The capability to uniformlyinduce flowering in cranberry (Vaccinium macrocarpon Ait. `Stevens') in < 1 year from microculture was investigated to accelerate cranberry breeding and to study woody plant reproductive biotechnology. Flower buds were induced on newly micropropagated cranberry plants during the first growing season. A treatment of 2.5 mg of paclobutrazol applied as a soil drench per 2- to 3-month-old potted plant in midsummer, when the plants were grown in coldframes under natural daylength and air temperatures, resulted in 70% of the plants flowering. Plants not treated with paclobutrazol did not flower. Reduced but significant flower bud set was observed on plants treated with paclobutrazol but grown in the greenhouse under natural daylength. Flowering was stimulated by cold treatment coupled with gibberellin sprays and/or repotting to nonpaclobutrazol-treated medium. Chemical name used: β -[(4-chlorophenyl)methyl]-ct-(1,1-dimethylethyl)-1H-1,2,4-triazole-1-ethanol (paclobutrazol).
Rodney Serres and Brent McCown
Rodney Serres and Brent McCown
The gene encoding β-glucuronidase, GUS, has been inserted into cranberry and is expressed in various tissues. Detectable expression of the GUS gene is enhanced up to 15x when the phenol-adsorbing compound, polyvinylpolypyrrolidone, is included in the extraction buffer of the fluorometric MUG assay, indicating that an endogenous, probably phenolic, compound is inactivating the foreign enzyme. Extracts from in vitro-grown cranberry leaves reduce the activity of purified β-glucuronidase in fluorometric assays. This is in contrast to extracts from other plants which have no affect on the enzyme. Detectable expression of the GUS gene for an individual transclone varies with the age of the tissue and the environment in which the plant is grown. The BT gene, which encodes for the Bacillus thuringiensis δ-endotoxin, was also inserted into cranberry with the purpose of incorporating lepidopteran insect resistance. Bioassays using an important insect pest on cranberry show generally inconsistent feeding patterns on transgenic plants. These results may be due to the interaction of the endogenous compounds and the B.t. δ-endotoxin.
Eric L. Zeldin, Thomas P. Jury, Rodney A. Serres, and Brent H. McCown
The American cranberry (Vaccinium macrocarpon Ait.) was genetically transformed with the bar gene, conferring tolerance to the phosphinothricin-based herbicide glufosinate. Plants of one `Pilgrim' transclone grown under greenhouse conditions were significantly injured by foliar treatments of 100 mg·L-1 glufosinate, although the injury was less severe when compared to untransformed plants. However, the same transclone grown outdoors in coldframes survived foliar sprays of 500 mg·L-1 glufosinate and higher, while untransformed plants were killed at 300 mg·L-1. Actively growing shoot tips were the most sensitive part of the plants and at higher dosages of glufosinate, shoot-tip injury was evident on the transclone. Injured transgenic plants quickly regrew new shoots. Shoots of goldenrod (Solidago sp.) and creeping sedge (Carex chordorrhizia), two weeds common to cranberry production areas, were seriously injured or killed at 400 mg·L-1 glufosinate when grown in either the greenhouse or coldframe environment. Stable transmission and expression of herbicide tolerance was observed in both inbred and outcrossed progeny of the above cranberry transclone. Expected segregation ratios were observed in the outcrossed progeny and some outcrossed individuals demonstrated significantly enhanced tolerance over the original transclone, with no tip death at levels up to 8000 mg·L-1. Southern analysis of the original transclone and two progeny selections with enhanced tolerance showed an identical banding pattern, indicating that the difference in tolerance levels was not due to rearrangement of the transgene. The enhanced tolerance of these first generation progeny was retained when second generation selfed progeny were tested.
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