An Agrobacterium-mediated transformation system was developed for chokecherry (Prunus virginiana L.), one of the most popular native small tree or large shrub species for resource conservation and wildlife habitat in North America. Leaf tissues from in vitro plants previously maintained in MS medium with 2.5 μm BA were co-cultivated on woody plant medium (WPM) containing 10 μm BA and 200 μm acetosyringone with Agrobacterium tumefaciens strain EHA105 harboring the binary Ti plasmid pBI121 carrying the uid A gene encoding for β-glucuronidase (GUS) and the npt II gene encoding neomycin phosphotransferase II. Infected leaf explants were disinfected in sterile water and antibiotics and then transferred to WPM containing 10 μM BA and the antibiotics cefotaxime, carbenicillin, and kanamycin (CCK) for shoot regeneration at 25 °C with a 16-hour photoperiod. Agrobacterium concentration, pre-conditioning of explants, application of acetosyringone, infection time, and kanamycin tolerance of leaf tissues were evaluated for effects on transformation efficiency. Regeneration of chokecherry shoots on kanamycin-containing medium and screening by GUS histochemical assays showed that both the npt II and the uid A genes were successfully transferred into chokecherry. The transformation will be further confirmed by polymerase chain reaction (PCR) and Southern blot analyses.
Wenhao Dai*, Christopher P. Johnson, Victoria A. Jacques, and James A. Walla
Zoran Jeknic, Stephen P. Lee, Joel Davis, Richard C. Ernst, and Tony H.H. Chen
A protocol was developed for production of transgenic iris plants (Iris germanica L. `Skating Party') from regenerable suspension cultures via Agrobacterium-mediated transformation. We tested a series of selection agents, and identified hygromycin and geneticin as the most suitable for selecting transformed iris cells. Suspension cultures of iris were cocultured for 3 days with A. tumefaciens LBA 4404(pTOK233) carrying an intron-interrupted uidA (GUS) gene encoding β-glucuronidase, and hpt (hygromycin) and nptII (geneticin) selectable marker genes. Hygromycin- or geneticin-resistant calli having GUS enzyme activity were identified and used to induce plant regeneration. More than 300 morphologically normal transgenic iris plants were obtained in ≈6 months. About 80% of the transformants were GUS-positive and NPTII-positive (paromomycin-resistant). Integration of transgenes into the nuclear genome of iris plants was confirmed by Southern blot analysis. We have, therefore, developed an efficient A. tumefaciens-mediated transformation system for Iris germanica, which will allow future improvement of this horticulturally important ornamental monocot via genetic engineering.
Jane E. Knapp and Mark H. Brand
Horticultural improvements in Rhododendron require long periods of time to produce flowering plants by traditional breeding methods. In addition, new trait development by conventional genetics is limited to existing germplasm. Genetic engineering approaches to horticultural improvement offer the possibility for introduction of new traits using foreign DNA from any source. To this end, we have developed a system for the genetic transformation of Rhododendron based on microprojectile bombardment. Leaves from in vitro-grown plantlets of R. `Catawbiense Album' L. were bombarded with the marker genes uidA (GUS) in combination with nptII or hph. Two days post-bombardment, explants were transferred to shoot iniation medium containing either 50 mg/L kanamycin or 2.5 mg/L hygromycin. After 4 weeks, proliferating tissues were transferred to media containing increased levels of selective agent (100 mg/L kanamycin or 5 mg/L hygromycin, respectively). Shoots that regenerated were then excised from necrotic tissues and transferred to shoot proliferation medium containing the high level of selective agent. PCR analysis of putative transformants revealed the presence of the transgenes. Southern blot hybridization confirmed stable transgene integration. Histochemical GUS assays of transformed tissues indicated uniform expression throughout the transgenic plant. With the development of an efficient transformation system, the introduction of genes to confer useful horticultural traits becomes feasible.
Sharon Billings, Gojko Jelenkovic, Chee-Kok Chin, and Jodi Eberhardt
A protocol with a high rate of transformation and regeneration of `Hibush' eggplant (Solanum melongena L.) has been developed. This protocol used leaves of in vitro-grown seedlings as a source of explants. The shoot regeneration culture medium contained 0.1 μm thidiazuron (TDZ) combined with 10 to 20 μm N6-[isopentyl] adenine (2iP). Adding TDZ significantly improved regeneration efficiency and produced a mean of 15 buds and 3 to 4 shoots per explant. When explants were cocultivated with Agrobacterium tumefaciens strains Q10, Q20, Q30, Q40, Q201, Q202, Q203, or Q204 containing the native cryIIIB Bacillus thuringiensis (Bt), neomycin phosphotransferase (NPTII), and β-glucuronidase (uidA) genes, a callus/bud regeneration frequency of 38.8% was observed on the selection medium. Kanamycin at 50 μg·mL-1 was most effective in selecting for transgenic buds and shoots. Augmentin at 300 μg·mL-1 was used to eliminate A. tumefaciens. Augmentin also enhanced shoot proliferation. A transformation/regeneration efficiency of 20.8% was observed for shoot production. More than 400 putative transgenic plants have been produced with this method. From 50 putative transgenic plants, gene integration has been confirmed with Southern blot analysis and progeny tests.
Rudaina Alrefai, Schuyler S. Korban, and Leslie L. Domier
Two strains of Agrobacterium tumefaciens carrying a disarmed Ti-binary vector, pZA-7, were used as vectors for transformation of apple leaf segments, EHA101:pZA-7 carries a helper plasmid derived from pTiB0542, and C58Z707:pZA-7 carries a helper plasmid derived from pTiC58. The binary vector provides two selection markers, neomycin phosphotransferase (nptII) and hygromycin resistance genes, and a screening marker, β-glucuronidase (GUS) gene. Preliminary experiments were conducted to determine the effect of different concentrations of kanamycin, carbenicillin, and cefotaxime on regeneration of apple leaf sections and inhibition of A. tumefaciens strains. In vitro-derived leaf sections of `Royal Gala' apple were grown on a regeneration medium containing thidiazuron and NAA; these were then dipped into a suspension culture of A. tumefaciens and transferred to a fresh regeneration medium. Callus lines exhibiting kanamycin and hygromycin resistance were obtained mostly with agrobacterium strain EHA101:pZA-7. Expression of GUS activity was also determined in putative transformed calli. Southern blot analysis was used for confirming integrative transformation in transgenic lines.
The amount of phosphorus (P) accumulated by vegetable crops is relatively small compared to nitrogen and potassium. However, large amounts of P fertilizer are often required for optimal yield and quality. Typically P added to soils is quickly converted to unavailable forms resulting in low crop utilization efficiencies. These low P uptake efficiencies have long been of economic concern and a major focus of agronomic and horticultural research. Additionally, in certain regions where crop production areas are hydrologically linked to wetland ecosystems, P fertilization is also of environmental concern. This presentation will summarize important P soil transformations, biotic and abiotic factors influencing the availability of P to crops, and P fertilizer management strategies for improved crop utilization efficiency and reduced environmental impact.
Christine Berry, J. Van Eck, S. Kitto, and A. Smigocki
Orangemint leaf disks were infected with three strains of Agrobacterium tumefaciens: A281, a hyper-virulent strain containing plasmid pTiBo542; C58, a strain containing nopaline Ti plasmid pTiC58; and A136, a derivative of C58 lacking the nopaline Ti plasmid. After a 24 or 48 hr cocultivation, leaf disks were placed on a medium containing MS salts and vitamins, 2% sucrose, and 200 μgm l-1 cefotaxime. Callus formed only on those leaf disks infected with A281. Five callus lines (R-12, -71, -73, -81, -83) were putatively transformed based on succinopine production. Definitive transformations were confirmed via DNA slot blot analysis. All callus lines assayed (R-11. -12, -41, -73, -83) hybridized to a 7.7 KB fragment from the T-DNA region.
Shanqiang Ke, Chiwon W. Lee, and Zong-Ming Cheng
Coleoptile tissues excised from young seedlings of `Touchdown' Kentucky bluegrass (Poa pratensis L.) were bombarded with the disarmed Agrobacterium tumefaciens strain EHA 101 carrying rolC (from A. rhizogenes), NPT II and GUS genes. These tissues were then cultured on Murashige and Skoog (MS) medium containing 0.2 mg·L–1 picloram, 0.01 mg·L–1 naphthaleneacetic acid (NAA), 150 mg·L–1 kanamycin, and 50 m acetosyringone. Calli formed on this medium within 2 weeks. The regenerated plants from these calli were analyzed for the presence of the GUS and rolC genes by histochemical GUS assay, PCR, and Southern hybridization. Only 3.7% of the regenerants were transformed when determined by the GUS assay. A similar frequency of transformation in the regenerated plants was obtained after bombarding the coleoptile tissues with the DNA isolated from the pGA-GUSGF-rolC plasmid. Most of the putative transformants were either albinos or variegated plants that are composed of both albino and green tissues.
Richard L. Bell, Ralph Scorza, Chinnathambi Srinivasan, and Kevin Webb
`Beurre Bosc' pear (Pyrus communis L.) was transformed with Agrobacterium tumefaciens (E.F. Smith & Townsend) Conn strain EHA101 containing the binary vector pGA-GUSGF into which the rolC gene had been inserted. Leaf explants from in vitro shoot tip cultures were wounded, Agrobacterium-inoculated, and cultured on kanamycin selection medium. Regenerating shoots were transferred to proliferation medium without antibiotics. Three clones tested positive for GUS and nptII enzyme activity. Transformation with the rolC gene was confirmed by DNA, RNA, and protein blot analyses. The number of copies of the rolC transgene varied from one to three. Plantlets of the three transgenic clones were acclimated and transferred to the greenhouse. Preliminary observations of phenotype indicate that the rolC gene reduced height, number of nodes, and leaf area of transgenic `Beurre Bosc'.
S. Jayashankar, Suman Bagga, and Gregory C. Phillips
In vitro genetic transformation of chile pepper, Capsicum annuum var. New Mexico 6-4, was achieved. Seeds of `New Mexico 6-4' were grown aseptically on Murashige and Skoog medium. Seedlings 22 days old were wounded on the hypocotyl region using a sterile hypodermic needle. A. rhizogenes strain K 599 harboring the plasmid p35S GUS Intron was inoculated on the wound site. Three days later the seedlings were transferred onto MS media with antibiotics (Cefotaxime, Carbenicillin, Amoxicillin, Clavulanic acid, and Kanamycin). New roots were seen to initiate from the wound site 15 to 20 days after inoculation. The roots were morphologically identified as “hairy roots.” Glucuronidase (Gus) assay performed 40 days after inoculation on randomly chosen roots that had grown into the selection medium, showed that 6/25 (24%) of the inoculated seedlings had roots that showed intense blue coloration. Presence of an intron makes it impossible for the bacteria to express the reporter gene. The seedlings that had transformed roots had a different morphology with wrinkled leaves and short internodes. The pattern of expression of the introduced gene varied greatly. Some positive tissues had the root tips alone being blue; a few had the vascular tissues and the root tips blue; and others had the vascular tissues, the surrounding parenchyma cells, root tips, and the root hairs turn very dark blue. The transformed roots did not need to grow into the selection media to be Gus positive. Isolated roots cultured on MS media supplemented with 0.2 mg/L IAA were maintained for 120 days and continued to express the reporter gene. Currently, methods to regenerate transformed shoots from roots are being tested. The “hairy root” transformation system in pepper could have application in the testing of root-expressible constructs for transgene expression assays.