| During a gene targeting project: (1) an efficient negative selection marker was created, (2) gene targeting vectors containing extended left border homology to the Arabidopsis thaliana Nia2 gene were created for Agrobacterium-mediated gene transfer experiments, (3) the frequent collinear transfer and integration of the entire binary vector following Agrobacterium-mediated gene transfer demonstrated that Agrobacterium can transfer long DNA molecules to the plant.;Use of a chimeric Rhodococcus indole oxidase gene controlled by an enhanced 35S cauliflower mosaic virus promoter, resulted in a 85, 91 and 100% reduction in transformation efficiency following Agrobacterium-mediated gene transfer of Nicotiana tabacum (leaf disk and NT1 suspension cells) and Arabidopsis thaliana root explants, respectively. Its stringent negative selection makes this marker a useful tool for gene targeting experiments.;Gene targeting vectors containing extended left end homology to the nitrate reductase (Nia2) gene were created for Agrobacterium-mediated gene transfer of A. thaliana. The longest homology was 1.97 kbp fused to a promoterless neomycin phosphotransferase gene. A hygromycin phosphotransferase gene was also present for initial screening. Transformants were produced by either root co-cultivation or in planta vacuum infiltration. Although a high frequency of kanamycin resistant transformants were obtained (up to 39%), no gene targeting events were detected in the greater than 1500 hygromycin transformants produced as determined by selection, PCR, Southern and genetic analysis. However, random T-DNA insertion mutants were detected including one chlorophyll deficient mutant, three embryo lethal mutants and one delayed germination mutant.;Further analysis of transformants revealed that a large proportion (up to 62%) of them contained the whole binary vector integrated into the genome and not merely the defined T-region. Transfer of the whole binary vector is a frequent occurrence and draws into question the definition of a T-region and left end.;Gene targeting in plants has not been obtained but the new negative selection marker and a better understanding of the transformation process may lead to gene targeting in the near future. |
