BigPatents India

The present invention deals with the development of an efficient tissue culture system for rapid development of callus cultures of Taxus sp. and Agrobacterium tumefaciens - mediated genetic transformation of callus cultures of Taxus sp. using a super binary vector, pTOK 233 (in A. tumefaciens strain LBA 4404) harbouring p-glucuronidase (GUS), hygromycin phosphotransferase (///J7) and neomycin phosphotransferase (NPT-IT) chimeric genes. Several critical factors were tested for standardization of callus transformation, and the factors like bacterial O.D., co-culture period, addition of 100 |iM acetosyringone and 3% sorbitol in the co-culture medium were found to be critical for efficient transformation. Several transformed callus lines were obtained and these lines were checked for GUS assay by histochemical method and many of the callus lines were found to be GUS positive. The molecular analyses, based on PCR and Southern analysis of several of the transformed callus lines, confirmed the integration of the transgenes. The developed protocol can be used to introduce the genes involved in the Paclitaxel biosynthetic pathway and other phyto-pharmaceutical pathways for enhancement of the anti-carcinogenic drug Paclitaxel) and any other gene for introducing new traits. No. of Pages : 21 No. of Claims : 8