Cry1Ac Expression Research Articles

Selective Feeding of Helicoverpa armigera (Hübner) and Spodoptera litura (Fabricius) on Meridic Diet with Bacillus thuringiensis Toxins

Laboratory experiments were conducted to evaluate the behavior of Helicoverpa armigera (Hubner) and Spodoptera litura (Fabricius) larvae on meridic diet with different concentrations of Bt spray formulation Delfin or isolated Cry1Ac protein or the foliage and bolls from transgenic cotton, Bollgard hybrid RCH-317 Bt. Both insect species selectively fed on nontreated diet compared with the diet treated with Delfin. While H. armigera exhibited concentration response with Cry1Ac, this protein did not affect S. litura larvae. In general Helicoverpa selected diet with low concentrations (EC20 and EC50 levels) of Cry1Ac compared with higher concentrations of Cry1Ac. In order to develop appropriate management strategies, a thorough understanding of the behavioral mechanisms leading to the responses of insects to the proteins in transgenic varieties is required. Thus, based on results of the insects fed individually on the leaf discs or bolls from transgenic cotton plants alone or under choice situation with meridic diet revealed that H. armigera larvae preferred meridic diet to transgenic leaves or bolls expressing Cry1Ac protein. H. armigera larvae preferred meridic diet to plant material; more than 70% larvae were seen on the meridic diet, and average larval weight gain was in the range of 121.7–130.5 mg. However, in case of S. litura the larvae showed no significant discrimination between meridic diet and the leaf discs. In fact more than 60% larvae preferred leaf discs for feeding, though Cry1Ac expression in leaf discs was in the range of 0.9–2.18 μg/g. Thus differences in behavioral response could potentially impact the level of efficacy of crop cultivars that have been genetically engineered to produce these proteins.

Yield, Leaf Senescence, and Cry1Ac Expression in Response to Removal of Early Fruiting Branches in Transgenic Bt Cotton

Two-year field experiments were conducted at Linqing, Yellow River valley of China, to study the plant response to the removal of early fruiting branches in transgenic Bt ( Bacillus thuringiensis) cotton ( Gossypium hirsutum L.) from 2003 to 2004. Plants were undamaged and treated by removing two basal fruiting branches (FB) at squaring to form the control and the removal treatment, respectively. The plant height, leaf area (LA), dry weight of fruiting forms (DWFF), the number of fruiting nodes (NFN), photosynthetic (Pn) rate, and levels of leaf chlorophyll (Chl), N, P, K, and Cry1Ac protein in main-stem leaves were measured at a 10- or 20-d interval after FB removal, and the sink/source ratio as indicated by NFN/LA and DWFF/LA was determined. FB removal significantly increased the plant height, LA, and plant biomass in both years. Lint yields were increased 7.5 and 5.2% by removal compared with their controls in 2003 and 2004, respectively. Significant increases in boll size (5.7 and 5.1%) were also observed in removal than in control for both years. Either NFN/LA or DWFF/LA was significantly reduced by removal before 40 d after removal; however, both NFN/LA and DWFF/LA were significantly enhanced by FB removal at 80 d after removal compared to the untreated control. There was no significant difference in fiber quality in the first two harvests between removal and control, but fiber strength and micronarie in the third harvest were significantly improved by FB removal. In terms of leaf Chl, Pn rate, levels of total N, P, and K in late season, leaf senescence was considerably delayed by FB removal. Levels of Cry1Ac protein in the fully expanded young leaves were considerably higher in FB-excised plants than in control, indicating FB removal enhanced Cry1Ac expression. It is suggested that the yield and quality improvement with FB removal may be attributed to the increased NFN/LA or DWFF/LA in late season and delayed leaf senescence, respectively. FB removal can be a potential practice incorporated into the intensive cultivation system for enhancing transgenic Bt cotton production.

Dual Insecticidal Activity of Spodoptera-Toxic Bacillus thuringiensis Strain Transformed with Lepidopteran-Specific Cry Toxin

The E. coli-B. thuringiensis shuttle vector for expression of cry1Ac, pHT1K-1Ac plasmid was introduced into acrystalliferous B. thuringiensis Cry −B and Spodoptera toxic STB-3 strain. The presence of a recombinant plasmid in transformants after electroporation was confirmed by PCR. The 1K-1Ac/Cry −B(Cry −B transformant) and 1K-1Ac/STB-3 (STB-3 transformant) produced bipyramidal-shaped parasporal inclusion that was 130 kDa in size as like B. thuringiensis subsp. kurstaki HD-73. In P. xylostella bioassay, these transformants showed significantly high toxicity than the wild-type recipients and further, in case of B. thuringiensis STB-3 transformant still had original Spodoptera toxicity. These results suggested that the pHT1K could be successfully applied for generating individual B. thuringiensis strains that produce various combinations of insecticidal proteins to expand their host spectrum and enhance insecticidal activity.

Expression of Bt cry1Ac in transgenic oilseed rape in China and transgenic performance of intraspecific hybrids against Helicoverpa armigera larvae

The expression of a synthetic Bacillus thuringiensis (Bt) cry1Ac gene in oilseed rape (OSR, Brassica napus) was monitored under field conditions in China, and performance against Helicoverpa armigera larvae was compared in intraspecific hybrids with a Chinese OSR variety. Leaf samples from transgenic OSR were collected at various developmental stages in two separate field experiments. The Bt Cry1Ac concentrations in the third uppermost leaves increased before pod formation stage and either increased or decreased after pod formation stage whereas the total soluble protein increased before and decreased after pod-fill in the later growing season. Spontaneously formed intraspecific hybrids between transgenic OSR and a Chinese conventional OSR were obtained in the field and transgenic status was confirmed by a green fluorescent protein (GFP) phenotype and polymerase chain reaction. A bioassay on the neonate larvae of a susceptible strain of H. armigera was performed to test the efficacy of Bt Cry1Ac toxin in hybrid OSR plants. Both the original transgenic OSR and hybrid plants had a negative effect on body-weight gain of insect larvae. It was assumed that Bt Cry1Ac toxin concentration was similar in hybrids compared to the original transgenic OSR at the investigated developmental stages. The frequency of hybrid production and volunteerism could potentially enhance the evolution of insect pest tolerance in the field.

Comparative study on effect of different promoters on expression of cry1Ac in Bacillus thuringiensis chromosome

The aim of this work was to investigate the effect of cry3A promoter on the expression of cry1Ac in Bacillus thuringiensis chromosome and stably enhance the production of different cry genes under the control of cry3A promoter. The cry1Ac gene, which is specific to Lepidopteran larvae, was integrated into the chromosome of a B. thuringiensis plasmid-free and acrystalliferous strain BMB171, under the control of cry3A promoter and cry1Ac promoter, respectively. The expression of cry1Ac genes in the chromosome of host strain was investigated. The results from sodium dodecyl sulfate-polyacrymide gel electrophoresis, crystal observation and bioassay showed that either integrated with cry3A promoter (cry3Apro-cry1Ac) or with its native promoter (cry1Acpro-cry1Ac), cry1Ac gene could efficiently and stably express in the chromosome. The production of cry3Apro-cry1Ac gene was higher than that of cry1Acpro-cry1Ac gene. The cry3A promoter enhanced the expression of cry1Ac gene efficiently either on the chromosome or on the plasmid in B. thuringiensis strain. So far, the comparative studies on cry3A promoter and other cry promoters were carried on B. thuringiensis plasmids. This system offers an additional method for potentially improving the efficacy of B. thuringiensis insecticidal proteins efficiently, stably and safely.

Correlated Expression of gfp and Bt cry1Ac Gene Facilitates Quantification of Transgenic Hybridization between Brassicas

Gene flow from transgenic oilseed rape (BRASSICA NAPUS) might not be avoidable, thus, it is important to detect and quantify hybridization events with its relatives in real time. Data are presented showing the correlation between genetically linked green fluorescent protein (GFP) with BACILLUS THURINGIENSIS (Bt) CRY1AC gene expression in hybrids formed between transgenic B. NAPUS "Westar" and a wild Chinese accession of wild mustard (B. JUNCEA) and hybridization between transgenic B. NAPUS and a conspecific Chinese landrace oilseed rape. Hybrids were obtained either by spontaneous hybridization in the field or by hand-crossing in a greenhouse. In all cases, transgenic hybrids were selected by GFP fluorescence among seedlings originating from seeds harvested from B. JUNCEA and the Chinese oilseed rape plants. Transgenicity was confirmed by PCR detection of transgenes. GFP fluorescence was easily and rapidly detected in the hybrids under greenhouse and field conditions. Results showed that both GFP fluorescence and Bt protein synthesis decreased as either plant or leaf aged, and GFP fluorescence intensity was closely correlated with Bt protein concentration during the entire vegetative lifetime in hybrids. These findings allow the use of GFP fluorescence as an accurate tool to detect gene-flow in time in the field and to conveniently estimate BT CRY1AC expression in hybrids on-the-plant.

Regeneration and transformation of an elite inbred line of maize (Zea mays L.), with a gene from Bacillus thuringiensis

Abstract Tissue culture conditions for regeneration of fertile plants from immature embryos were optimized using four local inbred lines of Zea mays . Immature embryos were cultured on five different media. The highest regeneration efficiency of 86.40% was observed for BR-6 at N6 modified media (supplemented with 1.0 mg L − 1 2,4-D, 25 mM L-Proline, and 100 mg L − 1 Casein Hydrolysate) while B-46, EX-285 and EX-295 gave a regeneration percentage of 57%, 77%, and 63%, respectively. A total number of 1646 immature embryos were bombarded by a gene gun using tungsten micro projectiles coated with a multi-gene plasmid pCMAC, containing gusA (reporter gene), hpt (hygromycin phosphotransferase), and cry 1Ac genes driven by maize ubiquitin promoter. Regenerates were selected on a medium containing 40 μg/L hygromycin and then screened with GUS assay. Thirty-five transgenic lines were obtained with a transformation frequency of 2.21%. The integration and expression of cry 1Ac was confirmed with PCR, Southern Blot analysis, ELISA, and Western Blot analysis. The studies confirmed that cry 1Ac gene has been stably transformed into the local inbred line (BR-6) and caused 80–100% mortality to armyworm ( Spodoptera frugiperda ) in insect feeding bioassay.

Changes in Cry1Ac Bt Transgenic Cotton in Response to Two Environmental Factors: Temperature and Insect Damage

The efficacy of Cry1Ac Bacillus thuringiensis (Bt) cotton plants against field populations of Helicoverpa armigera (Hübner) has been inconsistent over the growing season. Any reduction in efficacy (where efficacy is the capacity of the plant to affect the survival of the insect) increases the opportunities for H. armigera to evolve resistance to Bt toxin. Changes in efficacy could be due to changes at the level of gene expression and/or in the physiological makeup of the plant and may be induced by environmental conditions. Two environmental factors, temperature and insect damage, were investigated. Temperature was found to affect efficacy, whether plants were grown at different temperatures continuously or were exposed to a change in temperature for a short period. Damage caused by chewing insects (H. armigera larvae) produced a dramatic increase in the efficacy of presquare Bt cotton. In contrast, damage by sucking insects (aphids) did not induce changes in efficacy. Changes in efficacy seemed to be mediated through modification of the physiological background of the plant rather than changes in the level of Cry1Ac expression or in the concentration of the Bt toxin. The impact of the non-Bt responses of plants on strains of H. armigera should be evaluated. It is possible that by enhancing existing defensive mechanisms of plants, the rate of evolution of resistance to Bt toxins could be retarded by increasing the plants overall toxicity through the additive effects of the toxins and plant defenses.

Signal peptide of potato PinII enhances the expression of Cry1Ac in transgenic tobacco.

The modified Cry1Ac was expressed in transgenic tobacco plants. To allow secretion of the Cry1Ac protein into the intercellular space, the signal peptide sequence of potato proteinase inhibitor II (pinII) was N-terminally fused to the Cry1Ac encoding region. Expression of Cry1Ac in transgenic tobacco plants was assayed with ELISA. The results showed that pinII signal peptide sequence enhanced the expression of Cry1Ac protein and led to the secretion of the Cry1Ac protein in transgenic tobacco plants. GFP gene was also fused to the signal peptide sequence and transformed to tobacco. The results of fluorescent detection showed that GFP had localized in the apoplast of transgenic plants.

Expression of synthetic Cry1Ab and Cry1Ac genes in basmati rice (Oryza sativa L.) variety 370 via Agrobacterium-mediated transformation for the control of the european corn borer (Ostrinia nubilalis)

Movement of pests to non-host erops due to resistance of their host plants is diseussed. We report the simultaneous control of the European corn borer (Ostrinia nubilalis) and protection of transgenic rice (Oryza sativa) from the damages of this non-host pest. Indica rice Basmati 370 variety was genetically engineered via the Agrobacterium-mediated system for expression of Bacillus thuringiensis (cry1Ab and cry1Ac), hygromyein resistance (hpt) and β-glucuronidase (gus) genes. Molecular analysis of R0 and R1 progeny plants confirmed the integration, transeription and translation of all transgenes, with a 100% cointegration of linked genes. Southern blot analysis revealed the integration of transgenes in the range of one to three copy numbers. Northern blots revealed the presence of intact, full-length transcripts of the cry1Ab and cry1Ac. The expression of Cry1Ab and Cry1Ac was estimated up to 0.1% of total soluble protein. Inheritance of the introduced genes to R1 progeny was found to be in agreement with the Mendelian ratio in most of the transgenic lines. Bioassays, feeding of R1 progeny, of four independent transgenic lines, showed high levels of resistance to the European corn borer. Transgenic lines showed 100% mortality 5 d after the infestation in the bioassay experiments.

Field efficacy and seasonal expression profiles for terminal leaves of single and double Bacillus thuringiensis toxin cotton genotypes.

Examination of commercial Cry1Ac transgenic Bacillus thuringiensis Berliner (Bt) cotton varieties (Bollgard, Monsanto, St. Louis, MO) and an experimental Cry1Ac + Cry2Ab transgenic Bt cotton variety (Bollgard II, Monsanto) for lepidopteran field efficacy was conducted during the 2000 growing season. In addition, a commercially available (Envirologix, Portland, ME) quantification assay (ELISA) was used to measure and profile the expression levels of Cry proteins in two of these varieties ['DP50B, Bollgard'; 'DP50BII, Bollgard II' (Delta & Pine Land, Scott, MS)]. Populations of beet army worms, Spodoptera exigua (Hübner), and soybean loopers, Pseudoplusia includens (Walker), were significantly lower (P < 0.05) in Bollgard II plots compared with Bollgard. Population numbers for fall army worms, Spodoptera frugiperda (J. E. Smith), and salt marsh caterpillars, Estigmene acrea (Drury), were lower in Bollgard II plots compared with Bollgard but means did not differ significantly (P > 0.05). Single and dual-toxin genotypes remained superior (P < 0.05) compared with conventional cotton against the tobacco budworm, Heliothis virescens (F.). The addition of Cry2Ab had no significant (P > 0.05) impact on Cry1Ac expression in Bollgard II compared with Cry1Ac expression in Bollgard. Furthermore, throughout the season Cry2Ab was present at much higher levels in the plant compared with Cry1Ac for Bollgard II plants. Possible species-specific reasons for increased efficacy of Bollgard II over Bollgard are discussed.

Potential factors impacting season-long expression of Cry1Ac in 13 commercial varieties of Bollgard &lt;sup&gt;®&lt;/sup&gt; cotton.

Thirteen commercial varieties of transgenic Cry1Ac Bacillus thuringiensis Berliner (Bt) cotton were examined across two sites in 2000 for potential factors that impact endotoxin expression. In all cases, two varieties (NuCOTN 33B and DP 458B/RR, Delta and Pineland Co., Scott, MS) expressed more Cry1Ac than the other 11 varieties in various plant structures. These two varieties share the same parental background (DP 5415). Furthermore, when the next generation of plants were tested in the greenhouse, the same varietal patterns were exhibited. These data strongly suggest that factors such as parental background had a stronger impact on the expression of Cry1Ac than the environment.

Transgenic rice as a system to study the stability of transgene expression: multiple heterologous transgenes show similar behaviour in diverse genetic backgrounds

The success of contemporary breeding programmes involving genetic engineering depends on the stability of transgene expression over many generations. We studied the stability of transgene expression in 40 independent rice plant lines representing 11 diverse cultivated varieties. Each line contained three or four different transgenes delivered by particle bombardment, either by cotransformation or in the form of a cointegrate vector. Approximately 75% of the lines (29/40) demonstrated Mendelian inheritance of all transgenes, suggesting integration at a single locus. We found that levels of transgene expression varied among different lines, but primary transformants showing high-level expression of the gna, gusA, hpt and bar transgenes faithfully transmitted these traits to progeny. Furthermore, we found that cry1Ac and cry2A transgene expression was stably inherited when primary transformants showed moderate or low-level expression. Our results show that six transgenes (three markers and three insect-resistance genes) were stably expressed over four generations of transgenic rice plants. We showed that transgene expression was stable in lines of all the rice genotypes we analysed. Our data represent a step forward in the transfer of rice genetic engineering technology from model varieties to elite breeding lines grown in different parts of the world.

Preventing insect adaptation to insect-resistant crops: are seed mixtures or refugia the best strategy?

Transgenic crops expressing insecticidal toxins could soon provide safe, clean and effective means of pest control, but their usefulness will be short-lived if insects adapt to the toxins. Two planting strategies are among those that have been recommended to delay crop failure: susceptible insects could be conserved by planting either ‘refugia’, i.e. separate fields of toxic and toxin-free crop, or ‘seed mixtures’ of toxic and toxin-free plants in the same fields. However, we show that if insects can move from plant to plant, seed mixtures may actually hasten insect resistance compared with pure stands of toxic plants. Insect movement causes an increase in effective genetic dominance which can counteract reduced selection due to the mixture. This failure of seed mixtures is likely under just those conditions, low genetic dominance of resistance, which predict a good chance for resistance to the toxin to evolve slowly. Seed mixtures, unlike refugia, are therefore failure prone. This result also suggests potential problems with a third strategy, tissue-specific expression of toxins, which essentially provides a mixture of toxin-free and toxin-containing tissues on the same plant. However, better information and modelling are urgently required to evaluate alternative means of slowing insect adaptation to resistant crop plants. Legislation for toxinfree refugia may provide one of the best available means for conserving insect susceptibility.