Transgenic Acacia sinuata from Agrobacterium tumefaciens-mediated transformation of hypocotyls

Abstract

Transgenic herbicide tolerant Acacia sinuata plants were produced by transformation with the bar gene conferring phosphinothricin resistance. Precultured hypocotyl explants were infected with Agrobacterium tumefaciens strain EHA105 in the presence of 100 microM acetosyringone and shoots regenerated on MS (Murashige and Skoog, 1962, Physiol Plant 15:473-497) medium with 13.3 microM benzylaminopurine, 2.6 microM indole-3-acetic acid, 1 g l(-1) activated charcoal, 1.5 mg l(-1) phosphinothricin, and 300 mg l(-1) cefotaxime. Phosphinothricin at 1.5 mg l(-1) was used for the selection. Shoots surviving selection on medium with phosphinothricin expressed GUS. Following Southern hybridization, eight independent shoots regenerated of 500 cocultivated explants were demonstrated to be transgenic, which represented transformation frequency of 1.6%. The transgenics carried one to four copies of the transgene. Transgenic shoots were propagated as microcuttings in MS medium with 6.6 microM 6-benzylaminopurine and 1.5 mg l(-1) phosphinothricin. Shoots elongated and rooted in MS medium with gibberellic acid and indole-3-butyric acid, respectively both supplemented with 1.5 mg l(-1) phosphinothricin. Micropropagation of transgenic plants by microcuttings proved to be a simple means to bulk up the material. Several transgenic plants were found to be resistant to leaf painting with the herbicide Basta.