Seed Viability Tetrazolium Test or TZ Test

Seed Viability Tetrazolium Test: The topographic tetrazolium test was developed by Lakon (1942) in Germany. It is a quick, easy, and popular method of testing Seed viability, and requires to be known within a day or two, certain chemicals such as 2, 3, 5 – triphenyl tetrazolium chloride (TZ) or bromide, in the presence of dehydrogenase enzyme, these enzymes are essential for seed respiration and, hence, viability.

Seed Viability Tetrazolium Test or TZ Test: Methodology

The tetrazolium test requires that the chemical must come in contact with the embryo, which is the only respiring part of the seed.

Thus, preconditioned seeds may be handled as follows depending on the plant species.

• Seeds are placed directly in tetrazolium solution, e.g., in the case of peas and beans.
• Alternatively, prior to placing the seeds in tetrazolium solution, they may be bisected longitudinally, e.g., in the case of cereals and some grasses.
• Glumes of the seeds may be removed, e.g., in the case of oats, before they are placed in TTC solution.
• The seeds may be subjected to pricking below the embryo or they may be bisected laterally, e.g., in the case of very small seeds of grasses.
• The seed coat may be removed, e.g., in the case of melon, cotton, etc.
• The seed coat may be scratched above the embryo, e.g., in lettuce.
• A piece of the embryo may be excised and stained, e.g., in the case of pines. 

The procedure of the TZ test

• In this test, seeds are soaked in water overnight or on moist paper for 6-18 hours to prepare them for the test (i.e., for the removal of testa as in soybean or to cut the seed longitudinally as in wheat) and also for Clarity of stain, which would facilitate interpretation.
• Seeds are then soaked in 0.5 to 1.0% aqueous solution of 2, 3, 5-triphenyl tetrazolium chloride at 30°C in the dark. If the pH of the water available is not within 6.0-7.5, the solution should be made in phosphate buffer because solutions with progressively lower pH values than 6 stain weakly or may even fail to stain seeds. On the other hand solutions with higher pH values than optimum show darker staining. (commonly used, pH 6-8, temperature 30-35 o c).
• Evaluation problems may arise at exceedingly high pH values also. Seed coats of pulses may or may not be removed depending upon the permeability of the seed coat and the ability of cotyledons to stay together during the soaking time of the seed in tetrazolium solution.
• When the viable part of the seed like an embryo in monocot or root-shoot axis and cotyledons in dicot seeds turn red, the tetrazolium solution is drained off. Seeds are then rinsed. 3-4 times with distilled water and then individual seeds are evaluated for viability. Seed coats must be removed before interpretation.

Interpretation of Result

The living embryo or root-shoot axis of seeds converts the absorbed colorless tetrazolium solution into a clearly visible red stain (triphenyl formazan). Staining of seeds determines whether seeds are to be classified as viable, completely stained seeds are viable, partially stained, and completely unstained non-viable.

Accurate interpretation of the tetrazolium test depends upon:

1. Knowledge of seed and seedling structure.

2. Understanding of the mechanism of the test and its limitations.

3. Combining interpretation of staining patterns with other visible aspects of seed quality.

4. Interest and experience.

The analyst should be familiar with the areas of cell division in embryos while interpreting the results of the tetrazolium (TZ) test.

In monocots active areas are (i) tips of radicle and seminal roots, (i) the base of plumule, and (i) the point of attachment of the embryo to scutellum, and in dicots (i) the Point of attachment of cotyledons and hypocotyl and (i) the growing points.

Principle of Tetrazolium Test

• The tetrazolium-salt solution is imbibed by the seed and it interacts with the reduction process of living cells and accepts hydrogen from dehydrogenase.
• Dehydrogenases are a group of enzymes involved in the reaction by participating in the H+ transfer. When hydrogen is accepted by tetrazolium solution, a red, stable, and non-diffusible substance, triphenyl formazan, is formed in cells. This makes it possible to distinguish the red-colored living parts of seeds from the colorless dead ones. The reaction is as follows.
• Therefore, the tetrazolium test is a test for the activity of a particular enzyme system. Loss or activity of these enzyme systems tends to parallel loss in seed viability. Many seeds are neither completely dead nor completely alive, therefore, staining patterns should be correlated with the seedling description.
• Discrepancies between the TZ test and germination test results may occur due to several reasons. These are sample differences, improper germination-testing technique, improper tetrazolium-testing technique, dormancy in seeds, presence of hard seeds, presence of seed-borne organisms, and chemical injury to seeds during treatment or during fumigation. The chemical damage that prevents normal germination may not inhibit tetrazolium staining. (For more updates visit the ISTA website).

Also, visit Types of Seed Dormancy, Seed Pelleting, Seed Storage, Seed Testing, and AI in Seed Industry.

Relationship between TZ and Germination Test

• Properly conducted TZ and germination test results are generally in close agreement; differences in results are usually smaller with high-quality seeds than with low-quality seeds.
• The TZ test has been tried on many crop seeds and the results have been very good. A significant correlation between germination percentage and viability percentage by TZ was obtained in wheat, rice, and maize. Regression equations were also developed and the equations were used for predicting germination percentage. The predicted germination percentage was found to be very close to the actual germination percentage.

Lactophenol Solution

When the pigment within the seed coat or lemma prevents clear vision a few drops of the clearing solution can be used. Removal of pigmentation from the covering structure requires about 10-30 minutes.

ADVANTAGES OF TETRAZOLIUM TEST

1. A quick estimate of viability can be obtained (within 12-20 hours).
2. When the seed is dormant or very slow in germinating the TZ test is extremely useful.
3. Seeds are not damaged (in dicot only) in analysis, therefore, they could be germinated.

DISADVANTAGES OF TETRAZOLIUM TEST

1. It is difficult to distinguish between normal and abnormal seedlings.
2. It does not differentiate between dormant and non-dormant seeds.
3. Since the TZ test does not involve germination, microorganisms harmful to germinating seedlings are not detected.