IMPROVED EFFICIENCY FOR T-DNA-MEDIATED TRANSFORMATION AND PLASMID RESCUE IN ARABIDOPSIS-THALIANA

A vector was constructed for the isolation of gene fusions to the lacZ reporter gene following T-DNA integration into the genome of Arabidop sis thaliana. To facilitate the generation of tagged A. thaliana plant s, we established a modified method for high-frequency transformation of A. thaliana by Agrobacterium tumefaciens. The main modification req uired was to inhibit the methylation of T-DNA in the transformed calli . Apparently, cytosine residues of the nos-nptII gene used as a select able marker were methylated, and the expression of this gene was suppr essed. Treatment of the calli with the cytosine methylation inhibitor 5-azacytidine led to a dramatic increase (from 3% to 96%) in the regen eration of transformed (kanamycin-resistant) shoots. A total of 150 tr ansgenic plants were isolated, and in 17 of these expression of the la cZ reporter was detected by in situ staining. The T-DNA insert togethe r with flanking plant DNA sequences was cloned into Escherichia coli b y plasmid rescue from some of the T3 transformants that harbored one c opy of the integrated T-DNA. Comparison of the rescued DNA with the co rresponding DNA of the transgenic plant showed that most of the rescue d plasmids had undergone rearrangements. These rearrangements could be totally avoided if an mcrAB (modified cytosine restriction) mutant of E. coli was used as the recipient in plasmid rescue.