Seed Testing
	Objective & Importance
	Role of Seed Testing Laboratories (STL) Sampling Physical Purity Germination Moisture test

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Seed Testing

Seed testing is determining the standards of a seed lot viz., physical purity, moisture, germination and ODV and thereby enabling the farming community to get quality seeds.

The Seed Testing Laboratory is the hub of seed quality control. Seed testing services are required from time to time to gain information regarding planting value of seed lots. Seed testing is possible for all those who produce, sell and use seeds.

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Objective & Importance of Seed Testing

Seed testing is required to achieve the following objectives for minimising the risks of planting low quality seeds.

1. To identify the quality problem and their probable cause
   
2. To determine their quality, that is, their suitability for planting
   
3. To determine the need for drying and processing and specific procedures that should be used
   
4. To determine if seed meets established quality standards or labelling specifications.
   
5. To establish quality and provide a basis for price and consumer discrimination among lots in the market. The primary aim of the seed testing is to obtain accurate and reproducible results regarding the quality status of the seed samples submitted to the Seed Testing Laboratories.

Importance

• The importance of seed testing was realized more than 100 years ago for assured planting values. The adulteration of vegetable seeds by stone dust which was packed in some parts of the world particularly in Europe.              
•  Seed testing has been developed to aid agriculture to avoid some of the hazards of crop production by furnishing the needed information about different quality attributes viz., purity, moisture, germination, vigour and health.
•  Quality control of seed depends on the different seed testing protocols which determine the genuineness of the cultivar.
• Testing of seed to evaluate the planting value and the authenticity of the certified lot.
• Seed testing is required to assess the seed quality attributes of the seed lots which have to be offered for sale.
• These quality attributes are seed moisture content, germination and vigour, physical and genetic purity, freedom from seed borne diseases and insect infestation. In India, seed testing is done mainly for moisture, germination and physical purity of seeds.
• Standard seed testing procedures for the evaluation of the seeds were developed by ISTA. It is obligatory on the part of the seed analyst to follow rules prescribed by 1STA (1STA, 1985) if the seed is moving to the International trade. 
• The seed testing procedures which are described below are based mostly on the international rules because most of our rules (Chalam et al.. 1967) are based on, 1STA, 1996. Economic yield of a crop depends on the quality of seeds which can be evaluated by seed testing (1STA, 1996).
• The testing of seed quality is carried out on seed samples drawn from seed lot to be used for cultivation. The quantity of seed sample taken for testing in laboratory is minute compared to that of seed lot it represents.

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Role of Seed Testing Laboratories

Seed testing laboratories are essential organization in seed certification and seed quality control programmes. The main objective is to serve the producer, the consumer and the seed industry by providing information on seed quality. Test results may cause rejection of poor seed multiplication or low grade seed in a count of law.             

Analysis of seed in the laboratory: Seed testing is possible for all those who produce, sell and use seeds.Seed testing is highly specialized and technical job. With a view to maintain uniformity in quality control the seed analysis laboratory includes for distinct sections.

1. Section for purity testing: Purity analysis of seed lot is considered under two factors
   a) Testing the cleanliness of seed lot and
   b) Testing the geneuiness of the cultivar
2. Section for moisture testing
3. Section for viability, germination and section for vigour testing.

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Sampling in Seed Testing Laboratory

The seed samples received in the laboratory (submitted sample) are required to be reduced to obtain working samples for carrying out various tests. A number of methods are available for obtaining working samples.	Model seed samples received from producer
Mixing and dividing of seeds The main objective of mixing and dividing of seeds is to obtain the representative homogenous seed sample for analysis by reducing the submitted sample to the desired size of working sample. Method of mixing and dividing Mechanical dividing            Modified halving method Hand halving method Random cup method Spoon method

 Mechanical method

The reduction of sample size is carried out by the mechanical dividers suitable for all seeds except for chaffy and fuzzy seeds.

Objective of mechanical dividing

• To mix the seed sample and make homogenous as far as possible. To reduce the seed sample to the required size without any bias.
• The submitted sample can be thoroughly mixed by passing it through the divider to get 2 parts and passing the whole sample second time and 3rd time if necessary to make the seeds mixed and blended so as to get homogenous seed sample when the same seeds are passed through it into approximately equal parts.
• The sample is reduced to desired size by passing the seeds through the dividers repeat­edly with one half remain at each occasion.

 Types of mechanical dividers

Boerner divider It consists of a hopper, a cone and series of baffles directing the seeds into 2 spouts. The baffles are of equal size and equally spaced and every alternate one leading to one spout. They are arranged in circle and are directed inward. A valve at the base of the hopper retains the seeds in the hopper. When the valve is opened, the seeds fall by gravity over the cone where it is equally distributed and approximately equal quantity of seeds will be collected in each spout. A disadvantage of this divider is that it is difficult to check for cleanliness.

Soil divider It is a sample divider built on the same principles as the Boerner divider. Here the channels are arranged in a straight row. It consists of a hopper with attached channels, a frame work to hold the hopper, two receiving pans and a pouring pan. It is suitable for large seeds and chaffy seeds.

Random cup method

This is the method suitable for seeds requiring working sample upto 10 grams provided that they are not extremely chaffy and do not bounce or roll (e.g.) Brassica spp. Six to eight small cups are placed at random on a tray. After a preliminary mixing the seed is poured uniformly over the tray. The seeds that fall into the cup is taken as the working sample.

Modified halving method

The apparatus consists of a tray into which is fitted a grid of equal sized cubical cups open at the top and every alternate one having no bottom. After preliminary mixing the seed is poured evenly over the grid. When the grid is lifted, approximately half the sample remains on the tray. The submitted sample is successively halved in this method until a working sample size is obtained.

Spoon method

This is suitable for samples of single small seeded species. A tray, spatula and a spoon with a straight edge are required. After preliminary mixing, the seed is poured evenly over the tray. The tray should not be shaked thereafter. With the spoon in one hand, the spatula in the other and using both small portions of seed from not less than 5 random places on the tray should be removed. Sufficient portions of seed are taken to estimate a working sample approximately but not less than the required size.

Hand halving method

This method is restricted to the chaffy seeds. The seed is poured evenly on to a smooth clean surface and thoroughly mixed into a mound. The mound is then divided into 1/2 and each half is mound again and halved into 4 portions. Each of the 4 portions is halved again giving 8 portions. The halved portions are arranged in rows and alternate portions are combined and retained. The process is repeated until the sample of required weight is obtained.

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Physical Purity

Purity analysis

The purity analysis of a seed sample in the seed testing laboratory refers to the determination of the different components of the purity viz., pure seeds, other crop seeds, weed seeds and inert matter.

Objective

The objective of the purity analysis is to determine whether the submitted sample conforms to the prescribed physical quality standards with regard to physical components.

Method

The working sample

The purity analysis is done on the working sample of prescribed weight drawn from submitted sample. The analysis may be made on one working sample of the prescribed weight or on two sub-samples of atleast half of this weight, each independently drawn.

Weighing the working sample

The number of decimal places to which the working sample and the componenets of the working sample should be weighed is given below.

Purity separation

The working sample after weighing is separated into its components viz., pure seed, other seed crop, weed seed and inert matter.

Pure seed

The seeds of kind / species stated by the sender. It includes all botanical varieties of that kind / species. Immature, undersized, shrivelled, diseased or germinated seeds are also pure seeds. It also includes broken seeds, if the size is >1/2 of the original size except in leguminacea, and cruciferae where the seed coat entirely removed are regarded as inert matter.

Other crop seed

It refers to the seeds of crops other than the kind being examined.

Weed Seed

It includes seeds of those species normally recognized as weeds or specified under Seed Act as a noxious weed.

Inert matter

It includes seed like structures, stem pieces, leaves, sand particles, stone particles, empty glumes, lemmas, paleas, chaff, awns, stalks longer than florets and spikelets.

Method of purity separation

Place the sample on the purity work board after sieving / blowing operations and separate into other crop seeds and inert matter. After separation, identify each kind of weed seeds, other crop seeds as to genus and species. The names and number of each are recorded. The type of inert matter present should also be noted.

Seed Blower

Purity Work Board

Calculation

All the four components must be weighed to the required number of decimal places. The percentages of the components are determined as follows.
                                                            Weight of individual component
% of components                   =     __________________________ X 100
                                                          Total weight of all components                                                       
If there is a gain or loss between the weight of the original samples and the sum of all the components is in excess of one percent, another analysis should be made.

Duplicate tests

If the analysis result is near the border line in relation to the seed standards, one more test is done and the average is reported. However, if a duplicate analysis is made of two half sample or whole samples, the difference between the two must not exceed the permissible tolerance. If the difference is in excess of the tolerance, analyze further (but not more than 4 pairs in all) until a pair is obtained which has its member within tolerance.

Purity analysis in groundnut

It should be carried out on pods and the size of working sample is 1000.

Determination of huskless seeds

It is required in certain crops like sunflower and paddy. 400 seeds taken from the pure seed and the number of seeds without husk are counted (partly huskless seeds are excluded) and the % is calculated as

                                                            Number of huskless seeds
% of huskless seeds      =    ________________________       X100

                                                                                 400

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Seed germination test

Germination is defined as the emergence and development from the seed embryo, of those essential structures, for the kind of seed in question, indicates its ability to produce a normal plant under favourable conditions.

Principles

Germination tests shall be conducted with a pure seed fraction. A minimum of 400 seeds are required in four replicates of 100 seeds each or 8 replicates of 50 seeds each or 16 replicates of 25 seeds each depending on the size of seed and size of containers of substrate.

The test is conducted under favourable conditions of moisture, temperature, suitable substratum and light if necessary. No pretreatment to the seed is given except for those recommended by ISTA.

Materials required
Substratum

The substratum serves as moisture reservoir and provides a surface or medium for which the seeds can germinate and the seedlings grow. The commonly used substrate are sand, germination paper and soil.

1. Sand

Size of sand particle

Sand particles should not be too large or too small. The sand particles should pass through 0.80 mm sieve and retained by 0.05mm sieve.

Toxicity

Sand should not have any toxic material or any pathogen. If there is presence of any pathogen found then the sand should be sterilized in an autoclave.

Germination tray

When we use the sand, germination trays are used to carry out the test. The normal size of the tray is 22.5 x 22.5 x 4 cm. The tray may either zinc or stainless steel.

Germination tray

Method of seed placement

Seed in sand(S)

Seeds are planted in a uniform layer of moist sand and then covered to a depth of 1 to 2 cm with sand.

Sand method

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Top of sand (TS)

Seeds are pressed in to the surface of the sand.

Spacing

We must give equal spacing on all sides to facilitate normal growth of seedling and to avoid entangling of seed and spread of disease. Spacing should be 1-5 times the width or diameter of the seed.

Water

The amount of water to be added to the sand will depend on size of the seed. For cereals, except maize, the sand can be moistened to 50% of its water holding capacity. For large seeded legumes and maize sand is moistened to 60% water holding capacity.    

Most widely used paper substrates are filter paper, blotter or towel (kraft paper). It should have capillary movement of water, at vertical direction (30 mm rise / min.). It should be free from toxic substances and free from fungi or bacteria. It should hold sufficient moisture during the period of test. The texture should be such that the roots of germinating seedlings will grow on and not into the paper.

Methods

Top of paper (TP)

Seeds are placed on one or more layers of moist filter paper or blotter paper in petriplates. These petriplates are covered with lid and placed inside the germination cabinet. This is suitable for those seeds which require light.

Petriplate method

Between paper (BP)

The seeds are germinated between two layers of paper. The seeds are placed between two layers of paper and rolled in towels. The rolled towels are placed in the germinator in an upright position.

Germination paper	Seeds germinated on paper	Roll towel method

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Germination apparatus

Germination cabinet / Germination room

This is called chamber where in temperature and relative humidity are controlled. We can maintain the temperature, relative humidity and light required for different crops.

Room germinator

It works with same principle as that of germinator. This is a modified chamber of larger one and the worker can enter into it and evaluate the seedlings. Provisions are made to maintain the temperature and relative humidity. This is used widely in practice.

Seed germinator	Plant Growth Chamber
Seed counting board This is used for accurate counting and spacing of seeds. This consists of 2 plates. The basal one is stationary and top one is movable. Both top and basal plates are having uniform number of holes viz., 50/100, when the plates are in different position. After taking the sample, the top plate is pulled in such a way that the holes are in one line so that the fixed number of seeds falls on the substratum.	Seed Counting Board
Vacuum seed counter Consists of a head, pipe and wall. There are plates of 50 or 100 holes which can be fitted to the head. When vacuum is created the plate absorbs seeds and once the vacuum is released the seeds fall on the substrate.	Vacuum seed counter
Impression board                 Made of plastic / wood with 50 or 100 holes / pins. Here the knobs are arranged in equal length and space. By giving impression on the sand it makes uniform depth and spacing for seed.	Impression board

Evaluation of germination test

The germination test is evaluated as

• Normal seedlings
• Abnormal seedlings
• Hard seeds
• Fresh and ungerminated seeds
• Dead seeds

 ISTA classified the seedlings into different categories based on the development of essential structures.

Normal seedlings

Seedlings which has the capacity for continued development into normal plant when grown in favourable conditions of soil, water, temperature and light.

Characters of normal seedlings A well developed root system with primary root except in certain species of graminae which normally produce seminal root or secondary root. A well developed shoot axis consisting of elongated hypocotyls in seedlings of epigeal germination. A well developed epicotyl in seedlings of hypogeal germination. One cotyledon in monocotyledon and two in dicotyledons. A well developed coleoptiles in graminae containing a green leaf. A well developed plumule in dicotyledons.      Normal seedlings Seedlings with following slight defects are also taken as normal seedlings. Primary root with limited damage but well developed secondary roots in leguminaceae (Phaseolus, Pisum), graminae (Maize), cucurbitaceae (Cucumis) and malvaceae (cotton) Seedlings with limited damage or decay to essential structures but no damage to conducting tissue. Seedlings which are decayed by a pathogen with a clear evidence that the parent seed is not the source of infection.

Abnormal seedlings

Seedlings which do not show the capacity for continued development into normal plant when grown in favourable condition of soil, water, temperature and light.

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Types of abnormal seedlings

Retesting           

If the results of a test are considered unsatisfactory it will not be reported and a second test will be made by the same method or by alternative method under the following circumstances.

1. Replicates performance is out of tolerance
2. Results being inaccurate due to wrong evaluating of seedlings or counting or errors in test conditions
3. Dormancy persistence or phytotoxicity or spread of fungi or bacteria. The average of the two test shall be reported.

Use of tolerances

The result of a germination test can be relied upon only if the difference between the highest and the lowest replicates is within accepted tolerances.

To decide if two test results of the same sample are compatible again the tolerance table is used.

Reporting results

The result of the germination test is calculated as the average of 4x100 seed replicates. It is expressed as percentage by number of normal seedlings. The percentage is calculated to the nearest whole number. The percentage of abnormal seedlings, hard, fresh and dead seeds is calculated in the same way. These should be entered on the analysis of certificate under appropriate space. If the result is 'nil' for any of these categories it shall be reported as ‘0’.

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 Determination of moisture content

Objective

To determine the moisture content of seeds by methods suitable for routine use.

Definition

The moisture content of a seed sample is the loss in weight when it is dried. It is expressed as a percentage of the weight of the original sample. It is one of the most important factors in the maintenance of seed quality.

Method of moisture determination

1. Air oven method

In this method, seed moisture is removed by drying the seed sample at a specified temperature for a specified duration.

2. Moisture meters

Moisture meters estimate seed moisture quickly but the estimation is not as precise as by the air oven method.

Weight of the submitted sample

100 g for species that have to be ground. 50 g for all other species. The sample should be submitted in polythene bag of 700 gauge.

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Air oven method for seed moisture estimation

Materials required

Grinding mill It should be constructed of non-absorbent material. It should grind evenly and should be operated at such a speed that during grinding, it should not cause heating of the ground material. Air currents that might cause loss of moisture must be reduced to a minimum. The fineness of grinding should be adjustable. Container Container of glass or non-corrosive metal (e.g.) stainless steel should be used.		Grinding mill
Oven A good quality electric air oven with a thermostatic electronic temperature control for maintaining temperature within ±1°C is required.
Desiccator, Analytical balance, Sieves. A set of wire mesh sieves with meshes of 0.5 mm, 1.0 mm and 4.0 mm.
Desiccators	Balance	Sieves

Grinding

For some seeds (e.g. Cereals and Cotton) fine grinding is essential before the moisture content is determined. In such cases, at least 50% of the ground material should pass through a wire sieve with meshes of 0.5 mm and not more than 10% remain on a wire sieve with a mesh of 1.0 mm. For leguminous seeds, coarse grinding is recommended; at least 50% of the ground material shall pass through a wire sieve with meshes of 4.0 mm.

Pre drying

If the species is one for which grinding is necessary and the moisture content is more than 17%. (or 10% in the case of soy bean and 13% in rice) pre drying before grinding is necessary. For this purpose, two 50 g portions are weighed and placed on open trays at 130°C for 5-10 min. If seed moisture content is about 25% or more it should be pre-dried at 70° C for 2-5 hours, depending on the initial water content. The pre dried seeds should be kept in a closed desiccator for cooling. Then each of the duplicate quantities is weighed separately and about 20 g is ground. The ground material is then subjected to moisture testing using a hot air-oven as described below.

Moisture estimation

It should be carried out in duplicate on two independently drawn 5-10 g working samples, weighed with an accuracy of 1 mg. Most species are dried for 1 hr at 130° C, cereals for 2 hours (130°C) and maize for 4 hours (130°C). Seeds containing high percentage of oil should be dried at 103°C for 17 hours.

 F.G. : Fine grinding         ; C.G. : Coarse grinding

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Steps

m₂-m₃

m = _________  x 100

m₂-m₁

Where, m = Seed moisture content
m₁ = Weight of the empty container with its cover
m₂= Weight of the container with its cover and seeds before drying
m₃ = Weight of the container with its cover and seeds after drying
The duplicate result of the determination may not differ by more than 0.2% other­wise the analysis should be repeated.
If pre dried, the moisture content is calculated from the results obtained in the first (pre-drying) and second stages of the procedure. IfSI is the moisture lost in the first stage and S2 is the moisture lost in the second stage, each calculated as above and expressed as a percentage, the original moisture content of the sample is calculated as below.

                                                      S₁ x S₂
           m= S₁ + S₂   -              _________
                                                       100
m= moisture content
S₁ =Moisture percentage lost in predrying stage
S₂= Moisture percentage lost in drying stage

Moisture meters: Universal (OSAW) digital moisture meters
The principle involved in these moisture meters is that wet grains are good conductors while dry grains are less conductors of electricity. So, the moisture content is directly proportional to the electrical conductivity of the seed.

It consists of a compression unit to compress the sample to pre -determined thickness. The thickness setting is very easily read on a vertical and circular scale. The seed material on test is taken in a test cup and is compressed. Then press the push type switch till the reading comes in the display. Here no temperature reading and correlated dial are required. The computer version of digital moisture meter automatically compensate for temperature corrections.

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