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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

:// >. Goldson, S.L. Emberson, R.M. 1985 The potato moth Phthorimaea operculella (Zeller): Its habits, damage potential and management Special Publ., Agron. Soc N.Z., Christchurch, New Zealand 61 66

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

frequently applied to protect this crop. Potato tuberworm, Phthorimaea operculella (Zeller), is an important and destructive insect pest to the potato worldwide ( Flanders et al., 1999 ). The tuberworm is a common pest in tropical and subtropical regions

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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

.S. 2000 Evaluation of Bt-cry1Ia1 ( cryV ) transgenic potatoes on two species of potato tuber moth, Phthorimaea operculella and Symmetrischema tangolias (Lepidoptera: Gelechiidae) in Peru 117 121 Program Report 1999–2000 International Potato

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Hector Quemada, Kelly Zarka, Walter Pett, Gurling Bothma, Kimberly Felcher, Hope Mirendil, Muffy Koch, Johan Brink, and David Douches

The transgenic potato ‘SpuntaG2’ (Solanum tuberosum), which is resistant to potato tuber moth (Phthorimaea operculella), was subjected to protein safety evaluations including protein equivalency tests for the Cry1Ia1 protein from ‘SpuntaG2’ and bacterially produced Cry1Ia1, toxicity and allergenicity evaluations of Cry1Ia1 protein, and compositional equivalency of ‘SpuntaG2’ compared with non-transgenic ‘Spunta’. Western blot analysis and biological activity assays showed molecular and functional equivalency between ‘SpuntaG2’-derived Cry1Ia1 protein and bacteria-derived Cry1Ia1 protein. Comparison of the Cry1Ia1 amino acid sequence to known amino acid sequences revealed no significant homology to known toxins or known allergens. Acute toxicity studies using rodents were used to calculate an acceptable daily intake (ADI) value of 20 mg·kg−1 body weight per day. The ADI value was then used to calculate a margin of exposure (MOE) of 2,222,222, which is more than 22,000 times greater than the commonly used target MOE of 100. Digestibility and thermostability assays determined that Cry1Ia1 was fully digested within 30 s of exposure to pepsin and inactive after 3 to 4 minutes at 100 °C, indicating that it would not be a potential allergen. Compositional analyses revealed no difference between ‘SpuntaG2’ and non-transgenic ‘Spunta’. These results strongly indicate that the Cry1Ia protein and the transgenic potato ‘SpuntaG2’ is not a human health risk.

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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.

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D.S. Douches, W. Li, K. Zarka, J. Coombs, W. Pett, E. Grafius, and T. El-Nasr

The potato tuber moth (Phthorimaea operculella Zeller) is the primary insect pest of cultivated potato (Solanum tuberosum L.) in tropical and subtropical regions, causing both foliar and tuber damage. In contrast, the Colorado potato beetle (Leptinotarsa decemlineata Say) is the most important insect pest in the northern potato production latitudes. The codon-modified Bacillus thuringiensis Bt-cry5 gene (revised nomenclature cry1IaI), specifically toxic to Lepidoptera and Coleoptera, was transformed into cultivar Spunta using an Agrobacterium vector to provide resistance to both potato tuber moth and Colorado potato beetle. The Bt-cry5 gene was placed downstream from the constitutive CaMV35S promoter. Two transgenic 'Spunta' clones, G2 and G3, produced high levels of mortality in first instars of potato tuber moth in detached-leaf bioassays (80% to 83% mortality), laboratory tuber tests (100% mortality), and field trials in Egypt (99% to 100% undamaged tubers). Reduced feeding by Colorado potato beetle first instars was also observed in detached-leaf bioassays (80% to 90% reduction). Field trials in the United States demonstrated that the horticultural performance of the two transgenic lines was comparable to 'Spunta'. These Bt-cry5 transgenic potato plants with high potato tuber moth resistance have value in integrated pest management programs.

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D.S. Douches, A.L. Westedt, K. Zarka, B. Schroeter, and E.J. Grafius

Potato tuber moth (Phthorimaea operculella Zeller) is a highly destructive pest of the cultivated potato (Solanum tuberosum L.) in the tropics and subtropics and causes significant damage to both leaves and tubers. Development of host plant resistance is a central component of an integrated pest management (IPM) program for potato tuber moth. The purpose of this research was to augment natural resistance by transforming potato with a codon-modified CryV-Bt gene using Agrobacterium-mediated techniques. `Lemhi Russet' potato and two clones with different host plant resistance mechanisms, USDA8380-1 (leaf leptines) and L235-4 (glandular trichomes), were transformed with the CryV-Bt gene. Gene integration of regenerated plants was confirmed by polymerase chain reactions and Southern analyses; gene transcription was evaluated by northern analyses. Detached leaf bioassays showed that high levels of Bt expression occurred in the CryV-Bt transgenic lines (`Lemhi Russet' and L235-4), providing up to 96% control of potato tuber moth larvae, compared with 3% and 54% control in L235-4 and USDA8380-1, respectively. These transgenic lines can be used for breeding purposes to develop cultivars for (and eventual introduction into) IPM systems.

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Melanie M. Davidson, Jeanne M.E. Jacobs, Jill K. Reader, Ruth C. Butler, Christina M. Frater, Ngaire P. Markwick, Steve D. Wratten, and Anthony J. Conner

Transgenic potato (Solanum tuberosum L.) lines of two cultivars, Ilam Hardy and Iwa, were developed using Agrobacterium-mediated transformation to transfer a cry1Ac9 gene under the transcriptional control of the CaMV 35S promoter. PCR confirmed the presence of the nptII selectable marker gene in all recovered lines. All ten lines of Ilam Hardy and 14 of 15 Iwa lines were PCR-positive for the cry gene. In greenhouse trials, all Ilam Hardy transgenic lines produced phenotypically normal plants and significantly inhibited larval growth of potato tuber moth (Phthorimaea operculella Zeller). In contrast, only 60% of the Iwa transgenic lines produced phenotypically normal plants, but all lines positive for the cry gene significantly inhibited larval growth. All transgenic lines with a greenhouse appearance equivalent to the nontransgenic controls and improved resistance to potato tuber moth larvae were planted in the field. Three of the ten Ilam Hardy lines and two of the eight Iwa lines retained phenotypically normal appearance in the field and produced tuber yields equivalent to the nontransgenic controls. All five of these transgenic lines significantly inhibited larval growth of potato tuber moth on excised field-grown leaves. A high correlation was established between larval growth indices from the greenhouse and the field. A transgenic line from each cultivar inhibited larval growth by over 40%, and the line derived from Ilam Hardy prevented pupation of all larvae. Southern analysis on these five elite lines revealed that they contained either one or two copies of the cry1Ac9 gene. The amount of Cry protein in all transgenic lines tested was less than 60 ng·g-1 of fresh leaf tissue. A transgenic line from each cultivar was identified with comparable phenotypic appearance and yield to their parent cultivars coupled with high resistance to PTM.

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John L. Coffey, Alvin M. Simmons, B. Merle Shepard, Yaakov Tadmor, and Amnon Levi

. ( Seenivasan et al., 2005 ), and the potato tuber moth, Phthorimaea operculella Zell. ( Sharaby et al., 2009 ). There has not been any report of olfactometer assays of C. colocynthis and whiteflies. The objective of this study was to evaluate selected