Agrobacterium-mediated transformation of Australian rice cultivars Jarrah and Amaroo using modified promoters and selectable markers

We report the first successful Agrobacterium-mediated transformation of Aus tralian elite rice cultivars, Jarrah and Amaroo, using binary vectors with our improved promoters and selectable markers. Calli derived from mature em bryos were used as target tissues. The binary vectors contained hph (encodi ng hygromycin resistance) or bar (encoding herbicide resistance) as the sel ectable marker gene and uidA (gus) or sgfpS65T as the reporter gene driven by different promoters. Use of Agrobacterium strain AGL1 carrying derivativ es of an improved binary vector pWBVec8, wherein the CaMV35S driven hph gen e is interrupted by the castor bean catalase 1 intron, produced a 4-fold hi gher number of independent transgenic lines compared to that produced with the use of strain EHA101 carrying the binary vector pIG121-Hm wherein the C aMV35S driven hph is intronless. The Ubiquitin promoter produced 30-fold hi gher beta-glucuronidase (GUS) activity (derivatives of binary vector pWBVec 8) in transgenic plants than the CaMV35S promoter (pIG121-Hm). The two modi fied SCSV promoters produced GUS activity comparable to that produced by th e Ubiquitin promoter. Progeny analysis (R-1) for hygromycin resistance and GUS activity with selected lines showed both Mendelian and non-Mendelian se gregation. Lines showing very high levels of GUS activity in T-0 showed a r educed level of GUS activity in their T-1 progeny, while lines with moderat e levels of GUS activity showed increased levels in T1 progeny. Stable heri table green fluorescent protein (GFP) expression was also observed in few t ransgenic plants produced with the binary vector pTO134 which had the CaMV3 5S promoter-driven selectable marker gene bar and a modified CaMV35S promot er-driven reporter gene sgfpS65T.