Nonreciprocal homologous recombination between Agrobacterium transferred DNA and a plant chromosomal locus.

Abstract

Previously, we demonstrated the occurrence of gene targeting in tobacco cells after Agrobacterium-mediated transformation. In these experiments a defective kanamycin resistance (Kmr) gene residing at a chromosomal location was restored via homologous recombination with an incoming transferred DNA (T-DNA) repair construct (pSDM101) containing a different defective Kmr gene. In this article we describe gene targeting experiments with the same target line, but using an improved repair construct, pSDM321. In one of the Kmr calli obtained after transformation with pSDM321 (line A) the product of homologous recombination was detected using PCR. Further molecular analysis revealed that the defective Kmr gene present on the incoming T-DNA had been restored via homologous recombination with the target locus. The target locus was left unchanged and the corrected T-DNA was found to be inserted on the same chromosome but not close to the target locus. This paper presents molecular evidence in plants for the conversion of an introduced DNA molecule (in this case, T-DNA) by a homologous chromosomal locus.