Russet Burbank potato plants have been genetically improved to resist insect attack and damage by Colorado potato beetles (Leptinotarsa decemlineata (Say)) by the insertion of a cryIIIA gene encoding the insect control protein of Bacillus thuringiensis var. tenebrionis. A modified gene that dramatically improved plant expression of this protein was utilized. Its expression in Russet Burbank potato plants resulted in protection from damage by all insect stages in the laboratory and in dramatic levels of protection at multiple field locations. Analysis of these genetically modified potatoes indicated that they conform to the standards for Russet Burbank potatoes in terms of agronomic and quality characteristics including taste.

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A laboratory-selected colony of Heliothis virescens displaying a 20-to 70-fold level of resistance to Bacilus thuringiensis proteins was evaluated to identify mechanism(s) of resistance. Brush-border membrane vesicles were isolated from larval midgut epithelium from the susceptible and resistant strains ofH. virescens. Two B. thuringiensis proteins, CryIA(b) and CryIA(c), were iodinated and shown to specifically bind to brush-border membrane vesicles of both insect strains. Multiple changes in the receptor-binding parameters were seen in the resistant strain as compared with the susceptible strain. A 2-to 4-fold reduction in binding affinity was accompanied by a 4-to 6-fold increase in binding-site concentration for both proteins. Although these two B. thuringiensis proteins competed for the same high-affinity binding site, competition experiments revealed different receptor specificity toward these proteins in the resistant H. virescens line. The H. virescens strains were not sensitive to a coleopteran-active protein, CryMA, nor did these proteins compete with the CryLA proteins for binding. Complexity of the mechanism of resistance is consistent with the complex mode of action of B. thuringiensis proteins.Insect-control proteins from Bacillus thuringiensis ssp. kurstaki are active against a wide range of agronomically important lepidopteran larvae (1, 2). Use of these proteins in optimized microbial strains and genetically improved plants will become increasingly important for insect control (3), particularly as the popularity of many commercial chemical insecticides is declining due to the onset of resistance by target pests (4). Although commercial preparations of B. thuringiensis strains have been used for >25 yr, only recently have insects with reduced susceptibility been identified (5) and obtained in laboratory-selection experiments (6-8). Management strategies are being developed to prevent or delay the onset of insect resistance to assure the long-term efficacy of B. thuringiensis proteins. Biochemical characterization of B. thuringiensis proteins, their mode of action, and mechanisms of increased resistance are critical for the development of appropriate management strategies.The mode of action of B. thuringiensis protein insecticides is complex, as evidenced in a number of reports over the last few years (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). Upon ingestion by the insect, the proteins are proteolytically processed (9-14), cross the peritrophic membrane, and bind to high-affinity receptors on the midgut epithelium (15)(16)(17)(18)(19)(20). The membrane-bound B. thuringiensis protein disrupts the membrane integrity by forming a pore, causing an electrolyte imbalance that ultimately kills the insect (21,22). Receptor binding has been analyzed by usingmidgut brush-border vesicles from the gut epithelium from various lepidopteran larvae and for several B. thuringiensis proteins (16,(18)(19)(20)23). High-affinity binding sites were initially identified that directly correlated with the ob...

The European corn borer [ECB; Ostrinia nubilalis (Hübner)] is an economically significant pest of corn (Zea mays L.). The ability to routinely transform corn has broadened the control options available to include the introduction of resistance genes from sexually incompatible species. In this study, microprojectile bombardment was used to introduce synthetic versions of cryIA insecticidal protein genes from Bacillus thuringiensis subsp, kurstaki (Btk) into embryogenitcis sue of the Hi‐II] (A188/B73 derivative) genotype of corn. Of 715 independent transgenic calli produced, 314 (44%) had insecticidal activity against tobacco hornworm (Manduca sexta L.) larvae. Plants were regenerated, self‐pollinated when possible, and crossed to B73. First‐generation progeny of 173 independent Btk‐protein expressing calli were evaluated under field conditions with artificial ECB infestations in 1992 or 1993. Approximately half (89/173) segregated in single‐gene manner for resistance to first‐generation ECB leaf‐feeding damage. All of the 89 lines evaluated in 1992 or 1993 for resistance to second‐generation ECB exhibited less stalk tunneling damage than the non‐transgenic controls. In 1993, 44% (34177) of the lines tested had ≤2.5 cm of tunneling, compared to severe damage (mean = 45.7 cm) in the B73 × Hi‐II controls. Experiments are in progress to evaluate the effect of the introduced genes on yield and other agronomic properties.

We discuss assay approaches for monitoring the sensitivity of Lepidoptera to Bacillus thuringiensis (Bt) insecticidal proteins and compare the relative sensitivity of larval feeding bioassays in which, respectively, mortality or growth inhibition were scored. Heliothis virescens (F.) and Helicoverpa zea (Boddie), major lepidopteran pests targeted for control by transgenic cotton, were used for assay comparison. Larval growth inhibition assays using sublethal CryIA(c) protein concentrations were considerably more sensitive than dose-response mortality assays. Growth inhibition assays were easy to set-up and read, and could readily deliver a diagnostic dose allowing for visual discrimination of resistant from susceptible phenotypes. The ability of a larval growth assay, combined with a diagnostic dose, to unambiguously separate resistant from susceptible insects was validated using a CryIA(c) protein resistant strain of H. virescens and F 1 hybrids derived by crossing the resistant strain to a susceptible H. virescens strain.

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