Transgenic Research

, Volume 3, Issue 5, pp 317–325 | Cite as

T-DNA-insert-independent mutations induced in transformed plant cells duringAgrobacterium co-cultivation

  • László Márton
  • Milan Hrouda
  • Attila Pécsváradi
  • Mihály Czako
Papers

Abstract

Transformation frequencies were determined for 1n, 2n, and 4n Nicotiana plumbaginifolia protoplast cultures inAgrobacterium-mediated gene transfer experiments. An unexpected large drop (50%) in plating efficiencies was observed in the non-selected (control) 1n populations after transformation treatment with virulent strains. This effect was not observed in the 2n or 4n cultures or in the 1n cultures when treated with avirulent bacteria. The mortality was disproportionally high and could not be explained by the low (0.1–0.5%) transformation efficiency in the 1n population, indicating mutagenesis of the cell populations independently from the T-DNA insertions. Mutagenesis was also indicated in gene tagging experiments where nitrate reductase-deficient (NR) mutants were selected from haploidNicotiana plumbaginifolia protoplasts, as well as from leaf disc cultures or protoplasts of diploid plants that were heterozygotic for a mutation either in the NR apoenzyme gene (nia/wt) or one of the molybdenum-containing cofactor genes (cnxA/wt), afterAgrobacterium co-cultivation. The chlorate-resistant isolates were tested for the T-DNA-specific kanamycin resistance trait only after NR-deficiency had been established. Thirty-nine independent NR-deficient mutants were analysed further by Southern blot hybridization. There was no indication of integrated T-DNA sequences in the mutated NR genes, despite the fact that NR-deficient cells were found more frequently in cell populations which became transformed during the treatment than in the populations which did not. These observations suggest that transformation-competent cells undergo mutagenesis during theAgrobacterium gene transfer process not only as a result of stable integration 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.

Keywords

Agrobacterium mutagenesis Nicotiana plumbaginifolia nitrate reductase ploidy transformation 

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Copyright information

© Chapman & Hall 1994

Authors and Affiliations

  • László Márton
    • 1
  • Milan Hrouda
    • 2
  • Attila Pécsváradi
    • 3
  • Mihály Czako
    • 1
  1. 1.Department of Biological SciencesUniversity of South CarolinaColumbiaUSA
  2. 2.Institute for Crop ProductionPraha 6, RuzyneCzech Republic
  3. 3.Department of Plant PhysiologyAttila József UniversitySzegedHungary

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