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

<p style="text-align: justify;">Southern blot analyses of genomic DNA from line 1445 demonstrated the insertion at a single site on a 6.1 kb restriction fragment that included sequences encoding AAD, NPTII, CP4 EPSPS, and a partial <i>oriV</i> sequence. These analyses failed to detect any incorporation of the <i>gox</i> gene sequence.</p>

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

<p style="text-align: justify;">The stability of lines 1445 and 1698 was demonstrated for 5 generations. Southern blot analysis showed that the inserted DNA from the progeny of three successive homozygous generations (R3 through R5) was stably integrated into the cotton genome. Southern and genetic analyses of line 1445 demonstrated that a single copy of the insert was transferred at one single locus.</p>

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

<p style="text-align: justify;">Western immunoblot analysis of samples of line 1445 tissue demonstrated the presence of 48 kD immunoreactive species, which was the same apparent molecular weight (47.5 kD) as purified CP4 EPSPS protein isolated from <i>E. coli</i>. These studies, also verified that the CTP2 sequence (8.2 kD) was cleaved from the mature protein and rapidly degraded, following delivery of the CP4 EPSPS to the chloroplast.</p>

<p style="text-align: justify;">Expression levels of CP4 EPSPS protein in line 1445 were determined based on material from six different trial site locations in the United States during 1993 and 1994. Expression levels of CP4 EPSPS were measured in cottonseed and leaf, using enzyme linked immunosorbent assay (ELISA) and found to be low, ranging from 0.02% to 0.028% of total protein in cottonseed. In cottonseed the CP4 EPSPS concentration was approximately 0.08 &micro;g/mg fresh weight while in leaves the level was approximately 0.05 &micro;g/mg f.w. For line 1698 expression of the enzyme in the seed averaged 0.205 &micro;g/mg (f.w) while in the leaves the level averaged 0.311 &micro;g/mg (f.w). No expression was detected in the untransformed parental line Coker 312.</p>

<p style="text-align: justify;">Combed lint and brown linter stock (<i>i.e.</i> linters after the first processing step) were tested for the presence of CP4 EPSPS protein by Western blotting. CP4 EPSPS was detected in combed lint but not in brown linter stock, the first product in the sequence of processing linters for cellulose.</p>

<p style="text-align: justify;">The expression of the NPTII protein was determined by ELISA and detected at low levels in cottonseed and leaf material. The NPTII protein content of cottonseed was 0.0022% of total protein, present in whole seeds at concentrations of approximately 0.007 &micro;g/mg fresh weight. In leaf material, NPTII was present in concentrations of approximately 0.045 &micro;g/mg fresh weight. For line 1698, NPTII expression averaged 0.004 &micro;g/mg (f.w) in the seed and 0.031 &micro;g/mg (f.w.) in the leaf.</p>

<p style="text-align: justify;">The effect of application of glyphosate on levels of expression during plant growth was also investigated. There were no significant differences in expression levels of CP4 EPSPS and NPTII in cottonseed between plants treated with glyphosate and those that were not treated.</p>
