<p style="text-align: justify;">Cotton (<i>Gossypium hirsutum</i> L.) was grown commercially in over 80 countries with a combined production of 44.2 million metric tonnes of cotton seed and 24.8 million metric tonnes of cotton lint in 2006. The major producers of cotton seed and lint were China, the United States, India, Pakistan, Brazil, Uzbekistan and Turkey. Cotton is primarily grown for its seed bolls that produce fibres used in numerous textile products.</p>

<p style="text-align: justify;">About two thirds of the harvested cotton crop is seed, which is separated from the lint during ginning. The cotton seed is crushed to produce cottonseed oil, cottonseed cake (meal), and hulls. Cottonseed oil is used primarily as a cooking oil, in shortening, margarine and salad dressing, and is used extensively in the preparation of snack foods such as crackers, cookies and chips. The meal and hulls are an important protein concentrate for livestock, and may also serve as bedding and fuel. Linters, or fuzz, which are not removed in ginning, are used in felts, upholstery, mattresses, twine, wicks, carpets, surgical cottons, and in industrial products such as rayon, film, shatterproof glass, plastics, sausage skins, lacquers, and cellulose explosives.</p>

<p style="text-align: justify;">Tobacco budworm (<i>Heliothis virescens</i>), pink bollworm (<i>Pectinophora gossypiella</i>), and cotton bollworm (<i>Helicoverpa zea</i>) are three of the most destructive pests of cotton. These insects cause damage to squares and bolls (tobacco budworm, cotton bollworm) and staining of lint (pink bollworm). Species such as the soybean looper (<i>Pseudoplusia includens</i>) and beet armyworm (<i>Spodoptera exigua</i>) cause feeding damage to leaves. Insect damage to the plant also increases susceptibility to diseases (<i>e.g.</i>, boll rot), and ultimately results in the losses of yield and crop quality.</p>

<p style="text-align: justify;">Methods of insect control in cotton include the use of insecticides and integrated pest management methods, such as scouting and the use of economic thresholds, prior to application. In 2004, 64% of the cotton acreage in the United States was treated with insecticides; those most commonly used were malathion, acephate and aldicarb. However, the effectiveness of many insecticides has been reduced due to the development of resistance in some insect pests, such as the tobacco budworm.</p>

<p style="text-align: justify;">The cotton line COT102 was genetically engineered to resist attack from lepidopteran insect pests such as the cotton bollworm (<i>Helicoverpa zea</i>), tobacco budworm (<i>Heliothis virescens</i>), pink bollworm (<i>Pectinophora gossypiella</i>), fall armyworm (<i>Spodoptera frugiperda</i>), beet armyworm (<i>Spodoptera exigua</i>), soybean looper (<i>Pseudoplusia includens</i>), and cabbage looper (<i>Trichoplusia ni</i>), and cotton leaf perforator (<i>Bucculatrix thurberiella</i>). This insect resistance is conferred by the <i>vip3A(a)</i> gene, originally isolated from the common soil bacterium <i>Bacillus thuringiensis</i> (Bt) strain AB88. The <i>vip3A(a)</i> gene produces the insect control protein VIP3A in the plant tissues. VIP3A is a member of a class of recently discovered insecticidal proteins: VIP (vegetative insecticidal proteins) proteins are expressed by the bacterium during the vegetative stage, as well as during sporulation, the stage at which the delta-endotoxins (<i>i.e.</i>, Cry proteins) are expressed. VIP proteins have an insecticidal mode of action similar to that of the delta-endotoxins: the ingestion by targeted insects leads to feeding cessation, loss of gut peristalsis, insect paralysis, and death. As with Cry proteins, VIP proteins also possess an active proteolytic core, which is activated by insect gut proteases, and binds to specific sites localized on the midgut lining of susceptible insect species. However, the VIP3A protein targets different molecules (<i>i.e.</i>, receptors) in the mid-gut lining, and the binding results in the formation of ion channels distinct from those formed by delta endotoxins, such as Cry1Ab. VIP proteins are not expected to affect other invertebrate and vertebrate organisms, including beneficial arthropods, birds and mammals. Only lepidopteran insect species possess VIP binding sites on the surface of their gut epithelia, therefore, livestock animals and humans are not susceptible to these proteins. Also, since only lepidopteran insect species are targeted by the VIP proteins, species of other insect orders, including beneficial and pest species, are not expected to be affected by this insecticidal protein.</p>

<p style="text-align: justify;">COT102 cotton was developed as an alternative and novel insect control option for lepidopteran pests of cotton. COT102 was also developed to help prevent and manage resistance to Cry proteins due to the different insecticidal mode of action of VIP proteins. The <i>vip3A</i> gene was introduced into the cotton line &lsquo;Coker 312&rsquo; by <i>Agrobacterium</i>-mediated transformation and the successfully transformed event was termed COT102. Also introduced into COT102 was the <i>aph4</i> gene, coding for hygromycin-B phosphotransferase (APH4), and used as a selectable marker.</p>

<p style="text-align: justify;">The cotton line COT102 was field tested at several locations in the United States from 2000 to 2002. Characteristics such as yield, boll size, plant growth and morphology, seed germination, flowering and maturity were found to be similar to the parental line &lsquo;Coker 312,&rsquo; and also within the range of variability for commercial cotton cultivars. Susceptibility to diseases and non-lepidopteran insects was not altered compared to the parental line. COT102 did not demonstrate morphological, vegetative or reproductive growth characteristics such as those observed in weedy and invasive plant species.</p>

<p style="text-align: justify;">The effect of the cultivation of COT102, specifically that of the insecticidal protein VIP3A, on non-target organisms was investigated during field testing, as well as in ecotoxicological studies. Non-target insects were not detrimentally affected by the insecticidal protein in COT102. VIP3A protein was found to be insecticidal to several lepidopteran species, many of which are pests of cotton; however, VIP3A did not demonstrate insecticidal activity towards lepidopterans such as the European Corn Borer (<i>Ostrinia nubilalis&gt;</i>) and the Monarch butterfly (<i>Danaus plexipus</i>). Non-target organisms, such as arthropods, mammals, birds, and aquatic organisms were not negatively affected by the VIP3A protein in COT102. The cultivation of COT102 would therefore pose no greater risk to interacting, non-target organisms, than conventional cotton cultivars, with the exception of specific lepidopteran insect pests.</p>

<p style="text-align: justify;">The potential for introgression of the insect resistance trait from COT102 cotton into other cotton plants, or to wild relatives of cotton, was investigated. Cotton plants are primarily self-pollinating, but insects, especially bumblebees and honeybees, also distribute cotton pollen. Cotton can cross-pollinate with compatible species including <i>G. hirsutum</i> (wild or under cultivation) and <i>G. barbadense</i> (cultivated Pima cotton), and is genetically compatible with <i>G. tomentosum</i>. Overall, the probability of gene transfer to wild species in unmanaged ecosystems is low due to the relatively isolated distribution of <i>Gossypium</i> species, different breeding systems, and genomic incompatibility. Assuming proximity, synchronicity of flowering and presence of insects, COT012 could freely hybridize with other <i>G. hirsutum</i> varieties and wild plants. In the event of the formation of hybrids, there would no competitive advantage conferred on any hybrid progeny in the absence of infestations by certain Lepidopteran insect species.</p>

<p style="text-align: justify;">The potential for toxicity and allergenicity of the novel proteins VIP3A and APH4 was determined from results of ecotoxicological studies using VIP3A on non-target organisms, from acute feeding studies using mice, from <i>in-vitro</i> digestibility studies using simulated mammalian gastric fluids, and from the expression levels of in COT102 tissues. No adverse effects were observed in the non-target organisms, which were fed the VIP3A protein at levels that far exceeding the estimated environmental exposure, based on the expression levels in tissues. No adverse effects were observed in the acute feeding studies with mice. The intestinal epithelial tissues of VIP3A-fed mice were studied and no changes were observed in these tissues, compared to mice fed a diet without the VIP3A protein. No observed effects were observed in mice exposed to a very high oral dose of the APH4 protein; the exposure level to non-target organisms is also very low due to the levels of APH4 in most tissues that were below the levels of detection. The VIP3A protein was completely hydrolyzed in simulated mammalian gut fluids, which further demonstrated the lack of toxicity and allergenicity of the protein. The results of these studies led to the conclusion that the novel proteins VIP3A and APH4 in event COT102 cotton are neither toxic to non-target organisms, including mammalian species.</p>
