1. Resistance to the European Corn Borer (ECB)<br>Bacillus thuringiensis subsp. kurstaki (B.t.k.) is a common gram-positive soil-borne bacterium. In its spore forming stage, it produces several insecticidal protein crystals, including Cry1Ab that is active against lepidopteran insects such as ECB, corn earworm, spruce budworm, tent caterpillar, gypsy moth, diamondback moth, cabbage looper, tobacco budworm, and cabbage worm. The 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 and have a history of safe use. <br>The ECB resistance gene engineered into the corn hybrids X4334CBR and X4734CBR codes for a truncated version of Cry1Ab. Insecticidal activity results from the Cry1Ab protein binding to specific insect gut epithelium receptors, pore formation, loss of cations, and disruption of digestive processes following an osmotic pressure imbalance. Current knowledge indicates that only sensitive insects possess such receptors. <br>The cry1Ab gene, truncated and modified for enhanced plant expression, is linked to a strong constitutive promoter. Protein production (referred to as B.t.k. protein in the present document) was quantified in roots, leaves, stalk epidermis, stalk pith, tassel, pollen, silk, ear shank, husk, cob, brace root and kernel. It was found throughout the plant with highest levels detected in leaf tissues and at younger stages of development, and lowest levels detected in pollen grains. Expression was found to average 28.87 µg/g (f.w.) of young leaf tissue, 15.23 µg/g (f.w.) of mature leaf tissue and 4.76 µg/g (f.w.) of homozygous seed tissue. <br>The resulting B.t.k. proteins include a 69 kDa protein, a major proteolytic fragment of 65 kDa and two minor fragments of 40 and 15 kDa. These fragments result from trypsin digestion and are also released following tryptic digestion of the original bacterial B.t.k. Cry1Ab protein purified from Escherichia coli. <br>The expressed 69 kDa protein and the proteolytic fragments were compared to the bacterial proteins, and shown to be of similar molecular weight, primary amino acid sequences, immunological reactivity, trypsin resistance and potential glycosylation sites, indicating that there were no insertional or post-transcriptional modifications. <br>The biological activity of the trypsinized Cry1Ab protein from the modified corn was the same as that of the native B.t.k. Cry1Ab on ECB, with a LC50 of 0.46-0.51 µg/ml (f.w.) and an EC50 of 0.060-0.067 µg/g (f.w.). Studies have shown that B.t.k. proteins were rapidly inactivated when subjected to simulated mammalian gastric fluids. After one week incubation in soil, 99% of the leaf B.t.k. proteins' bioactivity was lost due to aerobic degradation. <br>The gene nucleotide sequence and the enzyme amino acid sequence were provided. <br>2. Glufosinate Ammonium Tolerance<br>Phosphinothricin (PPT), the active ingredient of glufosinate ammonium, inhibits glutamine synthetase, which results in the accumulation of lethal levels of ammonia in susceptible plants within hours of application. <br>The phosphinothricin tolerance gene engineered into hybrids X4334CBR and 4734CBR was isolated from a common aerobic soil actinomycete and codes for PPT-acetyltransferase (PAT). This enzyme detoxifies phosphinothricin by acetylation into an inactive compound. It has extremely high substrate specificity; experimental data clearly showed that neither L-PPT's analog L-glutamic acid, D-PPT, nor any protein amino acid can be acetylated by the PAT enzyme. <br>The gene, modified for enhanced gene expression, is linked to a constitutive promoter. Expression was estimated to average 0.049 µg/g (f.w.) of leaf tissue, 0.027 µg/g (f.w.) of tassel tissue and 0.005 µg/g (f.w.) in silks. It was not detected in roots, kernels nor pollen. <br>The expressed PAT enzyme was compared to the bacterial protein: molecular weights were similar, indicating that the protein had not been glycosylated nor had it undergone post transcriptional modifications. <br>Studies showed that the enzyme was inactivated within one minute when subjected to typical mammalian gastric conditions and is thus digested as a conventional dietary protein. <br>The gene nucleotide sequence and the enzyme amino acid sequence were provided. The nucleotide sequence showed no significant homology with the toxins or allergens entered in the GENEBANK DNA database.<br>3. Development Method<br>The original transformations were made through the use of a direct transfer system which did not involve the use of a DNA carrier. These transformations were conducted under contract for Northrup King by a third party and the exact method is considered to be confidential business information.<br>4. Stable Integration into the Plants'Genomes<br>The data provided showed that only one copy of the gene was integrated at one insertion site. The single insertion site was mapped to the long arm of chromosome 8 using Restriction Fragment Length Polymorphism (RFLP) markers. <br>Mendelian inheritance of the insect resistance and herbicide tolerance genes was shown over two generations.