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

<p style="text-align: justify;">Southern blot analysis determined a single site integration of a single copy each of the <i>cry2Ab2</i> and <i>uidA</i> gene cassettes. These cassettes contained the complete coding regions for each gene although the restriction site following the NOS 3&rsquo; polyadenylation sequence and the 260 bp of the 5&rsquo; end of the CaMV 35S promoter for the <i>uidA</i> gene were not present. The <i>uidA</i> promoter remained functional despite the truncation.</p>

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

<p style="text-align: justify;">Segregation data across four generations, including backcrosses to commercial cotton cultivars were statistically analysed, comparing frequency of observed numbers to expected numbers of progeny that expressed the Cry2Ab2 protein. Expression of the Cry2Ab2 protein was analysed using enzyme linked immunoabsorbent assay (ELISA). All generations segregated as expected for a single insertion site of the <i>cry2Ab2</i> gene. The presence of the Cry2Ab2 protein across multiple generations occurred as expected, consistent with a pattern of Mendelian inheritance. The presence of the <i>cry2Ab2</i> gene was confirmed using Southern blot analysis.</p>

<p style="text-align: justify;">The stability of the insert was also demonstrated using Southern blot analysis of genomic DNA samples from leaf tissues taken across five generations. There were no differences in the pattern of hybridizing fragments among DNA extracted from any of the five plant generations. These results demonstrated that the DNA insert from plasmid PV-GHBK11 is stable in the plant genome across five breeding generations.</p>

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

<p style="text-align: justify;">The expression levels of the Cry2Ab2 and GUS proteins were determined using ELISA. Cotton tissue samples were taken from eight field locations in the United States to determine the levels of Cry2Ab2 and GUS activity in a range of environments. Cry2Ab2 levels were examined in leaf, seed, pollen and whole plant tissues. GUS levels were determined in leaf and seed tissues.</p>

<p style="text-align: justify;">The mean levels of Cry2Ab2 in cottonseed, leaf tissue, and whole plants across all locations were 43.2 +/- 5.7 ug/g, 23.8 +/- 6.3 ug/g, 8.80 +/- 1.20 ug/g, and &lt; 0.25 ug/g respectively. The average GUS concentrations in cottonseed and leaf tissue were 106 +/- 32 ug/g, and 58.8 +/- 13.0 ug/g, respectively.</p>
