Crop Genebank Knowledge Base

Sample Processing

Sample processing of maize genetic resources

Contributors to this page: CIMMYT Maize, Mexico (Suketoshi Taba, Bonnie J. Furman), with inputs also received from IITA, Ibadan, Nigeria (Dominique Dumet); EMBRAPA (maize and sorghum genebank), Brazil (Flavia Teixeira); USDA (ARS/NC7, ISU); USA (Mark Millard).

Contents:
Seed cleaning
Seed drying
Seed moisture content

Seed cleaning

Cleaning is the removal of foreign material (debris) and low quality, infected and/ or infested seeds from the accession seed lot. The accession seed lot should match the original description in the passport. Pure quality seed should be preserved in the genebank.

Pollinated ears should be harvested from the regeneration plot.
Ears should be initially dried after harvest (see details below) for about 48 hours. Individual ears should be shelled (to separate the seeds from other plant parts, the cob and glumes) by a machine sheller, or hand-shelled to a pan. It is most convenient to use a corn sheller equipped with aspirator, feeding pan, cob ejection and bagging chute.
- Each ear should be shelled into an individual seed bag or envelope.
- The seeds should be cleaned, using an air-blown cleaner or sieves of various sizes.

Visual inspection of seeds

Visual inspection of the seeds is the most practical approach to ensure seed quality and accession integrity in the genebank. Inspection should be performed at the time of shelling ears, cleaning seeds and packaging seed for storage.

Visual inspection of seeds (photo: CIMMYT)

Infected and infested seeds and mechanically damaged seeds should be removed at shelling.
- It is important to look for the presence of mycelium, spores and larva on and inside the kernel.
- Good quality seeds are well-filled, plump and heavy.
When the shelled seeds are cleaned from debris, trash, damaged or low quality seeds, the accession purity of kernel type and colour should then be checked against the characteristics registered in the passport data.

Notes

Seed size may vary depending on where the regeneration is carried out.
The variation of primary and secondary seed colours and textures within the accession should be referenced with the passport data.
The pure seed definition (ISTA 1993) of more than a half of the original seed size should be valid to preserve quality seeds.
Very small seeds, as compared to the reference seed sample, should not be conserved.
A reference seed sample (50-100 seeds) of the original accession seed lot should be kept in the genebank for purity checking, as well as for identity of the genetic integrity. The reference seed sample should also be kept in the cold vault (base collection) and not used for seed distribution.

Special treatments

It is important that regenerated seeds are cleanly stored, without insect damage, during seed processing.

Maize ears should be dried on the plants in the regeneration field until harvest.
The harvested ears may carry insect eggs and larvae. To prevent infestation of the stored grain by pests such as weevils (Sitophilus zeamais), grain moth (Sitotroga cerealella) and grain borer (Prostephanus truncates), the ears should be dipped in a diluted insecticide solution (ex. marathon emulsion, DECIS 2.5 CE (Deltametrina) with surfactant chemical) and dried. The grain samples can also be fumigated by aluminum phosphide (57%) for 48 hours to avoid infestation, especially in the tropics.

Disposal of contaminated material

It is extremely important to correctly dispose of contaminated material to avoid contamination of the other material stored in active and base collections.

The maize seed bank operations need a system to dispose of the discarded seeds and cobs, as well as other plant material and trash that result from shelling and labeling.
The disposed seed, trash and plant material should be incinerated or buried at designated sites.
Old seeds should also be disposed of to save storage space (it is unnecessary and burdensome to preserve numerous original seeds of the same accession in a genebank).

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Seed drying

Seed drying is the most critical genebank operation to insure longevity of the maize seeds as an orthodox seed species. Refer to protocol seed storage behavior rules for seed testing (ISTA 2009) and keeping seeds alive (Probert and Hay 2000) for more information.

1^st stage of drying

Harvested ears should be dried in a hot air-brown drying room at 35^oC for about 48 hours to reduce the seed moisture content to 13-15% before shelling.

2^nd stage of drying

The cleaned seed lots should be transferred to the ultra-drying room with cold temperature and low humidity to attain 6-8% seed moisture to obtain an equilibrium seed moisture content of the individual accessions.
Desired equilibrium seed moisture content for long-term seed storage can be set by relative humidity and temperature in a drying room. For further information, refer to protocol seed storage behaviour and keeping seeds alive (Probert and Hay 2000). Most genebanks use an environment of 25% RH, between 5 and 15^oC. Drying time to reach equilibrium moisture content is about 2-3 months, depending on the type and volume of seeds and the air circulation or velocity inside the drying room.
- Silica gel can be used as an alternative method to dry the seeds for long-term storage.
Latin American maize races include seed types of dent, flint, floury, semi-dent, semi-flint, morocho, sugary and pop. The seed size of the racial accessions can vary a great deal. The time seeds take to reach an equilibrium seed moisture also depends on the amount of seeds loaded in the room.

Recording information during seed cleaning and drying

The following information should be recorded for each processing step:

Accession number or Introduction ID number.
Collection name or pedigree (name, pedigree).
Donor institute (the name of donor institution).
Donor identification number (the associated number).
Country of origin (country name).
Population type (landrace, synthetic, variety, breeding population, composite, inbred, hybrid).
Pollination type (half-sib, full-sib, selfing).
Race name or race classification (name of maize race, local name).
Grain type (dent, flint, floury, popcorn, morocho, sugary, opaque, white capped).
Grain colour (yellow, white, red, purple, variegated, sun-red, deep purple, brown, yellow-orange, blue, white cap, mottled).
Seed origin [location-year-plot number-(sample No)].
Seed viability (%).
Seed health data (good for storage or further testing or discarding or in need of further regeneration or collecting).

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Determination of seed moisture content

Seed moisture must be measured to make sure that they have the optimum moisture content for efficient preservation in active and/or base collections. Monitoring the seed moisture content during storage can be done with representative samples from the group of accessions that share the same seed regeneration origin.

Sampling frequency

Monitoring the moisture content of the seed accessions in the drying room can begin after one or two months.
The continuous monitoring of the moisture content in the drying room is usually carried out at 2-3 week intervals, until the moisture content falls below 8%.
The representative samples should be monitored from each of the groups of the accessions being dried.

Methods

Method 1

The seed moisture content of individual seed accessions can be determined most accurately by using the air-oven method, drying the previously ground seed samples at 130^oC in an oven for four hours. If the seeds have been pre-dried in the genebank to 8-10% before grinding, the samples can be dried for an hour to the constant moisture in an oven.
- Use 50-80 seeds (25-30 g) of each accession for grinding (as used at CIMMYT).
- Grind until reaching 0.5-1 mm medium fine particles.

Method 2

A rapid method for monitoring the moisture content is to use a Steinlite moisture meter SL 95 (Steinlite Cooperation, Atchison, Kansas, USA.).
- The experience at the CIMMYT maize genebank has shown that the moisture meter reading can be reliable to monitor the seed moisture content for packaging the seed samples when many seed accessions are processed in a short period of time.
- The difference of the moisture content between the oven method and the moisture meter is within 1%.

Recording information during the determination of seed moisture content

The following information should be recorded for each processing step:

Accession number (number) or Introduction ID number (number).
Collection name or pedigree (name, pedigree).
Seed origin (field book no., year, regeneration site, field plot no).
Initial seed moisture content (%).
Number of seeds or seed weight in grams processed (seed number or weight in g).

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References and further reading

Chang TT. 1985. Preservation of crop germplasm. Iowa State Journal of Research. Vol. 59. No.4. pp. 365-378.

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.

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.

Global Crop Diversity Trust. 2007. Global strategy for the ex situ conservation and utilization of maize germplasm [online]. Available from: www.croptrust.org/documents/web/Maize-Strategy-FINAL-18Sept07.pdf. Accessed: 4 August 2010.

Hanson J. 1985. Procedures for handling seeds in genebanks. Practical manuals for genebanks no. 1. International Board for Plant Genetic Resources, Rome.

Hong TD, Ellis RH. 1996. A protocol to determine seed storage behavior. IPGRI Technical Bulletin No. 1. International Plant Genetic Resources Institute. Rome. Italy. Available here.

International Seed Testing Association ISTA. [online]. Available from: http://www.seedtest.org/en/home.html. Date accessed: 30 January 2010.

Mezzalama M, Gilchrist L, McNab A. 2005. Seed Health: Rules and regulations for the safe movement of germplasm. Mexico. D.F., CIMMYT. Available from: http://libcatalog.cimmyt.org/download/cim/93586.pdf. Date accessed: 3 September 2010.

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.

Taba S, van Ginkel M, Hoisington D, Poland D. 2004a. Wellhausen-Anderson Plant Genetic Resources Center: Operations Manual, 2004. El Batan, Mexico: CIMMYT. Available here.

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