogeneous germplasm accessions. Germplasm regeneration is also very expensive.

Why should germplasm be regenerated

Germplasm is regenerated for the following purposes:

1. To increase the quantity of initial seeds or plant materials

In new collections or materials received as donations, the quantity of seeds or plant materials received by the genebank is often insufficient for direct conservation. Seeds or plant materials may also be of poor quality due to low viability or infection. All these materials require regeneration. Newly acquired germplasm of foreign origin may need to be initially regenerated under containment or in an isolation area under the supervision of the national plant quarantine authorities.

2. To replenish seed stocks or plant materials in active and base collections

Accessions that require regeneration are identified through monitoring viability and quantity during storage. Seed stocks or plant materials require regeneration for accessions that have:

• Low viability identified during periodic monitoring;
• Insufficient stocks for distribution or conservation. 

Active collections should be regenerated from original seeds or plant materials from a base collection; this is particularly important for out-breeding species to maintain the requested seeds as close as possible to the original sample. Using seeds from an active collection (click here to view the video showing this genebank procedure) for up to three regeneration cycles before returning to the original seeds or plant materials (base collection) is also acceptable (FAO 2013).

Base collections also require regeneration at very frequent intervals and should normally be regenerated using the seed or plant materials from the same sample.

Regeneration is expensive and time consuming, especially for perennial tree species which may not flower until over 20 years after planting, and comes with risks to genetic integrity and loss of material attached. Therefore, it is usual to harvest sufficient seeds to return seeds to the base collection for long term storage and the active collection to meet anticipated distribution needs as well as prepare subsamples for safety duplication from the same regeneration cycle.

How is it done

Each crop is different and requires its own environment and agronomic management during planting, growth and harvesting. Information provided in this section is general but specific regeneration guidelines have been developed for a range of crops and can be accessed by clicking here.

The following are the main factors to consider when regenerating germplasm accessions:

• Suitability of environment (click here to view the video showing this genebank procedure) to minimize natural selection;
• Suitability of site with adequate water and suitable soil type to minimize risk of loss of plants;
• Distance from other fields of the same crop to reduce unintentional geneflow and sources of diseases or pests;
• Adequate numbers of plants to maintain genetic integrity and retain genes in low frequency;
• Special requirements, if any, to break dormancy (click here to view the video showing this genebank procedure) and stimulate germination or promote flowering;
• Correct spacing (click here to view the video showing this genebank procedure) for optimum seed set;
• Breeding system of the plant and need for controlled pollination or isolation during field management;
• Harvesting (click here to view the video showing this genebank procedure) and mixing equal numbers of seeds from all plants to obtain representative samples.

If possible, regenerate germplasm in the ecological region of its origin or one to which it is adapted and likely to flower and produce seeds. Daylength or vernalization are important for some crops and wild species to trigger flowering and seed set. Seek an environment that does not select some genotypes in preference to others in a population to maintain genetic integrity. Examine the biotic environment in the context of prior information about the plants and past experience - an inappropriate biotic environment can be detrimental to the quality of plants, seed or propagation materials and the genetic integrity of an accession.

If no suitable site is found, seek collaboration with an institute that can provide a suitable site or regenerate in a controlled environment such as a greenhouse or growth room. The latter is important to control seed dispersal for wild species and crop relatives that may be considered as arable weeds (click here to view the video showing this genebank procedure).

Meeting special requirements

There may be special requirements for regeneration of accessions with special traits that breeders and researchers use frequently—such as high-yielding, pest-and disease-resistant accessions and genetic stocks — or if there are insufficient seeds (click here to view the video showing this genebank procedure) for safety duplication and repatriation. 

When should it be done

It should be done when either the quantity and/or the quality of a particular seed or plant material are not sufficient in a genebank.

The regeneration of accessions that have inadequate quality (low viability) should take priority over that of accessions with inadequate numbers of seeds or planting materials but with high viability.

The regeneration of accessions in base collections should take priority over regenerating those in active collections.

Regeneration guidelines for specific crops

Crop specific regeneration guidelines are available for the following crops:

• Introduction to regeneration guidelines
• Banana
• Barley (included in guidelines for small-grained cereals)
• Beans
• Breadfruit
• Cassava
• Chickpea
• Coconut
• Cowpea
• Fababean
• Finger millet
• Forage grasses
• Forage legumes
• Grasspea
• Lentil
• Maize
• Major aroids
• Oats (included in guidelines for small-grained cereals)
• Pearl millet
• Pigeon pea
• Rice
• Sorghum
• Sweet potato
• Wheat (included in guidelines for small-grained cereals)
• Wild potato
• Yam

References and further reading

Crossa J. 1995. Sample size and effective population size in seed regeneration of monoecious plants. In: Engels JMM, Rao RR, editors. Regeneration of seed crops and their wild relatives. Proceedings of a consultation meeting, 4–7 December 1995, ICRISAT, Hyderabad, India. IPGRI, Rome, Italy. pp.140–143.

Crossa J. 1989. Methodologies for estimating the sample size required for genetic conservation of outbreeding crops. Theoretical and Applied Genetics 77: 153-161.

Crossa J, Hernandez CM, Bretting P, Eberhart SA, Taba S. 1993. Practical considerations for maintaining germplasm in maize. Theoretical and Applied Genetics 86: 673–678.

Crossa J, Vencovsky R. 1994. Implications of the variance effective population size on the genetic conservation of monoecious species. Theoretical and Applied Genetics 89:936–942.

Crossa J, Vencovsky R. 1997. Variance effective population size for two-stage sampling of monoecious species. Crop Science 37:14–26.

Engels JMM, Visser L, editors. 2003. A guide to effective management of germplasm collections. IPGRI Handbooks for Genebanks No. 6. IPGRI, Rome, Italy. Available in English (1.4 MB) and Spanish (1.5 MB).

FAO. 2013. Genebank standards for plant genetic resources for food and agriculture. Food and Agriculture Organization of the United Nations, Rome. Available in English, Spanish, French, Arabic, Russian and Chinese here.

Rao NK, Hanson J, Dulloo ME, Ghosh K, Nowel D, Larinde M. 2006. Manual of seed handling in genebanks. Handbooks for Genebanks No. 8. Bioversity International, Rome, Italy. Available in English (1.5 MB),  Spanish (1.4 MB) and French (1.9 MB).

Sackville Hamilton NR, Chorlton KH. 1997. Regeneration of accessions in seed collections: a decision guide. Handbooks for Genebanks No. 5. IPGRI, Rome, Italy. Available here.

Thormann I, Metz T, Engels JMM. 2004. The Species Compendium (release 1.0; December 2004). [online]. Available from URL: http://www.bioversityinternational.org/scientific_information/information_sources/species_databases/species_compendium.html. Date accessed 30 March 2010

Vencovsky R, Crossa J. 1999. Variance effective population size under mixed self and random mating with applications to genetic conservation of species. Crop Science 39:1282–1294.

Distribution

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Page compiled by: Bioversity International/ILRI, Addis Ababa, Ethiopia (Alexandra Jorge); ILRI, Addis Ababa, Ethiopia (Jean Hanson) including information extracted from: Rao NK, Hanson J, Dulloo ME, Ghosh K, Nowel D and Larinde M. 2006. Manual of seed handling in genebanks. Handbooks for Genebanks No. 8. Bioversity International, Rome, Italy. 147pp.

What is germplasm dissemination

Germplasm distribution is the supply of representative samples of seeds or plant material accessions from a genebank in response to requests from germplasm users. In general, seeds or plant material is only distributed from active collections.

Why is germplasm distributed

The purpose of conserving germplasm in a genebank is to make it available for use by current and future generations, usually to improve crop varieties through plant breeding to meet the needs of farmers and communities, for research activities or to restore diversity lost on farm and in natural habitats. Genebanks can be more proactive in establishing links with germplasm users, breeders, researchers, farmers and other groups to enhance the use of the germplasm.

Legal considerations for distribution

Access to genetic resources for distribution is guided by international law, specifically the Convention on Biological Diversity and the International Treaty on Plant Genetic Resources for Food and Agriculture (Treaty), and is tied to sharing any benefits from the use of the material. Germplasm is usually distributed under a Material Transfer Agreement (MTA) that sets out the terms and conditions of access and responsibilities of recipients.

In the case of crop germplasm, the Treaty establishes a global system to provide farmers, plant breeders and scientists with no-cost, facilitated access to plant genetic material. It also ensures that users share any benefits they derive from genetic material used in plant breeding or biotechnology with the regions from which they originated. It recognizes a set of crops in Annex 1 of the Treaty with mutual dependence among countries and establishes a multilateral system for their conservation and use, using a standard Material Transfer Agreement setting out the terms of access and benefit sharing.

Several genebanks, including those of the International Agricultural Research Institutes, signed an agreement with the Governing Body of the Treaty placing their in-trust collections of Plant Genetic Resources for Food and Agriculture (PGRFA) within the purview of the Treaty. In accordance with this Agreement, all shipments of PGRFA of crops listed in Annex 1 to the Treaty (shipments of PGRFA under the Multilateral System) will be subject to the terms and conditions of the Standard Material Transfer Agreement (SMTA). In the event that the SMTA is used for the transfer of PGRFA other than those listed in Annex 1 of the Treaty:

• i) The references in the SMTA to the "Multilateral System" shall not be interpreted as limiting the application of the SMTA to Annex 1 Plant Genetic Resources for Food and Agriculture, and in the case of Article 6.2 of the SMTA shall mean "under this Agreement".

• ii) The reference in Article 6.11 and Annex 3 of the SMTA to "Plant Genetic Resources for Food and Agriculture belonging to the same crop, as set out in Annex 1 to the Treaty" shall be taken to mean "Plant Genetic Resources for Food and Agriculture belonging to the same crop".

The SMTA allows the recipient to utilize and conserve the material for research, breeding and training and distribute it to other parties provided such parties accept the terms and conditions of the SMTA and also to agree to a system of benefit sharing from any commercial use of the germplasm.

Those genebanks from countries which have signed the Treaty or have signed an agreement with the Governing Body of the Treaty will require recipients to agree to the terms of access under the SMTA before any germplasm can be distributed and will provide documents with the germplasm to clarify the conditions for its use.

How should germplasm be distributed

Germplasm should be distributed in a way that ensures it reaches its destination in good condition. Environmental conditions during transport can be detrimental to seed or plant material quality and viability, so seeds or plant material should be carefully packed and distributed in adequate containers for protection during transit using the most direct and fastest route. Mail or courier services with a tracking system are very useful to follow the package to its destination. 

The scope and extent of distribution varies with each genebank. Germplasm may be distributed within or outside the country, depending on the genebank’s mandate and whether its collection is national, regional or global.

Dissemination of germplasm in detailed steps

Click here to open a PDF document that shows how to disseminate germplasm in detailed steps.

 References and further reading

Rao NK, Hanson J, Dulloo ME, Ghosh K, Nowel D, Larinde M. 2006. Manual of seed handling in genebanks. Handbooks for Genebanks No. 8. Bioversity International, Rome, Italy. Available in English (1.5 MB),  Spanish (1.4 MB) and French (1.9 MB).

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Safety duplication

Contact person for Safety duplication: Jean Hanson, ILRI, Ethiopia

Contributors to this page: CIP, Lima, Peru (David Tay, Ana Panta, Catherine Espinosa); IFPRI, Washington (Daniela Horna); ILRI, Addis Ababa, Ethiopia (Jean Hanson); independent consultant (Juvy B. Cantrell); Bioversity International/ILRI, Addis Ababa, Ethiopia (Alexandra Jorge) including information extracted from: Rao NK, Hanson J, Dulloo ME, Ghosh K, Nowel D and Larinde M. 2006. Manual of seed handling in genebanks. Handbooks for Genebanks No. 8. Bioversity International, Rome, Italy. 147pp.

What is safety duplication?

This is the duplication of a genetically identical sub-sample of the accession to mitigate the risk of its partial or total loss caused by natural or man-made catastrophes. The safety duplicates are genetically identical to the base collection and are referred to as the secondary most original sample (Engels and Visser, 2003). Safety duplicates include both the duplication of material and its related information, and are deposited in a base collection at a different location, usually in another country. The location is chosen to minimize possible risks and provides the best possible storage facilities.

Safety duplication is generally under a ‘black-box’ approach. This means that the repository genebank has no entitlement to the use and distribution of the germplasm. It is the depositor’s responsibility to ensure that the deposited material is of high quality, to monitor seed viability over time and to use their own base collection to regenerate the collections when they begin to lose viability. The germplasm is not touched without permission from the depositor and is only returned on request when the original collection is lost or destroyed.

Safety duplication is done for all original seeds collected by the genebank or only held by the genebank. Seeds which are duplicates from other collections can usually be retrieved from those collections and do not require safety duplication unless there is doubt about their security in the other collection.

Information on the principles and best practices for safety duplication are spread through the literature. Recommendations based on scientific evidence and principles are presented as best practices for safety duplication of crop germplasm. Some general principles for safety duplication are given below.

• Maintain at least one duplicate of each accession as a safety back-up.
• Select a location with a suitable environment, good security and low risk for the duplicate samples.
• Consider risks to loss of genetic integrity within accessions and loss of accessions in selection of the location.
• Consider costs and practical arrangements.

How is it done?

Samples are prepared for safety duplication in the same way as is the base collection:

• Sample size should be sufficient to conduct at least three regenerations.
• Material should be clean and healthy.
• Material should be placed in appropriate containers.

Safety duplication of seeds should be under a black-box agreement while duplication of in vitro and cryopreserved samples may be under different agreements that require the recipient to assist in monitoring and reporting. If the storage conditions for the backup collection are the same as for the base collection, loss of viability can be predicted from the results of base collection monitoring.

Seed genebanks

• At least 500 viable seeds for outbreeders and heterogeneous accessions with high diversity.
• A minimum of 300 seeds for genetically uniform accessions.
• More seeds for accessions with seeds of low viability.
• An initial percentage viability of 85% and 3-7% seed moisture content.
• Packaging material of 12 µm of polyester, 30 µm of aluminium foil and an inner layer of high-density polythene of 80 µm thickness.
• Self-adhesive labels that are resistant to below zero temperatures outside and inside each packet of seeds.
• Storage temperatures of -18°C to -20°C.
• Samples should be renewed from the sender when the viability of the samples in similar storage conditions in the sender's base collection starts to decline. The samples in black-box storage can be either destroyed or returned to the sender and replaced with a new batch.

Field genebanks

• A minimum of three to five replicates per accession.
• Renewed when 30% of the collection is no longer viable.

In vitro genebanks

• A minimum of three to five replicates per accession.
• Stored in glass test tubes or gas-permeable, heat-sealable polyethylene bags.
• Samples should be renewed from the sender (sub-cultured from remaining replicates in sender genebank) when one replicate deteriorates or shows signs of senescence.

Cryo genebanks

• A minimum of 100 units or propagules per accession.
• Two batches of accessions must be cryopreserved separately to minimize risk.
• Polyester thermal transfer-printed label for cryovials.
• Polypropylene vials with a secure seal for cryosamples.
• Storage containers with narrow necks.
• Alarm system to indicate low liquid nitrogen level.
• Samples should be renewed from the sender when the viability of the samples in similar storage conditions in the sender's cryo collection starts to decline. The samples in black-box storage can be either destroyed or returned to the sender and replaced with a new batch.

Packing and shipping

Strong cold-resistant boxes (thick carton or polypropylene box) are the best options for transporting and storing seeds. Boxes should be sealed properly. Shipment should consider the fastest means of transport available either by air freight, courier or by land to avoid deterioration of seed quality or cultures during transit. In vitro cultures in sealed test tubes can be packed in strong thick cardboard or plastic boxes while cryopreserved materials should be transported using liquid nitrogen.

For black-box duplication outside the country, special permission is required to export seeds without phytosanitary certificates from the originating country. Similarly, the phytosanitary authority in the destination country must permit the recipient to import seeds without the routine quarantine examination. Material sent as in vitro or cryo plant cultures may require disease indexing before shipping for major diseases so that only certified disease-free germplasm is exported (see pages on Safe Transfer of Germplasm).

It is recommended to ship by the most direct and fastest route in the cooler seasons and label boxes clearly to indicate the need for temperature control and proper handling during shipment. It is desirable to include a data logger outside the box to monitor temperature changes during transport.

More information and a detailed analysis of options and current practices for safety duplication of seeds and vegetative material assessed against technical and economic considerations that mitigate risk, as well as recommended best practices based on scientific evidence and principles, can be found in: Safety duplication principles and strategy (December 2008).

Legal arrangements

Any safety duplication arrangement requires a clear signed legal agreement between the depositor and the recipient of the safety duplicate that sets out the responsibilities of the parties and terms and conditions under which the material is maintained. A draft standard depositor's agreement that can be customized to meet the needs of safety backup of seeds or vegetative collections is available at:

Safety deposit agreement - user's guide
Standard safety deposit agreement

Documentation

Suggested information for safety duplication includes the following:

• Depositor code.
• Depositor’s accession number/identifier.
• Crop and full scientific name: genus, species, subspecies, including authority.
• Storage conditions/collection type.
• Type of container.
• Container identification number.
• Total amount of seeds stored (by weight or number) or number of replicates/accessions (test tubes, cryovials, number of plants per container).
• Date (month/year) of viability testing & regeneration (seeds) or subculture (in vitro).
• Date of freezing (cryopreservation).
• Collecting number.
• Country of collection or source.
• Date of storage.
• Location in genebank.

The Svalbard Global Seed Vault

The Svalbard Global Seed Vault in Norway is an example of a secure facility for safety duplication of crop genetic resources. Located far beyond the Arctic Circle and 130 m deep inside a frozen mountain, permafrost provides an environmentally friendly solution to long-term secure conservation of crop diversity as a third safety duplicate that is only accessed in case of disaster or loss of the samples from the main safety backup. The vault can hold 4.5 million seed samples of crop diversity at storage temperatures of -18°C. Currently over 420 000 samples have been deposited there for safety. For more information visit the learning resources and Svalbard Global Seed Vault website.

Experience with using the safety duplication principles

Safety duplication tends to be done on a routine basis, but at irregular intervals, when a batch of material has been regenerated and is ready for duplication, therefore experience with using these principles and recommendations will take time. Some general issues that should be considered include:

• The principles are very generic and may be difficult to implement for some species due to the inherent biology of the samples, e.g. short-lived seeds, large seeded species where space and cost may be limiting.
• More careful tracking of shipments and monitoring of viability is important to ensure that seeds of high viability reach the duplicate site. However, since most samples are in black-box storage and viability cannot be checked on arrival, this raises a logistic problem. It is proposed that monitoring the samples should be routinely included in the shipment and agreements reached with the recipient institution on monitoring viability or returning samples for monitoring to the sender.
• Issues of liability may occur related to sending samples in sealed black-box conditions. One issue is on liability for contents of the sealed box and handling by customs and other authorities for entry into a country. In some cases boxes are opened and special seals are applied by the authorities to confirm that the samples are not medicinal or other prohibited plants. Another issue raised has been on the liability of the recipient institution should material be damaged or lose viability earlier than expected due to poor storage conditions.
• Genebanks may be more likely to use the new recommendations and agreement if approved by the Secretariat of the International Treaty on Plant Genetic Resources for Food and Agriculture.
• Some additional public awareness on the recommended best practices and draft agreement is needed to promote their more widespread use.

Current status of adoption of the safety duplication principles and lessons learnt from their application.

References and further reading

Dussert S, Engelmann F, Noirot M. 2003. Development of probabilistic tools to assist in the establishment and management of cryopreserved plant germplasm collections. Cryoletters 24:149-160.

Engelmann F. 2004. Plant cryopreservation: Progress and prospects. In vitro Cellular and Developmental Biology plant 40:427-433.

Engels JMM, Visser L, editors. 2003. A guide to effective management of germplasm collections. IPGRI Handbooks for Genebanks No. 6. IPGRI, Rome, Italy. Available in English (1.4 MB) and Spanish (1.5 MB). 

FAO. 2013. Genebank standards for plant genetic resources for food and agriculture. Food and Agriculture Organization of the United Nations, Rome. Available in English, Spanish, French, Arabic, Russian and Chinese here.

Harding K. 2004. Genetic integrity of cryopreserved plant cells: A review. CryoLetters 25:3-22.

Nordgen. 2008. Agreement between (depositor) and the Royal Norwegian Ministry of Agriculture and Food concerning the deposit of seeds in the Svalbard Global Seed Vault. The Svalbard Gloal Seed Vault. [online] The Nordic Genetic Resource Centre, ALNARP. Available from:  http://www.nordgen.org/sgsv/scope/sgsv/files/SGSV_Deposit_Agreement.pdf.

Reed BM. 2008. Plant Cryopreservation: A practical guide. Springer Science and Business Media, USA 513 pp.

Reed BM. 1991. Application of gas-permeable bags for in vitro cold storage of strawberry germplasm. Plant Cell Reports 10:431-434.

Reed BM. 1992. Cold storage of strawberries in vitro: A comparison of three storage systems. Fruit varieties journal 46:98-102.

Reed BM, Engelmann F, Dulloo ME, Engels JMM. 2004. Technical guidelines for the management of field and in vitro germplasm collections. IPGRI Handbooks for genebanks No. 7. International Plant Genetic Resources Institute, Rome, Italy. Available here.

Reed BM, Paynter C, Bartlett B. 2002. Shipping procedures for plant tissue cultures. USDA-ARS-NCGR. Available from: http://www.ars-grin.gov/cor/presentations/shipping2001/sld001.htm. Date accessed: 26 March 2010.

Tay DCS, Liu CR. 1992. Using hard agar medium and grooved tubes for the distribution of sweet potato tissue culture. Plant Genetic Resources Newsletter-FAO/IBPGR no. 88/89:23-25.

Documentation