ISHS Acta Horticulturae 528: VII International Symposium on Grapevine Genetics and Breeding
TRANSGENIC GRAPEVINE PLANTS (VITIS VINIFERA L.) PRODUCED BY SELECTING SECONDARY EMBRYOS AFTER COCULTIVATION OF EMBRYOGENIC CALLUS WITH AGROBACTERIUM TUMEFACIENS |
| Authors: | Y. Hoshino, Y.-M. Zhu, M. Mii, E. Takahashi, M. Nakano |
| Keywords: | Embryogenesis, β-glucuronidase, kanamycin, transformation |
| Abstract:
Embryogenic calli induced from leaf segments of grapevine (Vitis vinifera L. 'Koshusanjaku') were co-cultivated for 5 days with Agrobacterium tumefaciens strains EHA101 (pIG121Hm) or LBA4404 (pTOK233), both of which contained the plasmid carrying neomycin phosphotransferase II (NPTII), hygromycin phosphotransferase (HPT) and  -glucuronidase (GUS) genes.
Putative transgenic calli were selected on 2.0 g.1-1 gellan gum-solidified Nitsch's medium (1969) containing 50 mg 1-1 kanamycin and 20 g 1-1 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 NPTII genes.
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