Seed testing is an important aspect of an efficient seed programme. For proper management of a seed production programme it is necessary to provide appropriate facilities for testing seed scientifically. This process infact confirms the planting value of the seed. Quality control of seed depends on the different seed testing protocols which determine the geniuses of the cultivar in respect of its physical, physiological, genetical and biochemical properties.
The ‘International Seed Testing Association’ commonly known as ISTA, has developed uniform rules and regulation for testing since its establishment in 1924. The effort of the ISTA has become successful and its recommendations are being followed throughout the world uniformly.
Vegetable seed testing is very essential operation required to assess the seed quality attributes of the vegetable seed lots which have to be offered for sale or storage. The quality attributes which need to be evaluated are seed germination, seed moisture content, vigour, physical and genetic purity, freedom from seed borne diseases and insect infestation.
ISTA Background:
International Seed Testing Association (ISTA) was founded in 1924 and has ever since tried to develop the standardized seed testing methods to promote uniformity to seed testing methods in order to maintain a puring assessment of seed quality parameters throughout the world. It is obligatory on the part of the seed analysts to follow the seed testing rules prescribed from time to time by the ISTA.
The objectives of the association are:
i. To develop and publish standard procedures for sampling and testing of seeds and to promote uniform application of these procedures for evaluation of seeds moving in international trade.
ii. To actively promote research in all areas of seed science and technology, to encourage variety certification, to participate in conference and training courses.
Seed Testing Analysis:
Seed testing is used for control of quality parameters during seed handling, and test results are submitted to customers as documentation on seed quality. Seed test is carried out in the testing laboratories of each state. Random samples are carefully drawn from each seed lot and mixed together. From this mixture random samples are drawn; these samples are called working samples and are used for seed test. The guidelines on timing of seed testing are summarized in table 12.2.
The following are the observations to be taken during seed testing:
1. Seed Moisture (%):
Seed moisture content is one of the most important factors influencing the seed quality in storability. Therefore, it’s estimation during seed quality determination is important. Seed moisture content can be determined either by hot air oven or moisture meter.
(A) Hot Air Oven Method:
In this method, seed moisture is removed by drying at a specified temperature for a specified duration. It is the most common and standard method for seed moisture determination.
Steps involved in moisture determination:
i. Empty container alongwith its cover should be weighted.
ii. Two small portions of submitted samples should be weighted directly into the container.
iii. After weighing remove the lid of the container and the open container should be kept in the container which has already been heated to the prescribed high temperature.
iv. At the end of the drying period, container should be closed with it’s lid.
v. The container should be transferred into desiccators. The desiccator should be closed and the sample should be allowed to cool.
vi. The sample should be weighted again and per cent moisture content can be calculated to one decimal point by the following formula:
Where,
M = Seed moisture content in percent
M1 = Weight of the empty container with it’s cover
M2 = Weight of the container with its cover and seed before drying
M3 = Weight of the container with its cover and seed after drying
(B) Moisture Meter:
The use of moisture meter is a very convenient and rapid method for determining the seed moisture content. The moisture meter is calibrated for each species which should be carried out against the hot air oven method.
2. Purity Test:
Purity denotes the percentage of seeds (by weight) belonging to the variety under certification. The working sample is closely examined, usually with the help of a magnifying glass, and the following components are separated from it: seeds of other varieties, seeds of other crops, seeds of weeds/objectionable weeds, inert matter, defective seeds.
The purity of seed is estimated as follows:
Physical Purity:
Physical purity is the proportion of pure seed component in the seed lots as well as the proportion of the other seed, weed seed and inert matter by weight in per cent for which seed standards have been prescribed.
The primary objective of physical purity analysis is to determine:
i. The per cent composition by weight of the sample is being tested and by inference to the composition of the lot.
ii. Identification of various species of seeds and inert particles constituting the samples.
Pure Seed:
The pure seed shall refers to the seeds started by the sender, or found to be predominant in the test, and shall include all botanical varieties and cultivars of that specie (even if immature, under sized, shriveled, diseased or germinated provided that they can be definitely identified as of that specie) unless transformed into visible sclerotic, smut bolls or nematode galls. Pure seed shall include intact seed units as defined for each genus or species of seed units larger than one half their original size.
Other Crop Seed:
Other crop seed shall include seed units of any plant species other than of pure seed grown as crop. Multiple structures, capsules, pods are opened and the seeds are taken out and the non-seed material is placed in the matter.
Inert Matter:
Inert matter shall include seed units and all other matters and structure not defined as pure seeds excluding other crop seeds and weed seeds.
3. Germination Percentage:
Germination is determined as per cent of seeds that produce seedlings under a suitable environment. Thus germination is of great importance because the sole function of seed is to produce healthy seedlings for raising a good crop.
Germination test determines the percentage of seeds that produce healthy root and shoot. In most of the cases, seeds are germinated on wet filter papers placed in petri dishes. The petri dishes are kept under controlled conditions in an incubator. For most species a temperature between 18-25 °C is adequate; however, for some species a specific temperature may be required.
The duration of germination test varies from 7-28 days depending upon the crops. Germinated seeds are counted at regular intervals and are removed from the petri dishes. The total number of germinated seeds would be the sum of the number of seeds that germinated at different observations.
The per cent germination is calculated as follows:
For convenience, 100 seeds are plated in each sample. From each seed lot, 4 or more samples are plated for a reliable germination estimate. If there is a difference of 10 percent or more in the germination of different samples from the same seed lot, it is desirable to repeat the germination test.
Basic Procedure:
To perform a simple germination test, count out 100 seeds and place them somewhat equidistant from each other, usually not closer than half inch, spreading them out on the upper half of the moist blotter or paper towel. The bottom half of the blotter is folded over the top, and then the towel is folded over with a fold about every two to three inches and held in place loosely by a rubber band.
The towels or blotters are then placed vertically in plastic trays refrigerator containers, which are placed in the germination chamber or suitable location. The trays or containers should be covered loosely with zip-lock or plastic bags to keep the moisture in the towels, but they should not be completely closed because seeds need air. Towels are inspected twice a day and misted as necessary. Most vegetable seeds germinate in about 7 to 14 days.
During the first count remove the seeds that have germinated and when the final count is done, the remaining seeds are scored and the paper towel discarded. Seeds that require light are placed on blotter paper within plastic honey boxes. The light from the germination chamber will be sufficient to satisfy the light requirement.
Evaluation of Germination:
1. Normal Seedlings:
Seedlings, which show the potential for continue development into satisfactory plants when grown in good quality soil and under favourable conditions of moisture, temperature and light. These normal seedlings include intact seedlings, seedlings with slight defects and seedlings without secondary infection.
2. Abnormal Seedlings:
Seedlings which do not show the potential to develop into a normal plant when grown in good quality soil and under favourable conditions of moisture, temperature and light. The abnormal seedlings include damaged seedlings, deformed and decayed seedlings.
3. Hard Seeds:
Seeds that cannot absorb water due to an impermeable seed coat.
4. Fresh Ungerminated Seeds:
Seeds other than hard seeds which remain apparently viable at the end of test period.
5. Dead Seeds:
Seeds at the end of the test period are neither hard nor fresh and have not produced any part of the seedling.
6. Shoot Length:
For the normal germination test 10 normal seedlings will be selected randomly from each replication on the day of final count. A shoot length will be measured from the base of the primary root to the base of hypocotyls and the mean shoot length will be expressed in cm.
7. Root Length:
From the normal germination test 10 normal seedlings will be selected randomly from each replication on the day of final count. A root length will be measured and expressed in cm.
8. Seedling Vigour Index:
Seedling vigour index will be calculated adopting the following formula:
Seedling Vigour Index (SVI) = (Shoot length + root length) x Germination %
9. Seedling Dry Weight:
Ten seedlings which show earlier root and shoot length will be kept in butter paper and kept in oven maintained at 70°C for 24 hrs. The weight of dried seedlings will be recorded and mean value will be expressed in mg.
Seed Vigour Test:
The vigour tests are generally classified into four categories viz.:
(1) Physical test
(2) Biochemical tests
(3) Stress tests
(4) Seedling growth and evaluation test.
1. Physical Tests:
i. Seed Size (Weight):
Variation in seed size can also affect vigour test evaluations. Large seeds, usually produce heavier seedling making them appear more vigorous in tests where seedling weight is used as a measure of vigour.
ii. Radiographic Analysis of Seed:
The X-ray photographic method for analyzing seed quality developed by Simak and Gustafsson (1953) and Simak (1957) is based on the principle that the different parts of a seed, such as testa endosperm and embryo, absorb soft X-rays to different extent and can consequently be differentiated on X-ray photograph. A club to the vigour of a particular seed lot could be obtained from the impregnation pattern.
Since the extent of impregnation of contrast agent is directly proportional to the vigour of the seed tissues, it is possible to standardize seed vigour attributes. A large number of seeds could be analysed individually for impregnation and the analysis could be correlated with the extent of seedling vigour.
2. Biochemical Test:
Radicle protrusion and seedling growth during seed germination are the end result of a series of biochemical changes. The levels of metabolic activity determine seedling growth rate. The biochemical tests which measure some metabolic events in seeds associated with germination can be applied to measure seed vigour. These tests require less testing time than the other methods. However, biochemical tests necessitate more specialized equipment and training of the personnel than the other tests.
i. Tetrazolium (TZ) Test:
The tetrazolium is based on the measurement of dehydrogenase enzyme activity. The enzyme reduces the colourless tetrazolium chloride salt to form a water insoluble red compound-formazon which stains the living cells red, while the dead cells remains colourless. Lakon (1942) developed this technique for seed testing. This method determines the percentage of viable seeds which may be expected to germinate.
The chemical 1,3,5-tetrazolium chloride or tetrazolium chloride in short, is colourless but it develops intense red colour when it is reduced by living cells. This phenomenon is used to determine the percentage of viable seeds in a seed sample. Seeds are soaked in tap water overnight and are split longitudinally with the help of a scalpel so that a protein of embryo is attached with each half of the seed.
One half of each seed is placed in a petri dish and covered with 1% aqueous solution of tetrazolium chloride for 4 hours. The seeds are then washed in tap water and the number of seeds in which the embryo is stained red is determined.
The per cent of viable seed is computed as follows:
ii. Conductivity Test:
Poor membrane structure and weak cells are usually associated with low vigour seeds. These would results in a greater loss of electrolytes such as amino acid and organic acids from imbibing seeds and increase the conductivity of seed leachates. A higher leachate conductivity, may indicate a low seed vigour.
One important limitation of this test is that it expresses the result as an average for 25 seeds which presumes that all seeds are equally deteriorated. A seed lot is however, composed of population of individuals, each with its own unique potential to perform in the field.
Conductivity test results, therefore, would better reflect vigour capability of seed lots, if they are presented on individual basis. A commercial instrument called as “Automatic seed analyzer” is now available which monitors the electrolyte leakage of individual seeds. Increase leaching of inorganic salts and organic compounds from mechanically damaged and naturally aged seeds has been suggested as an index of seed viability or field performance.
iii. Respiration:
Seed germination and seedling growth require use of metabolic energy acquired from respiration. A decrease in the rate of respiration of germinating seeds has been showed to precede a decrease in the rate of seedling growth. Respiration tests are rapid and quantitative but require a spirometer and trained personnel. The mechanical injury which lowers seed vigour, may increase rather than decrease respiration rate.
3. Stress Tests:
Usually field conditions are less than optimum when the seeds are sown. Under sub-optimal conditions high vigour seeds will have greater potential for emergence and establishment. Some vigour tests such as accelerated ageing, cold test, cool germination test are applied under stress conditions.
i. Accelerated Test:
In this test, the seeds are stressed prior to the germination test. Seeds are placed in temperature of 40-45°C and nearly 100 per cent RH for varying lengths of time depending upon kind of seeds after which the germination test is made.
The basis for this test is that higher vigour seeds tolerate the high temperature high humidity treatment and thus retain their capability to produce normal seedling in germination test. This test was developed by Delouche (1965) for predicting the relative storability of seeds which he considered to be manifestation of vigour.
ii. Cold Test:
Seed are placed in a plastic box or in paper towel lined with soil and incubated at 10 °C for specified period. At the end of cold period, the tests are transferred to favourable temperature for germination. The emergence per cent is considered as an indication of seed vigour. The cold test has been traditionally used as vigour testing of corn. Testing for other kind of seeds are also been reported.
iii. Osmotic Stress:
Seed when sown would face drought stress which results in poor field emergence. Such a drought condition can be stimulated in a laboratory test. The basis of a stress test is that a vigours seed can tolerate a more severe osmotic stress and consequently the osmotic stress method has been suggested as a vigour test.
4. Seedling Growth Evaluation Test:
These tests are conducted under the same condition as the standard germination test. They are inexpensive, relatively rapid and require no specialized component and most importantly do not necessitate additional technical training.
i. Seedling Vigour Classification:
This vigour test is similar to standard germination test. The only difference between these two tests is that normal seedlings are further classified as ‘strong’ and ‘weak’.
ii. Seedling Growth Rate (SGR):
Rapid and uniform emergence is an important component of seed vigour definition. The measurement of plumule growth as vigour test was first suggested by Germ (1949). The measurement of seedling dry weight has been suggested by Woodstock (1976).
iii. Speed of Germination:
Seed lots with similar total germination often vary in their rate of germination and growth. A number of methods such as number of days a lot requires to reach 90 per cent germination; to reach 50 percent germination value (for lot with low germination percentage) and without selecting a percentage value.