Roundup ReadyTM Research Articles

Integration of omics analyses into GMO risk assessment in Europe: a case study from soybean field trials

In Europe, genetically modified organisms (GMOs) are subject to an authorization process including a mandatory risk assessment. According to the respective guidance by the European Food Safety Authority (EFSA), one of the pillars of this GMO risk assessment is a comparative analysis of the compositional and agronomic characteristics. This targeted approach has been criticized for its limitations, as it only considers pre-determined compounds, being insufficient to assess a comprehensive range of relevant compounds, including toxins and anti-nutrients, on a case-specific basis. Strategies based on advanced untargeted omics technologies have been proposed as a potential broader approach to be implemented into the initial step of the risk assessment framework. Here, we provide an example of a step-by-step omics analysis based on systems biology approach to fit into the context of European GMO regulation. We have performed field trial experiments with genetically modified (GM) Intacta™ Roundup Ready™ 2 Pro soybean containing both cry1Ac and cp4epsps transgenic inserts and analyzed its proteomic profile against the non-GM counterpart and reference varieties. Based on EFSA’s comparative endpoint-by-endpoint approach, the proteomics analysis revealed six proteins from the GMO outside the 99% tolerance intervals of reference varieties (RVs) in the equivalence test. Interestingly, from the near-isogenic (non-GM) comparator we found as many as ten proteins to be outside of the said RVs’ equivalence limits. According to EFSA’s statistical guidelines, differences found in metabolite abundance between a GMO and its non-GM comparator would not be considered biologically relevant as all compounds of concern remained within the equivalence limits of commercial RVs. By assessing the proteomic and metabolomic data through our proposed systems biology approach, we found 70 proteins, and the metabolite xylobiose as differentially expressed between the GMO and its non-GM comparator. Biological relevance of such results was revealed through a functional biological network analysis, where we found alterations in several metabolic pathways related to protein synthesis and protein processing. Moreover, the allergenicity analysis identified 43 proteins with allergenic potential being differentially expressed in the GM soybean variety. Our results demonstrate that implementation of advanced untargeted omics technologies in the risk assessment of GMOs will enable early and holistic assessment of possible adverse effects. The proposed approach can provide a better understanding of the specific unintended effects of the genetic modification on the plant’s metabolism, the involved biological networks, and their interactions, and allows to formulate and investigate dedicated risk hypotheses in the first place. We draw conclusions on a detailed comparison with the comparative assessment according to EFSA and provide scientific arguments and examples on how the current comparative approach is not fit for purpose.

Monitoring and traceability of genetically modified soya bean event GTS 40-3-2 during soya bean protein concentrate and isolate preparation.

To evaluate DNA fragmentation and GMO quantification during soya bean protein concentrate and isolate preparation, genetically modified soya bean event GTS 40-3-2 (Roundup ReadyTM soya bean, RRS) was blended with conventional soya beans at mass percentages of 0.9%, 2%, 3%, 5% and 10%. Qualitative PCR and real-time PCR were used to monitor the taxon-specific lectin and exogenous cp4 epsps target levels in all of the main products and by-products, which has practical significance for RRS labelling threshold and traceability. Along the preparation chain, the majority of DNA was distributed in main products, and the DNA degradation was noticed. From a holistic perspective, the lectin target degraded more than cp4 epsps target during both of the two soya bean proteins preparations. Therefore, the transgenic contents in the final protein products were higher than the actual mass percentages of RRS in raw materials. Our results are beneficial to the improvement of GMO labelling legislation and the protection of consumer rights.

Development and Testing of an Individual Kernel Detection System for Genetically Modified Soybean Events in Non-identity-preserved Soybean Samples.

A genetically modified (GM) soybean kernel detection system using combination of DNA preparation from individual soybean kernels and event-specific real-time PCR was developed to simultaneously identify GM soybean events authorized for food after safety assessments in Japan. Over 100 kernels in the non-identity-preserved soybean samples imported from the United States of America (two U.S.A. lots) and Brazil (one lot) were randomly selected and examined. In total, 98 and 96% of the two independent U.S.A. lots, and 100% of the Brazilian lot contained GM soybean kernels. Herbicide-tolerant events, MON89788 (trade name Genuity® Roundup Ready 2 Yield™), GTS 40-3-2 (trade name Roundup Ready™ soybean) and A2704-12 (trade name Liberty Link® soybean), were detected similarly in both U.S.A. lots. In the Brazilian lot, in addition to GTS 40-3-2, a stacked GM event, MON87701 × MON89788, having insect-resistance and herbicide-tolerance, was detected. There were no unauthorized GM soybeans comingled, and the ratio of GM soybean events detected was consistent with statistical reports on the cultivated GM soybean events in both countries.

Detection of genetically modified soybeans by PCR method and immunoassay kits

The suitability of detecting genetically modified (GM) soybeans by polymerase chain reaction (PCR) and immunoassay kits is determined. Primers specific for inserted genes in the Roundup Ready™ soybean (RRS, Monsanto company) were applied. Four pairs of primers, namely, 35S (35S-promoter, originated from cauliflower mosaic virus), NOS (nopaline synthase-terminator, derived from Agrobacterium tumefaciens), EPSPS (5-enolpyruvylshikimate-3-phosphate synthase, obtained from A. tumefaciens strain CP4) and LE (endogenous gene lectin) were used to identify the GM soybeans. The detection limit of PCR was 0.1% (w/w) GM soybeans when primers 35S and EPSPS were used, and 1% when primers NOS were used. All soybean samples were evidenced by LE primer-PCR as soybean products. Results of Immunoassays by two kinds of kits, strip and ELISA were matched with PCR's, and feasibility of quantitation detecting GM soybeans is determined among 0∼2.5%. The data further revealed that the PCR method and immunoassay kits can sufficiently differentiate GM soybeans from non-GM products.

The Yeast Atlas of Appalachia: Species and Phenotypic Diversity of Herbicide Resistance in Wild Yeast

Glyphosate and copper-based herbicides/fungicides affect non-target organisms, and these incidental exposures can impact microbial populations. In this study, glyphosate resistance was found in the historical collection of S. cerevisiae, which was collected over the last century, but only in yeast isolated after the introduction of glyphosate. Although herbicide application was not recorded, the highest glyphosate-resistant S. cerevisiae were isolated from agricultural sites. In an effort to assess glyphosate resistance and impact on non-target microorganisms, different yeast species were harvested from 15 areas with known herbicidal histories, including an organic farm, conventional farm, remediated coal mine, suburban locations, state park, and a national forest. Yeast representing 23 genera were isolated from 237 samples of plant, soil, spontaneous fermentation, nut, flower, fruit, feces, and tree material samples. Saccharomyces, Candida, Metschnikowia, Kluyveromyces, Hanseniaspora, and Pichia were other genera commonly found across our sampled environments. Managed areas had less species diversity, and at the brewery only Saccharomyces and Pichia were isolated. A conventional farm growing RoundUp Ready™ corn had the lowest phylogenetic diversity and the highest glyphosate resistance. The mine was sprayed with multiple herbicides including a commercial formulation of glyphosate; however, the S. cerevisiae did not have elevated glyphosate resistance. In contrast to the conventional farm, the mine was exposed to glyphosate only one year prior to sample isolation. Glyphosate resistance is an example of the anthropogenic selection of nontarget organisms.

Chronoamperometric magnetogenosensing for simultaneous detection of two Roundup Ready™ soybean lines: GTS 40-3-2 and MON89788

Development of expeditious analytical methods for the detection of genetically modified organisms (GMOs) is increasingly necessary, not only to verify compliance with labelling, but also to help industry to efficiently control the reception of raw materials. On the basis of this, a disposable electrochemical magnetogenoassay is proposed for simultaneous detection of two Roundup Ready (RR) soybean lines GTS 40-3-2 and MON89788, using gold-coated magnetic nanoparticles (Fe3O4@Au) as nanosupport. To perform this magnetogenoassay, a sandwich-type hybridization assay was used with different enzymatic labelling systems (fluorescein isothiocyanate and digoxigenin) and dual screen-printed carbon electrodes (SPdCEs), which allowed the simultaneous readout of each target. A linear relationship ranging from 0.1 to 2.5 nM and from 0.1 to 1.0 nM was achieved for GTS 40-3-2 and MON89788 events, respectively, and both assays showed a similar detection limit of about 0.1 nM. Furthermore, a good performance in terms of precision and selectivity was achieved. The proposed approach is a step forward for event-specific multiplex detection.

Glyphosate: A review of its global use, environmental impact, and potential health effects on humans and other species

Glyphosate, [N-(phosphonomethyl) glycine], was synthesized in 1950 and patented as a chemical chelator, capable of binding metals such as calcium, magnesium, and manganese. Glyphosate’s ability to bind to manganese was later found to inhibit an enzyme used by plants and bacteria for biosynthesis of three amino acids found in all proteins, and the commercial value of this property led to the development and marketing of glyphosate as a broad-spectrum herbicide. In 1974, the Monsanto Chemical Company introduced the herbicide as Roundup™, a formulation of glyphosate and adjuvants. Roundup™ was originally used for weed control in specific farming and landscaping operations and around power lines and train tracks. Following introduction of Roundup Ready™ seeds, in the 1990s, glyphosate use increased significantly. Although Monsanto’s patent on glyphosate expired in 2002, the widespread and growing use of Roundup Ready™ seed globally and competitive glyphosate marketing by other chemical companies have led to glyphosate’s significant increase in the environment. Concerns about potential adverse effects have also grown. While, at present, many regulatory agencies have determined that there is little risk of adverse health effects to the general public or to farmworkers using proper handling techniques, the International Agency for Research on Cancer (IARC) assessing hazard data on glyphosate identified it in 2016 as a category 2A carcinogen (likely to cause human cancer). Response to this classification has been divided: The agribusiness industry has been forceful in its opposition, while other experts support IARC’s classification. The following article examines these issues. It also examines the basis for regulatory decisions, controversies involved, and questions of environmental justice that may or may not be addressed as glyphosate continues to be used.

Tracing two Roundup Ready™ soybean lines (GTS 40-3-2 and MON89788) in foods commercialised in Portugal

Soybean (Glycine max L.) is the most important genetically modified (GM) crop, from which the glyphosate tolerant soybean, also known as Roundup Ready (RR), is one of the most representative. The present work intends to assess the presence of two RR soybean lines, namely GTS 40-3-2 and MON89788, in a wide range of processed foods commercialised in Portugal. For this purpose, two quantitative real-time PCR approaches were proposed using available and newly designed primers. The results showed that, from 90 food samples, 73 confirmed the presence of soybean, while 15 others suggest their incompliance with labelled soybean. RR soybean was detected in 9 samples, from which 5 were identified as being the GTS-40-3-2 event, 3 the MON89788 event and one contained both events. The developed real-time PCR methods were successfully validated through the participation in an interlaboratory proficiency program and applied to processed foods. The estimated RR soybean contents of 8 samples ranged between 0.01 and 0.39%, but the sample containing both events accounted for 23.9% of GM ingredients. Such high level above the threshold for labelling regulations suggests the need of more strict control of GMO in foods.

Detection of genetically modified organisms in soy products sold in Turkish market

PCR-based technique for GMO detection is the most reliable choice because of its high sensitivity and specificity. As a candidate of the European Union, Turkey must comply with the rules for launching into the market, traceability, and labeling of GMOs as established by EU legislation. Therefore, the objective of this study is to assess soybean products in the Turkish market to verify compliance with legislation using qualitative Polymerase Chain Reaction (PCR) assay to detect the presence of GM soybean and to quantify its amount of GM soybean in the samples tested positive using real-time PCR. DNA extracted by the modified CTAB method was properly used for PCR amplification of food materials. The amplification of a 118 bp DNA fragment of the lectin gene from soybean by PCR was successfully achieved in all samples. The GMO screening was based on the detection of 35S promoter and NOS terminator sequences. The GM positive samples were subjected to detection of Roundup ReadyTM soybean (RR) using quantitative real-time PCR. It was found that 100% of the tested food samples contained less than 0.1 per cent of EPSPS gene.

HYHEAR 1 hybrid Roundup Ready™ high erucic acid, low glucosinolate summer rape

McVetty, P. B. E., Cuthbert, J. L., Marwede, V., Paulmann, W., Sass, O., Duncan, R. W., Fernando, W. G. D., Li, G. and Zelmer, C. D. 2014. HYHEAR 1 hybrid Roundup Ready™ high erucic acid, low glucosinolate summer rape. Can. J. Plant Sci. 94: 453–455. HYHEAR 1 summer rape (Brassica napus L.) is the world's first hybrid Roundup Ready™ high erucic acid, low glucosinolate cultivar. On average, HYHEAR 1 yielded 33% more seed, 23 g kg−1 more seed oil but 17 g kg −1 less meal protein than MillenniUM 03 high erucic acid, low glucosinolate, open pollinated population summer rape. HYHEAR 1 has an erucic acid content of 52.2% in isolated field trials of HEAR lines and is adapted to the southern B. napus growing regions of western Canada.

Red River 1861 Roundup Ready™ high erucic acid, low glucosinolate summer rape

McVetty, P. B. E., Duncan, R. W., Fernando, W. G. D., Li, G. and Zelmer, C. D. 2012. Red River 1861 Roundup Ready™ high erucic acid, low glucosinolate summer rape. Can. J. Plant Sci. 92: 1407–1409. Red River 1861 summer rape (Brassica napus L.) is the world's fourth Roundup Ready™ high erucic acid, low glucosinolate cultivar. On average, Red River 1861 yielded 19% more seed, 7 g kg−1 more seed oil and 7 g kg −1 more meal protein than MillenniUM 03 high erucic acid, low glucosinolate summer rape. Red River 1861 has an erucic acid content of 52.6% in isolated field trials of HEAR lines and is adapted to the southern B. napus growing regions of western Canada.

Detection of genetically modified material in feed and foodstuffs containing soy and maize in Jordan

Food and feed products with genetically modified (GM) materials are not subject to labelling regulations in Jordan. The number of GM foods being developed to cope with growing food demand, locally and internationally, is growing. This situation may result in more consumer requests for traceability and assurance of food quality and safety with respect to GM foods. To this end, the main aim of this study was to investigate at what level Jordanian consumers are exposed to GM foods. DNA was extracted from 200 food and 80 feed samples bought in Jordanian markets, screened by polymerase chain reaction (PCR) and quantified by real-time PCR. The GM screening and quantification were based on the presence of Roundup Ready™ (RR) soybean, Bt 176 or 35S target sequence genes for the GM elements. GM positive samples were quantified by real-time PCR. Fifteen food and feed samples were found positive for maize Bt-176 or RR soybean genes, representing 5.4% of the total food tested samples. The GM elements were detected in more than 62.5% with less than 1% modification, compared to less than 37.5% containing more than 1% GM.

Relação entre densidade de plantas e genótipos de soja roundup readyTM

A presente pesquisa teve por objetivo estudar a influência de diferentes densidades de plantas e genótipos de soja Roundup ReadyTM (RR) em algumas características morfoagronômicas da soja, semeada em duas safras distintas. O experimento foi conduzido na região dos Campos de Cima da Serra, no Estado do Rio Grande do Sul, nas safras agrícolas 2006/07 e 2007/08, com os cultivares CD 213RR e BRS 255RR, nas densidades de 12, 18, 24, 30 e 36 plantas aptas por m². O delineamento experimental utilizado foi de blocos ao acaso, com quatro repetições. Para isso, foram avaliados altura de plantas, diâmetro da haste, número de ramificações por planta, número de vagens por planta, número de grãos por planta, número de grãos por vagem, massa de mil grãos e rendimento de grãos por hectare. O manejo das plantas daninhas com glyphosate em duas etapas (pré-semeadura e em V4) evitou eficientemente a matocompetição. O aumento da densidade de plantas de soja RR promoveu incremento linear da altura das plantas (0,5 a 0,9 cm por planta m-2), influenciada pelo genótipo e pelo ano de cultivo. O diâmetro da haste foi diminuído com o aumento da densidade de plantas de soja RR de forma linear (-0,03 a -0,2 mm por planta m-2). O número de ramos por planta reduziu com o aumento da densidade (-0,05 a -0,19 ramos por planta). Na segunda safra, o rendimento de grãos e a massa de mil grãos foram menores, afetados por um período de estiagem e também por uma geada no final do ciclo, mais acentuadamente para o CD 213RR. A combinação de maior número de plantas m-2 levou a uma redução do número de vagens por planta e de grãos por vagem, embora de magnitudes distintas entre as cultivares, o que foi discutido pela interação entre os fatores; no entanto, o rendimento de grãos não foi alterado nas diferentes densidades testadas, e sim sofreu influência da safra de cultivo.

Application of QUIZ for GM quantification in food

The quantification of GM in samples can be performed without the need for certified reference materials (CRMs) using QUIZ (quantitation using informative zeros). In this work we have applied QUIZ to estimate the contents of RoundUp Ready™ soya and MON810 in processed food containing one or both GMs. Results show good agreement between derived values and known input of GM material and compare favourably with quantitative real-time PCR.

Red River 1997 Roundup ReadyTM high erucic acid, low glucosinolate summer rape

Red River 1997 summer rape (Brassica napus L.) is the world’s third Roundup ReadyTM high erucic acid, low glucosinolate cultivar. On average, Red River 1997 yielded 13% more seed and 22 g kg–1 more seed oil, but 4 g kg–1 less meal protein than MillenniUM 03 high erucic acid, low glucosinolate summer rape. Red River 1997 has an erucic acid content of 51.4% in isolated field trials of HEAR lines and is adapted to the southern B. napus growing regions of western Canada. Key words: Rape, Roundup ReadyTM high erucic acid, low glucosinolate, cultivar description

Produtividade de grãos da soja em função do manejo de herbicida e fungicidas

Com a introdução da soja resistente ao glifosato, ocorreu mudança no manejo da cultura, principalmente com relação ao manejo das plantas invasoras. Este trabalho foi desenvolvido com o objetivo de avaliar os efeitos da aplicação de diferentes manejos de herbicidas e fungicidas sobre o rendimento de grãos e os componentes do rendimento da soja. O experimento foi conduzido no ano agrícola 2006/2007, na área experimental do Departamento de Fitotecnia da Universidade Federal de Santa Maria. Foram utilizadas quatro cultivares de soja Roundup ReadyTM ('NK Mireya 4.2 RR', 'Fundacep 55 RR', 'NK Mágica 7.3 RR' e 'A 8100 RG'), dois manejos de herbicida (Clethodim + Bentazon e outro com glifosato), dois manejos de doenças constituídos com a aplicação de fungicida (Tebuconazole + Tiofanato Metílico) e sem o tratamento. O delineamento experimental utilizado foi o de blocos ao acaso com parcela subsubdivididas, com quatro repetições. Foram avaliados o rendimento de grãos e os componentes do rendimento. As cultivares responderam diferentemente ao manejo de herbicidas e fungicidas para produção de grãos; as cultivares que foram afetadas pelo manejo de herbicida obtiveram maior produtividade com a aplicação de glifosato, efeito que também foi observado com a aplicação de fungicida. As cultivares mais e menos produtivas apresentaram o maior e o menor peso de grãos, respectivamente. A aplicação de fungicida aumentou o índice de colheita.

Características morfológicas de cultivares de soja convencionais e Roundup ReadyTM em função da época e densidade de semeadura

O trabalho tem como objetivo verificar a influência de diferentes densidades e épocas de semeadura em genótipos convencionais e Roundup ReadyTM (RR), nas características morfológicas de plantas de soja. O experimento foi conduzido em Jari, Rio Grande do Sul (RS). A primeira semeadura foi realizada dentro da época indicada para a cultura (novembro), e a segunda, após a época indicada (janeiro), com as cultivares 'CEP/CD 41', 'CD 201', 'Fundacep 39',' Fundacep 44', 'CD 205', 'BRS 133', 'Fundacep 45', 'A 6001 RG', 'Mágica', 'AL72' e 'A 8100 RG', nas densidades de 250, 400 e 550 mil sementes aptas ha-1, na safra 2005/2006. O delineamento experimental utilizado foi blocos ao acaso, com parcelas subsubdivididas e quatro repetições. Foram avaliados o ciclo das cultivares, a altura de plantas, a altura de inserção do primeiro legume, o número de ramos planta-1 e de ramos m-2, o número de nós na haste principal planta-1 e na haste principal m-2, o rendimento biológico (RB) e o índice de colheita (IC). A semeadura em janeiro ocasiona redução no ciclo da cultura, na altura de plantas, na altura de inserção do primeiro legume, no número de nós planta-1 e de RB e no atraso da semeadura. O aumento da densidade de sementes não proporcionou aumento na estatura de plantas e altura de inserção do primeiro legume, nas cultivares RR. O período do ciclo que é mais afetado com atraso da semeadura é o vegetativo. O IC foi menor na semeadura em novembro; no entanto, o RB foi maior.

Monitoring of GMO in Brazilian processed meat and soy-based products from 2007 to 2008

Roundup Ready™ (RR) soybean is the first genetically modified organism (GMO) approved in Brazil. In this country, the labeling of foodstuffs containing GMOs is mandatory if the GMO content exceeds 1% (10 g kg −1). In order to monitor and quantify the presence of RR soybean in soy-based and processed meat products on the Brazilian food market, DNA was extracted from 59 food samples and analyzed by polymerase chain reaction (PCR) to amplify the soybean lectin gene, and by nested PCR to amplify the recombinant DNA present in RR soybean. Positive samples for the presence of RR soybean were subjected to a real-time quantification of GMO using a duplex real-time PCR. The results showed that out of 59 samples, 54 were positive for lectin gene and six were positive for RR soybean, 5 samples contained less than10 g kg −1 GMO and one sample contained more than 10 g kg −1 GMO.

Tensão superficial estática de misturas em tanque de glyphosate + chlorimuron-ethyl isoladas ou associadas com adjuvantes

Objetivou-se neste estudo avaliar a tensão superficial estática, o pH e a produção da espuma de misturas em tanque de glyphosate + chlorimuron-ethyl, associadas ou não com adjuvantes. Dois trabalhos foram realizados, sendo a primeira etapa desenvolvida em delineamento inteiramente casualizado com seis tratamentos e 20 repetições, representados por soluções dos herbicidas glyphosate (540,0 g e.a. ha-1) e chlorimuron-ethyl (7,5 g ha-1), isolados e em mistura em tanque nas formulações: Polaris®; Roundup Ready®; Classic®; Polaris® + Classic®; Roundup Ready® + Classic® e testemunha (água). Na segunda etapa foram estudados em DIC 70 tratamentos e 20 repetições, em fatorial 2 x 5 x 7, constituído por duas misturas de glyphosate com chlorimuron-ethyl (Polaris® + Classic® e Roundup Ready ® + Classic®), cinco adjuvantes (Joint Oil®; Nimbus®; Assist®; Natur' Oil® e Agr' Óleo®) e sete doses dos adjuvantes (0,000; 0,031; 0,062; 0,125; 0,250; 0,500 e 1,000% de v/v). As soluções de Polaris® e Roundup Ready®; isoladas ou em misturas com Classic®; caracterizaram-se por tensões superficiais estáticas de 43,2 e 35,9 mN m-1 e pH médios em torno de 4,5 a 4,6, respectivamente. As misturas de Polaris®+Classic®; Roundup Ready®+Classic® e Classic® apresentaram a maior quantidade e persistência da espuma ao longo do tempo, quando comparadas às formulações de Polaris® e Roundup Ready®. Os adjuvantes promoveram redução da tensão superficial quando associados às misturas de Polaris® + Classic® e Roundup Ready® + Classic®; caracterizando-se em termos de eficiência pela ordem decrescente: Nimbus® (36,5% e 25,8%) < Joint Oil® (32,2% e 25,2%) < Agr' Oil® (21,2% e 13,4%) < Natur' Oil® (17,4% e 10,6%) < Assist® (8,5% e 10,6%). Os adjuvantes promoveram redução da quantidade e persistência da espuma das misturas de Polaris® + Classic® e Roundup Ready® + Classic®; com pequenas variações dos níveis médios de pH das soluções inferiores a 0,5.

Quantitation using informative zeros (QUIZ): Application for GMO detection and quantification without recourse to certified reference material

The amount of genetically modified RoundUp Ready™ soya in admixture samples was determined using a method, quantitation using informative zeros that employs most probable number statistics to estimate molecule numbers. By measuring the numbers of lectin, acting as a reference marker, and GMO-specific targets within these samples, we were able to determine their GMO contents. Estimates derived from QUIZ were comparable with results obtained using conventional real-time Taqman® PCR. Results of GMO content determination using QUIZ is based on a ‘per genome’ units and estimations can be obtained using very small amounts of DNA. Certified reference materials, constructed using weight measurements, were not required for quantification.