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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. obtained in many crops and often employs the resistance gene, bar. Bar was derived from a common soil bacterium Streptomy- ces hygroscopicus and encodes an enzyme that acetylates phosphinothricin, thus inactivating the herbicidal activity (Th- ompson et al., 1987). This paper reports the successful transformation of cranberry with the bar gene. One transclone showed moderate tolerance to the glufosinate (the ammonium salt of phosphinothricin) herbi- cide Liberty, and some outcrossed progeny showed significantly elevated tolerance over the original transclone, reaching com- mercially useful levels.

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/lit er. 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 The American cranberry is a woody, low-growing perennial vine native to North America and found growing in temperate lowland marsh areas. The cranberry industry is important in the few states that have conditions conducive to its culture. Eco- nomically, cranberry is the most valuable fruit crop in both Wisconsin and Massachusetts. Despite improvements in cranberry cultural practices, includ- ing the use of integrated pest management (Roper and Planer, 1989; Wis. Agr. Stat. Serv., 1989), insect and weed pests still account for significant losses in yield, and control of these pests has been estimated to account for ≈ 45% of the grower's direct field costs. Substantial amounts of carbamate and organophos- phate insecticides are used throughout the growing season for insect control in cranberry marshes (Mahr et al., 1988). Genetic transformation technology is being applied to cran- berry with the goal of increasing productivity while reducing the potential environmental impacts of cranberry production in sensitive wetland areas. Successful transformation of other fruit crops, including strawberry (Fragaria × annanassa) (Nehra et al., 1990), apple (Mahs pumila Mill.) (James et al., 1989), Rubus spp.) (Graham et al., 1990), and orange (Citrus sinensis Osh.) (Kobayashi and Uchimiya, 1989), has been reported; however, we found no reports of the incorporation of an eco

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). Cranberry is a vegetatively propagated perennial fruit crop cultivated in cool climates and lowland areas. Flowering occurs in early spring at the base of new growth of upright stems. Flower bud induction for the following year normally occurs in mid-summer (Main- land, 1985). Mixed vegetative/reproductive terminal buds are evident by fall on flowering uprights. Rapidly growing horizontal runners normally do not set flower buds. As is the case with most woody perennials, cranberry seedlings have an extended juvenile period. Seedlings produce vigorous vegeta- tive growth (mostly runners) their first 2 years; during the second growing season, flower buds may be induced at the terminals of new upright stems, resulting in flowering during the spring of the third growing season. Microcultured cranberry plants act physiologi- cally similar to seedlings in that they also produce mainly runners during their first sea- 6 -(2-isopentenyl) adenine(2ip), 0.13% D-gluconic acid (calcium gluconate), 0.3% agar (Sigma, St. Louis), 0.170 Gelrite (Scott, Fiskeville, R.I.), and 2% sucrose. In May of each of two subsequent years, rooted shoots were removed from the culture vessels and placed in plasticized peat plugs (Grow-Tech, Watsonville, Calif.) for continued root growth and acclimation to laboratory humidity levels. After 3 weeks, the plants were moved to the

`Stevens' cranberry was genetically engineered to confer tolerance to the broad spectrum herbicide glufosinate. Initially, herbicide tolerance was verified by spraying greenhouse plants with the commercial formulation Liberty. Although one transformant showed significant tolerance, the tolerance level was below that required to kill goldenrod, a common weed of cranberry beds. This transformant was propagated and the plants established outdoors in a coldframe, yielding a growth form more typical of field-grown plants than that of greenhouse-grown plants. These plants, as well as untransformed cranberry and goldenrod plants, were sprayed with various levels of the herbicide. The transformed plants were not killed at glufosinate concentrations up to 1000 ppm, although delayed growth did occur. Some runner tip injury was observed at 500 ppm as well as widespread shoot tip death at higher levels. The above-ground parts of goldenrod plants were killed at 400 ppm with significant injury at 200 ppm. Untransformed cranberry plants were killed at 300 ppm and had extensive tip death even at 100 ppm. Transformed cranberry plants with confirmed “field” tolerance were re-established in the greenhouse and new vegetative growth was forced. When these plants were sprayed with glufosinate, significant shoot tip injury was observed at levels as low as 100 ppm. The degree of herbicide tolerance of transformed cranberry appears to be modulated by the growth environment, which may affect the expression of the inserted genes or the physiological sensitivity of the impacted tissues.

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.