Bermudagrass (Cynodon L.C. Rich.) is grown on more than 4 million ha in the southern United States. The black cutworm (Agrotis ipsilon Hufnagel) is the most commonly encountered pest of bermudagrass, especially on golf course greens. Developing insect-resistant cultivars is a very desirable substitute, both environmentally and economically, to using current synthetic pesticides. Here we report, for the first time, Agrobacterium-mediated transformation of `Arizona Common' common bermudagrass [Cynodon dactylon (L.) Pers.] with the Bacillus thuringiensis Berliner cry1Ac gene encoding an endotoxin active against black cutworm. Mature seeds were used for producing embryogenic callus, and calli were transformed with a plasmid containing a synthetic cry1Ac and the kanamycin resistance (nptII) genes. Putative transgenic calli and plantlets were selected on media containing 100 and 50 mg·L-1 G418, respectively. RNA-blot analysis of PCR-positive lines revealed the expression of the cry1Ac transgene in three out of five putative transgenic lines. The larvae fed on transgenic plant leaves experienced highly significant (over 80%) mortality.
Hassan Salehi, Zahra Seddighi, Alexandra N. Kravchenko, and Mariam B. Sticklen
Ana Cristina M. Brasileiro, Francisco J. Lima Aragão, Sílvia Rossi, Diva Maria A. Dusi, Leila M. Gomes Barros, and Elíbio L. Rech
To develop an efficient protocol for Agrobacterium-mediated transformation of common bean (Phaseolus vulgaris L.) and tepary bean (P. acutifolius A. Gray), we have tested the susceptibility of six genotypes to eight Agrobacterium tumefaciens and two A. rhizogenes strains. The virulence of the Agrobacterium strains was shown to be genotype dependent. In general, the tumors observed on common bean cultivars were larger than those observed on tepary bean cultivars. The A. tumefaciens AT8196 and Ach5 strains and the A. rhizogenes 8196 strain induced the best responses in all genotypes tested. Polymerase chain reaction (PCR) analysis confirmed the presence of T-DNA in tumors derived from inoculation with three A. tumefaciens strains in common beans. Apical meristems of P. vulgaris cv. Jalo were bombarded with tungsten microprojectiles and then inoculated with an A. tumefaciens wild-type strain (Ach5). One month later, the explants showed a high frequency of tumor formation (50% to 70%). Similarly, when bombarded meristems were inoculated with an A. tumefaciens disarmed strain (LBA4404/p35SGUSINT), 44% of them showed substantial sectors of GUS activity, suggesting the expression of introduced gene. The bombardment/Agrobacterium system appears to be a promising method to stably transform bean through the regeneration of plants directly from transformed apical meristems.
Ralph Scorza, Laurene Levy, Vern Damsteegt, Luz Marcel Yepes, John Cordts, Ahmed Hadidi, Jerry Slightom, and Dennis Gonsalves
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.
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.
Kimberly J. Felcher, D.S. Douches, W.W. Kirk, R. Hammerschmidt, and W. Li
Research was done to determine if enhanced resistance to potato (Solanum tuberosum L.) late blight could be obtained by combining host plant resistance and engineered resistance. Late blight susceptible cultivars, Atlantic, and Spunta and the partially resistant cultivar Libertas were transformed with a fungal glucose oxidase gene, resulting in lines which ranged in transgene copy number from 1 to 8. Glucose oxidase enzyme activity ranged from 0.00 to 96.74×10-5 units/mg plant tissue. There was no correlation between copy number and level of transgene mRNA, level of transgene mRNA and enzyme activity, or between level of enzyme activity and disease resistance. Field and growth chamber evaluation of late blight response demonstrated little to no effect of the glucose oxidase transgene in either late blight susceptible or partially late blight resistant cultivars. However, enzyme activity levels were much lower than levels reported in previous research, which may account for the lack of effect of glucose oxidase against Phytophthora infestans. Twenty-one percent of the transgenic lines were phenotypically off-type compared to nontransgenic controls. Most of the off-type transgenic lines (four out of seven) were derived from `Libertas'. Because several off-type lines did not express the glucose oxidase protein, this phenomenon could not be attributed solely to the glucose oxidase transgene. Based on these results, transgenic lines produced for this study do not increase resistance to P. infestans even in combination with moderate host plant resistance. However, production of greater numbers of transgenic lines with the current construct or, production of transgenic lines in which a different constitutive promoter drives the expression of the glucose oxidase gene might result in greater disease resistance. However, the usefulness of any small increase in resistance would need to be evaluated against the time and cost required for development of transgenic potato cultivars and the potential for off-type tubers and plants.