Agrobacterium-mediated transformation of White Ponni, a non-basmati variety of indica rice (Oryza sativa L.)

We report successful Agrobacterium-mediated transformation of a popular rice variety White Ponni, a non-basmati indica rice (Oryza sativa L.). Scutellumderived calluses of White Ponni were transformed with Agrobacterium strain LBA4404 (pSB1) harbouring the binary vector pMKU-RF2 with rice chi11 gene. Five independent transgenic White Ponni plants were generated from hygromycin-resistant calluses. Stable integration of the transgene was confirmed and copy numbers were determined by Southern analysis. Among the five plants, four possessed single-copy T-DNA integration events while one was found to have two integrated copies of T-DNA. Western analysis revealed a higher level of chitinase accumulation in all the five T0 plants. Progeny analysis of T0 plants confirmed the inheritance of the transgene to the next generation

The current status of plant transformation technologies

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Kanamycin sensitivity of cultured tissues of Piper nigrum L.

The kanamycin sensitivity for callus growth was studied in vitro in a cultivar of black pepper (Piper nigrum) using cotyledons as explants to investigate the suitability of kanamycin resistance as a selectable marker for Agrobacterium mediated transformation. Callus formation was completely inhibited at 50 ug ml-1 and above concentrations of kanamycin suggesting that 50 ug ml-1 is the minimum concentration needed to select the transformed tissues. &nbsp

Kanamycin sensitivity of cultured tissues of Piper nigrum L.

The kanamycin sensitivity for callus growth was studied in vitro in a cultivar of black pepper (Piper nigrum) using cotyledons as explants to investigate the suitability of kanamycin resistance as a selectable marker for Agrobacterium mediated transformation. Callus formation was completely inhibited at 50 ug ml-1 and above concentrations of kanamycin suggesting that 50 ug ml-1 is the minimum concentration needed to select the transformed tissues. &nbsp

Opines stimulate induction of the vir genes of the Agrobacterium tumefaciens Ti plasmid.

Upon incubation of Agrobacterium tumefaciens A348 with acetosyringone, the vir genes encoded by the Ti (tumor-inducing) plasmid are induced. The addition of certain opines, including octopine, nopaline, leucinopine, and succinamopine, enhanced this induction 2- to 10-fold. The compounds mannopine, acetopine, arginine, pyruvate, and leucine did not stimulate the induction of the vir genes to such an extent. The enhancement of vir gene induction by opines depended on acetosyringone and the genes virA and virG. Opines stimulated the activity of the vir genes, the double-stranded cleavage of the T (transferred)-DNA at the border repeat sequences, and the production of T-strands by the bacterium. The transformation efficiency of cotton shoot tips was markedly increased by the addition of acetosyringone and nopaline at the time of infection

Dimerization of VirD2 Binding Protein Is Essential for Agrobacterium Induced Tumor Formation in Plants

10.1371/journal.ppat.1003948PLoS Pathogens103

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

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