1. A chemical test for seed viability.
Ionizing radiation has a lethal effect upon seeds if given in sufficient dose. In every experiment involving killing of seeds, it is usually necessary to test the germination of a certain number of seeds. However, viability can be determined much more quickly by using a chemical, 2‑3‑5‑triphenyltetrazolium chloride, commercially known as TPTZ. This is not a new compound, having been first prepared by Pechman and Runge in 1894. However, the application of TPTZ to testing seed viability is fairly recent, dating from George Lakon's work in Germany in 1942. Prior to 1942, Lakon analyzed TPTZ and found that it was a non‑toxic, water soluble compound which, when reduced, formed triphenylformazan, a red precipitate, insoluble in water, but soluble in alcohol. He also noted that, when in solution, the chemical was sensitive to light. In his studies of seed viability, Lakon used seeds of barley, wheat, rye, oats, and corn. In corn, he observed a direct relationship between the regions of the embryo in which the red precipitate was formed and the organs of the seedling which would develop. Recently, many modifications of Lakon's method have been used on both animal and plant tissues.
In general steps in testing seed viability are: (1) soak the seeds in tap water; (2) bisect each seed medianly through the embryo; (3) soak one‑half of each seed in TPTZ. The presoaking of the seeds is required only to facilitate the sectioning, as the initiation of germination is not necessary for the test. Presoaking times of one to eighteen hours were tried and it was found that, although the seeds which had been soaked for longer periods of time were easier to bisect, in general, comparable results were obtained when using seeds which had been presoaked for six to eighteen hours. All of the seeds were presoaked at room temperature (23‑24íC.)
After presoaking, each kernel is cut longitudinally so that the embryo is bisected medianly through its entire length and one-half of each kernel is submerged immediately in TPTZ. Prolonged contact of the cut seed with the air and of the TPTZ to the light should be avoided. The TPTZ may be contained in small dishes, similar to Petri dishes, which have been painted on the outside with aluminum or black paint or in similar unpainted dishes which can be placed in the dark. Tests of whole, uncut seeds showed that the embryo of the seed contained some of the red precipitate, indicating that the TPTZ molecules are able to penetrate the pericarp. Treating whole seeds has a limited and questionable use, since only seeds with colorless pericarp could be used and it is impossible to identify which regions of the embryo contain the precipitate, whole seeds after treatment of this type were planted in the greenhouse, but few of them produced seedlings. Further work on this is planned.
In the final step of the procedure, seeds were soaked in solutions of TPTZ varying in concentration from 0.0001% to 2.0%, in pH level from 2.0 to 11.0. and in length of time soaking from 15 minutes to 4 hours at room temperature and then examined. It was observed that accurate, reproducible results could be obtained under a wide range of conditions. The factors, pH, concentration, and length of time in TPTZ, are closely related in the effect upon the formation of the red precipitate. Solutions with pH levels of 4.0 to 10.0 and concentrations of 0.05% to 1.0% consistently formed precipitates in the viable embryos; the solutions with the lowest concentrations and lowest pH levels requiring the longest reaction time. Optimum conditions for the reaction were observed when the seeds were soaked in solutions of TPTZ ranging in concentration from 0.05% to 0.25% and pH levels from 6.0 to 9.0 for periods of time varying from one‑half to one and one‑half hours. The kermels which indicate viability possessed an embryo which was stained throughout, or at least stained in the regions of the coleoptile, plumule, secondary radicles, transition region, and scutellum.
The general conclusions drawn from this preliminary study are that the procedures used to test seed viability in corn with 2‑3‑5 triphenyltetrazolium chloride are not complicated or subject to rigid requirements of observation or chemical preparation.
Mary L. Kooster
*All research reported in this contribution was carried out under the auspices of the Atomic Energy Commission.