Development of osmotin transgenics in <i>Hevea brasiliensis</i> Muell. Arg. using explants of zygotic origin
Keywords:
Agrobacterium, GM, Genetic transformation, Osmotin, Rubber, TransgenicAbstract
Hevea brasiliensis (para rubber tree) is a perennial outbreeding species and is the major source of commercial natural rubber (cis-1, 4-polyisoprene). In order to impart tolerance to the abiotic stresses such as drought and cold inHevea, Agrobacterium mediated transformation was attempted with the gene coding for osmotin protein. Agrobacterium strain GV 2260 harboring the plasmid osm/BinAR with osmotin gene under the control of CaMV35S promoter was employed for genetic transformation. Neomycin phosphotransferase gene (nptII) imparting kanamycin resistance was used as the selectable marker. The target explant used in this study was embryogenic callus induced from immature zygotic embryo following half ovulo embryo culture technique. A transformation frequency of about 44.8 per cent was achieved. Putative transgenic lines were proliferated and the gene integration was confirmed by PCR analysis. The sequence of the cloned PCR product showed 100 per cent similarity with the inserted gene sequence. Successful embryo induction (43.5%), maturation (46.0%) and plant regeneration (23.6%) were achieved. The regenerated plants were acclimatized in growth chamber and maintained in the containment facility in big polybags. Transgene integration and expression have been confirmed by PCR, Southern blot and RT-PCR.Downloads
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