Crop Genebank Knowledge Base

Sample processing

Sample processing in seed banks of cultivated rice genetic resources

Contributors to this page: T.T. Chang Genetic Resources Centre-IRRI, Los Baños, Philippines (Ruaraidh Sackville Hamilton, Ken McNally, Flora de Guzman, Renato Reaño, Soccie Almazan, Adelaida Alcantara, Elizabeth Naredo); WARDA, Cotonou, Benin (Ines Sánchez); UPLB-University of the Philippines at Los Baños (Teresita Borromeo).

Contents:
Seed cleaning
Seed drying
Seed moisture content

Cleaning

Cleaning is the removal of physical contamination from the plant material after harvest, before it can be stored. The exposure to unfavourable conditions should be minimized for greater seed longevity in storage.

Immediately after harvesting, thresh and blow the seeds and place them in the drying room within the day (see details below).
Pre-clean the dried seeds by blowing to separate unfilled grains and low density materials.
Manually clean the seeds.

Visual inspection of seeds

Verify the sample by comparing it with the seed file.
Determine the selection to be carried out based on the recommendation during the verification process.
Examine the seeds and hand sieve them with graded mesh sizes (if mixtures/off-types vary in size) to separate slender and bold grains (manual cleaning limits contamination and damage especially when the seeds are very dry).
Remove discolored, deformed, infected, soiled, immature, damaged, germinated seeds and off-types.
Determine the actual action to be taken based on the quantity of clean seed (this will determine the packing system to use).
Prepare and label all the necessary envelopes for use in seed testing, viability testing and for storage in the base, active and temporary storage.
Submit the selected samples together with the seed file, pre-labelled envelopes and the original seed container for double checking and quality control.
Check the selected sample against the seed file and the pre-labelled envelopes against the original container (to minimize labelling errors).
Mix the selected samples and divide using the pre-labelled envelopes as follows in order of priority:

IRRI staff selecting good seeds for storage to the IRRI Genebank facility (photo: IRRI)

For Base Collection

2 x 60 g sample.
2 x 100 grains for initial viability testing.

For Active Collection

Bulk sample for active collection

2 (for normal accessions) up to 5 (for the most popular accessions) x 10 g samples to be pre-packed ready for distribution.
2 x 200 grains for initial seed health evaluation.

For black-box safety backup storage

2 x 15 g for safety backup storage (one for Svalbard and one for another recognized genebank with long-term storage accepting black-box safety backups).

For temporary storage to repeat regeneration of harvests with insufficient seeds (<30 g) or inadequate initial germination rate (< 85%):

1-5 x 10 g samples for paper pre-packs.
Place the cleaned samples again in the drying room while waiting for the viability and seed health test results.
Encode all related information.

Special treatments

If the recommended best practices are followed for seed drying, no extra treatment is necessary (drying seeds to the recommended level inhibits fungal growth and insect infestation).
If seeds cannot be dried and kept dry sufficiently, fumigate as required whenever a build up of storage insects becomes noticeable.

Disposal of contaminated material

Incinerate contaminated material.

Inspection and certification (purity analysis of seeds)

Retrieve the accession number of all the harvested material.
Retrieve the corresponding seed files (the seed file provides information of the sample composition and guides the selection process).
Determine and compare the composition of the harvest with the seed file or the remnants of the planting material if the seed file is not available.
Identify the obvious mixtures/off-types, if present.
If the harvest does not represent the original sample, trace back for possible errors. If there are doubts on the sample composition, place the harvest on hold and repeat planting, making additional observations during the growing period, especially of the traits that differ in the initial harvest. Genotype DNA if needed.
If the new seed lot does not totally represent the original sample, discard the seed and record WH (Wrong Harvest) as the harvest status in the documentation system.

Notes

Certain changes in seed phenotype may occur even though the seeds remain genetically true-to-type:

Seed introduced from other environments may produce grain of different size in the seed multiplication environment.
Hull pigmentation is more intense when grown under moist conditions.
Harvests from screen house and field differ in hull colour intensity.

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Seed drying

Methods

1st stage of drying (during seed cleaning)

Immediately after threshing place the seeds in net bags and store in a drying room maintained at 15^oC and 15% relative humidity (RH).
Spread the samples evenly on the drying shelves.
Mix the samples by turning the bags daily.
Monitor the moisture level.
Transfer the dried samples to paper bags when the moisture is less than 10% moisture content (MC).

2nd stage of drying (after seed cleaning)

After cleaning, and sub-sampling for testing, place the selected samples back in the same drying room for 1-2 weeks while waiting for the viability and seed health results (during the seed selection process, seeds take in moisture).
Test the moisture content.
Monitor conditions in the drying room (with pre-set humidity and temperature, moisture testing is needed only infrequently).
Test more often if fluctuations in RH are observed.

Alternative drying

If facilities are not available for drying at 15^oC and 15% RH or if the drying equipment is not working reliably, dry with self-indicating silica gel in moisture-proof containers.
Repeatedly replace the silica gel until it stops absorbing moisture.


Dissecators with silica gel (photos: Bioversity International/ILRI, by kind permission of RDA genebank, National Agrobiodiversity Center, Suwon, Republic of Korea)

Drying time

Minimum of three weeks.

Moisture content before drying

9-20%.

Moisture content for storage

6-7%.

Critical moisture content

8% (below this level no insects or fungi affect the seed).

Recording information during seed cleaning and drying

The following information should be recorded for each processing step:

AccID [Unique Genebank Accession ID. Note a technical distinction between accession number and accession ID.
- A number, unique within a collection, (the accession number) is often used by genebanks and is sufficient for genebank management purposes.
- However, for all public documentation that number should be prefixed with a code to identify the collection and thus create an ID that is unique across genebanks: the combination of prefix and accession number then is a public accession ID].
Cropping season (year and season of seed multiplication).
Germination % (2 replications; initial % of germination of freshly prepared seed).
Germination date (date of germination test).
Packaging date (month and year of packing).
Amount_bulk [amount (g) stored in bulk package in the active collection].
Number_paper packs [number of paper packs retained for repeat multiplication – (for temporary storage only, if the viability or Seed Health test results are below the acceptable limit)].
Number_apack (number of small aluminium foil packets for storage in the active collection, pre-packed with 10 g dried seed ready for distribution).
Amount_base [amount (g) stored in base collection].
Plant_mat (sufficiency of planting material for harvests that requires another round of seed multiplication).
No. of Rows (approximate number of rows that could be planted for seed multiplication using the available planting material).
Harvest_stat [status of harvest (insufficient, mixed, diseased)].
Active tray ID (ID of the tray where the sample is stored in the active collection).
Base tray ID (ID of the tray where the sample is stored in the base collection).
Amount for duplicate storage1 (mark X if a sample has been prepared for the primary black-box safety duplicate storage genebank).
Amount for duplicate storage 2 (mark X if a sample has been prepared for duplicate storage in Svalbard, Norway).
Insects and pests (degree of insect and fungal damage).

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Determination of seed moisture content

Using a moisture meter to check the moisture content of seeds (photo: IRRI)

Methods

The recommended method follows ISTA rules.

Sampling frequency

Before storage

Fresh harvest - measure moisture content on representative samples one week and two weeks after harvesting (the MC reading will determine if the seed is safe and could be transferred to paper bags to provide enough space for the incoming harvest).
For the dried seeds - measure moisture content on representative samples five times/year and on every major fluctuation in RH reading.
When the drying room is kept at constant 15% RH, expected equilibrium MC is maintained.

During storage

No MC test is needed, provided best practice is followed for storing seed in moisture-proof containers.
Seeds for base collection should be vacuum packed in one piece aluminium and rubberized lid.
Seeds for active collection should be packed in high grade aluminium foil bags with small silica bag inside.
Repacking should be done in low RH packing rooms and small silica bag replaced with the activated sample.

Before germplasm movement

No MC test needed.

Sample size

5 g per sample.
A sample of five representative accessions spanning a range of grain sizes.

Grinding

Grind the sample until 50% of the sample is about 0.5 mm particle size and less than 10% of the sample has particle size > 1 mm.
Weigh the metal container with cover and add 5 g ground sample.

Oven drying

Set the oven at constant 130°C for two hours. The counting of the time should start when the oven stabilizes at 130^oC.
Take the seeds out of the oven and place them in desiccators for one hour.
Take the final weight.
Calculate the percentage of moisture content by: % MC = (Initial Wt - Final Wt) *100/Initial Wt.

Recording information during determination of seed moisture content