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

<p style="text-align: justify;">Southern blot and PCR analysis were used to demonstrate that there was a single DNA insertion in line GT73 consisting of the T-DNA containing one complete copy of the CP4 EPSPS gene and a complete copy of the <i>gox</i> gene and their respective regulatory sequences.</p>

<p style="text-align: justify;">The CP4 EPSPS gene encodes a protein of 47.6 KDa consisting of a single polypeptide of 455 amino acids. It was determined that once translocated to the chloroplast, the chloroplast transit signal sequence was cleaved and rapidly degraded <i>in vivo</i> leaving only the only newly expressed CP4 EPSPS.</p>

<p style="text-align: justify;">The <i>gox</i> gene encodes a single polypeptide of 431 amino acids with a molecular mass of 46.1 KDa. Nucleotide sequencing comparing the <i>gox</i> variant protein with normal version determined that <i>goxv247</i> differs by three amino acid substitutions and the two proteins were greater than 99% identical.</p>

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

<p style="text-align: justify;">Southern blot analysis of inserted DNA from the third generation and fifth generation demonstrated that the CP4 EPSPS and <i>gox</i> genes were stably transferred into the plant genome and stably inherited from parent to offspring over several generations.</p>

<p style="text-align: justify;">The insertion site was characterized by backcrossing studies. <i>B. napus</i> is an amphidiploid composed of two genomes <i>B. rapa/B. oleracea</i>. Backcrossing studies with <i>B. rapa</i> were successful and suggested that the genetic insert was integrated within the <i>B. rapa</i> portion of the genome.</p>

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

<p style="text-align: justify;">The expression of both CP4 EPSPS and glyphosate oxidoreductase was detected in the leaves and seeds of transgenic GT73 canola. Enzyme linked immunosorbent assay (ELISA) of seed and leaf tissue demonstrated that the introduced proteins were expressed in GT73 at very low levels, constituting less than 0.02% of the seed on a fresh weight basis. No detectable CP4 EPSPS or GOX protein was measured in non-transgenic control &#39;Westar&#39; seed or leaf tissue from any year. In line GT73, expression of both CP4 EPSPS and GOX proteins in the seed was stable when observed in different trials. Levels of CP4 EPSPS ranged from 18 - 34 &micro;g/g fresh weight and levels of GOX protein ranged from 108 - 193 &micro;g/g fresh weight.</p>
