<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 cry1A(b) gene was developed for maximum expression in corn, and introduced into line MON810. 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 9.35 &micro;g/g (f.w.) in leaves and 0.31 &micro;g/g (f.w.) in seeds. Protein expression evaluated at one site, was 4.15 &micro;g/g (f.w.) in the whole plant and 0.09 &micro;g/g (f.w.) in pollen, as determined from a single sample. Protein expression ranged from 7.93 to 10.34 &micro;g/g (f.w.) in leaves, from 0.19 to 0.39 &micro;g/g (f.w.) in grain and from 3.65 to 4.65 &micro;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 similarly 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 native HD-1 protein has a molecular weight of 131 kD. The inserted, plant expressed cryIA(b) gene codes for a truncated protein with a molecular weight of 91 kD. After activation by trypsin to the insecticidal form, 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 were originally introduced into the original transformant to provide tolerance to glyphosate, the active ingredient in Roundup&reg; herbicide. The tolerance was used to select transformed plants. Molecular data was presented which showed that the genes conferring glyphosate tolerance were not present in line MON810. The genes were presumed to have inserted at genetic loci away from the cryIA(b) gene and then segregated out during subsequent crossing leading to line MON810. 3. Development Method MON810 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 truncated copy of the cry1A(b) gene, inserted without rearrangement. The corn line has been crossed into several diverse corn genotypes for 4 generations and the protection against ECB has been maintained. MON810 was derived from the third generation of backcrossing and therefore the single insert appears to be stably inherited.</p>

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