Tetrazolium testing was started in 1941 by the late Georg Lakon of Germany, who used it as a substitute for a poisonous selenium salt that was used in studies of seed. Tetrazolium became available in the United States about 4 years later. It is now used in many countries and States. In the United States, it is largely used as a rapid, nonofficial testing supplement to standard methods. A few laboratories make a specialty of chemical seed testing.

The TZ test makes it possible to determine the potential viability and vigor ratings within 15 minutes to 24 hours. It also provides a different approach to testing and new insights into seed evaluation. It can reveal causes for seed troubles that may remain concealed or uncertain in growth tests. These informative insights make the test useful in predicting or diagnosing reasons for failures of germination due to storage, laboratory, or field conditions. The TZ test is especially useful in evaluating dormant seeds at harvest or seeds that require long or uncertain testing periods otherwise.

Another good opportunity to use TZ testing is in the evaluation of seeds that remain firm at the end of growth tests. Producers of seed of corn and sorghum use the test before processing to evaluate the seriousness of injury by early frosts.

TZ testing is not infallible and is not a final answer to all testing problems. It is merely another useful tool. The test can only reveal. The accuracy of results depends on the qualifications of the analysts who interpret it.

One objection to the widespread use of TZ tests in trade channels is that a uniform system of standards is yet to be established and coordinated for most kinds of seed. Furthermore, unless TZ testing is approved as official for given kinds of seeds, the results are not acceptable in labeling for legal requirements. Another objection is the extra time needed to prepare and examine individual seeds.

Another consideration is that TZ tests reveal potential germination percentages based strictly on internal seed condition, but growth tests reveal a combined performance of seed quality and a given growing condition. Germination evaluations from the two approaches to testing are often similar, especially if disease-susceptible seed lots are treated with a fungicide prior to growth testing under favorable conditions. TZ tests may not detect recent applications of toxic levels of fungicides.

When TZ tests are properly evaluated, the limitations usually are not so serious as they appear at first, especially if they are evaluated along with weaknesses of any other single type of test. The limitations should not discourage a person from taking full advantage of the potential usefulness of TZ testing for vigor and viability.

A knowledge of internal embryo parts as they relate to the development of seedling structures is essential for a proper evaluation of TZ tests. Most agricultural seeds are characterized by one cotyledon, or seed leaf, as in grasses, or by two cotyledons, as in legumes. A knowledge of embryo structures of the two groups is a guide for understanding many kinds of seeds.

In one test of quality, a colorless solution produces a red pigment in strong, healthy tissues. In these drawings the shaded parts represent the red staining and show the live cells in embryos. a, corn; b, sorghum; c, wheat cut surface of upper half of embryo; d, bluegrass non-cut surface; e, soybean noncut surface.

Testing techniques require that certain basic procedures be followed. Seeds should first be softened in water to activate enzyme systems, and to promote clean cutting and uniform absorption of tetrazolium. Seeds with fragile structures, such as snap beans, should be preconditioned first by slow absorption of water from a moist medium to avoid critical fracturing.

Seeds are prepared for absorption of tetrazolium by methods appropriate for the kind involved. Corn and large-seeded grasses are cut longitudinally through the center of germs to expose embryo leaves and root buds. Fine-seeded grasses are punctured or cut crosswise immediately back of the germ, or under it. Seeds of legumes and other crops, which absorb tetrazolium through seedcoats, are stained without previous physical alteration.

A satisfactory testing solution consists of 0.5 gram of 2,3,5-tripheynl tetrazolium chloride in 200 cubic centimeters of water.

Staining should occur in darkness and at temperatures near 70 to 90 F. Approximate staining times at 86 are: Corn and other cut seeds, 2 to 4 hours; cotton, peanuts, and other seeds with exposed non-cut embryos, 4 to 6 hours; beans, grasses, and other crops in seedcoats, 6 to 18 hours.

When adequate staining has occurred, the solution should be discarded, and the seeds covered with water and refrigerated at 40 to 50 until analyzed. Seeds should be kept moist until evaluated.

SATISFACTORY interpretation of potential germination can best be acquired by an inexperienced analyst by comparing results from growth tests with observations from TZ tests.

Evaluation for vigor and viability requires observation of individual structures of each embryo and the relating of the condition of the decisive parts to the potential formation of normal or acceptable seedlings. Embryos should be observed individually, preferably under 5 to 10 X magnification, for the presence, extent, and seriousness of trouble areas. Variations in color patterns, texture, bruises, fractures, abnormal structures, infected areas, and insect infestation are all of potential importance. Firm, hard, or dormant seeds react normally to TZ staining and may be either germinative or nongerminative.

It is informative to classify individual germinative seeds within a lot on the basis of 1 to 5 and nongerminative 6 to 8. The physical condition of the embryo is used for these ratings. The percentage of seed in each class establishes different levels of vigor which is useful in predicting relative storage life of seed lots and response to adverse germinating conditions.

Advanced degrees of a localized or general aging, revealed as pale-red, mottled, necrotic, or flaccid tissues, cause more difficulty in interpretation than most other seed conditions. Interpretations of aging symptoms can be mastered with experience, and with this mastery will come a deeper appreciation of the gradual process of aging and the formation and enlargement of necrotic areas that tend to lead first to a nongerminative condition and later to complete death.

The TZ test has given us many insights into the mysteries of seeds. We expect to get more as its techniques are refined to reveal the causes of weaknesses of seeds and failures in germination. The advantages commend its use by agencies that need nonofficial evaluations quickly.

VERA L. COLBRY is a seed technologist in the Seed Branch, Grain Division, Agricultural Marketing Service, Agricultural Research Center, Beltsville, Md. She holds degrees from Oregon State Agricultural College and George Washington University. She has worked in the field of seed technology since 1931.

THOMAS F. SWOFFORD is in charge of the Region 8 Tree Seed Testing Laboratory, Macon, Ga. Other assignments have been administrative positions at various national forests in the South and Pacific Northwest. Mr. Swofford is a graduate in forestry of the University of Washington and Yale.

ROBERT P. MOORE is professor of research, crop stands, North Carolina Agricultural Experiment Station, Raleigh. He was formerly a teacher of field crop courses at the Oklahoma State University and the University of Tennessee; a graduate assistant with the Ohio Seed Improvement Association, Columbus; and Director in charge of the North Carolina Seed Improvement Association.