<p style="text-align: justify;">Yellow crookneck squash (<i>Cucurbita pepo</i> L.), together with pumpkins, gourds, and other squash, were grown in 87 countries in 2004, with a combined harvest of 19 million metric tonnes. The major producers of these vegetables were China, India, Ukraine, the United States, Egypt, Mexico, the Islamic Republic of Iran and Cuba. Yellow crookneck squash is grown primarily for the fresh market and the processed squash industry, which includes frozen foods, dehydrated and canned products.</p>

<p style="text-align: justify;">Squashes, pumpkins and gourds are mostly annual, warm season species in the genus Cucurbita. They are closely related to cucumber, muskmelon and watermelon. There are four major species in the Cucurbita genus: pepo, mixta, moschata, and maxima, and one minor species, ficifolia, which is a perennial plant that grows in the Andes. The terms pumpkin and squash have no precise botanical meaning and may refer to any of the above New World species. Squashes are also called vine crops because, originally, the cultivars in this family all grew on vines. While most of these crops still grow on vines, some of the newest cultivars have a bush growth habit, making them easier to grow in smaller spaces. The fruits come in an astonishing assortment of shapes and sizes, from tiny, marble-sized pumpkins of the Caribbean islands to giant gourds more than seven feet long. All of the New World species, except ficifolia, are annuals.</p>

<p style="text-align: justify;">Viral diseases, such as mosaic viruses, are a limiting factor to squash production, particularly during summer and fall months. Mosaic viruses include the cucumber mosaic cucumovirus (CMV), zucchini yellow mosaic potyvirus (ZYMV), and watermelon mosaic potyvirus (WMV2). It is often hard to differentiate virus infections from one another based on visual symptoms, which include mosaic patterns (light green, dark green) in leaves, puckering, leaf distortion, stunting, shortened internodes and misshapen fruit. Aphids vector each of the viruses mentioned above and CMV is also seed-transmitted. Often two or more viruses are detected in a single plant, and the viruses generally overwinter in weed hosts. Preventative control measures include avoiding late season planting and removing weeds around fields. Insect control has not proven effective. Other than removal of virus-infected plants, there are no other effective control measures for these crops once infected.</p>

<p style="text-align: justify;">The CZW-3 squash line was developed using recombinant DNA techniques to resist infection by CMV, ZYMV, and WMV2 by inserting virus-derived sequences that encode the coat proteins (CPs) from each of these viruses. The introduced viral sequences do not result in the formation of any infectious particles, nor does their expression result in any disease pathology.</p>

<p style="text-align: justify;">This transgenic 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).</p>

<p style="text-align: justify;">CZW-3 squash was tested in field trials in the United States (1993&ndash;1996). These tests demonstrated that CZW-3 plants exhibited the typical agronomic characteristics of conventional crookneck squash, with the addition of resistance to CMV, ZYMV and WMV2 infection. CZW-3 was comparable to conventional squash varieties and did not exhibit weedy characteristics, and had no effect on nontarget organisms or the general environment.</p>

<p style="text-align: justify;">Squash (<i>C. pepo</i>) and all other species of Cucurbita are monoecious, such that the male and female flowers are separate structures but still on the same plant. Pollination requires an insect vector, usually a bee, to transmit the pollen from the male flower to the female flower. Squash varieties, including CZW-3, will cross-pollinate with other varieties within the same species, such as zucchini squash and acorn squash.</p>

<p style="text-align: justify;">There are several related species to cultivated squash, which include <i>C. mixta, C. moschata</i>, and <i>C. maxima</i> and wild populations of <i>C. pepo</i> referred to as free living Cucurbita pepo (FLCP). There is no evidence that squash can naturally cross-pollinate with relatives from <i>C. mixta, C. moschata</i>, and <i>C. maxima</i>, despite the fact that they have been grown side by side under cultivation for many generations. Cultivated squash can cross-pollinate with FLCP and form fertile hybrids. Observations of FCLP plants indicate that disease resistance traits have not be transferred to FCLP. If virus resistance genes from CZW-3 were transferred to FLCP plants, the selective pressure in natural populations of FLCP plants to maintain the virus resistance gene would be minimal. Evidence supports the conclusion that FLCP populations are not under significant environmental stress from viral infection.</p>

<p style="text-align: justify;">The food and livestock feed safety of squash line CZW-3 was established based on several standard criteria. As part of the safety assessment, the nutritional composition of squash fruit was found to be equivalent to conventional varieties by the analyses of protein, moisture, total fat, ash, dietary fibre, total carbohydrates, calories, sugar profile (fructose, glucose, sucrose, lactose, maltose), vitamin C, beta-carotene, vitamin A, calcium, iron, and sodium. Cucurbit plants, such as squash, produce alkaloids classified as cucurbitacins, which are bitter-tasting compounds that discourage feeding by herbivores. A standard test in plant breeding for the presence of cucurbitacins involves tasting the product to determine its bitterness and is able to detect levels between 1 &ndash;10 ppb. The transgenic squash line CZW-3 and the parent variety were both non-bitter, indicating that the cucurbitacin level in each line was less than 10 ppb.</p>

<p style="text-align: justify;">The CMV, ZYMV, and WMV2 coat proteins do not possess characteristics typical of known protein allergens or toxins such as heat stability and resistance to digestion by simulated gastric fluids. Comparisons of the deduced amino acid sequence of the plant-expressed CMV, ZYMV, and WMV2 CPs did not reveal any homology to known protein allergens and toxins. Furthermore, CMV-, ZYMV-, and WMV2-infected squash naturally contains higher levels of viral CP than expressed in this transgenic squash, and there has been no evidence of adverse effects linked to the consumption of virus-infected squash.</p>
