Maize NK603 Research Articles

Development of genetically modified maize (<italic>Zea mays </italic>L.) NK603 matrix reference materials

<p id="C3">The implementation of genetically modified organisms (GMO) safety management and labeling system requires standardized testing methods and reference materials (RMs). Furthermore, the RMs are the guarantee of obtaining accurate, reliable and comparable testing results. At present, genetically modified (GM) maize (<italic>Zea mays</italic> L.) NK603 has been approved to be imported as processing raw material in China, and its safety supervision urgently needs to prepare RMs. In this study, the heterozygous seeds of GM maize NK603 and the non-GM maize counterpart after identification were well milled and then gravimetrically mixed to prepare three levels of powder RMs according to the mass fractions of 60.0, 100.0, and 1000 mg g^-1. The homogeneity and stability test by duplex ddPCR method showed that the RMs of GM maize NK603 were homogenous and stable for not less than six months. The property values were collaboratively characterized by eight laboratories using duplex ddPCR method. The certified values together with their expanded uncertainties were (2.75±0.26)%, (4.68±0.39)%, and (51.8±3.7)%, respectively. The minimum sample intake was determined to be 100 mg. This batch of RMs can be used for the qualitative and quantitative detection of NK603 event in food and feed, as well as for the evaluation of GM maize NK603-specific assays and laboratory quality control.

Assessment of genetically modified maize NK603 × T25 × DAS-40278-9 and subcombinations, for food and feed uses, under Regulation (EC) No 1829/2003 (application EFSA-GMO-NL-2019-164).

Maize NK603 × T25 × DAS‐40278‐9 (three‐event stack maize) was produced by conventional crossing to combine three single events: NK603, T25 and DAS‐40278‐9. The GMO Panel previously assessed the three single maize events and two of the subcombinations and did not identify safety concerns. No new data on the single maize events or the two subcombinations were identified that could lead to modification of the original conclusions on their safety. The molecular characterisation, comparative analysis (agronomic, phenotypic and compositional characteristics) and the outcome of the toxicological, allergenicity and nutritional assessment indicate that the combination of the single maize events and of the newly expressed proteins in the three‐event stack maize does not give rise to food and feed safety and nutritional concerns. The GMO Panel concludes that the three‐event stack maize, as described in this application, is as safe as the non‐GM comparator and the selected non‐GM reference varieties. In the case of accidental release of viable grains of the three‐event stack maize into the environment, this would not raise environmental safety concerns. The GMO Panel assessed the likelihood of interactions among the single events in one of the maize subcombinations not previously assessed and concludes that these are expected to be as safe as the single events, the previously assessed subcombinations and the three‐event stack maize. The post‐market environmental monitoring plan and reporting intervals are in line with the intended uses of the three‐event stack maize. Post‐market monitoring of food/feed is not considered necessary. The GMO Panel concludes that the three‐event stack maize and its subcombinations are as safe as the non‐GM comparator and the selected non‐GM reference varieties with respect to potential effects on human and animal health and the environment.

A sensitive immunosensor based on graphene-PAMAM composites for rapid detection of the CP4-EPSPS protein in genetically modified crops

A simple electrochemical immunosensor based on nitrogen-doped graphene and polyamide-amine (GN-PAM) composites was proposed for the detection of the CP4-EPSPS protein in genetically modified (GM) crops. In this immunosensor, the amplification of the detection signal was realized through antibodies labeled with gold nanoparticles (AuNPs). The electrochemical responses of the immunosensor were linear (R2 = 0.9935 and 0.9912) when the GM soybean RRS and maize NK603 content ranged from 0.025% to 1.0% and 0.05% to 1.5%, respectively. The limits of detection for the GM soybean RRS and maize NK603 were as low as 0.01% and 0.03%, respectively. The immunosensor also exhibited high specificity, and satisfactory stability, reproducibility, and accuracy. Our findings indicated that the constructed immunosensor provides a new approach for the sensitive detection of the CP4-EPSPS protein. Notably, the sensor may be applied to other proteins or pathogenic bacteria by simply changing the antibodies, and may also be used for multi-component analysis.

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

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

Analysis of transcriptomic differences between NK603 maize and near-isogenic varieties using RNA sequencing and RT-qPCR

BackgroundThe insertion of a transgene into a plant organism can, in addition to the intended effects, lead to unintended effects in the plants. To uncover such effects, we compared maize grains of two genetically modified varieties containing NK603 (AG8025RR2, AG9045RR2) to their non-transgenic counterparts (AG8025conv, AG9045conv) using high-throughput RNA sequencing. Moreover, in-depth analysis of these data was performed to reveal the biological meaning of detected differences.ResultsUniquely mapped reads corresponded to 29,146 and 33,420 counts in the AG8025 and AG9045 varieties, respectively. An analysis using the R-Bioconductor package EdgeR revealed 3534 and 694 DEGs (significant differentially expressed genes) between the varieties AG8025RR2 and AG9045RR2, respectively, and their non-transgenic counterparts. Furthermore, a Deseq2 package revealed 2477 and 440 DEGs between AG8025RR2 and AG9045RR2, respectively, and their counterparts. We were able to confirm the RNA-seq results by the analysis of two randomly selected genes using RT-qPCR (reverse transcription quantitative PCR). PCA and heatmap analysis confirmed a robust data set that differentiates the genotypes even by transgenic event. A detailed analysis of the DEGs was performed by the functional annotation of GO (Gene Ontology), annotation/enrichment analysis of KEGG (Kyoto Encyclopedia of Genes and Genomes) ontologies and functional classification of resulting key genes using the DAVID Bioinformatics Package. Several biological processes and metabolic pathways were found to be significantly different in both variety pairs.ConclusionOverall, our data clearly demonstrate substantial differences between the analyzed transgenic varieties and their non-transgenic counterparts. These differences indicate that several unintended effects have occurred as a result of NK603 integration. Heatmap data imply that most of the transgenic insert effects are variety-dependent. However, identified key genes involved in affected pathways of both variety pairs show that transgenic independent effects cannot be excluded. Further research of different NK603 varieties is necessary to clarify the role of internal and external influences on gene expression. Nevertheless, our study suggests that RNA-seq analysis can be utilized as a tool to characterize unintended genetic effects in transgenic plants and may also be useful in the safety assessment and authorization of genetically modified (GM) plants.

Correction to: Lack of adverse effects in subchronic and chronic toxicity/carcinogenicity studies on the glyphosate-resistant genetically modified maize NK603 in Wistar Han RCC rats.

In the original publication, the starting point in time for the three feeding trials.

Food and Environmental Risk Assessment of Herbicide-tolerant Genetically Modified Maize NK603 for Food Uses, Import and Processing under Directive 2001/18/EC (Notification C/ES/00/01)

Food and Environmental Risk Assessment of Herbicide-tolerant Genetically Modified Maize NK603 for Food Uses, Import and Processing under Directive 2001/18/EC (Notification C/ES/00/01)

Food/feed and Environmental Risk Assessment of Insect-resistant and Herbicide-tolerant Genetically Modified Maize 59122 x NK603 for Food and Feed Uses, Import and Processing under Regulation (EC) No 1829/2003 (EFSA/GMO/UK/2005/20)

Food/feed and Environmental Risk Assessment of Insect-resistant and Herbicide-tolerant Genetically Modified Maize 59122 x NK603 for Food and Feed Uses, Import and Processing under Regulation (EC) No 1829/2003 (EFSA/GMO/UK/2005/20)

Food/Feed and Environmental Risk Assessment of Insect-resistant and Herbicide-tolerant Genetically Modified Maize 59122 x 1507 x NK603 for Food and Feed Uses, Import and Processing under Regulation (EC) No 1829/2003 (EFSA/GMO/UK/2005/21)

Food/Feed and Environmental Risk Assessment of Insect-resistant and Herbicide-tolerant Genetically Modified Maize 59122 x 1507 x NK603 for Food and Feed Uses, Import and Processing under Regulation (EC) No 1829/2003 (EFSA/GMO/UK/2005/21)

Food/Feed and Environmental Risk Assessment of Insect-resistant and Herbicide-tolerant Genetically Modified Maize 1507 x NK603 for Food and Feed Uses, Import and Processing under Regulation (EC) No 1829/2003 (EFSA/GMO/UK/2004/05)

Food/Feed and Environmental Risk Assessment of Insect-resistant and Herbicide-tolerant Genetically Modified Maize 1507 x NK603 for Food and Feed Uses, Import and Processing under Regulation (EC) No 1829/2003 (EFSA/GMO/UK/2004/05)

Relationship between faecal microbiota and plasma metabolome in rats fed NK603 and MON810 GM maize from the GMO90+ study

Safety concerns arising from the consumption of foods derived from genetically modified (GM) crops remains a controversial subject. We report here a faecal microbiota compositional analysis in Wistar rats from the GMO90 + study, which fed glyphosate-tolerant NK603 (+/− Roundup application) and Bt toxin MON810 GM maize for 6 months in comparison to their closest non-GM isogenic lines. We first integrated the faecal microbiota compositional data with results from plasma metabolomics to understand which bacterial species can influence host metabolism. Coriobacteriaceae and Acetatifactor significantly predicted plasma metabolic profile in males, while Bifidobacterium and Ruminococcus were able to predict female plasma metabolites. We then investigated the differences in fecal microbiota composition between group of rats fed MON810 or NK603 GM maize in comparison to their isogenic lines. Bacterial community richness was not altered by the test diets. There were no statistically significant differences in taxa abundance in the rat faecal microbiota that we could attribute to the consumption of either MON810 or NK603. We show that the consumption of the widely cultivated GM maize varieties NK603 and MON810 even up to 33% of the total diet had no effect on the status of the faecal microbiota compared to non-GM near isogenic lines.

Lack of adverse effects in subchronic and chronic toxicity/carcinogenicity studies on the glyphosate-resistant genetically modified maize NK603 in Wistar Han RCC rats

In 2012, a controversial study on the long-term toxicity of a Roundup herbicide and the glyphosate-tolerant genetically modified (GM) maize NK603 was published. The EC-funded G-TwYST research consortium tested the potential subchronic and chronic toxicity as well as the carcinogenicity of the glyphosate-resistant genetically modified maize NK603 by performing two 90-day feeding trials, one with GM maize inclusion rates of 11 and 33% and one with inclusion rates of up to 50%, as well as a 2-year feeding trial with inclusion rates of 11 and 33% in male and female Wistar Han RCC rats by taking into account OECD Guidelines for the testing of chemicals and EFSA recommendations on the safety testing of whole-food/feed in laboratory animals. In all three trials, the NK603 maize, untreated and treated once with Roundup during its cultivation, and the conventional counterpart were tested. Differences between each test group and the control group were evaluated. Equivalence was assessed by comparing the observed difference to differences between non-GM reference groups in previous studies. In case of significant differences, whether the effects were dose-related and/or accompanied by changes in related parameters including histopathological findings was evaluated. It is concluded that no adverse effects related to the feeding of the NK603 maize cultivated with or without Roundup for up to 2 years were observed. Based on the outcome of the subchronic and combined chronic toxicity/carcinogenicity studies, recommendations on the scientific justification and added value of long-term feeding trials in the GM plant risk assessment process are presented.

Equivalence limit scaled differences for untargeted safety assessments: Comparative analyses to guard against unintended effects on the environment or human health of genetically modified maize

Safety assessments guard against unintended effects for human health and the environment. When new products are compared with accepted reference products by broad arrays of measurements, statistical analyses are usually summarised by significance tests or confidence intervals per endpoint. The traditional approach is to test for statistical significance of differences. However, absence or presence of significant differences is not a statement about safety. Equivalence limits are essential for safety assessment. We propose graphs to present the results of equivalence tests over the array of endpoints. It is argued that plots of the equivalence limit scaled difference (ELSD) are preferable over plots of the standardised effect size (SES) used previously for similar assessments. The ELSD method can be used either with externally specified equivalence limits or with equivalence limits estimated from (historical) data. The method is illustrated with two examples: first, environmental safety of MON810 Bt maize was assessed using field trial count data of arthropods; second, human safety of herbicide tolerant NK603 maize was assessed using haematological, biochemical and organ weight data from a 90-day rat feeding study. All assessed endpoints were classified in EFSA equivalence categories I or II, implying full equivalence or equivalence more likely than not.

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.

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.

Assessment of genetically modified maize NK603 x MON810 for renewal of authorisation under Regulation (EC) No1829/2003 (application EFSA-GMO-RX-007).

Following the submission of application EFSA‐GMO‐RX‐007 under Regulation (EC) No 1829/2003 from Monsanto, the Panel on Genetically Modified Organisms of the European Food Safety Authority (GMO Panel) was asked to deliver a scientific risk assessment on the data submitted in the context of the renewal of authorisation application of the herbicide‐tolerant and insect‐resistant genetically modified maize NK603 x MON810. The data received in the context of this renewal application contained post‐market environmental monitoring reports, a systematic search and evaluation of literature, updated bioinformatic analyses, and additional documents or studies performed by or on behalf of the applicant. The GMO Panel assessed these data for possible new hazards, modified exposure or new scientific uncertainties identified during the authorisation period and not previously assessed in the context of the original application. Under the assumption that the DNA sequence of the events in maize NK603 x MON810 considered for renewal is identical to the sequence of the originally assessed events, the GMO Panel concludes that there is no evidence in the renewal application EFSA‐GMO‐RX‐007 for new hazards, modified exposure or scientific uncertainties that would change the conclusions of the original risk assessment on maize NK603 x MON810.

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.

Split application of glyphosate in herbicide-tolerant maize provides efficient weed control and favors beneficial epigeic arthropods

Wide deployment of genetically modified herbicide-tolerant (GMHT) maize may affect the efficiency of weed control methods and impair ecosystem functioning. We examined these potential threats using glyphosate-tolerant maize NK603 (Monsanto Technology LLC, St. Louis, MO, USA) planted in 54 randomly distributed plots in 2013 and 2014. Maize was grown in three herbicide treatments combined with six tillage regimes. Conventional, single post-emergence application of a selective herbicide MaisTer (Bayer CropScience GmbH, Frankfurt am Main, Germany; hereafter as MT) was compared with two herbicide strategies used in the glyphosate-tolerant crops: split application of Roundup Rapid (Monsanto Europe S.A./N.V., Brussels, Belgium; hereafter as RRRR) and application of this herbicide mixed with the soil residual herbicide Guardian Extra (Monsanto Europe S.A./N.V.; hereafter as RRGE). MT proved unreliable, whereas RRRR and especially RRGE provided efficient weed control and affected weed performance during the season. RRRR permitted restoration of weed cover during the latter half of the cultivation season. Conventional, reduced, and conservation (mulching with Hordeum vulgare, Phacelia tanacetifolia, Sinapis alba, and Trifolium incarnatum) tillage had minor effect on the weed performance. Carabids, staphylinids, and spiders were monitored to assess environmental impact of tested weed management practices. Carabid communities were not affected by the type of tillage but responded to the herbicide treatments. The plots treated with MT harbored the highest carabid activity abundance and species richness, followed by RRRR, and then by RRGE. RRRR and RRGE treatments also reduced the rise of staphylinid abundance and species richness after harvest, while conventional tillage negatively affected staphylinids at the start of the cultivation season. Spider and carabid activity abundance was similar, but spider species richness was highest in the RRRR plots. Neither herbicides nor tillage strongly affected arthropod species evenness. Multivariate analysis showed that weed species richness was significantly correlated with the species activity abundance of all three arthropod groups; weed coverage had a similar but smaller effect but the effect of herbicides and tillage was negligible. We concluded that herbicide treatments curbed weed performance which consequently influenced associated arthropods.The RRRR herbicide treatment adequately regulates weeds and exerts restoration of weed cover later in growing season that is beneficial to the arthropods. Thus, GMHT crops have a potential to combine economic and environmental advantages for agroecosystem sustainability and can be recommended for implementation in European crop production systems.

Detección molecular de secuencias de adn transgénico en alimentos de consumo humano y animal en Costa Rica

Desde la introducción de los cultivos genéticamente modificados (GM), ha habido un crecimiento continuo en adopción de la tecnología y en la comercialización de alimentos y piensos derivados de cultivos GM alrededor del mundo. Gobiernos y organizaciones han desarrollado métodos para detectar rápidamente y con alto rendimiento los alimentos y piensos derivados de cultivos GM, y así verificar el cumplimiento de las regulaciones dirigidas a proveer información al consumidor. El objetivo de este estudio fue determinar la presencia de secuencias de ADN derivadas de maíz y soya GM, en una gama de piensos y alimentos, sin procesar y procesados, comercializados en Costa Rica, un mercado sin regulaciones que indiquen contenido transgénico en el etiquetado. Se empleó el método cualitativo estándar, de reacción en cadena de la polimerasa (PCR) en punto final, para la detección de material GM a partir las regiones comunes del promotor 35S y el terminador NOS, seguido de la detección específica de los eventos de maíz Bt11, MON810, GA21 y NK603, así como el evento de soya GTS 40-3-2, en una selección de alimentos y piensos disponibles en Costa Rica. Los resultados generales de la detección de secuencias derivadas de cultivos GM fueron: 86% para el promotor 35S, 72% para el terminador NOS y 40% para los eventos identificados. Los eventos más frecuentemente detectados fueron MON810, NK603 y Bt11. Los resultados demostraron que existen alimentos y piensos derivados de cultivos GM en el mercado local y que la significancia y viabilidad del etiquetado de los productos, para proveer información a los consumidores, debería ser abordado por las autoridades competentes. Sin embargo, todavía falta realizar estudios cuantitativos en los análisis de rutina, para detectar si el límite de material GM, establecido por la regulación sobre alimentos y piensos GM de la Unión Europea, se ha extralimitado.

Relevance of a new scientific publication (Mesnage et al., 2016†) on previous EFSA GMO Panel conclusions on the risk assessment of maize NK603

Relevance of a new scientific publication (Mesnage et al., 2016†) on previous EFSA GMO Panel conclusions on the risk assessment of maize NK603