Cry1Ac Research Articles

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.

Binding of Cry delta-endotoxins to brush border membrane vesicles of Helicoverpa armigera and Helicoverpa punctigera (Lepidoptera: Noctuidae)

The relatively low susceptibility of Helicoverpa armigera to Cry 1 Ac, its history of resistance to chemical insecticides and the seasonal decline in expression of Cry 1 Ac in transgenic cotton necessitated the development of cotton expressing two insecticidal proteins to provide sustainable control of this multinational pest. To manage the resistance issue, it was essential that the second insecticidal protein have a significantly different mode of action to Cry1 Ac. A common feature of resistance to Cry1 A proteins in several species as well as H. armigera has been a change in the binding site. A study of binding sites for some Cry proteins in the brush border membrane vesicles (BBMV) of H. armigera and Helicoverpa punctigera was undertaken. The binding affinity for Cry1Ac was higher than for Cry1Ab, matching their relative toxicities, and Cry1Ac and Cry1Ab were found to share at least one binding site in both H. armigera and H. punctigera. However Cry2Aa did not compete with Cry 1 Ac for binding and so could be used in transgenic cotton in combination with Cry 1 Ac to control H. armigera and manage resistance. Variation in the susceptibilities of three different H. armigera strains to Cry 1 Ac correlated with the parameter Bmax/Kcom.

Cry 1Ab and Cry 9C Transgenic Corn Hybrid Effects on Laboratory Populations of Indianmeal Moth (Lepidoptera: Pyralidae) and Angoumois Grain Moth (Lepidoptera: Gelechiidae)

Several Bacillus thuringiensis Berliner (Bt) Cry proteins including Cry 1Ab, Cry 1Ac, and Cry 9C, have been observed at relatively high levels in Bt corn grain using the CaMV 35S promoter. Thus, a laboratory experiment was conducted to quantify the impact of DeKalb 679 BTY Cry 1 Ab (MON 810) and Garst 8600 BLT Cry 9C (CBH 351) transgenic grain on Indianmeal moth, Plodia interpunctella (Hübner), and Angoumois grain moth, Sitotroga cerealella (Olivier), survival, development and fecundity. Eggs of Indianmeal moth or Angoumois grain moth were added to cracked or whole kernel corn. Emergence and fecundity were recorded for 5 wks. Emergence and fecundity of both moth species was reduced on both Cry 1Ab and Cry 9C-transformed corn, but only Cry 1 Ab-transformed corn delayed development of Indianmeal moth. Results indicate that populations of these moths may be negatively impacted in grain bins by Bt corn hybrids and that lepidopteran populations should be monitored in field-scale assays to examine the effects of the presence of Bt corn hybrids on insects in storage environments.

Effects of Starvation and the <I>Bacillus thuringiensis</I> Endotoxin Cry1Ac on the Midgut Cells, Feeding Behavior, and Growth of Lightbrown Apple Moth Larvae

Abstract Effects of ingesting Cry 1Ac, a δ-endotoxin from Bacillus thuringiensis, on the larval midgut of lightbrown apple moth, Epiphyas postvittana (Walker), were studied using light and electron microscopy and related to feeding behavior and growth. After larvae had ingested Cry 1Ac, epithelial midgut cells displayed fine protrusions from microvilli and loss of microvilli, expulsion of columnar cells into the midgut lumen, and, finally, rupture and disintegration of these cells. All of these effects were observed within 1 h of larvae being placed on the Cry 1Ac diet. As larvae continued to feed on this diet for 3 d, the midgut underwent disruption and recovery cycles. Midgut changes, feeding behavior, and growth of these larvae were compared with those of larvae on control diets and larvae starved for 3 d. Starvation induced a decrease in the size of midgut cells but no cell lysis, rupture, or disintegration. Larvae on the Cry 1Ac diet produced more frass than starved larvae but ate very little during ...

Protease Digestion and Role of N-acetyl Galactosamine in the Binding Characteristics of Bacillus thuringiensis Delta-endotoxin ( Cry 1Ac ) to Purified Receptor of Helicoverpa armigera

Protease Digestion and Role of N-acetyl Galactosamine in the Binding Characteristics of Bacillus thuringiensis Delta-endotoxin ( Cry 1Ac ) to Purified Receptor of Helicoverpa armigera

Interactions of Elevated Co2and Nitrogen Fertilization: Effects on Production ofBacillus thuringiensisToxins in Transgenic Plants

Elevated atmospheric CO2 concentrations will cause plants to grow faster, lower nitrogen content per unit of plant tissue, and generate higher carbon to nitrogen (C/N) ratios. We hypothesize that production of transgenic proteins will be reduced, thus reducing the efficiency of Bacillus thuringiensis (Bt) transgenes against insect populations. Commercially available transgenic cotton plants expressing the Cry 1Ac gene from Bt were compared with a near isogenic non-Bt cotton line in a split-plot design with two levels of atmospheric CO2 (ambient, 370 ppm and elevated, 900 ppm) incorporating a 2 × 2 factorial design with two nitrogen (N) fertilization regimes (low, 30 mg N/kg soil/wk and high, 130 mg N/kg soil/wk), and two levels of Bt (presence or absence). Bioassays using Spodoptera exigua (Hubner) and quantitative enzyme-linked immunosorbent assays for toxin content indicated reduced Bt protein production in elevated CO2. The tendency for test insects to consume more foliage from plants with lower N, caused by the elevated CO2, did not compensate for the reduction in toxin production. N fertilization regime interacted with CO2 concentration, showing that plants growing in N limited systems would produce substantially less toxin. The use of transgenic plants is becoming increasingly important and will continue to be so in the next decades. At the same time, atmospheric CO2 increase will affect the effectiveness of this strategy. These observations have implications not only for agricultural use of transgenic plants, but also for the ecological consequences of transfer of Bt toxins to closely related wild plant genotypes.

Transgenic tomato plants resistant to fruit borer ( Helicoverpa armigera Hubner)

A synthetic cry1 Ac gene coding for an insecticidal crystal protein (ICP) of Bacillus thuringiensis ( Bt) was transferred to tomato by cocultivating cotyledonary explants with Agrobacterium tumefaciens. Transformant plants resistant to kanamycin were regenerated. Hybridization experiments demonstrated gene integration and gene copy number in the transgenic plants. Double-antibody sandwich ELISA analysis revealed high levels of Bt ICP expression in the leaves of transgenic plants. The expression resulted in a high level of protection of transgenic plant leaves and fruits against the larvae of tomato fruit borer ( Helicoverpa armigera). Limited field trial of the transgenic plants (T 1 generation) confirmed the high levels of insect protection.

Genetically modified coffee plants expressing the Bacillus thuringiensis cry1Ac gene for resistance to leaf miner.

A synthetic version of the cry1Ac gene of Bacillus thuringiensis has been used for the transformation of coffee species (Coffea canephora and C. arabica) to confer resistance to an important pest, the coffee leaf miner (Perileucoptera coffeella and other Leucoptera spp). Somatic embryos were co-cultivated with the LBA4404 strain of Agrobacterium tumefaciens containing the cry1Ac gene. More than 100 transformed plants from independent transformation events were obtained for each coffee genotype. The integration and expression of the cry1Ac gene was studied, and effective resistance of transgenic plants against leaf miner was verified in bioassays with the insects. These plants could represent a good opportunity to analyse the impact of genetic engineering of perennial crops for sustainable resistance to an obligate endocarpic pest using a B. thuringiensis insecticidal protein.

Partial purification of Cry 1A toxin-binding proteins from Helicoverpa armigera larval midgut.

Toxicity of insecticidal endotoxins produced by Bacillus thuringiensis correlates with the presence of specific proteins in the midgut of susceptible larvae. This study was aimed at identifying and purifying Cry 1A binding proteins from Helicoverpa armigera, an important crop pest of India. B. thuringiensis strain HD 73 which produces Cry 1Ac toxin, specific for H. armigera was used in this study. Toxin-binding proteins from insect larvae were detected by employing a toxin overlay assay using both radiolabelled as well as unlabelled toxin. Detergent-solubilized fractions of larval brush border membranes were subjected to soybean agglutinin (SBA) chromatography, from which N-acetylgalactosamine (NAG)-containing proteins were eluted. Analysis of the SBA-purified proteins indicated that four proteins of approximately 97, 120, 170 and 200 kDa could bind to Cry 1Ac toxin, and three proteins of 97, 170 and 200 kDa proteins could bind to Cry 1Ab. Furthermore, in the presence of excess Cry 1Ab toxin, the labelled Cry 1Ac toxin could bind only to 170 and 200 kDa proteins, implying that Cry 1Ab can also bind the 120 kDa protein. This study therefore demonstrates that in H. armigera, midgut proteins of 97, 120, 170 and 200 kDa have the ability to bind both Cry 1Ab and Cry 1Ac. Furthermore, while the 170 and 200 kDa proteins have higher affinity for Cry 1Ac, the 97 kDa has higher affinity for Cry1 Ab.

Evaluation of transgenic tobacco expressing two insecticidal genes to delay resistance development ofHelicoverpa armigera

The resistance ratio ofHelicoverpa armigera to Cry1 Ac insecticidal protein fromBacillus thuringiensis (Bt) is 13.1- and 3.02-fold after 18 generations of selection by transgenic tobacco expressing Bt or two (Bt and CpTI) insecticidal protein genes, in which the average corrected mortality for each selection treatments is about 60%. The mortality of selected population by transgenic Bt gene tobacco is significantly lower than the control strain when fed on transgenic tobacco plants. The mortaltty of the selected population by transgenic two genes tobacco was not significantly different from the control strain. This is the first experiment under laboratory condition which has proved that transgenic two genes tobacco could significantly delay resistance development ofH. armigera compared with one gene.

BINDING ASSAY OF BACILLUS THURINGIENSIS CRY1AC INSECTICIDAL CRYSTAL PROTEINS IN DIAMONDBACK MOTH*

Abstract Binding assay was performed with NTP‐labeled Bacillus thuringiensis activated toxin Cry 1Ac on midgut brush border membrane vesicles (BBMVs) prepared from the whole larvae of dia‐mondback moth Plutella xylostella. We conclude that the BBMVs can be isolated properly and Cry 1Ac toxin binds saturably to BBMVs.

Bacillus thuringiensis HD-73 Spores Have Surface-Localized Cry1Ac Toxin: Physiological and Pathogenic Consequences

Spores from Cry(sup+) strains of Bacillus thuringiensis bound fluorescein isothiocyanate-labeled antibodies specific for the 65-kDa activated Cry 1Ac toxin, whereas spores from Bacillus cereus and Cry(sup-) strains of B. thuringiensis did not. The Cry(sup+) spores could be activated for germination by alkaline conditions (pH 10.3), whereas Cry(sup-) spores could not. Once the surrounding exosporia had been removed or permeabilized, Cry(sup+) spores were able to bind the toxin receptor(s) from insect gut brush border membrane vesicle preparations, and their germination rates were increased ca. threefold in the presence of brush border membrane vesicles. A model is presented whereby in the soil the Cry toxins on the spore surface are protected by the exosporium while in the gut they are exposed and available for binding to the insect receptors. This model explains why the disulfide-rich C terminus of the cry genes is so highly conserved even though it is removed during the processing of the protoxin to the activated toxin. It also highlights the trade-off resulting from having Cry toxins located on the spore surface, i.e., decreased spore resistance versus enhanced insect pathogenesis.

Binding of Bacillus thuringiensis Cry1 Toxins to the Midgut Brush Border Membrane Vesicles of Chilo suppressalis (Lepidoptera: Pyralidae): Evidence of Shared Binding Sites

Volume 62, no. 5, p. 1544, Abstract, line 4: "Cry1Aa" should read "Cry1Ac." [This corrects the article on p. 1544 in vol. 62.].