<p style="text-align: justify;">The yellow crookneck squash (<i>Curcurbita pepo</i> L.), line CZW-3 was developed using recombinant DNA techniques to be resistant to infection by three plant viruses that frequently infect commercial squash varieties, namely cucumber mosaic cucumovirus (CMV), zucchini yellow mosaic potyvirus (ZYMV), and watermelon mosaic potyvirus (WMV2). The novel variety was developed by insertion of the coat protein (CP) encoding sequences from these three single-stranded RNA viruses. The expression of viral CPs in this transgenic squash variety does not result in the formation of any infectious particles, or in the manifestation of any disease pathology, but rather enables the plant to resist infection by CMV, ZYMV and WMV2. These viruses are major pathogens of all cucurbit crops (squash, cucumbers, and their relatives) and are present in most regions where these crops are grown.</p>

<p style="text-align: justify;">ZYMV and WMV2 are members of the potyvirus group, one of the largest groups of plant viruses, while CMV is the type member of the cucumovirus group. ZYMV produces a severe disease consisting of mosaic (patchwork of yellow chlorotic tissues and green uninfected tissues), yellowing, shoestringing, stunting, and fruit and seed deformations on zucchini squash, muskmelon, cucumber, and watermelon. WMV2 causes mosaic and mottle diseases of cantaloupe, pumpkin, squash, and watermelon. On a given cucurbit host, ZYMV usually causes more severe symptoms than WMV2.<br />
CZW-3 squash exhibits &ldquo;pathogen derived resistance&rdquo; to infection and subsequent disease caused by CMV, ZYMV and WMV2 through a process that is related to viral cross-protection. Although the exact mechanism by which the viral protection occurs is unknown, most evidence suggests that expression of viral CP by a plant interferes with one of the first steps in viral replication, uncoating (removal of CP) from the incoming virus (Register &amp; Nelson, 1992). Other modes of action of cross protection have also been suggested (Matthews, 1991). Transgenic CZW-3 squash displayed a high level of resistance to systemic infection with CMV, ZYMV, and WMV 2, although 64% exhibited localized chlorotic dots that were mainly confined to older leaves. CZW-3 plants had a 50-fold increase in marketable yield compared to non-transgenic control plants.</p>

<p style="text-align: justify;">An antibiotic resistance marker gene (<i>neo</i>) encoding the enzyme neomycin phosphotransferase II (NPTII), which inactivates aminoglycoside antibiotics such as kanamycin and neomycin, was also introduced into the genome of this variety. This gene was derived from a bacterial transposon (Tn5 transposable element from <i>Escherichia coli</i>) and was included as a selectable marker to identify transformed plants during tissue culture regeneration and multiplication. The expression of the <i>neo</i> gene in these plants has no agronomic significance and the safety of the NPTII enzyme as a food additive was evaluated by the United States Food and Drug Administration in 1994 (US FDA, 1994).</p>
