The Supervirulence Plasmid pToK47 from Agrobacterium tumefaciens A281 Improves Transformation Efficiency of Hevea brasiliensis

Abstract

Problem statement: The present study investigates the ability of the pTok47 supervirulence plasmid form Agrobacterium tumefaciens A281 to enhance genetic transformation in Hevea brasiliensis. Approach: Hevea anther callus was transformed via Agrobacterium-mediated genetic transformation using two strains of Agrobacterium (GV2260 and GV3850) harboring the human serum albumin cDNA and the supervirulent plasmid pToK47 from Agrobacterium tumefaciens A281. The transformed callus was selected using kanamycin as the selection agent Results: The Agrobacterium strain GV2260 benefited from the presence of the supervirulence plasmid in giving a higher frequency of 7.4% transformed callus, 344.8% embryogenesis and 11.6% plantlet production compared to the corresponding strain on its own giving 0.9% transformed callus, 204.5% embryogenesis and 4.4% plantlet production. Similarly, Agrobacterium strain GV3850 conferred a higher frequency using the supervirulent plasmid, resulting in 3.5% transformed callus, 138.5% embryogenesis and 3.5% plantlet production compared to the corresponding strain on its own giving 0.7% transformed callus, 137.5% embryogenesis and 9.0% plantlet production. These findings were confirmed by the Wilcoxon Signed Rank Test that compared the effectiveness of the supervirulence plasmid in increasing the rate of genetic transformation in the calli surviving in kanamycin growth medium for GV2260 (p<0.001) and for GV3850 (p<0.05). Conclusion: This study showed that both Agrobacterium strains benefited from the presence of the supervirulence plasmid in giving a higher frequency of transformed callus, embryoids and plantlets. These results suggest that a highly virulent binary vector system might prove especially useful in generating high frequency transformation of Hevea.