<p><span style="font-size:12px;"><span style="font-family: arial,helvetica,sans-serif;"><font color="#003300"><strong>1. Resistance to the Colorado Potato Beetle (CPB) </strong><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 Atlantic 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<br>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 light activated promoter. Protein production (referred to as B.t.t. protein in the present document) was quantified for the five lines and found to average from 15.7 to 59.3 µg/g (f.w.) of leaf tissue and from 0.09 to 0.53 µg/g (f.w.) of tuber tissue, corresponding to 0.10 to 0.37% of total foliage protein and 0.0004 to 0.003% of total tuber protein (assuming protein levels of 1.6% in leaves and 2% in tubers, f.w.). These expression levels correspond to a two fold increase in leaves and a four fold decrease in tubers, compared to the NewLeaftm Russet Burbank potatoes previously authorized for release (please see DD96-06). Foliage expression levels were highest at six weeks and gradually decreased over the season.<br>The resulting B.t.t. proteins include a 68 kDa protein and a major proteolytic fragment of 55 kDa. 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>Western blot data was presented which showed that the 68 kDa and 55 kDa proteins expressed by B.thuringiensis, the Russet Burbank NewLeaftm potato and the Atlantic NewLeaftm potato had the same immunological reactivity and molecular weight. Data, previously submitted for the Russet Burbank NewLeaftm potato, showed the proteins had not been glycosylated or undergone other post translational modification and possessed similar bioactivity and host range specificity to the native protein.<br>Studies have shown that B.t.t. 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 was shown to be complete after nine hours.<br>The gene nucleotide sequence and the amino acid sequence were provided. A search for amino acid sequence similarity between the B.t.t. proteins and known allergens, using a database assembled from the public domain databases GenBank, EMBL, Pir and SwissProt, revealed no significant amino acid sequence homologies. </font><br><font color="#003300"><strong>2. Kanamycin Resistance </strong><br>Kanamycin is an aminoglycosidic antibiotic that binds to bacterial ribosomes thus disrupting normal protein synthesis and killing the bacterial cell. 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. The gene is linked to a constitutive promoter. The average expression levels for all five lines were estimated to range from 4.4 to 36.6 µg/g (f.w.) of leaf tissue, and from 0.5 to 2.9 µg/g (f.w.) of tuber tissue, corresponding to 0.028-0.23% of foliage protein and 0.0025-0.014% of tuber protein (assuming protein levels of 1.6% in leaves and 2% in tubers, f.w.). The expressed NPTII protein was compared to the bacterial protein, and shown to be of similar molecular weight, indicating that there were no insertional or post- transcriptional modifications. This protein is ubiquitous in the environment. It degrades rapidly in vitro in simulated mammalian gastric and intestinal fluids. The nucleotide sequence showed no significant homology with the toxins or allergens entered in the GENEBANK DNA database. The full nucleotide sequence and corresponding amino acid sequence were provided. </font><br><font color="#003300"><strong>3. Development Method </strong><br>Atlantic potatoes were transformed using a disarmed non-pathogenic <i>Agrobacterium tumefaciens</i> vector; the vector contained the transfer DNA (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&#39;s genome and the insertion is usually stable, as was demonstrated in these NewLeaftm Atlantic potatoes.</font><br><font color="#003300"><strong>4. Stable Integration into the Plant&#39;s Genome </strong><br>The data provided showed various integration patterns depending on the line: the T- DNA was integrated at three sites for line ATBT04-6 and at one site for lines ATBT04-30 and ATBT04-31. No coding region from outside the T-DNA borders was incorporated into the genotype of these lines. Two complete copies of the T-DNA were found at two insertion sites in line ATBT04-27, with one site also containing a bacterial gene coding for resistance to streptomycin and an other partial copy of the cry3A gene. The streptomycin resistance gene is driven by a bacterial promoter and is not expressed in plants, as was demonstrated in these NewLeaftm Atlantic potatoes. Finally, the transgenes were integrated at three insertion sites in line ATBT04-36, with one of the sites containing the whole <i>A. tumefaciens</i> plasmid. The transformed plants were propagated vegetatively, and CPB resistance was expressed for at least three vegetative generations.</font></span></span></p><br>