Pressure from insect-resistant maize on protected butterflies and moths.

Intensification in agriculture affects many insect species, including butterflies. Insect-resistant crops, such as Bt (Bacillus thuringiensis) maize, which produces a toxin active against Lepidoptera, are an alternative to insecticide sprays. Genetically modified crops are regulated in most countries and require an environmental risk assessment. In the European Union, such assessments include the use of simulation models to predict the effects on nontarget Lepidoptera (NTL). To support the assessment of protected NTL, we extended an individual-based, stochastic, spatially explicit mathematical model (LepiX) to include a wider range of exposure scenarios, a species-sensitivity distribution, and an option for repeated exposure of individuals. We applied the model to transgenic maize DAS-1507, which expresses a high concentration of Bt toxin in pollen that may be consumed by NTL larvae on their host plants nearby. Even in the most conservative scenario without repeated exposure, mortality estimates for highly sensitive species ranged from 41% to 6% at distances of 10-1000 m from the nearest maize field. Repeated exposure can cause additional mortality and thus is relevant for the overall risk assessment. Uncertainties in both exposure and ecotoxicity estimates strongly influenced the predicted mortalities. Care should be taken to include these uncertainties in the model scenarios used for decision-making. In accordance with other modeling results, our simulations demonstrated that mean mortality may not be safe for protected species. With its high pollen expression, DAS-1507 maize may pose risks to sensitive and protected butterfly and moth species that may be difficult to manage. High expression of Bt toxin in pollen is unnecessary for controlling target pests. Consequently, we suggest that Bt maize with high pollen expression not be cultivated in regions where protected butterflies are to be conserved.

La intensificación en la agricultura afecta a muchas especies de insectos, incluyendo a las mariposas. Los cultivos resistentes a los insectos, como el maíz Bt (Bacillus thuringiensis), el cual produce una toxina activa contra los lepidópteros, son una alternativa a los insecticidas. Los cultivos genéticamente modificados (GM) están regulados en la mayoría de los países y requieren de una evaluación de riesgo ambiental. En la Unión Europea (EU), dichas evaluaciones incluyen el uso de modelos de simulación para pronosticar los efectos sobre los lepidópteros no objetivo (LNO). Para apoyar a la evaluación de LNO protegidos, extendimos un modelo matemático espacialmente explícito, estocástico y basado en el individuo (LepiX) para incluir una mayor gama de escenarios de exposición, una distribución de la sensibilidad de las especies y una opción para la exposición repetida de los individuos. Aplicamos el modelo al maíz transgénico DAS­1507, el cual expresa una alta concentración de toxina Bt en el polen que puede ser consumido por las larvas de LNO en una planta hospedera cercana. Incluso en el escenario más conservador sin una exposición repetida, las estimaciones de mortalidad para las especies altamente sensibles variaron entre el 41% y el 6% en distancias de 10­1000 m a partir del campo de maíz más cercano. La exposición repetida puede causar mortalidad adicional y por lo tanto es relevante para la evaluación general del riesgo. La incertidumbre en las estimaciones de la exposición y la ecotoxicidad influyeron fuertemente sobre la mortalidad pronosticada. Se debe tener cuidado de incluir estas incertidumbres en los escenarios modelados usados para la toma de decisiones. De acuerdo con los resultados de otros modelos, nuestras simulaciones demostraron que la mortalidad media podría no ser segura para las especies protegidas. Con su alta producción de polen, el maíz DAS­1507 podría representar un riesgo difícil de manejar para las especies de mariposas y polillas sensibles y protegidas. No se necesita una expresión elevada de la toxina Bt en el polen para controlar a las plagas. En consecuencia, sugerimos que no se cultive el maíz Bt con una alta producción de polen en las regiones en donde se busca conservar a las mariposas protegidas. Presión del maíz resistente a insectos sobre mariposas y polillas protegidas.

Belgian dietitians' knowledge, perceptions and willingness-to-recommend of genetically modified food and organisms.

BACKGROUND: Dietitians play a critical role in the public's relationship with food and are often overlooked as an important stakeholder group in the general debate about sustainable food. Genetically modified organisms (GMOs) are one type of modern food source that could contribute to a more sustainable food system. This case study is the first to examine the knowledge, perception and willingness-to-recommend (WTR) genetically modified (GM) foods by dietitians in Europe. METHODS: An online survey was addressed to all members of the Flemish Association of Dietitians (Belgium) in 2021, resulting in a sample of 98 valid responses. Multivariate linear regression included sociodemographic, knowledge, and attitudinal factors as the independent variables to explain dietitians' WTR. RESULTS: Flemish dietitians had limited knowledge of GMOs; only about half of the GM questions were answered correctly. Most dietitians (53%-76%) would recommend GMOs with positive effects on human nutrition or sustainability, whereas few dietitians (19%-27%) would recommend other GMO applications. Trust in GMO information sources and perceived GM benefits significantly influenced a positive WTR of GM foods. Predominant negative information about GM foods was significantly associated with dietitians' low trust and WTR such foods. CONCLUSIONS: Countering the predominantly negative portrayal with more neutral and factual information could improve trust, which in turn could positively influence dietitians' perceptions towards GMOs. By further examining the knowledge and perception of dietitians worldwide GMOs and gene-edited products, new insights could be could gathered into the positioning of this underexposed stakeholder group.

Strategic transgene-free approaches of CRISPR-based genome editing in plants.

Genome editing through the alteration of nucleotide sequence has already revolutionized the field of site-directed mutagenesis for a decade. However, research in terms of precision and efficacy in targeting the loci and reduction in off-target mutation has always been a priority when DNA is involved. Therefore, recent research interest lies in utilizing the same precision technology but results in non-transgenic. In this review article, different technological advancements have been explained, which may provide a holistic concept of and need for transgene-free genome editing. The advantage and lacunas of each technology have been critically discussed to deliver a transparent view to the readers. A systematic analysis and evaluation of published research articles implied that researchers across the globe are putting continuous efforts in this direction to eliminate the hindrance of transgenic regulation. Nevertheless, this approach has severe implications legitimate for mitigating the conflict of acceptance, reliability, and generosity of gene-editing technology and sustainably retorting to the expanding global population feeding challenges.

Mitigating climate change and pandemic impacts on global food security: dual sustainable agriculture approach (2S approach).

MAIN CONCLUSION: Simultaneous application of two sustainability approaches such as the application of biofertilizers to GM plants and microbe bioengineering to enhance physiological response and beneficial interaction with GM plants may have a significant impact on strengthening global food security amid climate change and the pandemic. The second sustainable development goal (SDG 02, Zero Hunger) aims global agricultural sustainability and food security challenges. The agriculture sector has been an integral part of developing countries for millions of farmers and their families. Their contribution provides stability of raw matter related to food availability. But climate change, higher population growth and worldwide pandemics are the main obstacles to food quality, higher crop productivity and global food security. Scientists are concerned with the manifestation of agriculture sustainability in the modern crop management approach to resolving the issues. It is the only way to higher yield productivity by protecting the environment, conserving natural resources, and slowing climate change. Several strategies can be an option to implement, yet the proposed two sustainability approach or 2S approach will be the significant way toward the goal of zero hunger. The first sustainability approach is an application of genetically modified (S1: GMO) Plants and the other is an application of beneficiary plant growth-promoting microbes (S2: Biofertilizers) to the plants for both higher crops and maintenance of the environment. This study summarizes the essential points of S1 and S2 for the widespread utilization of the 2S approach in agriculture and recommends the potential alternatives to be implemented to produce food for all. Simultaneous application of the 2S approach can defeat all threats to gain sustainability in agriculture.

Survivorship and food consumption of immatures and adults of Apis mellifera and Scaptotrigona bipunctata exposed to genetically modified eucalyptus pollen.

Eucalyptus comprises the largest planted area of cultivated production forest in Brazil. Genetic modification (GM) of eucalyptus can provide additional characteristics for increasing productivity and protecting wood yield, as well as potentially altering fiber for a diversity of industrial uses. However, prior to releasing a new GM plant, risk assessments studies with non-target organisms must be undertaken. Bees are prominent biological models since they play an important role in varied ecosystems, including for Eucalyptus pollination. The main goal of this study was to evaluate whether a novel event (Eucalyptus 751K032), which carries the cp4-epsps gene that encodes the protein CP4-EPSPS and nptII gene that encodes the protein NPTII, might adversely affect honey bees (Apis mellifera) and stingless bees (Scaptotrigona bipunctata). The experiments were performed in southern Brazil, as follows: (i) larvae and adults were separately investigated, (ii) three or four different pollen diets were offered to bees, depending on larval or adult status, and (iii) two biological attributes, i.e., survivorship of larvae and adults and food intake by adults were evaluated. The diets were prepared with pollen from GM Eucalyptus 751K032; pollen from conventional Eucalyptus clone FGN-K, multifloral pollen or pure larval food. The insecticide dimethoate was used to evaluate the sensitivity of bees to toxic substances. Datasets were analyzed with Chi-square test, survival curves and repeated measures ANOVA. Results indicated no evidence of adverse effects of Eucalyptus pollen 751K032 on either honey bees or stingless bees assessed here. Therefore, the main findings suggest that the novel event may be considered harmless to these organisms since neither survivorship nor food consumption by bees were affected by it.

The Regulation of Cell Therapy and Gene Therapy Products in Switzerland.

This chapter describes the regulation of cell and gene therapy products (CGTPs) in Switzerland and its legal basis. The Swiss Agency for Therapeutic Products, Swissmedic, is the lead Regulatory Authority and its ATMP Division is responsible for the regulation of these products at the level of clinical trials and marketing authorization. CGTPs are regulated similarly to medicinal products. The legal basis is set by the Therapeutic Product Act, the Transplantation Act, the Human Research Act, and associated ordinances. The ATMP Division is involved in processes such as scientific advice meetings, presubmission advice meetings, pharmacovigilance, market surveillance, import/export approvals, manufacturing license approval, and inspections. In Switzerland, guidance documents relevant for cell and gene therapy provided by PIC/S, OECD, ICH, Ph.Eur., EMA, or FDA are considered. In order to harmonize requirements for CGTPs, the ATMP Division is in constant exchange of information with foreign Regulatory Authorities and part of working groups of ICH, IPRP, and Ph.Eur. As CGTPs are biologically and technically complex, a risk-based approach is applied on a case-by-case basis for the evaluation of clinical trial and marketing applications. A substantial part of this chapter will provide requirements with respect to the manufacturing and quality, nonclinical and clinical evaluation of CGTPs. Furthermore, information will be provided regarding the use of real-world evidence in evaluation of clinical long-term efficacy and safety in case of rare diseases where the numbers of patients are too small for statistically meaningful analysis during clinical trials. Finally, the chapter will provide information on a health technology assessment (HTA) program that was launched in 2015 in Switzerland by the federal authorities.

Reduction of Escherichia coli O157:H7 in Finishing Cattle Fed Corn Genetically Modified to Produce Increased Concentrations of Alpha Amylase in the Corn Kernel.

Cattle are recognized as the principal reservoir for Escherichia coli O157:H7 and preharvest food safety efforts often focus on decreasing shedding of this pathogen in cattle feces. Enogen® corn (EC; Syngenta Seeds, LLC) is genetically modified to produce enhanced concentrations of α-amylase in the corn kernel endosperm. Research has demonstrated improvements in feed efficiency for cattle fed EC and research has not yet explored whether improved digestion impacts foodborne pathogen populations in cattle. Therefore, this study explored effects of finishing diets containing EC on Escherichia coli O157:H7 prevalence in cattle. A 2 × 2 factorial experiment was conducted with steers (n = 960) fed diets consisting of 2 types of silage (EC or Control) and grain (EC or Control), fed daily ad libitum. Steers were grouped into 12 blocks by incoming body weight, blocks were randomly assigned to one of four pens, and pens were randomly assigned to one diet. Cattle were sampled using rectoanal mucosal swabs in cohorts of 298-337 cattle per day, for a total of 3 sampling days (15-16 days apart). Escherichia coli O157:H7 prevalence rates ranged from not detected (0/75) to 10.0% (8/80) depending on sampling day. Tests for the silage × corn interaction, and the main effects of silage and corn, were not significant (p > 0.05); however, EC reduced the odds of Escherichia coli O157:H7 prevalence by 43% compared to the control corn diet (p = 0.07). Diets containing EC tended to decrease Escherichia coli O157:H7 prevalence in feedlot cattle; however, this reduction was not significant. Before a conclusion can be drawn about impact of EC on Escherichia coli O157:H7 in cattle, further research is necessary to (1) determine if this tendency is due to increased alpha amylase activity and (2) elucidate impact on Escherichia coli O157:H7 prevalence and concentration, as well as a possible mechanism of action.

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.

Novel Field-Based Protein Detection Method Using Light Transmission Spectroscopy and Antibody Functionalized Gold Nanoparticles.

Protein detection is a universal tool critical to many applications in medicine, agriculture, and biotechnology. We developed a novel protein detection method combining light transmission spectroscopy and particle-size analysis of gold nanospheres monovalently functionalized with polyclonal antibodies and applied it to an emerging challenge for such technologies─the monitoring of environmental proteins (eProteins) present in natural aquatic systems. These are an underreported source of pollution and include the pseudopersistent Cry toxins that enter aquatic ecosystems from surrounding genetically engineered crops. The assay is capable of detecting proteins in complex matrices, such as water samples collected in the field, making it a competitive assay for eProtein detection. It is sensitive, reaching 1.25 ng mL-1, and we demonstrate its application to the detection of Cry1Ab from subsurface tile-drain and streamwater samples from agricultural waterways. The assay can also be quickly adapted for other protein detection applications in the future.

Oligo-Not Only for Silencing: Overlooked Potential for Multidirectional Action in Plants.

Oligo technology is a low-cost and easy-to-implement method for direct manipulation of gene activity. The major advantage of this method is that gene expression can be changed without requiring stable transformation. Oligo technology is mainly used for animal cells. However, the use of oligos in plants seems to be even easier. The oligo effect could be similar to that induced by endogenous miRNAs. In general, the action of exogenously introduced nucleic acids (Oligo) can be divided into a direct interaction with nucleic acids (genomic DNA, hnRNA, transcript) and an indirect interaction via the induction of processes regulating gene expression (at the transcriptional and translational levels) involving regulatory proteins using endogenous cellular mechanisms. Presumed mechanisms of oligonucleotides' action in plant cells (including differences from animal cells) are described in this review. Basic principles of oligo action in plants that allow bidirectional changes in gene activity and even those that lead to heritable epigenetic changes in gene expression are presented. The effect of oligos is related to the target sequence at which they are directed. This paper also compares different delivery methods and provides a quick guide to using IT tools to help design oligonucleotides.

Assessing the environmental fate of rAAV in activated sludge and water: Implications for environmental risk assessments and GMO regulatory frameworks.

During the past +20 years, recombinant adeno-associated virus (rAAV) vectors have emerged as the primary vehicle of choice for in vivo gene therapy. rAAV vectors are classified as genetically modified organisms (GMOs), therefore specific biosafety laws apply regarding their use. Environmental agencies participating in the review of clinical trial applications involving viral-based gene therapies (eg based on AAV) focus among other phenomena especially on shedding, a mechanism by which rAAV vectors exit a patient's body and enter the natural environment. For example, following patient use, shed viral particles excreted in urine and feces enter the wastewater treatment facility (WWTF) and subsequently may be released into the natural environment through wastewater effluent discharges. Based on basic molecular biology, it is generally assumed by the scientific community that shed rAAV particles will undergo degradation during the wastewater treatment process. However, despite their importance and increase in use during the last few decades, actual data to support our understanding of the environmental fate of shed rAAV vector particles is unavailable. Data to support this assumption would greatly enhance our knowledge and understanding of degradation kinetics associated with rAAV in the environment. Such data would also provide strong scientific support for changes in current legislation regarding the medicinal use of GMOs. Therefore, the goal of this research was to conduct laboratory experiments to assess the actual environmental fate of rAAV virions. In this study the stability of 4 different rAAV vectors (based on wildtype (wt) AAV serotypes 2,3,6,9) was assessed during incubation in activated sludge (containing live microorganisms). This setting corresponds to conditions as encountered in WWTFs, and has been used in order to assess rAAV fate under environmentally relevant conditions, to gain a better understanding of the general environmental risk posed by shed rAAV particles. The amount of detectable virions in the supernatant, as measured by sensitive and specific qPCR, rapidly decreased within hours and continued to decline, reaching the lower limit of quantitation prior to or by study termination on day 7. Furthermore, a half-life of approximately 7 days for rAAV virions was determined under abiotic conditions, during a room temperature incubation experiment of rAAV vectors in water in the absence of any microbiota or sludge. The findings from this study provide the first insight of its kind into the actual environmental fate of shed rAAV particles, and help the community to better understand the potential impact of rAAVs on the environment. It has become evident now that shed particles are not equipped to remain stable and/or soluble once entering a typical WWTF and therefore do not pose a threat to the natural environment. These findings support a data-driven approach towards a simplified, risk-based regulation of medicinal GMOs in the EU and other regions.

Eurasian consumers' food safety beliefs and trust issues in the age of COVID-19: evidence from an online survey in 15 countries.

BACKGROUND: This investigation provides an important insight into Eurasian consumers' food safety beliefs and trust issues influenced by the COVID-19 pandemic. An online survey was conducted in 15 European and Asian countries involving more than 4000 consumers. RESULTS: It has confirmed that different socioeconomic characteristics, cultural aspects and education levels shape food safety perceptions within Eurasian countries. The COVID-19 pandemic influenced their beliefs and trust in food safety, which is relatively low on average. However, it is significantly higher for European consumers (especially European Union ones) compared to their Asian counterparts. Both Asian and European respondents agreed that food fraud and climate changes represent a food safety issue. However, European consumers were less concerned regarding the food safety of genetically modified foods and meat and dairy analogs/hybrids. Asian consumers were, to a greater extent, worried about the risk of getting COVID-19 from food, restaurants, food retail establishments and home food deliveries. CONCLUSION: Eurasian consumers have put their greatest extent of trust, when food safety assurance is concerned, into food scientists and food producers holding a food safety certificate. Broadly, they are uncertain to what extent their federal governments and food inspectors are competent, able and efficient in ensuring food safety. Higher education of Eurasian consumers was followed by increased food safety confidence in all parts of the food chain. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Assessing Effects of Genetically Modified Plant Material on the Aquatic Environment Using higher-tier Studies.

Genetically modified organisms are used extensively in agriculture. To assess potential side effects of genetically modified (GM) plant material on aquatic ecosystems, only a very small number of higher-tier studies have been performed. At the same time, these studies are particularly important for comprehensive risk assessment covering complex ecological relationships. Here we evaluate the methods of experimental higher-tier effect studies with GM plant material (or Bt toxin) in comparison to those well-established for pesticides. A major difference is that nominal test concentrations and thus dose-response relationships cannot easily be produced with GM plant material. Another important difference, particularly to non-systemic pesticides, is that aquatic organisms are exposed to GM plant material primarily through their feed. These and further differences in test requirements, compared with pesticides, call for a standardisation for GM-specific higher-tier study designs to assess their potentially complex effects in the aquatic ecosystems comprehensively.

A novel, non-GMO surface display in Limosilactobacillus fermentum mediated by cell surface hydrolase without anchor motif.

Recent studies have demonstrated the potential of surface display technology in therapeutic development and enzyme immobilization. Utilization of lactic acid bacteria in non-GMO surface display applications is advantageous due to its GRAS status. This study aimed to develop a novel, non-GMO cell wall anchoring system for lactic acid bacteria using a cell-surface hydrolase (CshA) from Lactiplantibacillus plantarum SK156 for potential industrial and biomedical applications. Analysis of the CshA revealed that it does not contain any known classical anchor domains. Although CshA lacks a classical anchor domain, it successfully displayed the reporter protein superfolder GFP on the surface of several lactic acid bacteria in host dependent manner. CshA-sfGFP fusion protein was displayed greatest on Limosilactobacillus fermentum SK152. Pretreatment with trichloroacetic acid further enhanced the binding of CshA to Lm. fermentum. The binding conditions of CshA on pretreated Lm. fermentum (NaCl, pH, time, and temperature) were also optimized, resulting in a maximum binding of up to 106 CshA molecules per pretreated Lm. fermentum cell. Finally, this study demonstrated that CshA-decorated pretreated Lm. fermentum cells tolerates gastrointestinal stress, such as low pH and presence of bile acid. To our knowledge, this study is the first to characterize and demonstrate the cell-surface display ability of CshA. The potential application of CshA in non-GMO antigen delivery system and enzyme immobilization remains to be tested.

Sustainable use of CRISPR/Cas in fish aquaculture: the biosafety perspective.

Aquaculture is becoming the primary source of seafood for human diets, and farmed fish aquaculture is one of its fastest growing sectors. The industry currently faces several challenges including infectious and parasitic diseases, reduced viability, fertility reduction, slow growth, escapee fish and environmental pollution. The commercialization of the growth-enhanced AquAdvantage salmon and the CRISPR/Cas9-developed tilapia (Oreochromis niloticus) proffers genetic engineering and genome editing tools, e.g. CRISPR/Cas, as potential solutions to these challenges. Future traits being developed in different fish species include disease resistance, sterility, and enhanced growth. Despite these notable advances, off-target effect and non-clarification of trait-related genes among other technical challenges hinder full realization of CRISPR/Cas potentials in fish breeding. In addition, current regulatory and risk assessment frameworks are not fit-for purpose regarding the challenges of CRISPR/Cas notwithstanding that public and regulatory acceptance are key to commercialization of products of the new technology. In this study, we discuss how CRISPR/Cas can be used to overcome some of these limitations focusing on diseases and environmental release in farmed fish aquaculture. We further present technical limitations, regulatory and risk assessment challenges of the use of CRISPR/Cas, and proffer research strategies that will provide much-needed data for regulatory decisions, risk assessments, increased public awareness and sustainable applications of CRISPR/Cas in fish aquaculture with emphasis on Atlantic salmon (Salmo salar) breeding.

Genetically modified organisms: adapting regulatory frameworks for evolving genome editing technologies.

Genetic modification of living organisms has been a prosperous activity for research and development of agricultural, industrial and biomedical applications. Three decades have passed since the first genetically modified products, obtained by transgenesis, become available to the market. The regulatory frameworks across the world have not been able to keep up to date with new technologies, monitoring and safety concerns. New genome editing techniques are opening new avenues to genetic modification development and uses, putting pressure on these frameworks. Here we discuss the implications of definitions of living/genetically modified organisms, the evolving genome editing tools to obtain them and how the regulatory frameworks around the world have taken these technologies into account, with a focus on agricultural crops. Finally, we expand this review beyond commercial crops to address living modified organism uses in food industry, biomedical applications and climate change-oriented solutions.

Food engineers' attitudes and purchasing intentions towards genetically modified organism products.

Background: Biotechnological developments have resulted in the modification of the genetic structures of many organisms. However, the possibility of risks in terms of human health has caused consumers to approach products containing genetically modified organisms (GMOs) with suspicion. Objective: In this study, we aimed to determine the attitudes of food engineers towards GMO products and their effects on purchase intentions. Methods: For this purpose, an attitude scale towards GMO products was adapted, and a multivariate regression analysis was performed by applying the adapted questionnaire. Results: It has been determined that the negative attitudes of food engineers towards GMO products and their purchasing intentions have an effect. Attitudes toward the use of gene technology in production were determined with 17% as the most effective dimension of purchase intention, and it was found to have a significant effect (p < 0.05). Conclusions: Overall, it was determined that food engineers were concerned about the potential risk of GMO foods. In order to overcome these concerns, it is thought that eliminating the lack of knowledge of this professional group on gene technology should be planned as a strategic goal.

Target Lines for in Planta Gene Stacking in Japonica Rice.

The clustering of transgenes at a chromosome location minimizes the number of segregating loci that needs to be introgressed to field cultivars. Transgenes could be efficiently stacked through site-specific recombination and a recombinase-mediated in planta gene stacking process was described previously in tobacco based on the Mycobacteriophage Bxb1 site-specific integration system. Since this process requires a recombination site in the genome, this work describes the generation of target sites in the Japonica rice genome. Agrobacterium-mediated gene transfer yielded ~4000 random-insertion lines. Seven lines met the criteria of being single copy, not close to a centromere, not inserted within or close to a known gene or repetitive DNA, having precise recombination site sequences on both ends, and able to express the reporter gene. Each target line tested was able to accept the site-specific integration of a new gfp-containing plasmid and in three of those lines, we regenerated fertile plants. These target lines could be used as foundation lines for stacking new traits into Japonica rice.

Qualitative and Quantitative Real-Time PCR Methods for Assessing False-Positive Rates in Genetically Modified Organisms Based on the Microbial-Infection-Linked HPT Gene.

The hygromycin phosphotransferase (HPT) gene as a selective marker is normally used in screening tests as a first step in detecting and quantifying genetically modified organisms (GMOs) in seeds, food, and feed materials. Nevertheless, if researchers only focus on the HPT gene, it is difficult to distinguish genetically modified (GM) crops from microbial infection, leading to miscalculation of the rate of GM materials in a given sample set. Here, we cloned the 7259 bp sequence carrying the HPT gene from soybean sprouts using the genome walking strategy. BLAST analysis revealed that this sequence was derived from plasmids naturally occurring in microorganisms, such as Escherichia coli, Klebsiella pneumoniae or Salmonella sp. Using the reconstructed plasmid pFP-hpt, qualitative PCR and quantitative real-time PCR (qPCR) methods were established, and 261 bp and 156 bp products were produced. The specificity of these assays was assessed against related pFP-hpt plasmids, plant species with important agronomic traits, and GM crops containing the HPT gene. No unexpected results were observed between samples using these qualitative PCR and qPCR methods. The sensitivity of this qualitative PCR assay was determined at 20 copies, while the limit of detection (LOD) and limit of quantification (LOQ) of qPCR were both 5 copies per reaction. Our in-house validation indicated that the amplification efficiency, linearity, and repeatability of this qPCR assay were in line with performance requirements. Furthermore, a qualitative and quantitative duplex PCR showed high reliability for the simultaneous detection of the HPT gene in a plant sample and environmental micro-organisms harboring the HPT gene in one PCR reaction. These qualitative PCR and qPCR assays were able to differentiate between plants infected with E. coli harboring the HPT gene from GM plants, indicating that these two methods are broadly applicable for routine GMO testing.

GMO Genetic Elements Thesaurus (GMO-GET): a controlled vocabulary for the consensus designation of introduced or modified genetic elements in genetically modified organisms.

BACKGROUND: Various databases on genetically modified organisms (GMOs) exist, all with their specific focus to facilitate access to information needed for, e. g., the assistance in risk assessment, the development of detection and identification strategies or inspection and control activities. Each database has its unique approach towards the subject. Often these databases use different terminology to describe the GMOs. For adequate GMO addressing and identification and exchange of GMO-related information it is necessary to use commonly agreed upon concepts and terminology. RESULT: A hierarchically structured controlled vocabulary describing the genetic elements inserted into conventional GMOs, and GMOs developed by the use of gen(om)e-editing is presented: the GMO genetic element thesaurus (GMO-GET). GMO-GET can be used for GMO-related documentation, including GMO-related databases. It has initially been developed on the basis of two GMO databases, i.e. the Biosafety Clearing-House and the EUginius database. CONCLUSION: The use of GMO-GET will enable consistent and compatible information (harmonisation), also allowing an accurate exchange of information between the different data systems and thereby facilitating their interoperability. GMO-GET can also be used to describe genetic elements that are altered in organisms obtained through current targeted genome-editing techniques.