Bohorova, N; Zhang, W; Julstrum, P; McLean, S; Luna, B; Brito, RM; Diaz, L; Ramos, ME; Estanol, P; Pacheco, M; Salgado, M; Hoisington, D

Theoretical and Applied Genetics [Theor. Appl. Genet.], vol. 99, no. 3/4, pp. 437-444, 24 Aug 1999

To enhance the level of resistance to insects in tropical maize germplasm we have developed techniques to successfully transform elite tropical maize inbred based on the activity of specific cryI proteins against four major maize pests - corn earworm, fall armyworm, southwestern corn borer and sugarcane borer. Constructs containing cryIAb or cryIAc synthetic genes were used. To generate transgenic plants we have established methods for biolistic bombardment and the selection and regeneration of immature embryos and calli from the elite tropical lines CML72, CML216, CML323, CML327 and hybrids. Transgenic plants resistant to the herbicide Basta super(TM) contained the bands for the cry, bar and gus genes as detected by Southern blot analyses. A simple leaf bioassay presented varying levels of resistance to Southwestern corn borer of transgenic tropical maize carrying the cryIAc gene. Analyses of the progenies confirmed the sexual transmission of the introduced genes and their stable expression.

Saxena, D; Flores, S; Stotzky, G*

Nature, vol. 402, no. 6761, p. 480, 2 Dec 1999

Bt corn is corn (Zea mays) that has been genetically modified to express insecticidal toxins derived from the bacterium Bacillus thuringiensis to kill lepidopteran pests feeding on these plants. Here we show that Bt toxin is released into the rhizosphere soil in root exudates from Bt corn.

Hukuhara, T; Hayakawa, T; Wijonarko, A

Nature Biotechnology [Nat. Biotechnol.], vol. 17, no. 11, pp. 1122-1124, Nov 1999

We have introduced an entomopoxvirus gene encoding a virus enhancing factor (EF) into rice, which resulted in high-level accumulation of the EF in the transgenic plants. The introduced gene was stably inherited in the progeny of the primary transformants, as shown by analysis of their genomic DNA. Bioassays for insect susceptibility to baculovirus infection showed that armyworm larvae feeding on the transgenic rice had increased susceptibility to a Nucleopolyhedrovirus. Thus, introduction of the EF gene into plants can be used as a strategy to increase the effectiveness of baculoviruses in insect pest management.

Liu, Yong-Biao; Tabashnik, BE; Dennehy, TJ; Patin, AL; Bartlett, AC

Nature, vol. 400, no. 6744, p. 519, 5 Aug 1999

Crop plants genetically engineered to produce insecticidal toxins derived from the bacterium Bacillus thuringiensis (Bt) are being grown on millions of hectares, but their success will be short-lived if pests adapt to them quickly. The primary strategy for delaying insect resistance to transgenic Bt plants is to provide refuges of host plants that do not produce Bt toxins. This potentially delays the development of insect resistance to Bt crops by providing susceptible insects for mating with resistant insects. But our laboratory results with a worldwide pest of cotton, pink bollworm moths (Pectinophora gossypiella), contradict an important assumption of the refugee strategy. We find that a resistant strain of larvae on Bt cotton takes longer to develop than susceptible larvae on non-Bt cotton. This developmental asynchrony favours non-random mating that could reduce the expected benefits of the refuge strategy.

ffrench-Constant, R; Bowen, D

Current Opinion in Microbiology [Curr. Opin. Microbiol.], vol. 2, no. 3, pp. 284-288, Jun 1999

Following concerns over the potential for insect resistance to insecticidal Bacillus thuringiensis toxins expressed in transgenic plants, there has been recent interest in novel biological insecticides. Over the past year there has been considerable progress in the cloning of several alternative toxin genes from the bacteria Photorhabdus luminescens and Xenorhabdus nematophilus. These genes encode large insecticidal toxin complexes with little homology to other known toxins.

Schuler, TH; Poppy, GM; Kerry, BR; Denholm, I

Trends in Biotechnology [Trends Biotechnol.], vol. 17, no. 5, pp. 210-216, May 1999

Engineering genes encoding insecticidal proteins into crop plants offers numerous benefits to agriculture. However, like many conventional insecticides, this new technology has the potential to disrupt natural biological control through both direct and indirect side effects of the plants on the fitness or behavior of arthropod predators and parasitoids. Interactions between transgenic plants and these beneficial insects are being assessed to avoid incompatibility.

Kota, M; Daniell, H; Varma, S; Garczynski, SF; Gould, F; Moar, WJ

Proceedings of the National Academy of Sciences, USA [Proc. Natl. Acad. Sci. USA], vol. 96, no. 05, pp. 1840-1845, 2 Mar 1999

Evolving levels of resistance in insects to the bioinsecticide Bacillus thuringiensis (Bt) can be dramatically reduced through the genetic engineering of chloroplasts in plants. When transgenic tobacco leaves expressing Cry2Aa2 protoxin in chloroplasts were fed to susceptible, Cry1A-resistant (20,000- to 40,000-fold) and Cry2Aa2-resistant (330- to 393-fold) tobacco budworm Heliothis virescens, cotton bollworm Helicoverpa zea, and the beet armyworm Spodoptera exigua, 100% mortality was observed against all insect species and strains. Cry2Aa2 was chosen for this study because of its toxicity to many economically important insect pests, relatively low levels of cross- resistance against Cry1A-resistant insects, and its expression as a protoxin instead of a toxin because of its relatively small size (65 kDa). Southern blot analysis confirmed stable integration of cry2Aa2 into all of the chloroplast genomes (5,000-10,000 copies per cell) of transgenic plants. Transformed tobacco leaves expressed Cry2Aa2 protoxin at levels between 2% and 3% of total soluble protein, 20- to 30-fold higher levels than current commercial nuclear transgenic plants. These results suggest that plants expressing high levels of a nonhomologous Bt protein should be able to overcome or at the very least, significantly delay, broad spectrum Bt- resistance development in the field.

Van Mellaert, H; Botterman, J; Van Rie, J; Joos, H

US Patent 5866784, , 2 Feb 1999

Plants made resistant to insects by transforming their nuclear genome with two or more DNA sequences, each encoding a different non-competitively binding B. thuringiensis protoxin or insecticidal part thereof, preferably the toxin thereof.

Bonade-Bottino, M; Lerin, J; Zaccomer, B; Jouanin, L

Insect Biochemistry and Molecular Biology [Insect Biochem. Mol. Biol.], vol. 29, no. 2, pp. 131-138, Feb 1999

Larvae of Baris coerulescens Scop. (Coleoptera: Curculionidae) exhibit a complex array of gut proteinase activities comprising cysteine and serine proteinases. The major cysteine proteinase activity, showing an optimum at pH 6.0, corresponds to at least 4 different proteinases. On the contrary, the minor serine proteinase activity, with an optimum at pH 9.0, seems to be due essentially to a single proteinase. The cysteine proteinase inhibitor oryzacystatin I (OC-I) inhibits completely the cysteine proteinase activity in vitro. However, larval growth and survival were not significantly different on control and transgenic oilseed rape plants expressing high levels of active OC-I. In larvae grown on transgenic plants, cysteine proteinase activity was dramatically decreased, whereas serine proteinase activity was increased by more than 2-fold, when compared to larvae raised on control plants. For both activities, no new proteinase was detected in insects fed plants expressing OC-I. These results suggest that partial compensation of the inhibition of cysteine proteinase activity by the increase in serine proteinase activity allowed the larvae to overcome the effects of OC-I consumption. This case illustrates problems that could arise when trying to achieve high levels of protection for plants against Coleopteran pests possessing a complex digestive proteinase pool.

Lee, Soo In; Lee, Sung-Ho; Koo, Ja Choon; Chun, Hyun Jin; Lim, Chae Oh; Mun, Jeom Hee; Song, Yoo Han; Cho, Moo Je*

Molecular Breeding [Mol. Breed.], vol. 5, no. 1, pp. 1-9, 1999

Proteinase inhibitors are widely distributed in animals, plants and microorganisms and their roles in plants are associated with defense against pests. The utilization of proteinase inhibitors for crop protection has been actively investigated with a variety of proteinase inhibitors. Soybean Kunitz trypsin inhibitor (SKTI), one of the major seed storage protein, is synthesized for a short period during seed development. To investigate the role of SKTI in a plant's defense system against insect predation, a recombinant plasmid containing the full-length cDNA of SKTI under control of the CaMV 35S promoter was introduced into rice protoplasts by using the PEG direct gene transfer method and a large number of transgenic rice plants were regenerated. The integration, expression, and inheritance of this gene was demonstrated in R sub(1) and R sub(2) generations by Southern, northern, and western analyses. Accumulation levels (0.05-2.5% of soluble proteins) of SKTI protein were detected in R sub(1) and R sub(2) plants. Bioassay with R sub(1) and R sub(2) transgenic plants revealed that transgenic plants are more resistant to destructive insect pest of rice, brown planthopper (Nilaparvata lugens Stael), than the control plants. Thus, introduction of SKTI into rice plants can be used to control insect pests.

Altpeter, F; Diaz, I; McAuslane, H; Gaddour, K; Carbonero, P; Vasil, IK*

Molecular Breeding [Mol. Breed.], vol. 5, no. 1, pp. 53-63, 1999

Proteinase inhibitors have been proposed to function as plant defence agents against herbivorous pests. We have introduced the barley trypsin inhibitor CMe (BTI-CMe) into wheat (Triticum aestivum L.) by biolistic bombardment of cultured immature embryos. Of the 30 independent transgenic wheat lines selected, 16 expressed BTI-CMe. BTI-CMe was properly transcribed and translated as indicated by northern and western blot, with a level of expression in transgenic wheat seeds up to 1.1% of total extracted protein. No expression was detected in untransformed wheat seeds. Functional integrity of BTI-CMe was confirmed by trypsin inhibitor activity assay. The significant reduction of the survival rate of the Angoumois grain moth (Sitotroga cerealella, Lepidoptera: Gelechiidae), reared on transgenic wheat seeds expressing the trypsin inhibitor BTI-CMe, compared to the untransformed control confirmed the potential of BTI-CMe for the increase of insect resistance. However, only early-instar larvae were inhibited in transgenic seeds and expression of BTI-CMe protein in transgenic leaves did not have a significant protective effect against leaf-feeding insects.

Birch, ANE; Geoghegan, IE; Majerus, MEN; McNicol, JW; Hackett, CA; Gatehouse, AMR; Gatehouse, JA

Molecular Breeding [Mol. Breed.], vol. 5, no. 1, pp. 75-83, 1999

Transgenic crops genetically engineered for enhanced insect resistance should be compatible with other components of IPM for the pest resistance to be durable and effective. An experimental potato line was genetically engineered to express an anti-aphid plant protein (snowdrop lectin, GNA), and assessed for possible interactions of the insect resistance gene with a beneficial pest predator. These extended laboratory studies are the first to demonstrate adverse tri-trophic interactions involving a lectin-expressing transgenic crop, a target pest aphid and a beneficial aphidophagous predator. When adult 2-spot ladybirds (Adalia bipunctata [L.]) were fed for 12 days on peach-potato aphids (Myzus persicae Sulzer) colonising transgenic potatoes expressing GNA in leaves, ladybird fecundity, egg viability and longevity significantly decreased over the following 2-3 weeks. No acute toxicity due to the transgenic plants was observed, although female ladybird longevity was reduced by up to 51%. Adverse effects on ladybird reproduction, caused by eating peach-potato aphids from transgenic potatoes, were reversed after switching ladybirds to feeding on pea aphids from non-transgenic bean plants. These results demonstrate that expression of a lectin gene for insect resistance in a transgenic potato line can cause adverse effects to a predatory ladybird via aphids in its food chain. The significance of these potential ecological risks under field conditions need to be further evaluated.

Frutos, R; Rang, C; Royer, M

Critical Reviews in Biotechnology [Crit. Rev. Biotechnol.], vol. 19, no. 3, pp. 227-276, 1999

Insect-resistant transgenic plants have become an important tool for the protection of crops against insect pests. The acreage of insecticidal transgenic plants is expected to increase significantly in the near future. The bacterium Bacillus thuringiensis is currently the source of insecticidal proteins in commercial insect-resistant transgenic plants and will remain the most important source during the next decade. Insect resistance to B. thuringiensis Cry toxins is the main problem. Only one species, the diamondback moth, has evolved a resistance to B. thuringiensis-based formulations under field conditions. However, many other insect species were selected for resistance under laboratory conditions, indicating that there is a potential for evolution of resistance in most major pests. Many studies were conducted to elucidate the mode of action of the Cry toxins, the mechanisms and genetics of resistance, and the various factors influencing its development. This article reviews insect resistance to B. thuringiensis insecticidal proteins and related aspects, including the development of insect-resistant transgenic plants, B. thuringiensis toxins, their mode of action, mechanisms, stability, and genetics of resistance and management strategies for delaying resistance.

Smigocki, A; Neal, Jr J

US Patent 5792934, , 11 Aug 1998

A transgenic plant into which a chimeric gene comprising a wound inducible promoter and a gene for an enzyme involved in cytokinin biosynthesis has been introduced shows enhanced resistance to insect infestation.

Andow, DA; Alstad, DN; Pang, Y-H; Bolin, PC; Hutchison, WD

Journal of Economic Entomology [J. Econ. Entomol.], vol. 91, no. 3, pp. 579-584, Jun 1998

We present an application of an F sub(2) screening method for recovering and estimating the frequencies of rare alleles that confer insect resistance to a transgenic corn variety producing Bacillus thuringiensis Berliner crystal protein toxin (Bt corn). Based on a sample of 91 female Ostrinia nubilalis (Huebner) we show with 95% confidence that the frequency of B. thuringiensis resistance alleles from a wild Minnesota population is <0.013. This is an upper limit to the estimated allele frequency and does not provide clear evidence that 1 of the assumptions of the refuge plus high-dose strategy will or will not be met. With additional sampling, a more precise estimate of resistance allele frequency could be obtained that would clearly support or refute 1 of the assumptions of the refuge plus high-dose strategy. Variable costs of the screening method were $19.70 per female line, but these could be reduced by improved collecting, rearing, and handling methods.

Williams, WP; Buckley, PM; Sagers, JB; Hanten, JA

Journal of Agricultural Entomology [J. Agric. Entomol.], vol. 15, no. 2, pp. 105-112, Apr 1998

The development of crop plants expressing genes that encode insecticidal proteins isolated from the bacterium Bacillus thuringiensis Berliner (Bt) has provided a tremendous opportunity for improved control of insect pests. Although corn, Zea mays L., germplasm with resistance in the whorl stage to fall armyworm, Spodoptera frugiperda, and southwestern corn borer, Diatraea grandiosella Dyar, larval feeding has been identified and released, little progress has been made in identifying resistance to ear damage by these insects. Husks and silks were collected 3 to 4 d after anthesis from nontransgenic and transgenic corn hybrids and used in laboratory bioassays to determine the effects of the Bt insecticidal protein on larval survival and growth of fall armyworm, southwestern corn borer, and corn earworm, Helicoverpa zea. Southwestern corn borer larvae that fed on diets containing husks of transgenic plants did not survive. Corn earworm larvae fed on diets containing husks or silks from transgenic plants died, and those that fed on diets containing silks harvested from transgenic plants exhibited reduced survival and growth. Fall armyworm larvae were least susceptible to the Bt protein, but larval survival and growth on diets containing husks of transgenic plants were reduced. Larval growth also was reduced on diets containing silks from transgenic plants. The bioassays indicated that expression in husks and silks of genes encoding Bt insecticidal proteins could be useful in reducing insect damage in ears of corn.

Kumar, PA; Mandaokar, A; Sreenivasu, K; Chakrabarti, SK; Bisaria, S; Sharma, SR; Kaur, S; Sharma, RP*

Molecular Breeding [Mol. Breed.], vol. 4, no. 1, pp. 33-37, 1998

A synthetic cry1Ab gene coding for an insecticidal crystal protein (ICP) of Bacillus thuringiensis (Bt) was transferred to brinjal (eggplant) by cocultivating cotyledonary explants with Agrobacterium tumefaciens. Transformant plants resistant to kanamycin were regenerated. Hybridization experiments demonstrated gene integration and mRNA expression. Double-antibody sandwich ELISA analysis revealed Bt toxin protein expression in the transgenic plants. The expression resulted in a significant insecticidal activity of transgenic brinjal fruits against the larvae of fruit borer (Leucinodes orbonalis). The results also demonstrated that a synthetic gene based on monocot codon usage can be expressed in dicotyledonous plants for insect control.

Losey, J.E., Rayor, L.S., Carter, M.E.

Nature, vol. 399, no. 6733, p. 214, 1999

Although plants transformed with genetic material from the bacterium @@iBacillus thuringiensis@ (Bt) are generally thought to have negligible impact on non-target organisms, Bt corn plants might represent a risk because most hybrids express the Bt toxin in pollen, and corn pollen is dispersed over at least 60 metres by wind. Corn pollen is deposited on other plants near corn fields and can be ingested by the non-target organisms that consume these plants. In a laboratory assay we found that larvae of the monarch butterfly, @@iDanaus plexippus@, reared on milkweed leaves dusted with pollen from Bt corn, ate less, grew more slowly and suffered higher mortality than larvae reared on leaves dusted with untransformed corn pollen or on leaves without pollen.