Genetic transformation of Hevea brasiliensis with the gene coding for superoxide dismutase with FMV 34S promoter

Abstract

An efficient and reproducible protocol for Agrobacterium tumefaciens-mediated genetic transformation and plant regeneration of Hevea brasiliensis with the gene coding for superoxide dismutase under the control of Figwort Mosaic Virus (FMV) 34S promoter has been developed. H. brasiliensis (clone RRII 105) callus derived from immature anther was transformed with A. tumefaciens strain EHA 101, harbouring the binary vector containing the sequence coding for neomycin phosphotransferase (nptII) as the selectable marker gene, β-glucuronidase (GUS) as the reporter gene and the sequence coding for Mn-superoxide dismutase (Mn-SOD) under the control of FMV 34S promoter. The kanamycin-resistant GUS positive calli were selected and proliferated in Murashige and Skoog medium fortified with 2,4-D (4.5 μM), BAP (2.2 μM) and NAA (1.1 μM). High-frequency embryo induction was obtained in modified MS medium supplemented with BAP (1.5 μM), Kn (1.4 μM), NAA (1.1 μM) and GA 3 (2.0 μM). Embryo maturation was obtained in modified MS medium containing GA 3 (1.2 μM) and TDZ (1.5 μM). Twenty per cent of the mature transgenic embryos developed into plantlets. These plantlets were planted in polybags after hardening and maintained in a greenhouse. The transformation event was confirmed by PCR amplification with SOD and nptII-specific primers and Southern hybridization with nptII specific probe using DNA isolated from transgenic plants as the template.