1. Resistance to European Corn Borer (ECB)<br>Bacillus thuringiensis subsp. kurstaki (B.t.k.) is an endospore-forming, gram-positive, soil-borne bacterium. In its spore forming stage, it produces several insecticidal protein crystals, including the ð-endotoxin Cry1Ab which is active against certain lepidopteran insects such as ECB, Spruce Budworm, Tent caterpillar, Gypsy moth, Diamondback moth, Cabbage looper, Tobacco budworm, and Cabbage worm. The protein has been repeatedly 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. <br>A synthetic cry1Ab gene, derived from B.t.k. strain HD-1, coding for a truncated form of the Cry1Ab ð-endotoxin, and modified to enhance its expression in corn was developed. The synthetic gene has approximately 65% homology at the nucleotide level with the native gene. The truncated Cry1Ab protein contains the insecticidal region of the native Cry1Ab. The insecticidal protein binds to receptor sites in the epithelium of the susceptible insect's mid-gut, resulting in the formation of pores, disruption of the osmotic potential of the cells, cessation of feeding and the eventual death of the insect. <br>Event 176 was transformed with two of these synthetic cry1Ab genes. One gene is linked to a specific promoter which confers expression in green tissue, while the other is linked to a pollen-specific promoter, resulting in expression in pollen. Expression of the Cry1Ab protein in green tissues is intended to render the plant resistant to first generation ECB larvae feeding on leaves. Expression in pollen is intended to target second generation ECB larvae, which are known to feed on pollen. <br>Cry1Ab production was quantified in leaves and pollen among three genotypes. Expression in leaves, across genotypes, ranged from 0.596 to 1.159 µg/g (fresh weight) in seedlings, 0.530 to 3.029 µg/g (f.w.) at anthesis, 0.442 to 0.471 µg/g (f.w.) at physiological maturity, and 0.066 to 0.225 µg/g (f.w.) at senescence. Maximum expression in leaves was detected at either the vegetative or anthesis stages, depending on the genotype tested, and expression in all genotypes declined as plants senesced. Expression in pollen, across genotypes, ranged from 1.137 to 2.348 µg/g (f.w.). Expression of Cry1Ab in roots (<0.008 µg/g f.w.), pith (<0.008 µg/g f.w.), and kernels (<0.005 µg/g f.w) was below levels of quantification. Whole plant expression at anthesis, as a percentage of total protein ranged from 0.00025 to 0.0014 % (f.w.). <br>Biological activity of the truncated Cry1Ab protein produced in Event 176 leaves was compared to that of the native B.t.k. Cry1Ab on ECB, and other lepidopteran species. While quantitative comparisons were not possible, a similar rank order of sensitivity was observed to the Event 176 and native B.t.k. proteins among the tested lepidopteran species, other than Southwestern Corn Borer which did not feed on the Event 176 leaf extract. Non-susceptible species were unaffected by either the Event 176-expressed or native Cry1Ab proteins. <br>Western blot analyses of Event 176-expressed Cry1Ab, and of the native protein, displayed similar bands at approximately 65 kDa. Three additional immunoreactive proteins weighing approximately 60, 40 and 36 kDa were also detected in the leaves, but not in the pollen. It was suggested that these may represent breakdown products resulting from intrinsic proteolysis in Event 176 leaves. Toxicological studies were conducted using corn-expressed leaf and pollen Cry1Ab protein, thus the potential toxicity of the additional peptides detected in the leaves was assessed. <br>No evidence was found of post-translational modifications such as acetylation, glycosylation or phosphorylation in Event 176-expressed cry1Ab gene. <br>Cry1Ab protein from Event 176 leaves was subjected to in vitro digestibility studies under simulated mammalian gastric conditions and was shown to be digested as conventional dietary protein.<br>2. Glufosinate Ammonium Herbicide Tolerance<br>The glufosinate ammonium tolerance gene (bar gene), derived from the common soil bacterium Streptomyces hygroscopicus, codes for phosphinothricin acetyltransferase (PAT), and is linked to a constitutive promoter active in all plant tissues except pollen. Phosphinothricin, a glutamine synthetase inhibitor, is the active moiety of glufosinate ammonium. The herbicidal activity of phosphinothricin is characterized by the inhibition of glutamine synthetase resulting in the accumulation of lethal amounts of ammonia in the plant. PAT catalyses the acetylation of phosphinothricin, thus eliminating its herbicidal activity. <br>PAT was shown to act specifically on phosphinothricin, since no other activity was observed on other common acetyltransferase substrates, including pyruvate, choline or serine. <br>A sequence similarity search for the PAT protein revealed similarity only to other phosphinothricin resistance gene products and no similarity was found to known sequenced toxins. <br>Southern blot analysis revealed that the bar gene is present in transformed tissue, however, PAT protein levels were below the limit of quantification in tested tissues (&lt; 0.2 ppm in leaf, root, pith and whole plant samples). <br>In vitro digestibility studies, under simulated mammalian gastric conditions, were conducted with E. coli expressed PAT and revealed that this protein is digested as conventional dietary protein. <br>This trait was introduced to permit the identification of transformed embryos on selective medium and is also used to track genes during plant breeding.<br>3. Development Method<br>Two synthetic cry1Ab genes were cloned into a single plasmid vector. A second vector contained the herbicide tolerance gene (bar). These genes were introduced into the corn by microprojectile bombardment of immature embryos. Southern blot analysis indicated that there may be two or more copies of each plasmid integrated into the genome of Event 176. An ampicillin resistance gene, regulated by a bacterial promoter, also present in the vectors used to transform Event 176, was not expressed in either leaf tissue or pollen as confirmed by assays and Northern blot analysis. Other regulatory DNA elements inserted to enhance expression levels of cry1Ab did not code for any other protein.