Storage of bread wheat, durum wheat, triticale and related cereals

Contributors to this page: CIMMYT - Wheat, Mexico (Thomas Payne), ICARDA, Syria (Ahmed Amri) with inputs also received from CIMMYT - Maize, Mexico (Suketoshi Taba), USDA - National Small Grains Collection, Aberdeen, Idaho (Harold Bockelmann), CGN, Wageningen, The Netherlands (website) and IPK, Gatersleben, Germany (Helmut Knűpffer).

Base collection

Cold and dry storage rooms for conservation of seed. Notice the warm clothes of the genebank staff (photos: CIMMYT)

Definition

The base collection is a long-term storage facility. The initially collected seed accession sample is used as a bench mark for monitoring changes in the genetic identity and diversity of the accession, over time and after cycles of regeneration, that may arise due to genetic drift. The facility should insure that an accession’s seed viability remains at acceptable levels for at least 50 years, or at least one cycle of the regeneration of the accession.

When should base collection samples be used

• To hold seed samples for long-term conservation.
• To serve as a duplicate, safety collection of the active collection and for long-term conservation.
• To reduce the number of regeneration cycles, thereby better maintaining the genetic integrity of the accessions.
• To provide foundation seeds for regeneration of the genebank accessions.
• To provide seed samples of accessions requested for exchange with other genebanks.
• When blackbox sets are prepared for safety duplication deposit at other cooperative genebanks.
• Its function and operation should be supported by long-term institutional funding.

Sample specifications

Minimum sample size for storage

A representative sample should be used to avoid genetic drift. There are a few options that could be chosen:

• For cultivated cereals: two packets of 25 g each; for wild cereal species: one packet of 25 g (as followed at ICARDA).
• For wheat in general use 100 g, equivalent to about 3000 seeds (as followed at CIMMYT).

Minimum viability for storage

• For cultivated cereals: > 90%; for wild cereal species: > 80% (as followed at ICARDA).
• For wheat in general use >85% (as followed at CIMMYT).

Moisture content

To avoid any deterioration to the stored seed samples.

• 5-7% seed moisture content (as used at ICARDA).
• Or 3-5% seed moisture content (as used at CIMMYT).

Container specifications

Seed packaging method

• Use aluminum foil packets sealed hermetically under vacuum with a heat sealer.
• The packets should be re-sealable and impermeable to air and moisture.
• Packing is best carried out in an air-conditioned room with controlled humidity, as quickly as possible after drying.

Specifications of packaging material

• Three-ply aluminum foil bags should be used comprising:
  • An inner layer of polyethylene, needed for sealing.
  • An intermediate layer of aluminum foil, which prevents moisture penetration.
  • An outer layer of polyester providing mechanical strength.

Storage specifications

Assigning location codes

For easy access and location of the accessions.

• The seed accessions should be stored in an orderly fashion.
• A standard nomenclature for shelf location within a collection should be used allowing each accession in the collection to be quickly located. Use coordinates such as unit/block/cart number, row shelf number, shelf number, and tray/box number, packet number.

Storage conditions

Low temperatures and seed moisture content are important to maintain seed viability.

• -18 to -22^oC.

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).

Back to top

Active collection

Definition

The active collection consists of accessions that are designated as available for distribution and hence are accessed more frequently than those in the base collection. 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 should active collection samples be used

• The active collection should be used to hold seed samples for mid-term conservation.
• Accession samples from the active collection are used to:
  • Conserve those accessions that are most actively used, or those that are expected to be used, such as elite lines and populations, core subsets and reference seeds sets.
  • Distribute seed samples requested by clients.
  • Store introductions and the regenerated seeds prior to processing and formally inventorying them into the base and active collections.

Sample specifications

Minimum sample size

• For cultivated cereals: 1500 seeds; for wild cereal species: 1500 seeds (as used in ICARDA).
• Or 250 g (about 7000 seeds), as used in CIMMYT.

Viability for storage

• For cultivated cereals: > 90%; for wild cereal species: > 80% (as used in ICARDA).
• Or >85% (as used in CIMMYT).

Moisture content

To avoid any deterioration to the stored seed samples.

• 5-7% seed moisture content (as used in ICARDA).
• 3-5% seed moisture content (as used in CIMMYT).

Container specifications

Seed packaging method

• Hermetically sealed and re-sealable containers.

Specifications of packaging material

The packaging material must be moisture resistant and withstand storage at low temperature for long periods of time without cracking or any other physical deterioration.

• Some collections prefer to use three-ply aluminum foil bags comprising:

  Wheat seeds in vacuum packed alluminium bags (photo: Bioversity/ILRI by kind permission of Belarus genebank)

  • An inner layer of polyethylene, needed for sealing.
  • An intermediate layer of aluminum foil, which prevents moisture penetration.
  • An outer layer of polyester providing mechanical strength.
• Other collections prefer the easier access of square-based bottles with a screw cap made of pure, high quality, plastic materials, that are semi transparent or white in colour, with a volume of 400 ml, or clear glass jars.

Storage specifications

For easy access and location of the accessions.

Assigning location codes

• The seed accessions should be stored in an orderly fashion.
• A standard nomenclature for shelf location within a collection should be used allowing each accession in the collection to be quickly located. Use coordinates such as unit/block/cart number, row shelf number, shelf number, and tray/box number, packet number.

Storage conditions

The temperature can be between 0^oC and 10^oC with a relative humidity (RH) of 25% to 35% to ensure the seed viability for more than 20 years. Low relative humidity in the storage room is more important to the seed longevity than low temperature.

• 3 ± 2^oC (as used in ICARDA - it extends storage life and breaks any seed dormancy that may exist).
• 0^oC (as used in CIMMYT).

Recording information during storage – Active 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).
• Number of  'user packets' (if applicable) (number).
• 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 (coordinates).
• Flag indicating distribution status.

Back to top

Storage management

Storage space arrangement

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

• Minimum space should be used to maximize packing efficiency, while allowing convenient access to each sample.
• A standard nomenclature for shelf location within a collection should be used allowing each accession in the collection to be quickly located. Use coordinates such as unit/block/cart number, row shelf number, shelf number, and tray/box number, packet number.
• Movable shelves or racks are space saving designs in genebanks. They can be constructed in row and column arrangements for inventorying the samples in an orderly fashion.
• Fixed shelves are common, leaving spaces to easily access the seed samples. Each shelf may have several levels, depending on the size of the seed containers and boxes. 

System for tracking material/inventory system

Samples must be quickly accessed.

A good system as used at CGN Wageningen, The Netherlands:

• Prepare in advance and maintain as inventory items 'user bags' that are used for distribution of requested accessions and germination testing.
• When insufficient ‘user bags’ are identified by the inventory management system, new bags should be made.
• For production of new ‘user bags’, a sample from the active collection should be taken.
• The 'user bags' should be maintained in the active collection.

Back to top

Viability of bread wheat, durum wheat, triticale and related cereals genetic resources

Contributors to this page: CIMMYT - Wheat, Mexico (Thomas Payne) and ICARDA, Syria (Ahmed Amri) with inputs also received from CIMMYT - Maize, Mexico (Suketoshi Taba); USDA - National Small Grains Collection, Aberdeen, Idaho, USA (Harold Bockelmann); CGN, Wageningen, The Netherlands (website) and IPK, Gatersleben, Germany (Helmut Knűpffer).

Viability testing

Laboratory methods

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

Type of test

• Some collections advocate the use of externally contracted, accredited parties to conduct unbiased seed quality assessments.
• ISTA methods and rules of determining seed viability should be routinely and widely practiced.

Number of seeds and replicates

• Normally, replicates of 50 seeds totaling 100-200 seeds should be tested for monitoring seed viability.
• Genetically heterogeneous populations may need 200 seeds to determine viability, whereas more homozygous accessions may need fewer seeds (50-100) to measure viability.
• If the variability between the replicates is high, another germination test should be conducted with a further 100 seeds.

Pre-treatment

• Viability testing should be performed with seeds that have been cleaned and dried.
• Seeds may be surface sterilized with a 0.5% sodium hypochlorite solution for ten minutes to avoid fungal contamination.

Media

• Petri dishes lined with filter paper that has been wet with tap water should be used for germination tests.

Temperature

• Use a germinator incubator at 25˚C.

Light

• 12/12 hours dark/light regime.

Duration of test

• Normal, abnormal and seeds lacking germination should be tallied after four and seven days to determine percent of germination.

Monitoring intervals

To ensure the viability of the seed accessions in both base and active collection.

Below four different approaches to viability monitoring intervals are given. They are all valid, but the USDA approach seems to be the most innovative!

ICARDA

• An initial assessment of viability is made before an accession is added to a collection.
• After five years of the initial storage, a random sub-sample from stored accessions will be subjected to a new germination test.
• The above results are compared with the initial germination test results.
• If the results match, the monitoring interval can be extended.
• If a decline is observed, a larger random seed sample should be re-tested, and the regeneration procedures and storage facilities should be reviewed to avoid any improper conditions and procedures.

CIMMYT

• Monitoring of seed viability occurs with an initial assessment, repeated at subsequent ten-year intervals.
• Sequential seed viability monitoring may be reduced to five-year intervals depending on preceding seed vigour and the percentage of the germination test results (Ellis et al., 1985).
• Seed viability should be monitored in both base and active collection.
• Seed viability in a base collection is expected to maintain for much longer than in the active collection (Walters et al., 2005).

USDA National Small Grains Collection

• Accessions are scheduled for regeneration based on a priority matrix where each accession is assigned a regeneration priority number between 1 and 9, where 1 is the highest priority for regeneration and 9 the lowest as shown in the following table:

• Viability tests are scheduled for each accession every five years.

CGN Wageningen

• Testing initial germination capacity: the first test is carried out on a sample to be accessed to the genebank.
• Repeating germination test: monitoring of germination capacity, after some time, of numbers already accessed to the genebank. This repeated test is done on samples at intervals specific to each crop. Each regeneration of an accession can be tested several times.
• Controlling germination test: 5% of the above two germination test samples are tested twice to test the reproducibility of the test method.
• Ten years after the last germination test, the test should be repeated for:
  • All material with a germination capacity lower than 80% during the last determination.
  • Material of crops of which: seeds reportedly have a short life span, seeds in short-term storage that show a poor storability (on the basis of breeder’s experience), where data from literature are contradictory or lacking.
• After a minimum of 20 years after the last germination test and/or a maximum of 30 years after the last multiplication, these actions should be repeated for all remaining material.

Recording information during viability testing

The following information should be recorded for each processing step:

On the petri dishes

• Accession Number (unique identifier).
• Replication number (number).

On the data sheets and database

• Accession number (unique identifier).
• Taxonomic name (taxon).
• Replication number (number).
• Start and end trial dates (dates).
• Test method used (standard test procedure used).
• Sample size (number).
• Number of seeds (seedlings) germinated (results) in each category (normal, abnormal, non-germinated).
• Germination percentage (counted on normal seedlings only). Seedlings should be judged as normal according to specific criteria [ISTA (2003, 2005), Association of Seed Analysts (AOSA) (2005)].
   

Back to top

Routine monitoring

The genebank curator must continually monitor the general conditions of the genebank infrastructure and operating procedures to ensure that conserved seed quantity and seed viability is maintained to international standards. Safety from vandalism, robbery, adverse weather, electrical failure and lack of off-site safety duplication must all be considered when reviewing the security of a collection.

• Periodic monitoring of the collection’s seed inventory and seed viability is essential.
• Computerized stock control systems should be used to monitor the quality and quantity of seed accessions in cold store.
• Quality control flags should be used to determine when an accession is:
  • (A) = Available for distribution.
  • (L) = Low in seed stock, allowing an accession to be distributed but alerting the curation that a regeneration of the accession is needed.
  • (S) = Low number of seeds whereby no seed distribution is allowed; and sample regeneration should occur immediately.
  • (V) = When viability falls below prescribed levels.
• Inventory management through use of barcoding applications may result in increased managerial efficiency and accuracy. The process of barcoding an entire collection also provides an opportunity for the entire collection’s current inventory to be taken and confirmed.

Monitoring frequency

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

Critical quantity

• For both cultivated and wild species: 1000 seeds (to keep the integrity of the population samples as at ICARDA).
• Or set at 500 seeds (as followed at IPK-Gatersleben).

Critical germination level

• For cultivated wheat: 85%; for wild cereal species: 75% (or 10% less than the average value of fresh seeds, as suggested at ICARDA).
• For wheat in general, set at 70% (as used at IPK-Gatersleben).
• For wheat in general, set at 85%, based on the initial seed viability of 100% (as followed by CIMMYT, to ensure a large enough sample size is used to maintain the genetic integrity during the cycles of regeneration.

Recording information during routine monitoring

The following information should be recorded for each step:

• Accession number (unique identification number).
• Date of initial viability test (date).
• Germination %, initial test (percentage).
• Date of each subsequent viability test (update database with a date).
• Germination percentage of each subsequent viability test update database (%).
• Date of the initial storage (date).
• The initial amount of seeds stored (g).
• The current amount of seeds stored (g).
• Thousand grain weight (g).
• Date of next required viability test (one year if it is close to critical value; five years when it is above 90%).
• Regeneration flag (Yes – must go for regeneration; No – 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.

Ellis RH, Hong TD, Roberts EH. 1985. Handbook of seed technology for genebanks volume I. Principles and methodology. Handbooks for Genebanks no. 2. International Board for Plant Genetic Resources, Rome, Italy.

ISTA. 2005. International rules for seed testing. ISTA Secretariat, Switzerland.

ISTA. 2003. International rules for seed testing. ISTA Secretariat, Switzerland.

Walters C, Wheeler LM, Grotenhuis JM. 2005. Longevity of seeds stored in a genebank: species characteristics. Seed Science Research 15:1-20.

Back to top

Seed health diagnosis of bread wheat, durum wheat, triticale and related cereals genetic resources

Contributors to this page: CIMMYT - Wheat, Mexico (Thomas Payne), ICARDA, Syria (Ahmed Amri) with inputs also received from CIMMYT - Maize, Mexico (Suketoshi Taba), USDA - National Small Grains Collection, Aberdeen, Idaho, USA (Harold Bockelmann), CGN, Wageningen, The Netherlands (website) and IPK, Gatersleben, Germany (Helmut Knűpffer).

Recommended procedures to detect important quarantine seed pathogens

Bacteria

Xanthomonas translucens pv. undulosa (YDC agar and semi-selective XTS agar).

Fungi

Tilletia spp. (seed washing, filter test).
Ustilago spp. (seed washing, filter test).

Virus

Barley stripe mosaic virus (BSMV), [ELISA and TBIA (tissue-blot immunoassay)].

Testing protocols

Applying the latest laboratory methods, CIMMYT staff insure that seed entering and leaving the center is disease-free (photo: CIMMYT)

Test the accession seed samples before the accession is processed for inclusion in the active genebank collection, or prior to planting in the field to prevent the transfer or establishment of diseases or pests.

• Seed health should be tested by an independent, internal seed health laboratory, or via contracted, accredited external laboratories. 
• Policies and procedures for the detection of seed-borne pathogens should be undertaken in collaboration with the relevant national plant protection and quarantine authorities. 
• Only material that is free of quarantined seed-borne pathogens should be permitted for entry into a collection, as this practice will allow the subsequent distribution of disease-free material.

Recording information during seed health diagnosis

The following information should be recorded for each step:

• Accession number.
• Date of diagnosis.
• Diagnosis record number.
• Pathogen(s) tested.
• Method of pathogen test(s).
• Result of test (using appropriate scoring methods).
• Overall pathogen assessment (approved, conditionally approved, or rejected).

Other information

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.

Sample processing of bread wheat, durum wheat, triticale and related cereals genetic resources

Contributors to this page: CIMMYT - Wheat, Mexico (Thomas Payne) and ICARDA, Syria (Ahmed Amri) with inputs also received from CIMMYT - Maize, Mexico (Suketoshi Taba); USDA - National Small Grains Collection, Aberdeen, Idaho, USA (Harold Bockelmann); CGN, Wageningen, The Netherlands (website) and IPK, Gatersleben, Germany (Helmut Knűpffer).

Seed cleaning

Cleaning harvested seed using course sieves (photo: CIMMYT)

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

• Prepare labelled harvest cloth bags or paper sacks in advance of harvest.
• Label both outside and inside the seed bags with the plot number, accession number and accession name.
• Commence harvest when grain moisture content has reached 13-15%.
• Harvest and thresh with plot combine (seeds are hard and there is no physical damage). Allow the plot combine to thoroughly clean the area between plots to avoid any mixtures and contamination.
• Use a cylinder type machine to de-awn or fully thresh accessions.
• Clean harvested seed with a wind-blown separator and course sieves or screens (to separate the chaff, dust and other inert materials from the samples, as well as light and empty seeds), 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 allowing visual inspection for seed purity (mixtures, weed seed, diseased seed, chaff, soil, insects, etc.) is advisable (to remove contaminants and ensure initial degrees of integrity).
• Dry the seeds initially, for short-term storage (see details below, in seed drying).

Visual inspection of seeds

This is a quality control that must be done after harvesting the seeds.

• Spread the seeds on a flat well lit surface of contrasting colour.
• An illuminated table can be used if available.
• Examine dry seeds with the naked eye or under a microscope (visual inspection of the seeds is the most practical approach to ensure the seed quality and the accession integrity in a genebank).
• Isolate, remove and destroy the infested/infected seeds (good quality seeds are well filled and plump and heavy).

Special treatments

• Annually fumigate all temporary seed storage and seed preparation rooms and equipment against grain insect infestations.
• Prevent rodents from entering preparation and storage facilities, and maintain rodent deterrents at all times.

Disposal of contaminated material

• Seed, trash and plant material should be disposed of by incineration or burial at a designated site (to eliminate contaminated seed, preventing the spread and cross-contamination of insects and diseases).

Inspection and certification

A seed conservation manager or curator should be in charge of the final inspection on purity of seed material (the multistage seed inspection by the genebank curator or personnel should be required to ensure the purity of the seed accession and the genetic traits present in the germplasm accessions).

• If the seed material is of insufficient purity, the deviation should be recorded in the multiplication logbook.
• The curator should re-clean the material, or schedule a subsequent regeneration of the material.

Back to top

Seed drying

Methods

1st stage drying (during cleaning)

• In open, well ventilated areas, allow seeds to dry to a maximum of 12% moisture content, for short-term storage (to prevent seed moulding and to preserve viability).

2nd stage drying (after cleaning)

• Cleaned seed samples should be dried in a hermetically sealed, temperature and humidity controlled drying room [seed drying is the most critical genebank operation to insure seed longevity (ISTA 1993, Thormann et al. 2004, Probert and Hay 2000)].
• The temperature in the room should be set at 10-20°C and relative humidity (RH) at 13-15%.
• The seed samples should be dried in paper or other ‘breathing’ material bags.

Drying time

• Depending on the original moisture content and the seed quantity, it may take 3–5 weeks.

Moisture content for storage

• The desired moisture content to be reached is 3–7%.
• Replicated, representative seed samples should be monitored for moisture content during the drying process.

Critical moisture content

• Short-term storage requires a grain moisture content of 12% or less.
• Long-term storage requires a grain moisture content of less than 5%.

Recording information during seed cleaning and drying

The following information should be recorded for each processing step:

• All packets, sacks and bags containing seeds must be clearly labelled, 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.
• Inclusion of seed and/or glume colour may be desirable to enable verification of sample identity during seed processing.
• Accession number (the genebank accession number: the accessions are inventoried for long-term conservation and seed distribution).
• Year of regeneration (year of regeneration of the bagged sample).
• Location of regeneration (name of the experimental station, farm or field in which the regeneration trial took place).
• Trial and plot number of regeneration (trial and plot number identifying the origin of the seed lot).
• Taxonomy, genus and species.
• Accession name (name, pedigree).
• Grain or glume colour.

TIP: In the seed storage facilities, the type and the status of a seed lot must be clearly distinguishable so that seed material cannot enter a wrong process stream or activity. Some collections use coloured labels to identify different processes within their schema of accession management. CGN The Netherlands recommends that the type and/or status of seed lots be marked by means of coloured labels, which are attached to the boxes in which the material is stored. Relevant data should be noted on the labels, thus revealing the identity of the material. In addition, the location of further information about the material and where the material is stored should also be given.

• Red: deviant material.
• Pink: newly acquired material.
• Yellow: material from third parties.
• Blue: waiting for seed testing.
• Green: ready for accession.
• White: all accepted material.

Back to top

Determination of seed moisture content

Methods

• The seed moisture content of seed accessions can be determined most accurately by the use of the air-oven method (to ensure that dried seeds have reached the optimum moisture content required for long-term conservation).
• Rapid measure of whole grain moisture content is also possible using electronic moisture testers. Experience at CIMMYT indicates that electronic moisture meters are a rapid and reliable means to monitor the seed moisture content, with a difference in moisture content with the oven method of less than 1%.

Sampling frequency

• Prefered method is using the electronic meter.
• Sample as required to reach the desired target moisture content.

Sample size and grinding

• Obtain two random samples from the seed lot to be tested, 4.0-5.0 g each and grind separately.
• From each ground sample, make two 1.0-2.0 g sub-samples, treating each as two independent replicates.
• Use analytical scales to weigh the samples before and after oven drying.

Oven drying temperature

• Seeds with an initial moisture content of <10% before grinding can be oven dried for one hour to the constant moisture in an oven.
• Dry the ground seed samples (0.5-1 mm of medium fine particles) at 130^oC in an oven for four hours (ISTA, 1993).

Recording information during determination of seed moisture content

The following information should be recorded for each processing step:

• Accession number.
• Moisture content determination method (oven or electronic moisture meter).
• Net fresh weight in grams (g).
• Drying temperature.
• Time and date.
• Net dry weight in grams (g).
• Moisture content (%).

References and further reading

International Seed Testing Association ISTA. 1993. International rules for seed testing. Seed Science and Technology 21, Supplement.

Hong TD, Linington S, Ellis RH. 1996. Seed storage behaviour: a compendium. Handbooks for genebanks no 4. International Plant Genetic Resources Institute, Rome.

Probert RJ, Hay FR. 2000. Keeping seeds alive. In: Black M, Bewley JD, editors. Seed technology and its biological basis. CRC Press LLC. USA and Canada. pp. 375-404.

Thormann I, Metz T, Engels JMM. 2004. The Species Compendium (release 1.0; December 2004). [online]. Available from: http://www.bioversityinternational.org/scientific_information/Information_Sources/Species_Databases /Species_Compendium.html. Date accessed: 31 March 2010.

Back to top