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 investi gated. Leaf protoplasts of five transgenic tobacco lines, containing a T-DNA insertion with a defective npt-II gene at different positions i n the plant genome, were transformed via Agrobacterium with a T-DNA co ntaining a npt-II repair gene. After selection for kanamycin resistanc e and PCR analysis six recombinants were derived from four of the targ et lines. The recombination frequencies were similar for the different target lines with one recombinant from approximately 3x10(5) transfor mants. Apparently gene targeting is more or less independent of the lo cation of the target construct in the plant genome. Molecular analysis revealed that gene targeting had occurred in five of the six recombin ant lines. However precise recombination had occurred in only one line , while in the other four lines restoration of the npt-ll gene was acc ompanied by a deletion of part of the target locus. The sixth recombin ant line showed restoration of the npt-ll gene of the incoming T-DNA c onstruct which was inserted in the plant genome at a position closely linked to the target locus. The different recombination products favou r a model in which recombination is via gene conversion followed by re integration of the synthesized DNA via homologous or illegitimate reco mbination rather than a reciprocal exchange of DNA between two cross-o vers.