Induction of forward mutation at the yellow-green-2 (yg2) locus in Zea mays has been used as the genetic endpoint in mutation studies examining both physical and chemical mutagens. The yg2 assay has proved to be a rapid, inexpensive, and sensitive assay in screening agents for mutagenic activity. We have used the yg2 assay to study the kinetics of mutation induction following acute exposure to N-ethyl-N-nitrosourea (ENU). ENU is mutagenic in higher plants and animals. ENU is a monofunctional alkylating agent and was found to be the most potent chemical mutagen in the Mouse Specific Locus test (W. L. Russell et al., 1979, Proc. Natl. Acad. Sci. USA 76:5818).
Kernels heterozygous for yg2 were surface sterilized for 5 min in a 0.5% solution of sodium hypochlorite and rinsed for 10 min in running tap water. The kernels were soaked in aerated distilled water for 72 hr at 20 C. This allows the kernels to become metabolically active, completing one DNA replication during this period. Subsequently, the kernels were treated for 8 hr in various concentrations of aerated solutions of ENU at 20 C. A concurrent control of distilled water was also included. Following treatment the kernels were rinsed in running tap water for 30 min and were planted in soil in 10 cm plastic pots. The pots were placed in a plant growth chamber at 20 C with a 17 hr photoperiod (300 uE m-2 sec-1 PRR). Between 20 and 25 days after planting, leaves four and five were excised and scored for yellow-green sectors using a fluorescent light box to illuminate the leaves. Only sectors greater than 1 mm in length were counted. The mean frequency of mutant sectors per leaf was calculated for leaves four and five at each concentration of ENU. These data are presented in Table 1.
The data indicate that concentrations of 535 uM ENU or greater induce a significant increase in the frequency of mutant sectors over control values. The data from four experiments were compiled and exhibit linear kinetics for mutation induction for leaves four and five (Figure 1). The slopes of the dose-response curves for leaves four and five were 1.40 and 0.50, respectively. The induction of forward mutation at the yg2 locus is highly correlated with the concentration of ENU for both leaves (r = 0.97). Clearly, ENU induces forward mutation in somatic cells of maize. (This research was funded, in part, by NIEHS Grant No. 5 R01 ES01895 GEN.)
William E. Schy and Michael J. Plewa
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