345 J. AMER. SOC. HORT. SCI. 119(2):345–355. 1994. J. AMER. SOC. HORT. SCI. 119(2):345–355. 1994. Transferring Cucumber Mosaic Virus–White Leaf Strain Coat Protein Gene into Cucumis melo L. and Evaluating Transgenic Plants for Protection against Infections Carol Gonsalves, Baodi Xue, Marcela Yepes, Marc Fuchs, Kaishu Ling, and Shigetou Namba 1 Department of Plant Pathology, Cornell University, New York State Agricultural Experiment Station, Geneva, NY 14456 Paula Chee 2 and Jerry L. Slightom 2 Molecular Biology Unit 7242, The Upjohn Company, 301 Henrietta Street, Kalamazoo, MI 49007 Dennis Gonsalves Department of Plant Pathology, Cornell University, New York State Agricultural Experiment Station, Geneva, NY 14456 Additional index words. coat protein mediated-protection, gene gun, ploidy, regeneration, somaclonal variation, tissue culture, transformation Abstract. 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 μ M 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. R 0 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. R 1 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 R 0 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. Cucurbits, such as melons, cucumbers, pumpkins, squash, and gourds, are consumed in large quantities throughout the world. More than 58 million metric tons of cucurbits were produced in 1990, with nearly 54% of the world’s crop grown in Asia and >26% in Europe, with Africa and North, Central, and South America comprising the bulk of the remaining portion (Food and Agricul- ture Organization, 1990). Cucumber mosaic virus (CMV) is found virtually everywhere cucurbits are grown, and it is most active in temperate and subtropical zones. It has a host range of >800 plant species, is transmitted by aphids in a nonpersistent manner, and its visible symptoms include mosaic and stunting of growth (Palukaitis et al., 1992). Worldwide, CMV is one of the five major viruses affecting field-grown vegetables (Tomlinson, 1987). CMV is one of the most important cucurbit and pepper viruses in New York (T. Zitter, personal communication), where the virus is routinely the first to occur in cucurbits due to early insect vectoring from perennial weed hosts (Banik and Zitter, 1990). Resistance to CMV has been reported for some cucurbits and incorporated into a number of species (Pierce and Wehner, 1990; Robinson et al., 1982); CMV-resistant cucumbers are now widely grown (Munger, 1985; Wehner and Robinson, 1991). Resistance to CMV in muskmelons was identified as early as 1943 (Enzie, 1943), but, as far as we know, no commercial cultivars are currently available. Recently, studies have shown that genetically engineered plants expressing a plant viral coat protein (CP) gene can resist infection by the homologous and in some cases heterolo- gous viruses (Beachy et al., 1990; Ling et al., 1991; Namba et al., 1992; Stark and Beachy, 1989). This promising alternate approach to developing virus-resistant plants, known as CP-mediated pro- tection (CPMP), has been applied to some commercial crops in extensive field trials. Among vegetable crops, practical applica- tion of CPMP has been demonstrated in a 3-year field trial of transgenic cucumbers containing the CP gene of CMV–C strain (Gonsalves et al., 1992). This study showed that CMV resistance in transgenic cucumbers is comparable to that in ‘Marketmore’, the cucumber industry’s standard cultivar for CMV resistance. The family Cucurbitaceae meets the two major requirements for obtaining transgenic plants. First, tissue-culture procedures have been established for several species. These include crops Received for publication 9 Feb. 1993. Accepted for publication 21 June 1993. We gratefully thank R. Provvidenti and R.W. Robinson for advice, J.C. Sanford for use of a helium-driven particle accelerator, T. Zitter for the CMV–FNY strain, and N. Lu and S.E. Ecker-Day for excellent technical assistance. Mention of a trademark, proprietary product, or vendor does not imply its approval to the exclusion of other products or vendors that also may be suitable. The cost of publishing this paper was defrayed in part by the payment of page charges. Under postal regulations, this paper therefore must be hereby marked advertisement solely to indicate this fact. 1 Permanent address: Laboratory of Plant Pathology, Faculty of Agriculture, Univ. of Tokyo, Bunkyo-Ku, Tokyo, Japan.