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

<p style="text-align: justify;">Southern blot analysis of genomic DNA from lines MS8 and RF3 demonstrated that each line contained a single site of insertion for the T-DNA. The barnase and <i>bar</i> genes were integrated into MS8 and similarly, the barstar and <i>bar</i> genes were integrated in RF3.</p>

<p style="text-align: justify;">Further characterization of MS8 revealed that the inserted T-DNA was arranged in an inverted repeat structure with a second, incomplete T-DNA copy. The incomplete copy included a functional part of promoter PTA29, the coding region of barstar, the 3&acute;<i>nos</i> and a non-functional part of promoter PSsuAra.</p>

<p style="text-align: justify;">The insertion site of each line was well characterized. <i>Brassica napus</i> is an amphidiploid composed of two genomes <i>B. rapa/B. oleracea</i>. In line MS8 the insertion site was located in the <i>B. rapa</i> portion of the genome, while in line RF3 the insertion site was located in the <i>B. oleracea</i> portion of the genome.</p>

<p style="text-align: justify;">Based on Southern blots and detailed PCR analyses it was confirmed that no sequences outside of the T-DNA region from plasmids pTHW107 or pTHW118 were integrated into the plant genome. There were no marker genes for antibiotic resistance present in the transformed plants.</p>

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

<p style="text-align: justify;">Based on phenotypic and molecular techniques it was shown that the genes were stable and followed standard Mendelian inheritance. Segregation analysis demonstrated that in both transformation events the DNA was integrated at a single dominant locus.</p>

<p style="text-align: justify;">MS8 and RF3 are several generations removed from the original transformants. Comparisons to the original transformants demonstrated the novel traits were stably inserted and stably inherited into lines MS8 and RF3.</p>

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

<p style="text-align: justify;">Transgene expression and cryptic expression were addressed using Northern blot techniques. The PAT protein activity (the product of the <i>bar</i> gene that confers tolerance to herbicide) was detected in line MS8 in leaves and flower buds but not in dry seeds. In line RF3, PAT protein was detected in leaves and flower buds but not in dry seeds or pollen.</p>
