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.