Targeted recombination in plants using Agrobacterium coincides with additional rearrangements at the target locus.

The use of Agrobacterium for gene targeting in plants has been investigated. Leaf protoplasts of five transgenic tobacco lines, containing a T-DNA insertion with a defective npt-II gene at different positions in the plant genome, were transformed via Agrobacterium with a T-DNA containing a npt-II repair gene. After selection for kanamycin resistance and PCR analysis six recombinants were derived from four of the target lines. The recombination frequencies were similar for the different target lines with one recombinant from approximately 3×105 transformants. Apparently gene targeting is more or less independent of the location of the target construct in the plant genome. Molecular analysis revealed that gene targeting had occurred in five of the six recombinant lines. However precise recombination had occurred in only one line, while in the other four lines restoration of the npt-II gene was accompanied by a deletion of part of the target locus. The sixth recombinant line showed restoration of the npt-II gene of the incoming T-DNA construct which was inserted in the plant genome at a position closely linked to the target locus. The different recombination products favour a model in which recombination is via gene conversion followed by reintegration of the synthesized DNA via homologous or illegitimate recombination rather than a reciprocal exchange of DNA between two cross-overs.