Sample processing of cultivated chickpea and wild relatives genetic resources
Contributors to this page: ICRISAT, Patancheru, India (Hari D Upadhyaya, Shivali Sharma, Cholenahalli L Laxmipathi Gowda, Dintyala Sastry, Sube Singh); NBPGR, New Delhi, India (Shyam Sharma); ICARDA, Aleppo, Syria (Ahmed Amri, Kenneth Street, Natalya Rukhkyan), SARC-RIPP, Piestany, Slovak Republic (Gabriela Antalikova); Institute of Plant Genetic Resources ‘K.Malkov’, Sadovo, Bulgaria (Siyka Stoyanova); Department of Primary Industries, Victoria, Australia (Bob Redden); IPK, Gatersleben, Germany (Andreas Börner).
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Cleaning is the removal of physical contamination from the plant material after harvest, before it can be stored. The ICRISAT Genebank Manual (Upadhyaya and Laxmipathi, 2009) recommends:
- Hand threshing and manual cleaning as one seed lot for cultivated type and for wild Cicer species.
- Periodic harvesting, threshing and manual cleaning of seeds.
Visual inspection of seeds
This is a quality control that must be done after harvesting the seeds. ICRISAT's Genebank Manual Procedures (Upadhyaya and Laxmipathi, 2009) recommends:
- Spread seeds on a flat surface and examine for damage/infestation.
- Remove damaged and infected seeds and store healthy seeds.
- Use a light wind blower or remove manually shriveled and damaged seeds.
Special treatments
- No chemical seed treatment is recommended (chemicals induce mutations over long time).
- Fumigation can be carried out, however, to control insects.
Disposal of contaminated material
The Germplasm Exchange and Quarantine of ICRISAT Mandate Crops recommends the following procedures:
- Autoclave and incinerate contaminated material.
- Contaminants should be placed in secure containers and buried deep (to assure that pests and disease risks associated with contaminated seeds are neutralized).
Inspection and certification
- Visually observe the samples and compare them with original seed and database information.
- Perform the purity analysis (following the ISTA Standards).
Seed drying
Methods
Most genebanks follow FAO/IPGRI Standards as well as the ICRISAT Genebank Manual (Upadhyaya and Laxmipathi, 2009):
 Controlled environment facility for germplasm seed drying at ICRISAT (photo: ICRISAT) |
- Dehumidified drying (temperature 15-20ºC and RH 15%) and silica gel drying.
IPK (Gatersleben, Germany) uses procedures based on requirements of the IPK quality management system (DIN EN ISO 9001:2000):
- Computer controlled drying chamber.
Drying time
According to procedures of the ICRISAT Genebank Manual (Upadhyaya and Laxmipathi, 2009):
- Depends on size of samples, initial moisture content and method of drying – usually one to four weeks.
Moisture content before drying
According to procedures of FAO/IPGRI Standards, as well as the ICRISAT Genebank Manual (Upadhyaya and Laxmipathi, 2009):
- About 8-12% (based on practical experience).
Moisture content for storage
- 8-10% for active collection and 5-7% for base collections.
Critical moisture content
The equilibrium relationship between seed moisture content and RH for chickpea storage is described in the ICRISAT Genebank Manual (Upadhyaya and Laxmipathi, 2009).
- It can vary between 3 and 7%.
Recording information during seed cleaning and drying
The following information should be recorded for each processing step:
- Accession identifier; National identity; Alternative identity (unique identification of the accession).
- Reference to seed source (to trace the origin of the sample).
- Seed moisture at harvest (% of moisture content of samples collected during harvesting.
- Threshing method employed.
- Drying method employed.
- Plot yield (g) (harvested seed yield).
- Seed samples moisture % after drying.
- Purity (%).
- Seed quantity (g) (cleaned seed yield).
- 100-seed weight (g) (weight of 100 randomly selected seeds).
- Germination % (seed germination from a standard test).
- Date of storage (date, month, year as dd/mm/yyyy).
- *Photographic record of seeds for each accession (100 seeds in petri dish with ID label).
- Remarks (any other information).
* This could be restricted to being done once, during characterization and does not need to be repeated at every regeneration cycle.
Determination of seed moisture content
Methods
According to the ISTA Standards:
- Use a high, constant temperature oven-drying.
Sampling frequency
This is important to cover the diversity in the accessions especially the seed traits.
- 5% of total number of seed samples/accessions.
- Or a minimum of ten accessions.
Sample size
According to the ISTA Standards:
- Use two replicates of 1.5-2.0 g of seeds.
Grinding
According to the ISTA Standards:
- It is recommended to coarse grind the samples.
Oven drying temperature
According to the ISTA Standards:
- 130±2ºC for one hour.
Others
According to the ISTA Standards:
- Use two replicates per sample.
Recording information during determination of seed moisture content
The following information should be recorded for each step:
- Accession number (unique identification of the accession).
- Replication number.
- W1 [weight of empty dish (g)].
- W2 [weight of dish + fresh sample (g)].
- W3 [weight of dish + dried sample (g)].
- Drying temperature.
- Time and date.
- (W2-W3)/(W2-W1) x100 [% of moisture content (on wet basis) of the replication].
- Mean moisture content (%) of the replications.
- Additionally, data field names are mentioned in Handling Seed in Genebanks (Hanson 1985) and Manual of Seed Handling in Genebanks (Rao et al., 2006).
References and further reading
Chakrabarty SK, Anitha K, Girish AG, Sarath BB, Prasada RRDVJ, Varaprasad KS, Khetarpal RK, Thakur RP. 2005. Germplasm Exchange and Quarantine of ICRISAT Mandate Crops. Information Bulletin No. 69. Rajendranagar 500 030, Andhra Pradesh, India: National Bureau of Plant Genetic Resources; and Patancheru 502 324, Andhra Pradesh, India: International Crops Research Institute for the Semi-Arid Tropics. 80 pp. ISBN 92-9066-481-9.
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.
Hanson J. 1985. Procedures for handling seeds in genebanks. Practical manuals for genebanks no. 1. International Board for Plant Genetic Resources, Rome. http://www.bioversityinternational.org/index.php?id=19&user_bioversitypublications_pi1[showUid]=2235.
ISTA. 1993. International rules for seed testing. International Seed Testing Association. Bassersdorf, Switzerland. Seed Science and Technology 21 (Supplement):1-288.
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.
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).
Stoyanova SD. 1992. Drying chickpea seeds for long-term preservation in genebanks. PGR-Newsletter 90:19-27.
Upadhyaya HD, Laxmipathi Gowda CL. 2009. Managing and Enhancing the Use of Germplasm – Strategies and Methodologies. Technical Manual no. 10. Patancheru 502 324, Andhra Pradesh, India: International Crops Research Institute for the Semi-Arid Tropics. 236 pp.
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