Triphenyltetrazolium chloride for viability determination of frozen seed corn

A limited number of tests on nondried frozen seed failed to give satisfactory readings with the tetrazolium solutions. Although differences in staining level were noted, classification for viability was difficult and uncertain. A majority of the seeds tested immediately after freezing were classified as viable. Subsequent testing dried seed from the same lots gave greatly reduced tetrazolium and germination percentages. Apparently, an interval of time is necessary for a breakdown in the enzyme system of seeds injured by freezing before an accurate viability test may be made with tetrazolium. Drying seed to 12% or less moisture content in the grain by use of circulated air heated to 110 degrees F was adopted as part of the testing procedure. During this interval, seeds injured by freezing lost their capacity for reducing tetrazolium. Tetrazolium percentages were generally higher than standard germination percentages for the same samples. As an example, an estimate on the regression line shown in Fig. 3 gives a germination of 74.5% when the tetrazolium reading is 80%. The germination percentage should be regarded as approximately 95% of the tetrazolium readings. The slightly higher readings for tetrazolium percentages may have been the result of classifying "weak" seed as viable. In counting seedlings for germination percentages, a seedling which failed to produce a well-developed plumule, radicle, and secondary roots was rejected, yet there may have been enough activity in a similar seed to give a strong tetrazolium reduction. In samples frozen at high moisture level, there may have been present a small percentage of seeds which were very immature and which contained active reducing compounds other than those associated with high germinability. In one instance the tetrazolium readings were poor indexes to viability. The samples for the B hybrid listed in Table 1 gave much higher percentages by tetrazolium staining than were secured in the standard germination tests. Counts on "weak" seeds in the germination tests failed to account for the differences between tetrazolium percentages and germination percentages. Seed from the same samples were tested 3 months after the initial testing and the same differences between tetrazolium percentages and germination percentages were found. The seed of the B hybrid contained 51% moisture when frozen. Substances, other than those associated with the germination process. which are capable of reducing tetrazolium, may have been present in the very immature seeds and may have been "fixed" by the rapid drying process. Since the tetrazolium solution used was pH 12 or more, the presence of reducing sugars may account for at least a portion of the high readings. Mattson, Jensen, and Dutcher (4) found that reducing sugars form the red formazan only above pH 11.0. Temperatures at which freezing damage may occur in seed fields usually range from 17 degrees to 25 degrees F, but temperatures used for artificially freezing samples for trials reported in this paper were 10 degrees F or lower. Damage to vital tissues may not have been of the same degree or nature under artificial freezing conditions. In trial IV, freezing of different samples was terminated when the internal temperature of ears was between 17.0 degrees and 25.0 degrees F. Ear temperatures between these levels may occur in seed fields while the moisture in the grain is still above 25%. Under such conditions, loss in germinability takes place. Data not shown in this paper but used to plot Fig. 3 show that seed above 25 degrees F moisture when frozen to below 25 degrees F was reduced in viability. The temperature and moisture conditions for trial IV may be considered as similar to those frequently occurring in seed fields during unfavorable seasons. The viability percentages shown for the one set of naturally frozen samples collected in 1947 are fairly characteristic of germination percentages for large quantities of hybrid seed corn produced in the season of 1945. During the latter part of September 1945, temperatures over a large area in northern Iowa and southern Minnesota dropped to a critical low level. Reduction in germination varied from 10 to 50% or more as a result of freeze damage to immature seed. The tetrazolium percentages for the 1947 samples are good indexes to germinability of the same samples. High positive correlation between tetrazolium readings and germination percentages is indicated by the scatter diagram shown in Fig. 3 and by the correlation coefficients r=0.99 and 0.97 for trials III and IV, respectively. The correlation between tetrazolium percentages and standard germination percentages is linear, although individual ears varied from 19.0 to 34.0% moisture in the grain and were frozen at temperatures varying from 17.0 degrees to 25.0 degrees F. The linearity and high degree of correlation indicates that viability determined by tetrazolium is a good index to what the sample will germinate in a standard test, regardless of the amount of freeze injury which may have taken place. The evidence presented indicates that seed corn below 49% moisture

in the grain when frozen may be satisfactorily tested with tetrazolium for minimal damage at least within 48 hours after the end of a freezing period. The tetrazolium method is only a means of making all early appraisal of minimal freeze damage to seed corn. Samples from seed intended for planting purposes should be carefully retested by suitable germination tests for any subsequent deterioration in viability.