<p style="text-align: justify;">Argentine or oilseed rape (<i>Brassica napus</i>) was grown as a commercial crop in over 50 countries, with a combined harvest of 48.9 million metric tonnes in 2006. The major producers of rapeseed are China, Canada, India, Germany, France, the United Kingdom and Australia. Canola is a genetic variation of <i>B. napus</i> that was developed through conventional breeding to contain low levels of the natural rapeseed toxins, glucosinolate and erucic acid. Canola is grown for its seed, which represents a major source of edible vegetable oil and is also used in livestock feeds.<br />
The only food use of canola is as a refined oil. Typically, canola oil is used by itself as a salad oil or cooking oil, or blended with other vegetable oils in the manufacture of margarine, shortenings, cooking and salad oils. Canola meal, a byproduct of the oil production process, is added to livestock feed rations. An increasing amount of oil is being used for biodiesel production, especially in Europe.</p>

<p style="text-align: justify;">The canola lines MS8 and RF3 were developed using genetic engineering techniques to provide a pollination control system for the production of hybrid oilseed rape (MS8xRF3) expressing male sterility and tolerance to glufosinate ammonium. The novel hybridization system involves the use of two parental lines, a male sterile line MS8 and a fertility restorer line RF3. The transgenic MS8 plants do not produce viable pollen grains and cannot self-pollinate. In order to completely restore fertility in the hybrid progeny, line MS8 must be pollinated by a modified plant containing a fertility restorer gene, such as line RF3. The resultant F1 hybrid seed, derived from the cross between MS8 x RF3, generates hybrid plants that produce pollen and are completely fertile.</p>

<p style="text-align: justify;">The male-sterile trait was introduced in MS8 by inserting the <i>barnase</i> gene, isolated from <i>Bacillus amyloliquefaciens</i>, a common soil bacterium that is frequently used as a source for industrial enzymes. The <i>barnase</i> gene encodes for a ribonuclease enzyme (RNAse) that is expressed only in the tapetum cells of the pollen sac during anther development. The RNAse affects RNA production, disrupting normal cell functioning and arresting early anther development, thus leading to male sterility.</p>

<p style="text-align: justify;">The transgenic line RF3 was produced by genetically engineering plants to restore fertility in the hybrid line. Transgenic RF3 plants contain the <i>barstar</i> gene, also isolated from <i>Bacillus amyloliquefaciens</i>. The <i>barstar</i> gene codes for a ribonuclease inhibitor (barstar enzyme) expressed only in the tapetum cells of the pollen sac during anther development. The ribonuclease inhibitor (barstar enzyme) specifically inhibits barnase RNAse expressed by the MS8 line. Together, the RNAse and the ribonuclease inhibitor form a very stable one-to-one complex, in which the RNAse is inactivated. As a result, when pollen from the restorer line RF3 is crossed to the male sterile line MS8, the resultant progeny express the RNAse inhibitor in the tapetum cells of the anthers, allowing hybrid plants to develop normal anthers and restoring fertility.</p>

<p style="text-align: justify;">Both transgenic lines MS8 and RF3 were also engineered to express tolerance to glufosinate ammonium, the active ingredient in phosphinothricin herbicides (Basta&reg;, Rely&reg;, Finale&reg;, and Liberty&reg;). Glufosinate chemically resembles the amino acid glutamate and acts to inhibit an enzyme, called glutamine synthetase, which is involved in the synthesis of glutamine. Essentially, glufosinate acts enough like glutamate, the molecule used by glutamine synthetase to make glutamine, that it blocks the enzyme&#39;s usual activity. Glutamine synthetase is also involved in ammonia detoxification. The action of glufosinate results in reduced glutamine levels and a corresponding increase in concentrations of ammonia in plant tissues, leading to cell membrane disruption and cessation of photosynthesis resulting in plant withering and death.</p>

<p style="text-align: justify;">Glufosinate tolerance in these canola lines was the result of introducing a gene encoding the enzyme phosphinothricin-N-acetyltransferase (PAT) isolated from the common aerobic soil actinomycete, <i>Streptomyces hygroscopicus</i>. The PAT enzyme catalyzes the acetylation of phosphinothricin, detoxifying it into an inactive compound. The PAT enzyme is not known to have any toxic properties.</p>

<p style="text-align: justify;">The PAT enzyme was used as a selectable marker enabling identification of transformed plants during tissue culture regeneration, and as a field selection method to obtain 100% hybrid seed.</p>

<p style="text-align: justify;">The transgenic canola lines MS8, RF3, and derived hybrids, have been extensively field tested in Canada from 1994 to 1996, in the United States beginning in 1997, and in Europe. Important agronomic characteristics such as germination, vegetative vigour, flowering period, time to maturity and seed production were within the normal range of expression found in unmodified canola varieties. Flowers of the MS8 line had undeveloped anthers, slightly smaller petals and did not produce fertile pollen, but nectar production remained unchanged and normal insect pollination was observed. Stress adaptation was evaluated, including disease and pest susceptibilities, and determined to fall within the ranges currently displayed by commercial varieties. It was demonstrated that the transformed canola lines did not exhibit weedy characteristics, or negatively affect beneficial or non-target organisms. Canola lines MS8, RF3 and their progeny were not expected to impact on threatened or endangered species.</p>

<p style="text-align: justify;"><i>Brassica napus</i> may outcross (up to 30% of the time) with plants of the same species, and potentially with plants of related species <i>B. rapa, B. juncea, B. carinata, B. nigra, Diplotaxis muralis, Raphanus raphanistrum</i>, and <i>Erucastrum gallicum</i>. Previous studies have demonstrated that cross breeding is most likely to occur with <i>B. rapa</i>. Due to the ability of canola to outcross with related plants, the formation of glufosinate-tolerant hybrids is possible. However, the glufosinate-tolerance trait is not expected to provide a competitive advantage to hybrid plants unless grown in managed environments routinely subjected to glufosinate applications. Additionally, multiple barriers, including the male-sterility of the canola line MS8, ensured that gene flow from this transformed line into wild or cultivated sexually compatible plants was extremely unlikely. In the event that a glufosinate-tolerant hybrid survived, the herbicide-tolerant individual could be easily controlled using mechanical and/or other available chemical means.</p>

<p style="text-align: justify;">The human consumption of canola products is limited to the refined oil. Refined edible canola oil consists of purified triglycerides (96-97%) and does not contain any detectable protein, hence no amounts of the introduced proteins were detected in the refined oil of MS8 or RF3 canola. Furthermore processing would destroy the enzymatic activity of the introduced gene products. As such, there will be no human exposure to these proteins as a result of the consumption of refined oil from these transgenic lines, or their hybrids.</p>

<p style="text-align: justify;">The composition of refined canola oil from MS8xRF3 hybrid canola was compared to that of non-transgenic canola. Some statistical differences in fatty acid composition were revealed, but the fatty acid composition of the oil from the transgenic line was still within the normal range for canola oil. Processing according to protocols mimicking industrial practices further demonstrated the composition and physical characteristics of oil from the MS8xRF3 canola line was equivalent to that of non-transgenic canola. It was determined that the consumption of refined oil from MS8, RF3 or hybrids derived from these lines would have no significant impact on the nutritional quality of the food supply in Canada.</p>

<p style="text-align: justify;">Potential toxicity and allergenicity of the transformed canola lines was further investigated through examination of the amino acid sequences and physiochemical characteristics of the novel proteins RNAse (<i>barnase</i>), RNAse inhibitor (<i>barstar</i>), and PAT (<i>bar</i>). No significant homologies between the amino acid sequences of these novel proteins and those of known toxins or allergens were detected. Further, these proteins were shown not to possess the proteolytic or heat stability characteristic of toxic compounds, and were readily digested under conditions simulating mammalian digestion. It was concluded that these proteins, and thus MS8, RF3 and MS8xRF3 hybrid canola lines, possessed little or no potential for allergenicity or toxicity.</p>
