An Efficient Protocol for Agrobacterium-mediated Genetic Transformation in Rubber Tree (Hevea brasiliensis)

We report high frequency genetic transformation in Hevea brasiliensis (Hb) using anther derived embryogenic calli as the target tissue which is one of the important factors to yield maximum transformation events. Agrobacterium-mediated genetic transformation was carried out by infecting intact explants such as immature anther and ovule, sixty day old callus and embryogenic callus derived from immature anther and ovule explants. Two binary vectors (EHA101 Agrobacterium strain used) containing the sequence coding for Hb MnSOD gene in the sense and antisense orientation under the control of CaMV 35S promoter were used for genetic transformation. Both the vectors contained nptII gene for kanamycin selection and GUS as the reporter gene. Among different explants used, maximum transformation frequency (62%) was obtained with embryogenic callus (anther). Histochemical GUS assay revealed the expression of the uidA gene in the transgenic callus lines. Molecular confirmation of the transgene integration with Hevea genome was further confirmed by polymerase chain reaction.