Agrobacterium-mediated transformation of indica rice cv. ADT 43

A reproducible and highly efficient protocol for Agrobacterium tumefaciens-mediated transformation of indica rice (Oryza sativa L. subsp. indica cv. ADT 43) was established. Prior to transformation, embryogenic callus were induced from mature seeds incubated on Linsmaier and Skoog (LS) medium supplemented with 2.5 mg l(-1) 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 mg l(-1) thiamine-HCl. Callus, intact mature seeds, and other in vitro derived explants (leaf bases, leaf blades, coleoptiles, and root-tips) were immersed in a bacterial suspension culture of A. tumefaciens strain EHA 105, OD600 of 0.8, and co-cultivated on LS medium for 2 days in the dark at 25 +/- A 2A degrees C. Based on GUS expression analysis, 10 min incubation time of explants on a co-cultivation medium containing 100 mu M acetosyringone was optimum. Following beta-glucuronidase (GUS) assay and polymerase chain reaction (PCR) analysis, transformants were identified. Stable integration of the transgene was confirmed in four putatively transformed T-0 plants by Southern blot analysis. The copy number of the transgene in these lines, one to two, was then determined. Among the observations made, necrosis of co-cultivated explants was a problem, as well as sensitivity of callus to Agrobacterium infection. Levels of necrosis could be minimized following co-cultivation of explants in a medium consisting of 30% LS and containing 10 g l(-1) (14), polyvinyl pyrrolidone, 10% coconut water, and 250 mg l(-1) timentin (15:1). This latter medium also increased the final transformation efficiency to 15.33%.