<p style="text-align: justify;"><b>The Introduced DNA:&nbsp;&nbsp;</b>Southern blot analysis and Polymerase Chain Reaction (PCR) amplification of the genomic DNA of LLCotton25 demonstrated one site of integration of a single copy of the T-DNA of pGSV71. Southern blot analysis also confirmed the integrity of the <i>bar</i> gene along with its promoter and terminator sequences and, that no sequences of the vector backbone were integrated into the genome of LLCotton25. The P35S promoter and the 3&rsquo; <i>nos</i> terminator sequences were derived from plant pathogenic organisms. Data from observations on several generations of LLCotton25 confirmed that these non-coding sequences did not cause diseases in the plants.</p>

<p style="text-align: justify;"><b>Genetic Stability of the Trait:</b>&nbsp;&nbsp;The stability of the <i>bar </i>gene, along with its promoter and terminator sequences (collectively termed the &ldquo;<i>bar</i> gene cassette&rdquo;), were assessed over multiple generations of conventional breeding at several locations, and in different genetic backgrounds. The results of genomic DNA blot analysis confirmed the stable inheritance of the <i>bar</i> gene cassette. Mendelian segregation data confirmed the stable inheritance of a single integrated gene.</p>

<p style="text-align: justify;"><b>Expressed Material:</b>&nbsp;&nbsp;The PAT protein was expected to be expressed in all tissues of the LLCotton25 plant since the <i>bar</i> gene was linked to a constitutive promoter (P35S from cauliflower mosaic virus). The PAT protein was quantitatively determined from LLCotton25 plants grown at various locations in the U.S.A. Plant tissues (<i>i.e.</i>, roots, leaves, stems and pollen) and seed and fibre fractions (cleaned seed, fuzzy seed, and lint) were analyzed using the enzyme linked immunoabsorbant assay technique (ELISA). The concentrations of PAT protein were determined on a fresh weight basis. In plant tissues other than the seed and lint, PAT protein averaged 7.97 &mu;g/g in roots, 59.2 &mu;g/g in leaves, 38.8 &mu;g/g in stems, and 19,2 &mu;g/g in pollen. The average expression of PAT protein in cleaned seed was 127 &mu;g/g and 69.9 &mu;g/g in fuzzy seed. PAT protein was detected in all fractions of the seed <i>(i.e</i>., hulls, solvent extracted meal, toasted meal). The fibre components (<i>i.e.,</i> lint coat and lint fractions) contained less than 1.5% of the total PAT protein expressed in the plant. There was a negligible amount of PAT protein expressed in the oil.</p>
