Transgenic grape plants were regenerated from somatic embryos derived from leaves of in vitro-grown plants of `Thompson Seedless' grape (Vitis vinifera L.) plants. Somatic embryos were either exposed directly to engineered Agrobacterium tumefaciens or they were bombarded twice with 1-μm gold particles and then exposed to A. tumefaciens. Somatic embryos were transformed with either the lytic peptide Shiva-1 gene or the tomato ringspot virus (TomRSV) coat protein (CP) gene. After cocultivation, secondary embryos proliferated on Emershad/Ramming proliferation (ERP) medium for 6 weeks before selection on ERP medium containing 40 μg·mL-1 kanamycin (kan). Transgenic embryos were identified after 3 to 5 months under selection and allowed to germinate and develop into rooted plants on woody plant medium containing 1 μm 6-benzylaminopurine, 1.5% sucrose, 0.3% activated charcoal, and 0.75% agar. Integration of the foreign genes into these grapevines was verified by growth in the presence of kanamycin (kan), positive β-glucuronidase (GUS) and polymerase chain-reaction (PCR) assays, and Southern analysis.
R. Scorza, J.M. Cordts, D.J. Gray, D. Gonsalves, R.L. Emershad, and D.W. Ramming
R. Scorza, J.M. Cordts, D.J. Gray, D.W. Ramming, and R.L. Emershad
Transgenic grapevines were regenerated from somatic embryos produced from immature zygotic embryos of two seedless grape selections and from leaves of in vitro-grown plants of `Thompson Seedless'. Somatic embryos were bombarded with gold microparticles using the Biolistic PDS-1000/He device (Bio-Rad Labs) and then exposed to engineered A. tumefaciens EHA101 (E. Hood, WSU). Alternately, somatic embryos were exposed to A. tumefaciens without bombardment. Following cocultivation, secondary embryos multiplied on Emershad and Ramming proliferation medium under kan selection. Transgenic embryos were identified after 3 to 5 months and developed into rooted plants on woody plant medium with 1 mM N6-benzyladenine, 1.5% sucrose, and 0.3% activated charcoal. Seedless selections were transformed with plasmids pGA482GG (J. Slightom, Upjohn) and pCGN7314 (Calgene), which carry GUS and NPTII genes. `Thompson Seedless' was transformed with pGA482GG and pGA482GG/TomRSVcp-15 (D. Gonsalves, Cornell Univ.) containing the tomato ringspot virus coat protein gene. Integration of foreign genes into grapevines was verified by growth on kan, GUS, and PCR assays, and Southern analyses.
D.W. Ramming, R.L. Emershad, P. Spiegel-Roy, N. Sahar, and I. Baron
Immature grape embryos from early ripening genotypes of Vitis vinifera were successfully cultured in vitro on Difco orchid agar or a modified White's agar medium. Germination was increased in vitro for five genotypes from 0%, 7%, 11%, 12%, and 16% in vivo to 15%, 24%, 23%, 34%, and 24%, respectively. Subculturing embryos onto liquid culture from seeds that failed to germinate on agar also was possible. Differences in germination rates, as affected by pollen, were significant. This method will allow accelerated development of early ripening cultivars by allowing breeders to use such genotypes as females, as well as males.