Production of Transgenic Grapevine (Vitis vinifera L. cv. Koshusanjaku) Plants by Co-cultivation of Embryogenic Calli with Agrobacterium tumefaciens and Selecting Secondary Embryos

Abstract

Embryogenic calli induced from leaf segments of grapevine (Vitis vinifera L. cv. Koshusanjaku) were co-cultivated for 5 days with Agrobacterium tumefaciens strains EHA101 (pIG121Hm) or LBA4404 (pTOK233), both of which contained the plasmid carrying the neomycin phosphotransferase II (NPT II), hygromycin phosphotransferase (HPT) and the β-glucuronidase (GUS) genes. Putative transgenic calli were selected on 2g/l gellan gum-solidified Nitsch's medium (1969) containing 50mg/l kanamycin and 20g/l sucrose after co-cultivation with A. tumefaciens. Transformation frequency of the embryogenic calli evaluated by GUS histochemical assay was increased by the addition of acetosyringone to co-culture medium. Complete transgenic plants were selected among secondary embryos formed on the surface of embryos in the presence of kanamycin. Finally, kanamycin-resistant plants expressing GUS gene were obtained. PCR analysis confirmed their transgenic nature by detecting GUS and NPT II genes.