Bt Gene Research Articles

Registration of ‘NuMex COT 15 GLS’ Glandless Cotton

The New Mexico Agricultural Experiment Station announces the release of ‘NuMex COT 15 GLS’ (Reg. CV‐132, PI 678371), a glandless cotton (Gossypium hirsutum L.) cultivar that carries the incomplete dominant glandless allele Gl2e. NuMex COT 15 GLS was derived from an advanced backcross progeny of a cross between the Gl2e allele donor ‘Bahtim 110’ (G. barbadense) and glanded Upland cotton ‘CRI 12’, followed by five backcrosses with glanded Upland ‘CRI 35’ as the recurrent parent. In the advanced backcross population, the conventional NuMex COT 15 GLS was selected as a glandless progeny out of a mixture of glandless and glanded plants, followed by further testing for adventitious presence of genetically engineered biotech traits controlled by insect‐resistant Bt gene(s) and herbicide‐tolerant gene(s). This new cultivar was tested in four replicated field trials in New Mexico in 2013 to 2014 and in 11 tests across nine states in 2014. As compared to glandless ‘Acala GLS’, NuMex COT 15 GLS yielded 50% more lint, and it produced 90% of that of glanded ‘Acala 1517‐08’. Its fiber quality is classified as a medium to long staple with strong fiber strength and high uniformity, similar to other commercial medium staple cultivars, but it was inferior to both Acala cultivars. It had higher fiber elongation than most commercial cultivars tested in New Mexico. NuMex COT 15 GLS responded to thrips (Thrips or Frankliniella spp.) and Verticillium wilt (caused by Verticillium dahliae Kleb.) similarly to Acala 1517‐08 and Acala GLS. The release of NuMex GLS represents the first glandless Upland cotton cultivar carrying the introgressed incomplete dominant glandless allele Gl2e from G. barbadense with acceptable yield and fiber quality traits in the United States.

Genetic transformation and evaluation of two sweet sorghum genotypes for resistance to spotted stemborer, Chilo partellus (Swinhoe)

Sweet sorghum is a climate smart crop with multiple uses. The crop is susceptible to attack by the spotted stemborer, Chilo partellus (Swinhoe). This causes deadheart formation, leading to lodging of plants and consequent high economic losses. Lack of stable sources of resistance make any genetic enhancement through breeding difficult. We report a study to build up host plant resistance using transgenic technology by introducing two different classes of Bt genes (cry1Aa and cry1B) into two elite sweet sorghum genotypes of India (SSV84 and RSSV9). We devised tissue culture methods to suit the genotypes of our interest, SSV84 and RSSV9, and employed two methods of genetic transformation: the particle bombardment and in planta method of Agrobacterium. Modification of in vitro culture methods involved subculture every 3 days in the initial stages of culture and the use of precultured embryos as target tissues. For the in planta method, a floral dip for 1 h in Agrobacterium suspension supplemented with l-cysteine and Tween-20 was used. Sixteen transgenic events were generated; inheritance, integration and stable expression of the transgenes till the T4 generation were confirmed. The amount of Bt Cry1Aa protein at 25–30 days of growth ranged from 24.8 to 72.8 ng/g of fresh leaf tissue. We recorded 78.4 % larval mortality, reduced leaf damage (3.0 out of 9.0) and reduced feeding (41.0 %) over the controls in insect feed assays. Stable inheritance and expression in the in planta-derived transgenics are presented.

A Method to Determine Lethal and Sublethal Concentrations of Recombinant Bioinsecticides Derived from Bacillus thuringiensis Against Larvae of Spodoptera litura

The increased awareness of environmental pollution have led to the growing interests of the application of biological agents to control insects such as lepidopteran family .Thus , this study aimed to produce genetically improved bioinsecticide agents to be used to control Spodoptera litura which would be useful in reducing environmental pollution ,preventing resistance to bioinsecticides and for safe food production .Four Bacillus thuringensis strains were used as recipients ' while ' Seratia marcescens was used as a doner strain in the conjugation to induce new recombinants in Bacillus thuringiensis over expressed chitinase.Four matings were conducted in this study between Serratia marcescence and the four Bacillus thuringiensis strains which having opposite genetic markers.Two efficient transconjugants based on chitin hydrolysis on agar medium were selected from each conjugation to be evaluated for toxicites against the larvae of cotton leafworm in relation to their parents .The efficient LC50 was appeared by Tr4 ( 1.93 x105 ppm ) followed by Tr6 and Tr7 ( 2x105 ppm). However , more than 995% toxicity index was recorded by Tr4,Tr6 and Tr7. Higher toxicity index observed by these transconjugants may be the related to the overexpression of chitinase genes.Therefore , the lethal concentration values obtained was necessary to kill 50% of larval population from Spodoptera litura in a certain period .The virulence of Bt strains showed that a lower concentration from Tr4 was required to kill 100% of larval population .It must be pointed out that higher toxicity index obtained by transconjugants reflected the interaction between crystal genes from Bt and chitinase genes from Seratia marcescens which enhanced the possibility of larval mortality as a genetic helpful tool in integrated pest management in cotton fields.

Evaluation of Foliar Damage bySpodoptera frugiperda(Lepidoptera: Noctuidae) to Genetically Modified Corn (Poales: Poaceae) in Mexico

Abstract The fall armyworm, Spodoptera frugiperda Smith & Abbot (Lepidoptera: Noctuidae), is a key pest of corn, Zea mays L. (Poales: Poaceae), in Mexico. The development of genetically modified (GM) corn hybrids for resistance to this insect, with the inclusion of several genes coding for proteins Cry1Ab, Vip3Aa20, and mCry3A of Bacillus thuringiensis Berliner (Bacillales: Bacillaceae) (Bt), offer an alternative to conventional insecticides to control this pest. Resistance to fall armyworms of the GM corn hybrids Agrisure 3000 GT, Agrisure Viptera 3110, and Agrisure Viptera 3111 was evaluated in 4 locations at Sinaloa for a 3 yr period. Damage evaluation showed that the maize hybrids with the Bt gene insertion were not affected by the fall armyworm as compared with their respective isolines, which were seriously damaged. The results reaffirm the insect control benefits provided by this technology and provide a baseline for resistance management.

The impact ofBacillus thuringiensis technology on the occurrence of fumonisins and other mycotoxins in maize

In many developing countries, maize is both a staple food crop and a widely-used animal feed. However, adventitious colonisation or damage caused by insect pests allows fungi to penetrate the vegetative parts of the plant and the kernels, the latter resulting in mycotoxin contamination. Maize seeds contaminated with fumonisins and other mycotoxins pose a serious threat to both humans and livestock. However, numerous studies have reported a significant reduction in pest damage, disease symptoms and fumonisin levels in maize hybrids expressing theBacillus thuringiensis (Bt) genecry1Ab, particularly in areas where the European corn borer is prevalent. When other pests are also present, thecry1Ab gene alone offers insufficient protection, and combinations of insecticidal genes are required to reduce damage to plants caused by insects. The combination of Cry1Ab protein with other Cry proteins (such as Cry1F) or Vip proteins has reduced the incidence of pests and, indirectly, mycotoxin levels. Maize hybrids expressing multiple Bt genes, such as SmartStax®, are less susceptible to damage by insects, but mycotoxin levels are not routinely and consistently compared in these crops. Bt maize has a greater economic impact onFusarium toxins than aflatoxins. The main factors that determine the effectiveness of Bt hybrids are the type of pest and the environmental conditions, but the different fungal infection pathways must also be considered. An alternative strategy to reduce mycotoxin levels in crops is the development of transgenic plants expressing genes that protect against fungal infection or reduce mycotoxin levels byin situ detoxification. In this review article, we summarise what is known about the relationship between the cultivation of Bt maize hybrids and contamination levels with different types of mycotoxins.

Members of BTB Gene Family of Scaffold Proteins Suppress Nitrate Uptake and Nitrogen Use Efficiency.

Development of crops with improved nitrogen use efficiency (NUE) is essential for sustainable agriculture. However, achieving this goal has proven difficult since NUE is a complex trait encompassing physiological and developmental processes. We thought to tackle this problem by taking a systems biology approach to identify candidate target genes. First, we used a supervised machine-learning algorithm to predict a NUE gene network in Arabidopsis (Arabidopsis thaliana). Second, we identified BT2, a member of the Bric-a-Brac/Tramtrack/Broad gene family, as the most central and connected gene in the NUE network. Third, we experimentally tested BT2 for a role in NUE. We found NUE decreased in plants overexpressing BT2 gene compared to wild-type plants under limiting nitrate conditions. In addition, NUE increased compared to wild-type plants under low nitrate conditions in double mutant plants in bt2 and its closely related homolog bt1, indicating a functional redundancy of BT1 and BT2 for NUE. Expression of the nitrate transporter genes NRT2.1 and NRT2.4 increased in the bt1/bt2 double mutant compared to wild-type plants, with a concomitant 65% increase in nitrate uptake under low nitrate conditions. Similar to Arabidopsis, we found that mutation of the BT1/BT2 ortholog gene in rice (Oryza sativa) OsBT increased NUE by 20% compared to wild-type rice plants under low nitrogen conditions. These results indicate BT gene family members act as conserved negative regulators of nitrate uptake genes and NUE in plants and highlight them as prime targets for future strategies to improve NUE in crops.

Transgenic Bt (Cry1Ab/Ac) rice lines with different genetic backgrounds exhibit superior field performance under pesticide-free environment

Transgenic rice with Cry Bacillus thuringiensis (Bt) genes have been developed in the last few decades. The performance of transgenic rice, especially the yield and resistance, must be meticulously evaluated prior to commercial production. In this study, yield and resistance performance of newly bred transgenic Bt (Cry1Ab/Ac) rice lines with different genetic backgrounds were first investigated under pesticide-free field environment. Transgenic Bt (Cry1Ab/Ac) rice lines showed high insect resistances and superior agronomic traits, resulting in obvious yield advantages ranging from 9.1% to 38.0% over their respective non-Bt control lines. Bt protein content (BTC) was similar among the Bt (Cry1Ab/Ac) rice lines in different years, while it was much higher in leaves than in stems of the Bt (Cry1Ab/Ac) rice lines. Different genetic backgrounds of Bt (Cry1Ab/Ac) rice lines affected the yield advantage but had no influence on the resistance and BTC. The results suggested that Bt (Cry1Ab/Ac) rice lines have gained obvious yield advantages and high resistances regardless of genetic background under pesticide-free environment.

Transgenic rice expressing the cry2AX1 gene confers resistance to multiple lepidopteran pests.

A chimeric Bacillus thuringiensis toxin (Bt) gene, cry2AX1was cloned in a bi-selectable marker free binary vector construct. The cry2AX1 gene, driven by the Chrysanthemum rbcS1 promoter, was introduced into JK1044R, the restorer line (Oryza sativa L. ssp. Indica) of a notified commercially grown rice hybrid in India, by Agrobacterium-mediated transformation. Its effect against two major lepidopteran insect pests viz., yellow stem borer (YSB) Scirpophaga incertulas, rice leaf folder (RLF) Cnaphalocrocis medinalis and one minor insect pest, oriental army worm (OAW) Mythimna separata was demonstrated through bioassays of transgenic rice plants under laboratory and greenhouse conditions. The rbcS1 promoter with chloroplast signal peptide was used to avoid Cry2AX1 protein expression in rice seed endosperm tissue. A total of 37 independent transformants were generated, of which after preliminary molecular characterization and YSB bioassay screening, five events were selected for their protein expression and bioefficacy against all three rice insect. One elite transgenic rice line, BtE15, was identified with Cry2AX1 expression ranging from 0.68 to 1.34µgg(-1) leaf fresh weight and with 80-92% levels of resistance against rice pests at the vegetative and reproductive stages. Increase in Cry2AX1 protein concentration was also observed with crop maturity. The Cry2AX1protein concentration in the de-husked seeds was negligible (as low as 2.7-3.6ngg(-1)). These results indicate the potential application of cry2AX1 gene in rice for protection against YSB, RLF and OAW.

Risk assessment of Bt crops on the non-target plant-associated insects and soil organisms.

Transgenic plants containing Bacillus thuringiensis (Bt) genes are being cultivated worldwide to express toxic insecticidal proteins. However, the commercial utilisation of Bt crops greatly highlights biosafety issues worldwide. Therefore, assessing the risks caused by genetically modified crops prior to their commercial cultivation is a critical issue to be addressed. In agricultural biotechnology, the goal of safety assessment is not just to identify the safety of a genetically modified (GM) plant, rather to demonstrate its impact on the ecosystem. Various experimental studies have been made worldwide during the last 20 years to investigate the risks and fears associated with non-target organisms (NTOs). The NTOs include beneficial insects, natural pest controllers, rhizobacteria, growth promoting microbes, pollinators, soil dwellers, aquatic and terrestrial vertebrates, mammals and humans. To highlight all the possible risks associated with different GM events, information has been gathered from a total of 76 articles, regarding non-target plant and soil inhabiting organisms, and summarised in the form of the current review article. No significant harmful impact has been reported in any case study related to approved GM events, although critical risk assessments are still needed before commercialisation of these crops. © 2016 Society of Chemical Industry.

Towards better insect management strategy: restriction of insecticidal gene expression to biting sites in transgenic cotton

Most of the commercialized Bt crops express cry genes under 35S promoter that induces strong gene expression in all plant parts. However, targeted foreign gene expression in plants is esteemed more important as public may be likely to accept ‘less intrusive’ expression of transgene. We developed plant expression constructs harboring cry1Ac gene under control of wound-inducible promoter (AoPR1) to confine Bt gene expression in insect wounding parts of the plants in comparison with cry1Ac gene under the control of 35S promoter. The constructs were used to transform four Turkish cotton cultivars (GSN-12, STN-468, Ozbek-100 and Ayhan-107) through Agrobacterium tumefaciens strains GV2260 containing binary vectors p35SAcBAR.101 and AoPR1AcBAR.101 harboring cry1Ac gene under control of 35S and AoPR1, respectively. Phosphinothricin (PPT) was used at concentration of 5 mg L−1 for selection of primary transformants. The primary transformants were analyzed for transgene presence and expression standard molecular techniques. The transformants exhibited appreciable mortality rates against larvae of Spodoptera exigua and S. littoralis. It was found that mechanical wounding of T 1 transgenic plants was effective in inducing expression of cry1Ac protein as accumulated levels of cry1Ac protein increased during post-wounding period. We conclude that use of wound-inducible promoter to drive insecticidal gene(s) can be regarded as a valuable insect-resistant management strategy since the promoter activity is limited to insect biting sites of plant. There is no Bt toxin accumulation in unwounded plant organs, seed and crop residues, cotton products and by-products, thus minimizing food and environmental concerns.

Frequency of Bacillus thuringiensis Cry1A.105 resistance alleles in field populations of the fall armyworm, Spodoptera frugiperda, in Louisiana and Florida

Fall armyworm, Spodoptera frugiperda (J.E. Smith), is a major pest of many crops and a cross-crop target of transgenic maize, cotton, and soybean containing Bacillus thuringiensis (Bt) genes. Some of the current Bt maize products for controlling lepidopteran species contain the Bt event MON 89034. The objective of this study was to determine the frequency of resistance alleles in field populations of S. frugiperda collected from Louisiana and Florida, U.S. to Cry1A.105, one of the two Bt genes in MON 89034. A total of 150 F2 two-parent families of S. frugiperda were established using single-pair mating of field-collected individuals in 2011, which included 79 families from two locations in Louisiana and 71 families from one location in Florida. F2 screen was conducted to detect resistance alleles in these families to Cry1A.105 protein in maize plants. Four out of the 79 Louisiana and 14 out of the 71 Florida families were identified to possess resistance alleles to the Cry1A.105 maize plants. Thus, the corresponding frequency of resistance alleles to Cry1A.105 maize was estimated to be 0.0158 with a 95% credibility interval (CI) of 0.0052–0.0323 for the Louisiana populations and 0.0559 with a 95% CI of 0.0319–0.0868 for the Florida populations. The resistant families survived on whole Cry1A.105 maize plants and demonstrated a significant level (>116-fold) of resistance to the Cry1A.105 protein in a diet-incorporated bioassay. These findings suggest that resistance allele frequency in S. frugiperda to single-gene Cry1A.105 maize in the U.S. southeast region apparently is not rare, most likely due to the selection of Cry1F resistance and its cross-resistance to Cry1A.105.

Evaluation of two cotton varieties CRSP1 and CRSP2 for genetic transformation efficiency, expression of transgenes Cry1Ac + Cry2A, GT gene and insect mortality

Expression of the transgene with a desirable character in crop plant is the ultimate goal of transgenic research. Transformation of two Bt genes namely Cry1Ac and Cry2A cloned as separate cassette under 35S promoter in pKHG4 plant expression vector was done by using shoot apex cut method of Agrobacterium. Molecular confirmation of putative transgenic cotton plants for Cry1Ac, Cry2A and GT gene was done through PCR and ELISA. Transformation efficiency of CRSP-1 and CRSP-2 was calculated to be 1.2 and 0.8% for Cry1Ac while 0.9 and 0.6% for Cry2A and 1.5 and 0.7% for GTG respectively. CRSP-1 was found to adopt natural environment (acclimatized) earlier than CRSP-2 when exposed to sunlight for one month. Expression of Cry1Ac, Cry2A and GTG was found to be 1.2, 1 and 1.3ng/μl respectively for CRSP-1 as compared to CRSP-2 where expression was recorded to be 0.9, 0.5 and 0.9ng/μl respectively. FISH analysis of the transgenic CRSP-1 and CRSP-2 demonstrated the presence of one and two copy numbers respectively. Similarly, the response of CRSP-1 against Glyphosate @1900ml/acre was far better with almost negligible necrotic spot and efficient growth after spray as compared to CRSP-2 where some plants were found to have necrosis and negative control where the complete decay of plant was observed after seven days of spray assay. Similarly, almost 100% mortality of 2nd instar larvae of Heliothis armigera was recorded after three days in CRSP-1 as compared CRSP-2 where insect mortality was found to be less than 90%. Quantitatively speaking non transgenic plants were found with 23–90% leaf damage by insect, while CRSP-1 was with less than 5% and CRSP-2 with 17%. Taken together CRSP1 was found to have better insect control and weedicide resistance along with its natural ability of genetic modification and can be employed by the valuable farmers for better insect control and simultaneously for better production.

Pyramids of QTLs enhance host-plant resistance and Bt-mediated resistance to leaf-chewing insects in soybean.

Key messageQTL-M and QTL-E enhance soybean resistance to insects. Pyramiding these QTLs withcry1Ac increases protection against Bt-tolerant pests, presenting an opportunity to effectively deploy Bt with host–plant resistance genes.Plant resistance to leaf-chewing insects minimizes the need for insecticide applications, reducing crop production costs and pesticide concerns. In soybean [Glycine max (L.) Merr.], resistance to a broad range of leaf-chewing insects is found in PI 229358 and PI 227687. PI 229358’s resistance is conferred by three quantitative trait loci (QTLs): M, G, and H. PI 227687’s resistance is conferred by QTL-E. The letters indicate the soybean Linkage groups (LGs) on which the QTLs are located. This study aimed to determine if pyramiding PI 229358 and PI 227687 QTLs would enhance soybean resistance to leaf-chewing insects, and if pyramiding these QTLs with Bt (cry1Ac) enhances resistance against Bt-tolerant pests. The near-isogenic lines (NILs): BenningME, BenningMGHE, and BenningME+cry1Ac were developed. BenningME and BenningMGHE were evaluated in detached-leaf and greenhouse assays with soybean looper [SBL, Chrysodeixis includens (Walker)], corn earworm [CEW, Helicoverpa zea (Boddie)], fall armyworm [FAW, Spodoptera frugiperda (J.E. Smith)], and velvetbean caterpillar [VBC, Anticarsia gemmatalis (Hübner)]; and in field-cage assays with SBL. BenningME+cry1Ac was tested in detached-leaf assays against SBL, VBC, and Southern armyworm [SAW, Spodoptera eridania (Cramer)]. In the detached-leaf assay, BenningME showed the strongest antibiosis against CEW, FAW, and VBC. In field-cage conditions, BenningME and BenningMGHE suffered 61 % less defoliation than Benning. BenningME+cry1Ac was more resistant than BenningME and Benningcry1Ac against SBL and SAW. Agriculturally relevant levels of resistance in soybean can be achieved with just two loci, QTL-M and QTL-E. ME+cry1Ac could present an opportunity to protect the durability of Bt genes in elite soybean cultivars. These results should assist the development of effective pest management strategies, and sustainable deployment of Bt genes in soybean.

Effect of dual Bt-expression transformation vectors on transgene expression in tobacco.

This study aimed to determine the influence of vector structure on dual Bt gene expression and establish an efficient expression vector using Cry1Ac and Cry3A genes. Four vectors (N4, N5, N10, and S23) were developed and used for genetic transformation of tobacco to obtain insect-resistant transgenic lines. The vectors were constructed using the MAR structure, applying different promoter and enhancer sequences, and changing the transgene open-reading frame sequence. The average Cry1Ac toxalbumin expression quantity was 67 times higher in N5 than in N4 transgenic lines (8.77 and 0.13 μg/g, respectively). In contrast, the average Cry3A toxalbumin expression quantity was 1.5 times higher in N4 than in N5 lines (12.70 and 8.21 μg/g, respectively). The sequences of both Bt genes significantly influenced toxalbumin expression, although upstream Bt genes presented lower expression levels. The average Cry1Ac toxalbumin content was 13 times higher in the transgenic lines of AtADH 5'-non-translated sequence N5 (8.77 mg/g) than in the omega N10 lines (0.67 mg/g). Furthermore, the average Cry1Ac toxalbumin content was 5 times higher in MAR N5 than in non-MAR S23 lines (8.77 and 1.63 mg/g, respectively). The average Cry3A toxalbumin content was 1.3 times higher in N5 than in S23 lines (8.21 and 6.48 mg/g, respectively). Moreover, toxalbumin expression levels differed significantly among the S23-transformed lines. The MAR structure applied on both ends of the genes increased both the level and stability of exogenous gene expression. In conclusion, N5 was the most optimal of the four tested vectors.

Genetic transformation and expression of transgenic lines of Populus x euramericana with insect-resistance and salt-tolerance genes.

We characterized new transgenic varieties of poplar with multiple insect-resistant and salt stress tolerant genes. Two insect-resistant Bacillus thuringiensis (Bt) genes, Cry1Ac and Cry3A, and a salt-tolerant gene, Betaine aldehyde dehydrogenase (BADH) were inserted into a vector, p209-Cry1Ac-Cry3A-BADH. The clone of Populus x euramericana was transformed by the vector using the Agrobacterium-mediated method. Three transgenic lines were assessed using genetic detection and resistance expression analysis. PCR revealed that exogenous genes Cry1Ac, Cry3A, BADH and selective marker gene NPTII were present in three transgenic lines. Quantitative real-time PCR (qPCR) showed significant differences in the transcriptional abundance of three exogenous genes in different lines. Results of assays for Bt toxic proteins showed that the Cry1Ac and Cry3A toxic protein content of each line was 12.83-26.32 and 2108.91-2724.79 ng/g, respectively. The Cry1Ac toxic protein content of different lines was significantly different; the Cry3A toxic protein content was about 100 times higher than that of the Cry1Ac toxic protein. The insect-resistance test revealed the mortality rate of transgenic lines to Hyphantria cunea L1 larvae varied by 42.2-66.7%, which was significantly higher than non-transgenic lines. The mortality rate of L1 and L2 Plagiodera versicolora larvae was 100%. The insecticidal effect of transgenic lines to P. versicolora larvae was higher than that to H. cunea larvae. NaCl stress tolerance of three transgenic lines under 3-6% NaCl concentration was significantly higher than that of non-transgenic lines.

Transformation and Evaluation of Cry1Ac+Cry2A and GTGene in Gossypium hirsutum L.

More than 50 countries around the globe cultivate cotton on a large scale. It is a major cash crop of Pakistan and is considered “white gold” because it is highly important to the economy of Pakistan. In addition to its importance, cotton cultivation faces several problems, such as insect pests, weeds, and viruses. In the past, insects have been controlled by insecticides, but this method caused a severe loss to the economy. However, conventional breeding methods have provided considerable breakthroughs in the improvement of cotton, but it also has several limitations. In comparison with conventional methods, biotechnology has the potential to create genetically modified plants that are environmentally safe and economically viable. In this study, a local cotton variety VH 289 was transformed with two Bt genes (Cry1Ac and Cry2A) and a herbicide resistant gene (cp4 EPSPS) using the Agrobacterium mediated transformation method. The constitutive CaMV 35S promoter was attached to the genes taken from Bacillus thuringiensis (Bt) and to an herbicide resistant gene during cloning, and this promoter was used for the expression of the genes in cotton plants. This construct was used to develop the Glyphosate Tolerance Gene (GTGene) for herbicide tolerance and insecticidal gene (Cry1Ac and Cry2A) for insect tolerance in the cotton variety VH 289. The transgenic cotton variety performed 85% better compared with the non-transgenic variety. The study results suggest that farmers should use the transgenic cotton variety for general cultivation to improve the production of cotton.

Degradation and detection of transgenic Bacillus thuringiensis DNA and proteins in flour of three genetically modified rice events submitted to a set of thermal processes

This study aimed to investigate the degradation of three transgenic Bacillus thuringiensis (Bt) genes (Cry1Ab, Cry1Ac, and Cry1Ab/Ac) and the corresponding encoded Bt proteins in KMD1, KF6, and TT51-1 rice powder, respectively, following autoclaving, cooking, baking, or microwaving. Exogenous Bt genes were more stable than the endogenous sucrose phosphate synthase (SPS) gene, and short DNA fragments were detected more frequently than long DNA fragments in both the Bt and SPS genes. Autoclaving, cooking (boiling in water, 30 min), and baking (200 °C, 30 min) induced the most severe Bt protein degradation effects, and Cry1Ab protein was more stable than Cry1Ac and Cry1Ab/Ac protein, which was further confirmed by baking samples at 180 °C for different periods of time. Microwaving induced mild degradation of the Bt and SPS genes, and Bt proteins, whereas baking (180 °C, 15 min), cooking and autoclaving led to further degradation, and baking (200 °C, 30 min) induced the most severe degradation. The findings of the study indicated that degradation of the Bt genes and proteins somewhat correlated with the treatment intensity. Polymerase chain reaction, enzyme-linked immunosorbent assay, and lateral flow tests were used to detect the corresponding transgenic components. Strategies for detecting transgenic ingredients in highly processed foods are discussed.

Relevance of Bt toxin interaction studies for environmental risk assessment of genetically modified crops.

In recent years, different Bacillus thuringiensis (Bt) toxin-encoding genes have been combined or 'stacked' in genetically modified (GM) crops. Synergism between Bt proteins may occur and thereby increase the impact of the stacked GM event on nontarget invertebrates compared to plants expressing a single Bt gene. On the basis of bioassay data available for Bt toxins alone or in combination, we argue that the current knowledge of Bt protein interactions is of limited relevance in environmental risk assessment (ERA).

Safe use of Cry genes in genetically modified crops

Genomic technologies have been used to improve cultivated crop species. For example, Bt genes such as Cry1Ac, Cry2Ab, Cry1F and Cry3Bb1 are derived from Bacillus thuringiensis, a soil bacterium. Such genes provide protection against lepidopteran insect pests. Bt genes have been introduced in corn, cotton, soybean, rice, potato and canola. Genetically modified (GM)-cotton, containing the Cry1Ac gene, was released for cultivation in the mid-1990s in the USA and later in 28 countries including China and India. Potential harmful effects of the Bt-crops on non-targets were assessed before release into the environment. Most commonly, cultivation of the Bt-crops was found safe. Safety was tested using various experiments including: the insertional impact of transgene and its regulatory elements on plant phenotype and agronomic performance; effect on non-target organisms; and nutritional impacts on multiple experimental models, albeit the studies were conducted for limited durations. However, skeptics always claim for conducting extensive clinical as well as field trials and also cast doubt on methods and procedures of calculating the ecological risks. This debate got further momentum especially after the publication of reports on substantial reduction in monarch butterfly caterpillars when exposed to Bt-maize pollen—though later nullified—and detection of traces of transgene in various tissues of experimental animals. It is generally accepted that procedures, methods and protocols for evaluating the potential risks of GM-crops and foods should be standardized for building confidence of all stakeholders. Efforts should be exerted in deploying genes of interest, marker genes and regulatory sequences invoking no or little issues of potential risks to the ecosystem.

The effect of genetically modified feeds on productivity, milk composition, serum metabolite profiles and transfer of tDNA into milk of cows

The experiment was performed to determine whether transgenic maize containing the Bt gene (MON 810) and soyabean meal produced from glyphosate-tolerant plants (Roundup Ready, MON 40-3-2) compared with non- transgenic plants can affect the performance parameters, milk composition, blood serum metabolite profiles and transfer of tDNA into milk of cows. The experiment was carried out from the third week before parturition to the 305 th day of lactation on 40 Polish Holstein-Friesian cows, which were allotted to 4 groups of 10 animals according to body weight, milk yield and parity. The cows in all groups were fed a total mixed ration (TMR) formulated according to IZ-INRA (2009). The concentrate mixtures added to TMR contained: non- modified (traditional) maize and soyabean meal (group TM G /TS); traditional maize and genetically modified (GM) soyabean meal (group TMG/MS); GM maize and traditional soyabean meal (group MM G /TS); or GM maize and GM soyabean meal (group MM G /MS). There were no significant differences be- tween transgenic and non-transgenic feeds in productivity, milk composition and blood metabolite profiles such as: β-hydroxybutyric acid (BHBA), free fatty acids (FFA), glucose, insulin and progesterone. The transgenic DNA sequenc- es of MON 810 and RR soyabean meal were not detectable by PCR in milk.