The yellow crookneck squash (Curcurbita pepo L.), line ZW-20, was developed through a specific genetic modification of variety YC77E to be resistant to infection by two plant viruses WMV2 and ZYMV, which are members of the potyvirus group. The novel variety was developed by insertion of the coat protein (CP) encoding sequences from these two single-stranded RNA viruses. The introduced viral sequences do not result in the formation of any infectious particles, nor does their expression result in any disease pathology. The genetically modified squash exhibits the trait of resistance to infection and subsequent disease caused by these viruses through a process that is related to viral cross-protection.<br>The ZW-20 squash line was created by Agrobacterium-mediated transformation in which the transfer-DNA (T-DNA) contained the coat protein genes from each of the two viruses. The constitutive expression of these genes was regulated by the 35S promoter from cauliflower mosaic virus (CaMV). In addition, the sequence of the WMV2 coat protein was introduced as a 5’-chimera that included the sequence encoding the N-terminal 16 amino acids of the CaMV coat protein. While the gene encoding neomycin phosphotransferase (NPTII) was incorporated into the Ti plasmid vector used for transformation, only plants lacking this gene were selected for commercialization. The gene encoding NPTII is, therefore, not present in the ZW-20 genome. There was no incorporation of plasmid DNA sequences outside of the T-DNA region as verified by Southern blot analysis.<br>Constitutive expression of the ZYMV and WMV2 coat proteins in transgenic squash were determined by enzyme linked immunosorbent assay (ELISA) and found to range between 73 – 1218 mg/kg fresh weight and 47-112 mg/kg fresh weight, respectively. This level of expression was significantly lower than measured levels of ZYMV and WMV2 coat protein in zucchini squash, yellow crookneck squash, cantaloupe and honeydew melons from supermarket shelves. These latter levels have been estimated to be up to 268 and 421 fold greater than the corresponding amounts of ZYMV and WMV2 CP produced in transgenic squash. Human consumption of viral coat proteins occurs whenever virus-infected plant products are consumed, or where crops have been protected from virus infection by classical cross-protection using a mild or symptomless strain of the virus. Other than susceptibility to virus infection, the disease, pest and other agronomic characteristics of line ZW-20 were comparable to non-transgenic counterparts.