TAILIEUCHUNG - An efficient protocol for Agrobacterium mediated transformation of gus/gusplus gene into cassava plants (Manihot esculenta Crantz)
In this study, to evaluate the ability to accept transgenes of the two cassava cultivars KM94 and KM140, which are grown widely in Vietnam, A. tumefaciens bacterial strains C58/pGV2260, EHA105 and LBA4404 containing vector pCB-gusplus or pPIPRA558 haboring selectable marker gene gus/gusplus, were co-inoculated with explants from four selected sources, including (1) immature leaves, (2) shoot apexes, (3) callus, and (4) somatic embryo cotyledons. Transgenic explants were selected using three antibiotics kanamycin, neomycin, and paromomycin, at concentrations of 25, 50, 75, and 100 mg/l, based on nptII gene. | TAPAn CHI SINHprotocol HOC 2016, 38(4): 505-514 efficient for Agrobacterium DOI: AN EFFICIENT PROTOCOL FOR Agrobacterium-MEDIATED TRANSFORMATION OF GUS/GUSPLUS GENE INTO CASSAVA PLANTS (Manihot esculenta Crantz) Do Hai Lan1*, Le Van Son2, Le Tran Binh2,3 1 Faculty of Biology-Chemistry, Tay Bac University Institute of Biotechnology, Vietnam Academy of Science and Technology 3 University of Science and Technology of Ha Noi 2 ABSTRACT: In this study, to evaluate the ability to accept transgenes of the two cassava cultivars KM94 and KM140, which are grown widely in Vietnam, A. tumefaciens bacterial strains C58/pGV2260, EHA105 and LBA4404 containing vector pCB-gusplus or pPIPRA558 haboring selectable marker gene gus/gusplus, were co-inoculated with explants from four selected sources, including (1) immature leaves, (2) shoot apexes, (3) callus, and (4) somatic embryo cotyledons. Transgenic explants were selected using three antibiotics kanamycin, neomycin, and paromomycin, at concentrations of 25, 50, 75, and 100 mg/l, based on nptII gene. These experiments were conducted to optimize conditions for transferring gus gene to cassava plant. The transformation efficiency was evaluated based on the percentage of X-gluc positive stained explants 10 days after infection, somatic embryos, regenerated shoots and whole regenerated plants. The highest transformation efficiency was achieved when using A. tumefaciens C58/pGV2260, carrying expression vector pCB-gusplus, and cotyledons of cultivars KM94. In this protocol, cotyledons were cut into small pieces, then cultured on the callus induction medium for 2 days and submerged in bacterial suspension, supplemented with 100 μM AS, with shaking at 50 rpm for 15 minutes. Explants were then co-cultured on somatic embryo induction medium supplemented with 150 μM AS in the darkness for 2 days. Explants were then transferred to selective callus induction medium with 50 mg/l kanamycin for 3-4 weeks, .
đang nạp các trang xem trước
