Reduction of Chloroplast DNA Content in Solanum nigrum Suspension Cells by Treatment with Chloroplast DNA Synthesis Inhibitors

Suspension cell cultures of Solanum nigrum were grown in the presence of six different chloroplast DNA synthesis inhibitors in order to determine whether the pool size of chloroplast DNA (cpDNA) could be selectively reduced relative to the nuclear DNA content. One of the effects of the inhibitors was a reduction in cell growth and viability. Cell growth (fresh weight) was reduced 50% (in 8 day cultures) by: 100 micromolar bisbenzimide, 8 micromolar ethidium bromide, 0.3 micromolar 5-fluordeoxyuridine (Fudr), 200 micromolar nalidixic acid, 30 micromolar novobiocin, or 10 micrograms per milliliter rifampicin. At these concentrations, three of the inhibitors, ethidium bromide, Fudr, and rifampicin, also substantially reduced the viability of the cultures. Analyses of the chloroplast and nuclear DNA content per gram fresh weight by dot blot hybridizations indicated that the reduction of cpDNA content was greatest at inhibitor concentrations which reduced cell growth by more than 50% but this depended on the culture conditions. For example, the two DNA gyrase inhibitors, nalidixic acid and novobiocin, were more effective in lowering cpDNA content in cultures which were transferred (2 × 4 days) once during the eight day incubation. Because several inhibitors were toxic to cell growth, the DNA content of treated cells was also determined on the basis of cell (protoplasts) number. Analyses of nuclear and cpDNA content per cell for each treatment indicated that only the DNA gyrase inhibitors, nalidixic acid, and novobiocin reduced cpDNA content. Neither inhibitor reduced nuclear DNA content. These results suggest that DNA gyrases participate in cpDNA replication. The selective reduction of cpDNA content in regeneratable cultures may facilitate the generation and selection of cpDNA mutants or transformants from higher plants.