<p style="text-align: justify;">The MS8 and RF3 canola lines were both produced using <i>Agrobacterium</i>-mediated transformation of the <i>Brassica napus</i> cultivar &#39;Drakkar&#39;. The T-DNA region of the Ti plasmid was &quot;disarmed&quot; by removal of <i>virA</i> genes, normally associated with the pathogenicity and disease-causing properties of <i>A. tumefaciens</i>, and replaced with the genes of interest for each transgenic line. During transformation, the T-DNA portion of each plasmid was transferred into the plant cells and stably integrated into the plant genome of MS8 and RF3 respectively.</p>

<p style="text-align: justify;">Line MS8 was produced using plasmid pTHW107, which contained a copy of the barnase gene whose transcription was regulated with an anther specific promoter pTa29 from <i>Nicotiana tabacum</i>, terminated by part of the 3&acute;non-coding region (3&acute;<i>nos</i>) of the nopaline synthase gene of <i>A. tumefaciens</i>. Similarly, line RF3 was produced using plasmid pTHW118, which contained the barstar gene under the control of the pTa29 anther-specific promoter from <i>N. tabacum</i> and the <i>nos</i> termination signal.</p>

<p style="text-align: justify;">In addition, each T-DNA contained a copy of the <i>bar</i> gene from <i>S. hygroscopicus</i>, which encodes the PAT enzyme. Expression of the <i>bar</i> gene was regulated by the PSsuAra promoter from <i>Arabidopsis thaliana</i> and post-translational targeting of the gene product to the chloroplast organelles was accomplished by fusion of the 5&#39;-terminal coding sequence with the chloroplast transit peptide DNA sequence from <i>A. thaliana</i>.</p>

<p style="text-align: justify;">Sequences outside the T-DNA region contained: colE1 replication region from <i>Escherichia coli</i>; pVS1 replication region isolated from Pseudomonas; and a fragment of plasmid R751 from <i>Klebsiella aerogenes</i> comprising the streptomycin/spectinomycin (Sm/Sp) resistance gene with its own promoter.</p>
