Transgenic Soybean Research Articles

Improved salt-tolerance of transgenic soybean by stable over-expression of AhBADH gene from Atriplex hortensis.

The salt-tolerance of transgenic soybean cleared for environmental release was improved by stable over-expression of AhBADH gene from Atriplex hortensis, which was demonstrated through molecular analysis and field experiments. An effective strategy for increasing the productivity of major crops under salt stress conditions is the development of transgenics that harbor genes responsible for salinity tolerance. Betaine aldehyde dehydrogenase (BADH) is a key enzyme involved in the biosynthesis of the osmoprotectant, glycine betaine (GB), and osmotic balance in plants, and several plants transformed with BADH gene have shown significant improvements in salt tolerance. However, very few field-tested transgenic cultivars have been reported, as most of the transgenic studies are limited to laboratory or green house experiments. In this study, we demonstrated through field experiments that AhBADH from Atriplex hortensis confers salt tolerance when transformed into soybean (Glycine max L.). AhBADH was successfully introduced into soybean by Agrobacterium mediated transformation. A total of 256 transgenic plants were obtained, out of which 47 lines showed significant enhancement of salt tolerance compared to non-transgenic control plants. Molecular analyses of the transgenic line TL2 and TL7 with the highest salt tolerance exhibited stable inheritance and expression of AhBADH in progenies with a single copy insertion. TL1, TL2 and TL7 exhibited stable enhanced salt tolerance and improved agronomic traits when subjected to 300mM NaCl treatment. Currently, the transgenic line TL2 and TL7 with stable enhanced salt tolerance, which have been cleared for environmental release, are under biosafety assessment. TL 2 and TL7 stably expressing AhBADH could then be applied in commercial breeding experiments to genetically improve salt tolerance in soybean.

GmJAZ3 interacts with GmRR18a and GmMYC2a to regulate seed traits in soybean.

Seed weight is usually associated with seed size and is one of the important agronomic traits that determine yield. Understanding of seed weight control is limited, especially in soybean plants. Here we show that Glycine max JASMONATE-ZIM DOMAIN 3 (GmJAZ3), a gene identified through gene co-expression network analysis, regulates seed-related traits in soybean. Overexpression of GmJAZ3 promotes seed size/weight and other organ sizes in stable transgenic soybean plants likely by increasing cell proliferation. GmJAZ3 interacted with both G. max RESPONSE REGULATOR 18a (GmRR18a) and GmMYC2a to inhibit their transcriptional activation of cytokinin oxidase gene G. max CYTOKININ OXIDASE 3-4 (GmCKX3-4), which usually affects seed traits. Meanwhile, the GmRR18a binds to the promoter of GmMYC2a and activates GmMYC2a gene expression. In GmJAZ3-overexpressing soybean seeds, the protein contents were increased while the fatty acid contents were reduced compared to those in the control seeds, indicating that the GmJAZ3 affects seed size/weight and compositions. Natural variation in JAZ3 promoter region was further analyzed and Hap3 promoter correlates with higher promoter activity, higher gene expression and higher seed weight. The Hap3 promoter may be selected and fixed during soybean domestication. JAZ3 orthologs from other plants/crops may also control seed size and weight. Taken together, our study reveals a novel molecular module GmJAZ3-GmRR18a/GmMYC2a-GmCKXs for seed size and weight control, providing promising targets during soybean molecular breeding for better seed traits.

Transgenic soybean of GsMYB10 shapes rhizosphere microbes to promote resistance to aluminum (Al) toxicity

Plant resistance genes could affect rhizosphere microbiota, which in turn enhanced plant resistance to stresses. Our previous study found that overexpression of the GsMYB10 gene led to enhanced tolerance of soybean plants to aluminum (Al) toxicity. However, whether GsMYB10 gene could regulate rhizosphere microbiota to mitigate Al toxicity remains unclear. Here, we analyzed the rhizosphere microbiomes of HC6 soybean (WT) and transgenic soybean (trans-GsMYB10) at three Al concentrations, and constructed three different synthetic microbial communities (SynComs), including bacterial, fungal and cross-kingdom (bacteria and fungi) SynComs to verify their role in improving Al tolerance of soybean. Trans-GsMYB10 shaped the rhizosphere microbial communities and harbored some beneficial microbes, such as Bacillus, Aspergillus and Talaromyces under Al toxicity. Fungal and cross-kingdom SynComs showed a more effective role than the bacterial one in resistance to Al stress, and these SynComs helped soybean resist Al toxicity via affecting some functional genes that involved cell wall biosynthesis and organic acid transport etc. Overall, this study reveals the mechanism of soybean functional genes regulating the synergistic resistance of rhizosphere microbiota and plants to Al toxicity, and also highlights the possibility of focusing on the rhizobial microbial community as a potential molecular breeding target to produce crops.

Arbuscular mycorrhizal fungus Rhizophagus irregularis alleviates drought stress in soybean with overexpressing the GmSPL9d gene by promoting photosynthetic apparatus and regulating the antioxidant system

Drought is the most destructive abiotic stress and negatively affects crop growth and productivity. Modern breeding efforts have produced numerous cultivars with distinct genetic traits that improve crop growth and drought stress tolerance. Arbuscular mycorrhizal fungi (AMF) can enhance drought tolerance in soybean plants by directly providing nutrients to plants, promoting photosynthesis, or influencing interspecific plant interactions in natural communities. However, the interactions between AMF and wild and transgenic soybean genotypes remain unclear. Therefore, in the present study, we evaluated the effect of arbuscular mycorrhizal fungi on the growth performance of drought-stressed transgenic soybean lines (ZXOE-11 and ZXOE-13) overexpressing GmSPL9d gene and their wild soybean Tianlong 1 (TL1) at the seedling stage (45 d after sowing). The results showed that colonization of wild and transgenic soybean with Rhizophagus irregularis significantly decreased the adverse effects of drought on plant growth. AMF inoculation significantly increased plant biomass, root activity, chlorophyll metabolism, photosynthesis, and chlorophyll fluorescence in wild-type and transgenic plants under both control and drought stress conditions. Drought causes the production of ROS, such as hydrogen peroxide, which enhances MDA, thereby decreasing the membrane stability index (MSI). However, AMF-inoculated plants exhibited decreased ROS accumulation and increased MSI. Moreover, AMF treatment significantly improved osmolyte, nitrogen, and nitrate reductase activity under control and drought conditions, which increased the relative water content. Furthermore, AMF treatment enhanced the antioxidant systems of drought-stressed plants by increasing the activities of peroxidase, superoxide dismutase, catalase, and ascorbate peroxidase. AMF improved the growth performance, photosynthesis, and antioxidant activity of transgenic plants under drought stress conditions. The present findings indicate that the AMF contribution to soybean seedling drought tolerance was more significant for the transgenic plants than for the wild plants under drought conditions. The current findings emphasize the possibility of growth and photosynthetic variation in the degree of AMF-associated drought resistance in soybean plants. Our findings suggest that future crop breeding challenges include developing cultivars for sustainable production and maximizing crop cultivar and fungal species (AMF) combinations in drought-stressed regions.

Expression of Mn-sod, PAL1, aos1 and HPL genes in soybean plants overexpressing the NmDef02 defensin.

Plant defensins are a potential tool in crop improvement programs through biotechnology. Their antifungal action makes them attractive molecules for the production of transgenic plants. Information is currently lacking on what happens to the expression of defense genes in transgenic plants that overexpress a defensin. Here we show the relative expression of four defense-related genes: Mn-sod, PAL1, aos1 and HPL evaluated in two transgenic soybean events (Def1 and Def17) constitutively expressing the NmDef02 defensin gene from Nicotiana megalosiphon. The expression of these defense genes showed a differential profile in the transgenic events, with the increased expression of the aos1 gene and the repression of the Mn-sod gene in both events, when compared to the non-transgenic control. Furthermore, the expression of the PAL1 gene only increased in the Def17 event. The results indicate that although there were some changes in the expression of defense genes in transgenic plants overexpressing the defensin NmDef02; the morphoagronomic parameters evaluated were similar to the non-transgenic control. Understanding the molecular changes that occur in these transgenic plants could be of interest in the short, medium and long term.

Genetically modified crops do not present variations in pollen viability and morphology when compared to their conventional counterparts.

Modern agricultural biotechnologies, such as those derived from genetic modification, are solutions that can enable an increase in food production, lead to more efficient use of natural resources, and promote environmental impact reduction. Crops with altered genetic materials have been extensively subjected to safety assessments to fulfill regulatory requirements prior to commercialization. The Brazilian National Technical Biosafety Commission (CTNBio) provides provisions for commercial release of transgenic crops in Brazil, including requiring information on pollen dispersion ability as part of environmental risk assessment, which includes pollen viability and morphology studies. Here we present the pollen viability and morphology of non-transgenic conventional materials, single-event genetically modified (GM) products, and stacked GM products from soybean, maize and cotton cultivated in Brazil. Microscopical observation of stained pollen grain was conducted to determine the percentage of pollen viability as well as pollen morphology, which is assessed by measuring pollen grain diameter. The pollen viability and diameter of GM soybean, maize and cotton, evaluated across a number of GM events in each crop, were similar to the conventional non-GM counterparts. Pollen characterization data contributed to the detailed phenotypic description of GM crops, supporting the conclusion that the studied events were not fundamentally different from the conventional control.

In Situ Proteomic Analysis of Herbicide-Resistant Soybean and Hybrid Seeds via Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry Imaging

Transgenic soybean is the commercial crop with the largest cultivation area worldwide. During transgenic soybean cultivation, exogenous genes may be transferred to wild relatives through gene flow, posing unpredictable ecological risks. Accordingly, an environmental risk assessment should focus on fitness changes and underlying mechanisms in hybrids between transgenic and wild soybeans (Glycine soja). Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) was used for in situ detection and imaging of protein changes in the seeds of transgenic herbicide-resistant soybean harboring epsps and pat genes, non-transgenic soybean, wild soybean, and their F2 hybrid. Protein data clearly distinguished wild soybeans, while the F2 seeds had protein characteristics of both parents and were distinguished from wild soybean seeds. Using UPLC-Q-TOF-MS, 22 differentially expressed proteins (DEPs) were identified, including 13 specific to wild soybean. Sucrose synthase and stress response-related DEPs were differentially expressed in parental and offspring. Differences in these may underpin the greater adaptability of the latter. MSI revealed DEP distribution in transgenic, wild, and F2 seeds. Identifying DEPs related to fitness may elucidate mechanisms underlying fitness differences among the studied varieties. Our study shows that MALDI-MSI has the potential to become a visual method for transgenic soybean analysis.

Functional Characterization of a (E)-β-Ocimene Synthase Gene Contributing to the Defense against Spodoptera litura.

Soybean is a worldwide crop that offers valuable proteins, fatty acids, and phytonutrients to humans but is always damaged by insect pests or pathogens. Plants have captured sophisticated defense mechanisms in resisting the attack of insects and pathogens. How to protect soybean in an environment- or human-friendly way or how to develop plant-based pest control is a hotpot. Herbivore-induced plant volatiles that are released by multiple plant species have been assessed in multi-systems against various insects, of which (E)-β-ocimene has been reported to show anti-insect function in a variety of plants, including soybean. However, the responsible gene in soybean is unknown, and its mechanism of synthesis and anti-insect properties lacks comprehensive assessment. In this study, (E)-β-ocimene was confirmed to be induced by Spodoptera litura treatment. A plastidic localized monoterpene synthase gene, designated as GmOCS, was identified to be responsible for the biosynthesis of (E)-β-ocimene through genome-wide gene family screening and in vitro and in vivo assays. Results from transgenic soybean and tobacco confirmed that (E)-β-ocimene catalyzed by GmOCS had pivotal roles in repelling a S. litura attack. This study advances the understanding of (E)-β-ocimene synthesis and its function in crops, as well as provides a good candidate for further anti-insect soybean improvement.

“Blighted Futures: The Soybean Assemblage and Argentina’s Agro-extractive Turn”

Argentine GM soybean producers find themselves in an untenable situation. They must produce more even though pressures like weed resistances, global commodities price fluctuations, and an unpredictable tax regime pose ever greater challenges. Meanwhile, public sentiment has largely turned against the rural sector for its opposition to tax increases and the under-regulated use of agrochemicals. There are, however, no easy alternatives now that producers find themselves in the grips of this agro-economic complex. Argentine GM soybean producers' experiences of how they arrived at a point of agro-extractivist lock in is the focus of this article. To better understand their perspectives, this article elaborates the “soybean assemblage.” This consists of histories, politics, actors, and more-than-human agents that stabilized the GM soybean industry in Argentina, keep it in place, and render it fragile. This includes how autogamous soybeans and an intellectual property regime function in Argentina, the country’s famously fertile land and its position within the national imaginary, Kirchnerist politics that rely on soybean exports to fund social programs, producers' economic interests and sense of duty to the nation, the discipline of agronomy as a site of agrochemical knowledge production, and how agrochemicals, namely glyphosate, created long-term dependence under the guise of safe use.

Zinc-finger protein GmZF351 improves both salt and drought stress tolerance in soybean.

Abiotic stress is one of the most important factors reducing soybean yield. It is essential to identify regulatory factors contributing to stress responses. A previous study found that the tandem CCCH zinc-finger protein GmZF351 is an oil level regulator. In this study, we discovered that the GmZF351 gene is induced by stress and that the overexpression of GmZF351 confers stress tolerance to transgenic soybean. GmZF351 directly regulates the expression of GmCIPK9 and GmSnRK, leading to stomata closing, by binding to their promoter regions, which carry two CT(G/C)(T/A)AA elements. Stress induction of GmZF351 is mediated through reduction in the H3K27me3 level at the GmZF351 locus. Two JMJ30-demethylase-like genes, GmJMJ30-1 and GmJMJ30-2, are involved in this demethylation process. Overexpression of GmJMJ30-1/2 in transgenic hairy roots enhances GmZF351 expression mediated by histone demethylation and confers stress tolerance to soybean. Yield-related agronomic traits were evaluated in stable GmZF351-transgenic plants under mild drought stress conditions. Our study reveals a new mode of GmJMJ30-GmZF351 action in stress tolerance, in addition to that of GmZF351 in oil accumulation. Manipulation of the components in this pathway is expected to improve soybean traits and adaptation under unfavorable environments.

Safety assessment of subchronic feeding of insect-resistant and herbicide-resistant transgenic soybeans to juvenile channel catfish (Ictalurus punctatus)

Transgenic soybean is one of the most planted crops for human food and animal feed. The channel catfish (Ictalurus punctatus) is an important aquatic organism cultured worldwide. In this study, the effect of six different soybean diets containing: two transgenic soybeans expressing different types of cp4-epsps, Vip3Aa and pat genes (DBN9004 and DBN8002), their non-transgenic parent JACK, and three conventional soybean varieties (Dongsheng3, Dongsheng7, and Dongsheng9) was investigated in juvenile channel catfish for eight weeks, and a safety assessment was performed. During the experiment, no difference in survival rate was observed in six groups. The hepatosomatic index (HSI) and condition factor (CF) showed no significant difference. Moreover, comparable feed conversion (FC), feeding rate (FR), and feed conversion ratio (FCR) were found between transgenic soybean and JACK groups. Assessment of growth performance showed that the weight gain rate (WGR) and specific growth rate (SGR) of channel catfish were consistent. In addition, there were no changes in enzyme activity indexes (lactate dehydrogenase (LDH), total antioxidant capacity (T-AOC), aspartate aminotransferase (AST) and alanine aminotransferase (ALT)) in channel catfish among treatments. The research provided an experimental basis for the aquaculture feed industry to employ transgenic soybean DBN9004 and DBN8002 for commercial purposes.

Overexpression of GmNF-YA14 produced multiple phenotypes in soybean

Soybean is an important oil crop, and more and more soybean gene functions have been reported; however, one soybean gene with multiple functions is rarely seen. The NF-YA subfamily plays a broad regulatory role in plant growth and development, and GmNF-YA14 (Glyma.14G010000) is one of them. We found that the expression level of GmNF-YA14 in the root of soybean seedlings was significantly increased after a one-hour treatment with 200 mM salt. The transgenic soybean was obtained by Agrobacterium-mediated method, and all experiments were carried out using transgenic soybean homozygous lines. Under a 200 mM salt treatment, the leaf chlorophyll content of overexpressed GmNF-YA14 transgenic soybean seedlings was significantly decreased compared to the control. Moreover, the first flowering time of overexpressed GmNF-YA14 transgenic soybean was shortened by more than six days compared to the control, and the growth period structure changed significantly. Overexpressed GmNF-YA14 transgenic soybean plants had two or three branches, whereas control soybean plants had eight or nine branches. Compared to the control, the number of main stem nodes and plant height of overexpressed GmNF-YA14 transgenic soybean plants were significantly reduced. The 100-seed weight of GmNF-YA14 transgenic soybean line OE-8 was significantly higher than that of the control. Transcriptome analysis indicated that the multiple phenotypes of the overexpressed GmNF-YA14 transgenic soybean may be related to the mitogen-activated protein kinase (MAPK) signaling pathway. These results indicated that compared to the control, overexpressed GmNF-YA14 transgenic soybeans had changes in salt stress tolerance, growth period, plant type, and 100-seed weight, and these changes may be related to the MAPK signaling pathway.

Impacts of glyphosate-based herbicide on leaf stomatal density and biomass production of transgenic soybean (Glycine max [L.] Merr.) and corn (Zea mays L.)

Impacts of glyphosate-based herbicide on leaf stomatal density and biomass production of transgenic soybean (Glycine max [L.] Merr.) and corn (Zea mays L.)

Overexpression of the aldehyde dehydrogenase AhALDH3H1 from Arachis hypogaea in soybean increases saline-alkali stress tolerance.

Soybean production is severely hampered by saline-alkaline stress caused by saline-alkalization. Plants have aldehydrogenase (ALDH) family members that convert reactive aldehydes to carboxylic acids to remove active aldehyde molecules. However, little is known about the increased saline-alkali tolerance caused by the ALDH function in soybean. Here, we introduced a previously identified ALDH coding gene AhALDH3H1 from Arachis hypogaea into the soybean genome to investigate its critical role in response to saline-alkali stress. Transgenic soybean with increased aldehyde dehydrogenase activity showed significant tolerance to saline-alkali stress. It reduced malondialdehyde (MDA) content compared to its receptor, suggesting that over-expression of AhALDH3H1 accelerated soybean tolerance to saline-alkali stress by increasing aldehyde dehydrogenase activity, which is responsible for scavenging toxic MDA. To further analyze the inner mechanisms that allow transgenic plants to tolerate saline-alkali stress, we sequenced the transcriptome and metabolome of P3 (wild type, WT) and transgenic lines which were separately treated with water and a saline-alkali solution. When subjected to saline-alkali stress, the integrated analysis of the transcriptome and metabolome suggested that several genes related to cell wall structure crucial for preserving cell wall extensibility and plasticity were largely responsible for restoring homeostasis within the transgenic cells compared to WT. Metabolites, including both necessary ingredients for cell wall genesis and harmful production produced during the saline-alkali stress response, could be transported efficiently with the help of the ABC transporter, reducing the negative effects of saline-alkali stress. These findings suggest that introducing AhALDH3H1 increases transgenic soybean tolerance to saline-alkali stress may through cell wall structure maintenance and metabolites transport.

Expression of malic enzyme reveals subcellular carbon partitioning for storage reserve production in soybeans.

Central metabolism produces amino and fatty acids for protein and lipids that establish seed value. Biosynthesis of storage reserves occurs in multiple organelles that exchange central intermediates including two essential metabolites, malate, and pyruvate that are linked by malic enzyme. Malic enzyme can be active in multiple subcellular compartments, partitioning carbon and reducing equivalents for anabolic and catabolic requirements. Prior studies based on isotopic labeling and steady-state metabolic flux analyses indicated malic enzyme provides carbon for fatty acid biosynthesis in plants, though genetic evidence confirming this role is lacking. We hypothesized that increasing malic enzyme flux would alter carbon partitioning and result in increased lipid levels in soybeans. Homozygous transgenic soybean plants expressing Arabidopsis malic enzyme alleles, targeting the translational products to plastid or outside the plastid during seed development, were verified by transcript and enzyme activity analyses, organelle proteomics, and transient expression assays. Protein, oil, central metabolites, cofactors, and acyl-acyl carrier protein (ACPs) levels were quantified overdevelopment. Amino and fatty acid levels were altered resulting in an increase in lipids by 0.5-2% of seed biomass (i.e. 2-9% change in oil). Subcellular targeting of a single gene product in central metabolism impacts carbon and reducing equivalent partitioning for seed storage reserves in soybeans.

Overexpression of GmWRKY172 enhances cadmium tolerance in plants and reduces cadmium accumulation in soybean seeds.

Cadmium (Cd) stress is a significant threat to soybean production, and enhancing Cd tolerance in soybean is the focus of this study. The WRKY transcription factor family is associated with abiotic stress response processes. In this study, we aimed to identify a Cd-responsive WRKY transcription factor GmWRKY172 from soybean and investigate its potential for enhancing Cd tolerance in soybean. The characterization of GmWRKY172 involved analyzing its expression pattern, subcellular localization, and transcriptional activity. To assess the impact of GmWRKY172, transgenic Arabidopsis and soybean plants were generated and examined for their tolerance to Cd and Cd content in shoots. Additionally, transgenic soybean plants were evaluated for Cd translocation and various physiological stress indicators. RNA sequencing was performed to identify the potential biological pathways regulated by GmWRKY172. GmWRKY172 was significantly upregulated by Cd stress, highly expressed in leaves and flowers, and localized to the nucleus with transcriptional activity. Transgenic plants overexpressing GmWRKY172 showed enhanced Cd tolerance and reduced Cd content in shoots compared to WT. Lower Cd translocation from roots to shoots and seeds was also observed in transgenic soybean. Under Cd stress, transgenic soybean accumulated less malondialdehyde (MDA) and hydrogen peroxide (H2O2) than WT plants, with higher flavonoid and lignin contents, and peroxidase (POD) activity. RNA sequencing analysis revealed that many stress-related pathways were regulated by GmWRKY172 in transgenic soybean, including flavonoid biosynthesis, cell wall synthesis, and peroxidase activity. Our findings demonstrated that GmWRKY172 enhances Cd tolerance and reduces seed Cd accumulation in soybean by regulating multiple stress-related pathways, and could be a promising candidate for breeding Cd-tolerant and low Cd soybean varieties.

GMO analysis results from official food control laboratories in Germany from 2017 to 2021

In Germany, genetically modified organisms (GMO) analysis of food samples collected within the official food control is performed by the laboratories of the Federal States. The present report shows GMO analysis results from food samples of the years 2017 to 2021, including contaminations by unauthorized GMO, as well as genetically modified (GM) plant events authorized in the European Union. In addition to previous publications, evaluation of the aggregated food samples analysed for GMO components is shown. During this timeframe, 1077 (7.1%) out of 15,145 samples contained genetic modification. In 43 samples, DNA sequences of unauthorized GM plants were found. Additionally, for food derived from soybean, evaluations according to different product categories and the agronomic production (conventional and organic farming) are shown. Whereas in products from organic farming and in conventional soybeans labelled “without genetic engineering” GM soybeans were detected in 6.1% and 8.9%, of all tested samples, respectively, nearly 30% of all conventional soy samples yielded positive results below 0.1%. However, only in 0.7% of the overall analysed 5424 soybean samples GMO percentages of more than 0.1% were obtained. Generally, authorized GM plants were only found at low contamination levels. The labelling threshold of 0.9% for GM ingredients was exceeded only in 0.2% (maize) and 0.1% (soybean) samples, respectively. For monitoring purposes and risk evaluation, the data collection shall be continued.

Proficiency of European GMO control laboratories to quantify MON89788 soybean in a meat pâté matrix.

GMO control laboratories in the EU routinely monitor the presence and content of genetically modified organisms (GMOs) in food and feed products collected from the EU market. As the vast majority of GMOs comprize genetically modified plants, most control samples have a plant-based origin. For the first time, a pilot proficiency test was organised requiring the analysis of GMOs in a meat matrix. Meat pâté, a product in which soybean is occasionally identified, was spiked with GM soybean event MON89788, homogenised by mixing, aliquoted in sachets and frozen. The assigned value was determined by two independent expert laboratories. Several DNA extraction methods were tested and proved to be insufficient for the removal of PCR inhibitors present in the DNA extracts, resulting in a GM content underestimated by at least 30%. This problem was solved either by using hot-start qPCR chemistry or by applying the same method in a digital PCR format. A total of 52 laboratories participated in the study. They were requested to verify the presence of any GM soybean in the test item and to quantify the GM event(s) identified by their method of choice. All but one laboratory identified the MON89788 soybean event present in the pâté matrix. The majority of the quantitative results reported were below the assigned value, but did not deviate more than 50% from it. This study demonstrated the proficiency of most GMO control laboratories for the analysis of GMOs in a meat-based product. It also shows that method optimisation for GMO analysis in meat products is nevertheless advisable.

Combination of Hairy Root and Whole-Plant Transformation Protocols to Achieve Efficient CRISPR/Cas9 Genome Editing in Soybean

The new gene-editing technology CRISPR/Cas system has been widely used for genome engineering in various organisms. Since the CRISPR/Cas gene-editing system has a certain possibility of low efficiency and the whole plant transformation of soybean is time-consuming and laborious, it is important to evaluate the editing efficiency of designed CRISPR constructs before the stable whole plant transformation process starts. Here, we provide a modified protocol for generating transgenic hairy soybean roots to assess the efficiency of guide RNA (gRNA) sequences of the CRISPR/Cas constructs within 14 days. The cost- and space-effective protocol was first tested in transgenic soybean harboring the GUS reporter gene for the efficiency of different gRNA sequences. Targeted DNA mutations were detected in 71.43-97.62% of the transgenic hairy roots analyzed as evident by GUS staining and DNA sequencing of the target region. Among the four designed gene-editing sites, the highest editing efficiency occurred at the 3' terminal of the GUS gene. In addition to the reporter gene, the protocol was tested for the gene-editing of 26 soybean genes. Among the gRNAs selected for stable transformation, the editing efficiency of hairy root transformation and stable transformation ranged from 5% to 88.8% and 2.7% to 80%, respectively. The editing efficiencies of stable transformation were positively correlated with those of hairy root transformation with a Pearson correlation coefficient (r) of 0.83. Our results demonstrated that soybean hairy root transformation could rapidly assess the efficiency of designed gRNA sequences on genome editing. This method can not only be directly applied to the functional study of root-specific genes, but more importantly, it can be applied to the pre-screening of gRNA in CRISPR/Cas gene editing.

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.