<p>1. Resistance to the European Corn Borer (ECB) Bacillus thuringiensis var. kurstaki HD-1 (B.t.k.) is a common gram-positive soil-borne bacterium. In its spore forming stage, it produces several insecticidal protein crystals, including the &eth;-endotoxin Cry1A(b), which is active against certain lepidopteran insects, such as ECB. This protein has been shown to be non-toxic to humans, other vertebrates and beneficial insects. B.t.k. based foliar insecticides have been registered for over 30 years in Canada and have a long history of safe use. A synthetic cryIA(b) gene was developed for maximum expression in corn, and introduced into line MON809. The gene codes for a protein similar to the Bacillus thuringiensis var. kurstaki HD-1 insecticidal crystal protein. The protein is insecticidal to lepidopteran larvae after cleavage to a bio-active, trypsin resistant core. Insecticidal activity is believed to depend on the binding of the active fragment to specific receptors present in susceptible insects on midgut epithelial cells, forming pores which disrupt osmotic balance and eventually results in cell lysis. Specific Lepidopteran pests of corn sensitive to the protein are ECB and corn earworm. The cry1A(b) gene is linked to a strong constitutive promoter. Average protein expression evaluated at six locations was 1.63 g/g (f.w.) in leaves, 0.55 g/g (f.w.) in seeds and 1.23 g/g (f.w.)in the whole plant. Cry 1A(b) protein was not detected in pollen. Protein expression ranged from 0.88-2.37 g/g (f.w.) in leaves, from 0.28-0.73 g/g (f.w.) in grain and from 0.73-1.73 g/g (f.w.) in the whole plant. Protein expression declined over the growing season as indicated by the Cry1A(b) levels present in leaves assayed over the growing season. The Cry1A(b) protein was shown to degrade readily in the environment. The plant expressed protein had DT50 and DT90 values (time to degrade to 50% and 90 % of the original bioactivity) of 2 and 15 days respectively. Protein allergens are normally resistant to digestion unlike the Cry1A(b) protein which was shown to degrade readily in simulated gastric fluid. Unlike many known allergens the insecticidal protein is not glycosylated. A search for amino acid sequence similarity between the Cry1A(b) protein and known allergens, using a database assembled from the public domain databases GenBank, EMBL, Pir and SwissProt, revealed no significant amino acid sequence homologies. A search of a similarily constructed database of known toxins indicated no amino acid sequence homologies between known toxins and the Cry1A(b) protein, with the exception of homologies to other B.t. insecticidal proteins. Corn products are an important alternative to wheat flour for individuals afflicted with coeliac disease, an immune mediated food intolerance for which wheat gliadins have been implicated as the causal agent. In light of the importance of corn products to these individuals, a sequence similarity search was conducted and no amino acid sequence homologies between the Cry1A(b) protein and gliadins were detected. The full nucleotide sequence and corresponding amino acid sequence were provided. The 131 kDa protein expressed in plants and the resulting proteolytic fragments were compared to the bacterial proteins and shown to be of similar molecular weight, amino acid sequence, immunological reactivity and trypsin resistance. The protein was not glycosylated and showed similar bioactivity and host range specificity to the native protein. 2. Glyphosate Tolerance Two genes have been introduced into MON809 to provide tolerance to glyphosate, the active ingredient in Roundup&reg; herbicide. The tolerance, although not sufficient to provide field tolerance to the herbicide, was used to select transformed plants. The CP4 EPSPS gene expresses a bacterial version from Agrobacterium tumefaciens strain CP4, of 5-enolpyruvlshikimate-3-phosphate synthase, a plant enzyme involved in the shikimate biochemical pathway for the production of aromatic amino acids. Glyphosate inhibits the native plant EPSPS, thus blocking the shikimate pathway and halting the production of these necessary amino acids. The CP4-EPSPS is a glyphosate tolerant version of the enzyme with high catalytic activity. Under glyphosate selection, the addtion of the bacterial, glyphosate tolerant enzyme will permit the normal production of aromatic amino acids. The second gene, gox, is derived from Achromobacter sp., a ubiquitous soil bacteria species which expresses an enzyme that degrades glyphosate by conversion to aminomethlyphosphonic acid (AMPA) and glyoxylate. In line MON809 only a partial copy of the gene is inserted and no gene expression was detected. A plant-derived coding sequence expressing a chloroplast transit peptide was co-introduced with each of the glyphosate resistance genes. The chloroplast transit peptides facilitates the import of the protein products from the glyphosate resistance genes into the chloroplast, which is the location of the shikimate pathway and the site of action for glyphosate. Expression of the CP4 EPSPS gene averaged 21.68 &micro;g/g (f.w.) in leaves, 9.41 &micro;g/g (f.w.) in grain and 1.6 &micro;g/g (f.w.) in the whole plant. The plant expressed enzyme showed no significant homology with any known toxins or allergens. It is common in nature and no toxic or allergenic effects would be expected to occur. Data was presented which showed that the CP4 EPSPS enzyme is rapidly inactivated by heat and by digestion in simulated mammalian gastric fluid. 3. Development Method MON809 was co-transformed with two vectors, one carrying a synthetic cry1A(b) gene and the second bearing the two herbicide resistance genes. The two plasmid vectors were introduced by microprojectile bombardment into cultured plant cells. Glyphosate tolerant transformed cells were selected, then cultured in tissue culture medium for plant regeneration. 4. Stable Integration into the Plant&#39;s Genome Southern analysis indicated one integrated DNA segment which included a complete and a partial copy of the cryIA(b) gene, two complete copies of the CP4 EPSPS genes and a partial gox gene. Segregation data indicate a stable insertion, with the trait segregating according to Mendelian genetics, through five generations of crossing. 5. Nutrition The nutritional quality of grain from MON 809 is similar to that of grain from an equivalent non-transgenic line and to grain that is currently in commerce. Grain was analysed for proximate (i.e., ash, moisture content, crude protein, crude fat), amino acid and fatty acid composition. Some minor differences were observed between MON 809 and an equivalent non-transgenic line, but these components were within the range of variation normally observed in the commercial grain supply and are attributed to variations in the plant genotypes rather than to the presence of the introduced genes. 6. Safety The CryIA(b) and CP4 EPSPS proteins should pose no risks for consumption of products produced from insect resistant maize. The introduced proteins were compared to databases of known protein toxins and neither shows any significant amino acid sequence similarity to known protein toxins. Both proteins are rapidly broken down and their enzymatic activity lost under conditions that simulate mammalian digestion. Neither protein was toxic when fed to mice.</p><br>