GM Crop Database

The MS8 and RF3 canola lines (Brassica napus) were developed using genetic engineering techniques to provide a pollination control system for production of hybrid oilseed rape (MS8 x RF3). 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 cross between MS8 x RF3, produces hybrid plants that produce pollen and are completely fertile.

The transgenic line MS8 (DBN230-0028) was produced by genetically engineering plants to be male sterile and tolerant to the herbicide glufosinate ammonium (as a selectable marker). The parental line MS8 contains the barnase gene for male sterility, isolated from Bacillus amyloliquefaciens, a common soil bacterium that occurs naturally in the soil and is frequently used as a source for industrial enzymes. The barnase 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.

The transgenic line RF3 (DBN212-0005) was produced by genetically engineering plants to restore fertility in the hybrid line and to be tolerant to the herbicide glufosinate ammonium (as a selectable marker). Transgenic RF3 plants contain the barstar gene isolated from Bacillus amyloliquefaciens. The barstar gene codes for a ribonuclease inhibitor (barstar enzyme) that is expressed only in the tapetum cells of the pollen sac during anther development. The ribonuclease inhibitor 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 transferred 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 restore fertility.

Both trangenic canola lines MS8 and RF3 contain the bar gene that confers tolerance to the post-emergence, broad-spectrum phosphinothricin herbicides (Basta, Rely, Finale, and Liberty). The bar gene, isolated from the common soil microorganism Streptomyces hygroscopicus encodes a phosphinothricin acetyl transferase (PAT) enzyme. The active ingredient in phosphinothricin herbicides is glufosinate ammonium which acts by inhibiting the plant enzyme glutamine synthetase, leading to the accumulation of phytotoxic levels of ammonia killing the plant within hours of application. PAT detoxifies glufosinate ammonium by acetylation into an inactive compound, eliminating its herbicidal activity. The herbicide tolerance trait was introduced into the canola lines as a selectable marker to identify transformed plants during tissue culture regeneration, and as a field selection method to obtain 100% hybrid seed.

The MS8 and RF3 canola lines were both produced using Agrobacterium-mediated transformation of the Brassica napus cultivar 'Drakkar'. The T-DNA region of the Ti plasmid was "disarmed" by removal of virA genes, normally associated with the pathogenicity and disease-causing properties of A. tumefaciens, 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.

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 Nicotiana tabacum, terminated by part of the 3´non-coding region (3´nos) of the nopaline synthase gene of A. tumefaciens. Similarly, line RF3 was produced using plasmid pTHW118, which contained the barstar gene under the control of the pTa29 anther-specific promoter from N. tabacum and the nos termination signal.

In addition, each T-DNA contained a copy of the bar gene from S. hygroscopicus, which encodes the PAT enzyme. Expression of the bar gene was regulated by the PSsuAra promoter from Arabidopsis thaliana and post-translational targeting of the gene product to the chloroplast organelles was accomplished by fusion of the 5'-terminal coding sequence with the chloroplast transit peptide DNA sequence from A. thaliana.

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

The Introduced DNA
Southern blot analysis of genomic DNA from lines MS8 and RF3 demonstrated that each line contained a single site of insertion for the T-DNA. The barnase and bar genes were integrated into MS8 and similarly, the barstar and bar genes were integrated in RF3.

Further characterization of MS8 revealed that the inserted T-DNA was arranged in an inverted repeat structure with a second, incomplete T-DNA copy. The incomplete copy included a functional part of promoter PTA29, the coding region of barstar, the 3´nos and a non-functional part of promoter PSsuAra.

The insertion site of each line was well characterized. Brassica napus is an amphidiploid composed of two genomes B. rapa/B. oleracea. In line MS8 the insertion site was located in the B. rapa portion of the genome, while in line RF3 the insertion site was located in the B. oleracea portion of the genome.

Based on Southern blots and detailed PCR analyses it was confirmed that no sequences outside of the T-DNA region from plasmids pTHW107 or pTHW118 were integrated into the plant genome. There were no marker genes for antibiotic resistance present in the transformed plants.

Genetic Stability of the Introduced Trait
Based on phenotypic and molecular techniques it was shown that the genes were stable and followed standard Mendelian inheritance. Segregation analysis demonstrated that in both transformation events the DNA was integrated at a single dominant locus.

MS8 and RF3 are several generations removed from the original transformants. Comparisons to the original transformants demonstrated the novel traits were stably inserted and stably inherited into lines MS8 and RF3.

Expressed Material
Transgene expression and cryptic expression were addressed using Northern blot techniques. The PAT protein activity (the product of the bar gene that confers tolerance to herbicide) was detected in line MS8 in leaves and flower buds but not in dry seeds. In line RF3, PAT protein was detected in leaves and flower buds but not in dry seeds or pollen.

Field Testing
The transgenic canola lines MS8, RF3, and derived hybrids, have been extensively field tested in Canada (1994 to 1996), in the United States since 1997 and in Europe. Important agronomic characteristics such as germination, vegetative vigour, flowering period, time to maturity and seed production of both transgenic lines were within the normal range of expression of characteristics in unmodified B. napus counterparts. 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 its resistance or susceptibility to major B. napus pests and pathogens (e.g., blackleg, sclerotinia, flea beetles, diamondback moth larvae) and determined to fall within the ranges currently displayed by commercial varieties. The lines were tested under various environmental conditions, and showed no differences in agronomic performance when compared to unmodified counterparts under the same conditions. Overall the field data reports demonstrated that MS8 and RF3, and the hybrid MS8 x RF3 had no potential to pose a plant pest risk.

Outcrossing
The sterility of the B. napus line MS8 ensured that gene introgression from MS8 into wild or cultivated sexually-compatible plants was extremely unlikely. The MS8 plants can act as pollen recipients, but the progeny would be partially male sterile.

Transgenic line RF3 and hybrid plants displayed normal reproductive characteristics. Brassica napus plants are known to outcross up to 30% with other plants of the same species, and potentially with plants of related species B. rapa, B. juncea, B. carinata, B. nigra, Diplotaxis muralis, Raphanus raphanistrum, and Erucastrum gallicum. Previous studies have demonstrated that gene flow is most likely to occur with B. rapa, the other major canola species, and an occasional weed of cultivated land especially in the eastern provinces of Canada.

The genes coding for male sterility or fertility restoration do not confer any ecological advantage to potential hybrid offspring of MS8 or RF3 plants. If glufosinate ammonium tolerant individuals arose through interspecific or intergeneric hybridization, the novel traits would confer no competitive advantage to these plants unless these populations were routinely subject to herbicide treatments. In the event that a glufosinate ammonium tolerant B. napus survived, these herbicide-tolerant individuals would be easily controlled using mechanical and other available chemical means. It was concluded that gene flow from the transgenic lines MS8 and RF3 or their hybrids to canola relatives was possible, but would not result in increased weediness or invasiveness of these relatives.

Weediness Potential
Field studies on invasiveness and survival characteristics comparing the MS8, RF3 and hybrid MS8 x RF3 to unmodified plants determined that the transgenic lines were not different from their counterparts in these respects. It was determined that glufosinate ammonium did not confer a competitive advantage to the transgenic lines over unmodified varieties since herbicide resistance does not confer any pest resistance, alter reproductive biology or change any physiology related to survival. It was concluded that the traits controlling pollination in MS8, RF3 and their hybrid progeny had no altered weed or invasiveness potential compared to currently commercialized B. napus varieties and in fact, male sterility trait in MS8 would provide a competitive disadvantage.

Secondary and Non-Target Adverse Effects
It was determined that genetically modified lines MS8, RF3, and hybrid MS8 x RF3 did not have a significant adverse impact on organisms beneficial to plants or agriculture, nontarget organisms, and were not expected to impact on threatened or endangered species. The barnase and barstar proteins did not result in altered toxicity or allergenicity properties and are only produced in the tapetum cell layer of anthers at a specific developmental stage.

Impact on Biodiversity
The transgenic lines MS8, RF3 and their hybrids have no novel phenotypic characteristics which would extend their use beyond the current geographic range of canola/rapeseed production. Since outcross species are only found in disturbed habitats, transfer of novel traits would not have an impact on unmanaged natural environments. It was determined that the relative impact on biodiversity of MS8, RF3 and MS8 x RF3 was equivalent to that of currently commercialized canola lines.

Dietary Exposure
The human consumption of canola products is limited to the refined oil. Because virtually no protein is present in the oil extracted from the plants, the potential for human exposure is exceedingly low. Furthermore, the amounts of PAT protein present in seed-meal fed to animals would be too low to cause concern. Additionally, the barnase RNAse and its inhibitor encoded by barnase and barstar genes, respectively, were not detected in dry seeds. As the introduced gene products were not detectable in the refined oil produced from transgenic canola, there will be no human exposure to these proteins based on normal consumption patterns.

Nutritional Data
The composition of refined canola oil from MS8 x RF3 hybrid canola was compared to that for refined oil from non-transgenic canola. Some statistical differences in fatty acid composition were noted in the comparison, however, the fatty acids for the transgenic lines, including the erucic acid content of the oil, were within the normal range for canola oil fatty acids. Processing according to protocols mimicking industrial practices (including tempering, flaking, cooking, pressing, desolventizing oil and meal, oil blending, degumming, oil refining, water washing, bleaching, hydrogenation and deodorizing) further demonstrated that the composition and physical characteristics of the oil from MS8 x RF3 hybrids and control canola varieties were equivalent. The use of refined oil from MS8 x RF3 canola hybrids would therefore have no significant impact on the nutritional quality of the food supply. Similarly, the glucosinolate content of seed meal derived from transgenic canola hybrids was the same as that from non-transgenic control cultivars.

Toxicity and Allergenicity
Since only the processed oil from transgenic MS8, RF3, or hybrids derived therefrom (MS8 x RF3), are available for human consumption, and the processing removes proteinaceous material, there were no additional toxicity or allergenicity concerns regarding this product. This was further assessed by searching for amino acid sequence homologies with known protein toxins and allergens, and by examining the physiochemical characteristics of the introduced RNAse (barnase), RNAse inhibitor (barstar), and PAT (bar) proteins.

A comparative analysis using the FASTDB algorithm to search three amino acid sequence databases determined that amino acid sequence for each novel protein did not show significant homology with other proteins present in the databases, with the exception of other related compounds. For example, barnase RNAse was similar to ribonucleases from other bacilliform bacteria and PAT was similar to other phosphinothricin acetyltransferases originating from different organisms. No homologies with potential toxins or allergens were observed.

The RNAse, encoded by the barnase gene, was a small single-domain protein, containing no disulfide bonds, metal-ion cofactors or other non-peptide components. When heated, it unfolded completely into an inactive form.

No toxic or allergic effects were expected from PAT, as acetyltransferases are ubiquitous in nature, do not possess proteolytic or heat stability and are highly substrate specific. PAT has a extremely high substrate specificity for L-PPT and dimethylphosphinothricin (DMPT), and experimental data clearly showed that neither L-PPT's analog L-glutamic acid, D-PPT, nor any other amino acid can be acetylated by the PAT enzyme.