Abstract
Gene targeting (GT) by homologous recombination (HR) in higher plants is thought to be difficult because its targeting frequency is too low to be detected. We have developed an efficient HR-promoted GT procedure in rice by a large-scale Agrobacterium-mediated transformation with a strong positive-negative selection (PNS) and subsequent PCR screening and reproducibly succeeded in generating transgenic rice plants having one of eight different genes modified. In addition, we detected randomly integrated and truncated T-DNA segments containing our positive marker without intact negative markers in surviving calli with PNS. We also found both expected true gene targeting and one-sided invasion events. To look into the recombination crossover points, we performed GT with a vector having several point mutations in homologous regions and determined their sequences in a few targeted plants obtained. These results are discussed with regard to molecular processes of homologous and nonhomologous recombination.
