Cry1Ab Protein Research Articles

Integrative analysis identifies the safety of transgenic Cry1Ab rice to non-target spider Pardosa pseudoannulata

Cry1Ab toxin has been successfully integrated into crops (e.g., rice and cotton) for pest control. Concomitantly, the environmental safety assessment of non-target organisms exposed to transgenic crops has become an essential task. Spiders grant a new perspective on the influence of the transgenic toxin on invertebrates, but the mechanisms of Cry1Ab bioaccumulation, adaptation, and safety in spiders remain unclear. In this study, we identified that the wolf spider Pardosapseudoannulata, fed with Nilaparvata lugens enriched with Cry1Ab protein, suffered no significant toxicity in terms of body mass, carapace width, and survival rate, except the prolonged developmental duration in 7th ∼ 8th instars. The overall activities of four detoxifying enzymes (SOD, POD, CAT, and GSH-Px) were not altered in four organs of adults and spiderlings. Comparison analyses of whole-body transcriptomes revealed 1,480 differentially expressed genes (DEGs), and down-regulated cuticle encoding genes were considered as a segment for the prolonged developmental duration under Cry1Ab exposure. Further venom gland transcriptome sequencing analysis found that a total of 31 toxin encoding genes were up-regulated under Cry1Ab protein stress, indicating that the spiders needed to inject more venom to digest the external protein. Furthermore, gene expression profiles of ganglia transcriptome showed that Cry1Ab protein did not generate DEGs involved in neuron sensitivity in spiders. Behavioral experiments verified that both Cry1Ab and Cry1Ab-free spiders were in line with the Holling type II functional response model. In summary, the data provide multi-level views to assess the safety of genetically modified crops on non-target arthropods.

Spodoptera frugiperda (Noctuidae) fed on transgenic maize can transfer Bt proteins to Podisus nigrispinus (Pentatomidae)

An important concern with the use of genetically modified (GM) plants expressing Bacillus thuringiensis (Bt) insecticidal toxins is the deleterious effect on non–target organisms. The predatory stink bug Podisus nigrispinus (Dallas) (Hemiptera: Pentatomidae) is used in biological control programs and may be exposed to Bt toxins. This study evaluated the indirect effects of different Cry proteins on P. nigrispinus with the prey Spodoptera frugiperda (J.E. Smith, 1797) (Lepidoptera: Noctuidae), fed on simple or pyramided Bt maize genotypes. The experiment was carried out in a completely randomized design with three treatments: i) Isohybrid (not Bt), ii) Herculex® (transgenic maize encoding Cry1F protein) and iii) PowerCore® (pyramidal transgenic maize encoding the Cry1F, Cry1A.105, and Cry2Ab2 proteins), which were used to feed S. frugiperda for 48 h. The caterpillars were used as prey by P. nigrispinus females. We evaluated the presence of Cry proteins, consumed prey biomass (predation), oviposition period, number of postures, number of eggs, number of eggs per posture, number of nymphs, egg viability, embryonic period, female longevity and development, and survival rates of immature. The results show that different Cry proteins move through the food chain of P. nigrispinus and provide evidence that the ingestion of three different proteins does not lead to unexpected synergistic effects. However, Cry toxins promoted histopathological changes in midgut cells of P. nigrispinus.

Relationship between Serum Tumor-Related Markers and Genetically Modified Rice Expressing Cry1Ab Protein in Sprague-Dawley Rats

Genetically Modified (GM) foods are among the most important achievements of biotechnology. Given the safety importance of transgenic rice, this study was conducted to investigate the effect of GM rice consumption on serum concentrations of tumor markers in rats. In this experimental intervention, we used the blood serum samples from the Biobank taken from 60 males and 60 female Sprague-Dawley (SD) rats fed on three different diets, including rat’s standard food, non-GM rice, and GM rice after 90 day. Tumor markers including Carcinogenic embryonic antigen (CEA), Alpha-Fito protein (AFP), Cancer Antigen 19-9 (CA19-9), Cancer Antigen 125 (CA125), and Cancer Antigen15-3 (CA15-3) were assessed by enzyme-linked immune sorbent assay (ELISA) method. Statistical analysis was conducted via SPSS software. The results show that the concentrations of tumor markers were within the normal range in the SD rats treated with diet, and since the concentration of tumor markers was lower than the acceptable index determined, according to the kit standard in both groups, no obvious carcinogenic effect was found. However, these findings are not enough to make a final decision regarding the safety assessment of GM rice consumption.

MXene catalyzed Faraday cage-type electrochemiluminescence immunosensor for the detection of genetically modified crops

Herein, a MXene catalyzed Faraday cage-type electrochemiluminescence (ECL) immunosensor was developed for the detection of transgenic component Cry1Ab protein in genetically modified (GM) crops. The capture unit Fe3O4-Ab1 is Fe3O4 nanoparticles coated by capture antibody Ab1, while the signal unit MXene-PTCA-Ab2 is two-dimensional conductive material MXene with ECL labels 3,4,9,10-perylenetetracarboxylic acid (PTCA) and recognition antibody Ab2 simultaneously immobilized. In presence of the target Cry1Ab, the Faraday cage-type electrochemical immunosensor could be constructed by forming the capture unit-Cry1Ab-signal unit immunocomplex. In addition to the inherently high sensitivity brought by the Faraday cage-type sensor construction mode, MXene in the signal unit catalyzes the reduction of dissolved O2 to form reactive oxygen species which could accelerate the ECL reaction, resulting in a secondary ECL enhancement of PTCA and ensuing higher detection sensitivity. Under the optimized experimental conditions, Cry1Ab protein and GM maize MON810 could be quantitatively detected in the range of 0.005 ∼ 100 ng mL−1 and 0.005 % ∼ 2.0 %, with limit of detections (LODs) 0.001 ng mL−1 and 0.001 % respectively. Selectivity, stability, repeatability, precision, and accuracy are all satisfactory. Detection of actual samples is successful, showing its application prospect in the field of agriculture and food safety.

Transgene behavior in genetically modified teosinte hybrid plants: transcriptome expression, insecticidal protein production and bioactivity against a target insect pest

BackgroundIn 2009, Spanish farmers reported a novel weed, now identified as a relative of maize’s ancestor, teosinte, in their maize fields. Introgression of the insect resistance transgene cry1Ab from genetically modified (GM) maize into populations of this weedy Spanish teosinte could endow it with additional defense mechanisms. The aims of this study were: (1) to test if hybridization between GM maize and weedy plants from Spain is possible; (2) to understand the relationship between transgene transcription activity, concentrations of the expected transgene product (Cry1Ab protein) and the bioactivity of the latter on target insect pests following transgene flow from GM maize into Spanish teosinte plants.ResultsWe demonstrated that hybridization between GM maize and the weedy Spanish teosinte is possible, with no observable barrier to the formation of crop/weed hybrids when teosinte served as pollen donor. When GM maize plants were used as pollen donors, significant crossing incompatibility was observed: hybrid plants produced only few “normal” seeds. Nevertheless, viable F1 seeds from GM pollen crossed onto teosinte were indeed obtained. The cry1Ab transgene was stably expressed as mRNA in all crossings and backgrounds. Similarly, toxicity on neonate Ostrinia nubilalis, presumably due to Cry1Ab protein, was consistently expressed in teosinte hybrids, with mortality rates 95% or higher after only 4 days exposure, similar to rates on parental GM maize plants. Nevertheless, no strong correlations were observed between transgene transcription levels and Cry1Ab concentrations, nor between Cry1Ab concentrations and insect mortality rates across all of the different genetic backgrounds.ConclusionsOur results establish fundamental parameters for environmental risk assessments in the European context: first, we show that crop/weed hybridization in fields where maize and teosinte exist sympatrically can lead to potentially catastrophic transfer of resistance traits into an already noxious weed; second, our results question the viability of using gene dosage to model and predict ecological performance in either the intended crop plant or the undesired teosinte weed. Significant questions remain that should be addressed in order to provide a scientific, sound approach to the management of this novel weed.

Genetic basis and cross-resistance of Vip3Aa resistance in Spodoptera frugiperda (lepidoptera: Noctuidae) derived from Texas, USA

The fall armyworm, Spodoptera frugiperda (J.E. Smith), is an economically important crop pest and a major target of transgenic crops expressing Cry and/or Vip3Aa proteins. Widespread adoption of Bt crops has placed a strong selection pressure on the insect populations. Till now, seven lepidopteran and two coleopteran insect species have developed the field-evolved resistance to a Bt crop with practical consequences. Understanding the genetic basis of insect resistance to a Bt protein is essential for designing appropriate resistance management strategies. In this study, we firstly characterized the genetic basis of Vip3Aa resistance in S. frugiperda derived from Texas. Then, we compared the genetic basis of Vip3Aa resistance of this Texas population with a known Vip3Aa resistant population of S. frugiperda collected from Louisiana to determine whether the genetic basis of the resistance in these two resistant populations was the same. Finally, we evaluated the responses of the Vip3Aa-resistant population of S. frugiperda collected from Texas against Cry1F and Cry2Ab2 purified proteins. The results showed that the resistance to Vip3Aa protein in S. frugiperda collected from Texas was likely controlled by a single, functionally recessive and autosomal gene. In addition, the resistance to Vip3Aa in the Texas population of S. frugiperda was not cross-resistant to Cry1F and Cry2Ab2 proteins. The interstrain complementation tests between the Texas and Louisiana resistant populations indicated the genetic basis of the resistance to Vip3Aa in these two populations are different. Considering the rapid invasion of S. frugiperda and the adoption of Vip3Aa Bt crops in the United States and other countries, results generated from this study should provide important information for management of Vip3Aa resistance in S. frugiperda worldwide.

Baseline Susceptibility and Laboratory Selection of Resistance to Bt Cry1Ab Protein of Chinese Populations of Yellow Peach Moth, Conogethes punctiferalis (Guenée).

Yellow Peach Moth (YPM), Conogethes punctiferalis (Guenée), is one of the most destructive maize pests in the Huang-Huai-Hai summer maize region of China. Transgenic Bacillus thuringiensis (Bt) maize provides an effective means to control this insect pest in field trials. However, the establishment of Bt resistance to target pests is endangering the continued success of Bt crops. To use Bt maize against YPM, the baseline susceptibility of the local populations in the targeted areas needs to be verified. Diet-overlay bioassay results showed that all the fourteen YPM populations in China are highly susceptible to Cry1Ab. The LC50 values ranged from 0.35 to 2.38 ng/cm2 over the two years of the collection, and the difference between the most susceptible and most tolerant populations was sevenfold. The upper limit of the LC99 estimates of six pooled populations produced >99% larval mortality for representative eight populations collected in 2020 and was designated as diagnostic concentrations for monitoring susceptibility in YPM populations in China. Hence, we evaluated the laboratory selection of resistance in YPM to Cry1Ab using the diet-overlay bioassay method. Although the resistant ratio was generally low, YPM potentially could evolve resistance to Cry1Ab. The potential developmentof resistance by target pests points out the necessity to implement resistance management strategies for delaying the establishment of pest resistance to Bt crops.

Multiple and non-recessive resistance to Bt proteins in a Cry2Ab2-resistant population of Helicoverpa zea

Transgenic corn and cotton expressing Cry and/or Vip3Aa proteins from Bacillus thuringiensis (Bt) have been planted for managing lepidopteran and coleopteran insect pests for more than two decades in the U.S. Evolution of insect resistance to these Bt proteins is a major threat for the continued efficacy of Bt technology. Understanding patterns of cross-resistance and dominance of resistance in target insects is essential for resistance management and pest control. The bollworm, also called corn earworm, Helicoverpa zea (Boddie), is a major target pest of pyramided Bt crops in the U.S. In this study, we evaluated the responses of Cry2Ab2-resistant (RR), susceptible (SS), and heterozygous (RS) strains of H. zea to the Cry1Ac, Cry1A.105, Cry2Ab2 and Vip3Aa39 proteins using diet-overlay bioassays. The Cry2Ab2-resistant H. zea also possessed high levels of resistance to Cry1Ac and Cry1A.105, but were very susceptible to Vip3Aa. The resistance to Cry1 and Cry2 proteins in RR H. zea varied from incompletely recessive to completely dominant depending on the protein concentration. These data suggest that the RR H. zea possess multiple non-recessive resistance to Cry1/Cry2 proteins. These results have significant implications for understanding field-evolved resistance of H. zea to Cry1 and Cry2 proteins in U.S. Bt corn and cotton fields and should contribute to developing effective management strategies for control of H. zea.

A new generation of Bt maize for control of fall armyworm (Spodoptera frugiperda).

BACKGROUNDThe pyramided genetically modified maize (Zea mays [L.]) event MON 95379, expressing the Cry1B.868 and Cry1Da_7 proteins, was designed to protect against larval feeding damage by the fall armyworm, Spodoptera frugiperda (FAW). Here, we conducted laboratory, greenhouse, and field studies to assess the dose and field efficacy of MON 95379 against FAW and inform the development of insect resistance management plans.RESULTSThe Cry1B.868 and Cry1Da_7 proteins were active against susceptible FAW neonates in diet‐incorporation bioassays: median lethal concentration [LC50] (95% CI) = 62.8 (42.6–87.6) μg/ml diet for Cry1B.868 and 9.4 (5.3–18.6) μg/ml diet for Cry1Da_7. In laboratory leaf disc bioassays, MON 95379 maize and experimental maize lines expressing the individual components were effective in controlling susceptible FAW. In whole‐plant assays, MON 95379 controlled FAW resistant to the Cry1A.105 and Cry2Ab2 proteins. Likewise, under field conditions, MON 95379 maize expressing Cry1B.868 and Cry1Da_7 was highly effective at protecting plants against the larval feeding of FAW.CONCLUSIONSThe expression of Cry1B.868 and Cry1Da_7 in MON 95379 consistently protected maize plants against larval feeding by FAW and represents an alternative to manage trait resistance issues in South America. © 2021 Bayer Crop Science‐US. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Paper-Based Simplified Visual Detection of Cry2Ab Insecticide from Transgenic Cottonseed Samples Using Integrated Quantum Dots-IgY Antibodies.

In the present study, an easy to use field-deployable methodology was developed for onsite detection of pesticidal crystal protein Cry2Ab from transgenic cotton crops to reduce seed adulteration. Anti Cry2Ab IgG and IgY antibodies were developed against recombinant Cry2Ab protein in New Zealand white rabbits and in white leg horn chickens, respectively. Carboxyl-functionalized CdTe quantum dots (QDs) were used as revealing probes, and nitrocellulose paper was used as an assay matrix. Recombinant Cry2Ab was generated in the lab and used for immunization of chicken and rabbits. After successful immunization and attaining the desired titer values (1:32 000 for IgY and 1:64 000 for IgG), eggs and hyperimmune sera were collected. Anti Cry2Ab IgY was purified as per the standardized protocols, and anti Cry2Ab IgG was purified using protein A affinity chromatography. Sensitivity of the generated antibodies was examined using indirect ELISA methods against recombinant Cr2Ab protein. Specificity evaluation was carried out against other Cry proteins including Cry2Ab, Cry4b, Cry4a, Cry1Ec, and Cry1Ac. Functionalized CdTe QDs were characterized for structure and shape as well as fluorescence properties using standard laboratory techniques. A field-deployable paper-based detection methodology was developed where IgG acted as the capturing antibody and IgY-linked CdTe QDs were used as revealing probes. The limit of detection (LOD) and quantification (LOQ) were found to be 2.91 ng/mL and 9.71 ng/mL, respectively. The effect of matrix interference was assessed on the different plant crude extracts of cottonseed materials.

The implications of homozygous vip3Aa20- and cry1Ab-maize on Spodoptera frugiperda control

Historically, Bt maize hybrids have been produced with the Bt alleles in a hemizygous state. Homozygous Bt transgenes increase the expression of Bt proteins in plants; however, recent studies have only briefly explored the zygosity of Bt transgenes in maize hybrids and its effects on the control of lepidopteran pests. In this study, we investigated whether an additional allelic dose of the single Bt events Bt11 and MIR162, and a pyramided version (Bt11 + MIR162 + GA21) would impact Bt protein expression in leaves and increase the control of fall armyworm, Spodoptera frugiperda, in leaves and grains of near-isogenic maize hybrids. Our results revealed that homozygous Bt hybrids had higher Cry1Ab and Vip3Aa20 protein concentrations in leaves than their hemizygous versions. However, higher concentrations of Bt proteins in homozygous versions did not increase the control of S. frugiperda. Hybrids expressing Cry1Ab, hemizygous and homozygous for the transgene, had similar mortality of S. frugiperda fed on leaves and grains as the non-Bt near-isogenic version, likely due to resistance of the pest to Cry1Ab. High susceptibility to Vip3Aa20 caused all larvae to die on hemizygous and homozygous MIR162 leaves. Also, larvae that survived on Vip3Aa20 grains did not gain weight after four days of feeding, regardless of the Bt zygosity of Vip3Aa20-expressing grains. Additional implications of Bt zygosity in maize hybrids on insect resistance management strategies are discussed.

Food/Feed and Environmental Risk Assessment of Herbicide-tolerant and Insect-resistant Genetically Modified Maize NK603 x MON810 for Import, Processing, Food and Feed Uses under Regulation (EC) No 1829/2003 (EFSA/GMO/UK/2004/01)

Food/Feed and Environmental Risk Assessment of Herbicide-tolerant and Insect-resistant Genetically Modified Maize NK603 x MON810 for Import, Processing, Food and Feed Uses under Regulation (EC) No 1829/2003 (EFSA/GMO/UK/2004/01)

Extended investigation of field-evolved resistance of the corn earworm Helicoverpa zea (Lepidoptera: Noctuidae) to Bacillus thuringiensis Cry1A.105 and Cry2Ab2 proteins in the southeastern United States

Previous studies have reported that the corn earworm/bollworm, Helicoverpa zea (Boddie), has developed field resistance to pyramided Bacillus thuringiensis (Bt) Cry1A/Cry2A maize and cotton in certain areas of the southeastern United States. The objective of the current study was to determine the current status and distribution of the resistance to Cry1A.105 and Cry2Ab2 in H. zea. In the study, 31 H. zea populations were collected from major maize planting areas across seven southeastern states of the United States during 2018 and 2019 and assayed against the two Bt proteins. Diet over-lay bioassays showed that most of the populations collected during the two years were significantly resistant to the Cry1A.105 protein. Most of the populations collected during 2019 were also resistant to Cry2Ab2, while significant variances were observed in the susceptibility of the populations collected during 2018 to Cry2Ab2. The results showed that Cry1A.105 and Cry2Ab2 resistance in H. zea is widely distributed in the regions sampled. The resistance to Cry1A.105 appeared to have plateaued, while selection for Cry2Ab2 resistance is likely still occurring. Thus, effective measures for mitigating the Cry1A/Cry2A resistance need to be developed and implemented to ensure the sustainable use of Bt crop biotechnology.

Expression profiles of Cry1Ab protein and its insecticidal efficacy against the invasive fall armyworm for Chinese domestic GM maize DBN9936

The fall armyworm (FAW) Spodoptera frugiperda, which originated in the Americas, is advancing across China and threatening the nation's maize crops. Currently, one widely used tool for its control is genetically modified (GM) Bacillus thuringiensis (Bt) maize. Sufficient content of Bt protein in appropriate plant parts is crucial for enhancing resistance against insect pests. In this study, we conducted a systematic investigation of Cry1Ab levels in Chinese domestic GM maize DBN9936, which has recently obtained a biosafety certificate, and evaluated its efficacy against FAW. Quantification of expression levels of Cry1Ab, via ELISA, indicated a spatio-temporal dynamic, with significant variation of mean Cry1Ab, ranging from 0.76 to 8.48 μg g−1 FW with the Cry1Ab protein level ranked as: V6–V8 leaf>R1 leaf>R4 leaf>R1 silk>VT tassel>R4 kernel. Among the nine locations, the Cry1Ab levels in DBN9936 of the Xinxiang, Langfang, and Harbin fields were significantly lower than those from Wuhan and Shenyang, and were slightly, but not significantly lower than those from the other four fields. Furthermore, the artificial diet–Cry1Ab mixture and plant tissue feeding bioassays revealed that DBN9936 has high efficacy against FAW. The insecticidal efficacy of different tissues against FAW larvae reached 34–100% with a descending order of lethality as follows: VT leaf>R4 leaf>R1 husk>R1 silk>VT tassel>R4 kernel. Taken together, our results showed that Bt-Cry1Ab maize DBN9936 has potential as a promising strategy to manage FAW.

Baseline susceptibility of Philippine Ostrinia furnacalis (Lepidoptera: Crambidae) populations to insecticidal Cry1A.105 and Cry2Ab2 proteins and validation of candidate diagnostic concentration for monitoring resistance

Abstract. Alcantara EP, Atienza MM, Camacho L, Parimi S. 2021. Baseline susceptibility of Philippine Ostrinia furnacalis (Lepidoptera: Crambidae) populations to insecticidal Cry1A.105 and Cry2Ab2 proteins and validation of candidate diagnostic concentration for monitoring resistance. Biodiversitas 22: 956-960. This study estimated the baseline susceptibility of Ostrinia furnacalis populations from the Philippines, to purified insecticidal Cry1A.105 and Cry2Ab2 proteins and determined a diagnostic concentration (DC) through a validation experiment. The insect populations were collected from separate sites of corn farms in Northern and Central Luzon and in South Cotabato province of the island of Mindanao. Dose-response bioassays using artificial diet surface overlay method were conducted on eight populations. The bioassay results revealed that the LC50 of Cry1A.105 and Cry2Ab2 to O. furnacalis ranged from 0.03 ng/cm2 to 0.18 ng/cm2 and 1.40 ng/cm2 to 9.98 ng/cm2, respectively. The relative susceptibility ratios between the most susceptible and most tolerant populations were 6-fold for Cry1A.105 and about 7-fold for Cry2Ab2. The candidate diagnostic concentrations (DC) based on the LC99 were calculated using the baseline bioassay data for both Cry1A.105 and Cry2Ab2. The validation was performed on populations from the same locations used in the baseline susceptibility assay and a reference strain to produce at least 99% mortality for each protein. Data showed that populations tested with Cry1A.105 produced average mortality of at least 99% for the upper limit, while this was observed in the LC99 estimate for Cry2Ab2. The validated diagnostic concentration can be used for monitoring the resistance development of O. furnacalis exposed to Bt Corn, MON89034, in the Philippines.

Safety assessment of genetically modified rice expressing Cry1Ab protein in Sprague\u2013Dawley rats

Rice is considered one of the most important staple food crops. Genetically modified (GM) Bt rice, harbored cry1Ab gene expressing the insect-resistance protein has been developed to resistance to the insects. In this study, we assessed the safety of the GM Bt rice on Sprague–Dawley rats for 90 days. Totally, 120 rats in both sexes were used for three different diets, including 50% GM Bt rice, feeding with 50% rice, and standard feeding. Each 40 SD rats including 20 males and 20 females were considered as each diet. The clinical variables such as body weight and food consumption were measured and a range of clinical tests was examined, including hematology, serum chemistry parameters, urinalysis profile, thyroid, and sex hormone levels. Pathological assessments were also done. The results showed that the mean weekly feed utilization (%) had no significant difference among the studied groups. Also, blood biochemistry, hematological parameters, urine analysis, and hormonal levels had no significant differences among the groups. However, alanine aminotransferase was less in males versus female feeding with GM Bt rice. No histopathological changes were observed among the groups. In conclusion, this study demonstrated that GM Bt rice had no obvious adverse effects on rats' health.

Abundance and diversity of arthropods in transgenic Bt and non-Bt cotton fields under Indian conditions

Two-year field studies during Kharif 2015 and 2016 were conducted to assess the abundance and diversity of arthropod communities of Bt cotton carrying Cry1Ac and Cry2Ab proteins and non-Bt cotton under sprayed and unsprayed conditions. The experiment was conducted in a randomized block design with four treatments of BG-II and non-Bt cotton. The treatments were non- transgenic cotton with no insecticide treatment, non-transgenic cotton with insecticide treatment, transgenic cotton with no insecticide treatment and transgenic cotton with an insecticide treatment. Arthropod community categorized as target insect pests, sucking insect pests, natural enemies and non-target insects were recorded at different phases of the cotton crop. The sucking insect pests contributed the highest abundance among the total arthropods recorded during the period of study. The abundance and diversity of bollworms that were recorded from non-Bt cotton only suggested effectiveness of BG II cotton against target insect pests. The abundance, species richness and diversity indices with respect to populations of beneficial predaceous arthropods and non-target insects were similar in BG-II and its non-Bt counterpart under unsprayed conditions. On the contrary, these parameters were significantly on the lower side under sprayed regimes for both genotypes. The spray of insecticides in early season against sucking insect pests significantly reduced their abundance and diversity indices in both BGII and non-Bt cotton. As monocropping of BG-II cotton is common on large parts of India, there is a need to monitor the populations of non-target insect species with special emphasis on sucking insect pests on a continuous basis to understand the long-term impact of Bt crops on insect biodiversity for sustainable integrated pest management practices.

In planta genetic transformation in pigeonpea: Occurrence and analysis of chimerism in transformants

The recalcitrant pigeonpea was genetically transformed through Agrobacterium-mediated in planta gene transfer method. The agroinfection of pricked cotyledonary embryo axes from 2-day-old germinating seeds resulted in development of 16 cry1Ab PCR positive T0 plants. The transcription of transgene was detected in 11 plants by semi-quantitative RT-PCR. In planta gene transfer method is documented to produce chimeric plants due to transformation of differentiated and meristematic cells resulting in mixture of transformed and non-transformed shoots. The detection of Cry1Ab protein through ELISA in leaf tissues of some branches from a single T0 plant indicated presence of chimerism. Further, accumulation of Cry1Ab at variable levels in different leaf tissues revealed quantitative variation for recombinant protein within a single T0 plant. The distinction of this study pertains to its ability to detect quantitative variation within sectorial chimeras which is not possible through PCR and RT-PCR approaches.

Molecular and Toxicity Analyses of White Granulated Sugar and Other Processing Products Derived From Transgenic Sugarcane.

This study aimed to prepare the sugar industry for the possible introduction of genetically modified (GM) sugarcane and derived retail sugar products and to address several potential public concerns regarding the characteristics and safety of these products. GM sugarcane lines with integrated Cry1Ab and EPSPS foreign genes were used for GM sugar production. Traditional PCR, real-time fluorescent quantitative PCR (RT-qPCR), and enzyme-linked immunosorbent assay (ELISA) were performed in analyzing leaves, stems, and other derived materials during sugar production, such as fibers, clarified juices, filter mud, syrups, molasses, and final GM sugar product. The toxicity of GM sugar was examined with a feeding bioassay using Helicoverpa armigera larvae. PCR and RT-qPCR results showed that the leaves, stems, fibers, juices, syrups, filter mud, molasses, and white granulated sugar from GM sugarcane can be distinguished from those derived from non-GM sugarcane. The RT-qPCR detection method using short amplified product primers was more accurate than the traditional PCR method. Molecular analysis results indicated that trace amounts of DNA residues remain in GM sugar, and thus it can be accurately characterized using molecular analysis methods. ELISA results showed that only the leaves, stems, fibers, and juices sampled from the GM sugarcane differed from those derived from the non-GM sugarcane, indicating that filter mud, syrup, molasses, and white sugar did not contain detectable Cry1Ab and EPSPS proteins. Toxicity analysis showed that the GM sugar was not toxic to the H. armigera larvae. The final results showed that the GM sugar had no active proteins despite containing trace amounts of DNA residues. This finding will help to pave the way for the commercialization of GM sugarcane and production of GM sugar.

Bioinspired synthesis of protein-posnjakite organic-inorganic nanobiohybrid for biosensing applications

This study demonstrated a facile, green and bioinspired approach to synthesize protein-posnjakite nanobiohybrid with rod-assembled hollow shuttle-like structure. Through the one-pot mild coprecipitation process, the inorganic mineral posnjakite (Cu4(SO4) (OH)6·H2O) served as a nanocarrier to efficient co-immobilization of recognition protein (streptavidin) and enzyme (horseradish peroxidase) for signal amplification, which avoids tedious linking or purification procedures and significantly simplifies the synthetic process. This nanobiohybrid was then utilized as the signal tag for immunoassays and presented excellent performance for the detection of insecticidal crystalline (Cry) protein Cry1Ab, in the linear range of 0.1–40 ng mL−1, with the limit of detection of 63 pg mL−1. This proposed strategy is expected to the integration of a variety of biomolecules with posnjakite to design diverse multifunctional nanobiohybrids for multiple applications extending from biosensors, catalysis and biomedicine to environmental science and energy.