Extrachromosomal homologous recombination and gene targeting in plant cells after Agrobacterium mediated transformation

We determined whether T-DNA molecules introduced into plant cells using Agrobacterium are suitable substrates for homologous recombination. For the detection of such recombination events different mutant versions of a NPTII construct were used. In a first set of experiments protoplasts of Nicotiana tabacum SR1 were cocultivated with two Agrobacterium tumefaciens strains. Each strain contained a different T-DNA, one carrying a 5' deleted NPTII gene and the other a NPTII gene with a 3' deletion. A restored NPTII gene was found in 1-4% of the protoplasts that had been cotransformed with both T-DNAs. Restoration of the NPTII gene could only be the consequence of homologous recombination between the two different T-DNAs in the plant cell, since the possibility of recombination in Agrobacterium was excluded in control experiments. In subsequent experiments was investigated the potential use of Agrobacterium for gene targeting in plants. A transgenic tobacco line with a T-DNA insertion carrying a defective NPTII gene with a 3' deletion was transformed via Agrobacterium with a T-DNA containing a defective NPTII repair gene. Several kanamycin resistant plant lines were obtained with an intact NPTII gene integrated in their genome. In one of these lines the defective NPTII gene at the target locus had been properly restored. Our results show that in plants recombination can occur between a chromosomal locus and a homologous T-DNA introduced via A. tumefaciens. This opens the possibility of using the Agrobacterium transformation system for site directed mutagenesis of the plant genome.