<p style="text-align: justify;">The MS1 and RF1 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>vir</i>A 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 MS1 and RF1. During transformation, the T-DNA portion was transferred into the plant cells and stably integrated into the plant&#39;s genome.</p>

<p style="text-align: justify;">Line MS1 was produced from the insertion of T-DNA containing the <i>barnase</i> gene together 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 from <i>Agrobacterium tumefaciens</i>. Similarly, line RF1 was produced from T-DNA containing the <i>barstar</i> gene under the control of the pTa29 anther-specific promoter from <i>Nicotiana tabacum</i> and 3&#39;<i>nos</i> terminator.</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, and the neomycin phosphotransferase II (NPTII) encoding <i>neo</i> gene from the Tn5 transposon of <i>Escherichia coli</i>, strain K12. 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&rsquo;-terminal coding sequence with the chloroplast transit peptide DNA sequence from <i>A. thaliana</i>. The expression of NPTII activity, under control of the nopaline synthase promoter from <i>A. tumefaciens</i>, was used as a selectable trait for screening transformed plants for the presence of the barnase and barstar genes, respectively.</p>
