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R. Scorza, T.W. Zimmerman, J.M. Cordts, K.J. Footen, and M. Ravelonandro

`Wisconsin 38' tobacco (Nicotiana tabacum L.) leaf discs were transformed with the disarmed Agrobacterium tumefaciens strain EHA101 carrying the rolC gene from A. rhizogenes (Oono et al., 1987) and NPT II and GUS genes. Shoots that regenerated on kanamycin-containing medium were confirmed as transgenic through GUS assays, polymerase chain reaction (PCR), Southern blot analyses, and transmission of the foreign genes through the sexual cycle. Transgenic plants were as short as half the height of control plants; were earlier flowering by up to 35 days; and had smaller leaves, shorter internodes, smaller seed capsules, fewer seeds, smaller flowers, and reduced pollen viability. The number of seed capsules, leaf number, and specific root length were similar between transgenic and control plants. Transgenic clones varied in the expression of the rolC-induced growth alterations as did the first generation of seedlings from these clones. Such differences suggested the potential for selecting for different levels of expression. Transformation with the rolC gene presents a potentially useful method of genetically modifying horticultural crops, particularly for flowering date, height, and leaf and flower size. Chemical names used: neomycin phosphotransferase (NPTII), β-glucuronidase (GUS).

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R. Scorza, T.W. Zimmerman, J.M. Cordts, K.J. Footen, and M. Ravelonandro

Tobacco (Nicotiana tabacum cv Wisconsin 38) leaf discs were transformed with the disarmed Agrobacterium tumefaciens strain EHA101 carrying the Rol C gene from A. rhizogenes (Oono et al., Jpn. J. Genet. 62:501-505, 1987), NPT II and GUS. Shoots that regenerated on kanamycin-containing medium were confirmed transgenic through GUS assays, Southern analyses and transmission of foreign genes through the sexual cycle. Transgenic plants were as short as half the height of control plants, earlier flowering by up to 35 days, had smaller leaves, smaller seed capsules, fewer seeds, smaller flowers and reduced pollen viability. The number of seed capsules, leaf number and root density were similar between transgenic and control plants. Transgenic clones varied in the expression of the Rol C gene and transgenic plants similar or only slightly different from controls were identified. Transformation with the Rol C gene presents a potentially useful method of genetically modifying horticultural crops, particularly for flowering date, height, and leaf and flower size.

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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.

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R. Scorza, J.M. Cordts, D.J. Gray, D. Gonsalves, R.L. Emershad, and D.W. Ramming

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.