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

. 138 281 292 Chan, M. Chen, L. Chang, H. 1996 Expression of Bacillus thuringiensis (B.t.) insecticidal crystal protein gene in transgenic potato Bot. Bull. Acad. Sin. 37 17 23

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Abhava M. Dandekar, Gale H McGranahan, Sandra L. Uratsu, Charles Leslie, J. Steven Tebbets, and Patrick V. Vail

Insecticidal crystal protein fragments (ICPFs) of Bacillus thuringiensis (Bt) encoded by cryIA(c) gene were shown in diet incorporation studies to be lethal to codling moth (CM; Cydia pomonella) the key insect pest for walnut. However transformed walnut tissues expressing cryIA(c) with Bt codon usage patterns and native DNA sequence revealed very low levels of expression in planta. To correct this problem synthetic versions of one of these genes, cryIA(c) was used to transform walnut tissue. A total of 61 individual transgenic embryo lines were obtained. 34% of these lines (21/61) were high expressors (“class A”) demonstrating 80 to 100% mortality of first in star CM larvae and displaying no further larval development. Twelve clones (20%) were designated “class B” and these showed a marked retardation of larval development and a mortality between 40 to 79%. Embryos from the remaining 28 lines designated “class C” (46%). although transformed, were indistinguishable from the control (untransformed embryos) and showed a mortality of 0 to 39%.

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David R. Corbin, Frederick J. Perlak, David A. Fischhoff, John T. Greenplate, Zhen Shen, and John P. Purcell

Genetically modified potato and cotton crops that express insecticidal proteins from Bacillus thuringiensis (Bt) have recently been commercialized. These crops display autonomous resistance to specific insect pests, and thus offer major agricultural and environmental benefits. We have implemented a microbial screening program to discover new types of insecticidal proteins for use in transgenic crops. New proteins with diverse modes of action offer opportunities to control insect pests that are not susceptible to Bt insecticidal proteins and to delay or prevent the potential occurrence of resistance of insects to crops genetically modified with Bt genes. Cholesterol oxidase emerged from our screen as a new insecticidal protein with potent activity against the cotton boll weevil. Cholesterol oxidase was acutely toxic to boll weevil larvae, with an LC50 of 2–6 parts per million when ingested in artificial diet feeding assays, and caused marked reductions in fecundity when ingested by adult boll weevils. Cholesterol oxidase also exerted significant, though less severe, toxicity against several lepidopteran pests. The insecticidal action of cholesterol oxidase appears to be due to oxidation of midgut epithelial membrane cholesterol followed by membrane disruption. A cholesterol oxidase gene was cloned and expressed in transgenic tobacco plants to yield plant tissue that exerted potent activity against boll weevil. Expression of this cholesterol oxidase gene in cotton plants may offer significant protection against the cotton boll weevil and may also aid in the mitigation of resistance of cotton lepidopteran pests to Bt proteins.

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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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Brian A. Kahn and Lynn P. Brandenberger

Field studies were conducted in Oklahoma from 2010 to 2012 to develop protocols for fall sweet corn (Zea mays) production. Variables examined included a transgenic cultivar that expresses the CryIA(b) toxin from the bacterium Bacillus thuringiensis (Bt) and its nontransgenic near-isoline, seeding rates and planting dates, and various insecticide regimens. We found that, in eastern Oklahoma, a suitable planting window would correspond roughly to the last 2 weeks in July. Within this favorable period and given timely irrigation, it was possible to sow corn to an acceptable stand. A seeding rate ≈1½ times the desired final stand of one plant/ft appeared to be satisfactory. Use of a cultivar (GSS-0966) with genetic resistance to lepidopteran pests was a critical factor for successful production of fall sweet corn. Efforts to produce a crop with a nontransgenic cultivar using insecticides with relatively low mammalian toxicity were unsuccessful. Our experiments support previous recommendations for applying supplemental insecticides to transgenic Bt sweet corn to potentially increase production of “premium” ears by reducing the percentage of ears with severe insect damage (damage >1½ inches from the cob tip). We demonstrated that a spray schedule that rotated two insecticides with intermediate mammalian toxicity (carbaryl and permethrin) was as effective in reducing severe insect damage to ears of ‘GSS-0966’ as a similar schedule that rotated two highly toxic insecticides (esfenvalerate and methomyl).

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Sharon Billings, Gojko Jelenkovic, Chee-Kok Chin, and Jodi Eberhardt

A protocol with a high rate of transformation and regeneration of `Hibush' eggplant (Solanum melongena L.) has been developed. This protocol used leaves of in vitro-grown seedlings as a source of explants. The shoot regeneration culture medium contained 0.1 μm thidiazuron (TDZ) combined with 10 to 20 μm N6-[isopentyl] adenine (2iP). Adding TDZ significantly improved regeneration efficiency and produced a mean of 15 buds and 3 to 4 shoots per explant. When explants were cocultivated with Agrobacterium tumefaciens strains Q10, Q20, Q30, Q40, Q201, Q202, Q203, or Q204 containing the native cryIIIB Bacillus thuringiensis (Bt), neomycin phosphotransferase (NPTII), and β-glucuronidase (uidA) genes, a callus/bud regeneration frequency of 38.8% was observed on the selection medium. Kanamycin at 50 μg·mL-1 was most effective in selecting for transgenic buds and shoots. Augmentin at 300 μg·mL-1 was used to eliminate A. tumefaciens. Augmentin also enhanced shoot proliferation. A transformation/regeneration efficiency of 20.8% was observed for shoot production. More than 400 putative transgenic plants have been produced with this method. From 50 putative transgenic plants, gene integration has been confirmed with Southern blot analysis and progeny tests.

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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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Peter Juroszek and Hsing-Hua Tsai

), and mites (Acaridae); later during the cool-dry season, problems started with the fungal disease, anthracnose ( Colletotrichum spp.). A Bacillus thuringiensis ( Bt ) product [ B. thuringiensis ssp. aizawai strain ABTS1857 (XenTari®; Valent

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Nicholas D. Warren, Rebecca G. Sideman, and Richard G. Smith

( Spodoptera ornithogalli ) and tomato hornworm ( Manduca quinquemaculata ) occurred in all 3 years and were controlled by applying Bacillus thuringiensis [Bt (DiPel; Valent BioSciences, Libertyville, IL)] at the rate of 0.5 lb/acre or spinosad [spinosyn A