Abbreviations: GUS, β -glucuronidase; X-gluc, 5-bromo-4-chloro-3-indolylglucuronide. 1 Dept. of Horticulture, Oregon State Univ., Corvallis, OR 97331. 2 Agricultural Chemistry, Oregon State Univ., Corvallis, OR 97331. Present address: Dept. of
G.C. Martin, A.N. Miller, L.A. Castle, J.W. Morris, R.O. Morris, and A.M. Dandekar
Mark G. Bolyard and Mariam B. Stricklen
Amelanchier laevis shoots were co-cultured with Agrobacterium tumefaciens carrying the binary vector pBI121, which encodes the B-glucuronidase (GUS) and neomycin phosphotransferase genes. Shoots were then rinsed briefly in liquid MS medium and plated onto culture medium containing carbenicillin. After approximately three months of culture, adventitious shoots were assayed for the presences of GUS by Southern blotting and histochemical assays. Southern analysis revealed a signal when genomic DNA from putatively transformed plants was hybridized with a probe from a segment of pBI121. Regenerated shoots also showed a deep blue color when incubated with X-gluc, although the expression was chimeric, that is, the activity appeared only in a percentage of the cells in each shoot. Callus tissue at the base of each shoot also showed high levels of GUS expression using a fluorogenic assay.
Qingzhong Liu, Sarbagh Salih, and Freddi Hammerschlag
Factors influencing regeneration and ß-glucuronidase expression from apple (Malus × domestica Borkh.) stem internodes were studied as part of a program to develop transgenic `Royal Gala' apple with improved disease resistance. The early stages of the transformation process were monitored by counting the number of ß-glucuronidase (GUS) expressing zones immediately after co-cultivation of explants with Agrobacterium tumefaciens supervirulent strain EHA105 (p35SGUS_INT) and by counting the number of GUS-expressing calli developing on explants 2 weeks after co-cultivation. Etiolated shoots were produced from in vitro shoots cultured for 2 weeks in the light followed by 2 weeks in the dark and were compared with shoots cultured for 4 weeks in the light (green shoots). First internodes from etiolated shoots produced three, 10 and 100 times the number of shoots regenerated from second, third, and fourth internodal explants, respectively, and produced seven times the number of shoots compared with similar explants from green shoots. 100% of first internodes from etiolated shoots exhibited GUS-expressing zones and yielded twice as many GUS-expressing zones when compared with leaf explants from green shoots, which exhibited GUS-expressing zones in only 60% of the explants. An average of nine GUS-expressing calli per explant were produced on first internodes from etiolated shoots 2 weeks after co-cultivation.
Mark E. Lewis and Fred A. Bliss
Ten common bean (Phaseolus vulgaris L.) lines—including cultivars, breeding lines, and one wild line—were evaluated for susceptibility to Agrobacterium tumefaciens strain C58 by stab-inoculating intact shoot tips of germinating seeds. Significant differences for tumor frequency and size were found on the resulting 3-week-old seedlings. UW 325, a wild bean, had the highest rate of tumorigenesis; `Olathe', a dry bean cultivar, had the lowest. Uninoculated excised shoot tips cultured in media with BA or BA plus NAA exhibited differences in phytohormone sensitivity, as evidenced by callusing and root initiation. The cultivar Montcalm seemed to be highly sensitive, while `Olathe' was relatively insensitive. Fluorometric GUS assays of shoot tips from germinating seeds inoculated with the disarmed GUS-containing A. tumefaciens strain C58C1(pGV3850/pKIWI105) showed that UW 325 had the highest level of GUS activity. `Montcalm' had a high rate of tumorigenesis but a low level of GUS activity; this anomaly was attributed to its high phytohormone sensitivity. The use of the virulence-inducing compound acetosyringone in the inoculum culture medium did not alter genotypic differences (ranks) in susceptibility. Histochemical GUS assays of inoculated UW 325 shoot tips showed that 60% of the apexes exhibited one or more transformation events. Chemical names used: β-glucuronidase (GUS); α-naphthaleneacetic acid (NAA); N-(phenylmethyl)-1H-purin-6-amine (BA).
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
Jang R. Liu, Haeng S. Lee, Suk W. Kim, and Hyo W. Lee
β-Glucuronidase (GUS) gene of Escherichia coli was introduced into ginseng cells by an Agrobacterium binary vector system and expressed in somatic embryos derived from the cells. A binary vector pBI121 carrying CaMV 35S promoter-GUS gene fusion and a neomycin phosphotransferase gene as selection marker was transferred into Agrobacterium tumefaciens LBA4404. Zygotic embryo cotyledonary segments were co-cultivated with A. tumefaciens and transferred to the medium containg 1 mg 2,4-dichlorophenoxyacetic acid/liter, 0.5 mg kinetin/liter, and 100 mg kanamycin/liter. Kanamycin-resistant calli were formed after 3 to 4 weeks of culture. Southern analysis confirmed the resistant calli were transformed with GUS gene. High GUS activities were detected in somatic embryos developed from the calli.
Wenhao Dai, Zong-Ming Cheng, and Wayne Sargent
Transgenic hybrid aspens (Populus tremuloides × P. tremula) were produced by Agrobacterium-mediated transformation and confirmed by polymerase chain reaction. Three promoters (CaMV 35S, Heat shock, and Rol C) were used to drive transcription of chimeric genes -glucuronidase (GUS), npt-II, and rol B. Stem sections in ≈100 mm thick, leaf blades, and root tips of transgenic aspen were treated with X-Gluc solution for 2 to 12 h in a 37 °C incubator and fixed in a solution containing 5% formaldehyde, 5% acetic acid, and 20% ethanol (FAA) for 10 min. After washing with 50% ethanol twice and clearing with absolute ethanol until free of chlorophyll, the GUS expression (localization and intensity of blue staining) in leaf, stem, and root at different growth stages were evaluated and photographed under the light microscope. When CaMV35S and rol C were used as promoters, the GUS gene was expressed in all parts of mature stem except pith, with the strongest activity in phloem. The heat shock promoter gave rise to very strong expression only in epidermis and phloem. In the young stem, GUS activity was detected in epidermis, parenchyma, vascular cambium, and primary xylem in CaMV35S-GUS transformed aspen shoots. The rol C promoter produced GUS gene expression in all stem tissues. When the heat shock promoter was used, the GUS gene expressed in a more tissue-specific manner, especially in mature stems, with activity mainly in parenchyma. In young leaf tissues, the GUS activity was primarily located in veins and mesophyll. In the mature leaves, no blue staining was found in the main vein. In root tip, the GUS gene driven by CaMV35S and heat shock promoters were expressed in the columella, vascular, and root apical meristem with very strong expression in the root apical meristem. Aspen plants transformed by rol C-Gus construct showed less or no expression in the columella.
Aaron Brown and Harrison G. Hughes
Callus induction and regeneration of alkaligrass (Puccinellia distans) was developed in our laboratory for use in transformation studies of turfgrass. Particle bombardment of the embryogenic callus is being evaluated using a helium particle inflow gun constructed at Colorado State Univ., according to the design of Philippe et al. (Ohio State Univ., 1993). Its utility in delivering DNA to plant cells is being tested by measuring the frequency of transient gene expression of a reporter gene (GUS pBI121) in embryogenic callus of alkaligrass. Varying pressure of helium and the distance of the calli in the chamber are also being evaluated for efficiency in transformation.
Xiaojian Ye, Susan K. Brown, Ralph Scorza, John Cordts, and John C. Sanford
Abbreviations: GUS, ß-glucuronidase; NPTII, neomycin phosphotransferase; PCR, polymerase chain reaction 1 Graduate student. Present address: Biology Dept., Dartmouth College, Hanover, NH 03755. This research was funded through a USDA-Cornell Univ
Paula P. Chee and Jerry L. Slightom
Abbreviations: Cb, carbenicillin; CMV, cucumber mosaic virus; GUS, (β -glucuronidase; Km, kanamycin; MS, Murashige and Skoog NPT II, neomycin phosphotransferase II; NOS, nopaline synthase. We thank Krystal A. Fober for plant care in the greenhouse