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

<p style="text-align: justify;">Southern blot analysis of the genomic DNA from line 3006-210-23 indicated the incorporation of single intact copies of the <i>cry</i>1Ac and <i>pat</i> genes, along with intact copies of their respective regulatory sequences. Evidence was provided to show that none of the plasmid backbone sequences, including the erythromycin resistance gene, were incorporated into the genomic DNA.</p>

<p style="text-align: justify;">The <i>cry</i>1Ac gene is a synthetic, plant-optimized gene that codes for a full-length chimeric version of Cry1Ac1, originally from <i>Bacillus thuringiensis</i> var.<i> kurstaki</i> strain HD73. In this synthetic version, the first 612 amino acids are comprised of the toxic portion of Cry1Ac1; the remaining portion consists of C-terminal sequences from Cry1Ca3 (<i>B.t.</i> var.<i> aizawai </i>PS81I), and Cry1Ab1 (<i>B.t.</i> var. <i>Berliner</i> 1715). These last two fragments are part of the C-terminal portion of the crystal protein that is cleaved by proteases in the lepidopteran mid-gut. The molecular weight of the plant-expressed, protease-cleaved Cry1Ac protein is 65kDa, as confirmed by Western blot analysis.</p>

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

<p style="text-align: justify;">The stability of the inserted insect resistance trait in line 3006-210-23 was assessed within and across generations using Southern blot analysis. First generation plants, generated from backcrosses with one of the parental lines, segregated in a 1:1 ratio of resistant to non-resistant plants. The progeny resulting from the self-pollination of these first generation plants segregated in a 3:1 of resistant to non-resistant plants. These results were not significantly different from the expected Mendelian segregation ratios and confirmed the stable inheritance of a single dominant trait.</p>

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

<p style="text-align: justify;">The levels of Cry1Ac and PAT protein in the tissues and cottonseed processed products of 3006-210-23 cotton were quantified using enzyme-linked immunosorbant assay (ELISA) methods. Samples for analysis were obtained from field trials at 6 different locations in 2001. The average levels of Cry1Ac protein, on a dry matter basis, were 1.44 &ndash; 1.92 ng/mg in leaves, 1.84 ng/mg in squares, 1.92 ng/mg in flowers, 1.44 ng/mg in pollen, 0.77 ng/mg in bolls, 0.57 ng/mg in seeds, and 0.62 ng/mg in cottonseed (fresh-weight basis). Cry1Ac was not detected in toasted meal, hulls, or refined cottonseed oil.</p>

<p style="text-align: justify;">PAT protein was not detected in leaves, squares, flowers, pollen, bolls and refined oil. It was below the limit of quantification in seeds and cottonseed (0.06 and 0.09 ng/mg, respectively).</p>
