Storage of cultivated barley and wild relatives genetic resources

Contributors to this page: ICARDA, Syria (Ahmed Amri, Bilal Humeid, Kenneth Street, Natalya Rukhkyan, Jan Konopka, Siham Asaad, Adnan Omran and Fida Alô).

• Cultivated barley: two packets of 25 g each.
• Barley wild relatives: one packet of 25 g.

Minimum viability for storage

• Cultivated barley: >90%.
• Barley wild relatives: >80%.

Moisture content

• 5-7% (to avoid any deterioration of the stored seed samples).

Container specifications

Seed packaging method

• Aluminum foil packets sealed under vacuum (these are moisture-proof and occupy less space than other containers).
• Packing is best carried out in an air-conditioned room with controlled humidity, as quickly as possible after drying (to prevent the re-absorption of water).

Specifications of packaging material

• Aluminum foil packets consisting of the following layers:
  • Polyester 17 g/m^².
  • Alufoil 33 g/m^².
  • Polyethylene 63 g/m^².

Storage specifications

Assigning location codes

• It is preferable to use sliding shelves (for easy access to the accessions).
• Record the position of each accession in the store, e.g. unit/block number, row shelf number, shelf number and tray number.

Storage conditions

• -18 to -22^°C (maintains seed viability) in cold walk-in rooms or in freezers (preferably vertical freezers).

Recording information during storage - Base collection

The following information should be recorded for each step:

• Accession number (unique number).
• Weight of the sample (g).
• Estimated number of seeds (estimated from 1000 kernel weight).
• Year of regeneration (date).
• Viability (%).
• Viability test date (date).
• Recommended next viability test date (date).
• Phytosanitary status (approved, conditionally approved, rejected).
• Position of the sample in the storage facility (coordinates).

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Active collection

An active collection consists of accessions that are available for distribution and are accessed frequently. The active collection is intended to preserve germplasm viability in medium-term storage conditions for at least 20 years or for half of one regeneration cycle in the base collection.

When it should be used

• The active collection should be used to hold seed samples for medium-term conservation.
• The active collection is important to:
  • Conserve accessions most actively used or expected to be used such as elite lines and populations, core subsets and reference seed sets of all accessions.
  • Distribute seed samples requested by users.
  • Store introductions and the regenerated seeds before inventorying them.

Sample specifications

Minimum sample size for storage

• Cultivated barley: 1500 seeds (L flag) (this is a representative sample).
• Wild species: 1500 seeds (L flag).

Viability for storage

• Cultivated barley: > 90%.
• Wild species: > 80%.

Moisture content

• 5-7% (to avoid any deterioration of the stored seed samples).

Container specifications

Seed packaging method

Use permeable plastic containers (this will create the balance between seed moisture content and the ambient relative humidity).

Specifications of packaging material

• Square-based bottles with a screw cap made of pure plastic material (high quality), semi transparent, white in colour, volume (400 ml) (as used at ICARDA). These are moisture resistant and withstand storage at low temperature for a long period of time without cracking or any other physical deterioration.

Storage specifications

Assigning location codes

• It is preferable to use sliding shelves (for easy access to the accessions).
• Record the position of each accession in the store, e.g. unit/block number, row shelf number, shelf number and tray number.

Storage conditions:

• 3 ± 2^oC (extends the storage life and breaks any dormancy if present). Relative humidity controlled at 15-20% if seeds are stored in permeable plastic containers.

Recording information during storage - Active collection

The following information should be recorded for each step:

• Accession number.
• Weight of the sample.
• 1000 kernel weight.
• Number of seeds (estimated from weight using 1000 kernels).
• Number of packets.
• Year of production of seeds.
• Viability (%).
• Viability test date.
• Recommended next viability test date.
• Flag for regeneration (Y/N).
• Position of the sample in the storage.
• Flag indicating distribution status (A-available for distribution; L-limited stock; S-stop distribution).

For information on safety duplication in seed banks, click here.

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Storage management

Provides guidance on how to arrange the physical space, how to track seed material and record information during storage.

Storage space arrangement

It is very important to provide easy access to the accessions.

Room

• Use the minimum space possible (to reduce the cost of cooling the storeroom), but allow convenient access to each sample.

Shelves

• Sliding shelves should preferably be used (to maximize space between and on the racks in the store and to provide easy access to the accessions).
• Shelves should be spaced slightly higher than the height of the containers.
• Record the position of each accession in the store, e.g. unit/block number, row shelf number, shelf number and tray number.

System for tracking material/inventory system

• It is important to use a database to keep track of stock and location of material, and to use barcodes (databases are needed to keep track of information; barcoding helps to avoid errors in recording).

Recording information during storage

The following information must be recorded for each consignment:

• 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 1000 seeds).
• Number of packets (number of containers for seeds of one lot of each 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-available for distribution; L-limited stock; S-stop distribution).
• 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).

References and further reading

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

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Viability of cultivated barley and wild relatives genetic resources

Contributors to this page: ICARDA, Syria (Ahmed Amri, Bilal Humeid, Kenneth Street, Natalya Rukhkyan, Jan Konopka, Siham Asaad, Adnan Omran and Fida Alô).

Viability testing

Laboratory methods

Describes the various recommended options, to test the viability and quality of barley seeds. The percentage of germination of the stored seeds of the accessions determines when regeneration of the accessions should take place and if the accession can be distributed to the users.

Type of test

• Standard viability test, following the ISTA method (this one is more accurate, but more seeds are required and hence should be applied when there are enough seeds).
• Sequential viability test, following the Bioversity International method (this one is less accurate, but requires fewer seeds and hence should be applied in the case of limited number of seeds - See Bioversity's genebank manual No 8).
  • This test should be undertaken twice for accuracy.
  • If the results are less than the critical value, an additional test should be done.

Number of seeds and replicates

• Standard viability test - 100 seeds and 4 replications (ISTA method).
• Sequential viability test - 10 seeds and 4 replications (Bioversity International method).

Pre-treatment

• Sterilize the seeds using 0.5% solution of sodium hypochlorite for 10 minutes (to avoid any fungi infections).

Media

• Petri dishes, filter papers and tap water.

Temperature

• 20-25^oC.

Light

• No specific requirements.

Duration of test

• 5-10 days (most of the seeds germinate within this period).

Interpretation of results

Following ISTA (2003, 2005) and Association of Seed Analysts (AOSA), 2005:

• For each accession, consider the total number germinated in each replicate.
• Calculate the mean viability percentage of that accession from the results of all replicates.
• If it is above 90%, accept the test as valid. If the result is below 85% (75% for wild species) repeat the test. Calculate the mean viability percentage from the results of the two tests and use this as the overall test result.
• If the results are far below the germination standard, the accession should be sent to regeneration.
• Germination is complete when the seedling can be judged as normal according to specific criteria.

Monitoring intervals

Describes the recommended intervals for different storage conditions.

• After five years of the initial storage, a random sub-sample from stored accessions should be subjected to a new germination test (preferred sequential test) to ensure a high quality of seeds during storage and to meet international standards.
• The results should be compared with the figures from an initial germination test.
• If the results match, the monitoring interval should be expanded.
• If a decline is observed, a larger random seed sample should be re-tested along with checking the storage facilities to avoid any improper conditions and leakages.

Recording information during viability testing

On petri dishes

• Accession number and replication number.

On data sheets and database

• Taxonomic name.
• Number of replications.
• Date started.
• Date ended.
• Temperature.
• Number of seeds (seedlings) germinated in each category (normal, abnormal, non-germinated) (ISTA).
• Germination percentage (counted on normal seedlings only) (seedlings are judged as normal according to specific criteria (ISTA, 2003, 2005; Association of Seed Analysts [AOSA], 2005).

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Routine monitoring

Describes the recommended monitoring methods to assure minimum viability and quantity of seeds in storage.

Methods

• A computerized stock control system should be developed to monitor the quality and quantity of seed accessions in cold store.

To check the quantity

• Flags should be used to show that accessions are available for distribution.
• (L) Low stock: accession is available for distribution but multiplication should be planned.
• (S) Low number of seeds: no distribution should be allowed; sample should be multiplied. Distribution is permitted after multiplication.

To check the viability

• Random samples should be selected for viability testing. The results should be compared with previous viability test results.
• When viability is below the critical value the accession should be planned for regeneration.
• If the current viability is close to critical value, the date for the next test should be set for the following year; if viability is well above the critical value, the date of the next test should be set for five years after the current date.
• In the case of a significant decrease in viability, the genebank facilities should be checked for damage, leakage problems, etc.

Monitoring frequency

Lists the minimum quantity and minimum viability of seeds below which they need to be regenerated.

Critical quantity

• For both cultivated and wild relatives: 1000 seeds (to keep the integrity of the population samples).

Critical germination level

• Cultivated barley: 85%.
• Barley wild relative: 75%.
• Or 10% less than the average value of fresh seeds.

Recording information during routine monitoring

The following information should be recorded for each monitoring step:

• Accession number.
• Date of viability test (update database).
• Germination percentage (update database).
• Date of next test (one year if it is close to critical value; five years when it is above 90%).
• Regeneration flag: (Y – must go for regeneration; N – no need for regeneration).

References and further reading

AOSA (Association of Official Seed Analysts). 2005. Page 113 in: Rules for Testing Seeds (Capashew Ed.), 4-0, 4-11. Las Cruces, New Mexico, U.S.A.

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.

ISTA. 2003. International rules for seed testing. International Seed Testing Association, Bassersdorf, Switzerland.

ISTA. 2005. International rules for seed testing. International Seed Testing Association, Bassersdorf, Switzerland.

 

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Health diagnosis of cultivated barley and wild relatives genetic resources

Contributors to this page: ICARDA, Syria (Ahmed Amri, Bilal Humeid, Kenneth Street, Natalya Rukhkyan, Jan Konopka, Siham Asaad, Adnan Omran and Fida Alô).

List of pests and diseases of quarantine importance for barley

Click here for more detailed outputs from the page on the safe transfer of germplasm, on this website.

See also the list of pests and diseases for this crop listed in the crop regeneration guidelines.

Options for testing procedures

• Ustilago nuda (embryo count method).
• Ustilago hordei (seed washing test).
• Pyrenophora graminea (freezing blotter method).
• Xanthomonas translucens pv. undulosa (YDC agar and semi-selective XTS agar).
• Gibberella zeae and Monographella nivalis (freezing blotter method).
• Pyrenophora teres (freezing blotter method, PDA).
• Cochliobolus sativus (freezing blotter method, PDA).
• Barley stripe mosaic virus (ELISA and TBIA [tissue-blot immunoassay]).

Testing intervals/seasons

• Ustilago nuda, Ustilago hordei - Field inspection at flowering stage.
• Pyrenophora gramínea - Field inspection from seedling up to maturity and testing seed after harvest.
• Xanthomonas translucens pv. Undulosa - Field inspection at seedling and heading, and testing seed after harvest.
• Gibberella zeae and Monographella nivalis - Field inspection at maturity stage and testing seed after harvest.
• Pyrenophora teres, Cochliobolus sativus, Barley stripe mosaic virus (BCMV) - Field inspection from seedling to maturity and testing seed after harvest.

Recording information during health diagnosis

The following information should be recorded for each step:

• Accession number.
• Date (of diagnosis).
• Field number (to record the history of field).
• Experiment code or number.
• Plot number.
• Pathogen tested.
• Result of test (using appropriate scoring methods).

References and further reading

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

Sample processing of cultivated barley and wild relatives genetic resources

Contributors to this page: ICARDA, Syria (Ahmed Amri, Bilal Humeid, Kenneth Street, Natalya Rukhkyan, Jan Konopka, Siham Asaad, Adnan Omran and Fida Alô).

Seed cleaning

Cleaning is the removal of physical contamination from the plant materials after harvest, before they can be stored.

For cultivated barley:

• Harvest and thresh with a plot combine or stationary plot thresher, ensuring that the thresher and combine are clean before proceeding to the next accessions, to avoid any mixture between accessions. Barley seeds are hard and threshing does not cause any physical damage to the seeds, except for hull-less barley which should be threshed gently to avoid damaging the embryos.
• Remove the remaining awns with a di-awner machine.
• Clean with a wind blower separator (to separate the chaff, dust and other inert material from the samples, as well as light and empty seeds).
• Clean with a cylinder-type machine to remove broken seeds (caused by mechanical threshing), diseased seeds, soil particles and stones of the same size and weight of the grains.
• Hand cleaning is advisable as a final stage (to remove contaminants left after machine cleaning e.g. diseased grains).

For barley wild relatives:

• Harvest spikes only by hand (it is common practice to harvest before shattering).
• Remove the remaining awns with a di-awner machine.
• Clean with a wind blower separator (to separate the chaff, dust and other inert material from the samples, as well as light and empty seeds).
• Hand cleaning is advisable as a final stage (to remove contaminants left after machine cleaning e.g. diseased grains).

Visual inspection of seeds

This is a quality control process that must be carried out after harvesting the seeds.

• Spread the seeds on a flat, well-lit surface of contrasting colour. (This method reveals freely moving insects, eggs, mites, fungal fructifications, bacterial masses and infected plant debris. Examinations under or near ultraviolet light reveals infections of certain fungi and bacteria through emission of fluorescence).
• An illuminated table can be used if available.
• Examine dry seeds with the naked eye or under a microscope.

For insect and fungal damage

• Isolate the infested/infected samples (to prevent further spread of fungi or insects).
• Fumigation kills adult insects but does not kill eggs and fungi.
• Destroy samples when quarantine diseases and/or insects are found.
• Otherwise clean the seeds before storage using fumigation to eliminate insects, or wash samples using water and Clorox baths.

For mechanical damage and empty seeds

• Check incoming seed samples, using a light wind blower.
• Or manually clean shriveled and damaged seeds.

Special treatments

• Fumigate all harvested and incoming material (to eliminate insects).

Disposal of contaminated material

• Destroy samples contaminated with quarantine pests by burning (to prevent the spread of insects and diseases to other material and to ensure complete destruction of wasted material).

Inspection and certification

• Perform purity analysis of the seeds (following the ISTA standard method).

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Seed drying

Methods

• Use a mechanical slow process by drying samples in dehumidifying rooms at 20-25^oC and under relative humidity of 13-15% (to ensure embryo viability).
• The seed samples should be kept in paper or cotton bags on mesh racks during the drying period.

Drying time

• Minimum three weeks (the barley seed coat texture is not so hard thus the moisture is evaporates into ambient air).

Moisture content before drying

• 10-12% is a typical starting moisture content in dry areas. In other areas the range will be different (higher).

Moisture content for storage

• For both base and active collections moisture content for storage should be 5-7%.

Critical moisture content

• Should be 3-4% (for most of the crops it is recommended not to dry below 3% to avoid any adverse effects on the seed texture and viability).
• Recent research suggests 2-3% to extend the period of conservation.

Recording information during seed cleaning and drying

The following information should be recorded for each processing step:

• Accession Number (unique number in the collection).
• Reference to seed source (to trace the origin of the sample).
• Flags Y (Yes) and N (No) indicating if steps mentioned above have been completed (for checking).
• All packets, sacks and bags containing seed must be clearly labeled, identifying the accession with unique identifier(s).
• Durable labels should be affixed on, and inserted into, each seed bag.
• Data attributable to the accession sample should be printed on the label, including accession number, plot number, regeneration year and location, taxonomy and accession name.
• Remarks.

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Determination of seed moisture content

Methods

• High constant temperature oven-drying (following ISTA 2005 methods, that are accurate and acceptable).

Sampling frequency

• Randomly.

Sample size

• Take two random samples of 4.0-5.0 g each and grind them separately.
• Take 1.0-2.0 g from each sample as two independent replicates.

Grinding

• Fine grinding is required by using adjustable mechanical grinder (to promote uniform and complete drying and to prevent errors in moisture content determination through water loss or absorption during the process).

Oven drying temperature

• 175^oC for 90 minutes (following the ISTA rules).

Others

• Use analytical balance (to assure precision).
• Keep the dried samples in moisture-proof containers (desiccators) to cool down before weighing (to avoid changes in moisture content).

Recording information during determination of seed moisture content

The following information should be recorded for each step:

• Accession Number (unique number of sample in the collection).
• Net fresh weight in grams (g).
• Drying temperature.
• Time and date.
• Net dry weight in grams (g).
• Moisture content (%).

References and further reading

ISTA. 2005. International Rules for Seed Testing. Edition 2005. International Seed Testing Association, Bassersdorf, Switzerland.

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