T-DNA-INSERT-INDEPENDENT MUTATIONS INDUCED IN TRANSFORMED PLANT-CELLSDURING AGROBACTERIUM COCULTIVATION

Transformation frequencies were determined for 1n, 2n, and 4n Nicotian a plumbaginifolia protoplast cultures in Agrobacterium-mediated gene t ransfer experiments. An unexpected large drop (50%) in plating efficie ncies was observed in the non-selected (control) In populations after transformation treatment with virulent strains. This effect was not ob served in the 2n or 4n cultures or in the In cultures when treated wit h avirulent bacteria. The mortality was disproportionally high and cou ld not be explained by the low (0.1-0.5%) transformation efficiency in the In population, indicating mutagenesis of the cell populations ind ependently from the T-DNA insertions. Mutagenesis was also indicated i n gene tagging experiments where nitrate reductase-deficient (NR(-)) m utants were selected from haploid Nicotiana plumbaginifolia protoplast s, as well as from leaf disc cultures or protoplasts of diploid plants that were heterozygotic for a mutation either in the NR apoenzyme gen e (nia/wt) or one of the molybdenum-containing cofactor genes (cnxA/wt ), after Agrobacterium co-cultivation. The chlorate-resistant isolates were tested for the T-DNA-specific kanamycin resistance trait only af ter NR-deficiency had been established. Thirty-nine independent NR-def icient mutants were analysed further by Southern blot hybridization. T here was no indication of integrated T-DNA sequences in the mutated NR genes, despite the fact that NR-deficient cells were found more frequ ently in cell populations which became transformed during the treatmen t than in the populations which did not. These observations suggest th at transformation-competent cells undergo mutagenesis during the Agrob acterium gene transfer process not only as a result of stable integrat ion events, but also through accompanying events that do not result in major changes in the mutated loci. The nature of these changes at the molecular level remains to be elucidated.