<p style="text-align: justify;">The cotton lines 531, 757, and 1076 were produced by <i>Agrobacterium</i>-mediated transformation of the cotton (<i>Gossypium hirsutum</i>) line L. cv Coker C312 with plasmid PV-GHBKO4. Plasmid PV-GHBK04 contained the following elements: the 0.4 kb <i>oriV</i> fragment from the RK2 plasmid fused to the 3.4 kb segment of pBR322 allowing maintenance in <i>Escherichia coli</i> and in <i>Agrobacterium tumefaciens</i>. This was fused to the 360 bp DNA fragment from pTiT37 plasmid, which contained the nopaline T-DNA right border.</p>

<p style="text-align: justify;">The remaining portion consisted of two genes engineered for plant expression, the <i>cry1Ac</i> and the NPTII encoding <i>neo</i> gene. The <i>cry1Ac</i> gene was modified for optimal expression in plants and contained part of the 5&#39; end of the <i>cry1Ab</i> gene with a portion of the <i>cry1Ac</i> gene. Expression of the modified <i>cry1Ac</i> gene was regulated by cauliflower mosaic virus (CaMV) 35S promoter with a duplicated enhancer region and the nontranslated region of the soybean alpha subunit of the beta-conglycin gene which provided the mRNA polyadenylation signals (7S 3&rsquo; terminator sequence).<br />
The transformation plasmid also contained the <i>aad</i> gene isolated from <i>E. coli</i> bacterial transposon Tn7, which encodes the enzyme aminoglycoside adenyltransferase (AAD) that confers resistance to the antibiotics spectinomycin and streptomycin. The <i>aad</i> gene was under the control of its own bacterial promoter and terminator and was included in the construct as a marker to allow for selection of bacteria containing PV-GHBK04 prior to transformation of the plant cells. The <i>aad</i> gene has no plant regulatory sequences and was not expressed in plant tissues.</p>

<p style="text-align: justify;">The <i>neo</i> gene was located downstream of the <i>aad</i> gene and its expression was regulated using the CaMV 35S promoter and the non-translated region of the 3&rsquo; region of the nopaline synthase gene (<i>nos</i>) from the pTiT37 plasmid of <i>A. tumefaciens</i> strain T37.</p>
