Monitoring in field banks of forage grass genetic resources

Contributors to this page: ILRI, Addis Ababa, Ethiopia (Jean Hanson); CIAT, Cali, Colombia (Rainer Schultze-Kraft); Bioversity International/ILRI, Addis Ababa, Ethiopia (Alexandra Jorge).

Germplasm testing in field banks

• When field banks are established from seeds, use the same procedures as for viability in forage seed genebanks.

Health diagnosis in field banks

• It should be carried out annually or if any disease symptoms are noticed.
• When established from seeds use the same procedures as for health in forage seed genebanks.
• The same pests and disease apply for field grown plants.

Routine monitoring of field banks

Methods and frequency

• Survival rates - Check survival monthly (most forage grasses that have to be maintained in a field genebank are perennial and regular monitoring is important to ensure survival).
• Vigour - Record vigour monthly.
• Pests and diseases - Inspect for pests and diseases monthly (this is sufficient to identify any pests or diseases and rescue the plants if problems are detected).
• Replacement by other weeds - Rogue out plants of other species regularly and at flowering time. Check taxonomy at least once in a year.

Recording information during routine monitoring of field genebanks

The following information should be recorded for each step:

• Accession number (an ID number).
• Genus (genus name of the plant, entered in full).
• Species (species name of the plant, entered in full).
• Field genebank site name (a code to indicate the site of seed production).
• Plot number (the plot number at the production site).
• Date of monitoring (date when data is collected).
• Name of staff (name of staff recording the data).
• Damage [a score of 1-5 (where 5 is damaged) on amount of damage (specifying insect, disease etc)].
• Vigour [assessment of vigour of the plants on a scale of 1-5 (where 5 is high)].

Distribution of forage grass genetic resources

Contributors to this page: ILRI, Addis Ababa, Ethiopia (Jean Hanson); Bioversity International/ILRI, Addis Ababa, Ethiopia (Alexandra Jorge).

Policies and regulations for distribution

Common policies on distribution and access to plant material

• Follow the International Treaty on Plant Genetic Resources for Food and Agriculture (ITPGRFA)  and (SGRP) for in-trust germplasm using the Standard Material Transfer Agreement-SMTA (SMTA: ITPGRFA, SGRP) and Plant Breeders' Rights (click for more information from Australia, Canada and the UK) for varieties (CGIAR centres have signed an agreement with the ITPGRFA).

Policy exceptions

• Repatriation of germplasm is done whenever material is available (material should always be repatriated as a special case).

National laws and regulations

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

• Export permits.
• Phytosanitary certificate.
• Certificate of origin.

International laws and regulations

• The seed shipment should be sent with the Standard Material Transfer Agreement (SMTA: ITPGRFA, SGRP) even for non Annex I species using appropriate footnotes (CGIAR Centres have signed an agreement with the ITPGRFA which covers use of the SMTA).

Phytosanitary regulations

• Phytosanitary certificates are needed for most countries.
• See the Safe transfer of Germplasm (STOG) section on this website for seed health movement (this is essential to avoid spread of pests and diseases).

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User related issues for distribution

Feedback to users

Describes factors that can influence the delivery of plant material:

• Respond to requests with lists of material, forms and conditions of access, SMTA (ITPGRFA, SGRP) as soon as possible after receipt of request (users may not know about the conditions so it is better to inform them before proceeding with the request).
• Provide passport and germination data with requests.

Describes recommended procedures that ensure the material distributed matches the client request:

• On specific requests, match accession numbers with specified request.
• If accession numbers are not specified, match accessions to users' needs. These could include:
  • Species.
  • Plant habit to fit the crop system.
  • Environment.
  • Use (grazing, cut and carry, soil stabilization).

Feedback from users

Describes the most relevant information required to be received from users:

• Information on characterization and evaluation/use (information on performance in one area allows better selection of germplasm for similar areas).

Quantity of material recommended to be distributed

• It must be sufficient to cover the diversity in the accession and produce material for future use (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. The provision of small quantities maintains the stocks and reduces the regeneration frequency).
• Cultivated species: 100 seeds.
• Wild species: 50 seeds.

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Procedures for distribution

Checking availability

Availability in stock

• Check availability of seeds in stock or vegetative material in the field and verify their availability.
• Distribution of the requested seeds should not cause the accession to fall below the minimum stock or distribution of vegetative cuttings cause plant death.
• Accessions with low amounts of seeds in stock or little vegetative material should 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 germplasm by sending material that the requestor does not need or want).

Preparing samples for distribution

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 users' needs.

Checking requirements for material transfer agreements

• All Annex I and non-Annex I in-trust material must be sent with the SMTA (ITPGRFA, SGRP).
• Otherwise use appropriate MTA.

Generating labels

• Print labels with the most important information: accession number, taxonomic identification, origin.
• Use the database and print labels to avoid errors.

Labelling 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 the genebank and acclimatization procedures required

• Allow all seeds to warm to room temperature before opening containers and seal seed packets again (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

Check packets against lists to avoid error (photo: ILRI)

• Staff should double check all labels and seeds against lists to avoid errors (this ensures accuracy and avoids errors).
• The use of barcoding technology reduces 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 and containers closed as quickly as possible to avoid uptake of moisture.
• Place the weighed samples of seeds for distribution in clean labeled paper bags.

Preparing vegetative materials for distribution

Root splits

Take small pieces of healthy plants with strong roots. Prepare from 3-5 root splits per accession. Cut back the stems to about 5 cm and wash all soil from the roots.

Pack the roots in moist tissue paper or newspaper so they have moisture during shipping and cover the roots with plastic bags and tie the bag around the stem with string. Leave the stems/cut leaves outside the plastic so that they do not rot during shipping.

Stem cuttings

Cut short pieces of stem with three nodes per cutting from healthy plants. Prepare from 3-5 stem cuttings per accession.

Wash the surface of the stems with 10% bleach solution and allow to air dry.

Pack in perforated plastic bags to allow oxygen to enter and avoid build up of moisture in the bags so that they do not rot during shipping.

Preparing the information list to accompany the plant material

The following basic information is important for the user:

• Passport data:
  • Accession number.
  • Accession identification.
  • Crop name.
  • Taxonomic identification.
  • Country of origin.
  • Biological status.
  • Collecting location.
  • Source.
• Germination data showing viability and testing method.
• Characterization data used to verify accessions should be provided upon request.

Cover letter

• Remind users of the terms and conditions of access and request feedback (important to make contact with the user for future feedback).

Dispatching the plant material

Packing seeds for distribution (photo: ILRI)

Packaging

• Pack the seeds envelopes or laminated aluminium foil envelopes in plastic bags to avoid packets bursting and mixing seeds or getting wet during transit.
• Then pack seed envelopes or packs of vegetative cuttings and attached documents, e.g. the seed list, SMTA, Phytosanitary certificate, import permit, GMO-free certificate in a strong envelope or a cardboard box.
• Attach a copy of the phytosanitary certificate, import permit and list of materials to the outside of the box if material is being dispatched to another country.
• A copy of the SMTA must be attached to the outside of the envelope or box. Use of the material constitutes agreement with the terms of the SMTA.
• Label the envelope/box with the complete mailing address of the requester.

Reply form

• Include a reply form (a reply form should be used to acknowledge that the seeds have been received by requester in good conditions).

Sending the plant material

• Use courier or other rapid means of transit (method should avoid heating and delays in transport).

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).

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Recording information during distribution

The following information must be recorded for each consignment:

• Reference number.
• Crop name.
• Consignee’s name and designation.
• Name and address of organization.
• User information (type of organization requesting material).
• Date of request.
• Date of supply.
• Accession number and quantity of samples provided.
• Phytosanitary certificate.
• Export permit number.
• Reference number of SMTA.
• Classification of intended germplasm use.

System for tracking materials/inventory system for distribution

• Update related data tables in database management system (a database system allows easy and fast access to data and allows macros to be written for routine operations).
• Use labels with good adhesive and clear printing (this avoids errors and mixing during shipping).

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Establishment in field banks of forage grass genetic resources

Contributors to this page: ILRI, Addis Ababa, Ethiopia (Jean Hanson); CIAT, Cali, Colombia (Rainer Schultze-Kraft); Bioversity International/ILRI, Addis Ababa, Ethiopia (Alexandra Jorge).

Preparation of planting material

Cleaning propagules/seeds in field banks

Field genebanks for grasses are usually planted using root splits or stem cuttings. For shy seeders, where only few seeds are available, they can also be established from seeds that are first germinated in the laboratory and transplanted to the field using similar sample processing procedures to those for seed establishment for forage seed genebanks.

Source of planting material in field banks

• Use seeds, stem cuttings or root splits, depending on the reproductive mechanism of the species (seeds or stem cuttings are preferred to root splits to avoid spread of pests in the soil around the roots). Wash root splits carefully to remove any soil before planting.

Using seeds

Seeds are used in the case of recalcitrant seeds that are difficult to keep for a long time in a seed genebank or for shy seeders where few seeds are produced.

Seeds should be germinated in petri dishes in an incubator. The conditions vary with species (see table for use in germination testing).

• As soon as the radicals start to emerge, plant the young seedlings individually in seedling trays or pots using sterilized compost or forest soil.
• Maintain the pots in a warm place and cover at night to retain moisture.
• Keep the pots away from direct sun but with good light intensity or in a greenhouse.
• Water carefully using a spray bottle so the pots remain moist but not wet.
• Once seedlings are strong and growing well, place the pots outside for one week for the seedlings to harden off, watering carefully.
• Label the plot with the accession number, planting date and plot number.
• Peg out the plots at the chosen row spacing and make holes at 50 cm along the row.
• Transplant the seedlings to the field, one seedling per hole taking care not to damage the roots when the seedlings are transferred from the pots, and water after transplanting.

See also germplasm testing section in the viability page in forage seed, for more details.

Using stem cuttings

• They should usually be first planted in pots in screen houses, tested for diseases and only the clean plants transplanted into field plots.

Using new root splits

• All the plants from the plots should be removed and vigorous and healthy root splits chosen from healthy plants to be replanted, usually into the same plot.

Pre-treatments

• Fumigation.
• Insecticide application.
• Others - as per forage seed genebanks.

Visual inspection of plant material

• Establish plants from vegetative cuttings in an isolated screened area and check carefully for pests and diseases before release to the field (vegetative materials have a high risk of transferring pests and diseases and visual inspection is important to ensure only clean material goes to the field).
• Insect damage - Check with the microscope that there are no insects hiding in the leaf whorls (insect eggs can be carried in the leaf whorls and hatch after transplanting to the field).
• Fungal damage - Observe the plants carefully for pathogenic fungi before transferring to the field. Incinerate any infected material (vegetative material can be a source of pathogenic fungi. Look for symptoms before the material is released).
• Mechanical damage.
• Empty seeds - Check that the seeds have caryopses by rubbing gently with sandpaper to remove the glumes (empty glumes are common in some grasses and caryopses should be removed for germination).
• Others - as per forage seed genebanks.

Disposal of contaminated material

• Incinerate or autoclave contaminated material (to avoid spreading diseases and pests).

Recording information during the preparation of planting material

The following information should be recorded for each processing step:

• Accession number (an ID number).
• Lot number (an ID number).
• Seed weight (weight of seeds for storage) or number of propagules.
• Reference to seed source (to trace the origin of the sample).
• Flags (Y/N) indicating completion of steps mentioned above (checking).
• Remarks.

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 Field establishment

Field preparation

• Select the environment and soil type best suited for the species (see table).
• Soil should be ploughed and disked, and weeds and grasses removed. The soil should be prepared by tillage to obtain a well prepared and level seed bed prior to planting.

Field planting

• Planting should be done at the start of the rainy season (to aid establishment).
• Aim for a final plant number of 30 in plots of 10 m² for vegetatively propagated grasses.
• Plant in rows. Three rows of 5 m in length, each row 100 cm apart with within row spacing of 50 cm. Giving a density of 30 plants per plot is suitable for most grasses. See table, for more specific information about plot density of each species.

Empty plots waiting to be planted in the grass field genebank of Zwai, Ethiopia (photo: ILRI)	Grass field genebank in Zwai, Ethiopia (photo: ILRI)

Frequency of new establishment

• It depends on the species and their growth rate, but it should generally be done every 3-5 years.

Field maintenance and management

Weed management

• Early growth can be slow and hand weeding four weeks after establishment is recommended.
• Eliminate plants growing off-row. Rogue out plants that are genuine mixtures.

Irrigation

• Irrigate the field after sowing and when needed subsequently. Do not allow the leaves to wilt at any stage. Ensure enough moisture in the soil at the time of flowering.

Fertilization

• It is recommended to apply phosphorous at planting using DAP at 100 kg per hectare. An additional application of 50-60 kg N per ha as a top dressing at early flowering stage will ensure good seed quality.

Common pest and diseases

• Forage grasses are susceptible to many fungal diseases and a few viruses and phytoplasma (see table, for the most frequently detected pests and diseases).

Pest and disease control

• Spray with fungicide to control mildew during the rainy season or when using irrigation and with insecticide at the first sign of insect damage. Pay particular attention to army worm and spray at the first infestation.

Renovation of field bank

• Regular maintenance of an existing field of grasses should be done by weeding, roughing and cutting the plants.
• Cutting should be regularly done twice or three times a year (to control pests and diseases, to promote young growth and flowering).

Harvesting seeds from grass field banks

Sometimes, there is a need to collect seeds from already established field genebanks in order to increase some seed stocks (many grasses have short-lived seeds or shy seeds – i.e. they do seed, but rarely or have a few caryopses). Seeds may be used to overcome difficulties in distributing vegetative material (due to pests and diseases, quarantine regulations or high volume /cost of transportation). In these cases, there is a need to isolate the seeds so they do not cross pollinate, and there are two main options to harvest seeds for dispatch:

Isolation

• Spatial isolation - generally 100 m between any related species.
• Temporal isolation – cut back all neighbouring related species (within 100 m) to prevent flowering, during flowering period.

Bagging and tagging

• Bag the inflorescences (a bunch of them) to avoid contamination with foreign pollen during flowering time. Tag each stem for easier identification and remove the bags after flowering is completed to avoid condensation forming in the bags and causing rotting. Harvest the seeds when mature.
   

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Information management

System for tracking material/inventory system in field banks

• Use a database for stock and location.
• Use pegs, tags or bar codes for labeling.
• Use impermeable ink and write clearly (databases are needed to keep track of information).
• Plots must be well labeled to avoid errors.
• Bar codes help avoid errors in recording.

Recording information during field bank establishment

The following information should be recorded during storage:

• Accession number (an ID number).
• Genus (genus name of the plant, entered in full).
• Species (species name of the plant, entered in full).
• Field genebank site name (a code to indicate the site of seed production).
• Plot number (the plot number at the production site).
• Plot size (plot size in square meters).
• Date of planting (the date that the planting was carried out).
• Type of planting [a code for the method used for planting (seed, cutting etc)].
• Number of plants (the total number of plants in a plot).
• Spacing within row [the space (in cm) between plants in a row].
• Spacing between rows [the space (in cm) between rows in a plot].

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Storage in seed banks for forage grass genetic resources

Contributors to this page: ILRI, Addis Ababa, Ethiopia (Jean Hanson); Bioversity International/ILRI, Addis Ababa, Ethiopia (Alexandra Jorge).

Base collection

When it should be used

• All original samples should be placed in long-term storage (original samples are the best representative sample of that accession and storage conditions should be used to retain viability for as long as possible).
• In addition, representative samples of all accessions should be stored in base collections for long-term storage.

Sample specifications

Minimum sample size/viability for storage:

• 4000 seeds or 5 g for small seeds of 90% germination or 80% germination for wild species (see FAO/IPGRI, 1994).

Moisture content

• 3-7% (according to FAO/IPGRI, 1994).

Container specifications

Seed packaging method

• Laminated re-sealable aluminium foil packets are moisture proof and very practical, making good use of space.
• 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 (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, sufficiently flexible for handling and strong enough for forage grass seeds, which are usually smooth).

Storage specifications

The two commonly available options for seed storage are walk-in cold stores and freezers. The choice depends on the number of accessions to be stored, seed size and storage temperatures selected.

• When collections are small and sub-zero temperatures are required, chest or upright freezers are a cheaper option for seed storage.

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) or freezer (e.g.  freezer shelf and box number). The exact locations are very important to locate material in the store.
• Moveable shelves on tracks are preferred (moveable shelves make best use of space).

Storage conditions

• At -18^oC to -20^oC (FAO/IPGRI, 1994).

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Active collection

When it should be used

To store seeds for distribution and use (seed quantities for distribution are usually larger and are not usually stored for long periods, therefore storage conditions are less stringent).

Sample specifications

Minimum sample size/viability for storage:

• A minimum of 1500 seeds and up to 500 g and above 80% of original germination (sufficient seeds for distribution needs for period of high viability).

Moisture content:

• 3-7% (according to FAO/IPGRI, 1994).

Container specifications

Seed packaging method

• Laminated re-sealable aluminium foil packets are moisture proof and very practical, making good use of space.

• Plastic or metal containers if store is dehumidified (store must be dehumidified to avoid absorption of moisture from the air surrounding the seeds).

Specifications of packaging material

• Laminated aluminium foil: 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 grass seeds, which are usually smooth).
• Plastic or metal containers: Square-based bottles with a screw cap and inner rubber seal made of pure plastic materials (high quality), semi transparent, white in colour, adequate volume or metal cans of similar design.

Storage specifications

Assigning location codes

• Location codes should specify the exact location in the store, e.g. unit/block number, row number, shelf number and tray/box number (exact locations are important to locate and access material in the store).
• Moveable shelves on tracks are preferred (moveable shelves make best use of space).

Storage conditions:

• Between 0 to 10^oC (conditions to maintain seed viability for at least 25 years).

For information on safety duplication, click here.

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Storage management

Storage space arrangement

• Moveable racks (this maximizes space between and on the racks in the store and is the most economic way to utilize the space).
• Shelves should be spaced slightly more than the height of the containers.

System for tracking material/inventory system

• Database of stock and location and use of barcodes (databases are needed to keep track of information. Barcodes help avoid errors in recording).

Recording information during conservation

The following information must be recorded for each accession:

• 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 or hundred seed weight (weight of 1,000 or 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 seed availability and 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).

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References and further reading

Chin HF, Hanson J. 1999. Seed storage. In: Loch DS, Ferguson J, editors. Forage Seed Production Vol. II Tropical and Subtropical Species. CABI, Wallingford, UK. pp. 303-315.

FAO/IPGRI. 1994. Genebank standards. Food and Agriculture Organization of the United Nations, Rome and International Plant Genetic Resources Institute, Rome. Available in English, Spanish, French and Arabic.

Harty RL, Hopkinson JM, English BH, Alder J. 1983. Germination, dormancy and longevity in stored seeds of Panicum maximum. Seed Science and Technology 11:341-351.

Whiteman PC, Mendra K. 1982. Effects of storage and seed treatments on germination of Brachiaria decumbens. Seed Science and Technology 10:233-242.