Transgenic plum plants expressing the papaya ringspot virus (PRV) coat protein (CP) were produced by Agrobacterium-mediated transformation of hypocotyl slices. Hypocotyl slices were cocultivated with Agrobacterium tumefaciens strain C58/Z707 containing the plasmid pGA482GG/CPPRV-4. This plasmid carries the PRVCP gene construct and chimeric NPTII and GUS genes. Shoots were regenerated on Murashige and Skoog salts, vitamins, 2% sucrose, 2.5 μm indolebutyric acid, 7.5 μm thidiazuron, and appropriate antibiotics for selection. Integration of the foreign genes was verified through kanamycin resistance, GUS assays, polymerase chain reaction (PCR), and Southern blot analyses. Four transgenic clones were identified. Three were vegetatively propagated and graft-inoculated with plum pox virus (PPV)-infected budwood in a quarantine, containment greenhouse. PPV infection was evaluated over a 2- to 4-year period through visual symptoms, enzyme-linked immunosorbent assay, and reverse transcriptase PCR assays. While most plants showed signs of infection and systemic spread of PPV within l-6 months, one plant appeared to delay the spread of virus and the appearance of disease symptoms. Virus spread was limited to basal portions of this plant up to 19 months postinoculation, but, after 32 months symptoms were evident and virus was detected throughout the plant. Our results suggest that heterologous protection with PRVCP, while having the potential to delay PPV symptoms and spread throughout plum plants, may not provide an adequate level of long-term resistance.
Ralph Scorza, Laurene Levy, Vern Damsteegt, Luz Marcel Yepes, John Cordts, Ahmed Hadidi, Jerry Slightom, and Dennis Gonsalves
Mingbo Qin, Chiwon W. Lee, Alex Y. Borovkov, and Murray E. Duysen
A study was initiated to characterize key enzymes that influence sweetness in carrot (Daucus carota L.) roots. Sucrose synthase (SS), sucrose phosphate synthase (SPS), and UDP-glucose pyrophosphorylase (UDPL) genes were isolated from potato (Solanum tuberosum L.) and cloned in an anti-sense orientation into Agrobacterium tumefaciens Bin19, which has a CaMV 35S promoter. Seedling hypocotyl sections of selected carrot lines were pre-incubated on B5 medium for 2 days, co-cultivated with A. tumefaciens Bin 19 for additional 3 days, and then transferred to a modified B5 medium containing 50 g/mL kanamycin and 400 g/mL carbenicillin. In 4 weeks, 18.6%, 33.3%, and 26.7% of the cultures from a breeding line (W204-C) were found to be transformed, respectively, with SS, SPS, and UDPL as determined by kanamycin resistance. In contrast, no kanamycin-resistant calli were obtained from a commercial cultivar (Navajo) in these transformation studies. The transformed calli proliferated in the medium containing 50 g/mL kanamycin and 400 g/mL carbenicillin, whereas non-transformed calli died in the same medium. These transformed calli are currently being used to regenerate plants via asexual embryogenesis using a suspension culture. The influence of these additional genes on sugar metabolism and accumulation in root tissues of transformed carrots will be characterized in the future.
J.D. Chung, B.J. Lee, H.S. Lee, and C.K. Kim
Lettuce (Lactuca sativa L.) were transformed using microparticle bombardment with two different genes, alpha-glucuronidase (GUS) gene and Chinese cabbage Glutathione Reductase (GR) gene. The adventitious shoots of cotyledonary explant from 4-day-old seedlings were formed (46.7%) in MS basal media supplemented with 5.0 μm IAA and 1.0 μm 2ip. When 1100 psi helium pressure, 9 target distance, and coating with tungsten 10 microparticles were used and explants were treated with osmoticum-conditioning medium (0.6M sorbitol/mannitol), 4 h prior to and 16 h after bombardment, it was identified by GUS assay that these conditions were the most efficient for transformation of foreign genes into cotyledon tissue of lettuce with particle bombardment. PCR confirmed that the band observed in the transgenic plants were originated from T-DNA tranfer with strong hybridization. The genomic Southern analysis showed that the 1.5-kbp fragment was hybridized with radiolabeled 1.5-kbp GR probe. To know whether the expression of the GR gene can be stably maintained in the next generation, when T2 selfing seeds that were obtained from the transformed mother plants were sowed on MS medium supplemented with 200 μm kanamycin, 70% of seedlings were revealed resistance to kanamycin.
Francis Zvomuya and Carl J. Rosen
Current techniques used in genetic transformation can result in variation of numerous traits in addition to the transformed trait. Backcrossing to the standard genotype can eliminate this variation, but because of the heterozygous nature of potatoes (Solanum tuberosum L), backcrossing is not effective. Therefore, the chances of obtaining altered performance in transformed potato are high. `Superior' potato plants were recently genetically modified to resist attack and damage by the Colorado potato beetle [Leptinotarsa decemlineata (Say)]. The transformed clone, `NewLeaf Superior' (`NewLeaf'), has been shown in previous field trials to be more vigorous than the standard clone. The objective of this 2-year study was to evaluate the performance of `NewLeaf' relative to that of the standard clone at various fertilizer nitrogen (N) levels. The two clones were randomly assigned as subplots to main plots consisting of four N levels (28, 112, 224, or 336 kg·ha-1). Based on regression analysis, total yield was higher for `NewLeaf' than for `Superior' at N rates below 92 kg·ha-1 in 1997. At higher rates, however, `Superior' had higher yields than the transgenic clone. In 1998, the clon×N rate interaction was significant, but there was no consistent trend to the response of the two clones to N application. At the 112 kg·ha-1 N rate, total yield was higher for `NewLeaf' than for `Superior', but yields were similar for the two clones at other N rates investigated. Nitrogen and biomass accumulation in vines increased more for `NewLeaf' than for `Superior' as N rate was increased from 28 to 336 kg·ha-1. At equivalent N rates, these traits were higher for the transformed than for the standard clone within the range of N rates investigated. However, harvest index at equivalent N rates was higher for the standard clone than for `NewLeaf'. `Superior' and `NewLeaf' produced similar tuber dry weight yields per unit of N supplied and per unit of N absorbed by the plant. Nitrogen uptake efficiency (NUE) was 16% higher for `NewLeaf' than for the standard clone at the low N rate (112 kg·ha-1), whereas at higher N rates NUE was either lower for `NewLeaf' or similar for the two clones. This observation, together with the finding that yield for `NewLeaf' was maximized at lower N levels than the standard clone, suggests that `NewLeaf' may require lower N input than the standard clone. Results from the study indicate that the greater efficiency of `NewLeaf' at lower N levels was associated with acquisition of N from the soil rather than utilization of absorbed N in metabolism.
Xiaoling He, Susan C. Miyasaka, Yi Zou, Maureen M.M. Fitch, and Yun J. Zhu
have the potential to enhance disease resistance of crop plants without adversely affecting other important qualities. Genetic transformation involves the insertion of transgenes into totipotent cells that are then regenerated into whole plants. A
Jinpeng Xing, Yan Xu, Jiang Tian, Thomas Gianfagna, and Bingru Huang
application of products containing CK, or to overexpress genes controlling CK synthesis through genetic transformation. Transgenic plants with modified endogenous CK production have recently been used to study the involvement of CK in delaying leaf senescence
R. Scorza, J.M. Cordts, D.J. Gray, D. Gonsalves, R.L. Emershad, and D.W. Ramming
Transgenic grape plants were regenerated from somatic embryos derived from leaves of in vitro-grown plants of `Thompson Seedless' grape (Vitis vinifera L.) plants. Somatic embryos were either exposed directly to engineered Agrobacterium tumefaciens or they were bombarded twice with 1-μm gold particles and then exposed to A. tumefaciens. Somatic embryos were transformed with either the lytic peptide Shiva-1 gene or the tomato ringspot virus (TomRSV) coat protein (CP) gene. After cocultivation, secondary embryos proliferated on Emershad/Ramming proliferation (ERP) medium for 6 weeks before selection on ERP medium containing 40 μg·mL-1 kanamycin (kan). Transgenic embryos were identified after 3 to 5 months under selection and allowed to germinate and develop into rooted plants on woody plant medium containing 1 μm 6-benzylaminopurine, 1.5% sucrose, 0.3% activated charcoal, and 0.75% agar. Integration of the foreign genes into these grapevines was verified by growth in the presence of kanamycin (kan), positive β-glucuronidase (GUS) and polymerase chain-reaction (PCR) assays, and Southern analysis.
R. Scorza, J.M. Cordts, D.J. Gray, D.W. Ramming, and R.L. Emershad
Transgenic grapevines were regenerated from somatic embryos produced from immature zygotic embryos of two seedless grape selections and from leaves of in vitro-grown plants of `Thompson Seedless'. Somatic embryos were bombarded with gold microparticles using the Biolistic PDS-1000/He device (Bio-Rad Labs) and then exposed to engineered A. tumefaciens EHA101 (E. Hood, WSU). Alternately, somatic embryos were exposed to A. tumefaciens without bombardment. Following cocultivation, secondary embryos multiplied on Emershad and Ramming proliferation medium under kan selection. Transgenic embryos were identified after 3 to 5 months and developed into rooted plants on woody plant medium with 1 mM N6-benzyladenine, 1.5% sucrose, and 0.3% activated charcoal. Seedless selections were transformed with plasmids pGA482GG (J. Slightom, Upjohn) and pCGN7314 (Calgene), which carry GUS and NPTII genes. `Thompson Seedless' was transformed with pGA482GG and pGA482GG/TomRSVcp-15 (D. Gonsalves, Cornell Univ.) containing the tomato ringspot virus coat protein gene. Integration of foreign genes into grapevines was verified by growth on kan, GUS, and PCR assays, and Southern analyses.
Antonio C. Torres, Russell T. Nagata, Robert J. Ferl, Thomas A. Bewick, and Daniel J. Cantliffe
Glyphosate-resistant plants of `South Bay' lettuce (Lactuca sativa L.) were produced by using Agrobacterium tumefaciens containing a plasmid carrying glyphosate oxidase and EPSPS gene. An in vitro assay was performed to determine the sensitivity of `South Bay' leaf discs and seedling explants to varying glyphosate concentrations. The I50 for glyphosate leaf discs was 53.8 μm and for glyphosate seedlings 7.6 μm. There was a high correlation between the response of leaf discs and seedlings to glyphosate based on dry weight. These findings will allow identification of glyphosate-resistant transformants in an early stage of plant development, saving time and reducing the cost in generating an improved cultivar with the glyphosate resistance trait.