The NewLeaf-Y™ potato (Solanum tuberosum) lines SEMT15-02, SEMT15-15 and RBMT15-101 were developed through a specific genetic modification of cultivars Shepody and Russet Burbank to be CPB (Leptinotarsa decemlineata Say.) resistant and to resist infection by PVY. The novel lines produce a version of the insecticidal protein, CryIIIA, derived from Bacillus thuringiensis, as well as the coat protein (CP) from the ordinary (O) strain of potato virus Y (PVY-O). Delta-endotoxins, such as the CryIIIA protein expressed in Shepody and Russet Burbank NewLeaf-Y™ potatoes, act by selectively binding to specific receptors localized on the brush border midgut epithelium of susceptible insect species. Following binding, cation-specific pores are formed that disrupt midgut ion flow and thereby cause paralysis and death. CryIIIA and related endotoxins are insecticidal only to lepidopteran or coleopteran insects and their specificity of action is directly attributable to the presence of specific receptors in the target insects. There are no receptors for delta-endotoxins of B. thuringiensis on the surface of mammalian intestinal cells, therefore, livestock animals and humans are not susceptible to<br>these proteins. PVY is the type member of the potyvirus group and is an aphid-transmissible RNA virus that commonly infects potato causing serious disease and economic loss. The introduced viral sequences do not result in the formation of any infectious particles, nor does their expression result in any disease pathology. The genetically modified potato cultivars exhibit the trait of resistance to infection and subsequent disease caused by PVY through a process that is related to viral cross-protection.<br>The transgenic Shepody and Russet Burbank potato lines were created through two separate<br>Agrobacterium-mediated transformation events in which the transfer DNA (T-DNA) contained the genes encoding the CryIIIA protein from B. thuringiensis subsp. tenebrionis and the CP from PVY-O. In addition, the T-DNA contained sequences encoding the enzyme neomycin phosphotransferase II (NPTII). The expression of NPTII activity was used as a selectable trait for screening transformed plants for the presence of the cryIIIA and PVY-O CP genes. Additional DNA outside of the T-DNA border sequences was incorporated into the genome of Shepody lines SEMT15-02 and SEMT15-15. These lines also contain the aad gene that encodes the enzyme 3'’(9)-O-aminoglycoside adenylyltransferase, which confers bacterial resistance to spectinomycin and streptomycin. The aad gene was not expressed in plant tissue, but was present on the Ti plasmid as a selectable trait for screening bacterial colonies for the<br>presence of plasmid vector. The NewLeaf-Y™ Shepody and Russet Burbank cultivars produce three novel proteins: CryIIIA, PVY-O CP and NPTII.<br>The constitutive expression of CryIIIA protein was demonstrated in each of the transgenic NewLeaf-Y™ Shepody and Russet Burbank cultivars. On average, the amounts of CryIIIA protein produced in the leaves and tubers of SEMT15-02, SEMT15-15 and RBMT15-101 were comparable to the concentrations previously reported for NewLeaf™ Atlantic and Russet Burbank cultivars. The expression of PVY-O CP in either leaves or tubers from any of these transgenic lines was undetectable at a threshold of 2 ng/mg fresh weight tissue. In contrast, the accumulation of CP in plants naturally infected with PVY-O is readily detectable using enzyme linked immunosorbent assay (ELISA) or Western immunoblot analysis. It is not uncommon for commercial potato plantings to be significantly infected by PVY-O and thus the human<br>consumption of viral CP from these sources is likely to be much higher than the exposure due to<br>consumption of NewLeaf-Y™ potatoes. The presence of NPTII protein has been judged to be<br>insignificant with respect to any human health risk due to exposure. Solanine and chaconine are the principal glycoalkaloids commonly found in potato tubers. Analyses of total glycoalkaloid (TGA) levels in each of the transgenic lines demonstrated that in each case the level was below the administrative guideline of 20 mg/100g fresh weight that has previously been established for TGA in potato. Other than resistance to CPB and infection by PVY, the disease, pest and other agronomic characteristics of the NewLeaf-Y™ Shepody and Russet Burbank lines were comparable to their non-transgenic parental cultivars.