Sample processing

Page compiled by: Bioversity International/ILRI, Addis Ababa, Ethiopia (Alexandra Jorge); ILRI, Addis Ababa, Ethiopia (Jean Hanson) including information extracted from: Rao NK, Hanson J, Dulloo ME, Ghosh K, Nowel D and Larinde M. 2006. Manual of seed handling in genebanks. Handbooks for Genebanks No. 8. Bioversity International, Rome, Italy. 147pp.

What is sample processing

The processing of seeds or plant material is the preparation of the samples to be stored. This is directly linked to the method of conservation, i.e. seed cleaning and drying is related to seed conservation while extraction and disinfection of plant material is related to tissue culture or cryopreservation. Therefore, for practical purposes in this presentation this is incorporated within each conservation method.

 Seed processing is usually done in four main steps:

Seed cleaning

Seed cleaning is the removal of debris, physical contamination, inert material, damaged and infected seeds and seeds of other species. It is essential to improve the quality of samples and should be done immediately after harvesting or as soon as the material arrives at a genebank, before storage.

Why it should be done

• To reduce the volume of seeds/propagules to be transported and stored (removing extraneous material).
• To improve the purity of the samples (removing damaged seeds/planting material).
• To optimize storage space and reduce genebank costs.

Steps of seed cleaning

Threshing

Seed threshing is the separation of the grains from the straw either by impact, friction or combing action. It can be done manually or with threshing machines.

Cleaning

The first cleaning usually separates seeds from debris, physical contamination and inert material. It can be done manually or with cleaning machines.

Visual inspection

This is important to check and prevent further spread of insect and fungal damage and remove damaged or empty seeds.

Final cleaning

This is when damaged and empty seeds are identified and removed. It is usually done manually.

Purity analysis

Purity is an expression of how ‘clean’ the seed lot is.

ISTA (2005) specifies a pure seed faction to contain: 

• Intact seeds of actual species as well as dead, shrivelled, diseased, immature and pre-germinated seeds.
• Achenes and similar fruits, such as samara with or without perianth regardless of whether they contain a true seed, unless it is apparent that none is contained.
• Fractions of broken seeds, achenes, etc. that are more than half of the original size.

Information on actual seed lot composition is important; purity analysis serves as a guideline to determine the necessity of further cleaning. During purity analysis, each ‘pure’ seed fraction from the working sample is separated from the inert matter and other seeds.

• Weigh out a working sample of  a given weight (for example 250 g) of the total seed lot randomly using electronic scales.
• Spread the sample on the table and separate out all pure seeds manually with tweezers or remove impurities by blowing, sifting or letting seeds roll down a slanting surface.
• Weigh the ‘pure’ seed fraction and express purity as the percentage weight of pure seed over the total weight of the working sample, as shown below.

Purity (%)   =     Weight of pure seeds (g) x 100
                            Total weight of working sample (g)

Documentation of information about seed cleaning

It is important to file all the relevant information about the cleaning process for future reference. The information may be relevant to explain germination test results or contamination in the future.

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Seed drying

Seed drying is the reduction of seed moisture content to recommended levels for storage using techniques that are not detrimental to seed viability. It should be done as soon as possible after clean seeds arrive in a genebank, to avoid deterioration and to reduce the moisture content in the seed. High moisture promotes respiration and growth of seed embryos, insects and fungi.

Seed samples are usually kept in paper, mesh or cotton bags in well aerated and cool environments (with low relative humidity) for a few weeks to dry.

Why it should be done

• To dry the seeds to a safe moisture content level to prevent damage, heating and infestation during storage.

Steps of seed drying

• Determine the seed moisture content (this can be omitted if seed amounts are limiting and/or genebank staff have experience on expected seed moisture content at harvest).  
• Dry seed in porous containers.
• Determine seed moisture content.
• Document the information about seed drying.

For more detailed information on seed drying see pages 36-49 of the Handbooks for Genebanks No. 8. (Available here).

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Seed moisture content determination

Seed moisture content is the amount of water in a seed and is expressed in terms of the weight of water contained in a seed. The moisture content is one of the most important factors that determines the rate at which seeds will deteriorate in genebanks. It is usually carried out with oven-drying methods or with moisture meters.

Why it should be done

• Knowing the seed moisture and storage temperature allows genebanks to accurately predict the potential storage life of each seed sample.

Steps of moisture content determination

Pre-drying

Pre-drying is obligatory if seeds are wet and their moisture content is suspected to be above 17% (10% for soybean and 13% for rice); it should be conducted prior to moisture content determination by oven-drying.

If pre-drying is required, proceed as follows:

• Weigh two sub-samples of 4-5 g of seeds in their containers.
• Pre-dry the samples overnight in a warm, dry place such as a laboratory bench.
• Weigh them again in their containers and determine the loss of weight (loss of moisture) by subtraction.
• Calculate the moisture content on a fresh-weight basis.

Grinding

Some seeds require grinding into smaller particles to have uniform and complete drying. The table below provides a list of species for which grinding is obligatory (ISTA, 2005).

Moisture content methods

Recommended methods vary with each species. The table below provides the suggested methods of moisture determination for some important crops and forages (ISTA 2005).

Calculation of moisture content percentage

Calculate the moisture content on a wet-weight basis using the following formula:

Moisture content (%) = W2 - W3 x 100
                                             W2-W1

where,
          W1 = weight of container with lid;
          W2 = weight of container with lid and sample before drying; and
          W3 = weight of container with lid and sample after drying.

Documentation of information about seed moisture content

This is important to file all the relevant information about the seed moisture content for future reference.

For more detailed information on seed moisture content determination see pages 28-35 of the Handbooks for Genebanks No. 8. (Available here).

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Seed packing

Seeds are packaged to:

• prevent absorption of water from the atmosphere after drying.
• keep accessions separate and avoid mixing.
• prevent contamination from insects during storage.

For many crop species, keeping seeds at low moisture content improves longevity during storage. Maintain low moisture during packing by:

• Packing seeds in an air-conditioned room where the relative humidty is controlled in tropical humid environments.
• Exposing seeds taken from the drying room to ambient air for the shortest time possible, so that they do not re-absorb water.

Different types of containers are available for packaging; the choice depends on storage conditions and species (size, shape and sharpness). It is important that the packing and labelling material be completely impermeable to water, withstand freezing and is suitable for long-term use. Frequently used containers include glass bottles, aluminium cans, laminated aluminium foil packets and plastic bottles.

Different types of containers have advantages and disadvantages.

• Glass bottles with tight gaskets around sealed lids are moisture proof but can easily break.
• Aluminium cans are difficult to reseal once they have been opened.
• Aluminium foil packets can be resealed and occupy less space than other containers, but seeds with sharp projections can pierce them and moisture can leak in.
• Plastic bottles and aluminium cans with lids are moisture resistant but not moisture proof unless they have a tight rubber seal. They should be used with caution if the relative humidty of the storage room is not controlled.

Correct and clearly written labels are extremely important in germplasm collections to keep track of packets during storage:

• Label packets with a unique identification number, location and any other important information.
• Write clearly and carefully or print computer labels using indelible ink.
• Use labels that are as indestructible as possible with good sticking properties.
• Place labels inside and outside of each packet.

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References and further reading

ISTA. 2005. International Rules for Seed Testing. Edition 2005. International Seed Testing Association, Bassersdorf, Switzerland. ISTA homepage available from: www.seedtest.org/en/home.html.

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).

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