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R. Provvidenti and David M. Tricoli

In a yellow summer squash (Cucurbita pepo L.) experimental line developed by Seminis Vegetable Seeds, the coat protein gene of an American strain of squash mosaic virus (SqMV-M88), conferred resistance to Arizona, California, New Jersey, and New York strains belonging to the two pathotypes of the virus. An analysis of genetic populations derived from crosses and reciprocal backcrosses of a homozygous SqMV-resistant line A127-1-2 with the susceptible cultivar Butterbar revealed that the high level of resistance mimics the response of a single recessive gene.

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Manoel T. Souza Jr., Paula F. Tennant, and Dennis Gonsalves

Line 63-1 is a `Sunset'-derived transgenic papaya expressing the coat protein (CP) gene from a mild mutant of a Hawaiian isolate of Papaya ringspot virus (PRSV). Previous work showed that line 63-1 R1 plants exhibited a range of resistance to severe PRSV isolates from Hawaii (HA), Jamaica (JA), Thailand (TH), and Brazil (BR). Genetic and molecular data obtained in this study confirm that line 63-1 has two CP transgene insertion sites; segregation analysis shows that the CP and the npt II genes are present at both loci. To study the potential effect of gene dosage on resistance, various populations of R1, R2, and R3 seedlings were challenged by PRSV HA, BR, and TH. A R1 population obtained by self-pollination of line 63-1 hermaphrodite R0 plant exhibited resistance to all three isolates. The percentage of plants resistant to all three PRSV isolates increased in 63-1-derived populations as a result of recurrent selection. Additional genetic studies demonstrate that the number of resistant plants in a 63-1-derived population is directly correlated with the number of plants with multiple transgene copies. We conclude that transgene dosage plays a major role in affecting the resistance of 63-1 to PRSV isolates from various geographical locations.

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Jean-Michel Hily, Michel Ravelonandro, Vern Damsteegt, Carole Bassett, Cesar Petri, Zongrang Liu, and Ralph Scorza

promising approach to developing PPV-resistant cultivars is through the application of pathogen-derived resistance (PDR) ( Sanford and Johnston, 1985 ). This technology was first demonstrated by the expression of the coat protein ( CP ) gene of tobacco

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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.

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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.

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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.

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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.

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Elvia C. Palacios-Torres, M. Alejandra Gutièrrez-Espinosa, Gloria A. Moore, Gustavo Mora-Aguilera, Daniel L. Ochoa-Martínez, and Angel Villegas-Monter

Citrus Tristeza Closterovirus (CTV) induces mild and/or severe symptoms on Citrus species. It may cause death of trees if the rootstock-scion combination is susceptible. It has been found in other plant/virus combinations that transformation with partial or complete viral genes (e.g., coat protein genes) can confer resistance to the resulting transgenic plants. We previously reported A. tumefaciens mediated transformation and production of two sour orange (C. aurantium L.) plants expressing the coat protein gene of CTV, which was the first report of production of transgenic Citrus using a viral gene. However, in order to properly evaluate resistance, it is necessary to obtain as many transgenic Citrus plants from single transformation events as possible. Therefore, we are currently transforming grapefruit (Citrus paradisi) `Marsh' and `Star Ruby' and sweet orange (C. sinensis) `Valencia' with CTV coat protein genes. These species are susceptible to CTV and more amenable to transformation than sour orange. Epicotyl segments of etiolated seedlings were inoculated with A. tumefaciens strain EHA101 harboring binary plasmid pGA482GG containing the coat protein gene of mild Florida CTV strain T30 (CP-T30) or severe Florida strain T36 (CP-T36). Putatively transformed shoots were regenerated on selection medium containing kanamycin. Regenerated shoots were evaluated with GUS assays; those shoots positively identified by GUS were then evaluated with PCR. We have currently identified 17 `Marsh' grapefruit, 20 `Star Ruby' grapefruit, and seven sweet orange putatively transformed plants.

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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.

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Carol Gonsalves, Baodi Xue, Marcela Yepes, Marc Fuchs, Kaishu Ling, Shigetou Namba, Paula Chee, Jerry L. Slightom, and Dennis Gonsalves

A single regeneration procedure using cotyledon explants effectively regenerated five commercially grown muskmelon cultivars. This regeneration scheme was used to facilitate gene transfers using either Agrobacterium tumefaciens (using `Burpee Hybrid' and `Hales Best Jumbo') or microprojectile bombardment (using `Topmark') methods. In both cases, the transferred genes were from the T-DNA region of the binary vector plasmid pGA482GG/cp cucumber mosaic virus-white leaf strain (CMV-WL), which contains genes that encode neomycin phosphotransferase II (NPT II), β-glucuronidase (GUS), and the CMV-WL coat protein (CP). Explants treated with pGA482GG/cpCMV-WL regenerated shoots on Murashige and Skoog medium containing 4.4 μm 6-benzylaminopurine (BA), kanamycin (Km) at 150 mg·liter-1 and carbenicillin (Cb) at 500 mg·liter-1. Our comparison of A. tumefaciens- and microprojectile-mediated gene transfer procedures shows that both methods effectively produce nearly the same percentage of transgenic plants. R0 plants were first tested for GUS or NPT II expression, then the polymerase chain reaction (PCR) and other tests were used to verify the transfer of the NPT II, GUS, and CMV-WL CP genes. This analysis showed that plants transformed by A. tumefaciens contained all three genes, although co-transferring the genes into bombarded plants was not always successful. R1 plants were challenge inoculated with CMV-FNY, a destructive strain of CMV found in New York. Resistance levels varied according to the different transformed genotypes. Somaclonal variation was observed in a significant number of R0 transgenic plants. Flow cytometry analysis of leaf tissue revealed that a significant number of transgenic plants were tetraploid or mixoploid, whereas the commercial nontransformed cultivars were diploid. In a study of young, germinated cotyledons, however, a mixture of diploid, tetraploid, and octoploid cells were found at the shoot regeneration sites.