INDUCTION OF SOMATIC DNA-DAMAGE AS MEASURED BY SINGLE-CELL GEL-ELECTROPHORESIS AND POINT MUTATION IN LEAVES OF TOBACCO PLANTS

The induction and measurement of DNA damage in nuclei of plant tissues is a new area of study with the alkaline single cell gel electrophore sis/comet assay. Methods to isolate plant cell nuclei cause high level s of DNA damage which are detected by the comet assay. We developed a method to isolate nuclei from leaf tissue of Nicotiana tabacum (a(1)()/a(1); a(2)(+)/a(2)) in a modified Sorensen buffer that resulted in c onstant, low tail moment values for the negative controls. After treat ing intact tobacco plants with 1-8 mM ethyl methanesulfonate (EMS) we obtained a direct concentration-response with an average median tail m oment of 65.9 +/- 4.4 mu m for plants exposed to the highest EMS conce ntration as compared to the median control tail moment value of 4.1 +/ - 0.8. We found that the highest resolution was obtained with electrop horetic conditions of 0.74 V/cm at 300 mA for 20 min. Multiple leaves could be analyzed per plant within each treatment group and the tail m oments were not significantly different. Tobacco seedlings were treate d with EMS in the same manner as used for the comet assay and mutation s were induced in the leaf primordia, The mean mutant frequency for th e control was 1.46 +/- 0.20 mutant sectors/leaf. The mutant frequency increased in a concentration dependent manner; the mutant frequency in duced by 8 mM EMS was 37.89 +/- 2.37 mutant sectors/leaf, The comet ta il moment values and the leaf mutant frequency were highly correlated (r = 0.98), The genetic response factor was calculated by the ratio of the difference in the response within the linear portion of each conc entration-response curve divided by the slope of the curve, The geneti c response factor for the tail moment was 7.82 while the value for mut ation induction was 7.76. In this paper we describe a sensitive method with high resolution to apply the alkaline comet assay to plant leave s, The comet assay response was compared to that of induced point muta tion. With this sensitive method for nuclei isolation from plant leave s, the alkaline SCGE assay could be incorporated into in situ plant en vironmental monitoring. (C) 1998 Elsevier Science B,V. All rights rese rved.