GM Maize Research Articles

Disharmonization in the Regulation of Transgenic Plants in Europe

Disharmonization in the Regulation of Transgenic Plants in Europe

Environmental Risk Assessment of Insect Resistant Genetically Modified Maize MON810 for Cultivation, Seed Production, Import, Processing and Feed Uses under Directive 2001/18/EC (Notification C/F/95/12/02)

Environmental Risk Assessment of Insect Resistant Genetically Modified Maize MON810 for Cultivation, Seed Production, Import, Processing and Feed Uses under Directive 2001/18/EC (Notification C/F/95/12/02)

Resilience of Biocontrol for Aflatoxin Minimization Strategies: Climate Change Abiotic Factors May Affect Control in Non-GM and GM-Maize Cultivars.

There has been significant interest in the development of formulations of non-toxigenic strains of Aspergillus flavus for control of toxigenic strains to reduce the aflatoxin B1 (AFB1) contamination of maize. In the future, climate change (CC) abiotic conditions of temperature (+2–4°C), CO2 (existing levels of 400 vs. 800–1,200 ppb), and drought stress will impact on the agronomy and control of pests and diseases. This study has examined (1) the effect of two-way interacting factors of water activity × temperature on colonization and AFB1 contamination of maize cobs of different ripening ages; (2) the effect of non-toxigenic strains of A. flavus (50:50 inoculum ratio) on relative control of toxigenic A. flavus and AFB1 contamination of ripening cobs; (3) post-harvest control of AFB1 by non-toxigenic strains of A. flavus in non-GM and isogenic GM maize cultivars using the same inoculum ratio; and (4) the impact of three-way interacting CC factors on relative control of AFB1 in maize cobs pre-harvest and in stored non-GM/GM cultivars. Pre-harvest colonization and AFB1 production by a toxigenic A. flavus strain was conserved at 37°C when compared with 30°C, at the three ripening stages of cob development examined: milk ripe (R3), dough (R4), and dent (R5). However, pre-harvest biocontrol with a non-toxigenic strain was only effective at the R3 and R4 stages and not at the R5 stage. This was supported by relative expression of the aflR regulatory biosynthetic gene in the different treatments. When exposed to three-way interacting CC factors for control of AFB1 pre-harvest, the non-toxigenic A. flavus strain was effective at R3 and £4 stages but not at the R5 stage. Post-harvest storage of non-GM and GM cultivars showed that control was achievable at 30°C, with slightly better control in GM-cultivars in terms of the overall inhibition of AFB1 production. However, in stored maize, the non-toxigenic strains of A. flavus had conserved biocontrol of AFB1 contamination, especially in the GM-maize cultivars under three-way interacting CC conditions (37°C × 1,000 ppm CO2 and drought stress). This was supported by the relative expression of the aflR gene in these treatments. This study suggests that the choice of the biocontrol strains, for pre- or post-harvest control, needs to take into account their resilience in CC-related abiotic conditions to ensure that control of AFB1 contamination can be conserved.

Development of dwarfish and yield-effective GM maize through passivation of bioactive gibberellin.

During the Green Revolution in the 1960s, breeding dwarf cultivars turned out to be a landmark, leading to a significant increase in the global production of wheat and rice. The most direct and effective strategy for breeding dwarf crops, among others, would be to control endogenous gibberellin (GA) levels of the crops. GA 2-oxidases are a group of 2-oxoglutarate-dependent dioxygenases that catalyze the deactivation of bioactive GAs. The ArabidopsisAtGA2ox1 gene was transformed into maize with the aim of obtaining a height-reduced GM maize. The characterization of the GM maize revealed that the highest plant height reduction was accomplished by a 74% decline in GA1 level, and by approximately twofold increases in both chlorophyll content and root/shoot ratio over the wild-type (WT). Interestingly, the stem cells of the GM maize were condensed, and the typical vascular bundle structure was found to be deformed. Based on a 2-season field trial, the GM maize exhibited a higher harvest index (9-17%) and grain yield (10-14%) than the WT. The current results suggest that a modulation of the endogenous GA level would be a sensible approach for improving the crop architecture and grain yield in maize.

The Adoption Drivers of New Technology: The Case of Genetically Modified Crop Adoption by French Farmers

This work is the first study of adoption of GM crops by French farmers. The GM crop technology case was a fertile field for the study of technology adoption but existing literature mainly focuses on studies of these issues in USA, Africa and Asia. In France, main production area of maize in Europe, the agricultural sector is very atypical compared to the agricultural sector investigated in the existing literature on the GMO adoption. By a study of the GM crop adoption in the French context and within the main production area, the south-west region of France, this study reveals 4 determinant factors that shape the choice of GM or NGM maize cropping. Some of the factors pointed by this study are in line with the existing literature on technology adoption and output uncertainty and input uncertainty (uncertainty on the expected marginal benefits and costs to sustain the new technology). Other factors identified under the atypical French agriculture sector distinctive features are particularly novel. These new factors are linked to the implementation of the UE regulation on GM/NGM coexistence in the context of small fragmented farms (need of coordination between farmers to prevent GM dispersal) and importance of the market outlets proximity to federate farmers. These novel results pinpoint that in the French context there is a need of both vertical coordination (farmers with the operators downstream the supply chain as the outlet industrial) and horizontal coordination (among farmers in neighbourhood situation) to foster the technology adoption in such industry context.

Identification of T-DNA Insertion Site and Flanking Sequence of a Genetically Modified Maize Event IE09S034 Using Next-Generation Sequencing Technology.

Molecular characteristics including information of insertion site, flanking sequence, and copy numbers are the base for the safety assessment and subsequent monitoring of genetically modified organisms (GMOs), which has to be revealed thoroughly in a case-by-case manner. Although both polymerase chain reaction (PCR)-based and next-generation sequencing (NGS)-based approaches are proven to be effective in the molecular characterization of most of GM events, they often fail to work with GM maize events, mainly due to the genome complexity. In this study, by using NGS, we successfully identified the 3' end T-DNA insertion site and flanking sequence of a GM maize event IE09S034, which were confirmed by PCR amplification and Sanger sequencing. Notably, insertions of unintended exogenous elements were revealed in this event although the single copy of target exogenous genes was also confirmed by digital PCR. The output of this study provides novel and important genetic evidence for the safety assessment and monitoring of GM maize event IE09S034.

An overview of intellectual property within agricultural biotechnology in Brazil

Brazilian agricultural biotechnology has seen great advances in recent decades, especially in the development of GM crops, including soybean, cotton, and maize, which has placed Brazil in second place since 2013 in the ranking of countries with the greatest GM-cultivated area. However, patenting these technologies is somewhat more restrictive in Brazil than in other countries, such as the USA and Japan, especially concerning isolated biological material from nature. Hence, the intellectual protection of crops in Brazil is encompassed by sui generis rights and/or the patenting of only the development process. Given the current scenario and the importance of biotechnology for the Brazilian agriculture sector, it is necessary to deeply study the patent system for recently developed technologies to identify opportunities for enterprises and national institutes to act in this area. The application of novel biotechnological strategies to agriculture will contribute to the expanding agriculture sector and become part of the solution to global challenges. Through this study, we can identify the major companies developing and protecting their agrobiotechnologies. Additionally, a more detailed analysis verifies that although there are some restrictions in Brazilian laws, GM patent applicants find ways to obtain intellectual protection for the tools they use in the development of GM crops, which include regulatory sequences, gene constructs and production methodologies. Mechanisms to stimulate investment in Brazilian research companies and public policies must be consolidated, allowing investment and public–private partnerships in this sector, with the aim of applying biotechnological knowledge and turn it into products demanded by society.

Ultrafast, universal and visual screening of dual genetically modified elements based on dual super PCR and a lateral flow biosensor

In this study, a cascade screening system has been developed combining Dual Super Polymerase Chain Reaction (DSPCR) with the universal Lateral Flow Biosensor (LFB) for the ultrafast, universal and visual screening of dual GM elements, taking P-35s × T-nos for example. In the design of DSPCR for universal screening, gene-specific forward primers were labelled with biotin and gene-specific reverse primers were tagged with Cy5 and digoxin, respectively. In 2.5-min, DSPCR effectively amplified the dual target fragments through our prototype facility. Then, through specific antigen-antibody binding, a universal lateral flow biosensor exported visually dual-amplified results simultaneously without cross contamination. After optimization, the detection limit allowed 0.05% GM maize, corresponding to nine copies in maize. The entire detection process could be achieved in 10 min without any large-scale instrumentation. This method may be useful for the ultrafast, universal and visual screening of dual GM elements (P-35s × T-nos) in GM crop lines and is expected to be of great promise for rapid GMO screening and point-of-care tests.

Optimised LAMP allows single copy detection of 35Sp and NOSt in transgenic maize using Bioluminescent Assay in Real Time (BART)

Loop-mediated amplification (LAMP) has been widely used to amplify and hence detect nucleic acid target sequences from various pathogens, viruses and genetic modifications. Two distinct types of primer are required for LAMP; hairpin-forming LAMP and displacement. High specificity arises from this use of multiple primers, but without optimal conditions for LAMP, sensitivity can be poor. We confirm here the importance of LAMP primer design, concentrations and ratios for efficient LAMP amplification. We further show that displacement primers are non-essential to the LAMP reaction at certain concentrations providing accelerating loop primers are present. We investigate various methods to quantify DNA extracts from GM maize certified reference materials to calculate the target copy numbers of template presented to the LAMP reaction, and show that LAMP can amplify transgenic promoter/terminator sequences in DNA extracted from various maize GM events using primers designed to target the 35S promoter (35Sp) or NOS terminator (NOSt) sequences, detection with both bioluminescence in real-time (BART) and fluorescent methods. With prior denaturation and HPLC grade LAMP primers single copy detection was achieved, showing that optimised LAMP conditions can be combined with BART for single copy targets, with simple and cost efficient light detection electronics over fluorescent alternatives.

Systematic miRNome profiling reveals differential microRNAs in transgenic maize metabolism

BackgroundWhile some genetically modified organisms (GMOs) are created to produce new double-stranded RNA molecules (dsRNA), in others, such molecules may occur as an unintended effect of the genetic engineering process. Furthermore, GMOs might produce naturally occurring dsRNA molecules in higher or lower quantities than its non-transgenic counterpart. This study is the first to use high-throughput technology to characterize the miRNome of commercialized GM maize events and to investigate potential alterations in miRNA regulatory networks.ResultsThirteen different conserved miRNAs were found to be dys-regulated in GM samples. The insecticide Bt GM variety had the most distinct miRNome. These miRNAs target a range of endogenous transcripts, such as transcription factors and nucleic acid binding domains, which play key molecular functions in basic genetic regulation. In addition, we have identified 20 potential novel miRNAs with target transcripts involved in lipid metabolism in maize. isomiRs were also found in 96 conserved miRNAs sequences, as well as potential transgenic miRNA sequences, which both can be a source of potential off-target effects in the plant genome. We have also provided information on technical limitations and when to carry on additional in vivo experimental testing.ConclusionsThese findings do not reveal hazards per se but show that robust and reproducible miRNA profiling technique can strengthen the assessment of risk by detecting any new intended and unintended dsRNA molecules, regardless of the outcome, at any stage of GMO development.

Reply to \u2018Comments on two recent publications on GM maize and Roundup\u2019

The opinion expressed by Eriksson and colleagues’ fails to recognise that there are no standard experimental designs for academic investigations involving omics analyses of genetically modified crops and that the only valid comparator to determine the effect of the process of transgenesis is a near isogenic variety grown at the same time and location, as was the case in our investigation of NK603 maize. Eriksson does not acknowledge that the quality of the rat liver tissues in our chronic Roundup toxicity study has neither been questioned nor branded as unsuitable for further investigation. In addition, Eriksson fails to appreciate that the statistical methods we used to analyse the liver metabolomics dataset are recognised as appropriate as some of a number of approaches that can be taken. Moreover, Eriksson neglects to mention that the proteomics analysis of the liver tissues highlights structural and functional damage from Roundup exposure. Thus our results are sound and the claims by Eriksson and colleagues of experimental flaws are unfounded.Replying to: Eriksson et al. Sci Rep 8 (2018); https://doi.org/10.1038/s41598-018-30440-7.

Comments on two recent publications on GM maize and Roundup

Two -omics studies on genetically modified maize and Roundup-fed rats, recently published in the journal Scientific Reports, contain serious flaws in the experimental design, methodology and interpretation of results, which we point out here. The use of -omics technologies are of increasing importance in research, however we argue for a cautious approach to the potential application in food safety assessments as these exceptionally sensitive and complex methods require a thorough and detailed evaluation of the biological significance of obtained results. Arising from: Mesnage et al. Sci Rep 7:39328 (2017), Mesnage et al. Sci Rep 6:37855 (2016).

Monitoring of genetically modified food on the Czech food market and a cross-country comparison

The presence of genetically modified organisms (GMOs) was analysed in food samples from the Czech food markets. Four different types of food samples (soya beans, soya bean products, maize flour, and rice) were collected at twelve places in four terms in the years 2008–2013. It represents a total 1152 food samples. Soya and maize were chosen, because these are the major transgenic crops grown worldwide. Increased cultivation of GM rice in China, India, Indonesia, and the Philippines has been observed. Polymerase chain reaction-based methods were applied to detect GMOs. GMOs were detected in 107 samples (9.3%). The results show that in food from the Czech food market GMOs mainly in maize flour and rice were found. GM maize was detected in 63 (21.9%) maize flour samples. Maize lines MON810, NK603, and Bt176 were detected in 14 (22%), 9 (14%), and 1 (1.6%) maize flour samples, respectively. Unauthorised GM rice was detected in 39 (13.5%) rice samples. Unauthorised rice Bt63 was detected in one sample of rice. Roundup Ready soya was detected in 4 (1.4%) soya bean samples and in 1 (0.35%) soya product sample. These results were compared with results of the world´s studies.

Highly Sensitive GMO Detection Using Real-Time PCR with a Large Amount of DNA Template: Single-Laboratory Validation.

Current genetically modified organism (GMO) detection methods allow for sensitive detection. However, a further increase in sensitivity will enable more efficient testing for large grain samples and reliable testing for processed foods. In this study, we investigated real-time PCR-based GMO detection methods using a large amount of DNA template. We selected target sequences that are commonly introduced into many kinds of GM crops, i.e., 35S promoter and nopaline synthase (NOS) terminator. This makes the newly developed method applicable to a wide range of GMOs, including some unauthorized ones. The estimated LOD of the new method was 0.005% of GM maize events; to the best of our knowledge, this method is the most sensitive among the GM maize detection methods for which the LOD was evaluated in terms of GMO content. A 10-fold increase in the DNA amount as compared with the amount used under common testing conditions gave an approximately 10-fold reduction in the LOD without PCR inhibition. Our method is applicable to various analytical samples, including processed foods. The use of other primers and fluorescence probes would permit highly sensitive detection of various recombinant DNA sequences besides the 35S promoter and NOS terminator.

Multiplex Droplet Digital PCR Protocols for Quantification of GM Maize Events.

The standard-curve based simplex quantitative polymerase chain reaction (qPCR) has been the gold standard for DNA target quantification for more than a decade. The large and growing number of individual analyses needed to test for genetically modified organisms (GMOs) is reducing the cost-effectiveness of qPCR. Droplet digital PCR (ddPCR) enables absolute quantification without standard curves, avoids the amplification efficiency bias observed with qPCR, allows more accurate estimations at low target copy numbers and, in combination with multiplexing, significantly improves cost efficiency. Here we describe two protocols for multiplex quantification of GM maize events: (1) nondiscriminating, with multiplex quantification of targets as a group (12 GM maize lines) and (2) discriminating, with multiplex quantification of individual targets (events). The first enables the quantification of twelve European Union authorized GM maize events as a group with only two assays, but does not permit determination of the individual events present. The second protocol enables the quantification of four individual targets (three GM events and one endogene) in a single reaction. Both protocols can be modified for quantification of any other DNA target.

Development of PCR screening assays focused on gene-coding sequences for GMO detection

Description of the subject. This paper describes the development, evaluation and limitations of screening PCR assays based on genes and for use in GMO detection. Objectives. The aim of this research is to propose new PCR assays based on gene-coding sequences that will join a panel of existing screening assays to make better GMO detection possible. Method. Real-time PCR methods using double-dye probes were developed for genes frequently encountered in GM constructs and evaluated in terms of specificity and sensitivity. Results. Eight real-time PCR tests were designed based on the sequences of the bar, pat, EPSPS, gox, gus and hsp70 genes. Two of them proved of limited interest due to the presence of positive signals linked to the presence of the donor organisms: residual sequences of Escherichia coli in the master mixes for the gus PCR assay and while for the hsp70 PCR test, hsp70 being only used in GM maize at present, it is useless as hsp70 originates from maize. The assays for bar, pat, EPSPS and gox were found to successfully detect the corresponding structural elements introduced in GM constructs. Several PCR assays were proposed for the EPSPS gene in order to cover the different versions of the gene. Conclusions. Real-time PCR tests for bar, pat, EPSPS and gox were developed and met the expected performance criteria in terms of specificity and sensitivity. The targets can be amplified with the same PCR conditions as PCR assays already developed for the detection of promoters and terminators, and can be used in combination on the same PCR plate in order to provide wider coverage of GMOs and initial information concerning the GMO(s) present.

Моніторинг харчових продуктів, кормів та сільськогосподарської сировини в Україні на вміст генетично модифікованих інгредієнтів

Biotechnological agricultural crops are growing during the last 20 years and these crops take up about 180 million hectares (10% of arable areas). World GM plant growing focuses mostly at four agricultural crops – soybean, cotton, maize and rapeseed. These crops have an herbicide-and vermin resistance. Governments of many countries develop legal instruments for prevention of potential risks associated with GMO circulation. Since Ukraine is one of the main producers and exporters of grain crops, it is very important for our country. Gmo legislation of Ukraine is imperfect unfortunately and it requires considerable modifying. Thus, in spite of ban of GMO using in Ukraine, producing of GM soybean, maize and rapeseed is carried into practice. Data from different institutions demonstrates this fact. Monitoring of GM plants presence and propagation was the aim of this study. Study was performed at the molecular-genetic laboratory of SE «Ukrmetrtest standart». Food and agricultural samples of Ukrainian production were analyzed during 2007-2017. Samples of soybean, maize and rapeseed leaves, shoots and seed from different regions of Ukraine were analyzed. Kits of own production were used for analysis. Kits were designed on the basis of TaqMan Real-Time PCR method. 17 636 food and raw samples were analyzed. More than half of it was plant raw material and derivative products (soybean, maize, rapeseed, wheat, barley and rice). Biotechnological soybean, maize and rapeseed were detected in food and raw material. In 2007 our laboratory analyzed 413 food samples. We detected GMO in 22% cases. We detected GMO mostly in sausages and half-finished meats with soybean content. GM ingredients were detected in confectionary and food products, produced with soybean and maize. Tests quantity increased in 2008. Main part of tests was raw material. It indicates producers interest in GMO control. We analyzed 1177 samples, and 1080 (92%) were detected as non-GMO. In 2009 we tested 2126 samples generally. GMO was detected in 107 samples, what made up 5%. Tests quantity increased more in 2010.We tested 2570 samples. GMO was detected in 204 cases, what made up 7%. In 2011 quantity of samples has decreased significantly. We tested 1866 samples, and MO was detected in 59 samples, what made up 3 %. In 2012 року we tested 2001 samples, and GMO was detected in 67 samples. During 2013-2014 we tested 6040 food and agricultural samples. GM ingredients were detected in 66 (4 %) and 77 (5 %) samples, respectively. In 2015 quantity of GM-positive samples increased to 8 %, it was mostly agricultural raw material. We detected 5 % GM samples during 2016. Main part of GM-positive samples was soybean, maize and rapeseed. Percentage of detected GMO`s increased up to 8% again in 2017. Totally we detected 95 GM-samples. Designed test-systems detect most biotechnological crops without detailed identification. Data obtained from 2007-2017 shows biotech soy, maize and rape presence in quantities more than 0,9%, despite the real prohibition of biotechnological crops growing in the country. Key words: genetically modified organisms, Real-Time polymerase chain reaction, food products

Optimization and Verification of Droplet Digital PCR Even-Specific Methods for the Quantification of GM Maize DAS1507 and NK603.

In recent years, digital polymerase chain reaction (dPCR), a new molecular biology technique, has been gaining in popularity. Among many other applications, this technique can also be used for the detection and quantification of genetically modified organisms (GMOs) in food and feed. It might replace the currently widely used real-time PCR method (qPCR), by overcoming problems related to the PCR inhibition and the requirement of certified reference materials to be used as a calibrant. In theory, validated qPCR methods can be easily transferred to the dPCR platform. However, optimization of the PCR conditions might be necessary. In this study, we report the transfer of two validated qPCR methods for quantification of maize DAS1507 and NK603 events to the droplet dPCR (ddPCR) platform. After some optimization, both methods have been verified according to the guidance of the European Network of GMO Laboratories (ENGL) on analytical method verification (ENGL working group on "Method Verification." (2011) Verification of Analytical Methods for GMO Testing When Implementing Interlaboratory Validated Methods). Digital PCR methods performed equally or better than the qPCR methods. Optimized ddPCR methods confirm their suitability for GMO determination in food and feed.

Economic impacts of commercializing insect-resistant GM maize in China

PurposeThe purpose of this paper is to examine the potential economic impacts of China’s insect-resistant GM maize and provide new evidence for decision making concerning its commercialization.Design/methodology/approachThis study uses data drawn from the production trials of insect-resistant GM maize and expert interviews to determine the impacts of commercializing GM maize at farm level under three scenarios with varying severity of insect pest attacks in maize production. Economic impacts are simulated using a modified Global Trade Analysis Project model.FindingsIn farm terms, insect-resistant GM maize increases crop yield and reduces both pesticide and labor inputs. In national terms, China can increase its GDP by USD8.6 billion and maize self-sufficiency by about 2 percent given normal insect pest attacks if China commercializes GM maize. Additional beneficiaries include consumers and the livestock industry. Non-maize crops can also benefit from land saving through GM maize commercialization. Chemical is a sector with the decrease in its output because demand for pesticides will fall.Originality/valueAlthough China has announced a roadmap for commercializing GM crops for use as feed and in processing after nearly two decades of producing GM cotton, no clear timetable for producing GM maize as feed has been established due to several concerns, including the potential for economic gains from GM maize. This study is the first to assess the economic impacts of commercializing China’s GM maize. The findings should have significant policy implications for the development and commercialization of GM crops in general and GM maize in particular.

Identification and freedom to operate analysis of potential genes for drought tolerance in maize

Drought tolerance is an important character for agricultural crops, particularly corn. Genes confering this feature can be patented, thus hindering their use. From a thorough analysis, three genes (DREB, ZAT10 and CspB) were identified and their sequences were captured in the NCBI database. From these sequences and using free software tools, expression cassettes -including regulatory regions (promoters E35S + Pleader, Ubi-1, rab17; terminators Trub, Tnos)- were designed. Patent searches were conducted in international databases (The Lens and PATENTSCOPE). Four patents and an application were found. In the Colombian national database of the Superintendence of Industry and Commerce (SIC), only the application made through PCT was identified. The claims and nucleotide sequences contained in the application were analyzed and it was found that they do not affect the expression cassettes designed. There is freedom to operate for these constructs and it is possible to continue developing drought-tolerant GM maize lines for the domestic market.