<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.</p>

<p style="text-align: justify;">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 MS1 and RF1 were developed using genetic engineering techniques to provide a pollination control system for the production of hybrid oilseed rape (MS1xRF1) expressing male sterility and tolerance to glufosinate ammonium. The novel hybridization system involves the use of two parental lines, a male sterile line MS1 and a fertility restorer line RF1. The transgenic MS1 plants do not produce viable pollen grains and cannot self-pollinate. In order to completely restore fertility in the hybrid progeny, line MS1 must be pollinated by a modified plant containing a fertility restorer gene, such as line RF1. The resultant F1 hybrid seed, derived from the cross between MS1 x RF1, generates hybrid plants that produce pollen and are completely fertile.</p>

<p style="text-align: justify;">The male-sterile trait was introduced in MS1 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 RF1 was produced by genetically engineering plants to restore fertility in the hybrid line. Transgenic RF1 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 <i>barnase</i> RNAse expressed by the MS1 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 RF1 is crossed to the male sterile line MS1, 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 MS1 and RF1 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 canola lines MS1 and RF1, and the hybrid MS1xRF1, were field tested in Canada from 1991 to 1994. Agronomic characteristics such as vegetative vigour, overwintering capacity, flowering period, and seed germination and dormancy were within the normal range of expression found in unmodified canola varieties. As expected, seed production of the hybrid line was greater than that of the unmodified counterparts. Flowers of the MS1 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 susceptibilities to major insect pests and diseases, and was 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 MS1, RF1 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 MS1, 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 MS1 or RF1 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.</p>

<p style="text-align: justify;">The analysis of nutrients from transgenic canola lines MS1 and RF1 and non-transgenic canola did not reveal any significant differences in the levels crude protein, crude fat, crude fibre, ash and gross energy in either whole seed or processed meal. The fatty acid composition of oils extracted from both transgenic and non-transgenic canola was statistically the same, and within the normal range for canola oil. It was determined that the consumption of refined oil from MS1, RF1 or hybrids derived from these lines would have no significant impact on the nutritional quality of the food supply.</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. It was concluded that these proteins, and thus MS1, RF1 and MS1xRF1 hybrid canola lines, possessed little or no potential for allergenicity or toxicity.</p>
