1. Resistance to the Colorado Potato Beetle (CPB)<br>Bacillus thuringiensis subsp. tenebrionis (B.t.t.) is a common gram-positive soil-borne bacterium. In its spore forming stage, it produces several insecticidal crystal Cry3A ð-endotoxins that are non-toxic to humans, other vertebrates, and beneficial insects. These proteins are used as environmentally acceptable foliar insecticides against CPB. <br>The CPB resistance gene engineered into NewleafTM potatoes codes for one of these insecticidal crystal proteins, active against specific Coleoptera such as CPB, elm leaf beetle and yellow mealworm. Insecticidal activity results from the Cry3A 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 cry3A gene is linked to a strong constitutive promoter. Protein production (referred to as B.t.t. protein in the present document) was quantified for the seven lines and found to average from 14.4 to 19.1 µg/g (f.w.) of leaf tissue and 0.8 µg/g (f.w.) of tuber tissue, corresponding to 0.09 to 0.12% of total foliage protein and 0.004% of total tuber protein. The mean whole plant expression for all seven lines was 6.64 µg/g (f.w.) of tissue, corresponding to 0.04% of the total plant protein. B.t.t. was expressed in foliage with little variation throughout the season. <br>The resulting B.t.t. proteins include a 68 kDa protein and two major proteolytic fragments of 55 kDa each. These two fragments account for 20-30% of the tuber B.t.t. protein, while only trace amounts are found in the leaves. A similar 55 kDa protein is also released in some B.t.t. registered microbial insecticides, and following tryptic digestion of the bacterial B.t. Cry3A protein purified from Escherichia coli. <br>The expressed 68 kDa protein and the 55 kDa fragments were compared to the bacterial proteins, and shown to be of similar molecular weight and immunological reactivity, indicating that there were no insertional or post-transcriptional modifications. Protein-bound carbohydrate residues were not detected, showing lack of glycosylation. The 68 kDa protein was shown to be of similar biological activity against CPB as the 68 kDa B.t. derived protein; preliminary data suggested that the 55 kDa fragments are more active than the 68 kDa protein. <br>Studies showed that the proteins were rapidly inactivated when subjected to typical mammalian acidic stomach conditions, i.e. in simulated gastric fluids. Aerobic soil degradation of the B.t.t. proteins is complete after 9 hours. <br>The gene nucleotide sequence and the enzyme amino acid sequence were provided. An amino acid database of allergens was used to search for homology between the B.t.t. protein and known sequenced allergens. No homology was found. <br>2. Kanamycin Resistance<br>Kanamycin is an aminoglycosidic antibiotic that binds to bacterial ribosomes thus disrupting normal protein synthesis and killing the bacterial cell. <br>The kanamycin-resistance gene, isolated from the bacterium E. coli, codes for an enzyme that phosphorylates kanamycin, thereby preventing it from binding to ribosomes and rendering the cells resistant. <br>The gene is linked to a constitutive promoter. Expression was estimated to average 0.339 µg/g (f.w.) of leaf tissue, and 0.197 µg/g (f.w.) of tuber tissue, corresponding to 0.002% of foliage protein and 0.001% of tuber protein . <br>This protein is ubiquitous in the environment. It degrades rapidly in vitro in simulated mammalian gastric and intestinal fluids. <br>The nucleotide sequence showed no significant homology with the toxins or allergens entered in the GENEBANK DNA database. <br>The full nucleotide sequence and corresponding amino acid sequence were provided.<br>3. Development Method<br>Russet Burbank potatoes were transformed using a disarmed non-pathogenic Agrobacterium tumefaciens vector; the vector contained the T-DNA region of an Agrobacterium plasmid from which virulence and plant disease-causing genes were removed, and replaced with genes coding for CPB resistance and kanamycin resistance. The T-DNA portion of the plasmid is known to insert randomly into the plant's genome and the insertion is usually stable, as was demonstrated in these NewleafTM potatoes.<br>4. Stable Integration into the Plant's Genome<br>The data provided showed that there was no incorporation of any coding region from outside the T-DNA borders. Single copies of the T-DNA are integrated at a single insertion site in lines BT6, BT12, BT17, and BT23. Line BT10 contains two copies inserted in tandem and BT16 contains two copies inserted at separate genetic loci. <br>The transformed plants were propagated vegetatively, and CPB resistance was expressed for at least four vegetative generations.