Agrobacterium tumefaciens-mediated transformation of japonica and indica rice varieties

Genetic transformation of rice (Oryza sativa L.) mediated by Agrobacterium tumefaciens has been confirmed for japonica varieties and extended to include the more recalcitrant indica varieties. Immature embryos were inoculated with either A. tumefaciens At656 (pChTL56) or LBA4404 (pTOK233). Experimental conditions were developed initially for immature embryos treated with strain At656, based upon both transient and stable beta-glucuromdase (GUS) activities. However, plant regeneration following selection on G418 (pCNL56 contained the nptII gene) did not occur. Using the same basic protocol. but inoculating immature embryos of rice with LBA4404 (pTOK233), resulted in efficient (about 27%) production of transgenic plants of the japonica variety, Radon, and an acceptable efficiency (from 1-5%) for the indica varieties IR72 and TCS10. Transformation was based upon resistance to hygromycin (pTOK233 contains the hpt gene), the presence of GUS activity (from the gusA gene), Southern blots for detection of the integrated gusA gene, and transmission of GUS activity to progeny in a Mendelian 3:1 segregation ratio. Southern blots indicated two to three copies of the gene integrated in most transformants. Transgenic plants of both the japonica and indica varieties were self-fertile and comparable in this respect to seed-grown plants. Key factors facilitating the transformation of rice by Agrobacterium tumefaciens appeared to be the use of embryos as the explant, the use of hygromycin as the selection agent (which does not interfere with rice regeneration), the presence of extra copies of certain vir genes on the binary vector of pTOK233, and maintaining high concentrations of acetosyringone for inducing the vir genes during co-cultivation of embryos with Agrobacterium.