respectively. Table 1. Schematic drawing of intron-hairpin-RNA constructs carrying the plum pox virus–coat protein ( PPV-CP ) gene. Plum pox virus DNA sequences in B1 and B14 constructs were under the control of the 35S promoter derived from
Jean-Michel Hily, Michel Ravelonandro, Vern Damsteegt, Carole Bassett, Cesar Petri, Zongrang Liu and Ralph Scorza
Guowei Fang and Rebecca Grumet
Zucchini yellow mosaic virus (ZYMV), a potyvirus, can cause major losses in cucurbit crops. With the goal of genetically engineering resistance to this disease we have engineered the ZYMV coat protein gene into a plant expression vector. The complete coat protein coding sequence, or the conserved core portion of the capsid gene, was attached to the 5' untranslated region of tobacco etch virus (TEV) in the pTL37 vector (Carrington et al., 1987, Nucl. Acid Res. 15:10066) The capsid constructs were successfully expressed by in vitro transcription and translation systems as verified by SDS-PAGE and ZYMV coat protein antibody. The constructs were then subcloned using polymerase chain reaction and attached to the CaMV 35 S transcriptional promoter on the CIBA-GEIGY pCIB710 plasmid. The constructs containing the CaMV 35S promoter, the 5' untranslated leader of TEV, and ZYMV coat protein sequences were then put between the Agrobacterium tumefaciens left and right borders in the pCIB10 vector and transferred to A. tumefaciens strain LBA4404 by triparental mating. These vectors are now being used to transform muskmelon and cucumber; resultant transgenic plants will be tested for ZYMV coat protein expression.
Hyo-Won Seo, Jung-Yoon Yi, Young-Il Hahm, Hyun-Mook Cho and Kuen Woo Park
Three potato (Solanum tuberosum L.) cultivars `Superior', `Irish Cobbler', and `Jopung' were transformed by co-cultivation with tuber discs and disarmed Agrobacterium tumefaciens LBA4404 carrying modified vector pBI121, that contained PLRV coat protein (CP) gene and controlled by CaMV35S promoter. Putative transformants were selected and their genomic DNA and RNA transcripts were analyzed for the confirmation of genetic stability by RT-PCR, PCR, southern, and northern blot. The growth characteristics and viral resistance of progenies of transgenic potato plants were investigated. Twelve lines among the different seven-times manipulated transgenic lines were grown in greenhouse and isolates trial field. PLRV coat protein gene was stably inherited in `Superior', but not in `Jopung'. `Jopung' was less stable than `Irish Cobbler' and `Superior' at genetic stability of PLRV CP gene. And some of these transgenic lines were highly resisted in PLRV multiplication. The yield of transformants was reduced in `Irish Cobbler' but not in `Superior'. Possible explanations for these types of resistance are gene silencing and positional effects of transformed PLRV CP genes and that had cultivar specificity. We consider the appearance of escaped transformants in `Jopung' for emergence of chimeric explants from early selection stage.
Paula P. Chee and Jerry L. Slightom
Cotyledon explants of cucumber (Cucumis sativus L. cv. Poinsett 76) seedlings were cocultivated with disarmed Agrobacterium strain C58Z707 that contained the binary vector plasmid pGA482GG/cpCMV19. The T-DNA region of this binary vector contains plant-expressible genes for neomycin phosphotransferase II (NPT II), β -glucuronidase (GUS), and the coat protein of cucumber mosaic virus strain C (CMV-C). After infection, the cotyledons were placed on Murashige and Skoog medium containing 100 mg kanamycidliter. Putative transformed embryogenic calli were obtained, followed by the development of mature embryos and their germination to plants. All transformed RO cucumber plants appeared morphologically normal and tested positive for NPT IL Southern blot analysis of selected cucumber DNAs indicated that NPT II, GUS, and CMV-C coat protein genes were integrated into the genomes. Enzyme-linked immunosorbent assay and Western blot analysis indicated that the CMV-C coat protein is present in the protein extracts of progeny plants. These results show that the Agrobacterium-mediated gene transfer system and regeneration via somatic embryogenesis is an effective method for producing transgenic plants in Cucurbitaceae.
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.
Wenbin Li, Kelly A. Zarka, David S. Douches, Joseph J. Coombs, Walter L. Pett and Edward J. Grafius
The codon-modified cryV-Bt gene (cryV-Bt) from Bacillus thuringiensis subsp. kurstaki Berliner, which is specifically toxic to Lepidoptera and Coleoptera insects, and a potato virus Yo coat protein gene (PVYocp), in which the aphid transmission site was inactivated, were cotransformed into potato (Solanum tuberosum L.) `Spunta' via Agrobacterium tumefaciens Conn. We demonstrated the integration and expression of both genes by molecular analysis and bioassays. All cryV-Bt/PVYocp-transgenic lines were more resistant to potato tuber moth (Phthorimaea operculella Zeller) and PVYo infection than nontransgenic `Spunta'. Four cryV-Bt/PVYocp transgenic lines were equal in potato tuber moth mortality to a cryV-Bt transgenic line, but of these four only two lines were equivalent in PVYo titer levels to a PVYocp-transgenic line. We identified two transgenic lines, 6a-3 and 6a-5, which showed greater resistance to potato tuber moth and PVYo than the other cryV-Bt/PVYocp transgenic lines. This study indicated that multiple genes, conferring insect pest resistance and virus resistance, could be engineered into and expressed simultaneously in a potato cultivar.
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.
Ralph Scorza, Michel Ravelonandro, Ann Callahan, Ioan Zagrai, Jaroslav Polak, Tadeuz Malinowski, Mariano Cambra, Laurene Levy, Vern Damsteegt, Boris Krška, John Cordts, Dennis Gonsalves and Chris Dardick
, R. Ravelonandro, M. 2006 Field trials of plum clones transformed with the Plum pox virus coat protein (PPV-CP) gene Plant Dis. 90 1012 1018 Polak, J. Jarosova, J. Kumar, J. Sochor, J. Kizek, R. Krška, B. Gogolkova, K. Ravelonandro, M. Scorza, R
Tracie K. Matsumoto, Francis T.P. Zee, Jon Y. Suzuki, Savarni Tripathi, James Carr and Bruce Mackey
acid sequence of the N-terminal end of the Cauliflower mosaic virus coat protein that is translationally fused to the coat protein of PRSV HA 5-1. Expression is controlled by the 35S promoter and selection was based on the NPTII
Wenhao Dai, Victoria Magnusson and Chris Johnson
-Verlag Berlin, Heidelberg Scorza, R. Levy, L. Damsteegt, V.D. Yepes, L.M. Cordts, J.M. 1995b Transformation of plum with the papaya ringspot virus coat protein gene and reaction of transgenic plants to plum