We conducted an investigation using the yellow-green-2 (yg2) locus as the genetic endpoint for the induction of mutation in somatic cells following exposure to ionizing radiation. The purpose of this research was to determine the sensitivity of the yg2 locus to ionizing radiation and to develop a standard for use in comparing genetic damage induced by chemical mutagens.
Maize plants heterozygous at the yg2 locus have a normal green phenotype. Loss of the dominant allele (Yg2) by point mutation or a chromosome break will allow the expression of the recessive yg2 allele as a yellow-green sector in the leaf.
The fourth and fifth leaves are scored in this assay. Dormant maize embryos contain 1,500 primordial cells for leaf four and 250 primordial cells for leaf five (H. H. Smith and H. H. Rossi, Rad. Res. 28:302).
Kernels heterozygous at the yg2 locus were used in these experiments. The kernels were surface sterilized by soaking for 5 min in a 0.5% sodium hypochlorite solution and rinsing for 10 min in running tap water. For each treatment group 33 kernels were soaked for 72 hr at 20 C in aerated distilled water. This procedure allows for one cell division to occur, thus the target population size is approximately 3,000 and 500 cells for leaf four and five, respectively (B. V. Conger and J. V. Carabia, 1977, Mut. Res. 46:285). The kernels were treated by exposure to 137Cesium in a J. L. Shepard irradiator. The radiation doses were: 0 (control), 50, 100, 250, 500 and 1,000 rads of gamma radiation. Following treatment, the kernels were planted in soil in 10 cm diameter plastic pots, three kernels per pot. The pots were placed in a plant growth chamber at 20 C with a 17 hr photoperiod (300 uE m-2 sec-1 PRR) for 20 to 25 days. The fourth and fifth leaves were scored for the presence of yellow-green sectors with a minimum length of 1 mm. A fluorescent light box and magnifying lens were used as aids in scoring.
The results of the experiments are presented in Table 1. The data are expressed as the mean number of yellow-green sectors per leaf four or leaf five.
The variance within each group is expressed as the standard error (SE) of the mean. The data for leaves four and five are separately compiled and plotted in Figures 1 and 2. The primordial cells of both leaves are very sensitive to mutation induction by gamma rays.
For leaf four, the frequency of yellow-green sectors per leaf ranged from a mean of 0.11 + 0.04 for the control to 9.19 + 0.58 for kernels exposed to 500 rads. A radiation dose of 1,000 rads extensively damaged the leaves and they were unscorable. The dose-response curve for leaf four exhibited linear kinetics, especially within the range of 0 to 250 rads (Figure 1). The induction of yellow-green sectors and the dose of gamma were highly correlated (r = 0.97). The frequency of yellow-green sectors per leaf for leaf five ranged from 0 for the control to 1.22 + 0.16 for kernels exposed to 500 rads. As in the case of leaf four, a dose of 1,000 rads severely damaged the fifth leaf of the plants. Although the mean frequency of sectors per leaf five was considerably lower than leaf four, the dose-response curve exhibited linear kinetics throughout the dose range analyzed (Figure 2). The induction of leaf sectors and the radiation dose were highly correlated (r = 0.98).
The yg2 locus is a sensitive and a relatively rapid assay for studies in environmental mutagenesis involving higher organisms. The data on gamma radiation presented here shall serve as a standard to compare the effective mutagenicity of chemical agents. (This research was funded, in part, by NIEHS Grant No. ES01895 GEN.)
Patrick A. Dowd and Michael J. Plewa
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