Agrobacterium-mediated transformation of rice using immature embryos or calli induced from mature seed

Here, we provide comprehensive, highly efficient protocols for Agrobacterium tumefaciens-mediated transformation of a wide range of rice genotypes. Methods that use either immature embryos (japonica and indica rice) or calli (japonica cultivars and the indica cultivar, Kasalath) as a starting material for inoculation with Agrobacterium are described. Immature embryos are pretreated with heat and centrifugal force, which significantly enhances the efficiency of gene transfer, and then infected with Agrobacterium. Callus is induced from mature seeds and infected. Transformed cells proliferated from these tissues are selected on the basis of hygromycin resistance, and transgenic plants are eventually regenerated. A single immature japonica or Kasalath embryo will produce between 10 and 18 independent transgenic plants; for other non-Kasalath indica varieties, the number of transgenic plants expected will be between 5 and 13. For japonica and Kasalath, transformants should be obtained from between 50 and 90% of calli. From inoculation with Agrobacterium to transplanting to soil will take 55 d for japonica and Kasalath, and 74 d for indica other than Kasalath using the immature embryo method, and 50 d for japonica and Kasalath using the callus method.