Analysis of junctions between T-DNA and plant genome in transgenic Arabidopsis thaliana

To understand the mechanism for Agrobacterium-mediated transformation of plants, we analyzed the junctions between T-DNA and plant genome, using 12 individual transgenic lines transformed with 7 different plant expression constructs. After performing TAIL-PCR, we sequenced 42 PCR products for analysis. All of the RBs were nicked by VirD1/VirD2 proteins whereas only 62% of the LBs were. Additional deletions of the adjacent T-DNA region were found in 50% of the RBs. For the LBs, only two showed such additional deletions. Filler DNAs were observed in 60% of the RBs (ranging from 1 to 132 nucleotides) versus 54% for the LBs. We also found that only 25% of the RBs were integrated into the plant genome while the rest showed integration into the expression constructs. In comparison, all of the LBs were integrated, except for one that was considered intact. Our results suggest that the origin for a binary vector backbone (BVB) in the plant genome is due not only to a mistake in the VirD1/VirD2 proteins within the T-DNA borders but also because of the linkage of RBs to either the T-DNA or BVB.