<p style="text-align: justify;"><b>1. The Roundup-ReadyTM Genes</b></p>

<p style="text-align: justify;">Two genes were introduced, which in combination provided field level tolerance to glyphosate, the active ingredient in Roundup&reg; herbicide.</p>

<p style="text-align: justify;">The first gene expresses a bacterial derived version from <i>Agrobacteium tumefaciens</i> strain CP4, of the plant 5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS), an enzyme involved in the shikimate biochemical pathway for the production of the aromatic amino acids phenylalanine, tyrosine and tryptophan. Glyphosate inhibits the native plant EPSPS, thus blocking the shikimate pathway and halting the synthesis of these amino acids and leading to growth suppression or death of the plant. The CP4 EPSPS enzyme is highly insensitive to glyphosate and thus allows the aromatic amino acid needs of the plant.</p>

<p style="text-align: justify;">The second gene is derived from Achromobacter sp., a ubiquitous soil bacteria species which expresses an enzyme that degrades glyphosate by conversion to aminomethylphosphonic acid (AMPA) and glyoxylate, thereby deactivating the herbicidal effect. The coding sequence of this gene has been altered to enhance the efficiency of glyphosate degradation, compared to the original bacterial version.</p>

<p style="text-align: justify;">A plant-derived coding sequence expressing a chloroplast transit peptide was co- introduced with each of the Roundup-ReadyTM genes. This peptide facilitates the import of the newly translated Roundup-ReadyTM enzymes into the chloroplasts, the site of both the shikimate pathway and the site of action for glyphosate.</p>

<p style="text-align: justify;">Both genes associated with their transit peptide coding sequence, are linked to the same constitutive promoter. Expression of both Roundup-ReadyTM proteins in seed was quantified.</p>

<p style="text-align: justify;">The range of expression of both proteins in the seed was comparable to those of <i>B. napus</i> GT73. The average expression levels (in &micro;g/mg f.w.) of the first protein ranged from 0.032 to 0.053 for ZSR500; from 0.014 to 0.053 for ZSR502; and from 0.025 to 0.043 for ZSR503. The average expression levels (in &micro;g/mg f.w) of the second protein ranged from 0.064 to 0.124 for ZSR500; from 0.056 to 0.148 for ZSR502 and; from 0.061 to 0.135 for ZSR503.</p>

<p style="text-align: justify;">The Roundup-ReadyTM proteins showed no significant homology with any known toxins or allergens. The novel enzyme associated with the shikimate pathway, is a version of an enzyme that is ubiquitous in nature, and therefore would not be expected to be toxic or allergenic to non-target organisms. For the second novel enzyme, which degrades glyphosate, Monsanto describes experiments with a variety of substrates that show that it has a narrow substrate specificity, and appears not to affect any plant specific pathways. Both proteins are inactivated by heat, and by proteases in simulated mammalian gastric and intestinal fluids.</p>

<p style="text-align: justify;"><b>2. Development Method</b></p>

<p style="text-align: justify;">The <i>Brassica napus</i> line GT73 was developed by transforming the variety &quot;Westar&quot; using a disarmed non-pathogenic <i>Agrobacterium tumefaciens</i> vector; the vector contained the transfer DNA (T-DNA) region of an <i>A. tumefaciens</i> plasmid from which virulence and disease causing genes were removed, and replaced with the Roundup-ReadyTM genes. The T-DNA portion of <i>A. tumefaciens</i> plasmids are generally known to insert randomly into the plant&#39;s genome and the insertion is usually stable, as was shown to be the case in GT73.</p>

<p style="text-align: justify;">GT73 was then crossed with Hysyn Polish canola varieties. The mid-season <i>B. rapa</i> canola lines ZSR500, ZSR502 and ZSR503 were derived from four parental populations. Since populations were combined for their development, these lines are referred to as synthetic lines.</p>

<p style="text-align: justify;"><br />
<b>3. Stable Integration into the Plants&#39; Genomes</b></p>

<p style="text-align: justify;">Monsanto provided information on segregation and Southern blot analysis demonstrating that GT73 had a stable single genetic insert, consisting of single copies of the Roundup-ReadyTM genes (please see decision document DD95-02).</p>

<p style="text-align: justify;">Since Monsanto was able to cross GT73 with <i>B. rapa</i> and transfer the herbicide tolerance successfully, it is suggested that the genetic insert was located in the <i>B. rapa</i> portion (genome A) of the amphidiploid <i>B. rapa/B. oleracea</i> genome of <i>B. napus</i>.</p>
