Seed bank for wild rice and related genera genetic resources

Contributors to this page: T.T. Chang Genetic Resources Centre-IRRI, Los Baños, Philippines (Ruaraidh Sackville Hamilton, Ken McNally, Flora de Guzman, Renato Reaño, Soccie Almazan, Adelaida Alcantara, Elizabeth Naredo); WARDA, Cotonou, Benin (Ines Sánchez); UPLB-University of the Philippines at Los Baños (Teresita Borromeo).

When it should be used

Conservation of rice seed material under cold (between 2 and -20^oC) and dry (moisture content of 6-7% fresh weight) storage is the most common, efficient and cheapest means of conservation of rice genetic resources. There are about 20 000 samples of wild rice seeds and related genera conserved in seed banks worldwide. The largest collections of wild rice and related genera are housed at CAAS, China and IRRI, the Philippines.

• Should be used for any rice accessions and species that produce seeds that remain viable under the standard cold and dry seed storage conditions.

Sample processing in seed banks of wild rice and related genera genetic resources

Contributors to this page: T.T. Chang Genetic Resources Centre-IRRI, Los Baños, Philippines (Ruaraidh Sackville Hamilton, Ken McNally, Flora de Guzman, Renato Reaño, Soccie Almazan, Adelaida Alcantara, Elizabeth Naredo); WARDA, Cotonou, Benin (Ines Sánchez); UPLB-University of the Philippines at Los Baños (Teresita Borromeo).

Cleaning

Cleaning is the removal of physical contamination from the plant material after harvesting, before it can be stored. The exposure to unfavourable conditions should be minimized for greater seed longevity in storage.

• Immediately after harvesting, the panicles should be dried (see details below) and kept inside the drying room for two weeks.
• Wild species are highly shattering thus panicles should be bagged to collect the seeds.
• Then they should be carefully hand threshed (this is necessary to maximize seed production) and cleaned.

Visual inspection of seeds

IRRI staff selecting good seeds for storage to the IRRI Genebank facility (photo: IRRI)

• Verify the sample comparing it with the seed file.

• Determine the selection to be carried out based on the recommendation during the verification process.

• Examine the seeds and hand sieve them with graded mesh sizes (if mixtures/off-types vary in size) to separate slender and bold grains (manual cleaning limits contamination and damage especially when the seeds are very dry).

• Remove discolored, deformed, infected, soiled, immature, damaged, germinated seeds and off-types.

• Determine the actual action to be taken based on the quantity of clean seed (this will determine the packing system to use).

• Prepare and label all the necessary envelopes for use in seed testing, viability testing and for storage in the base, active and temporary storage.

• Submit the selected samples together with the seed file, pre-labelled envelopes and the original seed container for double checking and quality control.

• Check the selected sample against the seed file and the pre-labelled envelopes against the original container (to minimize labelling errors).

• Mix the selected samples and divide using the pre-labelled envelopes as follows in order of priority:

For base collection

• 100 grains or sufficient to plant them twice.

For active collection

Bulk sample for active collection.

• 100 grains.

For black-box safety backup storage:

• 2 x 100 grains for safety backup storage (one for Svalbard and one for another recognized genebank with long-term store accepting black-box safety backups).

For temporary storage

Often samples of wild rice do not produce sufficient material especially when there are limited incoming seeds.

• Place the cleaned samples again in the drying room.

• Regenerate when possible to obtain greater amounts of seed that would be sufficient for storage.

Disposal of contaminated material

This is important for discarded material, either from incoming samples or after harvest. Wild rice species are classified as noxious or invasive species, subject to rigorous biosafety containment procedures during all the stages of sample handling.

• All wild rice plant parts should be treated as if contaminated and processed in contained facilities separate from cultivated rice.
• All dried leaves/straws, unfilled grains, mixtures and off-types should be collected, burned and buried.
• A disposal area (a pit of about 3-4 m deep from the surface ground) should be designated for burying discarded and burnt samples.
• A modified incinerator or burning facility should be provided to accommodate burning activities especially during the rainy season.

Inspection and certification (purity analysis of seeds)

• Retrieve the accession number of all the harvested material.
• Retrieve the corresponding seed files (seed file provides information of the sample composition and guides the selection process).
• Determine and compare the composition of the harvest with the seed file or the remnants of the planting material if the seed file is not available.
• Identify the obvious mixtures/off-types, if present.
• If the harvest does not represent the original sample, trace back for possible errors.
• If there are doubts on the sample composition, place the harvest on hold and repeat planting, making additional observations during the growing period especially on the traits that differ in the initial harvest.
• Genotype the DNA if needed.
• If the new seed lot does not totally represent the original sample, discard the seed and record WH (Wrong Harvest) as the harvest status in the documentation system.

Note

Certain changes in seed phenotype may occur even though the seeds remain genetically true-to-type:

• Seed introduced from other environments may produce grain of different size in the seed multiplication environment.
• Hull pigmentation is more intense when grown under moist conditions.
• Harvests from screen house and field differ in hull colour intensity.

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Seed drying

Methods

1st stage of drying (during cleaning)

• Immediately after threshing, place the seeds in net bags and store in a drying room maintained at 15^oC and 15% RH.
• Spread the samples evenly on the drying shelves.
• Mix the samples by turning the bags daily.
• Monitor the moisture level.
• Transfer the dried samples to paper bags when the moisture is less than 10% MC.

2nd stage of drying (after cleaning)

• After cleaning, and sub-sampling for testing, place the selected samples back in the same drying room for 1-2 weeks while waiting for the viability and seed health results (during the seed selection process, seeds take in moisture).
• Test the moisture content.
• Monitor conditions in the drying room (with pre-set humidity and temperature, moisture testing is needed only infrequently. Test more often if fluctuations in RH are observed).

Alternative drying

• If facilities are not available for drying at 15^oC and 15% RH or if the drying equipment is not working reliably, dry with self-indicating silica gel in moisture-proof containers.
• Repeatedly replace the silica gel until it stops absorbing moisture.

Using a moisture meter to check the moisture content of seeds (photo: IRRI)

Drying time

• Minimum three weeks.

Moisture content before drying

• 9-20%.

Moisture content for storage

• 6-7%.

Critical moisture content

• 8% (below this level no insects or fungi affect the seed).

Determination of moisture content

• Wild rice species often have very few seeds in stock, therefore it is not recommended to perform any moisture content tests.

Recording information during seed cleaning and drying

The following information should be recorded for each processing step:

• AccID [Unique Genebank Accession ID. Note a technical distinction between accession number and accession ID. A number, unique within a collection, (the accession number) is often used by genebanks and is sufficient for genebank management purposes. However, for all public documentation that number should be prefixed with a code to identify the collection and thus create an ID that is unique across genebanks: the combination of prefix and accession number then is a public accession ID].
• Cropping season (year and season of seed multiplication).
• Germination % (two replications; initial % of germination of freshly prepared seed).
• Germination date (date of germination test).
• Packaging date (month and year of packing).
• Amount_bulk [amount (g) stored in bulk package in the active collection].
• Number_paper packs [number of paper packs retained for repeat multiplication – (for temporary storage only, if the viability or Seed Health tests result is below the acceptable limit)].
• Number_apack (number of small aluminium foil packets for storage in the active collection, pre-packed with 10 g dried seed ready for distribution).
• Amount_base [amount (g) stored in base collection].
• Plant_material (sufficiency of planting material for harvests that requires another round of seed multiplication).
• No. of Rows (approximate number of rows that could be planted for seed multiplication using the available planting material).
• Harvest_status [status of harvest (Insufficient, Mixed, Diseased)].
• Active tray ID (ID of the tray where the sample is stored in the active collection).
• Base tray ID (ID of the tray where the sample is stored in the base collection).
• Amount for duplicate storage1 (mark X if a sample has been prepared for the primary black-box safety duplicate storage genebank).
• Amount for duplicate storage 2 (mark X if a sample has been prepared for duplicate storage in Svalbard, Norway).
• Insects and pests (degree of insect and fungal damage).

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Viability of wild rice and related genera genetic resources

Contributors to this page: T.T. Chang Genetic Resources Centre-IRRI, Los Baños, Philippines (Ruaraidh Sackville Hamilton, Ken McNally, Flora de Guzman, Renato Reaño, Soccie Almazan, Adelaida Alcantara, Elizabeth Naredo); WARDA, Cotonou, Benin (Ines Sánchez); UPLB-University of the Philippines at Los Baños (Teresita Borromeo).

Viability testing

Laboratory methods

Type of test

• Standard germination test (modified from ISTA methods).

Number of seeds and replicates

• Active and base collections: 20 seeds with 1 or 2 replicates, depending on the amount harvested (some species may not produce enough seeds for storage; some of them may be highly sterile).

Pre-treatment

Viability is tested initially by placing seeds under conditions conducive to germination and recording the percentage that germinates (photo: IRRI)

Wild species generally have stronger dormancy than cultivated species and differ widely in response to procedures for breaking dormancy.

• Depending on the species, one or a combination of dormancy-breaking treatments including heat treatment, dehulling, exposure to alternating temperatures and, in some cases, chemical treatment.
• Treat dehulled seeds with fungicide (dehulled seeds are more susceptible to fungal attacks).

Media

• Use Petri dishes lined with moistured paper.

Temperature/ Humidity

• 30^oC and 100% RH.

Light

• 12/12 hours light/dark.

Duration of test

• 7-28 days.

Monitoring intervals

• No monitoring should be done during storage (most stocks do not have sufficient stocks for routine monitoring).
• The longevity of wild rice in storage is generally high.

Recording information during viability testing

The following information should be recorded for each testing step:

• Germination date (date of germination).
• No. Tested (number of seed tested).
• % germination rep 1 (germination % in replication 1).
• % germination rep 2 (germination % in replication 2).
• % germination mean (system-calculated mean for the % germination).
• Germination retest (germination % of the retested sample).
• Accession (list of accessions tested).
• Date counted (first count at 7 days and second count at 14 days).
• Normal seedling (seedlings that are free from decay, have well-developed primary root systems, have well-developed and intact coleoptiles).
• Abnormal seedling (seedlings not well developed).
• Assumed dormant seeds (hard but non-germinated seeds).
• Dead seeds (rotten seeds).
• Germination % (the percentage of normal seedlings).

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Routine monitoring

Methods

Minimum viability

• This is not monitored for wild rice due to generally low stocks of seeds.

Minimum quantity

• Each time an accession is removed from storage, for whatever purpose, weigh again and update the computerized inventory before replacing the accession back in storage.
• Use barcoded labels with computerized inventory management system linking the balance to the database.
• If the quantity remaining is less than 60 g, mark the accession in the database as unavailable for distribution and schedule it for regeneration.

Monitoring frequency

This section recommends the minimum quantity and minimum viability of seeds below which they need to be regenerated.

Critical quantity

• 100 grains (twice the amount needed for a regeneration planting).

Critical germination level

• Not applicable for wild rice (dormancy is very hard to break for some spp.).

Recording information during routine monitoring

The following information should be recorded for each step:

• Accession ID (ID of accession).
• Seed Lot ID (ID of this sample of the accession).
• Crop season (year and season the seed was harvested).
• Store – temporary, active bulk, active pre-pack, base, primary safety backup, secondary safety backup (type of storage and packing for this sample).
• Species (the scientific name of the species).
• Germination % (two replications) (germination % - separate values for each replication; new record for each test).
• Germination test No. (number of seed per replicate used for testing germination %).
• Germination date (date of germination test; new record for each test).
• Seed amount units – grams, number of packets (units used to record the amount of seed of this sample).
• Seed amount (amount of seed in store; new record for each update).
• Inventory date (date the amount was last updated; new record for each update).

Note: Health diagnosis (testing) is not recommended during storage of wild rice, due to the usually limited number of seeds in storage.

References and further reading

Naredo MEB, Juliani AB, Lu BR, Guzman F, Jackson MT. 1998. Responses to seed dormancy-breaking treatments in rice species (Oryza L.). Seed Science and Technology 26:675-689.

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Storage of wild rice and related genera genetic resources

Contributors to this page: T.T. Chang Genetic Resources Centre-IRRI, Los Baños, Philippines (Ruaraidh Sackville Hamilton, Ken McNally, Flora de Guzman, Renato Reaño, Soccie Almazan, Adelaida Alcantara, Elizabeth Naredo); WARDA, Cotonou, Benin (Ines Sánchez); UPLB-University of the Philippines at Los Baños (Teresita Borromeo).

Base collection

When it should be used

• A base collection should be held only by a genebank with a mandate and responsibility for secure long-term conservation.
• A sample of every unique accession should be stored in a base collection.
• A genebank with only an active collection and no facilities for long-term conservation should make arrangements for long-term conservation of its unique germplasm in the base collection of another suitable genebank.
• Accessions conserved in a base collection should never be used, except:
  • To regenerate seed for the base collection when viability decreases below the threshold.
  • To regenerate seed for the active collection when the only available source for planting in the active collection is four generations from the original seed, or when a problem in managing the active collection has resulted in the total loss of seed from the active collection.
  • For scheduled viability monitoring.
  • In the event of accidental loss of all seed from the active collection.

Sample specifications

Minimum sample size for storage

• Wild rice: 100 grains or equivalent to double the amount required for planting (many samples cannot produce sufficient material especially when there are limited incoming seeds. Further regeneration to obtain a greater amount will increase the regeneration cycle).

Minimum viability for storage

• Not applicable for wild rice.

Moisture content

• 6-7%.

Container specifications

Moisture-proof laminated aluminium foil with wide seam on all sides (small pouch for distribution) (photo: IRRI)

Seed packaging method

• Use a specially designated seed packing, labelling and weighing room, situated adjacent to the seed drying room and with humidity reduced using a dehumidifier.
• Prepare two aluminium pouches per accession (two separate bags per accession improves security and management, enabling half the accession to be kept packed in the event that it is necessary to remove seeds).
• Prepare barcoded labels with the following printed information: accession number, name, crop year, Seed Lot ID, tray ID for storage and barcode. The barcode itself contains the Seed Lot ID.
• Label each pouch with:
  • Accession ID and crop season written on the pouch using permanent marking pens.
  • Barcoded label affixed to the pouch.
  • Small paper labels printed with accession ID and crop season, placed inside the pouches.
• Arrange the pouches in the packing area, in the order of taking samples out of the drying room.
• Take only a few samples at a time from the drying room (to minimize reabsorption of water during packing).
• For each sample, find its two corresponding labelled pouches and double check that all labels match (i.e. the label inside the pouches, outside on the pouch and on the packet retrieved from the drying room).
• Pour seeds into the pouch.
• Vacuum-seal the pouch, using a high temperature constant heat sealer (HM 305 CTE Constant Twin Element laminate crimp sealer, supplied by Hulme-Martin Ltd.) with 1 cm seal width.
• Check for deficiencies in the pouch: if there is any deficiency, discard and repeat labelling and packing with a new pouch.
• Weigh the pouches (tare to the weight of an empty pouch) and record weight in the database using the inventory management system.

Specifications of packaging material

• Aluminium (corrosion and leak free) foil pouches, strong enough to withstand vacuum packing.
• Air-tight and moisture-proof seal.
• Appropriate size, to accommodate the required stocks.
• Special-purpose high-quality labels created with material, adhesive and print designed to survive 100 years at 0-100% humidity and extreme temperatures of - 20°C to +30°C, e.g. from CILS International.

Storage specifications

Assigning location codes

• Arrange the packed seeds side by side in numbered box according to accession number.
• Place and arrange the box consecutively in the Base Collection room.

Storage conditions

• -18 to -20°C.

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Active collection

When it should be used

• An active collection should be maintained by a genebank with a mandate and responsibility to distribute seeds to users.
• Samples conserved in the active collection should be the normal source of seed for use, including to:
  • Distribute seed to users.
  • Regenerate seed for the active collection, for up to three generations from the original seed.
  • All other genebank purposes, including characterization, viability testing and other genebank research activities.

Sample specifications

Minimum sample size for storage

• Sufficient to establish two plots for regeneration with a safety margin – an amount of 60 g.

Viability for storage

• Not applicable for wild rice.

Moisture content

• 6-7%.

Container specifications

Seed packaging method

• Use a specially designated seed packing, labelling and weighing room, situated adjacent to the seed drying room and with humidity reduced using a dehumidifier.
• Prepare containers (preparing ‘pre-packs’ for distribution in advance increases initial packing costs for storage but substantially reduces re-packing costs for distribution and re-storage. It is strongly advised for heavily used collections).
• For little used collections it is more effective to store only bulk samples (with a smaller than average amount than in heavily-used collections):
  • Normal accessions: one large pouch for the bulk and two pre-packs for distribution.
  • Popular accessions: two large pouches for the bulk and five pre-packs for distribution.

• Prepare barcoded labels for the bulk samples, with the following printed information: accession number, name, crop year, Seed Lot ID, tray ID for storage and barcode. The barcode itself contains the Seed Lot ID.
• Prepare labels for the aluminium pre-packs, labelled with the same information as for the bulk sample but without a barcode.
• Prepare small samples of activated self-indicating silica gel (one per bulk pouch) in a sealed plastic pack.
• Affix labels to the bulk and pre-packs, and arrange them in the packing area in the same order in which they will be withdrawn from the drying room.
• Take only a few samples at a time from the drying room (to minimize reabsorption of water).
• For each sample, find its corresponding labelled bulk and pre-pack pouches, and double check that all labels match.
• Pour 10 g aliquots of seed into each pre-pack and seal using high temperature constant heat sealer (HM 305 CTE Constant Twin Element laminate crimp sealer, supplied by Hulme-Martin Ltd.) with 1 cm seal width.
• Pour the rest of the sample in the pouch(s) for the bulk sample.
• Perforate one packet of silica gel and place it in the foil pouch just before sealing (the silica gel serves a double purpose, to remove moisture that may have been absorbed during the packing process, and to indicate cases of leakage during storage).
• Seal using the high temperature constant heat sealer.
• Check for packing deficiency. Reseal or replace if there is any deficiency.
• Weigh the bulk pouches and record weight in the database using the inventory management system.

Specifications of packaging material

• Moisture-proof laminated aluminium foil with wide seam on all sides.
• For bulk storage: use large pouches, approx 24 cm x 16 cm.
• For distribution use pre-packs: small pouches, approx 11 cm x 8 cm (pre-packs are small pouches containing a 10 g sample of rice pre-packed ready for use, before it is actually needed. Aluminium pre-packs are for distribution to users. Preparing them in advance substantially reduces packing and re-packing costs in a well-used genebank).
• Special-purpose high-quality labels created with material, adhesive and print designed to survive 40 years at 0-100% humidity and 2°C to +30°C, e.g. from CILS International.
• To reduce costs, lower quality labels may be used for the pre-packs.

Storage specifications

Assigning location codes

• Arrange the packed seeds side by side in a numbered tray according to accession number.
• Assign tray number corresponding to the accession number.
• Place and arrange the tray consecutively in the Active Collection room.
• For accessions without bulk samples, a temporary tray number is assigned until a bulk has been prepared.

Storage conditions

• 6-7%.

For information on safety duplication, click here.

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Storage management

Storage space arrangement

These practices are based on manual operations, not on robot-mediated deposits and withdrawals.

Room

• Walk-in cold store, height 3-5 m, heavily insulated.
• Redundant cooling equipment with backup failover mechanism, placed outside under shade to facilitate heat exchange.

Shelf stacks

• Parallel stacks, in two rows against opposite walls with 2 m passage between rows.
• Height to about 0.5-1 m below ceiling.
• Total depth about 1 m per shelf, with racks/slots on both sides for removable trays.
• Mobile shelves mounted on wheels on tracks, with rotating handles to move the shelves, geared to facilitate easy movement even when fully loaded, to enable denser packing of the cold store.
• When positioned to maximize space between one pair of shelves, the distance between them is about 1 m, to facilitate the withdrawal of trays.
• Labelled with a shelf ID corresponding to ID used in the database.

Trays

• About 0.5 m deep (half shelf depth).
• Height: enough to accommodate a single layer of seed packets with room to slide in and out.
• Labelled with a tray ID corresponding to the ID used in the database.

System for tracking material/inventory system

Preferred option: barcoded inventory

• Ideally use barcoded labels with a barcode reader and balance linked to the genebank database through a computerized inventory management system.
• For samples with the amount recorded in grams for the inventory (i.e. bulk sample for the active collection, and the sample for the base collection), before a newly-packed sample is placed in storage, and subsequently each time seed is removed from it and repacked, for whatever purpose, weigh or re-weigh the seed before (re-)placing the sample in storage. The inventory management system will automatically update the inventory.
• For samples with the amount recorded as number of packets (i.e. aluminium pre-packs for the active collection, aluminium pre-packs for safety backup, and paper pre-packs for other immediate uses), the number of packets added or remaining is automatically updated in the inventory by the inventory management system as part of the computer-controlled processes of storing new seeds and distributing samples to users.

Alternative option: inventory management without barcodes

• Bulk sample in the active collection:
  • Before a newly-packed bulk sample is placed in storage, weigh it and record the weight manually in the inventory.
  • Each time the bulk sample is removed to extract seed (e.g. to make new aluminium pre-packs’ or for direct local use), subtract the amount removed from the amount recorded in the inventory.
  • Once every ten years, undertake a complete manual inventory of the active collection, to correct the errors that gradually cumulate by recording amounts removed cumulatively rather than the amount replaced in storage.
• For samples with amount recorded as number of packets, the procedure is the same as described for the barcoded system.
• Base collection: record the amount as number of packets, not grams, and maintain inventory as for pre-packs.

Recording information during storage

The following information should be recorded for each step:

• Accession ID (ID of accession).
• Seed Lot ID (ID of this sample of the accession).
• Crop season (year and season the seed was harvested).
• Store type (temporary, active bulk, active pre-pack, base, primary safety backup, secondary safety backup (type of storage and packing for this sample).
• Location in storage (shelf and tray number (temporary, active, base).
• Species (the scientific name of the species).
• Germination % (two replications) (germination % - separate values for each replication; new record for each test).
• Germination test No. (number of seeds per replicate used for testing germination %).
• Germination date (date of germination test; new record for each test).
• Seed amount units – grams, number of packets (units used to record the amount of seed of this sample).
• Seed amount (amount of seed in store; new record for each update).
• Inventory date (date the amount was last updated; new record for each update).

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