Early-Early Synthetic (a rapidly maturing maize inbred) is being calibrated with physical and chemical mutagens to investigate the kinetics of mutation induction and as a monitor for environmental genotoxins. The genetic events under evaluation are forward mutation at the wx locus in pollen grains, forward mutation at the yg2 locus in leaves and chromosome aberrations resulting in micronuclei in root tip cells. Micronuclei are formed from acentric fragments, lagging chromosomes or multicentric chromosomes connected by bridges. A micronucleus test in root tip cells of Vicia faba has been developed by Degrassi and Rizzoni (1982, Mutation Res. 97:19-33)
Kernels heterozygous at the yg2 locus were surface sterilized by soaking in a 0.5% sodium hypochlorite solution for 5 min. Thirty-three kernels per group were soaked for 72 hr in aerated distilled water at 20 C. The kernels were treated for 8 hr at 20 C with aerated solutions of ethylmethanesulfonate (EMS). The concentrations tested were: 0 (control), 1 mM, 10 mM and 20 mM EMS. Following treatment, the kernels were rinsed for 30 min in running tap water and three kernels each were planted in soil in 10 cm diameter plastic pots. The pots were placed in a plant growth chamber at 20 C with a 17 hr photoperiod. Root tips were cut from each group 5 and 9 days after planting and placed in a fixative of ethanol and acetic acid (3:1 v/v). The root tips were fixed overnight. For analysis, the root tips were rinsed in deionized water and placed in 1 M HCl at 60 C for 7.5 min. The root tips were rinsed again and placed in Feulgen stain for 1 hr followed by a 5% pectinase treatment for 1 hr. Slides were made by squashing the root tips in Feulgen stain. The number of interphase cells counted on each slide was approximately 1,000 and five slides were analyzed from each control or treatment group.
The results are presented in Table 1. After 5 days, the micronucleus frequencies for all treatment groups were significantly higher than the control. The concentration of EMS and the induction of micronuclei are highly correlated (r = 0.94) and the dose-response curve exhibited linear kinetics (Figure 1).
After nine days, the micronucleus frequencies of the treatment groups were not different from the control. The decrease in the frequencies in the treatment groups may be due to two causes, cell death due to gross chromosomal aberrations and the eventual disappearance of micronuclei by the action of cytoplasmic nucleases and proteases. It is interesting to note that the control frequency of 9 days is four times greater than the control frequency of 5 days. There may or may not be an age factor in the spontaneous frequency of micronuclei. However, this demonstrates the importance of conducting concurrent controls with all experiments.
The advantages of the micronucleus test in maize root tip cells are many. It is a relatively easy assay that can be conducted under acute or chronic exposure regimens. It is sensitive to acute treatments with a classic mutagen, EMS. Experiments are currently underway to simultaneously investigate the relationships between forward mutation at the wx and yg2 loci and chromosome aberrations using identical plants. (This research was funded, in part, by NIEHS Grant No. ES01895 Gen.)
Elizabeth D. Wagner and Michael J. Plewa
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