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Tetrazolium test for determining viability of seeds

The performance of a seed can be determined by studying its germination and viability potential. One of the most significant advances in seed testing technology in recent years is the topographical tetrazolium method.

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Seeds form the most important, cheapest component and basic input of mass plant cultivation. Only seeds with assured quality will respond to fertilizers, water, pesticides and other inputs and pay required dividends. Progress in food production is assured only when good quality seeds can be identified, multiplied and marketed efficiently. The performance of a seed can be determined by studying it germination and viability potential. One of the most significant advances in seed testing technology in recent years is the topographical tetrazolium method. It's the fastest test available in testing seed viability for both dormant and non-dormant seeds, which is a boon to the seed industry for quicker decision making. The test provides for a rapid evaluation of seed lot in a short time of 2-24 hrs.

<br>Petridishes, Scalpel, Forceps, Magnifying lens or Stereoscopic microscope, Dispensing bottle, Distilled water, Filter paper, Oven or incubator to maintain temperature at 40-45 degree centigrade.

<br>Reagents -
<br>1. Tetrazolium solution: The concentration of the solution varies according to the seed type. A 1.0% solution maybe used for whole seeds [seeds that are not bisected through the embryo] or a 0.1% solution maybe used for excised seeds. A pH of 6 to 8 has to be maintained for best staining results.

<br>Phosphate buffer solution -
<br>Solution 1: Dissolve 9.078 g of KH2PO4 in 1000 ml distilled water.
<br>Solution 2: Dissolve 11.876 g of Na2HPO4.2H2O in 100 ml distilled water.

<br>400 ml solution 1 + 600 ml solution 2 gives 1 l buffer solution. Dissolve 10 g of tetrazolium salt in this buffer to get 1.0% tetrazolium solution of pH 7.0.
<br>Store solution in brown bottle to prevent deterioration in light.

<br>2. Lactophenol solution - Used for clearing grass seeds after staining so that the lemma and palea are clearly visible.
<br>Mix lactic acid, phenol, glycerin and distilled water in the ratio 1:1:2:1 respectively.

<br>Procedure: Seed samples in replicates of 50 or 100 are sufficient for the test.

<br>1. Hydrating -
The seeds have to be hydrated in order to facilitate dehydrogenase activity. This process releases hydrogen ions need for the staining reaction. Hence the seeds are conditioned overnight between folded moist blotters or soaked in warm water [30 deg C] for 3-4 hrs.

<br>2. Cutting or puncturing -
The seed may be bisected longitudinally, sectioned laterally or pierced with a needle. Seeds of peanut and cotton require removal of seed coat. In cotton seed, despite the removal of seed coat, a thin membrane is found attached to the cotyledons, which needs to be removed before staining. While seeds of clover for example does not require any conditioning as the seed coat is permeable to tetrazolium.

<br>3. Staining -
The prepared seed is then placed in a petridish and covered with tetrazolium solution and incubated in an oven or incubator at 40 deg centigrade. The staining time taken for complete coloration varies with the type of seed tested.

<br>4. Evaluation of the samples -
It is very important that one know the structure and anatomy of the seed tested so that the staining pattern of the embryo maybe analyzed. Normal tetrazolium stain appears cherry red to the naked eye. Under magnification, the scutellum appears to have many dark spots against a pink or light red background.

<br>E.g.: A wheat seed can be said to be viable or germinable if the entire embryo is stained bright red and extremities of the scutellum, radicle tip and coleorhizza are unstained. The seed is non-germinable if the stain is a very faint pink or the plumule, radicle, scutellum, embryonic axis, coloerrhiza, upper half or the entire embryo is unstained.

<br>Chemistry/Principle of the test:
<br>This biochemical test involves highlighting living cells by making it visible using a indicator dye tetrazolium. 2,3,5 triphenly tetrazolium chloride interferes with the reduction process in living cells of seeds and accepts hydrogen from the hydrogenases. The hydrogenation leads to the formation of a red, stable and non-diffusible triphenly formazan. This change in color makes it possible to distinguish the red color living cells from the colorless dead cells. The staining pattern differs with the position and size of the necrotic regions in the seed and this determines whether the seeds are viable or non viable. Since the color difference is the decisive factor in viability determination, this test is also called the topographical tetrazolium test.