PRIMISULFURON HERBICIDE-RESISTANT TOBACCO CELL-LINES - APPLICATION OF FLUCTUATION TEST DESIGN TO INVITRO MUTANT SELECTION WITH PLANT-CELLS

A fluctuation test experimental design was used to select tobacco cell cultures resistant to a new sulfonyl urea herbicide, primisulfuron (2-[3-(4,6-bis(difluoromethoxy)-pyrimidin-2-yl)-ureidosulfonyl]benzoic acid methylester). From approximately 3.95 × 109 plated cells a total of 2455 resistant colonies were obtained, 2362 from selection at 100 µg/L, another 93 from selection at 1000 µg/L. Selection at 100 µg/L yielded resistant colonies at a frequency about 60 times higher than did selection at 1000 µg/L. Following mutagenesis with N-ethyl-N-nitroso-urea, a bulk culture and 20 separate individual cultures were employed in a fluctuation test experiment to calculate mutation rates. No significant difference was found in the distribution of mutants among the individual and the bulk cultures, but the ratio of variance to mean (≫ 1) indicated a spontaneous origin of the mutants. The mutagen treatment increased the frequency of resistant lines about 25-fold. The mutation rate per cell generation was calculated to be 2.7 × 10-8 for the non-mutagenized bulk culture selected at 100 µg/L. Mutation rates were almost 30-times higher for the mutagen-treated bulk and the individual cultures selected at 100 µg/L. Mutagenized bulk and individual cultures selected at 1000 µg/L had mutation rates of 2.9 × 10-8 and 4.4 × 10-8 per cell generation, respectively. About 45 % of the lines originally selected at 100 µg/L grew vigorously upon transfer to 1000 NAg/L primisulfuron, indicating effective selection for high level resistance even though the initial selection pressure was comparatively low. © 1991, Gustav Fischer Verlag, Stuttgart. All rights reserved.