<p style="text-align: justify;"><b>Field Testing</b></p>

<p style="text-align: justify;">The transgenic squash line CZW-3 was field tested in the United States (1993 &ndash; 1996). CZW-3 was extensively tested in the laboratory, greenhouse, and field experiments and based on these assessments it was determined that the plants exhibited the typical agronomic characteristics of the parent crookneck squash, with the addition of resistance to CMV, ZYMV and WMV2 infection. Field trial reports demonstrated that CZW-3 did not exhibit weedy characteristics, and had no effect on nontarget organisms or the general environment.</p>

<p style="text-align: justify;"><b>Outcrossing</b></p>

<p style="text-align: justify;">Squash (<i>C. pepo</i>) and all other species of Cucurbita are monoecious (having both male and female flowers on the same plant) and produce heavy sticky pollen grains. Pollination requires an arthropod vector, usually a bee, to transmit the pollen from the staminate to the pistillate flower.</p>

<p style="text-align: justify;">Interspecific hybridization has been extensively investigated and is well understood in the four cultivated species of <i>C. pepo</i>, <i>C. mixta</i>, <i>C. moschata</i>, and <i>C. maxima</i>. It is reported that F1 hybrids can be obtained in breeding programs, but only with difficulty and such hybrids are usually sterile. There is no evidence of spontaneous hybridization among these four species despite that fact that they have been grown side by side under cultivation for many generations (Whitaker &amp; Robinson, 1986).</p>

<p style="text-align: justify;">Successful transmission of genetic material from CZW-3 squash via pollen is possible to a limited number of squash relatives (Wilson, 1993). In the United States, there exists two free-living subspecies of <i>C. pepo</i> that can cross with cultivated squash varieties without loss of fertility (designated as free-living Cucuribita pepo or FLCP). These include the free living gourds in many states including Texas (<i>C. pepo</i> ssp. ovifera var. texana) and free-living gourds in Illinois, Arkansas, and Oklahoma (<i>C. pepo</i> ssp. ovifera var. ozarkana.). FLCP plants have grown in proximity to new, improved cultivars of squash, and there have been no reports to suggest that disease resistance traits have introgressed into FLCP plants to produce hybrid populations that pose increased problems as weeds. Furthermore, field tests with hybrid plants derived from controlled crosses of CZW-3 with FLCP plants indicate that FLCP x CZW-3 hybrids do not appear to be strong competitors when growing in fields that have not been tilled to remove competing wild plants.</p>

<p style="text-align: justify;">Natural populations of FLCP appear to be largely free of infection by CMV, ZYMV and WMV2, suggesting that resistance to CMV, ZYMV and WMV2 would not provide any selective advantage. Should the virus resistance genes from CZW-3 transfer to FLCP plants, the selective pressure to maintain the virus resistance in natural populations of FLCP plants should be minimal, since all evidence supports the conclusion that FLCP populations are not under significant environmental stress from viral infection.</p>

<p style="text-align: justify;"><b>Weediness Potential</b></p>

<p style="text-align: justify;">The transgenic squash line CZW-3 was not likely to present a plant pest risk as a weed. The parent plant, yellow crookneck squash, is an agricultural crop plant that exhibits no appreciable weedy characteristics. In general there are no reports of squash to be a common or troublesome weed anywhere in the United States (Bridges &amp; Baumman, 1992). Squash volunteers are not uncommon in areas next to production fields, but they do not readily establish feral or free-living populations. Volunteers are controlled by mechanical means or herbicides.</p>

<p style="text-align: justify;">There are no indications that resistance to CMV, ZYMV and WMV2 would result in CZW-3 squash becoming a weed. During field trials, the transgenic plants were examined for any characteristics that may increase their weediness potential, and none were observed. There were no major changes in seed germination, cucurbitin levels, seed set viability, susceptibility or resistance to pathogens or insects (except CMV, ZYMV and WMV2), and there were no differences in overwintering survivability between CZW-3 squash and nontransgenic squash. It was concluded that CZW-3 was unlikely to increase the weediness of yellow crookneck squash and was no more likely to become a weed than virus-resistant plants.</p>

<p style="text-align: justify;"><b>Secondary and Non-Target Adverse Effects</b></p>

<p style="text-align: justify;">No direct pathogenic properties, nor any hypothetical mechanisms for pathogenesis toward beneficial organisms, such as bees and earthworms, were identified for CZW-3 squash. The coat proteins expressed in CZW-3 squash are not known to have any toxic properties. In fact, these viral coat proteins are routinely ingested by virtually all animals, including humans, when squash is consumed. Naturally occurring infections of susceptible squash varieties result in concentrations of coat proteins far higher than those that occur in the tissues of the CZW-3 squash. It was concluded that the genes inserted into the transgenic squash line CZW-3 would not result in any deleterious effects or significant impacts on nontarget organisms, including threatened and endangered species or beneficial organisms.</p>

<p style="text-align: justify;"><b>Impact on Biodiversity</b></p>

<p style="text-align: justify;">The squash line CZW-3 had no novel phenotypic characteristics that would extend its use beyond the current geographic range of squash production. The impact of cultivation of CZW-3 squash on biodiversity was likely to be comparable to that from non-transgenic varieties.</p>
