<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. In the United States alone the combined costs of control and yield loss attributed to these pests is approximately $476 million per year. In Egypt, China and Brazil, pink bollworm commonly causes cotton losses of up to 20 percent.</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 281-24-236 was genetically engineered to resist attack from Lepidopteran insect pests such as the tobacco budworm, cotton bollworm, beet armyworm, and soybean looper. This insect resistance is conferred by the <i>cry</i>1F gene, originally isolated from the common soil bacterium <i>Bacillus thuringiensis</i> (Bt) var. <i>aizawai</i>. The <i>cry</i>1F gene produces the insect control protein Cry1F, a delta-endotoxin, in the plant tissues. Cry proteins, of which Cry1F is only one, act by selectively binding to specific sites localized on the lining of the midgut of susceptible insect species. Following binding, pores are formed that disrupt midgut ion flow, causing gut paralysis and eventual death due to bacterial sepsis. Cry1F is insecticidal only when eaten by the larvae of lepidopteran insects (moths and butterflies), and its specificity of action is directly attributable to the presence of specific binding sites in the target insects. There are no binding sites for delta-endotoxins of <i>B. thuringiensis</i> on the surface of mammalian intestinal cells, therefore, livestock animals and humans are not susceptible to these proteins. The <i>cry</i>1F gene was introduced into the cotton line &lsquo;Germain&rsquo;s Acala GC510&rsquo; by <i>Agrobacterium</i>-mediated transformation.</p>

<p style="text-align: justify;">The cotton line 281-24-236 was field tested in the United States from 2000 to 2002, and in Puerto Rico in 2000-01. Agronomic characteristics such as yield, boll size, plant vigour, growth, morphology, germination and flowering were found to be within the range for commercial cotton lines. Susceptibility to diseases was not altered compared to conventional cotton varieties. The cotton line 281-24-236 did not demonstrate morphological or growth characteristics such as those observed in weedy and invasive plant species.</p>

<p style="text-align: justify;">The effect of the cultivation of line 281-24-236 on non-target organisms was investigated during field testing. Non-lepidopteran insects were not detrimentally affected by this cotton line; this was not unexpected, given the insecticidal specificity of the Cry proteins. Insecticidal effects on the monarch butterfly, a lepidopteran, although not directly assessed in the field, were not expected given the very low expression of the Cry protein in the pollen. Based on the results from dietary studies of monarch butterfly larvae with Cry1F protein, the estimated concentration of Cry1F necessary for a 50% reduction in larval growth is several hundred thousand-fold the concentration in pollen from line 281-24-236. This, combined with the low volume of pollen produced, would result in very little risk to non-target lepidopterans, such as the monarch butterfly. While cotton is mostly self-pollinated, any cross-pollination would be achieved by non-lepidopteran insects, such as bees, which are unaffected by Cry proteins. Other non-target organisms, such as arthropods, mammals, birds, and aquatic organisms were not negatively affected by the cultivation of line 281-24-236. It was concluded that, compared to conventional cotton varieties, the cultivation of line 281-24-236 would pose no greater risk to interacting organisms, with the exception of specific lepidopteran insect pests of cotton.</p>

<p style="text-align: justify;">The potential for introgression of the insect resistance trait from line 281-24-236 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, the cotton line 281-24-236 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 Lepidopteran insect species.<br />
The food and livestock safety of the cotton line 281-24-236 was based on: the equivalence of the Cry1F protein to the bacterially produced protein which has a history of safe use as Bt insecticidal treatments; the lack of amino acid sequence homology of both the Cry1F and PAT proteins to known toxins and allergens; results of feeding studies with mammals, birds, aquatic organisms and non-target arthropods, demonstrating the lack of toxicity of the Cry1F protein to organisms other than lepidopteran insects; the rapid digestibility of the proteins in simulated gastric fluids; the very low levels of expression of both novel proteins in all plant tissues, including seed and oil; and the equivalence in nutritional components and anti-nutrients, of the seed and oil compared to conventional cotton cultivars.</p>
