OTHER SEED VIABILITY TESTS
While the TZ test actually monitors the physiological condition of the seed, there are other seed vi8ability tests which focus primarily on the integrity of the external seed parts such as the seed coat. We now know that an intact seed coat is essential for optimum seed performance. This is because the seed coat plays an integral role in mediating the inrush of water into the seed during imbibition as well as protecting the sensitive embryo against injury from soil pathogens and herbicides. In addition , the intact seed coats are also ess3ential to reduce the amount of cellular leakage of metabolites from an imbibing seed. Such compounds serve as ready sources of nutrients for soil microflora which rapidly cause seed deterioration. The following are three rapid lab tests which can identify seed coat and mechanical damage.
1.indoxyl acetate test for seed coat damage of large-seeded legumes
Any damage to seed coats, particularly soybeans and other large-seeded legumes, is an indication of mechanical abuse during harvesting and conditioning and serves as an entry site for pathogen infestation. The indoxyl acetate test is a laboratory test that reveals seed coat damage in soybeans and other light-colored legume seeds.
A sample of 50 seeds are to be soaked for 10 seconds in a 0.1% solution of indoxyl acetate prepared in 95% ethanol. Following this soak treatment, the seeds are sprayed with a 20% ammonia solution for 10 seconds and allowed to air-dry. Lesions in the seed coats that are difficult to detect with the naked eye become immediately visible because they turn purplish green against the yellow or white seed cotyledon background.
2. Fast green test for seed coat damage of large seeded grasses
The fast green test has been a test utilized for a number of years to detect seed coat damage in corn. Again, your instructor will demonstrate this procedure. Two hundred corn seeds will be placed in a 250 ml beaker and covered with a 0.25% fast green solution ( 0.25 grams fast green in 100 ml distilled water) for 15 seconds. After this period, the fast green solution is removed and the seeds are rinsed under water. Seed coat damage is readily apparent as green lines against the yellow seed color.
3. Ferric chloride test for mechanical damage of legume seeds
Mechanically injured legume seeds turn black when placed in a solution of ferric chloride. This is a very fast and useful test that provides the farmer or seedsman a rapid estimatte of the percentage of abnormal seedlings he can expect from his crop. i.e 100 field beans in a 250 ml beaker, cover with a 20% ferric chloride (FeCl3) solution (20 grams FeCl3 in 100 ml water) for 15 minutes, at which time all black-stained seeds are separated.
TETRAZOLIUM SEED TESTING
The tetrazolium test possesses advantages and disadvantages. Among the advantages of this test is its rapidity of assessment. A tetrazolium test can generally be completed in less than 24 hours. Among the disadvantages are the subjectivity of assessments and the experience required to make correct interpretations of seed viability. However, despite these disadvantages the TZ test continues to increase in popularity and use – a strong testament to its importance in seed viability testing.
Preparation of seeds for testing
Conditioning: Interpretation of TZ staining patterns is facilitated when the seeds are softened in water before staining. This process permits the activation of germination enzymes and makes the seed tissues less fragile. To condition the two seed lots, place 100 seeds from each lot between two moistened paper towels and allow them to imbibe overnight at 30C. The conditioning process is completed when the seeds are fully imbibed and soft enough to permit a clean slice th rough the embryo.
Preparation for Staining: The seeds must be prepared in such a way that the TZ solution can come in contact with the embryo. Since the pericarp of grasses is not permeable to TZ, the pericarp must be sectioned. In the case of Pensacola Bahigrass, the student must bisect the seeds while they are still on the moistened conditioning paper by cutting longitudinally with a sharp razor blade near the center of the caryopsis above the embryo. Discard the apical portion of the bisected seed. In the case of the small seeded legume, crimson clover, TZ will penetrate these seed coats, so no additional preparation is necessary.
Staining: Following preparation for staining, the two seed lots are now ready for staining. The Bahigrass should be placed in a 0.1% TZ solution (0.1 grams of 2,3, 5-triphenyl tetrazolium chloride in 100 ml distilled water) in a 250 ml beaker at 35C for 2 hours. The crimson clover seeds should be placed in a 1.0% TZ solution (1.0 gram TZ in 100 ml distilled water) in a 250 ml beaker for 6 hours at 35C prior to evaluation. Ensure that the seeds are completely immersed in the TZ solution.
Evaluation: The TZ evaluation is the most crucial yet difficult stage for correct TZ assessments. Accurate interpretation of a TZ test is dependent on:
- Knowledge of seed and seedling structures and their role in seed germination
- Understanding of the physiological basis for this test and its limitations.
- Combining interpretation of staining patterns with other visible aspects of seed quality.
- Experience with making comparative germination tests
Since many seeds are neither completely alive nor completely dead, a knowledge of the relationships of seed structures to seedling structures is necessary to interpret the importance of unstained seed tissues. For example, to the beginning practitioner of TZ testing, the intensity of red (formazan) color formation which is a consequence of respiration is indicative of the quality of the seed lot. While this may be true in some instances, it is not always so. For example, a particular seed may be completely stained by TZ with the exception of a small necrotic lesion on the radicle tip. Even though the rest of the seed is completely viable, since the seed is incapable of producing a root, it would be considered nongerminable. This example illustrates that it is not the intensity of staining, but what is stained that is important. Thus one must be familiar with the function of the various seed parts in making a correct TZ test.