Plant activation of m-phenylenediamine by tobacco, cotton, and carrot cell suspension cultures

Tobacco, cotton, and carrot plant cell suspension cultures activated the promutagen m-phenylenediamine into a mutagen as detected by Salmonella typhimurium strain TA98 with the use of the plant cell/microbe coincubation assay. For each cell line, mid-log phase plant cells at a density of 100-150 mg/ml were coincubated for 1 hr with concentrations of m-phenylenediamine that ranged from 0.1 to 10 mumol per reaction tube in the preincubation test of the plant cell/microbe coincubation assay. Further experiments were conducted to optimize the activation response for each plant cell line. The density of plant cells in the reaction mixture, the time of coincubation of the reaction mixture, and the stage of the growth curve at which the plant cells were harvested for use in the reaction mixture all affected the response. Experiments that used the conditions determined as optimum for each plant cell line were then conducted. With each cell line, the optimized conditions enhanced the activation response that had been observed with the preliminary conditions. A ranking order based on the concentration-response curves indicated a relationship of tobacco cells much greater than carrot cells greater than cotton cells. Tobacco cells were able to activate m-phenylenediamine into a mutagen at concentrations of less than 10 nmol per plate when using TA98 as the genetic indicator organism. Finally, experiments to determine the type of genetic lesion induced by plant-activated m-phenylenediamine were conducted. By using five of the Ames strains, m-phenylenediamine was shown to be active in inducing revertants in strains TA1538 and TA98 following activation by tobacco cells under the optimized conditions. We conclude that m-phenylenediamine is activated by plant cells into a mutagen that primarily induces frameshift mutations.