Forward mutation at the yellow-green-2 (yg2) locus of Zea mays has been used as the endpoint in various studies on mutagenesis (B. V. Conger and J. V. Carabia, 1977, 46:285-296; T. Fujii, 1980, Japan. J. Genetics 55:241-245; T. Fujii, 1981, Environ. Exper. Bot. 21:127-131). We are interested in investigating the kinetics of mutation induction in somatic cells after the test organism has been exposed to a chemical mutagen under acute or chronic treatment regimens.
Maize kernels heterozygous at the yg2 locus were used in all experiments. The kernels were surface sterilized by soaking for 5 min in a 0.5% sodium hypochlorite solution and rinsing 10 min in running tap water. In the acute tests 10 to 25 kernels per treatment group were soaked for 72 hr in aerated distilled water at 20 C. The kernels were treated for 8 hr at 20 C in aerated solutions of ethylmethanesulfonate (EMS). The concentrations of EMS tested were: 0 (control), 1mM, 10 mM and 20 mM. Following treatment the kernels were rinsed in running tap water for 16 hr. Three kernels each were planted in soil in 10 cm diameter plastic pots. The pots were placed in a growth chamber at 20 C with a 17 hr photoperiod (300 uE/m-2sec-1 PRR) for 20 to 25 days. The fourth and fifth leaves were scored for the presence of yg2 sectors. Only yellow-green colored sectors with a minimum length of 1 mm were scored. A fluorescent light box and magnifying lens were used as aids in scoring.
In the chronic treatment regimen, the kernels were surface sterilized and soaked for 72 hr in distilled water as in the acute treatments. The kernels were planted in 8.5 cm diameter paper cups filled with sterile vermiculite. Each container received 150 ml of Hoagland's solution or a known concentration of EMS in Hoagland's solution. The concentrations tested were: 0 (control), 1 uM, 10 uM, 100 uM and 1 mM EMS with 10 to 25 kernels per treatment group. When the seedlings reached 3 to 5 cm in height they were transferred to foil-covered 1 liter glass jars filled with 800 ml of the appropriate control or treatment Hoagland's solution. Three to four plants were grown in each jar in a plant growth chamber at 20 C with a 17 h photoperiod (300 uE/m-2sec-1 PRR). Water-saturated air was continuously bubbled within each jar. The jars were replenished with the control or treatment solutions during the course of the experiment and the amounts of solution added were recorded. The fourth and fifth leaves were scored for the presence of yg2 sectors.
The results of the acute treatment experiments are presented in Table 1. Although at concentrations of EMS of 1 mM there is a slight increase in the mean number of yellow-green sectors in both leaf four and leaf five, the increase is not significant. At 10 mM EMS the increase in the mean number of sectors is apparent when compared to the control values. We analyzed the summary data for leaf four and five by linear regression and calculated the coefficient of determination (r2). For the leaf four data within the range of 0 to 20 mM EMS the r2 was 0.99. For the leaf five data within the range of 0 to 20 mM EMS the r2 was 0.99. Thus the increase in yellow-green sectors in both leaf four and leaf five is dose dependent and linear.
The results of the chronic treatment experiments are presented in Table 2. From the summary data an increased number of sectors over control was induced in leaf four with an EMS concentration of 1 uM. Within the range of 0 to 100 uM EMS the frequency of mutant sectors in leaf four increased in a dose dependent and linear manner (r2 = 0.96). However, a reduction in the number of sectors was observed when a concentration of 1 mM EMS was administered to the plants. For leaf five, a dose dependent and linear response (r2 = 0.93) was induced by EMS in the concentration range of 0 to 1 mM. However, the first discernible increase in the mean number of sectors required a concentration of 10 uM EMS.
The comparison of acute versus chronic treatments of EMS and the corresponding genetic effects indicate that both treatment regimens induce mutant yg2 sectors with dose dependent and linear kinetics. The data indicate that the plants are more sensitive to a lower molar concentration of EMS in the chronic treatment regimen versus the acute treatment regimen. However, this comparison does not account for the total moles of EMS that the plants were exposed to under the two treatment regimens. A dose of a mutagen should be expressed in terms of uMoles or mMoles per kilogram of tissue. Also the rate of degradation of the mutagen must be considered when computing dosage. Finally the time of exposure should be incorporated into an assessment of dose. We are presently trying to determine these parameters and hope to use them in comparing the kinetics of mutation induction after acute versus chronic treatment conditions. (This research was funded, in part, by NIEHS Grant No. ES01895 Gen.).
Michael Plewa and Patrick Dowd
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