Distribution and degradation of DNA from non-genetically and genetically modified soybean (Roundup Ready): Impact of soybean protein concentrate and soybean protein isolate preparation.

To improve genetically modified product labelling legislation and promote the development of genetically modified foods, the mass variations of genomic DNA and length distributions of DNA fragments in non-genetically and genetically modified soybean (Roundup Ready) and the variations in transgenic contents during soybean protein concentrate (SPC) and soybean protein isolate (SPI) preparation were monitored. The material masses throughout the process conformed to the law of mass conservation, and amounts of DNA recovered decreased with SPC and SPI preparation. The successive steps of ethanol extraction led to a decrease in the size distribution of the recovered DNA. For the LECTIN and CP4 EPSPS targets investigated, longer fragments (>800 bp) were more affected than smaller fragments (<200 bp), and both targets degraded slowly upon degradation into small fragments. DNA distribution and degradation thereby affected GMO quantification. After preparation procedures, the transgenic contents of SPC and SPI products were higher than that of raw soybean.

Fate of Glyphosate during Production and Processing of Glyphosate-Resistant Sugar Beet ( Beta vulgaris).

Glyphosate is a widely used herbicide in commercial crop production for both conventional and herbicide-resistant crops. Herbicide-resistant crops, like glyphosate-resistant sugar beet, are often exposed to multiple applications of glyphosate during the growing season. The fate of this herbicide in resistant crops has not been publicly documented. We investigated the fate of glyphosate and main metabolite aminomethylphosphonic acid in glyphosate-resistant sugar beet grown in northern Colorado. Glyphosate residues were measured via directed ultra-high-performance liquid chromatography tandem mass spectrometry analysis of sugar beet shoots and roots throughout the growing season, from samples collected at various steps during sugar beet processing, and from flow-through samples of greenhouse-grown beets. Sugar beet rapidly absorbed glyphosate after foliar application, and subsequently translocated the herbicide to its roots, with between 2 and 3 µg/g fresh weight measured in both tissue types within 1 week of application. However, only trace amounts of glyphosate remained in either the shoots or the roots 2 weeks after application. Analysis of irrigation flow-through in pot assays confirmed that the herbicide readily exuded out of the roots. Processing of the beets removed glyphosate and herbicide levels were below the limit of detection in the crystalline sugar final product.

Real-time PCR for traceability and quantification of genetically modified seeds in lots of non-transgenic soybean / PCR em tempo real para rastreabilidade e quantificação de sementes geneticamente modificados em lotes de soja não transgênica

The constant presence of genetically modified (GM) soybean in conventional seed lots has become a growing problem for international seed trade. In this context, seed companies have prompted the development of routine tests for accurate genetically modified soybean seeds detection. In this study, a quantitative PCR-based method was standardized in order to detect and quantify mixtures of seeds (i.e. certified seed) or GM grains (i.e. seeds came from field) into samples of non-GM soybean, in a way that soybean lots can be assessed within the standards established by legislation. The method involved the use of p35S-f2/petu-r1 primers targeting CP-4 enolpyruvylshikimate-3-phosphate synthase (cp4-epsps) gene (i.e. that confers herbicide tolerance in Roundup ReadyTM (RR)) for real-time PCR detection and quantification through mericon Quant GMO Detection Assay. The results revealed the method efficiency to detect and quantify the presence of even one soybean seed in batch used for routine evaluation of GM seeds. In addition, it was possible to detect of up to 0.1% of transgenic DNA relative to the soybean grains content. Thus, the sensitive GMO quantitative approach described in this study will provide support in supervising activities, and facilitate the process and control of GM soybean.

A constante presença da soja geneticamente modificada (GM) em lotes de sementes convencionais têm se tornado um grande problema para o comércio internacional de sementes. Neste contexto, as empresas de sementes estão em busca de testes de rotina extremamente precisos para a detecção de sementes de soja geneticamente modificadas. Neste estudo, um método baseado em PCR quantitativo foi padronizado para detectar e quantificar misturas de sementes (i.e. sementes certificadas) ou grãos geneticamente modificados (i.e. sementes oriundas do campo) dentro de lotes de soja não transgênica, de um modo que os lotes de soja possam ser avaliados dentro dos parâmetros estabelecidos pela legislação. O método envolveu o uso dos iniciadores p35S-f2/petu-r1 alvejando o gene CP-4 5-nolpiruvil-shikimato-3-fosfato sintase (cp4-epsps) (i.e. que confere a tolerância ao herbicida Roundup Ready® (RR)) para detecção e quantificação em PCR de tempo real via Ensaio de detecção Mericon Quant GMO. Os resultados revelaram um método eficiente para detectar e quantificar a presença de até mesmo uma única semente de soja no lote usado para a avaliação de rotina de sementes geneticamente modificadas. Adicionalmente, foi possível detectar até 0,1% de DNA transgênico relativo ao conteúdo de grãos de soja. Dessa forma, uma abordagem quantitativa sensível à soja geneticamente modificada foi descrita nesse estudo e poderá fornecer suporte em atividades de supervisão, além de facilitar o processo de controle da soja geneticamente modificada.

Targeting the middle region of CP4-EPSPS protein for its traceability in highly processed soy-related products.

Transgenic components in genetically modified organisms consist not only of the transgenic genes, but also the transgenic protein. However, compared with transgenic DNA, less attention has been paid to the detection of expressed protein, especially those degraded from genetically modified soybean after food processing. In this study, the full length 5-enolpyruvyl-shikimate-3-phosphate synthase (CP4-EPSPS, 47.6 kD) protein was probed with the SC-16 (S19-R33) and the DC-16 (D219-K233) polyclonal antibodies in immunoblots. Both antibodies were able to detect the full length CP4-EPSPS and its residues in soy powder made from Roundup-Ready soybeans after heating and microwaving treatments which also reduced the molecular weight of the protein to 45.8 and 38.7 kD, respectively. Taken together the immunoblot results suggest that the middle region of the CP4-EPSPS protein possessed better stability than its N-terminal during thermal processing. This deduction was further validated by autoclave treatment, where a 37.4 kD residue of the protein was recognized by DC-16. A similar result was obtained in processed smoked sausage containing Roundup Ready soybean protein isolate (as an extender). The additional use of a further polyclonal antibody CK-17 (C372-K388), showed that compared with only the one signal for CP4-EPSPS detected by the SC-16 and CK-17 antibodies, the DC-16 middle region antibody detected four signals for CP4-EPSPS from five market sourced soy protein concentrates. Taken together, the study suggested that the middle region of CP4-EPSPS was more useful than the N- and C-terminal for tracing transgenic CP4-EPSPS protein and its remnants in highly processed soy-related products.

Rapid screening of roundup ready soybean in food samples by a hand-held PCR device.

Insulated isothermal PCR (iiPCR) method was recently available for rapid on-site detection of roundup ready soybean (RRS; event GTS40-3-2) in food materials and products. Performance of this method was evaluated in this study. The 100% detection endpoint for the RRS by iiPCR was found in samples containing 0.1% RRS, equivalent to the results of the reference real-time PCR (rtPCR). Analysis of nucleic acids of soybean-based processed food products indicated 95% agreement between the iiPCR and rtPCR for RRS detection. By testing soybean milk and tofu samples using simple pretreatment methods, we found that the agreements between iiPCR and rtPCR methods of the aforementioned samples were 80% and 90%, respectively. Replicated tests of all discrepant samples implied that these samples had trace amounts of RRS, suggesting that the iiPCR system is more sensitive than the rtPCR method. In conclusion, the iiPCR technology can be a useful point-of-need tool to help make a timely decision in the consumption of genetically modified organisms.

Doses e épocas de aplicação de manganês via foliar no cultivo de soja convencional e em derivada transgênica RR / Rates and stages of foliar Mn application in the conventional soybean crop and its genetically modified RR descendants

O objetivo com este trabalho foi avaliar o efeito da aplicação foliar de manganês sobre as características agronômicas de cultivares de soja convencionais e em suas derivadas transgênicas RR (Roundup Ready®, evento GTS 40-3-2). O ensaio foi conduzido na Universidade Federal de Lavras (UFLA), Lavras, MG, em blocos casualizados, com três repetições e esquema fatorial 4 x 4 x 2, sendo quatro cultivares de soja, duas convencionais e suas derivadas RR (BRS Celeste e BRS Baliza RR; BRSGO Jataí e BRS Silvânia RR), quatro doses de Mn via foliar (0; 200; 400 e 600 g Mn ha-1) e dois estádios de aplicação (R1 ou R3). Foram avaliadas as características agronômicas: índice de acamamento; número de legumes por planta; peso de mil sementes e produtividade de sementes. A transgenia RR em soja não proporciona maior resposta à aplicação de Mn, sendo esta possivelmente condicionada ao genótipo. Há aumento na produtividade de sementes de soja das cultivares Celeste e Baliza RR em função da aplicação foliar de Mn, em solo com teor de Mn abaixo do nível crítico e produtividades máximas obtidas com 150 g Mn ha-1. A aplicação foliar de Mn realizada em R1 resulta em maior produtividade de sementes em relação à R3.

The aim of this study was to evaluate the influence of foliar Mn application in the agronomic characteristics of conventional cultivars soybean and its derived RR (Roundup Ready®, event GTS 40-3-2). The experiment was carried out at the Federal University of Lavras, MG state, using a randomized block design with three replications and a 4 x 4 x 2 factorial structure, with four soybean cultivars, two conventional and its derived RR (BRS Celeste and BRS Baliza RR; BRSGO Jataí and BRS Silvânia RR), four rates of foliar Mn (0; 200; 400 and 600 g Mn ha-1) and two stages of application (R1 or R3). It was evaluated the agronomic traits: lodging index, number of pods per plant, thousand seed weight and seeds yield. The response to the application of Mn might be conditioned to the genotype rather than to the RR genetic modification, with increase in seed yield of Baliza RR and Celeste with of foliar Mn application, in soil with Mn content below a critical level and maximum yields obtained with 150 g Mn ha-1. The foliar application of Mn in R1 results in a higher seed yield in relation to R3.

Efeitos do herbicida glifosato e da aplicação foliar de micronutrientes em soja transgênica / Effects of glyphosate and foliar application of micronutrients in transgenic soybean

A característica quelante do glifosato pode ocasionar a imobilização de nutrientes como Fe e Mn em soja transgênica resistente a este herbicida. No entanto, a intensidade deste efeito e o resultado de formas de mitigação deste processo são pouco conhecidos. O objetivo deste estudo foi identificar os efeitos do herbicida glifosato em interação com a fertilização foliar em cultivares de soja transgênica que apresentam a ocorrência de sintomatologia de amarelecimento relacionada à fitointoxicação deste herbicida. O primeiro experimento foi utilizado para a identificação de cultivares que mais apresentavam o sintoma de amarelecimento como consequência da aplicação do herbicida glifosato. O segundo experimento foi realizado a campo, onde os tratamentos constaram das cultivares CD 206RR e Fundacep 59RR, do herbicida glifosato aplicado nas doses de 0, 900 e 1440 g ha-1 e. a., e da presença ou ausência de adubação foliar na dose de 1,5 L ha-1. O efeito do herbicida glifosato e da utilização de adubo foliar não alterou o teor dos micronutrientes Fe e Mn e os parâmetros de fluorescência da clorofila nas cultivares de soja CD 206RR e Fundacep 59RR. O herbicida ghifosate diminui o rendimento de grãos da cultivar Fundacep 59 RR. A fertilização foliar não foi eficiente como forma de aumentar o rendimento de grãos de soja.

The chelant characteristic of glyphosate may result in the immobilization of micronutrients such as Fe and Mn in transgenic soybean resistant to this herbicide. However, the intensity of this effect and the results of mitigation practices are not fully understood. The objective of the study was to identify the effects of the herbicide glyphosate in interaction with the foliar application of micronutrients in two cultivars of transgenic soybean that demonstrated the occurrence of injury of this herbicide named yellow flashig. A preliminary experiment identified the larger intensity of yellow flashing in the cultivars CD 206RR and Fundacep 59 RR among seven cultivars evaluated. A field experiment was carried out where the treatments were the soybean cultivars described above, glyphosate at rates of 0,900 and 1440 g ha-1 e.a. and the presence or absence of the foliar fertilization of micronutrients at 1,5 L ha-1. The herbicide glyphosate and the foliar application of micronutrients did not change the concentration of Fe and Mn and the chlorophyll fluorescence in the cultivars CD 206RR e Fundacep 59RR. The herbicide glyphosate decrease the grain yield of the cultivar Fundacep 59RR and did not affect the cultvar CD 206RR. The foliar fertilization of micronutrients did not increase the soybean grain yield in the evaluated cultivars.

Parâmetros fisiológicos e nutricionais de cultivares de soja resistentes ao glifosato em comparação com cultivares isogênicas próximas / Physiological and nutritional evaluation of soybean resistant to glyphosate in comparison with near isogenic lines

Cultivares de soja resistentes ao herbicida glifosato podem apresentar variação de adaptação em função dos efeitos do processo de transformação e da consequência do herbicida glifosato sobre a disponibilidade de micronutrientes. O objetivo deste estudo foi avaliar a variação de adaptação em cultivares de soja resistentes a glifosato, em comparação com cultivares isogênicas próximas sobre parâmetros fisiológicos, nutricionais, de crescimento e no rendimento de grãos da cultura. Foi realizado experimento em campo, utilizando sete cultivares resistentes ao herbicida glifosato e seis cultivares convencionais, correspondentes a isolíneas próximas das cultivares transgênicas. O tratamento herbicida constou da ausência ou presença da aplicação do herbicida glifosato nas cultivares transgênicas, na dose de 900g ha-1 e. a. Os parâmetros de teores e fluorescência da clorofila não apresentaram relação com os tratamentos efetuados entre as cultivares avaliadas. A cultivar 'BRS Taura RR' apresentou menor acúmulo de Fe foliar, em comparação com a sua linha isogênica BRS 154, devido à aplicação do herbicida glifosato. A aplicação do herbicida glifosato reduziu o rendimento de grãos de três cultivares resistentes. Os resultados encontrados indicam a existência de variação entre cultivares de soja resistentes a glifosato em relação ao efeito deste herbicida sobre parâmetros nutricionais, de fluorescência da clorofila e rendimento de grãos.

Fitness penalty in glyphosate-resistant (GR) soybean can occur due to the effects of transformation process and as a consequence of the glyphosate on the availability of micronutrients. The aim of this study was to evaluate the effect of glyphosaste on physiological, nutritional, and growth parameters and grain yield in 'GR' soybean cultivars in comparison with near isogenenic lines. The field experiment was conducted using seven 'GR' cultivars and six near isogenic cultivars. The herbicide glyphosate was applied or not in 'GR' cultivars at a dose of 900g ha-1 i.a. The parameters of chlorophyll fluorescence were not related with the ghlyphosate effect on the evaluated cultivars. The cultivar 'BRS Taura RR' had lower leaf Fe accumulation compared with its isogenic line BRS 154 due to application of the herbicide glyphosate. The application of glyphosate at a dose of 900g ha-1 reduced the grain yield of three 'GR' cultivars. The obtained results indicate the occurance of variability among soybean cultivars resistant to ghyphosate in relation to the efect of this herbicide on nutrient availability, chlorophyll fluorescence and grain yield.

Métodos para detecção de soja Roundup Ready® em grãos e produtos de soja por reação em cadeia da polimerase: revisão e análise crítica das práticas de validação / Methods for detecting the Roundup Ready® soy in soybeans and soy products by polymerase chain reaction: a review and critical analysis of current practices of validation

A informação sobre a origem transgênica de alimentos é muito relevante. Conforme a legislação brasileira e de outros países, o consumidor deve ser informado da natureza transgênica dos alimentos ou ingredientes que contenham ou que sejam produzidos a partir de organismos geneticamente modificados (OGM), com presença acima de um limite estabelecido. A necessidade de monitorar a presença e determinar o percentual de OGM em alimentos tem gerado uma constante demanda pelo desenvolvimento de metodologias capazes de detectar, identificar e quantificar o DNA exógeno. Entretanto, esses métodos necessitam ser validados para garantir confiabilidade aos resultados. No presente trabalho, a validação de métodos para detecção de soja Roundup Ready® em grãos e produtos de soja por reação em cadeia de polimerase foi contextualizada.Considerando-se atuais tendências em validação de métodos, os guias para validação de métodos específicos para OGM não contemplam todos os parâmetros necessários para avaliar a adequação aos propósitos de uso dos métodos, principalmente no caso de métodos qualitativos. Os parâmetros de desempenho mais frequentemente citados na literatura foram precisão (repetitividade), sensibilidade, linearidade e veracidade para metodologias quantitativas e taxas de sensibilidade e seletividade para qualitativas. Contudo, importantes parâmetros têm sido negligenciados nos processos de validação de métodos deste escopo analítico...

Analysis of genetically modified organisms by pyrosequencing on a portable photodiode-based bioluminescence sequencer.

A portable bioluminescence analyser for detecting the DNA sequence of genetically modified organisms (GMOs) was developed by using a photodiode (PD) array. Pyrosequencing on eight genes (zSSIIb, Bt11 and Bt176 gene of genetically modified maize; Lectin, 35S-CTP4, CP4EPSPS, CaMV35S promoter and NOS terminator of the genetically modified Roundup ready soya) was successfully detected with this instrument. The corresponding limit of detection (LOD) was 0.01% with 35 PCR cycles. The maize and soya available from three different provenances in China were detected. The results indicate that pyrosequencing using the small size of the detector is a simple, inexpensive, and reliable way in a farm/field test of GMO analysis.

Differences between conventional and glyphosate tolerant soybeans and moisture effect in their discrimination by near infrared spectroscopy.

Previous studies showed that Near Infrared Spectroscopy (NIRS) could distinguish between Roundup Ready® (RR) and conventional soybeans at the bulk and single seed sample level, but it was not clear which compounds drove the classification. In this research the varieties used did not show significant differences in major compounds between RR and conventional beans, but moisture content had a big impact on classification accuracies. Four of the five RR samples had slightly higher moistures and had a higher water uptake than their conventional counterparts. This could be linked with differences in their hulls, being either compositional or morphological. Because water absorption occurs in the same region as main compounds in hulls (mainly carbohydrates) and water causes physical changes from swelling, variations in moisture cause a complex interaction resulting in a large impact on discrimination accuracies.

Presence of CP4-EPSPS component in roundup ready soybean-derived food products.

With the widespread use of Roundup Ready soya (event 40-3-2) (RRS), the traceability of transgenic components, especially protein residues, in different soya-related foodstuffs has become an important issue. In this report, transgenic components in commercial soya (including RRS) protein concentrates were firstly detected by using polymerase chain reaction (PCR) and western blot. The results illustrated the different degradation patterns of the cp4-epsps gene and corresponding protein in RRS-derived protein concentrates. Furthermore, western blot was applied to investigate the single factor of food processing and the matrix on the disintegration of CP4-EPSPS protein in RRS powder and soya-derived foodstuffs, and trace the degradation patterns during the food production chain. Our results suggested that the exogenous full length of CP4-EPSPS protein in RRS powder was distinctively sensitive to various heat treatments, including heat, microwave and autoclave (especially), and only one degradation fragment (23.4 kD) of CP4-EPSPS protein was apparently observed when autoclaving was applied. By tracing the protein degradation during RRS-related products, including tofu, tou-kan, and bean curd sheets, however, four degradation fragments (42.9, 38.2, 32.2 and 23.4 kD) were displayed, suggesting that both boiling and bittern adding procedures might have extensive effects on CP4-EPSPS protein degradation. Our results thus confirmed that the distinctive residues of the CP4-EPSPS component could be traced in RRS-related foodstuffs.

Desempenho de cultivares transgênicas de soja em sucessão a culturas de inverno em semeadura direta / Performance of transgenic soybean cultivars in succession to winter cover crops under no tillage system

Objetivou-se com esse trabalho avaliar o desempenho de cultivares de soja transgênica em sucessão a culturas de inverno na implantação do sistema de semeadura direta. O delineamento experimental utilizado foi o de blocos casualizados, em esquema de parcelas subdivididas, com quatro repetições. As parcelas foram representadas por cinco espécies cultivadas no inverno, aveia branca, nabo forrageiro, cevada, trigo e ervilha forrageira e área de pousio. As subparcelas foram constituídas por seis cultivares de soja (BRS 243 RR, BRS 244 RR, BRS 245 RR, BRS 247 RR, BRS 255 RR e BRS 256 RR). Para as características agronômicas e os componentes de produção da cultura da soja indicou que a interação culturas de inverno x cultivares de soja foi significativa para a população final de plantas, número de nódulos por planta, massa de matéria seca de nódulos por planta, número de vagens por planta, número de grãos por vagem e produtividade de grãos. O tratamento de pousio proporcionou os menores valores de nodulação da cultura da soja. Para produtividade de grãos, houve diferenças entre as culturas de inverno antecessoras para o cultivar BRS 243 RR, sendo que, a aveia proporcionou maiores valores.

The objective of this research was to evaluate crop yield and some characteristics and yield components of transgenic soybean cultivars sown after different winter cover crops in the first year under no tillage system. The experimental design was the completely randomized block with split plots and four replications. The main plots consisted of five winter cover crops, white oat (Avena sativa L.), forage turnip (Raphanus sativus L.), barley (Hordeum vulgare L.), wheat (Triticum aestivum L.) and ground pea (Pisum sativum L.) and an area under fallow (spontaneous vegetation). The subplots consisted of six soybean cultivars (BRS 243 RR, BRS 245 RR, BRS 247 RR, BRS 255 RR, BRS 256 RR and BRS 244 RR). Variance analysis for agronomic characteristics showed that soybean yield components were influenced by the interaction between winter crop and soybean cultivar. Thus, final population, number of nodes and pods per plant, nodes dry matter per plant, number of grains per pod and grain yield were affected significantly. When soybean nodulation was evaluated, the treatment with the area under fallow showed lower values. There was difference among winter crops for BRS 243 RR grain yield, white oat showed the highest values.

Application of DNA Hybridization Biosensor as a Screening Method for the Detection of Genetically Modified Food Components.

An electrochemical biosensor for the detection of genetically modified food components is presented. The biosensor was based on 21-mer single-stranded oligonucleotide (ssDNA probe) specific to either 35S promoter or nos terminator, which are frequently present in transgenic DNA cassettes. ssDNA probe was covalently attached by 5'-phosphate end to amino group of cysteamine self-assembled monolayer (SAM) on gold electrode surface with the use of activating reagents - water soluble 1-ethyl-3(3'- dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxy-sulfosuccinimide (NHS). The hybridization reaction on the electrode surface was detected via methylene blue (MB) presenting higher affinity to ssDNA probe than to DNA duplex. The electrode modification procedure was optimized using 19-mer oligoG and oligoC nucleotides. The biosensor enabled distinction between DNA samples isolated from soybean RoundupReady® (RR soybean) and non-genetically modified soybean. The frequent introduction of investigated DNA sequences in other genetically modified organisms (GMOs) give a broad perspectives for analytical application of the biosensor.