<p style="text-align: justify;"><b>The Introduced DNA</b></p>

<p style="text-align: justify;">The inserted <i>cry1Ac</i> gene encoded an insecticidal protein similar to a full length Cry1Ac protein with some minor changes arising from the design of the gene. The encoded Cry1Ac plant produced protein was 99.4% homologous to the native protein from <i>B. thuringiensis</i> subsp. <i>kurstaki</i> HD73.</p>

<p style="text-align: justify;">Southern blot analyses of genome DNA from line 531 demonstrated that two copies of the T-DNA insert were integrated in a head-to-tail arrangement. One T-DNA insert contained a full-length <i>cry1Ac</i> gene and the NPTII encoding gene, and the second insert contained an inactive 3&rsquo; portion of the <i>cry1Ac</i> gene. The two inserts were linked and segregated as a single locus. Similar analyses demonstrated that the <i>ori322</i> region, present in PV-GHBK04, was not transferred into the 531 genome. The <i>aad</i> gene was present but was not expressed since it was under the control of a bacterial promoter.</p>

<p style="text-align: justify;"><b>Genetic Stability of the Introduced Trait</b></p>

<p style="text-align: justify;">The stability of the inserted genes for lines 531 and 1076 was demonstrated over 4 generations. Stability for line 757 was tested and confirmed across 2 generations.</p>

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

<p style="text-align: justify;">Cry1Ac protein expression levels in each of the three transgenic cotton lines were quantitated using enzyme linked immunosorbent assay (ELISA). Mean Cry1Ac expression in line 531 was 1.56 &micro;g protein/g and 0.86 &micro;g protein/g fresh weight in leaf and seed tissue respectively. In the whole plant the Cry1Ac protein averaged 0.044 &micro;g protein/g fresh weight, (25 &micro;g/ plant) for a total of about 1.44 g/ acre. Expression varied approximately 3 fold over the growing season, with the levels peaking late in the season. Expression of the Cry1Ac protein was higher in line 757. Average values were 12.6 &micro;g protein/g fresh weight and 9.9 &micro;g protein/g fresh weight in leaves and seeds respectively. Expression varied less than 3 fold over the season and peaked early in the season in leaf tissue. Whole plant amounts were 1.1 &micro;g protein/g fresh weight (200 &micro;g/plant) for a total of 12.2 g/ acre. Line 1076 produced the cry1Ac protein at an average of 12.2 &micro;g protein/g fresh weight and 12.7 &micro;g protein/g fresh weight in leaves and seed respectively. Gene expression over the growing season varied less than 5 fold, with the highest concentration occurring early in the season. Whole plant totals were 1.1 &micro;g protein/g fresh weight (389 &micro;g/plant) for a total of 23.3 g/ acre. (Calculations per acre based on 60000 plants/acre). Expression in pollen and nectar was negligible in all three lines.</p>

<p style="text-align: justify;">Expression levels for the NPTII protein in line 531 were 3.14 &micro;g protein /g fresh weight and 2.45 &micro;g protein/g fresh weight in leaf and seed tissues respectively. Expression varied about 2 fold over the season. In line 757 NPTII expression was 6.9 &micro;g protein/g fresh weight and 3.3 &micro;g protein/g fresh weight in leaves and seeds respectively. For line 1076, the NPTII expression was 16.3 &micro;g protein/g fresh weight and 7.9 &micro;g protein/g fresh weight in leaves and seed respectively. Gene expression varied 2 to 3 fold over the growing season for line 757 and line 1076.</p>
