Genetic transformation and regeneration of rubber tree ( Hevea brasiliensis Muell. Arg) transgenic plants with a constitutive version of an anti-oxidative stress superoxide dismutase gene

Abstract

Agrobacterium tumefaciens-mediated genetic transformation and the regeneration of transgenic plants was achieved in Hevea brasiliensis. Immature anther-derived calli were used to develop transgenic plants. These calli were co-cultured with A. tumefaciens harboring a plasmid vector containing the H. brasiliensis superoxide dismutase gene (HbSOD) under the control of the CaMV 35S promoter. The beta-glucuronidase gene (uidA) was used for screening and the neomycin phosphotransferase gene (nptII) was used for selection of the transformed calli. Factors such as co-cultivation time, co-cultivation media and kanamycin concentration were assessed to establish optimal conditions for the selection of transformed callus lines. Transformed calli surviving on medium containing 300 mg l(-1) kanamycin showed a strong GUS-positive reaction. Somatic embryos were then regenerated from these transgenic calli on MS2 medium containing 2.0 mg l(-1) spermine and 0.1 mg l(-1) abscisic acid. Mature embryos were germinated and developed into plantlets on MS4 medium supplemented with 0.2 mg l(-1) gibberellic acid, 0.2 mg l(-1) kinetin (KIN) and 0.1 mg l(-1) indole-3-acetic acid. A transformation frequency of 4% was achieved. The morphology of the transgenic plants was similar to that of untransformed plants. Histochemical GUS assay revealed the expression of the uidA gene in embryos as well as leaves of transgenic plants. The presence of the uidA, nptII and HbSOD genes in the Hevea genome was confirmed by polymerase chain reaction amplification and genomic Southern blot hybridization analyses.

De novo assembly and characterization of bark transcriptome using Illumina sequencing and development of EST-SSR markers in rubber tree (Hevea brasiliensis Muell. Arg.)

“…Currently, there are only 37,745 rubber tree ESTs available in the national center for biotechnology information (NCBI) database (as of Dec 2011). Most of these ESTs were generated with the aim to identify the candidate genes involved in various abiotic and biotic stress responses and rubber biosynthesis [7-20], whereas only superoxide dismutase was further studied by using transgenic approaches [21-23]. Among these 37,745 ESTs, most were derived from the latex, and only a few from the bark and leaf.…”

Genomics of Hevea Rubber

“…Biochemical analysis of the transgenic embryogenic calli of Hevea with SOD indicated signi fi cant increase in the activity of superoxide dismutase, catalase, and peroxidase as compared to the control (Sobha et al 2003a, b ) . Jayashree et al ( 2003 ) reported successful development and establishment of transgenic rubber plant with SOD gene for their further evaluation. Genetic transformation experiment to overexpress hmgr1 gene, involved in latex biosynthesis, in Hevea was performed by Arokiaraj et al ( 1995 ) .…”

“…Subsequently, attempts were made to increase the SOD enzyme activity by overexpression of the same genes in Hevea . Transgenic plants were developed with SOD gene under the control of CaMV 35S and FMV 34S promoters (Jayashree et al 2003 ;Sobha et al 2003a ) . Biochemical analysis of the transgenic embryogenic calli of Hevea with SOD indicated signi fi cant increase in the activity of superoxide dismutase, catalase, and peroxidase as compared to the control (Sobha et al 2003a, b ) .…”

The effect of Agrobacterium densities and inoculation times on gene transformation efficiency in rubber tree

“…Agrobacterium tumefaciens-mediated genetic transformation technique has most widely been used for plant species due to its easy protocol without any special equipments. These techniques were developed on numerous Hevea clones, GL1, RRII 105 and PB 260 (Arokiaraj et al, 1994;Jayashree et al, 2003;Priya et al, 2006;Montoro et al, 2003;Lardet et al, 2011). Montoro et al (2003) developed genetic transformation protocol using A. tumefaciens in friable integument callus line (clone PB260) for a good frequency of integration of transgenic calli.…”

“…Nowadays, over-expression of endogenous genes involved in reactive oxygen species scavenging systems, such as MnSOD has been reported (Jayashree et al, 2003). The target of transgene expression in latex cells was also attempted using the promoter from gene HEV2.1, which was the major latex Hevein protein (Montoro et al, 2008).…”