Insertional mutagenesis in Arabidopsis thaliana

Recently, a number of mutant gene loci in the Arabidopsis thaliana plant genome have been identified through insertional mutagenesis. In this review, we evaluate different methods used for Agrobacterium tumefaciens-mediated T-DNA insertional mutagenesis with regard to their mutation frequencies and conclude that a major breakthrough in the isolation of genes involved in plant development has been acheived. To provide a specific example, we summarize recent progress made in the understanding of flower morphogenesis at the molecular level through the study of homeotic genes obtained via gene tagging. T-DNA gene fusion vectors are being discussed that will allow the isolation of plant regulatory sequences with particular cell or tissue specificity, or that are controlled by specific external stimuli. Finally, we report on the approaches followed to convert the maize transposons Ac/Ds into valuable gene tags for use in a heterologous host such as Arabidopsis.     

Mutagenesis by insertion of the drug resistance transposon Tn7 applied to the Ti plasmid of Agrobacterium tumefaciens

Insertions of the linked trimethoprim and streptomycin resistance transposon Tn7 were isolated in a cointegrated plasmid formed between the Ti-plasmid of Agrobacterium tumefaciens strain B6S3 and the broad host range resistance plasmid RP4. By using the spontaneous dissociation of this cointegrate, we could demonstrate that these insertions had occurred into the Ti as well as into the RP4 part of the cointegrated plasmid. Among the insertions in the Ti part, mutants affected in different Ti plasmid-determined phenotypes, including non-oncogenic mutants, were detected. These mutants are useful for the physical mapping of these plasmid markers.     

Agrobacterium-Mediated Gene Transfer Results Mainly in Transgenic Plants Transmitting T-DNA as a Single Mendelian Factor

Forty-four independent transformed tobacco plants were obtained from a cocultivation experiment with Agrobacterium tumefaciens strains carrying modified Ti-plasmids. The transformed plants were either self-fertilized or crossed with nontransformed plants or with other transformed plants. The segregation of a phenotypic marker (kanamycin resistance) in the progenies of these plants was determined. In 40 cases out of 44, the segregation of the kanamycin resistance marker is consistent with Mendelian genetics. Among these 40 clones, 35 contain a single kanamycin resistance locus. The five others segregate two independent resistance loci. In two of the single insert clones, the segregation ratio after selfing indicates that the T-DNA insertion may have caused a recessive lethal mutation.     

Transfer of Ti plasmids between Agrobacterium strains by mobilisation with the conjugative plasmid RP4

Summary The P type conjugative plasmid RP4 has been shown to be able to promote the transfer of the Agrobacterium Ti-plasmid. The results provide additional evidence that agrocin 84 sensitivity, exclusion of phage AP₁, ability to catabolize the guanidine derivatives octopine and nopaline and tumor inducing ability, are Ti-plasmid determined properties. Furthermore, the results strongly support the notion that at least part of the Ti-plasmid is transferred from the bacterium to the target plant cells, since it was demonstrated that Ti-plasmid linked genes specify the synthesis of octopine or nopaline by crown-gall tumor cells.