Analysis of octopine left border-directed DNA transfer from Agrobacterium to plants

Abstract

We constructed a binary plasmid, pVR30, with a neomycin phosphotransferase II (nptII) plant expression cassette flanked by a pTiA6 left border on its right and a pTiA6 right border on its left. This plasmid was used to study transfer of DNA to plants from a left border in the presence of a right border. Infection of tobacco leaf discs with a wild type octopine strain ofAgrobacterium tumefaciens harbouring the binary plasmid resulted in the generation of kanamycin resistant calli at 18 to 26% frequency. Southern hybridization analysis of DNA isolated from eight transformed lines to different probes indicated that left border could mediate DNA transfer to plants in the presence of a right border in cis. Our results also suggest that transfer events corresponding to transfer of T-centre DNA of octopine Ti plasmid pTiA6 do occur. We have shown the relevance of left border-initiated T-DNA transfer by specifically selecting for such events and have confirmed it by Southern hybridization analysis. We also found that a border could be skipped in a few T-DNA transsfer events.