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Polymerase chain reaction (PCR) technology has become a standard technique for the detection of genetically modified organisms (GMOs). However, this method requires a PCR amplification process which is both expensive and time-consuming. Herein, we propose electric field-induced release and measurement (EFIRM) technology as an alternative method for GMO screening. The specificity and sensitivity of the EFIRM assay were proven to be comparable to those of the real-time PCR method for detecting genetically modified soybeans. After all the parameters had been evaluated, the actual evaluation of soybean samples from soybean cargoes was performed. An actual EFIRM screening was performed on 157 soybean cargo samples, which had 102 transgenic soybean samples containing the GTS-40-3-2 gene, through a blind trial at the Dalian port of China. Our results showed that 101 transgenic soybean samples were correctly detected, with only one false-negative case, and 55 non-transgenic soybean samples were detected as negative; this demonstrates that the EFIRM assay is an effective, accurate, simple, and economical novel method for detecting transgenic products, which may have a positive impact on the development of rapid on-site GMO monitoring platforms., (© 2021. Springer-Verlag GmbH Germany, part of Springer Nature.)

The transgenic glyphosate-tolerant soybean MON87712 event was developed by the agrochemical and agricultural biotechnology company Monsanto (USA) and commercialized in 2013. Due to the absence of matrix-based and genomic DNA-positive reference material for MON87712, it is very difficult to detect and monitor this event. In this study, we developed a recombinant 760-bp linearized plasmid, including 150 bp of the soybean endogenous lectin gene and 610 bp of the exogenous BBX32 gene plus its 3' flanking sequence of MON87712 by In-Fusion cloning technology. In addition, a duplex real-time polymerase chain reaction for the detection of MON87712 and the soybean endogenous lectin gene was established. By using this method, we achieved specific and quantitative detection of MON87712 in 45 other kinds of crops, with a detection limit of 10 copies/μl. This method provides a new technical means for the accurate detection of transgenic soybean MON87712, as well as technical support for the supervision of agricultural transgenic organisms., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The releasing of transgenic soybeans (Glycine max (L.) Merr.) into farming systems raises concerns that transgenes might escape from the soybeans via pollen into their endemic wild relatives, the wild soybean (Glycine soja Sieb. et Zucc.). The fitness of F1 hybrids obtained from 10 wild soybean populations collected from China and transgenic glyphosate-resistant soybean was measured without weed competition, as well as one JLBC-1 F1 hybrid under weed competition. All crossed seeds emerged at a lower rate from 13.33-63.33%. Compared with those of their wild progenitors, most F1 hybrids were shorter, smaller, and with decreased aboveground dry biomass, pod number, and 100-seed weight. All F1 hybrids had lower pollen viability and filled seeds per plant. Finally, the composite fitness of nine F1 hybrids was significantly lower. One exceptional F1 hybrid was IMBT F1, in which the composite fitness was 1.28, which was similar to that of its wild progenitor due to the similarities in pod number, increased aboveground dry biomass, and 100-seed weight. Under weed competition, plant height, aboveground dry biomass, pod number per plant, filled seed number per plant, and 100-seed weight of JLBC-1 F1 were lower than those of the wild progenitor JLBC-1. JLBC-1 F1 hybrids produced 60 filled seeds per plant. Therefore, F1 hybrids could emerge and produce offspring. Thus, effective measures should be taken to prevent gene flow from transgenic soybean to wild soybean to avoid the production F1 hybrids when releasing transgenic soybean in fields in the future.

Low availability of inorganic phosphate (Pi) in soil is often a limiting factor for plant growth and productivity. The Pi transporter plays an important role in the absorption and utilization of phosphorus in plants. Eutrema salsugineum shows strong tolerance under Pi stress conditions, but the function of the E. salsugineum PHT1 genes has not yet been systematically studied. This study isolated a phosphate transporter gene (EsPHT1;1) from the halophyte E. salsugineum and functionally characterized it in the herbaceous model plant, Arabidopsis thaliana, and in an important oil crop species, soybean (Glycine max (L.) Merr.). Under Pi deficient conditions, transgenic Arabidopsis and transgenic soybean grew better and exhibited significant improvement in root growth, biomass accumulation and seed yield compared with wild-type (WT) plants. These phenotypic enhancements were more apparent under inadequate Pi conditions than under sufficient or no Pi conditions, which is in agreement with the observation that the transgenic plants accumulated higher amounts of Pi and total P in shoots and roots than WT plants only when inadequate Pi was supplied. The results of the present study indicate that overexpression of EsPHT1;1 can efficiently enhance the growth and reproductive performance of both Arabidopsis and soybean plants challenged by low P stress, which results confirm the important role of PHT1;1 in dealing with Pi deficiency., (© 2020 German Society for Plant Sciences and The Royal Botanical Society of the Netherlands.)

A new polyclonal antibody that recognizes the CP4 5-enolpyruvylshikimate-3-phosphate synthase (CP4-EPSPS), which provides resistance to glyphosate in soybean (Roundup Ready®, RR soybean), was produced. New Zealand rabbits were injected with a synthetic peptide (Pc_312-324, (PEP)) present in the soybean CP4-EPSPS protein. The anti-PEP antibodies production was evaluated by electrophoresis (SDS-PAGE) and an enzyme-linked immunosorbent assay (ELISA) was developed in order to study their specificity. The ELISA showed that the polyclonal antibody was specific to PEP. In addition, the anti- PEP was immobilized onto a gold disk electrode and the antigen-antibody interaction was evaluated using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Moreover, the EIS showed that the electron transfer resistance of the modified electrode increased after incubation with solutions containing CP4-EPSPS protein from RR transgenic soybean, while no changes were detected after incubation with no-RR soybean proteins. These results suggest that the CP4-EPSPS was immobilized onto the electrode, due to the specific interaction with the anti-PEP. These results show that this antigen-antibody interaction can be detected by electrochemical techniques, suggesting that the anti-PEP produced can be used in electrochemical immunosensors development to quantify transgenic soybean., (Copyright © 2018. Published by Elsevier B.V.)

Background: Cry8-like from Bacillus thuringiensis (Bt) encodes an insecticidal crystal (Cry) protein. Holotrichia parallela (Coleoptera: Scarabaeoidae), commonly known as the dark black chafer, is a troublesome pest of soybean (Glycine max). To test whether cry8-like can confer resistance against H. parallela to soybean, we introduced cry8-like from the Bt strain HBF-18 into soybean cultivar Jinong 28., Results: Quantitative reverse transcription-PCR analysis demonstrated that cry8-like was expressed most highly in soybean leaves. In addition, Southern blot assays revealed that one copy of the integrated fragment was present in the transformed plants. Eight independent cry8-like transgenic lines were subsequently fed on by H. parallela. Under H. parallela feeding stress, the survival rates of the non-transgenic plants were 92% lower than those of the transgenic plants. The mortality rate of H. parallela increased when the larvae fed on the roots of T 1 transgenic soybean plants. Moreover, the surviving larvae were deformed, and their growth was inhibited., Conclusions: Collectively, our data suggest that transgenic soybean plants expressing the cry8-like gene are more resistant to H. parallela than non-transgenic plants and that transgenic expression of the cry8-like gene may represent a promising strategy for engineering pest tolerance. The events generated in this study could thus be utilized in soybean breeding programs.

Xyloglucan endotransglycosylases/hydrolases (XTHs) are a class of enzymes involved in the construction and remodeling of cellulose/xyloglucan crosslinks and play an important role in regulating cell wall extensibility. However, little is known about this class of enzymes in soybean. Here, 61 soybean XTH genes ( GmXTH s) were identified and classified into three subgroups through comparative phylogenetic analysis. Genome duplication greatly contributed to the expansion of GmXTH genes in soybean. A conserved amino acid motif responsible for the catalytic activity was identified in all GmXTHs. Further expression analysis revealed that most GmXTHs exhibited a distinct organ-specific expression pattern, and the expression level of many GmXTH genes was significantly associated with ethylene and flooding stress. To illustrate a possible role of XTH genes in regulating stress responses, the Arabidopsis AtXTH31 gene was overexpressed in soybean. The generated transgenic plants exhibited improved tolerance to flooding stress, with a higher germination rate and longer roots/hypocotyls during the seedling stage and vegetative growth stages. In summary, our combined bioinformatics and gene expression pattern analyses suggest that GmXTH genes play a role in regulating soybean stress responses. The enhanced soybean flooding tolerance resulting from the expression of an Arabidopsis XTH also supports the role of XTH genes in regulating plant flooding stress responses.

The soybean pod borer [SPB; Leguminivora glycinivorella (Mats.) Obraztsov] is a major soybean pest in northeastern Asia. A useful method for addressing this problem is the generation of transgenic plants producing double-stranded RNA (dsRNA) that target essential insect genes. In this study, we confirmed that 18S ribosomal RNA is critical for SPB development. Downregulated Spb18S expression induced by dsRNA injection increased larval mortality rates and resulted in early pupation. We also assessed whether Spb18S is silenced in SPB larvae fed on transgenic soybean expressing Spb18S dsRNA. Transgenic plants downregulated Spb18S expression levels and second-instar larval survival rates. Moreover, such plants were less damaged by SPB larvae than control plants under field conditions., (© 2018 Wiley Periodicals, Inc.)

Event DAS-444Ø6-6 soybean is genetically modified (GM) to provide tolerance to 2,4-diclorophenoxyacetic acid (2,4-D), glyphosate, and glufosinate herbicides through expression of the AAD-12, 2mEPSPS, and PAT proteins, respectively. DAS-444Ø6-6 soybeans were evaluated for safety in subchronic rat feeding studies. The results from two previous subchronic rat feeding studies evaluating diets formulated with 20% inclusion of DAS-444Ø6-6 soybean meal (the latter also containing DAS-444Ø6-6 derived hulls and oil) did not show any treatment-related adverse effects. In 2017, to comply with recent guidance from EFSA, a third 90-day rat feeding study was conducted with Sprague-Dawley rats (16/sex/group) with diets formulated either with 15% or 30% w/w of toasted DAS-444Ø6-6 soybean meal. DAS-444Ø6-6 soybean hulls and oil were also added to the transgenic test diets at 1% or 2% w/w and 1.35% or 2.7%, respectively, for the low- and high-dose groups. No toxicologically significant effects were observed under the conditions of this study., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: The soybean pod borer [SPB; Leguminivora glycinivorella (Matsumura) (Lepidoptera: Tortricidae)] is the most important soybean pest in northeastern Asia. Silencing genes using plant-mediated RNA-interference is a promising strategy for controlling SPB infestations. The ribosomal protein P0 is important for protein translation and DNA repair in the SPB. Thus, transferring P0 double-stranded RNA (dsRNA) into plants may help prevent SPB-induced damage., Results: We investigated the effects of SpbP0 dsRNA injections and SpbP0 dsRNA-expressing transgenic soybean plants on the SPB. Larval mortality rates were greater for SpbP0 dsRNA-injected larvae (96%) than for the control larvae (31%) at 14 days after injections. Transgenic T 2 soybean plants expressing SpbP0 dsRNA sustained less damage from SPB larvae than control plants. In addition, the expression level of the SpbP0 gene decreased and the mortality rate increased when SPB larvae were fed on T 3 transgenic soybean pods. Moreover, the surviving larvae were deformed and exhibited inhibited growth., Conclusion: Silencing SpbP0 expression is lethal to the SPB. Transgenic soybean plants expressing SpbP0 dsRNA are more resistant to the SPB than wild-type plants. Thus, SpbP0 dsRNA-expressing transgenic plants may be useful for controlling insect pests. © 2017 Society of Chemical Industry., (© 2017 Society of Chemical Industry.)

A 90-day sub chronic toxicity study was conducted in rats to evaluate the safety of genetically modified DAS-444Ø6-6 soybeans expressing herbicide tolerant proteins when compared with its conventional comparators (non-transgenic near isoline control soybean and three commercially available non-transgenic line control soybeans). Rats were given diets formulated with either 10% or 20% w/w of soybean meal and 1% or 2% hulls of DAS-444Ø6-6 soybean with an equivalent amount of hulls from an isoline non-transgenic control soybean for at least 90 days. In addition, three separate 20% w/w non-transgenic commercially available soybean varieties were also given to groups of rats to serve as reference controls. Animals were evaluated by cage-side and hand-held detailed clinical observations, ophthalmic examinations, body weights/body weight gains, feed consumption, hematology, prothrombin time, urinalysis, clinical chemistry, selected organ weights, and gross and histopathologic examinations. Under the conditions of this study, the genetically modified DAS-444Ø6-6 diets did not cause any treatment-related effects in rats following 90 days of dietary administration as compared with rats fed diets with soybean of isoline control or commercial reference controls and are considered equivalent to the diets prepared from conventional comparators., (Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

With the increasing concern of potential health and environmental risk, it is essential to develop reliable methods for transgenic soybean detection. Herein, a simple, sensitive and selective assay was constructed based on homogeneous fluorescence resonance energy transfer (FRET) between CdTe quantum dots (QDs) and multiwalled carbon nanotubes@graphene oxide nanoribbons (MWCNTs@GONRs) to form the fluorescent "on-off-on" switching for simultaneous monitoring dual target DNAs of promoter cauliflower mosaic virus 35s (P35s) and terminator nopaline synthase (TNOS) from transgenic soybean. The capture DNAs were immobilized with corresponding QDs to obtain strong fluorescent signals (turning on). The strong π-π stacking interaction between single-stranded DNA (ssDNA) probes and MWCNTs@GONRs led to minimal background fluorescence due to the FRET process (turning off). The targets of P35s and TNOS were recognized by dual fluorescent probes to form double-stranded DNA (dsDNA) through the specific hybridization between target DNAs and ssDNA probes. And the dsDNA were released from the surface of MWCNTs@GONRs, which leaded the dual fluorescent probes to generate the strong fluorescent emissions (turning on). Therefore, this proposed homogeneous assay can be achieved to detect P35s and TNOS simultaneously by monitoring the relevant fluorescent emissions. Moreover, this assay can distinguish complementary and mismatched nucleic acid sequences with high sensitivity. The constructed approach has the potential to be a tool for daily detection of genetically modified organism with the merits of feasibility and reliability., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In this work, a novel homogeneous assay for DNA quantitative analysis based on förster resonance energy transfer (FRET) was developed for cauliflwer mosaic virus 35s (CaMV35S) promoter of transgenic soybean detection. The homogenous FRET of fluorescence signal was fabricated by DNA hybridization with probe modified nitrogen-doped graphene quantum dots (NGQDs) and silver nanoparticles (AgNPs), which acted the donor-acceptor pairs for the first time. The highly efficient FRET and unique properties of the NGQDs made the proposed FRET system as a functionalized detection platform for labelling of DNA. Upon the recognition of specific target DNA (tDNA), the FRET between NGQDs and AgNPs was triggered to produce fluorescence quenching, which could be used for tDNA detection. The fabricated homogeneous FRET assay displayed a wide linear range of 0.1-500.0 nM and a low limit of detection 0.03 nM for the detection of CaMV35S (S/N = 3). This proposed biosensor revealed high specificity to detect tDNA, with acceptable intra-assay precision and excellent stability. This method was successfully applied to identify the real sample of 0.5% containing transgenic soybean, which achieved the most of national law regulations. This assay was further validated by polymerase chain reaction as the genetically modified organisms, suggesting that the proposed FRET system is a feasible tool for the further daily genetically modified organism detection., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A methodology for detection of the Cauliflower Mosaic Virus 35S(CaMV35S) promoter was developed to distinguish transgenic from non-transgenic soybean samples by using photoelectrochemical (PEC) biosensor. In this PEC biosensing system, the as-prepared gold nanoparticles-reduced graphene oxide acted as a nanocarrier to immobilize the thiol-functional probe (probe1), and the SiO 2 @CdTe quantum dots (QDs) core-shell nanoparticles tagged with the amino-functional probe (probe2) acted as signal indicators, respectively. In the presence of target DNA (tDNA) of CaMV35S, the binding of tDNA with probe1 and probe2 through the high specific DNA hybridization led to the fabrication of sandwich structure, and thus the high loading of the signal indicators SiO 2 @CdTe QDs at the electrode surface, which increased the PEC signal. The increased PEC signal depended on the concentration of tDNA, and a wide linear range from 0.1pM to 0.5nM with low detection limit of 0.05pM was obtained. In addition, the PEC biosensor has been successfully used for discriminating transgenic soybean from non-transgenic samples, which was consistent with the polymerase chain reaction (PCR) results, suggesting the proposed PEC biosensor is a feasible tool for the further daily genetically modified organism detection., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Soybean (Glycine max (L.) Merr.) salicylic acid methyl transferase (GmSAMT1) catalyses the conversion of salicylic acid to methyl salicylate. Prior results showed that when GmSAMT1 was overexpressed in transgenic soybean hairy roots, resistance is conferred against soybean cyst nematode (SCN), Heterodera glycines Ichinohe. In this study, we produced transgenic soybean overexpressing GmSAMT1 and characterized their response to various SCN races. Transgenic plants conferred a significant reduction in the development of SCN HG type 1.2.5.7 (race 2), HG type 0 (race 3) and HG type 2.5.7 (race 5). Among transgenic lines, GmSAMT1 expression in roots was positively associated with SCN resistance. In some transgenic lines, there was a significant decrease in salicylic acid titer relative to control plants. No significant seed yield differences were observed between transgenics and control soybean plants grown in one greenhouse with 22 °C day/night temperature, whereas transgenic soybean had higher yield than controls grown a warmer greenhouse (27 °C day/23 °C night) temperature. In a 1-year field experiment in Knoxville, TN, there was no significant difference in seed yield between the transgenic and nontransgenic soybean under conditions with negligible SCN infection. We hypothesize that GmSAMT1 expression affects salicylic acid biosynthesis, which, in turn, attenuates SCN development, without negative consequences to soybean yield or other morphological traits. Thus, we conclude that GmSAMT1 overexpression confers broad resistance to multiple SCN races, which would be potentially applicable to commercial production., (© 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

The transgenic soybean [Glycine max (L.) Merrill] occupies about 80 % of the global area cropped with this legume, the majority comprising the glyphosate-resistant trait (Roundup Ready(®), GR or RR). However, concerns about possible impacts of transgenic crops on soil microbial communities are often raised. We investigated soil chemical, physical and microbiological properties, and grain yields in long-term field trials involving conventional and nearly isogenic RR transgenic genotypes. The trials were performed at two locations in Brazil, with different edaphoclimatic conditions. Large differences in physical, chemical and classic microbiological parameters (microbial biomass of C and N, basal respiration), as well as in grain production were observed between the sites. Some phyla (Proteobacteria, Actinobacteria, Acidobacteria), classes (Alphaproteobacteria, Actinomycetales, Solibacteres) and orders (Rhizobiales, Burkholderiales, Myxococcales, Pseudomonadales), as well as some functional subsystems (clustering-based subsystems, carbohydrates, amino acids and protein metabolism) were, in general, abundant in all treatments. However, bioindicators related to superior soil fertility and physical properties at Londrina were identified, among them a higher ratio of Proteobacteria:Acidobacteria. Regarding the transgene, the metagenomics showed differences in microbial taxonomic and functional abundances, but lower in magnitude than differences observed between the sites. Besides the site-specific differences, Proteobacteria, Firmicutes and Chlorophyta were higher in the transgenic treatment, as well as sequences related to protein metabolism, cell division and cycle. Although confirming effects of the transgenic trait on soil microbiome, no differences were recorded in grain yields, probably due to the buffering capacity associated with the high taxonomic and functional microbial diversity observed in all treatments.

Background: Genetically modified MON 87701 × MON 89788 soybean (Glycine max) that expresses the Cry1Ac and EPSP-synthase proteins is a new Integrated Pest Management (IPM) tool for controlling Heliothis virescens in Brazil. To Support Insect Resistance Management (IRM) programs and understand the value of this event for IPM, we conducted laboratory and field studies to assess the biological activity of Cry1Ac protein expressed on Bt soybean against this insect pest., Results: Heliothis virescens was highly susceptible to purified Cry1Ac protein [LC50 (FL 95%) = 0.026 (0.021-0.033) µg Cry1Ac mL(-1) diet]. In bioassays with freeze-dried MON 87701 × MON 89788 soybean tissue diluted 25 times in an artificial diet, there was 100% mortality of H. virescens. In bioassays with leaf disc, young trifoliate, flower buds, pods and high artificial infestation under greenhouse condition, MON 87701 × MON 89788 soybean showed a high level of control against H. virescens. Moreover, larvae from first through fifth instar were highly susceptible to MON 87701 × MON 89788 soybean., Conclusions: The MON 87701 × MON 89788 soybean provides an effective alternative in controlling Heliothis virescens and complies with the concept of high-dose for IRM programs., (© 2013 Society of Chemical Industry.)