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

<p style="text-align: justify;">Genomic DNA from leaf tissue of GHB614 cotton plants (identity confirmed by PCR) was analysed using Southern blot analysis to determine the insert number, the copy number, the integrity of the inserted 2mepsps gene cassette, and evaluate the presence or absence of plasmid backbone sequences. Isolated genomic DNA samples from GHB614 cotton and conventional cotton were digested with nine different restriction enzymes, separated on agarose gels and then subjected to Southern blot analysis. To determine the insert and copy number of the introduced DNA, the separated DNA fragments were transferred to a membrane and sequentially hybridized with different radioactively labelled probes: four probes containing each single genetic element present in the p-TEM2 vector used for the transformation, and the complete T-DNA probe.</p>

<p style="text-align: justify;">Based on a comparison of the size and pattern of observed fragments with the expected fragment sizes from digestion of genomic DNA, a single and unique site of insertion of the transgenic sequences is present in cotton line GHB614.<br />
The organisation of the genetic elements within the insert in GHB614 cotton was further characterised using PCR analysis by amplifying three overlapping regions of DNA spanning the entire length of the insert. The PCR products generated, following PCR of genomic DNA from GHB614 cotton, were all of the expected size.</p>

<p style="text-align: justify;">The PCR products generated from GHB614 cotton genomic DNA were subject to DNA sequencing to further confirm the organisation of genetic elements within the insert, as well as to determine the 5&rsquo; and 3&rsquo; insert-to-genomic DNA junctions, and the complete DNA sequence of the inserted DNA and adjacent genomic DNA regions. The sequence determination indicated that the size of the inserted DNA in GHB614 cotton is 3978 base pairs (bp) and the arrangement of the genetic elements within the insert is identical to the corresponding transformation vector, pTEM2. In addition to the insert sequence, 214 bp of Right Border flanking sequence (3&rsquo; end of insert) and 738 bp of Left Border flanking sequence (5&rsquo; end of insert) were found to be completely identical to the cotton genomic sequences present at the integration site before transformation.</p>

<p style="text-align: justify;"><b>Stability and Inheritance</b></p>

<p style="text-align: justify;">For breeding purposes and further evaluation of inheritance, various backcrosses were performed and evaluated in the greenhouse for segregation and glyphosate resistance. Mendelian inheritance for a single gene locus would predict one resistant plant for every one susceptible plant within BC2F1 progenies. Furthermore, BC2F2 progeny would be expected to show three resistant plants for every one susceptible plant. For the segregation analysis, data from a Chi-square test of inheritance were used to determine the heritability and stability of the new trait. All Chi-square values indicate no significant differences between observed and expected genetic ratios across all tested generations of GHB614 cotton. These results are consistent with a single site of insertion for the 2mepsps gene expression cassette.<br />
To determine the stability of the inserted DNA, Southern blot analyses were done using genomic DNA isolated from multiple generations of GHB614 cotton (T3, T4, T5 and T6). For these analyses, DNA samples from leaf tissue representing each generation were digested and probed to detect two integration fragments corresponding to ~4850 bp and ~9100 bp. In all tested samples, the expected 5&rsquo; and 3&rsquo; integration fragments were present. These results are consistent with bands detected in other Southern analyses of GHB614 cotton and confirm the stability of the insert across multiple generations of breeding.</p>

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

<p style="text-align: justify;">Concentrations of 2mEPSPS protein were determined in samples of fuzzy cottonseed collected from nine field trial locations in the southern United States. At each site, six plots were planted with transgenic cotton and three plots planted with the non-transgenic control. Three of the transgenic plots were sprayed three times with glyphosate herbicide at the level of 840 g/hectare, and three transgenic plots were untreated. Because the cottonseed (fuzzy seed) had been ginned but not delinted, it could not be ground into a homogeneous material. A procedure was developed to effectively remove the lint and the associated seed coat. This created two fractions, which were designated &lsquo;kernel&rsquo; and &lsquo;lint coat&rsquo;. These fractions were analysed separately for 2mEPSPS protein and total extractable protein and the respective values added to give values for the fuzzy seed as received from the field.</p>

<p style="text-align: justify;">The 2mEPSPS protein was found in all fractions of transgenic fuzzy seed (kernel and lint coat). As expected, more than 99.5% of the novel protein was found in the kernel samples. The lint coat generally contained less than 0.5% of the 2mEPSPS protein, and some samples were below the limit of detection. The levels of 2mEPSPS protein varied between different trial sites and between treatments with glyphosate. On a fresh weight basis, the 2mEPSPS protein content in fuzzy seed of GHB614 cotton, not sprayed with glyphosate, ranged from about 15.8 &micro;g/g to 25.5 &micro;g/g fresh weight, with an overall average value of 19.2 &plusmn;3.1 &micro;g/g. On a fresh weight basis, the fuzzy seed from GHB614 cotton plants, sprayed with a conventional herbicide regime, contained 2mEPSPS protein in the range 16.2 &micro;g/g to 30.5 &micro;g/g, with an overall average value of 21.2 &plusmn; 4.0 &micro;g/g. Using the average values for the amount of novel protein in unsprayed and sprayed fuzzy seed relative to the amount of crude protein, the 2mEPSPS protein comprised an average of 0.0093% &plusmn; 0.0018% and 0.0100% &plusmn; 0.0019% of the total crude protein respectively.</p>
