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Genetic transformation through the use of hyperhydric tobacco meristems

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Abstract

Exposed shoot meristems from normal and hyperhydric (vitrified) tobacco, Nicotiana tabacum, were bombarded with gold particles either coated with plasmid DNA containing neomycin phosphotransferase (NPTII), rolC and β-glucuronidase (GUS) genes (plasmid pGA-GUSGFrolC) or left uncoated. Meristems bombarded with uncoated particles were co-cultivated with Agrobacterium tumefaciens strain EHA 101 harboring the binary vector pGA-GUSGFrolC. Whole-plant transformants were produced from 4 of 40 hyperhydric meristems bombarded with uncoated particles followed by co-cultivation with A. tumefaciens. One transgenic plant was obtained from 40 normal, non-hyperhydric meristems treated. Transformation was verified by growth on kanamycin-containing medium, GUS assays, PCR, and Southern analysis. The plants tested through Southern analysis appeared to have 2 or more copies of the transgene insert. Seeds obtained from self-pollination of these transgenic plants segregated 3:1 or 15:1 (kanamycin resistant:sensitive) when germinated on medium containing 100 mg/l kanamycin, indicating transfer of foreign genes through the sexual cycle. Whole-plant transformants were not produced from 50 normal tobacco meristems bombarded with plasmid-coated gold particles and not exposed to engineered A. tumefaciens, but 1 plant of 60 bombarded hyperhydric meristems produced transgenic roots, the result of a chimera. We suggest that hyperhydric meristems are more readily transformed.

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Author notes

  1. Thomas W. Zimmerman

    Present address: Agricultural Experiment Station, University of the Virgin Islands, RR2 Box 10000, USVI 00850, Kingshill, USA

Authors and Affiliations

  1. Appalachian Fruit Research Station, USDA-ARS, 45Wiltshire Rd., 25430, Kearneysville, WV, USA

    Thomas W. Zimmerman & Ralph Scorza

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  1. Thomas W. Zimmerman
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  2. Ralph Scorza
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Zimmerman, T.W., Scorza, R. Genetic transformation through the use of hyperhydric tobacco meristems. Mol Breeding 2, 73–80 (1996). https://doi.org/10.1007/BF00171353

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  • DOI: https://doi.org/10.1007/BF00171353

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