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Joseph C. Kuhl, Kelly Zarka, Joseph Coombs, William W. Kirk and David S. Douches

Late blight of potato (Solanum tuberosum L.), incited by Phytophthora infestans (Mont.) de Bary, is a devastating disease affecting tuber yield and storage. Recent work has isolated a resistance gene, RB, from the wild species Solanum bulbocastanum Dun. Earlier work in Toluca, Mexico, observed significant levels of field resistance under intense disease pressure in a somatic hybrid containing RB. In this study, five transgenic RB lines were recovered from the late blight susceptible line MSE149-5Y, from the Michigan State University (MSU) potato breeding program. Transgenic lines were molecularly characterized for the RB transgene, RB transcript, and insertion number of the kanamycin resistance gene NPTII. Transgenic lines and the parent line were evaluated for resistance in field and laboratory tests. Molecular characterization alone did not predict which lines were resistant. Three of the RB transformed MSE149-5Y lines showed increased resistance under field conditions at MSU and increased resistance in detached leaf evaluations using multiple isolates individually (US-1, US-1.7, US-8, US-10, and US-14). Transfer of RB into late blight susceptible and resistant lines could provide increased protection to potato late blight. The use of the RB gene for transformation in this way creates a partially cisgenic event in potato because the gene's native promoter and terminator are used. This type of transformation provides a chance to generate greater public acceptance of engineered approaches to trait introgression in food crops.

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

The Colorado potato beetle [Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae)] is a destructive pest of the cultivated potato (Solanum tuberosum L.) in northern latitudes. Combining resistance mechanisms of leptine glycoalkaloids and glandular trichomes with the synthetic Bacillus thuringiensis Berliner (Bt) cry3A gene in potato may be an effective strategy for controlling the Colorado potato beetle. Bt-cry3A transgenic plants were developed for three potato lines with differing levels of resistance to Colorado potato beetle ['Yukon Gold' (susceptible control), USDA8380-1 (leptine glycoalkaloids), and NYL235-4 (glandular trichomes)]. Polymerase chain reaction, and Southern and northern blot analyses confirmed integration and transcription of the cry3A gene in the transgenic lines. Detached-leaf bioassays of the cry3A engineered transgenic lines demonstrated that resistance effectively controlled feeding by first instar Colorado potato beetles. The susceptible `Yukon Gold' control suffered 32.3% defoliation, the nontransformed high foliar leptine line (USDA8380-1) had 3.0% defoliation, and the nontransformed glandular trichome line (NYL235-4) had 32.9% defoliation. Mean percentage defoliation for all transgenic lines ranged between 0.1% and 1.9%. Mean mortality ranged from 0.0% to 98.9% among the Bt-cry3A transgenic lines, compared to 20% for the susceptible `Yukon Gold' control, 32.2% for USDA8380-1, and 16.4% for NYL235-4. Results indicate that genetic engineering and the availability of natural resistance mechanisms of potato provide the ability to readily combine host plant resistance factors with different mechanisms in potato.

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

The colorado potato beetle, Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae), is the leading insect pest of potato (Solanum tuberosum L.) in northern latitudes. Host plant resistance has the potential use in an integrated pest management program for control of colorado potato beetle. During the 1998 and 1999 seasons, field studies were conducted to compare natural (leptine glycoalkaloids and glandular trichomes), engineered (Bt-cry3A and Bt-cry5 transgenic potato lines), and combined (Bt-cry5+glandular trichomes) plant resistance mechanisms of potato for control of colorado potato beetle. Nine different potato clones representing five different host plant resistance mechanisms were evaluated under natural colorado potato beetle infestation at the Montcalm Research Farm in Entrican, Michigan. The Bt-cry3A transgenic lines, the high leptine line (USDA8380-1), and the high foliar glycoalkaloid line (ND5873-15) were most effective for controlling defoliation by colorado potato beetle adults and larvae. The Bt-cry5 line (SPc5-G2) was not as effective as the Bt-cry3A transgenic lines ('Russet Burbank Newleaf,' RBN15, and YGc3.1). The glandular trichome (NYL235-4) and Bt-cry5+glandular trichome lines proved to be ineffective. Significant rank correlations for the potato lines between the two years were observed for egg masses, second and third instar, and fourth instar seasonal cumulative mean number of individuals per plant, and defoliation. Egg mass and first instar seasonal cumulative mean number of individuals per plant were not strong indicators of host plant resistance in contrast to second and third instars or adults. Based on these results, the Bt-cry3A transgenic lines, the high leptine line, and the high total glycoalkaloid line are effective host plant resistance mechanisms for control of colorado potato beetle.

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Joseph J. Coombs, David S. Douches, Susannah G. Cooper, Edward J. Grafius, Walter L. Pett and Dale D. Moyer

Colorado potato beetle (Leptinotarsa decemlineata Say) is the leading insect pest of potato (Solanum tuberosum L.) in northern latitudes. Host plant resistance is an important tool in an integrated pest management program for controlling insect pests. Field studies were conducted to compare natural host plant resistance mechanisms (glandular trichomes and Solanum chacoense Bitter-derived resistance), engineered [Bacillus thuringiensis (Bt) Berliner Bt-cry3A], and combined (glandular trichomes + Bt-cry3A and S. chacoense-derived resistance + Bt-cry3A transgenic potato lines) sources of resistance for control of colorado potato beetle. Six different potato clones representing five different host plant resistance mechanisms were evaluated for 2 years in a field situation under natural colorado potato beetle pressure in Michigan and New York, and in a no-choice field cage study in Michigan. In the field studies, the S. chacoense-derived resistance line, Bt-cry3A transgenic, and combined resistance lines were effective in controlling defoliation by colorado potato beetle adults and larvae. Effectively no feeding was observed in the Bt-cry3A transgenic lines. The glandular trichome line suffered less defoliation than the susceptible control, but had greater defoliation than the Bt-cry3A transgenic lines and the S. chacoense-derived resistance line. In the no-choice cage study, the Bt-cry3A transgenic lines and the combined resistance lines were effective in controlling feeding by colorado potato beetle adults and larvae with no defoliation observed. The S. chacoense-derived resistance line and the glandular trichome line suffered less defoliation than the susceptible control. Based on the results of the field trials and no-choice field cage studies, these host plant resistance mechanisms could be used to develop potato varieties for use in a resistance management program for control of colorado potato beetle.

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David Douches, Walter Pett, Diedrich Visser, Joseph Coombs, Kelly Zarka, Kimberly Felcher, Gurling Bothma, Johan Brink, Muffy Koch and Hector Quemada

‘SpuntaG2’ is a transgenic potato (Solanum tuberosum) cultivar that contains the cry1Ia1 gene for resistance to potato tuber moth (Phthorimaea operculella), which is a serious pest of potato in many parts of the world. Previous studies have characterized ‘SpuntaG2’ at the molecular level and evaluated it for safety as a human food source. The objective here was to determine the efficacy of ‘SpuntaG2’ against the potato tuber moth in the field and in storage and to evaluate its agronomic performance. Efficacy trials at seven South African locations over 5 years indicated that ‘SpuntaG2’ gave complete control of potato tuber moth in the field and storage. The agronomic performance (tuber size and yield) of ‘SpuntaG2’ was not statistically different from ‘Spunta’ or was better than ‘Spunta’ at all locations/years with two exceptions. Three years of trials in Michigan further support these conclusions. Evaluations done during the 2006–07 South African season showed that ‘SpuntaG2’ did not differ from non-transgenic ‘Spunta’ for the following traits: maturity, growth habit, eye morphology, tuber size distribution, tuber shape, skin and flesh color, growth cracks, internal defects, specific gravity, chip color, cooking quality, disease resistance, and pollen fertility. Based on the results of these trials, it was concluded that ‘SpuntaG2’ provides the agronomic performance of ‘Spunta’ with the added benefit of resistance to potato tuber moth.

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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.