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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Field 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 are field banks (in vivo collections) used

• For wild rice species or related genera that produce recalcitrant seed or no seed.
  • This is the case of O. longistaminata, O. neocaledonica, O. granulalta, O. meyeriana and related genera such as Leersia at IRRI, that do not produce enough seeds for storage.
  • Porteresia coarctata has recalcitrant seeds.
• When wild rice or related genera do not flower in genebank conditions.
  • This is the case of O. schlechteri and related genera such as Potamophila and Zizaniopsis at IRRI.
• When wild rice relatives have special needs, where different species require different cultural practices.
  • Oryza granulata and Oryza meyeriana for example, need partial shading and special soils, because they are originally from forest regions, whereas most of the other species need to be kept soaked because they are originally from swampy areas.

There are more than 20 different species of wild rice scattered across tropical Asia, Africa, Latin America and the Caribbean that need to be well conserved. These species are from a rich pool of diversity from different habitats, from sunny open lands to shady forests.

At the IRRI genebank there are more than 3000 potted wild rice specimens being conserved in a large screen house as living plants.

Establishment of field genebanks (in vivo collections)

• Perennial species should be maintained as living plants when seeds are difficult to produce.
• Plants must only be grown inside contained facilities (e.g. glasshouse or screen house), in compliance with national biosafety regulations, to prevent their escape as a weed.

Sample processing and viability in field banks (wild rice and related genera)

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

Provides guidelines and recommendations about the best practices for the daily management of genebanks, describing the most important procedures for sample processing and viability in field genebanks for wild rice and related genera. Best practices are shown in a pale blue color with bullets. Justification for each best practice is given within brackets (…).

Cleaning propagules/seeds in field banks

Source of planting materials in field banks

• Use seeds treated with additional care to assure germination and survival.

Visual inspection of plant materials

• Check the materials carefully for pest or disease contamination.
• Consult the health diagnosis menu for specific details about pests and diseases.

Disposal of contaminated materials

To ensure plants do not spread by seeds or rhizomes, specific measures should be followed:

• Do the seed multiplication of all wild rices inside the screenhouse in pots.
• Designate a disposal area (a pit about 3-4 meters deep from the surface ground) for burying discarded and burnt samples.
• Provide a modified incinerator or burning facility to accommodate burning activities especially during the rainy season.
• Cover all drainage canals inside the screenhouse with fine-mesh screens to further control dissemination of seeds through water.
• Waste materials from the canals should be regularly hauled, burned, and buried.
• If sufficient seeds are obtained, old plants should be discarded, burned and buried into the pit.
• Excess planting materials (seeds, seedlings, rattooned tillers) should be collected, burned and buried after seeding, transplanting and/or replanting.
• Discarded soil used in growing should be treated with herbicide and buried in the designated area.
• Before filling up all the discarded materials, the disposal area should be treated with a non-selective herbicide (e.g. glyphosate).
• Screenhouse staff should be advised to change their working clothes to minimize dispersal of seeds when they leave.
• Hand threshing and seed cleaning should be done in a specified room in the Seed Processing Area.
• All dried leaves/straws, unfilled grains, mixtures and off-types must be collected, burned and buried.
• The access to screenhouses should be regulated depending on the nature/importance of the visit.

Routine monitoring for field banks

Methods and frequency

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

Recording information during sample processing and routine monitoring in field banks

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 bank 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 (insect, disease etc)].
• Vigor [assessment of vigor of the plants on a scale of 1-5 (where 5 is high)].
• Details of plants removed or destroyed (due to type mixtures or pests or diseases contamination).

Regeneration guidelines for wild rice and related genera

Contributors to this page: T.T. Chang Genetic Resources Centre-IRRI, Los Baños, Philippines (Ruaraidh 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).

Provides guidelines and recommendations about the best practices for the daily management of genebanks, describing the most important procedures to regenerate wild rice and related genera. Best practices are shown in a pale blue color with bullets. Justification for each best practice is given within brackets (…).

Storage specifications

Choice of environment and planting season

Climatic conditions

Preparation for regeneration

When regenerate?

• When stocks are below??

Pre-treatments

• Apply .... to break dormancy??

Method of regeneration

Sowing method

• Plant the germinated seeds 1-2 cm apart, in a seed box containing moist, fine, clean (preferably sterilized) soil mixed well with appropriate amount of ammonium sulfate.
• Apply a granular insecticide (e.g. Furadan) 3-4 days after planting to protect the seedlings from ants and other insects.
• Water the seedlings carefully with a fine spray, and grown them under partial shade until a week before transplanting.

Transplanting

• Transplant the seedlings 30 days after sowing to water-tight pots with good quality soil mixed with about 5 g of complete fertilizer.
• Maintain the water level to at least 1 cm depth.
• A granular insecticide (e.g. Carbofuran) should be applied 7 and 14 days after transplant to protect the plants against hoppers and defoliators.
• For species of the O. meyeriana complex, the seedlings should be transplanted into pots with light soil and good internal drainage to prevent water logging as they thrive best in mesophytic conditions.
• For the highly stoloniferous species, such as O. schlechteri and some related genera like Luziola, Leersia, and Hygroryza, a modified flat bed should be constructed and used for growing and maintaining a single accession.
• The pots should be laid-out at least 100 cm apart to provide sufficient ventilation between plants and enough space for plant management.
• All species of the genus Oryza grow well under full sunlight except members of the O. meyeriana complex and O. ridleyi complexes which are best maintained in partial shade.

Different varieties of rice being grown inside an IRRI green house (photo: IRRI)

Crop management

Fertilization

Irrigation

Pest and disease control

• Plant health should be monitored regularly.
• Appropriate control measures should be applied to specific pest and diseases once symptoms appear.
• Maintaining the cleanliness of plants also helps preventing the spread of diseases.

Thinning

• When purelines are to be developed, only 1 plant per pot should be maintained and spaced widely, preferably alternating species of different complexes.
• If a bulk population of seeds is required, 2-3 seedlings per pot should be transplanted and all the plants should be maintained.

Harvesting

Panicle bagging

• At the late vegetative stage, (about 60 days after transplant) the tillers should be tied loosely with, for example, an abaca twine to a bamboo stake (5 cm x 2 m) to prevent plants from encroaching from one pot to another, at the late reproductive stage, to facilitate panicle bagging.
• Panicle bagging is necessary for handling wild rices to minimize outcrossing, to prevent seed loss due to shattering, and to prevent mixtures at harvesting.
• Panicles should be bagged a week after full panicle emergence using nylon net bags which provides ample ventilation to facilitate anther dehiscence and prevent mold formation on glumes.
• For species with shorter panicles, glassine bags are a good substitute.
• The net bag should be pinned to the bamboo pole.
• Prior to bagging, labels should be prepared using shipping tags written with plot number and date of bagging with indelible ink.
• The labels must be attached inside the net bags.

Rice being grown inside an IRRI screen house. Notice the bagged panicles (photo: IRRI)

Panicle harvesting

• The panicles should be harvested 30 days after bagging or when most of the seeds have shattered.
• If sufficient seeds are obtained, the plants should be discarded and disposed through burning.
• However, for species with low seed set like O. rufipogon and O. longistaminata, the plants should be ratooned by cutting about 20-25 cm from the culms base, a little amount of ammonium sulfate should be applied and plants should be maintained until the next flowering to maximize seed production.
• After harvesting, the panicles should be dried and kept inside the drying room for two weeks, before carefully hand-threshing and cleaning the seeds.

Post harvest management

Disposal of contaminated material

Storage management

Documentation of information during regeneration

System for tracking material/inventory system during regeneration

• Use databases of stock and location (databases are needed to keep track of information).
• Use pegs, tags or barcodes for labelling:
  • Use impermeable ink and write clearly.
  • Plots must be well labelled to avoid errors.
  • Barcodes help avoid errors in recording.

Recording information during regeneration

The following information should be recorded for each step:

• Site name and map/GPS reference.
• Name of collaborator.
• Regeneration site name (a code to identify the site location).
• Plot reference (the plot number at the field site).
• Accession number; population identification.
• Name of staff (name of staff recording the data).
• Method of planting, date and spacing.
• Screen house layout used.
• Screen house management details (watering, fertilizer, weeding, pest and disease control, stresses recorded, others).
• Environmental conditions (altitude, precipitation, temperature, soil type and others).
• Number of plants established.
• Days from planting to flowering (note: this will only be important if seed collection is anticipated).
• Harvest date and method.
• Number of plants harvested.
• Comparisons with reference materials (record any identification numbers or references of any samples taken from the plots).
• Any evaluation undertaken during the growing period or at harvest.
• Post harvest (describe any relevant procedures).
• Others.

References and further reading