Viability of forage grass genetic resources
Contributors to this page: ILRI, Addis Ababa, Ethiopia (Jean Hanson); CIAT, Cali, Colombia (Daniel Debouck); Bioversity International/ILRI, Addis Ababa, Ethiopia (Alexandra Jorge).
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Laboratory methods
Viability test methods are specific for different species. International Seed Testing Association rules should be used when available. Specific information is available for many species (see Species Compendium).
Type of test
- Standard germination tests (see list for species specific information).
or
- Sequential germination tests (these are less accurate, but require less seeds and are suitable in case of a limited number of seeds). Use at least 40 seeds per test. If fewer than 30 seeds germinate, programme the accession for regeneration.
Number of seeds and replicates
International Seed Testing Association rules should be used when available.
- ISTA recommends four replications of 100 seeds.
- A minimum number of four replications of 50 seeds is acceptable, when seeds are limited.
- The number of seeds should be practical (depending on the seeds available) and enough to provide statistically significant results.
- Wild species often have limited numbers of seeds to use for testing.
- For the sequential test use 40 seeds.
Pre-treatment
See the attached list with recommendations for some grass species (viability test methods are specific for different species. International Seed Testing Association rules should be used when available).
For Brachiaria seeds, scarification is done by immersing in a 2% solution of sulphuric acid for 15 minutes and washing well to remove all traces of acid before setting for germination.
A 0.2% potassium nitrate solution is also used to break dormancy in several species of tropical grasses. Seeds can be soaked in the solution overnight or the solution can be used with the germination media or incorporated into water agar.
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Media
- See the attached list with recommendations for some grass species (viability test methods are specific for different species. International Seed Testing Association rules should be used when available).
- Media that hold moisture such as water agar are recommended for grass seeds that take time to germinate.
River sand that has been treated previously with steam at 100°C for two hours is recommended for grass seeds so that they can grow into strong seedlings. Seeds are usually planted in rows 3-4 cm deep and then covered with sand to 2 cm depth. The sand is kept moistened, usually twice a day with a spray. Viable seeds germinate in 6-10 days.
Temperature
- See the attached list with recommendations for some grass species (viability test methods are specific for different species. International Seed Testing Association rules should be used when available).
Light
- A 12 hour light period should be used to simulate tropical day length conditions (International Seed Testing Association rules should be used when available).
Duration of test
- See the attached list with recommendations for some grass species (viability test methods are specific for different species. International Seed Testing Association rules should be used when available).
Others
- Germination is complete when the seedling can be judged as normal with a well developed root and shoot (ISTA, 2006).
Monitoring intervals
Some species of grass seeds are very short-lived so the sampling interval should be sufficient to measure loss of viability without being so frequent as to use valuable germplasm for testing:
- Every five years above 50% germination.
- Every three years below 50% germination.
Recording information during viability testing
The picture above shows abnormal (left) and normal (right) grass seedlings. (photo: ILRI) |
The following information must be recorded for each testing step:
- Accession number (ID number).
- Lot number (ID number).
- Taxonomic identification.
- Date of test (the date that the test was commenced).
- Number of replications (the number of replicates in the test).
- Number of seeds per replication (the number of seeds in each replicate of the test).
- Pre-treatment (pre-treatments used for the test. More than one can be used in conjunction).
- Media (the media for the test).
- Temperature (the temperature used during the test in degrees centigrade. If an alternate temperature is used this temperature usually refers to the temperature during the day period, unless otherwise stated).
- Alternate temperature (if more than one temperature is used, the temperature in degrees centigrade of the alternate period. The alternate temperature usually corresponds to the dark period).
- Length of dark (the number of hours with no light. This usually corresponds to the period at lower temperature, when two temperatures are used).
- Length of test (the number of days during which the test was conducted). Percentage germination (the average percentage germination over all replicates, calculated from the number germinated per replicate).
- Tolerance (a measure of the statistical accuracy of the test according to the standard tolerance tables).
- Percentage dormancy (the average percentage of hard seeds which are not rotten, over all replicates).
- Test reference (a consecutive number given each time that seed lot is tested over time).
Methods
- Do routine tests for seed viability at regular intervals (monitoring methods for viability should minimize use of seeds while providing a good indication of reduction of viability).
- The stock control system should be queried to determine accessions for regeneration.
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Accessions can be flagged (marking accessions in the database helps to avoid distribution of accessions by mistake and quickly identify accessions for regeneration) in the database as:
- (A) Accession is available for distribution.
- (L) Low stock; Accession is available for distribution but regeneration should be planned.
- (S) Low number of seeds; no distribution is allowed; sample should be regenerated.
- Genebank facilities should be checked for working order or damage and preventive maintenance should be done on equipment (regular monitoring of equipment and buildings allows early identification of problems and preventive maintenance before failure).
Monitoring frequency
This is set according to the minimum quantity and minimum viability of seeds below which they need to be regenerated.
Critical quantity
- A minimum of 600 seeds (this quantity allows two regeneration attempts using 300 seeds giving a high probability to have 100 plants to maintain genetic integrity of accessions, even for seeds with 50% viability).
- Where possible it is better to regenerate when seed quantities reach 1000, to mitigate risks.
Critical germination level
- 65% germination percentage for seeds of many species and 50% for seeds of grasses which have short to medium longevity in storage (recommended by FAO/IPGRI, 1994).
Recording information during routine monitoring
The following information should be recorded for each step:
- Accession number (ID number).
- Lot number (ID number).
- Weight of seeds (weight of seeds on hand).
- 1000 seed weight (weight of one thousand seeds to be used for calculating number of seeds from weights).
- Percentage of germination (the average percentage germination over all replicates, calculated from the number germinated per replicate).
- Stock control flag (code to indicate the stock level and distribution).
- Availability of material flagged (code to indicate the stock level – see above).
- Regeneration flag (code to indicate if regeneration is required).
References and further reading
Butler JE. 1985. Germination of Buffel grass (Cenchrus ciliaris). Seed Science and Technology 13:583-591.
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. 2008. International Rules for Seed Testing. International Seed Testing Association. ISTA secretariat, CH-Switzerland. Available from: www.seedtest.org/.
Smith RL. 1979. Seed dormancy in Panicum maximum Jacq. Tropical Agriculture 56:233-239.
Thormann I, Metz T, Engels JMM. 2004. The Species Compendium (release 1.0; December 2004). [online] Available from: http://www.bioversityinternational.org/databases/species_compendium_database/index.html. Date accessed: 09 April 2013.
Whiteman PC, Mendra K. 1982. Effects of storage and seed treatments on germination of Brachiaria decumbens. Seed Science and Technology 10:233-242.
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