Gene Transfer Using Electric Discharge Particle Bombardment and Recovery of Transformed Cranberry Plants

Abstract

<jats:p>Genetic transformation of the American cranberry, <jats:italic>Vaccinium macrocarpon</jats:italic> Ait., was accomplished using electric discharge particle acceleration. Plasmid DNA containing the genes <jats:italic>GUS</jats:italic> (β-glucuronidase), <jats:italic>NPTII</jats:italic> (neomycin phosphotransferase II), and <jats:italic>BT (Bacillus thuringiensis</jats:italic> subsp. <jats:italic>kurstaki</jats:italic> 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. <jats:italic>GUS</jats:italic> expression varied markedly among various transformed plants. Preliminary bioassays for efficacy of the <jats:italic>BT</jats:italic> 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</jats:p>

Adventitious shoot regeneration from leaf explants of southern highbush blueberry cultivars

“…Recombinant DNA and transformation techniques have been reported that can supplement and extend conventional breeding methods for cranberry (Serres et al 1992(Serres et al , 1997Polashock and Vorsa 2002a;Zeldin et al 2002) and blueberry (Graham et al 1996;Song and Sink 2004;Song et al 2008). While transgenic breeding can play an important role in introducing desirable agronomic traits not possible by conventional breeding into commercial Vaccinium varieties, it is obvious that reliable, efficient transformation protocols must first be developed (Polashock and Vorsa 2002a;McCown and Zeldin 2005;Rowland and Hammerschlag 2005;Hancock et al 2008b).…”

“…(Nickerson and Hall 1976;Nickerson 1978Nickerson , 1980Dweikat and Lyrene 1988), highbush blueberry (V. corymbosum L.) (Billings et al 1988;Callow et al 1989;Ogden 1992, 1993;Hruskoci and Read 1993;Cao and Hammerschlag 2000;Song and Sink 2004), large cranberry (V. macrocarpon Ait.) (Serres et al 1992(Serres et al , 1997Marcotrigiano et al 1996;Qu et al 2000), lingonberry (V. vitis-idaea L.) (Debnath and McRae 2002;Debnath 2005;Meiners et al 2007), and bilberry (V. myrtillus L.) (Shibli and Smith 1996). Most of the regeneration occurred via organogenesis.…”

Shoot regeneration from tissue-cultured leaves of the American cranberry (Vaccinium macrocarpon)

“…), however, there has been only one report of the use of tissue culture to induce adventitious shoots. Serres et al (1992) regenerated shoots from internode segments of micropropagated shoots as a part of a gene transfer protocol using particle bombardment. The efficiency and reproducibility of the system was not reported.…”

Strategies for variety-independent genetic transformation of important cereals, legumes and woody species utilizing particle bombardment

“…was accomplished via electric discharge particle bombardment (Serres et al ., 1992) . Stem sections derived from in vitro cultures 23 were bombarded with gus, npt II and bt genes.…”