cry1Ac Gene Research Articles

Final Health and Environmental Risk Assessment of Genetically Modified Soybean MON 87701

Soybean MON 87701 expresses the cry1Ac gene from Bacillus thuringiensis. The encoded Cry1Ac protein confers resistance against specific lepidopteran pests. Updated bioinformatics analyses of the inserted DNA and flanking sequences in soybean MON 87701 have not indicated a potential production of harmful toxins and allergens or polypeptides caused by the genetic modification. Genomic stability of the functional insert and consistent expression of the cry1Ac gene, have been shown over several generations of soybean MON 87701. Data from several field trials performed in USA, Canada, Chile and Argentina during 2005-2006 show that soybean MON 87701 is compositionally, morphologically and agronomically equivalent to its conventional counterpart and other commercial soybean cultivars. Subchronic feeding studies with rats as well as nutritional assessment with broilers have not revealed relevant adverse effects of MON 87701. These studies indicate that MON 87701 is nutritionally equivalent to and as safe as conventional soybean cultivars. The Cry1Ac protein produced in soybean MON 87701 do not show sequence resemblance to known toxins or IgE-dependent allergens, nor has the whole GM plant been reported to cause changes in IgE-mediated allergic reactions in patients reactive to soybean or in non-ectopic control individuals. Soybean is not cultivated in Norway, and there are no cross-compatible wild or weedy relatives of soybean in Europe. Based on current knowledge and considering the intended uses, which exclude cultivation, the VKM GMO Panel concludes that soybean MON 87701 with the Cry1Ac protein:
 
 - Is compositionally, morphologically and agronomically equivalent to its conventional counterpart and other commercial soybean cultivars
 - Is unlikely to introduce a toxic or allergenic potential in food or feed compared to conventional soybean cultivars
 - Is nutritionally equivalent to and as safe as its conventional counterpart and other conventional soybean cultivars
 - Does not represent an environmental risk in Norway.

Frequency of resistance alleles to Cry1Ac toxin from cotton bollworm, Helicoverpa armigera (Hübner) collected from Bt-cotton growing areas of Telangana state of India

Transgenic cotton expressing Bacillus thuringiensis (Bt) cry1Ac and cry2Ab toxin genes is widely cultivated to manage bollworm complex in India. Cotton bollworm Helicoverpa armigera (Hübner) is one of the most serious of this complex. It is likely to evolve resistance to Cry toxins in view of continual selection pressure due to extensive cultivation of Bt cotton. Monitoring susceptibility of cotton bollworm using conventional bioassays is reported to have shown its increasing tolerance to Cry1Ac over the years. We report using an F2 screen Cry1Ac resistance allele frequencies of 0.050 (95% CI 0.022–0.076) and 0.056 (95% CI 0.035–0.075) in the insect populations collected from pigeon pea grown alongside Bt cotton in the respective years of 2016 and 2017 in the Telangana state of India. Compared to our earlier studies for 2013 and 2014, resistance allele frequency to Cry1Ac in the cotton bollworm in the following two years remains unchanged. The significance of these results is discussed in the context of non-Bt host crops acting as refuge for cotton bollworm for ensuring sustainable resistance management.

Growth and arthropod community characteristics of transgenic poplar 741 in an experimental forest

Use of the Bt insect-resistance gene has provided substantial economic and ecological benefits. Full utilization of transgenic poplars requires a comprehensive understanding of the growth-related phenotypic characteristics and ecological risks associated with of genetically modified trees. In the present study, we assessed the effects of transgenic poplar 741 with different insect-resistant genes on plant growth and arthropod communities in the field. Transgenic lines carrying the Cry1Ac and Cry3A genes were used as model lines, and non-transgenic poplar 741 was used as a control. Over a period of 6 years, we analyzed plant growth and transcriptomic data, and monitored arthropod richness and abundance in an experimental forest comparing transgenic and control lines. Our results indicate that the Pb29 line had a similar plant height to CK, but a significantly lower diameter at breast height (DBH) than CK. The plant height and DBH of the CC84 line were slightly higher than those of CK, but the differences were not significant. Transcriptome data analysis showed downregulated and upregulated expression of growth hormone-related genes and lignin synthesis-related genes, respectively, in the Pb29 line compared with CK and the CC84 line. The survey data obtained in this study showed that annual trends in arthropod numbers in the ecosystem were consistent among transgenic poplar 741 and control lines, gradually decreasing with time since planting. The Cry1Ac line has selective resistance to Lepidoptera and exerted strong toxic effects on Arctiidae, Tortricidae, and Notodontidae, but had no obvious effects on Lyonetiidae or Geometridae. The Cry3A line exhibited significant resistance to coleopterans only in 2016 when the abundance of coleopteran insects was high. Both lines exhibited inhibitory effects on non-target pests. The two transgenic lines were similar to the control with respect to arthropod community structure, and there were no significant differences in arthropod diversity indices between transgenic and control lines, although richness was slightly higher for the Cry3A line than for the control. The results of this study showed that the insertion of Bt genes had no apparent inhibitory effect on the growth of poplar 741. Also, there was no adverse environmental impact of transgenic poplar 741 on ecosystem arthropod communities compared with conventional poplar 741.

Development of event-specific detection method for identification of insect resistant NIBGE-1601 cotton harboring double gene Cry1Ac-Cry2Ab construct

Bt cotton expressing Cry1Ac is being cultivated in Pakistan. It has been observed that pink bollworm may have developed resistance against single Bt gene (Cry1Ac). For durable resistance, insect resistant NIBGE-1601 cotton harboring double gene Cry1Ac-Cry2Ab construct was developed. There was a need to characterize NIBGE-1601 event for intellectual property rights protection. The Presence of NIBGE Cry1Ac and NIBGE Cry2Ab genes was checked in NIBGE-1601 cotton plants through PCR, while there was no amplification using primers specific for Monsanto events (MON531, MON15985, MON1445). Using genome walking technology, NIBGE-601 event has been characterized. Event-specific primers of NIBGE-1601 were designed and evaluated to differentiate it from other cotton events mentioned above. NIBGE-1601 event detection primers are highly specific, therefore, can detect NIBGE 1601 event at different conditions using single or multiplex PCR. In the qualitative PCR, using NIBGE-1601 event specific primers, 0.05 ng was the limit of detection for NIBGE-1601double gene cotton genomic DNA. Thus event characterization and development of event-specific diagnostics will help in breeding new cotton varieties resistant to cotton bollworms.

Effects of transgenic oilseed rape harboring the Cry1Ac gene on microbial communities in the rhizosphere soil

Soil microorganisms play important roles in the rhizosphere in various biological activities in natural and agricultural ecosystems. We performed a two-year field experiment that featured the cultivation of three Bt transgenic oilseed rape (Brassica napus L.) plant lines to evaluate their effects on rhizosphere microorganisms using phospholipid fatty acid (PLFA) profiling. A total of 27 fatty acids were detected in the rhizosphere soil of transgenic oilseed rape during the two consecutive planting years. During plant growth stages in the first year, there were no significant changes in the microbial community structure and diversity in rhizosphere soil samples, except a lower total fungi content (4.09 μmol g−1) at the bolting stage. During the second year, the soil microbial community associated with transgenic plants varied significantly with developmental stage, such as total PLFAs (43.70 μmol·g−1), PLFAs of bacteria (26.00 μmol g−1) and fungi (11.84 μmol g−1). The Shannon's diversity, richness, and evenness of the soil microbial community increased significantly with transgenic GT1 and GT5 lines during the flowering stage. PCA analysis of the PLFA profile explain 87.41% variation of PLFAs in 2013 and 86.96% in 2014, showing that the different transgenic lines might have a minor impact on soil microbial structure. This study demonstrated that field cultivation of transgenic oilseed rape may cause changes in soil microbial structure and diversity for consecutive seasons and at different growth stages.

Transformation and evaluation of Broad-Spectrum insect and weedicide resistant genes in Gossypium arboreum (Desi Cotton)

ABSTRACT Gossypium arboreum (Desi Cotton) holds a special place in cotton industry because of its inherent ability to withstand drought, salinity, and remarkable resistance to sucking pests and cotton leaf curl virus. However, it suffers yield losses due to weeds and bollworm infestation. Genetic modification of G. arboreum variety FBD-1 was attempted in the current study to combat insect and weedicide resistance by incorporating cry1Ac, cry2A and cp4-EPSPS genes under control of 35S promoter in two different cassettes using kanamycin and GUS as markers through Agrobacterium-mediated shoot apex cut method of cotton transformation. The efficiency of transformation was found to be 1.57%. Amplification of 1700 bp for cry1Ac, 167 bp for cry2A and 111 bp for cp4-EPSPS confirmed the presence of transgenes in cotton plants. The maximum mRNA expression of cry1Ac and cp4-EPSPS was observed in transgenic cotton line L3 while minimum in transgenic cotton line L1. The maximum protein concentrations of Cry1Ac, Cry2A and Cp4-EPSPS of 3.534 µg g−1, 2.534 µg g−1 and 3.58 µg-g−1 respectively were observed for transgenic cotton line L3 as compared to control cotton line. On leaf-feed-based insect bioassay, almost 99% mortality was observed for Helicoverpa armigera on the transgenic cotton plant (L3). It completely survived the 1900 ml hectare−1 glyphosate spray assay as compared to non-transgenic cotton plants. The necrotic spots appeared on the third day, leading to the complete death of control plants on the fifth day of assay. The successful multiple gene-stacking in G. arboreum FBD-1 variety could be further used for qualitative improvement of cotton fiber through plant breeding techniques.

New entomopathogenic strain of Bacillus thuringiensis is able to solubilize different sources of inorganic phosphates

Phosphate-solubilizing strain INTA 293-1 from a soil sample of Corrientes province (Argentina) was identified as Bacillus thuringiensis based on its faculty to produce crystalline inclusions during sporulation and 16S rDNA sequence similarity. INTA 293-1 was found to produce bipyramidal and cuboidal crystals, containing major proteins of ca. 130 and 65 kDa respectively, resembling those present in Lepidoptera- and Diptera-active strains. Accordingly, INTA 293-1 showed insecticidal activity against lepidopteran and dipteran larvae. No insecticidal activity nor evident effects on the normal development of coleopteran larvae or nematodes were observed. Screening of cry, cyt and vip genes showed that INTA 293-1 contains cry1Ac, cry1Ia, cry2Aa, cry2Ab and vip3Aa genes. Furthermore, this new entomopathogenic strain of B. thuringiensis was able to solubilize different sources of inorganic phosphates in liquid media following similar solubilization kinetics. INTA 293-1 was effective in solubilizing hydroxyapatite, patagonic phosphate rock, and to a lesser extent the aluminium phosphate. The differential behaviour between them may be explained by differences in INTA 293-1 survival. Phosphate solubilization was associated with pH reduction. This study reveals a little-explored aspect of B. thuringiensis and paves the way for the development of new agrobiological products with multiple modes of action.

Bt cotton seed purity in Burkina Faso: status and lessons learnt

BackgroundSince the commercial release of Bt cotton in Burkina Faso in 2009, the issue of seed purity in producers’ fields has rarely been addressed in an unbiased and objective manner. The potential for contamination of conventional seed varieties with Bt traits and the consequent threat to the continuation of organic cotton production has been documented. However, studies are rare on the varietal purity of Bt cotton seeds, despite the implications for the effectiveness and sustainability of their use.This paper compensates for the lack of research on the varietal purity of cotton seeds in Burkina Faso by reporting the results of Enzyme linked immunosorbent assay tests collected in 2015 on samples of both conventional and Bt varieties from 646 fields.ResultsAccording to the conservative criteria used to declare the presence of a Bt gene in a given variety (more than 10% of seeds of conventional variety exhibit Bt traits, and at least 90% of seeds of Bt variety exhibit Bt traits), seed purity was very questionable for both types of variety. For the supposedly conventional variety, the Cry1Ac gene was observed in 63.6% of samples, the Cry2Ab gene was observed in 59.3% of samples, and both genes were detected in 52.2% of the seed samples. Only 29.3% of the seeds that were supposed to be of conventional type contained no Bt genes. Conversely, for the labeled Bt variety, the Cry1Ac gene was found in only 59.6% of samples, the Cry2Ab gene was found in 53.6% of the samples, and both genes were found in 40.4% of the samples. Finally, for the seeds that were supposed to contain both genes (Bollguard 2), both Cry1Ac and Cry2Ab genes were found in only 40.4% of the samples, only one of the genes was found in 32.4% of the samples, and 27.2% of the seeds in the samples contained neither.Two factors are responsible for the severe lack of seed purity. First, conventional varieties are being contaminated with Bt traits because of a failure to revise the seed production scheme in Burkina Faso to prevent cross-pollination. Second, the original Bt seeds provided to Burkina Faso lacked varietal purity.The organic sector plays a very minor role in the cotton sector of Burkina Faso (production of organic cotton totaled 453 t in 2018/2019, out of national cotton production of 183 000 t). Nevertheless, the lack of purity in conventional seed varieties is a threat to efforts to expand certified organic cotton production. The poor presence of Bt proteins in supposed Bt varieties undermines their effectiveness in controlling pests and increases the likelihood of the development of resistance among pest populations.ConclusionOur results show the extent of purity loss when inadequate attention is paid to the preservation of seed purity. Pure conventional seeds could vanish in Burkina Faso, while Bt seeds do not carry the combination of the expected Bt traits. Any country wishing to embark on the use of Bt cotton, or to resume its use, as in the case of Burkina Faso, must first adjust its national seed production scheme to ensure that procedures to preserve varietal purity are enforced. The preservation of varietal purity is necessary to enable the launch or the continuation of identity-cotton production. In addition, the preservation of varietal purity is necessary to ensure the sustainable effectiveness of Bt cotton. In order to ensure that procedures to preserve varietal purity are observed, seed purity must be tested regularly, and test results must be published.

Enhancing the insecticidal activity of new Bacillus thuringiensis X023 by copper ions

BackgroundA new Bacillus thuringiensis X023 (BtX023) with high insecticidal activity was isolated in Hunan Province, China. The addition of metals (Cu, Fe, Mg and Mn) to the medium could influence the formation of spores and/or insecticidal crystal proteins (ICPs). In previous studies, Cu ions considerably increased the synthesis of ICPs by enhancing the synthesis of poly-β-hydroxy butyrate. However, the present study could provide new insights into the function of Cu ions in ICPs.ResultsBioassay results showed that wild strain BtX023 exhibited high insecticidal activity against Plutella xylostella. The addition of 1 × 10−5 M Cu2+ could considerably increase the expression of cry1Ac and vip3Aa, and the insecticidal activity was enhanced. Quantitative real-time polymerase chain reaction (qRT-PCR) and proteomic analyses revealed that the upregulated proteins included amino acid synthesis, the glyoxylate pathway, oxidative phosphorylation, and poly-β-hydroxy butyrate synthesis. The Cu ions enhanced energy metabolism and primary amino acid synthesis, will providing abundant raw material accumulation for ICP synthesis.ConclusionThe new strain BtX023 exerted a strong insecticidal effect on P. xylostella by producing ICPs. The addition of 1 × 10−5 M Cu2+ in the medium could considerably enhance the expression of the cry1Ac and vip3Aa genes, thereby further increasing the toxicity of BtX023 to Helicoverpa armigera and P. xylostella by enhancing energy synthesis, the glyoxylate cycle, and branched-chain amino acids synthesis, but not poly-β-hydroxy butyrate synthesis.

Screening brinjal (Solanum melongena) accessions conserved in the National Genebank collected from states adjoining Bangladesh for adventitious presence of EE1 event

Systematic management of Plant Genetic Resources (PGRs) is the key to sustainable agriculture for food and nutritional security and in mitigating climate change. The National Genebank (NGB) at ICAR-National Bureau of Plant Genetic Resources (NBPGR), New Delhi, acts as a repository of PGRs for future use. This study aimed at screening for the adventitious presence of transgenes in brinjal (Solanum melongena L.) accessions conserved in the NGB. The study targeted the collections made during 2007-2016 from areas adjacent to Bangladesh (Assam, Meghalaya, Mizoram, Tripura and West Bengal), where field trials of Bt brinjal event EE1 were conducted during 2005-2012 and commercial cultivation of this event was permitted in 2013.There could be an apprehension of both unintentional introgression and transboundary movement through borders. Adventitious presence of transgenes was checked in a set of 96 accessions of brinjal employing Polymerase Chain Reaction (PCR) and real-time PCR assays. As event EE1 carries cry1Ac gene for insect resistance with Cauliflower Mosaic Virus (CaMV) promoter (P-35S) and marker genes (nptII and aadA), so these genetic elements were targeted for qualitative GM testing. Based on the test results, transgenes were not detected in brinjal accessions conserved in NGB. Our study showed that brinjal and wild species collected from adjoining areas of Bangladesh, post field trials and release, do not contain the event EE1. The study presents an efficient and reliable method to ensure conservation of GM-free germplasm in the NGB.

Improvement of sugarcane for borer resistance using Agrobacterium mediated transformation of cry1Ac gene

ABSTRACT The sugarcane (Saccharum X officinarum) is one of the most important crops used to produce sugar and raw material for biofuel in the world. One of the main causes for sucrose content and yield losses is the attack by insect. In this investigation, cry1Ac gene was introduced into sugarcane variety GT54-9(C9) using the Agrobacterium tumefaciens transformation method for transgenic sugarcane production presenting insect-resistance. The A. tumefaciens strain GV1303 including pARTcry1Ac vector was used for the production of transformed sugarcane. The Bacillus thuringiensis cry gene were successfully used to produce transgenic plants used for the improvement of both agronomic efficiency and product quality by acquiring insect resistance. PCR and Southern hybridization techniques were used to confirm the cry1Ac gene incorporation into sugarcane genome. Transformation percentage was 22.2% using PCR analysis with specific primers for cry1Ac and npt-II (Neomycin phosphotransferase) genes. The expression of cry1Ac gene was determined using reverse transcriptase polymerase chain reaction (RT-PCR), QuickStix test, and insect bioassays. Bioassays for transformed sugarcane plants showed high level of toxicity to Sesamia cretica giving 100% mortality of the larvae. Sugarcane insect resistance was improved significantly by using cry1Ac gene transformation.

Compositional analysis of transgenic Bt-chickpea resistant to Helicoverpa armigera

ABSTRACT Transgenic chickpeas expressing high levels of a truncated version of the cry1Ac (trcry1Ac) gene conferred complete protection to Helicoverpa armigera in the greenhouse. Homozygous progeny of two lines, Cry1Ac.1 and Cry1Ac.2, had similar growth pattern and other morphological characteristics, including seed yield, compared to the non-transgenic counterpart; therefore, seed compositional analysis was carried out. These selected homozygous chickpea lines were selfed for ten generations along with the non-transgenic parent under contained conditions. A comparative seed composition assessment, seed storage proteins profiling, and in vitro protein digestibility were performed to confirm that these lines do not have significant alterations in seed composition compared to the parent. Our analyses showed no significant difference in primary nutritional composition between transgenic and non-transgenic chickpeas. In addition, the seed storage protein profile also showed no variation between the transgenic chickpea lines. Seed protein digestibility assays using simulated gastric fluid revealed a similar rate of digestion of proteins from the transgenic trcry1Ac lines compared to the non-transgenic line. Thus, our data suggest no unintended changes in the seed composition of transgenic chickpea expressing a trcry1Ac gene.

Effect of natural high temperature and flooding conditions on cry1ac gene expression in different transgenic Bt cotton (Gossypium hirsutum L.) cultivars

Effect of natural high temperature and flooding conditions on cry1ac gene expression in different transgenic Bt cotton (Gossypium hirsutum L.) cultivars

The signal peptide of Cry1Ia can improve the expression of eGFP or mCherry in Escherichia coli and Bacillus thuringiensis and enhance the host\u2019s fluorescent intensity

BackgroundThe signal peptides (SPs) of secretory proteins are frequently used or modified to guide recombinant proteins outside the cytoplasm of prokaryotic cells. In the periplasmic space and extracellular environment, recombinant proteins are kept away from the intracellular proteases and often they can fold correctly and efficiently. Consequently, expression levels of the recombinant protein can be enhanced by the presence of a SP. However, little attention has been paid to the use of SPs with low translocation efficiency for recombinant protein production. In this paper, the function of the signal peptide of Bacillus thuringiensis (Bt) Cry1Ia toxin (Iasp), which is speculated to be a weak translocation signal, on regulation of protein expression was investigated using fluorescent proteins as reporters.ResultsWhen fused to the N-terminal of eGFP or mCherry, the Iasp can improve the expression of the fluorescent proteins and as a consequence enhance the fluorescent intensity of both Escherichia coli and Bt host cells. Real-time quantitative PCR analysis revealed the higher transcript levels of Iegfp over those of egfp gene in E. coli TG1 cells. By immunoblot analysis and confocal microscope observation, lower translocation efficiency of IeGFP was demonstrated. The novel fluorescent fusion protein IeGFP was then used to compare the relative strengths of cry1Ia (Pi) and cry1Ac (Pac) gene promoters in Bt strain, the latter promoter proving the stronger. The eGFP reporter, by contrast, cannot indicate unambiguously the regulation pattern of Pi at the same level of sensitivity. The fluorescent signals of E. coli and Bt cells expressing the Iasp fused mCherry (ImCherry) were also enhanced. Importantly, the Iasp can also enhanced the expression of two difficult-to-express proteins, matrix metalloprotease-13 (MMP13) and myostatin (growth differentiating factor-8, GDF8) in E. coli BL21-star (DE3) strain.ConclusionsWe identified the positive effects of a weak signal peptide, Iasp, on the expression of fluorescent proteins and other recombinant proteins in bacteria. The produced IeGFP and ImCherry can be used as novel fluorescent protein variants in prokaryotic cells. The results suggested the potential application of Iasp as a novel fusion tag for improving the recombinant protein expression.

Construct-Specific Loop-Mediated Isothermal Amplification: Rapid Detection of Genetically Modified Crops with Insect Resistance or Herbicide Tolerance.

Insect resistant and herbicide tolerant genetically modified (GM) events have been approved in many countries. Screening methods could facilitate preliminary testing to check the GM status, which may target control elements, transgenes, and marker genes or construct regions. Among these, methods targeting the construct region, i.e., the junction between two genetic elements of a transgenic cassette are more specific. Loop-mediated isothermal amplification (LAMP) assays targeting three construct regions were developed; between Cauliflower Mosaic Virus 35S promoter and cry1Ac gene (p35S-cry1Ac), cry2Ab2 gene and nos terminator (cry2Ab2-tnos), and cp4-epsps gene and nos terminator (cp4epsps-tnos). LAMP assays were performed by incubation at constant temperatures for selected targets. Positive amplification was detected as a change in color from orange to green on addition of SYBR® Green dye in visual LAMP and as real-time amplification curves in real-time LAMP. These assays showed acceptable specificity and sensitivity. Visual LAMP was found to be sensitive enough to detect as low as 0.005%, equivalent to two target copies. Real-time LAMP assays were able to detect as low as four copies of the target within 40 min, making them suitable for rapid on-site testing for GM organisms (GMO). Practical utility was also verified using spiked test samples. These assays could be employed to address some of the biosafety or post-release monitoring issues, as well as to check for approved and unapproved GM events in a country. LAMP assays targeting three construct regions have been developed, enabling screening for approved or unapproved GMO.

Monitoring Adventitious Presence of Transgenes in Cotton Collections from Genebank and Experimental Plots: Ensuring GM-Free Conservation and Cultivation of Genetic Resources

Bt cotton is the commercialized genetically modified (GM) crop in India with four approved GM events, namely MON531 (Bollgard® I), MON15985 (Bollgard® II), GFM and Event 1. Several GM events of cotton with insect-resistant or herbicide-tolerant GM traits were under field trials or imported for research purposes. With the increase in adoption of Bt cotton, unintentional introgression of transgenes in the conserved diversity-rich germplasm and in the field samples needs to be monitored in a systematic manner employing efficient GM diagnostics. Adventitious presence of transgenes was checked in 100 ex situ accessions of cotton being conserved in the National Genebank. These accessions belonging to Gossypium hirsutum, G. barbadense, G. arboreum, G. hirsutum × G. barbadense, represented seven Bt cotton growing states. Pentaplex PCR assay covering screening for over 90% of globally approved GM cotton events was employed, simultaneously targeting Cauliflower Mosaic Virus 35S promoter (P-35S), Agrobacterium tumefaciens nos terminator (T-nos), neomycin phosphotransferase (nptII) and phosphinothricin-N-acetyltransferase (pat) marker genes, and endogenous stearoyl acyl carrier protein desaturase (Sad1) gene as an internal control. Real-time PCR assays targeting P-35S and T-nos, with the limit of detection up to 0.01%, were employed for further confirmation. A study was also conducted on fifty cotton samples collected from the experimental plots, which were grown at distance of 50 and 10 metres from Bt cotton event MON15985. Adventitious presence of transgenes was checked using multiplex PCR assay targeting cry1Ac, cry2Ab2 and endogenous Sad1 gene to define a strategy for screening for specific GM events. Adventitious presence of transgenes was not detected in any of the tested samples based on the screening tests conducted. This strategy targeting specific GM traits would assist in GM-free cultivation of varieties in the experimental plots. The reported approach with wide GM coverage is reliable for checking adventitious presence of transgenes in genebanks, which could be applied to test conserved germplasm on a regular basis, for ensuring GM-free conservation of genetic resources.

Detection of mPing mobilization in transgenic rice plants.

Various kinds of transposable elements (TEs) constitute high proportions of eukaryotic genomes. Although most of these TEs are not actively mobile, genome stress can induce mobilization of dormant TEs. Transgenic plants undergo tissue culture and subsequent whole-plant regeneration, which can cause genomic stress and in turn induce mobilization of inactive TEs. To investigate the activation of transposable elements on the genome wide of the GM plant. Transposon activities were analyzed in three transgenic rice plants carrying the insect resistance gene Cry1Ac and an herbicide resistance gene by the transposon display technique. These three transgenic plants were derived from a leading Korean rice variety, Illmi. We detected seven mobile activities in the mPing element, which is a MITE family transposon. The identity of the novel fragments in the gel display was confirmed by checking TAA target site duplication via sequence analysis. The genomic integration sites were all on different chromosomes, and the integrations were specific to either one or two T1 transgenic lines, except for one common integration on chromosome 4. One integration was in the 5'-UTR of the Glycerol-3-phosphate acyltransferase 8 gene, two integrations were in introns of expressed genes, and the other four integrations were in intergenic regions. Thus, novel mobilization of dormant TEs occurs in transgenic plants, which must be considered in the generation of genetically modified crops (GM crops).

Enhanced expression of Arabidopsis rubisco small subunit gene promoter regulated Cry1Ac gene in chickpea conferred complete resistance to Helicoverpa armigera

A major pest of chickpea, Helicoverpa armigera, can be controlled by expressing genes from the bacterium Bacillus thuringiensis as an environmentally compatible option. Here we show that transgenic chickpeas containing a cry1Ac gene conferred a high degree of resistance to H. armigera. The Agrobacterium binary vector contained the nptII gene as the selectable marker and cry1Ac gene driven by the Arabidopsis rubisco small subunit gene (ats1A) promoter. We generated 54 and 47 independent transgenic lines using truncated (trcry1Ac) and full-length versions of the cry1Ac (flcry1Ac) gene, respectively. Of these lines, twelve transmitted the trcry1Ac transgene to the next generation at a 3:1 ratio, while only 8 flcry1Ac lines segregated in a 3:1 ratio. Five lines expressed trCry1Ac protein > 50 μg/g fresh weight, however, only one line accumulated about 30 μg/g flCry1Ac protein. Such high levels of trCry1Ac protein have not been reported before in chickpea. When trCry1Ac lines were challenged to whole plant bioassays in the greenhouse, lowest pod damage was observed in BS100B (1.4%) followed by BS81P (4.4%), and BS100E (6.2%) compared to the parental line (49.9%). The phenotypes of the lines expressing high levels of Cry1Ac protein were indistinguishable from their null segregants and controls. Thus, trCry1Ac lines could be suitable for crossing with our existing Cry2Aa lines for generation of a pyramided Bt chickpea for enhanced insect resistance management in the field.

The insect-protected CTC91087-6 sugarcane event expresses Cry1Ac protein preferentially in leaves and presents compositional equivalence to conventional sugarcane

ABSTRACTA Cry1Ac-expressing sugarcane cultivar, CTC91087-6, has been developed by Centro de Tecnologia Canavieira (CTC) to be resistant to the sugarcane borer (Diatraea saccharalis). This genetically modified event was developed using Agrobacterium-mediated transformation and the help of the selectable marker phosphinothricin N-acetyltransferase (PAT) expressed from bar gene. We describe here a detailed characterization of CTC91087-6 event with respect to protein expression, nutritional composition, and assessment of its derived DNA and proteins in raw sugar. Expression of the Cry1Ac and PAT (bar) proteins produced by CTC91087-6 was evaluated in different tissues and at different times during the growing season. The new proteins are preferentially expressed in leaves, are produced at low levels in stalks, and are near the limits of detection in root tissues. The levels of Cry1Ac were much higher than PAT in all evaluated tissues. Furthermore, Cry1Ac levels in CTC91087-6 leaves are stable at various times during sugarcane cultivation cycle, assuring borer control throughout the complete crop cycle. Assessment of CTC91087-6 tissues for key food and feed nutrients as recommended by OECD to assess the safety of new varieties of sugarcane showed compositional equivalence to the conventional counterpart CTC9001 and to other commercial sugarcane varieties used as references. Raw sugar samples produced from CTC91087-6 did not contain DNA corresponding to cry1Ac and bar genes nor DNA specifically derived from CTC91087-6. In a similar way, there is no detection of Cry1Ac and PAT proteins in raw sugar produced from CTC91087-6. Taken together these results show that CTC91087-6 stably expresses Cry1Ac and PAT proteins and is substantially equivalent to the conventional counterpart CTC9001.

Development and evaluation of double gene transgenic cotton lines expressing Cry toxins for protection against chewing insect pests

Cotton is the main fiber producing crop globally, with a significant impact on the economy of Pakistan. Bt cotton expressing a Cry1Ac gene is grown over a large area in Pakistan, however, there is a major concern that bollworms may develop resistance. Here we have used a durable resistance strategy against bollworms by developing a double gene construct containing Cry1Ac and Cry2Ab (pGA482-12R) for cotton transformation. Both Cry toxin genes have been cloned in the same T-DNA borders and transferred successfully into cotton via Agrobacterium-mediated transformation. Both genes are expressed in transgenic cotton plants and is likely to help breeders in developing new cotton cultivars by incorporating these genes in cotton lines having no Bt genes or expressing Cry1Ac gene (Mon 531). Positive transgenic cotton was identified by PCR using specific primers for the amplification of both Cry1Ac and Cry2Ab genes. Cry1Ac and Cry2Ab expression was confirmed with an immunostrip test and quantified using ELISA that showed significant spatio-temporal expression of Cry2Ab ranging from 3.28 to 7.72 µg/g of the tissue leaf. Insect bioassay with army worm (Spodoptera litura) was performed to check the efficacy of NIBGE (National Institute for Biotechnology and Genetic Engineering) double gene transgenic cotton plants and up to 93% insect mortality was observed.