THE MECHANISM OF THE TETRAZOLIUM REACTION IN CORN EMBRYOS

Abstract

The reduction of tetrazolium salts to insoluble, colored formazans by living tissues was pointed out by KUHN and JERCHEL in 1941 (18). The reaction has since been widely used as an indicator of viability or metabolic activity with a variety of tissues and tissue extracts from animals (6, 19, 28, 31), microorganisms (5, 11, 13, 22), and higher plants (9, 12, 27, 32), especially seeds (8, 10, 20, 25). The 2,3,5-triphenyltetrazolium salts (here-after called tetrazolium) have been used most commonly although several other derivatives have been introduced recently (1, 2, 28). The biological reduction of tetrazolium has generally been attributed to enzymatic action. This interpretation was first suggested by KUHN and JERCHEL (18) on the basis of the low oxidation-reduction potential (-0.080 volts) and the lack of reduction by dead tissues or by ascorbic acid, sulfhy-dryl compounds, or sugars at physiological pH values. MATSON et al. (24) first reported that a dehydrogenase preparation (the glucose-6-phosphate system) would catalyze reduction, and several others (19, 22, 28, 31) have used the reaction as a dehydrogenase indicator. Because of the increasing use of the tetrazolium test in the measurement of seed viability a more thorough study of the mechanism of the reaction in seed tissues was undertaken in 1949. This paper reports the determination of optimum conditions and the kinetics of tetrazolium reduction by malic dehydrogenase, the role of diaphorase, and the relative activities of several diphosphopyridine-nucleotide-linked dehydrogenases in corn embryos. Since this work was completed JENSEN et al. (16) have published further evidence that several pyridine-nucleotide-linked dehydrogenases would reduce tetrazolium, and BRODIE and GOTS (7) have shown that diaphorase may participate in the reaction. Materials and methods All enzyme preparations were made from seed of open pollinated WF9 x 38-11 hybrid corn. The seeds were soaked at 300 C for 18 to 20 hours with the embryo side down and half immersed in water. At this stage of germination there was visible radicle growth but seldom any emergence. Excised embryos were ground in cold 0.02 M phosphate buffer (pH 8) in a Potter-Elvehjem homogenizer to give a 10% suspension on a fresh weight basis. Cellular debris was removed by centrifuging cold for seven to eight