Best practices for forage legume genebank management

Best practices for forage legume genebank management

Crop

Forage legumes

Author(s)

Dr Jean Hanson, Dr. Ahmed Amri, Dr. Kenneth Street, Mr. Bilal Humeid, Mr. Fawzi Sweid, Dr. Ali Shehadeh, Mr. Jan Konopka, Ms. Natalya Rukhkyan, Dr. Siham Assad, Mr. Adnan Omran, Ms. Fida Alo

Organization(s)

ILRI, ICARDA

Other contributions (please list all genebanks contacted that provided information)

Genetic Resource Centre for Temperate Pasture Legumes, Perth

General guidelines: This format was developed to collect and gather updated and current recommendations on the best practices for genebank management for 8 target crops (please provide references whenever possible). Use one format per crop, first describing distinct procedures for each type of genebank (steps 2, 3 and 4 - add additional paragraphs for different methodologies) and secondly the common procedures (regardless of type of conservation, i.e. steps 1, 5, 6 and 7). Please specify special cases for genotypes or wild species that deviate from the normal crop behavior. This guideline was prepared for either seed or clonal crops; use only the subtitles relevant for your crop and add any additional information needed. This information must have enough detail so that the work can be repeated elsewhere. The recommendation(s) of the best practices should first be gathered from other genebanks, then compared, discussed and adapted and finally chosen and justified into this data format. This justification should be clear, concise and simple. The recommendations of best practices can result in more than one best practices, taking in consideration different scenarios (different conditions and facilities) if applicable. Recommended best practices are not necessarily what the CGIAR centres are currently doing, but how genebank activities should be carried out, with the current technical knowledge. The given format was developed in detailed boxes to prompt and facilitate the gathering of relevant information from the various contributors. This format will then be adapted/adjusted according to each crop, into the Knowledge Base website. There will be cases of headings that are not applicable for a specific crop. There will be therefore three choices when filing a heading (please write one of these choices for any heading): - Applicable, and therefore should be filled. - Not applicable for this crop. - Information not known. Please fill the information on the left column an the justification on the right column We recognize that many of the best practices currently in use are likely to be those recommended from the Genebank Standards, 1994. FAO, IBPGR. If this is the case, please just refer to this document (or any others) in the justification column. Ex situ conservation strategies: (Indicate which methods are used to conserve this crop and list the starting material for each) a) Seed bank Starting material (seed category i.e. orthodox, intermediate, recalcitrant)

Best practices: Orthodox seeds

Justification:

b) In vitro bank Starting material (ex vitro axilary buds, in vitro plant materials or seed category)

Best practices: Justification:

c) Cryo bank Starting material (specify)


d) DNA bank

Starting material (specify)


e) Field bank Starting material (specify)

Best practices:

Justification:

f) Others (specify) Starting material (specify)


References for all genebank steps (please list with full citations any relevant documents, web sites, movies, or publications. Attach also any relevant photos, drawings or diagrams you might have that illustrate these procedures) Citation Link (or file name) FAO/IPGRI. 1994. Genebank Standards. Food and Agriculture Organization of the United Nations

General standards

ISTA. 2008. International Rules for Seed Testing. International Seed Testing Association. ISTA secretariat

Viability testing

McIvor, J.G. and Bray, R.A. (eds) 1983. Genetic Resources of Forage Plants. CSIRO, Australia.

Forage genetic resources practices

Rao N.K., Hanson J., Dulloo M.E., Ghosh K., Nowell D. and Larinde M. 2006. Manual of seed handling in genebanks Handbook for Genebanks No.8. Bioversity International, Rome, Italy.

General genebank management

http://www.ilri.org/forage/ http://www.tropicalforages.info

Step 2 – Sample processing in seed banks (describe the general procedures used for this crop, to be stored under the above described method) 2.1 – Cleaning (removal of physical contamination from the plant materials before it can be store). Include threshing, washing, sterilization, disinfestations and pre-treatments such as pre drying of seeds, seed moisture content determination) Best practice: Machine threshing, followed by air screen cleaning. Final cleaning by blowing or hand cleaning. Hand harvesting and cleaning used

Justification: Legume seeds are hard and there is no physical damage. All contaminants should be removed because of space constraints and costs of storage. Many wild species have pods which shatter and require special handling.

2.2 - Visual inspection of seeds (insect and fungal damage, mechanical damage and empty seeds) Best practice: According to ISTA rules Spread the seeds on a flat well lit surface of contrasting color. An illuminated table can be used if available. Examine dry seeds with the naked eye or under binocular. This method reveals free moving insects, eggs, mites, fungal fructifications, bacterial masses, infected plant debris. Examinations under or near ultraviolet reveals infections of certain fungi and bacteria through emission of fluorescence. For insect and fungal damage Isolate the infested/infected samples: destroy when quarantine diseases and/or insects; otherwise clean theseeds before storage using fumigation to control insects. For mechanical damage and empty seeds Clean manually shriveled and damaged seeds

Justification: ISTA standard method To prevent further spread of fungi or insects. To kill adult insects but does not kill larva and eggs and fungi.

2.3 – Special treatments (fumigation, dipping) Best practice: If infested with insects, place seeds in deep freeze for 3 days. Fumigation

Justification: There is no information on the effects of agrochemicals on longevity during storage in genebanks, so it is better to avoid them. Also for staff health and safety

2.4 - Disposal of contaminated materials (procedures to deal with infected or contaminated plant samples) Best practice: Incinerated or autoclaved Justification:

To avoid spreading diseases and pests and destroy infected materials

2.5 - Inspection and certification (purity analysis of seeds) Best practice: According to ISTA rules Justification: ISTA standard method 2.6 - Seed drying (procedures, predicting drying time, predicting moisture content, critical moisture content, equilibrium moisture content) Best practice: Method: Dehumidified drying room 25% rH and 15oC. The

Justification: Recommended in genebank standards

seed samples are kept in a paper, mesh or cotton bags on a mesh racks during the drying period. Drying time: Depends on seed size, from 2- 8 weeks

N/A

Moisture content before drying: Depends on seed size and seed coat, 8-12% and up to 20% for Lupinus spp.

N/A

Moisture content for storage: 5% + 2% except soyabean not less than 8%

Recommended in genebank standards, experience that seeds remain with high viability for >20years

Critical moisture content: 3-4% for some temperate forage legumes

It is recommended for most of the crops not to dry below 3 % to avoid any adverse effects on the seed texture and viability. But recent findings are suggesting drying to 2% to extend storage period.

Others:

2.7 – Recording information during sample processing (list the data fields that should be recorded and the recommended methods to check possible errors or understand changes during storage) Data field name Content Accession number ID number Lot number ID number Seed weight Weight of seeds for storage Reference to seed source To trace the origin of the sample. Flags (Y/N) indicating completion of steps mentioned above

Checking.

Remarks Step 3 – Germplasm testing in seed banks (describe the tests for quality, genetic integrity and fitness for this crop, of each type of seeds or plant materials to be stored under the above described method) 3.1 - Seed moisture content in seed banks 3.1.1 - Seed moisture content determination methods Best practice: Justification: Sampling frequency: Sampled once at end of drying period to determine moisture for storage. Random sample during storage.

Standard drying periods under the drying conditions available in the genebank have been determined and so it is only necessary to measure moisture content on sample accessions once.

Sample size: Take 1.0-2.0 g (0.25-0.5 g for small seeded species) from each sample as two independent replicates

Follows a modified ISTA method using more replicates of smaller weight of seeds

Grinding: Follow ISTA rules. Coarse grinding is required by using adjustable mechanical grinder.

Standard and proven method

Oven drying temperature: Follow ISTA rules

Standard and proven method

Others:

3.1.2 - Recording information during seed moisture content (list the data fields that should be recorded and the

recommended methods to check possible errors or understand changes during storage) Data field name Content Accession number ID number Lot number ID number Genus Genus name of the plant, entered in full. Species Species name of the plant, entered in full. Date of test The date that the test was commenced Method Name of standard method Drying temperature Oven temperature Fresh weight in grams Weight of sample per rep Dry weight in grams Weight of sample per rep Moisture content Percentage moisture content after drying 3.2 – Viability testing in seed banks 3.2.1 – Viability testing laboratory methods (describe the various recommended options, taking into consideration various levels of technical expertise, equipment or laboratory conditions). Best practice: Justification: Type of test: Standard germination tests (see attached list) Sequential germination tests

Viability test methods are specific for different species. International Seed Testing Association rules are used when available. Less accurate, but requires less seeds and suitable in case of limited number of seeds. This test should be undertaken twice for accuracy. If the results are less than critical value then use an addition test.

No of seeds and replicates: A minimum of four replicates of 50 seeds is used. For the sequential test use 10 seeds in 4 replications

International Seed Testing Association rules are used when available.

Pre-treatment: See attached list

Viability test methods are specific for different species. International Seed Testing Association rules are used when available.

Media: See attached list


Temperature: See attached list


Light: A 12 hour light period is used to simulate tropical daylength conditions

International Seed Testing Association rules are used when available.

Duration of test: See attached list


Others: Germination is complete when the seedling can be judged as normal according to specific criteria.

(ISTA [2003, 2005], Association of Seed Analysts (AOSA), [2005]).

3.2.2 – Monitoring intervals (describe the recommended intervals for different storage conditions) Best practices: 10 years above 50% germination 5 years below 50% germination or when longevity not known

Justification: Legume seeds show long longevity so the sampling interval should be sufficient to measure loss of viability without being so frequent as to use valuable germplasm for testing

3.2.3 - Recording information during viability testing (list the data fields that should be recorded and the recommended methods to check possible errors or understand changes during storage) Data field name Content Accession number ID number Lot number ID number Genus Genus name of the plant, entered in full. Species Species name of the plant, entered in full. Date of test The date that the test was commenced Number of reps The number of replicates in the test Number of seeds per rep The number of seeds in each replicate of the test. Pre-treatment Pre-treatments used for the test. More than one can be used in

conjunction. Media The media for the test Temperature The temperature used during the test in degrees centigrade. If an

alternate temperature is used this temperature usually refers to the temperature during the day period, unless stated otherwise.

Alternate temperature If more than one temperature is used, the temperature in degrees centigrade of the alternate period. The alternate temperature usually corresponds to the dark period.

Length of dark The number of hours that there is no light. This usually corresponds to the period at lower temperature, when two temperatures are used.

Length of test The number of days during which the test was continued. Percentage germination The average percentage germination over all replicates, calculated

from the number germinated per replicate Tolerance A measure of the statistical accuracy of the test according to the

standard tolerance tables Percentage dormancy The average percentage of hard seeds which are not rotten, over

all replicates Test reference A consecutive number given each time that seed lot is tested over

time. 3.3 – Health diagnosis in seed banks (describe the recommended methods for each type of plant materials in storage) - If you have already provided this information to Cecilia Ynouye, please cut and paste the relevant information here. 3.3.1 – List pests and diseases of quarantine importance (website links will be inserted here to similar outputs from activity 3.1) Pest Region(s) Bacteria: Pseudomonas syringae pv. Phaseolicola, Curtobacterium flaccumfasciens Xanthomonas sp., Pseudomonas fluorescens

LAC

Virus: Bean common mosaic virus (BCMV), Bean southern mosaic virus (BSMV), Bean yellow mosaic virus (BYMV), alfalfa mosaic virus (AMV), Cowpea mosaic virus (CPMV),

SSA, WANA,

Peanut mottle virus (PeMoV), Soybean mosaic virus (SMV) Fungi: Cercospora canescens, Colletotrichum sp., Fusarium oxysporum, Macrophomina phaseoli, Phaeoisariopsis griseola, Phoma sp, Rhizoctonia solani, Penicillium sp. Sclerotium rolfsii, Drechslera sp., Curvularia sp., Helminthosporium sp., Pyricularia sp., Botrytis sp., Macrophoma sp., Phomosis sp.

SSA

3.3.2 – Provide options for testing procedures (considering various equipment and facilities scenarios, i.e. giving recommendations for various options) Pest Method Virus ELISA, TBIA Ascochyta spp. Freezing blotter method, PDA Phoma spp. Freezing blotter method, PDA Botrytis cinerea PDA Colletotrichum sp Blotter method Fusarium oxysporum Blotter method Rhizoctonia solanum Blotter method Penicillium sp. Blotter method Ditylenchus dipsaci Nematode test Orobanche spp. Filter wash test Cuscuta spp. Filter wash test 3.3.3 – Testing intervals/seasons (recommend the adequate times to perform each testing) Best practices: Virus: Test seedlings before transfer to the field for regeneration or during regeneration and rogue infected materials Fungus: Test seed lots on entry to genebank Ascochyta spp., Phoma spp., Botrytis cinerea Ditylenchus dipsaci Orobanche spp., Cuscuta spp

Justification: Testing before material goes into the genebank or to the field is important to reduce transfer of disease. Field inspection at all stages and rogue infected plants or/and seed. Field inspection at all stages and rogue infected plants or/and seed. Field inspection at flowering stage, rogue before seed dispersal and burn the plants.

3.3.4 - Recording information during health diagnosis (list the data fields that should be recorded and the recommended methods to check possible errors or understand changes during storage) Data field name Content Accession number ID number Lot number ID number Genus Genus name of the plant, entered in full. Species Species name of the plant, entered in full. Date of test The date that the test was commenced Number of reps The number of replicates in the test Number of seeds/plants per rep The number of seeds/plants in each replicate of the test. Pre-treatments Pre-treatments used for the test.

Media The media for the test (fungi) Pathogen tested Name of pathogen tested Test method Method used Plot numbers Plot ID number Percentage infection % of seeds or plants infected 3.4 – Transgenes detection in seed banks (only important for those crops that have undergone transgenic events and where transgenic varieties had been used in the area of collection) - If you have already provided this information to Monica Mezzalama, please cut and paste the relevant information here. 3.4.1 – List the current transgenes (for this crop in each major region of growth) (website links will be inserted here to similar outputs from activity 2.1.2) Transgene Region(s) 3.4.2 - Transgenes determination methods (describe the general required tests for the genetic integrity of this crop, to be stored under the above described method) Transgene Method 3.4.3 - Recording information during transgene detection (list the data fields that should be recorded and the recommended methods to check possible errors or understand changes during storage) Data field name Content 3.5 – Storage monitoring in seed banks 3.5.1 – Routine monitoring methods (describe the recommended monitoring methods for minimum viability and quantity of plant materials for the described storage method for this crop) Best practices: Routine testing for seed viability at regular intervals Stock control system queried to determine accessions for regeneration Accessions can be flagged in the database as: (A) Accession is available for distribution (L) Low stock; Accession is available for distribution but multiplication should be planned(S) Low number of seeds; no distribution is allowed; sample should be multiplied. Genebank facilities are checked for working order, damage or leaks and preventive

Justification: Monitoring methods for viability should minimize use of seeds while providing a good indication of reduction of viability. Marking accessions in the database helps to avoid distribution of accessions by mistake and quickly identify accessions for regeneration. Regular monitoring of equipment and buildings allows early

maintenance is done on equipment.

identification of problems and preventive maintenance before failure.

3.5.2 – Monitoring frequency (List the minimum quantity and minimum viability of seeds below which they need to be regenerated) Best practice: Justification: Critical quantity 600-1000 seeds

This allows two regeneration attempts using 300 seeds giving a high probability to have 100 plants to maintain genetic integrity of accessions, even for seeds of viability of 50%

Critical germination level 85% of the original germination percentage and 75 % for wild species

FAO/IPGRI standards

3.5.3 - Recording information during storage monitoring in seed banks (list the data fields that should be recorded and the recommended methods to check possible errors or understand changes during storage) Data field name Content Accession number ID number Lot number ID number Weight of seeds Weight of seeds on hand 1000 Seed weight Weight of one thousand seeds to be used for calculating number

of seeds from weights Percentage germination The average percentage germination over all replicates, calculated

from the number germinated per replicate Stock control flag Code to indicate stock level and distribution Regeneration flag Code to indicate if regeneration required Step 4 – Conservation - Types of storage in seed banks 4.1 - Base collections in seed banks (In this section describe when, why and how should it be done; fill the information below for conditions and requirements) Best practice: When should be used All original samples should be placed in long term storage

Justification: Original samples are the best representative sample of that accession and storage conditions should be used to retain viability as long as possible

4.1.1 - Sample specifications Best practice: Justification: Minimum sample size/viability for storage: 4000 seeds or 5g for small seeds of 90% germination or 80% germination for wild species

FAO/IPGRI standards

Moisture content: 3-7%

FAO/IPGRI standards

4.1.2 - Container specifications Best practice: Justification: Seed packaging method: Packets are moisture proof and very practical, making good use

Laminated aluminium foil. Use of vacuum for packing is optional Packing is best carried out in an air-conditioned room with controlled humidity as soon as possible after drying.

of space, and re-sealable. Rapid packing in a dry environment prevents reabsorption of moisture by the seeds during packing.

Specifications of packaging material: Minimum quality: Outer polyester of 12 µm thickness, middle aluminium layer of 9 µm thickness and inner polythene layer of 55 µm.

This thickness is impermeable and sufficiently flexible for handling and strong enough for forage legume seeds, which are usually smooth.

4.1.3 – Storage specifications Best practice: Justification: Assigning location codes: Location codes should specify the exact place in the store e.g. unit/block number, row number, shelf number and tray/box number. Moveable shelves on tracks are preferred.

Exact locations are important to locate material in the store. Moveable shelves make best use of space.

Storage conditions: -18oC to -20oC

FAO/IPGRI standards

4.2 - Active collections in seed banks (In this section describe when, why and how should it be done; fill the information below for conditions and requirements) Best practice: When should be used To store seeds for distribution and use

Justification: Seed quantities for distribution are usually larger and are not usually stored for long periods, therefore storage conditions are less stringent.

4.2.1 - Sample specifications Best practice: Justification: Minimum sample size/viability for storage: A minimum of 1500 seeds and up to 500g and above 85% of original germination

Sufficient seeds for distribution needs for period of high viability


FAO/IPGRI standards

4.2.2 - Container specifications Best practice: Justification: Seed packaging method: Laminated aluminium foil Plastic containers if store dehumidified.

Packets are moisture proof and very practical, making good use of space and re-sealable. Store must be dehumidified to avoid absorption of moisture from the air surrounding the seeds.


This thickness is impermeable and sufficiently flexible for handling and strong enough for forage legume seeds, which are usually smooth.

4.2.3 – Storage specifications Best practice: Justification: Assigning location codes: Exact locations are important to locate material in the store.

Location codes should specify the exact place in the store e.g. unit/block number, row number, shelf number and tray/box number. Moveable shelves on tracks are preferred.

Moveable shelves make best use of space.

Storage conditions: 0 to to 10 oC

Sufficient to maintain seed viability for at least 25 years

4.3 - Safety duplication in seed banks (In this section describe when, why and how should it be done; fill the information below for conditions and requirements) Best practice: When should it be used For all original seeds collected by the genebank or only held by the genebank.

Justification: 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.

4.3.1 - Sample specifications Best practice: Justification: Minimum sample size/viability for storage: Minimum of 600 seeds of more than 85% viability. 500 seeds acceptable for Svalbard. 1000 seeds preferable.

See activity on safety duplication


FAO/IPGRI standards

4.3.2 - Container specifications Best practice: Justification: Seed packaging method: Laminated aluminium foil. Use of vacuum for packing is optional Packing is best carried out in an air-conditioned room with controlled humidity as soon as possible after drying.

Packets are moisture proof and very practical, making good use of space, and re-sealable. Rapid packing in a dry environment prevents reabsorption of moisture by the seeds during packing.


4.3.3 – Storage specifications Best practice: Justification: Assigning location codes in boxes: Boxes are numbered and lists of seeds per box maintained in a database

Use of internal partitions increases weight and costs and in most cases entire boxes are returned to owner.

Storage conditions: Long term storage

Long term storage is needed to ensure the longevity of seeds in safety duplication and the costs of preparing and shipping safety duplicates is not economic if storage is for short periods.

Shipping method: Courier or air freight or any rapid method

Method should avoid heating and delays in transport.

Legal arrangements: Common CGIAR agreement should be used.

• Standard agreement • Special agreement for Svalbard • Phytosanitary certificate; • Certificate of origin; • Certificate of no-commercial value; • Electronic and hard copy of associated

passport information; • GMO-free certificate (if required);

4.4 - Storage space arrangement in seed banks (recommend the best options, suggestions and practical hints for this crop) Best practices: Moveable racks Shelves should be spaced slightly more than the height of the containers

Justification: This maximizes space between and on the racks in the store and is the most economic way to utilize the space.

4.5 - System for tracking materials/inventory system in seed banks (describe the best recommended methods as well as alternatives in case of limited conditions) Best practices: Database of stock and location and use of bar codes

Justification: Databases are needed to keep track of information. Bar codes help avoid errors in recording.

4.6 - Recording information during conservation in seed banks (list the data fields that should be recorded and the recommended methods to check possible errors or understand changes during storage) Data field name Content Accession number ID number Lot number ID number Weight of seeds Weight of seeds in store Number of seeds Number of seeds in store Thousand seed weight Weight of 100 seeds Number of packets Number of containers for seeds of one lot of one accession Location of containers Store, shelf or box number Type of container Material and size of container Year of production of seeds Indicates age of seeds Flag for regeneration [Y/N] Flag indicating distribution status [A,L,S] Year of safety-duplication Year Institute holding the duplicate Name of institute holding the safety duplication Location of duplicate sample Box label where the duplicate sample is placed E) Information required for Field banks Step 2 – Sample processing on field banks (describe the general procedures used for this crop, to be stored under the above described method) 2.1 - Source of material on field banks (briefly describe how the materials should be collected and cleaned) Best practices: Justification: Pre-treatment Fumigation Insecticide application

Others Propagation material to be planted Type of propagule

Cleaning Washing Threshing Sterilization Disinfestations Others

2.2 - Visual inspection of plant materials on field banks (describe recommended procedures) Best practice: Justification: Insect damage Fungal damage Mechanical damage Empty seeds Others

2.3 – Cleaning propagules on field banks (removal of physical contamination from the plant materials before it can be store) Best practices: Justification: Pre-treatment Fumigation Insecticide application Others

2.4 - Disposal of contaminated materials on field banks (procedures to deal with infected or contaminated plant samples) Best practices:

Justification:

2.5 - Recording information during sample processing on field banks (list the data fields that should be recorded and the recommended methods to check possible errors or understand changes during storage) Data field name Content Step 3 – Germplasm testing on field banks (describe the general required tests for quality, genetic integrity and fitness for this crop, of each type of plant materials to be stored under the above described method) Best practices:

Justification:

3.1 – Health diagnosis on field banks (describe the recommended methods for each type of plant materials in storage/planted) - If you have already provided this information to Cecilia Ynouye, please cut and paste the

relevant information here. 3.1.1 – List pests and diseases of quarantine importance on field banks (for this crop in each major region of growth) (website links will be inserted here to similar outputs from activity 3.1) Pests Region(s) 3.1.2 – Provide options for testing procedures on field banks (taking in consideration various equipment and facilities scenarios, i.e. giving recommendations for various options) (website links will be inserted here to similar outputs from activity 3.1) Pests

Method

3.1.3 – Testing intervals/seasons on field banks (recommend the adequate times to perform each testing) Best practices

Justification:

3.1.4 - Recording information during health diagnosis on field banks (list the data fields that should be recorded and the recommended methods to check possible errors or understand changes during storage) Data field name Content 3.2 – Transgenes detection on field banks (only important for crops that have undergone transgenic events and where transgenic varieties had been used in the area of collection) 3.2.1 – List the current transgenes on field banks (for this crop in each major region of growth) - If you have already provided this information to Monica Mezzalama, please cut and paste the relevant information here. Transgene Region(s) 3.2.2 - Transgenes determination methods on field banks (describe the general required tests for the genetic integrity of this crop, to be stored under the above described method) (website links will be inserted here to similar outputs from activity 2.1.2) Transgene Method 3.2.3 - Recording information during transgenes detection on field banks (list the data fields that should be recorded and the recommended methods to check possible errors or understand changes during storage) Data field name Content 3.3 – Storage monitoring for field banks

3.3.1 – Routine monitoring methods for field banks (describe the recommended monitoring methods to test the quality/viability of plant materials for the described storage method for this crop) Best practices: Justification: Survival rates Vigour Pests and diseases Replacement by other weeds

3.3.2 – Monitoring frequency on field banks (describe the recommended frequency monitoring should be done in each type of storage for this crop) Best practices

Justification:

3.3.3 - Recording information during storage monitoring on field banks (describe the type of information that should be recorded and the recommended methods to check possible errors or understand changes during storage) Data field name Content Step 4 – Conservation on field banks - If you have already provided this information to the regeneration guidelines, please indicate where applicable and add any additional relevant information in the boxes below. 4.1 – Sample specifications on field banks 4.1.1 – Sample preparation (describe the methodology to prepare the samples for storage/planting; propagation/germination techniques, pre-treatments, special care, procedures) Best practices: Justification: Pre-treatments Propagation/germination techniques Plant health requirements Special care 4.1.2 - Sample (explant) type and size Best practices: Justification: Propagule type Propagule size Propagule development stage Minimum/maximum numbers of replicates/ accession to plant

Others 4.2 – Storage specifications on field banks 4.2.1 – Field preparation Best practices: Justification: Field selection Planting season

Soil/substrate preparation Isolation Randomization Replication Special requirements 4.2.2 – Field planting Best practices: Justification: Spacing Design and layout Method 4.2.3 – Field maintenance and management Best practices: Justification: Weeding method Weeding frequency Irrigation Fertilizer application Pruning/coppicing 4.2.4 – Harvest for renewal Best practices: Justification: Method Frequency Health testing 4.2.5 – Post harvest handling (For those species where renewal of field genebank is required on a regular basis e.g cassava) Best practices:

Justification:

4.2.6 - System for tracking materials/inventory system in field banks (describe the best recommended methods as well as alternatives in case of limited conditions Best practices:

Justification:

4.2.7 - Recording information during storage/planting in field banks (list the data fields that should be recorded and the recommended methods to check possible errors or understand changes during storage) Data field name Content

Common steps (regardless of conservation strategy) Step 1 – Registration: Describe the registration procedures for this crop. Add additional steps if required. 1.1 – Verification accompanying documentation (list required documents and information including quarantine

regulations) Best practices: List of accessions Phytosanitary certificate Plant import permit GMO free certificate (if GMOs in crop) SMTA or germplasm acquisition agreement Passport data

Justification: Minimal documentation is essential to track material. Health and IPR certificates are very important for entry to the country.

1.2 – Verification of consignment (physical/ visual inspections required before accession numbers are assigned) Best practice: • Check all packets against the list provided

with the samples. • If no list is provided or seeds do not

correspond with the list, prepare a list and send to donor/provider for confirmation and obtaining the proper data.

• Check seeds for insect infestation or obvious signs of disease or damage during shipping.

• Isolate the infested/infected samples: destroy when quarantine diseases and/or insects; otherwise clean the seeds before storage using fumigation to control insects.

• If possible, ask donor to replace sample.

Justification: Verification. Avoid introduction of new pests and diseases and storing dead material. Only seeds that appear in good condition and have a high probability of being viable should be registered

Best practice: - Check genebank database for duplication, i.e. if the sample is already conserved in the genebank. a. If it is, assign a new seed lot under the original accession number (of suspected duplicate). The ‘duplication’ will be verified later (at first growth out and/or characterization). b. If sample appears ‘original’, assign a unique accession number. Write the accession number clearly on the packet using a permanent marker. - For each accession enter all passport data (plus any other information if provided) in genebank database.

Justification: Avoid conserving duplicates. To have a complete information on genebank accessions. Data enhances the value of the germplasm.

1.3 – Recording information (actual registration, labeling and methods to enter the information) Data field name Content

Accession number ID number Lot number ID number Source Country and institute where the seeds came from Date of registration Date of entry to genebank Step 5 - Regeneration (this information has been collected in a separate format and will be extracted from there) Step 6 - Characterization (describe the recommended procedures for this crop) 6.1 – Planting and cultural practices for characterization (detail if differ from regeneration procedures) Best practice: Justification: Environment The environment is selected on species adaptation. (See attached table)

The environment should be suited to the species for the traits to be expressed for characterization.

Soil type Soil type is selected on species adaptation (See attached table)

The soil type should be suited to the species for good growth.

Rainfall Rainfall of >350mm or 500mm with irrigation or 1000-1200mm rainfed for most forage species. Irrigate the field directly after sowing if first rain is delayed. Supplementary irrigation should be applied as necessary to ensure adequate seed yield to measure seed traits.

Sufficient rainfall or supplementary irrigation is essential for flowering and seed set to observe floral traits.

Season Data is usually taken at 50% flowering for forages. This may be end of the wet season for annuals and anytime that flowering occurs for perennials.

Morphological data is best taken at plant maturity when 50% of the plants are flowering.

Plot size Either plots of 2m2 or 25m2 or single rows of at least 1-2m long per accession with up to 90cm between rows depending on species.

Plot size should be large enough to accommodate at least 10 plants for data collection.

Sampling area/border area Quadrats of 50cm to 1m for yield and 10 plants from within the centre of the plot for morphological traits.

This has been shown to be statistically acceptable for forage crops

Plant density Plant density is selected on species adaptation (See attached table)


Replications A minimum of three reps and preferably 4 with data taken from at least 10 plants across reps.


Standard check cultivars None used since there are few cultivars for many forages

Frequency of standard checks Not applicable

Time of day for data collection Time of day is selected for each species for when flowers are open.

6.2 - Morphological descriptors for characterization (website links will be inserted here to Bioversity descriptors). Detail any additional descriptors not included in the Bioversity descriptors for this crop. If there are no Bioversity descriptors attach a list of descriptors used/recommended Descriptor Definition See lists for forages developed by Bioversity International

6.3 - Pictures for characterization (describe relevant plant parts to record) Best practice: Take images for character(s) which may be difficult to describe verbally. Images of whole plant, racemes (if applicable), flowers, pods. Store in a database file linked to other characterization data.

Justification: Sufficient detail should be captured in images to taxonomically identify the plant and demonstrate the traits that show variation.

6.4 - Herbarium samples for characterization (describe best procedures for your crop) Best practice: Take and store vouchers for future reference and taxonomic verification. Collect plants with leaves, flowers and pods, also roots if plants are small. Spread plants open before pressing.

Justification: Sufficient detail should be captured to taxonomically identify the plant and demonstrate the traits that show variation.

6.5 - Molecular descriptors for characterization (describe best procedures for your crop, if applicable) Best practice: SSR, EST-SSR, AFLP, RAPD

Justification: More efficient techniques.

6.6 - Cytological characterization (describe best procedures for your crop, if applicable) Best practice:

Justification:

6.7 – Nutritional traits for characterization (describe best procedures for your crop) Best practice: Crude protein Minerals (P, K, Ca) Palatability Digestibility Fibre (Neutral detergent fibre, Acid detergent fibre) Lignin Anti-nutritional factors (tannins, saponins, alkaloids, non-protein amino acids) ODAP content for Lathyrus spp

Justification: The main use for forages is for livestock feed. Nutritional traits are therefore very important.

6.8 – Others

Best practice:

Justification:

6.9 - Recording information during characterization (list the data fields in addition to descriptors that should be recorded and the recommended methods to check possible errors or understand changes during storage). Select from the list below and add any missing ones) Data field Content Site Name of site with coordinates if possible Planting date Date of sowing/planting Harvesting date Date when plot was harvested (for nutritional/yield traits) Data collection date Date when data was collected Data collector Full name Trial identifier Reference number for trial Step 7 – Distribution of plant materials 7.1 – Policies and regulations for distribution 7.1.1 - Common policies on distribution and access to plant materials (describe policies that are common for most institutions holding this crop) Best practice: Follow the International Treaty on Plant Genetic Resources for Food and Agriculture (ITPGRFA) for in trust germplasm using the SMTA and Plant Breeders Rights for varieties.

Justification: Centres have signed an agreement with the ITPGRFA.

7.1.2 - Policy exceptions (describe exceptions for relevant institutions holding this crop) Best practice: Repatriation of germplasm is done whenever material is available

Justification: Material should always be repatriated as a special case

7.1.3 - National laws and regulations (list relevant permits and certificates required in common for most countries holding this crop) Best practice: Export permits Phytosanitary certificate Certificate of origin

Justification: It is essential to follow the terms and conditions in the host country agreements

7.1.4 - International laws and regulations (list relevant international permits and certificates required for most countries holding this crop) Best practice: The seed shipment should be sent with the Standard Material Transfer Agreement (SMTA) even for non Annex 1 species using appropriate footnotes.

Justification: Centres have signed an agreement with the ITPGRFA which covers use of the SMTA.

7.1.5 - Phytosanitary regulations (list the important regulations for your crop or refer to health testing section) Best practice: Phytosanitary certificates needed for most countries. See general guidelines.

Justification: Essential to avoid spread of pests and diseases.

7.2 – Users related issues for distribution 7.2.1 - Feedback to users (give estimates of the time frame and other factors that can influence the delivery of plant materials of your crop)

Best practice: Respond to requests with lists of materials, forms and conditions of access, SMTA as soon as possible after receipt of request. Provide passport and germination data with requests.

Justification: Users may not know about the conditions so it is better to inform them before proceeding with the request.

7.2.2 - Customer assurance (recommend procedures that ensure the materials distributed match the client request) Best practice: Match accession numbers or match accessions to environment If no specific accessions requested provide a random sample of accessions or send the core collection.

Justification: Material will be better used if it meets the needs of the users.

7.2.3 - Feedback from users (list most relevant information required to be received from users) Best practice: Adaptation and any information on characterization and use.

Justification: Information on performance in one area allows better selection of germplasm for similar areas.

7.2.4 - Quantity of material recommended to be distributed Best practice: Sufficient to cover the diversity in the accession and produce material for future use. Minimum sample size of: Cultivated species: 100 seeds Wild species: 50 seeds

Justification: Users should have access to the full diversity within the accession and sufficient seeds to multiply to obtain a genetically similar sample for future use. Provision of small quantities maintains the stocks and reduces the regeneration frequency.

7.3 - Procedures for distribution (describe the recommended procedures and requirements for each heading; update or add any additional steps) 7.3.1 - Check availability Best practice: Justification: Availability in stock Check availability of seeds in stock and verify their availability. Distribution of the seeds required should not cause the accession to fall below the minimum stock if no long term collection.

Minimum stocks should be maintained. Accessions with low amount of seeds in stock will not be distributed.

Checking passport data Passport data can be checked to ensure the species is adapted to the requestor’s needs

This avoids waste of seeds by sending material that the requestor does not need or want.

7.3.2 - Preparing samples for distribution Best practice: Justification: Registering the request Give consecutive numbers to track requests

This allows requests to be handled on a first come first served basis.

Preparing list of accessions available Generate lists of accessions that meet their needs

Use the database and print lists to avoid errors.

Checking requirements for material transfer ILRI has signed an agreement with the ITPGRFA which covers

agreements All in trust material will be sent with the SMTA.

use of the SMTA.

Generating labels Print labels with full information

Use the database and print labels to avoid errors.

Labeling the accession containers Use labels with good adhesive and clear printing.

This avoids errors and mixing during shipping.

Checking inventory files and location of containers in the genebank Locations can be printed with stock amounts to quickly find the seeds.

This ensures accuracy and avoids errors.

Removing containers from genebank and acclimatization procedures required Allow all seeds to warm to room temperature before opening containers and seal seeds again as quickly as possible

Condensation will form on cold seeds and cause changes in moisture content. Packets should be sealed as quickly as possible to avoid uptake of moisture.

Assuring accuracy in identification Staff should double check all labels and seeds against lists to avoid errors.

This ensures accuracy and avoids errors.

Extracting samples from the original containers Use a clean spatula, take care not to mix samples and do not leave containers open for long periods.

Cleanliness and care is needed to avoid errors through mixing. Packets should be sealed as quickly as possible to avoid uptake of moisture.

7.3.3 - Preparing the information list to accompany the plant materials Best practice: Justification: Passport data Accession identification Source Biological status

Basic information is important for the user

Characterization data used to verify accessions Basic information is important for the user Cover letter Remind users of the terms and conditions of access and request feedback

Important to make contact with the user for future feedback.

7.3.4 - Type of information (list additional minimum information required to be sent to customers, in addition to the Multi-crop Passport Data, often agreed as best practice) Best practices:

Justification:

7.3.5 - Dispatching the plant materials Best practice: Justification: Packaging Packaging the seeds in paper envelops or laminated envelops or nylon bags. Then pack seeds in plastic bags and attach the phytosanitarycertificate, import permit, in a plastic bag and then in a strong envelope or a cardboard box outside the box. SMTA and list along with copies of all documents are put inside the box.

Plastic bags are suitable for short periods to avoid packets bursting and mixing seeds or getting wet during transit.

Reply form Include a reply card

A reply card can be used to know when the user receives the shipment.

Sending the plant materials Method should avoid heating and delays in transport.

Use courier or other rapid means of transit Recording shipping details Record date and method of shipping

Shipping details are important for tracking during shipping

Updating inventory of genebank Deduct the weight of seeds sent from the stock on hand.

Needed for up to date stock control.

7.3.6 - Recording information during distribution (list the data fields that should be recorded and the recommended methods to check possible errors or understand changes during distribution). Select from the list below and add any missing ones) Data field Content Reference number Crop name Consignee’s name and designation Name and address of organization User information (type of organization requesting materials)

Date of request Date of supply Number and quantity of samples provided Phyto-sanitary certificate Export permit number Reference number of SMTA Classification of intended germplasm use 7.3.7 - System for tracking materials/inventory system for distribution (describe the best recommended methods as well as alternatives in case of limited conditions) Best practices: Related data tables in database management system.

Justification: A database system allows easy and fast access to data and allows macros to be written to routine operations.

Best practices for forage legume genebank management

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