Effects of the herbicide paraquat on Nicotiana tabacum and isolation of paraquat resistant mutants

The purpose of this study was to determine the effects of the herbicide paraquat (l, l'-dimethyl-4, 4'-dipyridylium salt) on Nicotiana tabacum var. Wisconsin 38 in culture and to isolate paraquat-resistant mutants. Tobacco cells were grown in modified Linsmaier and Skoog media as callus and liquid suspension cultures. Paraquat caused a marked decrease in growth of tobacco callus at l0-5M and total inhibition of growth at 10-4M. Four methods were used for the isolation of resistant cell lines: 1) X-irradiation of callus tissue at 10,000R prior to selection at 10-5M paraquat, 2) selection of callus tissue at 10-5M paraquat, 3) selection of callus tissue at 5 x 10-6M paraquat prior to selection at 10-5M, and 4) plating of liquid cell cultures on agarized medium containing paraquat at 10-5M. Actively growing resistant tissues were selected, transferred to non-selective medium for a period of non-selective growth, and retested on selective medium. Cell lines which retained resistance to paraquat at 10-4M (lethal level) were transferred to regeneration medium. Plantlets from regenerating cell lines were tested for resistance by floating leaves on a paraquat solution at 10-6M, 10-5M, and 10-4M. Additionally, leaf derived callus from some cell lines was tested for resistance at 10-5M and 10-4M paraquat.

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iv A higher percentage of cell lines retaining the resistant phenotype was obtained using X-irradiation as a mutagenic agent as compared to no mutagen treatment. Sublethal selection at 5 x 10-6M paraquat prior to selection at 10-5M paraquat did not enhance recovery of resistant mutants as compared to selection only at 10-5m. Selection from plated cell suspension cultures did increase the percentage of resistant cell lines obtained as compared to selection using callus tissue. Some resistant cell lines were not capable of being regenerated. Approximately 35 percent of the regenerated plants retained resistance. Leaf derived callus from resistant plants of two cell lines retained resistance at l0-4M paraquat. Leaf derived callus from non-resistant plants of five cell lines retained resistance at 10-4M paraquat.

Possible protection mechanisms against paraquat toxicity were considered. Increased copper, a component of superoxide dismutase, in the growth medium enhanced growth of tobacco callus over that expected for paraquat and copper. Increased iron, a component of catalases and peroxidases, in the growth medium did not enhance growth of tobacco callus.