<i>Agrobacterium</i> mediated multiple gene integration in <i>Hevea brasiliensis</i> Muell. Arg.

Abstract

Hevea brasiliensis (Euphorbiaceae) is the major source of the world natural rubber. Susceptibility of Hevea brasiliensis to various biotic and abiotic stresses and incidence of tapping panel dryness are the major constraints in natural rubber production. Gene stacking permits the integration of multiple genes for complex traits simultaneously into the target plant. In the present study, gene stacking was achieved by repeated genetic transformation for the integration of two genes, manganese superoxide dismutase (MnSOD) for abiotic stress tolerance and 3-hydroxy-3-methyl-glutaryl-CoA reductase (hmgrI) for enhanced latex yield. Initially, using the embryogenic callus derived from immature zygotic embryo as the target tissue, Agrobacterium mediated transformation by vacuum infiltration was carried out with the binary vector harboring MnSOD gene and nptII as the selectable marker gene. High frequency (30%) transformation was obtained and was detected by GUS histochemical staining and PCR using MnSOD gene specific primer. This MnSOD transgenic callus was used as the target tissue for the integration ofhmgrI gene containing hpt as the selectable marker gene. PCR using hpt gene specific primer was performed for the detection of hmgrI gene integration. Somatic embryo induction (32%) was achieved from the multiple gene integrated callus lines in modified MS medium with BA and Kin (0.3 mg l-1 each) and GA3 (0.5 mg l-1). Fifteen percent of the embryos were matured and were further cultured for plant regeneration. This is the first report on multiple gene integration inHevea brasiliensis.