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Maria A. Estrada, Kelly Zarka, Susannah Cooper, Joseph Coombs, David S. Douches, and Edward J. Grafius

The potato tuberworm [Phthorimaea operculella (Zeller)] is one of the most destructive insect pests to potato (Solanum tuberosum L.) in tropical and subtropical regions, and it has recently become established in the Pacific Northwest of the United States. Combining natural resistance mechanisms with Bacillus thuringiensis (Bt) cry genes could be a potential solution to improve potato resistance to tuberworm. We have expressed Bt cry1Ac in two potato lines: Spunta, a susceptible potato line, and ND5873-15, a moderately resistant line with high foliar glycoalkaloids derived from Solanum chacoense. Putative transgenic lines of Spunta and ND5873-15 were developed using a vector construct pSPUD15 with the codon-modified Bt cry1Ac driven by the 35S CaMV promoter. Integration of Bt cry1Ac in Spunta and ND5873-15 transgenic lines was determined by PCR and Southern analysis. Protein expression in the transgenic lines (0–580 ng·g−1) was determined by ELISA. Plants expressing Bt cry1Ac were effective in controlling potato tuberworm first-instar larvae in the detached-leaf bioassays (up to 97% mortality) and in tuber bioassays (up to 99% mortality). Based on the assays conducted, the Bt cry1Ac Spunta lines were similar to the Bt cry1Ac ND5873-15 lines for potato tuberworm mortality. Constitutively expressed Bt cry1Ac would be a useful gene to use for host plant resistance to potato tuberworm.

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Kelly A. Zarka, Ria Greyling, Inge Gazendam, Dean Olefse, Kimberly Felcher, Gurling Bothma, Johan Brink, Hector Quemada, and David S. Douches

Potato tuber moth (Phthorimaea operculella) is a serious pest of potatoes in tropical and subtropical regions of the world, including South Africa. The cry1Ia1 gene (from Bacillus thuringiensis) under the control of the 35S cauliflower mosaic virus promoter was transformed into the potato (Solanum tuberosum) cultivar Spunta to develop a cultivar with resistance to potato tuber moth for release in South Africa. Two transformation events, ‘SpuntaG2’ and ‘SpuntaG3’, were selected and subjected to extensive molecular analyses as required by the regulatory agencies of South Africa. Southern hybridization experiments indicated that ‘SpuntaG2’ and ‘SpuntaG3’ had one and three copies of the cry1Ia1 gene, respectively, and that the gene insertion was stable through multiple clonal generations. Furthermore, the sequence of the cry1Ia1 gene in ‘SpuntaG2’ was compared with the known sequence of the cry1Ia1 gene and found to be identical. Polymerase chain reaction (PCR) amplification using primers for plasmid “backbone” genes demonstrated that ‘SpuntaG2’ contained no backbone plasmid genes, whereas ‘SpuntaG3’ contained several backbone plasmid genes. Therefore, further analyses were limited to ‘SpuntaG2’, and event-specific primers were developed for this cultivar. Analysis of the left and right border regions in ‘SpuntaG2’ demonstrated that the insertion of the cry1Ia1 gene did not disrupt any functional genes nor did it create new open reading frames that encoded proteins with a significant match to the non-redundant sequence database queried by the BLASTP program. Enzyme-linked immunoabsorbent assays (ELISA) tests indicate that the cry1Ia1 gene was expressed at a mean concentration of 2.24 μg·g−1 fresh weight in leaf tissue and 0.12 μg·g−1 fresh weight in tubers. This study demonstrates the extensive molecular characterization that is necessary to apply for deregulation of a genetically modified crop and these data have been used in a regulatory package for the general release of ‘SpuntaG2’.

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Wenbin Li, Kelly A. Zarka, David S. Douches, Joseph J. Coombs, Walter L. Pett, and Edward J. Grafius

The codon-modified cryV-Bt gene (cryV-Bt) from Bacillus thuringiensis subsp. kurstaki Berliner, which is specifically toxic to Lepidoptera and Coleoptera insects, and a potato virus Yo coat protein gene (PVYocp), in which the aphid transmission site was inactivated, were cotransformed into potato (Solanum tuberosum L.) `Spunta' via Agrobacterium tumefaciens Conn. We demonstrated the integration and expression of both genes by molecular analysis and bioassays. All cryV-Bt/PVYocp-transgenic lines were more resistant to potato tuber moth (Phthorimaea operculella Zeller) and PVYo infection than nontransgenic `Spunta'. Four cryV-Bt/PVYocp transgenic lines were equal in potato tuber moth mortality to a cryV-Bt transgenic line, but of these four only two lines were equivalent in PVYo titer levels to a PVYocp-transgenic line. We identified two transgenic lines, 6a-3 and 6a-5, which showed greater resistance to potato tuber moth and PVYo than the other cryV-Bt/PVYocp transgenic lines. This study indicated that multiple genes, conferring insect pest resistance and virus resistance, could be engineered into and expressed simultaneously in a potato cultivar.