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

<p style="text-align: justify;">In order to demonstrate the stable integration of the pat gene in T45 and its progeny, the genomic DNA from three different generations of event T45 (F5, F7 and R2) was analyzed by Southern blot. These analyses indicated that the original transformant (T45) contained a single copy of the <i>pat</i> gene integrated at a single site. Additional analyses to indentify the possible incorporation of plasmid DNA sequences outside of the T-DNA region were negative, indicating that the T-DNA was the only new DNA inserted into the host genome.</p>

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

<p style="text-align: justify;">HCN28 was at least four generations removed from the original transformant (T45) during which time the presence and expression of the <i>pat</i> gene remained stable.</p>

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

<p style="text-align: justify;">The only newly expressed material in T45 was the PAT protein. The identity and levels of expression of PAT enzyme were determined using a double antibody sandwich (DAS)-ELISA performed on total protein extracts obtained from seeds of transgenic T45 plants. Concentrations of PAT protein in canola seeds ranged from 95 to 246 ng/g of seed tissue.</p>

<p style="text-align: justify;">The levels of PAT protein detected by DAS-ELISA were highest in the untoasted canola meal, up to 38 &micro;g/g protein. The presence of the PAT protein was expected in the meal due to the use of a constitutive promoter (CaMV 35S), which drives PAT expression throughout the plant, but only minimally in pollen. The untoasted canola meal showed detectable enzymatic activity. After the meal was toasted, all activity was destroyed indicating that the enzyme was denatured during the first stages of processing. Levels of PAT protein detected in toasted canola meal were much lower than those in untoasted meal, and ranged from 2-5 &micro;g/g of canola meal. This reduction is likely due to a decrease in antibody reactivity as a result of PAT protein denaturation due to the meal toasting process where temperatures in excess of 90&deg;C are encountered.</p>

<p style="text-align: justify;">PAT protein was not detected in crude oil from the non-transgenic control, but was detected at a low level in crude oil samples from transgenic samples. Amounts detected ranged from 0.296 to 0.460 ng/g, well below the validated limit of quantitation of 250 ng/g. The PAT protein was not detected by DAS-ELISA in any of the refined oils obtained from transgenic or non-transgenic sources, and there were no detectable levels of PPT-acetyltransferase activity. Refined canola oil generally contains no protein of any kind.</p>
