<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 281-24-236 indicated the incorporation of single copies of the <i>cry</i>1F and <i>pat</i> genes, along with their respective regulatory sequences. Further analysis demonstrated a second, partial copy of the <i>pat</i> gene together with a second copy of the Ubi Zm1 promoter; however, this <i>pat</i> gene fragment was not associated with its terminator, ORF25 Poly A. No protein expression was detected from the gene fragment. Evidence was also provided to show that none of the plasmid backbone sequences, including the erythromycin gene, were incorporated into the genomic DNA.</p>

<p style="text-align: justify;">The <i>cry</i>1F gene is a synthetic, plant-optimized gene that codes for Cry1F, a full-length chimeric version of Cry1Fa2, originally from <i>Bacillus thuringiensis</i> var. <i>aizawai</i>. In this synthetic version, the first 694 amino acids are from the toxic portion of Cry1Fa2; 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 Cry1F 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 281-24-236 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 Cry1F and PAT protein in the tissues and cottonseed processed products of event 281-24-236 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 Cry1F protein, on a dry matter basis, were 6.46&ndash;7.67 ng/mg in leaves, 5.04 ng/mg in squares, 5.71 ng/mg in flowers, 4.02 ng/mg in bolls, 5.13 ng/mg in seeds, and 3.3 ng/mg in cottonseed (fresh-weight basis). The average level of Cry1F in pollen was below the level of quantification (0.09 ng/mg). The concentration of Cry1F in hulls was 0.22 ng/mg, and was not detected in toasted meal, or refined cottonseed oil.</p>

<p style="text-align: justify;">The average levels of PAT protein were considerably lower than those of the Cry1F protein. Average levels of PAT protein, on a dry matter basis, were 0.21 &ndash; 0.43 ng/mg in leaves, 0.51 ng/mg in squares, 0.22 ng/mg in bolls, 0.47 ng/mg in seeds, and 0.42 ng/mg in cottonseed (fresh-weight basis). The average level of Cry1F in pollen (0.09 ng/mg) was also below the level of quantification, and neither was it detectable in refined cottonseed oil.</p>
