Electric discharge particle acceleration was used to introduce three foreign genes into the American cranberry (Vaccinium macrocarpon Ait.). These genes were NPTII (conferring resistance to the antibiotic, kanamycin), GUS (allowing for visual verification), and B.t. (conferring resistance to lepidopteran insects). Adventitious buds were induced on stem sections prior to bombardment with DNA-coated gold pellets. Bombarded stem sections were then transferred to a selection medium containing kanamycin. The surface of the medium was overlaid with a thin layer of kanamycin solution. Approximately 35 days after blasting, proliferating cell masses and elongating shoots were observed amidst the mass of kanamycin-inhibited tissue. Seven weeks after blasting, a histochemical assay verified GUS expression in these tissues, and polymerase chain reaction was used to confirm the presence of the introduced genes.
Rod Serres, Brent McCown, Dennis McCabe, Elden Stang, Dave Russell, and Brian Martinell
Jim Sellmer, Dave Ellis, Brent McCown, Dennis McCabe, Dave Russell, and Brian Martinell
Successful recovery of plants transformed by direct gene transfer techniques relies on 3 factors: 1) a regenerable cell/tissue culture system, 2) a foreign DNA delivery system which can be fine tuned, and 3) a cell population receptive to the transfer and integration of foreign DNA into its genome. Cell receptivity to foreign DNA incorporation is being determined by bombarding poplar cells with gold particles coated with plasmid DNA containing a CaMV35s pro–meted β-glucuronidase chimeric gene construct. Histochemical GUS expression assays conducted after bombardment show that early log phase cells are most receptive with a transient transformation rate of 0.26% compared to 0.08–0.04% for mid log and stationary phase cells. Furthermore, cell receptivity in early log phase cells is decreased when cytokinin is removed from the culture medium for 3 growth cycles prior to bombardment. This response suggests that plant growth regulators may be used to enhance cell receptivity along with cell synchronizing agents.
Rodney Serres, Elden Stang, Dennis McCabe, David Russell, Daniel Mahr, and Brent McCown
Genetic transformation of the American cranberry, Vaccinium macrocarpon Ait., was accomplished using electric discharge particle acceleration. Plasmid DNA containing the genes GUS (β-glucuronidase), NPTII (neomycin phosphotransferase II), and BT (Bacillus thuringiensis subsp. kurstaki crystal protein) was introduced into stem sections, derived from in vitro cultures, that had been induced to form adventitious buds. The stage of development of these adventitious buds was critical for efficient initial expression. After exposure to electric discharge particle acceleration, stem sections were cultured on a solid-phase bud-inducing medium containing 300 mg kanamycin/liter. In addition, a thin overlay of 300 mg kanamycin/liter in water was added to inhibit growth of nontransformed cells. Within 7 weeks, green shoots emerged amidst kanamycin-inhibited tissue. No escape (nontransformed) shoots were recovered, and 90% of the transformed shoots were shown through PCR and Southern blot analysis to contain all three introduced genes. GUS expression varied markedly among various transformed plants. Preliminary bioassays for efficacy of the BT gene against the feeding of an economically important lepidopteran cranberry pest have shown no consistently effective control. Potential problems with the expression of the BT and GUS genes are discussed