cry1Ac Gene Research Articles

Development and Evaluation of the Cotton Leaf Curl Kokhran Virus-Burewala Bidirectional Promoter for Enhanced Cry1Ac Endotoxin Expression in Bt Transgenic Cotton

Fluctuation in Cry1Ac endotoxin levels expressed in transgenic Bacillus thuringiensis (Bt) cotton (Gossypium hirsutum L.) can result in a variation in efficacy throughout the growing season. Here, a green tissue-specific strong promoter of the cotton leaf curl Kokhran virus-Burewala (CLCuKoV-Bu) C1 gene is reported that can direct consistently high levels of Cry1Ac endotoxin expression in transformed cotton plants. The objective of this study was to investigate the capacities of the CLCuKoV-BuC1 promoter to drive transcription of Cry1Ac and stably express endotoxin in mature leaves and bolls of transgenic cotton plants, compared to the traditional CaMV35S promoter. The Cry1Ac gene expression cassettes were constructed under the control of a bidirectional promoter and transformed into cotton ‘MNK-786′. The expression of Cry1Ac constructs was evaluated in transient and stable expression systems using Nicotiana tabacum ‘Rustica’ and cotton plants, respectively. Accumulation of the Cry1Ac expressed in two resultant transgenic cotton plants harboring the constructs driven by the CLCuKoV-BuC1 and CaMV35S promoter, respectively, was analyzed using a commercially available enzyme-linked immunosorbent assay. In leaves and bolls of two cotton plants shown to express CLCuKoV-BuC1-Cry1Ac (CLCuV-Ac), the Cry1Ac protein accumulated at 400 and 300 ng g−1 per fresh tissue weight, respectively, whereas no toxin was detectable in the roots. In contrast, CaMV35S-Cry1Ac transgenic cotton plants accumulated three times less Cry1Ac protein than those transformed with CLCuV-Ac. Results indicate that the greatest amount of Cry1Ac endotoxin accumulated in transgenic cotton when expression was driven by the CLCuKoV-BuC1 compared to the CaMV35S promoter. Thus, the CLCuKoV-BuC1 promoter offered more robust transgene expression in cotton plants than the traditional CaMV35S promoter. The newly validated CLCuV-Ac promoter of begomoviral origin offers an exciting alternative as a robust promoter for genetic engineering of cotton and other plants.

Development and evaluation of triple gene transgenic cotton lines expressing three genes (Cry1Ac-Cry2Ab-EPSPS) for lepidopteran insect pests and herbicide tolerance

Cotton is an international agricultural commodity and the main cash crop of Pakistan of which quality and quantity are subject to various whims of nature. Climate change, insect pest complex, and weeds are reducing its productivity. Here, we have developed triple gene cotton containing EPSPS gene along with two Bt toxin genes Cry1Ac and Cry2Ab using a strategy where all three genes are cloned in the same T-DNA, followed by successful cotton transformation via Agrobacterium-mediated transformation. This strategy has been developed to help cotton breeders in developing new cultivars by incorporating these genes into the non-transgenic or single Bt (Cry1Ac) gene cotton background where all three genes will inherit together. The expression of all three proteins was confirmed through immunostrips and was quantified through enzyme-linked immunosorbent assay (ELISA). The spatio-temporal expression of Bt protein in different parts of triple gene NIBGE cotton plants was determined. Maximum expression was found in leaves followed by seeds and boll rinds. Insect bioassays with cotton bollworms (Helicoverpa armigera), armyworms (Spodoptera litura), and pink bollworms (Pectinophora gossypiella) showed more than 90% mortality. The best performing line (NIBGE-E2) on the basis of spatiotemporal expression, glyphosate assays, and insect mortality data, was used for event characterization by using the genome sequencing approach. The event was successfully characterized and named NIBGE 20-01. A diagnostics test based on event-specific PCR was developed and its ability to distinguish NIBGE 20-01 event from other commercial transgenic cotton events was confirmed. To confirm stable expression of all three proteins in the field conditions, homozygous transgenic lines were grown in the field and the expression was confirmed through immunostrip assays. It was found that all three genes are expressed under field conditions. To show that all three genes are inherited together upon crossing with local elite cotton lines, the F1 generation was grown under glasshouse and field conditions. The expression of all three genes was confirmed under field conditions. Our results showed that transgenic cotton with three genes cloned in the same T-DNA can express all genes and can be conveniently transferred into elite cotton lines through a single cross.

P07-45 Toxicity assessment of transgenic cotton containing double gene (Cry1Ac and Cry2Ab) and triple gene (Cry1Ac, Cry2Ab, and EPSPS) as plant incorporated protectants against insects and weed

P07-45 Toxicity assessment of transgenic cotton containing double gene (Cry1Ac and Cry2Ab) and triple gene (Cry1Ac, Cry2Ab, and EPSPS) as plant incorporated protectants against insects and weed

Resistance in Pink Bollworm Pectinophora gossypiella (Saunders) against Bt Cotton, a Major Threat to Cotton in India: A Brief Review

The pink bollworm, Pectinophora gossypiella (Lepidoptera: Gelechiidae) is a primary pest of cotton in many regions of the world. In many parts of India, pink bollworm (PBW) has become resistant to BG-I (Bollgard I®) that expresses a single Bt gene (Cry1Ac) in 2009 and BG-II (Cry1Ac and cry 2Ab) in 2015. The brief review addresses the possible reasons and solutions to resistance posed by this insect. The review addresses the following points: reasons for resistance in PBW is discussed; the life-history is discussed how it’s distinctness from other bollworms; the introduction of hybrid cotton lead to insecticide treadmill in India; reduced accomplishment of the refugia recommendations also contributed to the problem; poor compliance of the bio-safety laws also led to the problem; unfavorable crop management practices prove to be an add-on to the problem. Furthermore, the possible solutions of the problem are discussed; monitoring and mating disruption; cultivation of High-density short season (HD-SS) pure line varieties; case studies of sterile insect technique (SIT) proved to be supporting in control of PBW; RNA interference technique can overcome the problem of resistance; use of chemical insecticides in different manner can change the perspective; proper cultural or farming practices can make the difference; Bio-control agents can overcome the problem; extension functionaries can help in solving the PBW problem. Therefore the management of resistance in PBW requires a broader vision and development of technology.

Characterization and Evaluation of Indigenous Bacillus thuringiensis Isolate T352 against Fall Armyworm, Spodoptera frugiperda (J.E. Smith)

Bacillus thuringiensis (Bt) Berliner is a ubiquitous soil bacterium with commercial bio pesticidal value and widely used for effective control of various important agricultural insect pests. Invasion of Fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith) into India caused potential yield loss in maize production, and also it threats the cultivation of other related crops. This study was aimed to characterize and evaluate the pathogenic activity of indigenous Bt isolate T352 against fall armyworm (FAW), S. frugiperda. The Bt isolate T352 was creamy white in colour and had irregular shaped flat colonies with undulated margin. Bipyramidal shape of parasporal inclusions was found in isolate T352. The isolate produced protein bands of ̴ 130 kDa and ̴ 65 kDa size in SDS PAGE analysis. PCR screening also confirmed the presence of cry1Ab, cry1Ac, cry2Aa, cry2Ab and vip3A genes. During probit analysis, isolate T352 exhibited the LC50 of 1.927 µg/ml as against 0.421µg/ml in positive standard strain HD- 1 based on mortality observed at 72 h after treatment in leaf disc bioassay with spore crystal mixtures.

Biochemical analyses demonstrate that Bt maize has no adverse effects on Eisenia fetida.

The potential effects of Bt (Bacillus thuringiensis) maize on non-target organisms should be evaluated before such maize is commercially planted. Earthworms play an indispensable role in the soil ecosystem; act as important bio-indicators of soil quality and environmental pollution. Therefore, earthworms are often used as the object to evaluate the non-target effect of Bt maize. To accelerate the commercialization of transgenic maize in China, a 90-day Eisenia fetida feeding experiment was conducted to evaluate the potential effects of Bt maize line, BT799—which was developed by China Agricultural University and contains the Cry1Ac gene—and its non-Bt conventional isoline—Zheng 58—on E. fetida. Our results showed that the Bt maize line had no significant effects on the growth, reproduction, or enzymatic activities of these earthworms. In summary, Bt maize had no toxic effects on E. fetida.

Characterization of indigenous Bacillus thuringiensis isolate RM11 toxic to the diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae)

BackgroundBacillus thuringiensis (Bt) Berliner is an omnipresent soil bacterium used as world’s leading biopesticide to combat agriculturally important insect pests. This study was aimed at protein and gene profiling of an indigenous Bt isolate RM11, which was toxic to the larvae of diamondback moth, Plutella xylostella (L.) in laboratory bioassays.ResultsIndigenous Bt isolate RM11 was characterized along with the standard checks B. thuringiensis subsp. kurstaki (Btk) HD1 and 78/11, based on colony characters, protein profile and PCR screening. All three Bt colonies were fried egg type, white in color with flat elevation and undulated margin. PCR screening revealed the presence of cry1Ac and vip3A genes, which encode lepidopteran toxic proteins in RM11. SDS-PAGE results showed the presence of a prominent protein band of cry1Ac, vip3A with molecular weights 135 kDa, 88 kDa and other bands at 70, 50, 32 and 10 kDa. In leaf disk bioassay with spore crystal mixture, RM11 exhibited toxicity with LC50 of 4.51 µg/ml as against 0.07 µg/ml in positive standard HD1, based on mortality at 72 h after treatment. At LC50 of 4.51 µg/ml, solubilized and insolubilized protein of RM11 was found to produce 56 and 70% mortality.ConclusionsThe present study revealed that RM11 could be a viable alternative for consideration in developing a native Bt formulation and for inclusion in the integrated management of P. xylostella with other native isolates producing different toxins. Furthermore, these findings imply that RM11 could be a source of new cry toxin, which can be confirmed through whole-genome sequencing analysis.

Introgressing cry1Ac for Pod Borer Resistance in Chickpea Through Marker-Assisted Backcross Breeding.

The gram pod borer Helicoverpa armigera is a major constraint to chickpea (Cicer arietinum L.) production worldwide, reducing crop yield by up to 90%. The constraint is difficult to overcome as chickpea germplasm including wild species either lacks pod borer resistance or if possessing resistance is cross-incompatible. This study describes conversion of elite but pod borer-susceptible commercial chickpea cultivars into resistant cultivars through introgression of cry1Ac using marker-assisted backcross breeding. The chickpea cultivars (PBG7 and L552) were crossed with pod borer-resistant transgenic lines (BS 100B and BS 100E) carrying cry1Ac that led to the development of BC1F1, BC1F2, BC1F3, BC2F1, BC2F2, and BC2F3 populations from three cross combinations. The foreground selection revealed that 35.38% BC1F1 and 8.4% BC1F2 plants obtained from Cross A (PBG7 × BS 100B), 50% BC1F1 and 76.5% BC1F2 plants from Cross B (L552 × BS 100E), and 12.05% BC2F2 and 82.81% (average) BC2F3 plants derived from Cross C (PBG7 × BS 100E) carried the cry1Ac gene. The bioassay of backcross populations for toxicity to H. armigera displayed up to 100% larval mortality. BC1F1 and BC1F2 populations derived from Cross B and BC2F3 population from Cross C segregated in the Mendelian ratio for cry1Ac confirmed inheritance of a single copy of transgene, whereas BC1F1 and BC1F2 populations obtained from Cross A and BC2F2 population from Cross C exhibited distorted segregation ratios. BC1F1 plants of Cross A and Cross B accumulated Cry1Ac protein ranging from 11.03 to 11.71 µgg−1 in leaf tissue. Cry1Ac-positive BC2F2 plants from Cross C demonstrated high recurrent parent genome recovery (91.3%) through background selection using SSR markers and phenome recovery of 90.94%, amongst these 30% plants, were homozygous for transgene. The performance of BC2F3 progenies derived from homozygous plants was similar to that of the recurrent parent for main agronomic traits, such as number of pods and seed yield per plant. These progenies are a valuable source for H. armigera resistance in chickpea breeding programs.

Clean gene technology to develop selectable marker-free pod borer-resistant transgenic pigeon pea events involving the constitutive expression of Cry1Ac.

The most crucial yield constraint of pigeon pea is susceptibility to the pod borer Helicoverpa armigera, which causes extensive damage and severe economic losses every year. The Agrobacterium-mediated plumular meristem transformation technique was applied for the development of cry1Ac transgenic pigeon pea. Bioactivity of the cry1Ac gene was compared based on integration and expression driven by two promoters, the constitutive CaMV35S promoter and the green-tissue-specific ats1A promoter, in those transgenic events. The transgenic events also contained the selectable marker gene nptII flanked by loxP sites. Independent transgenic events expressing the Cre recombinase gene along with a linked bar selection marker were also developed. Integration and expression patterns of both cry1Ac and cre were confirmed through Southern and western blot analysis of T1 events. The constitutive expression of the Cry1Ac protein was found to be more effective for conferring resistant activity against H. armigera larvae in comparison to green-tissue-specific expression. Constitutively expressing Cry1Ac T1 events were crossed with Cre recombinase expressing T1 events. The crossing-based Cre/lox-mediated marker gene elimination strategy was demonstrated to generate nptII-free Cry1Ac-expressing T2 events. These events were subsequently analyzed in the T3 generation for the segregation of cre and bar genes. Five Cry1Ac-expressing T3 transgenic pigeon pea events were devoid of the nptII marker as well as cre-bar genes. H. armigera larval mortality in those marker-free T3 events was found to be 80-100%. The development of such nptII selectable marker-free Cry1Ac-expressing pigeon pea transgenics for the first time would greatly support the sustainable biotechnological breeding program for pod borer resistance in pigeon pea. KEY POINTS: • Constitutive expression of Cry1Ac conferred complete resistance against Helicoverpa armigera • Green-tissue-specific expression of Cry1Ac conferred partial pest resistance • Cre/lox-mediated nptII elimination was successful in constitutively expressing Cry1Ac transgenic pigeon pea events.

Recovery Characteristics of Cry1Ac Endotoxin Expression and Related Physiological Mechanisms in Bt Transgenic Cotton Squares after High-Temperature Stress Termination

High-temperature stress reduces the endotoxin expression of the insecticidal gene (Cry1Ac) in transgenic Bacillus thuringiensis (Bt) cotton and affects nitrogen metabolism. However, its effects on Cry1Ac endotoxin expression after high-temperature stress termination remain unclear. In order to investigate the effect of high-temperature stress on the expression of insecticidal proteins in Bt cotton squares, the conventional Sikang-1 cultivar and hybrid Sikang-3 cultivar were used as the experimental materials. The potted cotton plants in the squaring stage were moved to an artificial climatic chamber at 38 °C for 72 h and 96 h in 2017 and 2018, respectively, and plants were moved to the climate chamber where the control cotton plants were located (at 27 °C). Then, cotton squares were collected to measure the Bt protein concentration and nitrogen metabolism physiology at 0, 12, 24, 48, 72 and 96 h after high-temperature stress termination, respectively. The Cry1Ac endotoxin expression of the squares could be recovered to the corresponding control level and a longer recovery time was required as the high-temperature stress period increased. Therefore, the recovery degree of Cry1Ac endotoxin expression of cotton squares can be predicted according to the duration of high-temperature stress, which may provide a reference for the rational control of Helicoverpa armigera and related pests in cotton production.

Rifampicin Increases Expression of Plant Codon-Optimized Bacillus thuringiensis δ-Endotoxin Genes in Escherichia coli.

Transgenic crops expressing Cry δ-endotoxins of Bacillus thuringiensis for insect resistance have been commercialized worldwide with increased crop productivity and spectacular socioeconomic gains. To attain the enhanced level of protein expression, the cry genes have to be extensively modified for RNA stability and translation efficiency in the plant systems. However, such modifications in nucleotide sequences make it difficult to express the cry genes in Escherichia coli because of the presence of E. coli rare codons. Induction of gene expression through the T7 promoter/lac operator system results in high levels of transcription but limits the availability of activated tRNA corresponding to rare codons that leads to translation stalling at ribosomes. In the present study, an Isopropyl ß-D-1-thiogalactopyranoside (IPTG)/rifampicin combination-based approach was adopted to induce transcription of cry genes through T7 promoter/lac operator while simultaneously inhibiting the transcription of host genes through rifampicin. The results show that the IPTG/rifampicin combination leads to high-level expression of four plant codon-optimized cry genes (cry2Aa, cry1F, cry1Ac, and cry1AcF). Northern blot analysis of the cry gene expressing E. coli samples showed that the RNA expression level in the IPTG-induced samples was higher as compared to that in the IPTG/rifampicin-induced samples. Diet overlay insect bioassay of IPTG/rifampicin-induced Cry toxins with Helicoverpa armigera larvae showed bioactivity (measured as LC50) similar to the previous studies. The experiment has proved that recombinant synthetic gene (plant codon-optimized gene) with the combination of Rifampicin which inhibits DNA-dependent bacterial RNA polymerase and reduces the excessive baggage of translational machinery of the bacterial cell triggers the production of synthetic protein. Purification of protein using high pH buffer increases the solubility of the protein. Further, LC50 analysis shows no reduction of protein activity leads to protein stability. Further, purified cry toxin protein can be used for crop protection against pests and a purified form of the synthetic protein can be used for antibody production and perform the immunoassay for the identification of the transgenic plant. The crystallographic structure of synthetic protein could be used for interaction study with another insect to see insecticidal activity.

Expression profiling of transgenes (Cry1Ac and Cry2A) in cotton genotypes under different genetic backgrounds

Transgenic cotton carrying the Cry1Ac gene has revolutionized insect pest control since its adoption, although the development of resistance in insect pests has reduced its efficacy. After 10 years of cultivating Bacillus thuringiensis (Bt) cotton with a single Cry1Ac gene, growers are on the verge of adopting Bt cotton that carries the double gene (Cry1Ac+Cry2A) due to its better effectiveness against insect pests. Thus, the current study was designed to evaluate the role of each gene in the effectiveness of Bt cotton carrying the double gene. The expression levels of the Cry1Ac and Cry2A genes were evaluated in the leaves of 10 genotypes (2 parents and 8 F1 hybrids) at 30 days after sowing (DAS), while samples of leaves, bolls and flowers were taken from the upper and lower canopies at 70 and 110 DAS. The F1 hybrids were developed through reciprocal crosses between two Bt (CKC-1, CKC-2) and two non-Bt (MNH-786, FH-942) parents. The differential expression of transgenes was evaluated through Enzyme Linked Immuno-Sorbent Assay (ELISA). The results showed that the MNH786×CKC-1 hybrid had the highest concentrations of Cry1Ac gene at 30 DAS (3.08 μg g−1) and 110 DAS (1.01 μg g−1) in leaves. In contrast, the CKC-2×MNH-786 hybrid showed the lowest concentrations of Cry1Ac gene at 30 DAS (2.30 μg g−1) and 110 DAS (0.86 μg g−1). The F1 hybrid FH-942×CKC-2 showed the highest concentrations of Cry2A gene at 30 DAS (8.39 μg g−1) and 110 DAS (7.74 μg g−1) in leaves, while the CKC-1×MNH-786 hybrid expressed the lowest concentrations of Cry2A gene at 30 DAS (7.10 μg g−1) and 110 DAS (8.31 μg g−1). A comparison between the two stages of plant growth showed that leaves had the highest concentrations at 30 DAS, whereas the lowest concentrations were observed at 110 DAS for both genes in leaves. When the expression pattern was compared between various plant parts in genotype CKC-2, it was found that leaves had higher concentrations of Cry1Ac (3.12 μg g−1) and Cry2A (8.31 μg g−1) at 70 DAS, followed by bolls (Cry1Ac (1.66 μg g−1) and Cry2A (8.15 μg g−1)) and flowers (Cry1Ac (1.07 μg g−1) and Cry2A (7.99 μg g−1)). The genotype CKC-2 had higher concentrations of Cry1Ac (3.12 μg g−1) and Cry2A (8.31 μg g−1) in the upper canopy but less accumulation (2.66 μg g−1 of Cry1Ac, 8.09 μg g−1 of Cry2A) in the lower canopy at 70 DAS. Similarly, at 110 DAS, the expression levels of Cry1Ac and Cry2A in upper and lower canopy leaves were 1.52 and 7.92 μg g−1, and 0.99 and 7.54 μg g−1, respectively. Hence, the current study demonstrates that different genotypes showed variable expression for both of the Cry1Ac and Cry2A genes during plant growth due to different genetic backgrounds. The Cry2A gene had three-fold higher expression than Cry1Ac with significant differences in expression in different plant parts. The findings of this study will be helpful for breeding insect-resistant double-gene genotypes with better gene expression levels of Cry1Ac and Cry2A for sustainable cotton production worldwide.

Complete genome sequence of Bacillus thuringiensis BR145, a strain with insecticidal activity against Lepidoptera pests.

Bacillus thuringiensis BR145 isolated from a soybean field in Southern Brazil showed toxicity against two important insect pests from soybean crop, Helicoverpa armigera, and Chrysodeixis includens, with LC50 0.294 µg.cm-2 and 0.277 µg.cm-2, respectively. We analyzed the genome of this strain through sequences obtained by Next Generation DNA Sequencing and de novo assembly. The analysis of the genome revealed insecticidal genes cry1Aa, cry1Ab, cry1Ac, cry1Ia, cry2Ab, cyt1, and vip3Aa, suggesting the use of this strain in new strategies of biological control.

Enhancing the resilience of transgenic cotton for insect resistance.

The efficacy of Bt crystal proteins has been compromised due to their extensive utilization in the field. The second-generation Bt vegetative insecticidal proteins could be the best-suited alternative to combat resistance build-up due to their broad range affinity with midgut receptors of insects. The codon-optimized synthetic vegetative insecticidal proteins (Vip3Aa) gene under the control of CaMV35S promoter was transformed into a locally developed transgenic cotton variety (CKC-01) expressing cry1Ac and cry2A genes. Transformation efficiency of 1.63% was recorded. The highest Vip3Aa expression (51.98-fold) was found in MS3 transgenic cotton plant. Maximum Vip3Aa protein concentration (4.23µg/mL) was calculated in transgenic cotton plant MS3 through ELISA. The transgenic cotton plant (MS3) showed one copy number on both chromatids in the homozygous form at chromosome 8 at the telophase stage. Almost 99% mortality of H. armigera was recorded in transgenic cotton plants expressing double crystal proteins pyramided with Vip3Aa gene as contrasted to transgenic cotton plant expressing only double crystal protein with 70% mortality. The results obtained during this study suggest that the combination of Bt cry1Ac, cry2A, and Vip3Aa toxins is the best possible alternative approach to combat chewing insects.

Addition of proteinase K during the culture alter the physiology of Bacillus thuringiensis culture and the cry1Ac, nprX, nprA, and spo0A gene transcription.

Bacillus thuringiensis is the major bioinsecticide worldwide produced due to the Cry protein activity. Several studies have been done to improve the cost-productivity relation. The neutral protease A (NprA) is the major extracellular protein massively produced during the stationary phase by this bacterium, contributing to the Cry proteins' degradation. Also, the deletion of aprA and nprA genes enhanced the yield of Cry protein, stabilizing it. Therefore, to increase Cry production, one possibility is to degrade the NprA protease in the culture media. In the present study, proteinase K was used to hydrolyze the NprA to increase Cry production. Proteinase K was added during the exponential growth of B. thuringiensis culture. The bacilli and endospores were measured along all culture, while the Cry protein was measured at the end of the culture. The addition of PK affects the bacilli and spore kinetics positively but negatively to the Cry protein (there is no Cry protein detection). Therefore, the gene expression of the cry1Ac, nprX, nprA, and spo0A was measured. The expression of each gene was followed along all culture. Results demonstrated that PK alters both the transcriptional levels and the expression order of the genes.

Development of marker-free insect resistant transgenic okra (Abelmoschus esculentus L. Moench) expressing the cry1Ac gene and identification of vector backbone-free events.

Agrobacterium-mediated co-transformation method was used to generate marker-free insect resistant transgenic okraplants expressing the cry1Ac gene. The cry1Ac gene was borne on the T-DNA of one plasmid while nptII and uidA (GUS) marker genes were present on the T-DNA of a second plasmid. Putative transgenic plants were screened by histochemical GUS assay for expression of -glucuronidase and 32 transgenic events were positive for GUS in which 21 transgenic events were positive in ELISA for the presence of Cry1Ac protein. Out of 21 Cry1Ac positive T0 events, three events displayed Mendelian inheritance of the transgenes in (9:3:3:1 ratio) T1 generation for Cry1Ac and GUS. Selected events were chosen for further genetic and molecular analysis. The cry1Ac and marker genes were found to segregate independently, of each other in 10 events in T1 generation out of 11 Cry1Ac gene inheriting events analysed indicating that the two T-DNAs insertions were genetically unlinked and identification of marker-free plants were possible in these 10 events. The marker-free nature and vector backbone-free Bt events (clean T-DNA insertions carrying cry1Ac gene) were confirmed by Southern analysis using suitable probes. The plants from selected transgenic events were rigorously screened in whole plant insect bioassays using the larvae of shoot and fruit borer, Earias vittella, an important pest of okra. Insect bioassays indicated 100% larval mortality without any infestation in five of the transgenic events and two events showed 5 to 10 percent infestation establishing the insect resistant nature of the transgenic plants. Finally the events inheriting transgenes in Mendelian fashion were characterized further and marker-free and vector backbone-free events were identified showing complete protection from the target pest Earias vittella in whole-plant insect bioassays. Quantification of Cry1Ac protein levels in the plant parts of selected events (lines) wasconsistent with the results of bioassays. Further, two lines identified in this study met the criteria for inclusion in commercial breeding programs.

Effect of GM maize on metabolism and performance of chicken

Effect of GM maize on metabolism and performance of chicken

Exogenous Gene Expression and Insect Resistance in Dual Bt Toxin Populus × euramericana 'Neva' Transgenic Plants.

Bacillus thuringiensis (Bt) insecticidal protein genes are important tools in efforts to develop insect resistance in poplar. In this study, the Cry1Ac and Cry3A Bt toxin genes were simultaneously transformed into the poplar variety Populus × euramericana ‘Neva’ by Agrobacterium-mediated transformation to explore the exogenous gene expression and insect resistance, and to examine the effects of Bt toxin on the growth and development of Anoplophora glabripennis larvae after feeding on the transgenic plant. Integration and expression of the transgenes were determined by molecular analyses and the insect resistance of transgenic lines was evaluated in feeding experiments. Sixteen transgenic dual Bt toxin genes Populus × euramericana ‘Neva’ lines were obtained. The dual Bt toxin genes were expressed at both the transcriptional and translational levels; however, Cry3A protein levels were much higher than those of Cry1Ac. Some of the transgenic lines exhibited high resistance to the first instar larvae of Hyphantria cunea and Micromelalopha troglodyta, and the first and second instar larvae and adults of Plagiodera versicolora. Six transgenic lines inhibited the growth and development of A. glabripennis larvae. The differences in the transcriptomes of A. glabripennis larvae fed transgenic lines or non-transgenic control by RNA-seq analyses were determined to reveal the mechanism by which Bt toxin regulates the growth and development of longicorn beetle larvae. The expression of genes related to Bt prototoxin activation, digestive enzymes, binding receptors, and detoxification and protective enzymes showed significant changes in A. glabripennis larvae fed Bt toxin, indicating that the larvae responded by regulating the expression of genes related to their growth and development. This study lay a theoretical foundation for developing resistance to A. glabripennis in poplar, and provide a foundation for exploring the mechanism of Bt toxin action on Cerambycidae insects.

The first GM crop in Bangladesh – Bt Eggplant

Eggplant (Solanum melongena) commonly called Brinjal is the most popular vegetable in Bangladesh. It is extensively cultivated around homesteads and in commercial fields throughout the year. It is estimated that nearly 67 percent of the eggplant crop yield in Bangladesh is lost due to the damage caused by Leucinodes orbonalis also known as eggplant fruit and shoot borer (EFSB). EFSB is reportedly the most serious pest of eggplant. To overcome this EFSB problem Bt eggplant project has been initiated in 2004. The Agricultural Biotechnology Support Project II (ABSPII) is a USAID-supported project that has funded the development of Bt eggplant in Bangladesh under the Public Private Partnership (PPP) mode. Bangladesh Agricultural Research Institute (BARI) received the Event EE-1 eggplant in Mahyco and backcrossed into nine popular eggplant varieties of Bangladesh selected based on the regional consumer preference. The efficacy of cry1Ac gene in providing effective resistance to the target pest was also assessed in terms of productively (fruit yield increase) which was studied in 9 Bt eggplant lines for a period of 7 years from 2008 to 2015. To launch the best 4 varieties, BARI applied to the National Technical Committee on Crop Biotechnology (NTCCB). For release of GM crop variety, first of all follow the recommendation of NTCCB, then submitted to the National Technical Committee on Crop Biotechnology (NTCCB) Core Committee and then to the National Committee on Biosafety (NCB). The Bangladesh government approved four varieties namely, BARI Bt Begun-1 (Uttara), BARI Bt Begun-2 (Kazla), BARI Bt Begun-3 (Nayantara) and BARI Bt Begun-4 (BARI Begun-6 or ISD006) on 30 October 2013 and these got momentum countrywide. On 22 January 2014, Bt seedlings were distributed among 20 farmers in four districts of Bangladesh as the first genetically modified (GM) crops. After the government of Bangladesh approved the cultivation of Bt eggplant in 2013, its popularity is increasing day by day and the socio-economic condition of the farmers is also improving. Bt eggplant started its journey in 2014 with only 20 farmers, in seven years it has grown to about 27012 farmers and from 8 acres to about 5,000 acres. This advancement of Bt eggplant has also faced opposition from different quarters at different times. Today, the progress of Bt eggplant continues by overcoming all obstacles. Field days, trainings, various publications, documentary making, use of mass and social media, observations, workshops, stewardship activities are acting as important regulators of this progress.

English

DNA fingerprinting is rapid, easy, and efficient method for discrimination, identification and characterization of various genotypes for protection of plant breeder’s rights (PBRs). Present study was designed for DNA fingerprinting and genetic diversity assessment of 25 GM cotton genotypes (possessing Cry1Ac gene) using 297 SSR markers through conventional PCR and Polyacrylamide gel electrophoresis. Out of 297 SSR markers, 25 markers were not amplified, 28 were monomorphic and 244 were polymorphic. A total of 1537 alleles were amplified among which 1294 (84.18%) were polymorphic. PIC value in our study ranged from 0.08 to 0.93 with an average of 0.73. Unique allelic pattern was observed for nineteen genotypes whereas six genotypes were identified using two-step identification methods. The UPGMA dendrogram divided the genotypes into two distinct clusters. Cluster I was comprised of 20 genotypes whereas cluster II was comprised of four genotypes. MNH-1020 did not obey any clustering and remained separated. The results of the structure analysis were complementary to cluster analysis and the population was divided into two subgroups. Our results evidenced narrow genetic base of the cotton genotypes cultivated in Punjab Pakistan due to use of common parents in the pedigree/parentage. Further, we proposed a core set of markers for future DNA fingerprinting and genetic diversity studies. The information generated in this study will be helpful in variety registration and subsequent protection under PBRs. Further our findings will be useful in selection of SSR markers for future studies which are focused on DNA fingerprinting and genetic diversity assessment. © 2021 Friends Science Publishers