The science of evaluating the planting value of the seeds. By seed testing we can asses the quality attributes of the seed lots which have to be offered for sale and minimizing the risk of planting low quality seeds.

• To determine the seed quality i.e., their sustainability for planting.

• To identify seed quality problems and their probable causes.

• To determine if seed meets established quality standards or labeling specifications.

• To establish quality and provide a basis for price and consumer discrimination among lots in the market.

• To determine the need for drying and processing and specific procedures that should be used.

Historic Events

1869 1st Seed Testing Lab was established in Thrandt, Saxony, Germany by “Friedrich Nobbe”

1871 E. Moller Holst, Copenhagen, Denmark1876 1st Seed Testing station in US

1900 Europe 130 Seed Testing Stations

1924 International Seed Testing Association (ISTA)-to devlop procedures and to promote uniform application.

1939; Association of official Seed Analysts (AOSA)

1961 First Seed Testing Laboratory was establishedin India.

1967 First seed testing manual was published foruniform testing.

1.Seed sampling

2.Physical purity

3.Germination.

4.Seed moisture.

5.Seed viability.

6.Seed health

7. Seed Vigour Testing

Seed sampling: It is the process of obtaining a sample of required size for test in which the same

constituents are present as in the seed lot and in same proportion.

Types of samples received at STL

Service sample

Official sample

Charged sample

Seed sampling

Primary Sample: a fraction of seeds taken randomly from seed lot different places.

Composit Sample: pooled sample of all primary samples.

Submitted Sample: sample which is submitted by producer to seed testing laboratory. It is large in size.

Working Sample: subdivision of the submitted sample.

Equipments for sampling: SeedTriers-Stick or sleeve type trier, Bin sampler, Nobbe trier

Seed Dividers: Boerner seed divider, Gamet seed divider, Soil type seed devider

Random cup Method: Suitable for crops requiring sample up to 10g. Place 6-8 cups in the tray and pour seeds uniformly over tray and select the cup randomly.

Spoon Method: Permissible for sampling of small seeded seeds. Pour the seeds evenly over the tray. Remove small portion of seeds using spoon and spatula.

Hand Halving method: Restricted to specific genera of chaffy seeds. Pour the seeds on floor and make a mound. Mound is then divided into half and each half is halved again.

The primary objective of purity analysis

is to determine whether the submitted seed sample confirms to the prescribed quality

standards in regard to purity components, objectionable weed seeds, other inseparable

crop seeds and seeds of other distinguishable varieties

Purity table or working board, Seed divider, Forceps, Spatula, Brush

Aluminum purity dish, Magnifier (5 to 7x), Analytical balance, Hand screen (with ISI specifications), Top loading balance (as mentioned above), Stereoscopic binocular microscope, Seed blower, Table lamp, Watch glass

Pure seed: Seeds of kind/species stated by sander or found to predominant in the test.

Other crop seeds: seeds of plants which are as crops , other than main crop.

Inert matter: includes seed units and all other matter and structures not defined as pure seed, other crop seed or weed seed.

Weed seeds: Seeds of a weed species which are recognized as weeds by law/general usage.

• Weight by percentage Single decimal place

• All components should add to 100 %

• Less than 0.05% reported as trace

• Any component is found to be nil- reported as 0.0

• The components scientific names should be mentioned

It is the emergence and development from the seed embryo of those essential structures which for the kind of seeds being tested, indicate the ability to develop into a normal plant under favorable conditions in the soil

Objectives of the germination test

Sand (0.05 to 0.8 mm particle size)/Germination paper,Germinaton chamber,  Plastic boxes for seed germination in sand, Rubber band Glass Marking Pencil, Miscellaneous laboratory glass wares

Different substrata

Paper media

1. Between paper method

2. Top of the paper method

3. Sand method

According to ISTA 400 seeds, 100 seeds in each replication (4 repns) has to be used for seed germination test.

Spacing between seeds should be uniform and there should not be any over lapping of seeds

Temperature to that specific species should be maintained

Species which required light (Grasses) has to be provided

1. First count

2. Final count

Normal seedlings: It shows the potential for continued development in to satisfactory plant when grown in good quality soil and under favourable conditions of moisture, temperature and light.

Abnormal seedlings: Don’t shows the potential for continued development in to satisfactory plant when grown in good quality soil and under favourable conditions of moisture, temperature and light.

Fresh ungerminated seeds: Seeds which have not germinated by end of test period but remain clean and firm.

Hard seeds: Remain hard by end of test period.

Dead seeds: Seeds which are neither hard nor fresh nor produced any part of seedlings at end of test period.

The main purpose of moisture determination of seeds is to prevent loss of seed viability till it is planted for commercial purposes.

Rapid method using moisture meters

Hot air oven method

Rapid method using moisture meters

• Electronic moisture meter

• Universal moisture meter

• Infrared moisture balance

Tz is a biochemical test and one of the quick methods to predict seed viability developed by Lakon (1942) in Germany.

In this biochemical test, living cells are made visible by reduction of an indicatordye. Indicator dye 2,3 5 Tripheny tetrazolium chloride (soluble, colourless and diffusible) interfeares with the reduction process of living cells and accepts hydrogen from the hydrogenases. Red, stable and nondiffusabe Formazon is produced in living cells. Tjhis makes it possible to distinguish the red coloured living parta of seeds from the colour less dead ones.

Procedure: Preparation of seeds

1. Bisect longitudinally: eg. Sorghum, large seeded grasses

2. Bisect laterlly: eg.Small seeded grasses

3. Remove seed cot: eg. Dicots

4. Condtioning only: eg: large seeded legumes

Staining: Prepared seed should be placed in suitable container and cover with testing solution. Place the container in dark and worm place.

Evaluation:Should have good knowledge of staining pattern. If embryos and well stained thouse seeds are germinable.

Advantages of theTz test

The objective is to determine the health status of the seed sample i.e., whether the seeds

are infected with fungi or not. Seed borne inoculum may give rise to spread of disease in field and reduce the commercial value of the crop. Further the seeds imported from out side source may introduce new pests and diseases into new areas.

Seed health testing methods:

Inspecting dry seeds Seedling symptom test, Agar plate method, blotter method etc..

Seed Vigour; It’s a sum total of a all the attributes which resulted in quick, uniform and early emergence of seedlings even in unfavourable environmental conditions

The principle objective of a seed vigour test is to differentiate a range of quality levels, for example high, medium and low vigour seeds.

Direct Vigour tests Indirect vigour tests

Brick gravel tests (2-3mm size) Dry weight of seedlings

Paper piercing test Speed of germination

Seedling length measurement

RQ test

GADA test

TZ test

EC of seed leachates test

APPLICATION OF MOLECULAR MARKERS FOR HYBRID PURITY TESTING

Quality seeds has to meet the minimum seed certification standards and quality attributes viz., physical purity, germination per cent, moisture content, seed health and genetic purity. The genuineness of the variety is one of the most important characteristics of good quality seed. Genetic purity test is done to verify any deviation from genuineness of the variety during multiplication stages. For certification genetic purity test is compulsory for all foundation and certified hybrid seeds. Higher genetic purity is an essential requirement for the commercialization of any seed. Besides for the success of any hybrid technology depends on the quality of the seed supplied in time.

The genetic purity during multiplication stage plot is prone to contamination from presence of pollen shedders, out crossing with foreign pollen and physical admixtures due to careless handling during harvesting, threshing, processing and bagging. Using low genetically pure seeds will proportionately reduce the commercial yield. It is estimated that for every 1% impurity in the hybrid seed the yield reduction is 100 kg per hectare (Mao et al., 1996). Maintenance of parental line purity is a prerequisite to ensure high genetic purity of hybrid seeds.

A certification standard of genetic purity for foundation is 99 per cent. While, for certified hybrids it is 95 per cent except cotton (90 %) and castor (85%). A set of qualitative and quantitative characters known as descriptors are currently in use for variety identification and description. Some of these characters, particularly those showing quantitative inheritance, interact with the environment in which the variety is grown and thus make the process of variety identification subjective.

Marker

Marker is a ‘TAG’ for identification. Identifying a genotype based on some recognizable mark can be used for its identification and characterization. It may be morphological or biochemical or molecular markers. Ideal marker should be polymorphic, Co-dominant, distributed evenly through out genome, heritable, independent to environmental factors and stage of the crop, easy, quick and cost effective to conduct and results must be reproducible

Methods to assess genetic purity

Genetic purity of a given seed lot can be assessed by using one of the following methods

1. Conventional grow out test
2. Chemical test
3. Electrophoresis method
   1. Biochemical markers (Proteins and Isozymes)
   2. Molecular markers (DNA)

Molecular markers

Molecular markers, in contrast, being based on DNA sequence variation, provide an unbiased means of identifying crop varieties. The Biochemical and Molecular Techniques group of the International Union for the Protection of New Varieties of Plants (UPOV) is evaluating different DNA marker parameters prior to its routine use in establishing distinctness, uniformity and stability (DUS) of plant varieties.

This is a new approach to test the genetic purity of the seeds done at nucleotide level. This test screen through whole genome and produces enough polymorphism in closely related parental lines also. DNA markers has advantages over morphological and biochemical markers. Those are the resultant on genotype and environmental effect. While, DNA markers are resultant of only genotype of the cultivar and are independent to environmental conditions. The markers are dominant and Co-dominant and can differentiate between homozygous and heterozygous conditions of the cultivar. This is rapid to conduct and results are reproducible.

The banding pattern appeared at a particular molecular weight is used for the identification of parental lines, hybrid and offtypes.

Hybrid seed production

AA x aa Aa

Hybrid seed is the progeny of crossing between two genetypically different parents.

Commercial hybrid seed production is produced using different methods viz.,

• Hand emasculation and pollination: tomato, cotton, brinjal etc.
• Male sterility systems- CMS, GMS and CGMS: Rice, sorghum, sunflower, bajra, pigeon pea etc.
• Self incompatibility: Brassica
• Sex expressions: cucurbits

During production of hybrids using these methods seeds are prone to contamination due to Selfed seeds, sibed seeds, out crossing etc.

Hybrid seed production two steps

(i) Foundation seed production/Female line multiplication (A/B):

(Male sterile line) A x B (Maintainer line)

A line

(Male sterile line)

This is the stage of parental line multiplication. Sterile line (A line) is multiplied by crossing with maintainer line. During this stage CMS line seed lots are often contaminated by its respective maintainer line (shedders) as mechanical admixtures. And it is not possible to distinguish CMS and its respective maintainers based on seed morphology as they are iso-nuclear genotypes. So, have to conduct GOT till flowering and have to observe for pollen fertility to identify pollen shedders.

ii) Certified/Hybrid seed production (A/R).

A (Seed parent) x R (Restorer line)

F1 Hybrid

This is the stage of commercial hybrid seed production. CMS line (A line) is crossed with Restorer line (R line) to obtain F₁ hybrid seeds. During this stage the presence of B line in A line rows and if not roughed out before flowering will lead to production of genetically impure seeds i.e contaminated with A and B line seeds.

Importance - stable marker for genetic purity

CMS plants and its maintainer plants, which originated from female parent during multiplication, are major off-types in F1 hybrids. Maintenance of the purity of parent CMS lines is essential in achieving the purity of hybrid and thus commercial benefit. CMS purity during multiplication can only be assessed at heading stage by observing pollen fertility. The results are prone to be erratic due to different examiners and environments. So it is significant to develop a novel, simple, rapid and effective method to assess CMS seed purity during multiplication at seedling stage.

Hybridity confirmation

Identifying breeding lines and determining hybrid purity are major requirements in plant breeding and seed production. To test the conformity of hybrid seed, one must be able to distinguish the true hybrid resulting from cross between the male and female parents and one coming from self pollination of the female parent. The DNA finger printing of parental lines, hybrid and offtypes are used as a data base to identify offtypes in the seed lot under question.

Molecular markers which could clearly distinguish the hybrids form its parental lines have been identified and developed in major agriculture and horticulture crops are given in the following Table.

The molecular markers are more efficient in assessing genetic purity. Among markers RAPD and AFLP are dominant markers. Dominant markers unable to identify heterozygous condition (AA and aa only but not Aa).

Low reproducibility of RAPD and lengthy process of AFLP markers have made them impractical and difficult for their routine use in seed purity analysis. While SSR, SCAR, STMS are C0- dominant markers and are able to identify heterozygous condition. These markers are more popular because of their accuracy in results and are reproducible. Quick and simple process of these markers have made them practical for their routine use in hybrid conformity and seed purity analysis. Markers vary based on their ability to differentiate lines with the crop and hybrids and parental lines involved in developing particular hybrid.

CONCLUSION

Hybrid seed producer have to test every commercial hybrid seed lot produced. A fast and reliable method with capacity to screen large number of seed samples. With the availability of high throughput PCR machines and simpler DNA extraction techniques may surpass the current methods of grow out test. DNA is now routinely used for the plant variety breeding purposes and it is likely that hybrid seed producers can adapt these modern techniques for hybrid seed purity analysis purposes.