Non-uniform distribution pattern for differentially expressed genes of transgenic rice Huahui 1 at different developmental stages and environments

DNA microarray analysis is an effective method to detect unintended effects by detecting differentially expressed genes (DEG) in safety assessment of genetically modified (GM) crops. With the aim to reveal the distribution of DEG of GM crops under different conditions, we performed DNA microarray analysis using transgenic rice Huahui 1 (HH1) and its non-transgenic parent Minghui 63 (MH63) at different developmental stages and environmental conditions. Considerable DEG were selected in each group of HH1 under different conditions. For each group of HH1, the number of DEG was different; however, considerable common DEG were shared between different groups of HH1. These findings suggested that both DEG and common DEG were adequate for investigation of unintended effects. Furthermore, a number of significantly changed pathways were found in all groups of HH1, indicating genetic modification caused everlasting changes to plants. To our knowledge, our study for the first time provided the non-uniformly distributed pattern for DEG of GM crops at different developmental stages and environments. Our result also suggested that DEG selected in GM plants at specific developmental stage and environment could act as useful clues for further evaluation of unintended effects of GM plants.     

Development of selection marker-free transgenic potato plants with enhanced tolerance to oxidative stress

A binary vector devoid of a plant selection-marker gene (designated as pSSA-F) was constructed to overcome bio-safety concerns about genetically modified plants. This vector carried chloroplast-targeted superoxide dismutase (SOD) and ascorbate peroxidase (APX) genes under the control of an oxidative stress-inducible(SWPA2) promoter, and was utilized to transform potato (Solanum tuberosum L.). Integration of these foreign genes into transgenic plants was primarily performed via PCR with genomic DNA. Twelve marker-free transgenic lines were obtained by inoculating stem explants. The maximum transformation efficiency was 6.25% and averaged 2.2%. Successful integration of the SOD and APX genes rendered transgenic plants tolerant to methyl viologen-mediated oxidative stress at the leaf-disc and whole-plant levels. Our findings suggest that this technique for developing selection marker-free transgenic plants is feasible and can be employed with other crop species.     

Unintended effects of genetic modifications in plants and methods of their analysis

The problem of unintended effects caused by genetic modification of plants is analysed. Factors that can provoke the unintended effects in genetically engineered plants, their consequences and possibility of the avoiding of unintended effects with use of current methods of genetic modification are discussed. Modern methodological approaches applied to analyse the unintended effects during the safety assessment of transgenic plants, in particular methods of molecular profiling with different "-omic"-technologies are described.     

Assessment of the food safety issues related to genetically modified foods

International consensus has been reached on the principles regarding evaluation of the food safety of genetically modified plants. The concept of substantial equivalence has been developed as part of a safety evaluation framework, based on the idea that existing foods can serve as a basis for comparing the properties of genetically modified foods with the appropriate counterpart. Application of the concept is not a safety assessment per se, but helps to identify similarities and differences between the existing food and the new product, which are then subject to further toxicological investigation. Substantial equivalence is a starting point in the safety evaluation, rather than an endpoint of the assessment. Consensus on practical application of the principle should be further elaborated. Experiences with the safety testing of newly inserted proteins and of whole genetically modified foods are reviewed, and limitations of current test methodologies are discussed. The development and validation of new profiling methods such as DNA microarray technology, proteomics, and metabolomics for the identification and characterization of unintended effects, which may occur as a result of the genetic modification, is recommended. The assessment of the allergenicity of newly inserted proteins and of marker genes is discussed. An issue that will gain importance in the near future is that of post-marketing surveillance of the foods derived from genetically modified crops. It is concluded, among others that, that application of the principle of substantial equivalence has proven adequate, and that no alternative adequate safety assessment strategies are available.     

Development of transformation vectors based upon a modified plant alpha-tubulin gene as the selectable marker

A plant transformation and selection system has been developed utilizing a modified tubulin gene as a selectable marker. The vector constructs carrying a mutant alpha-tubulin gene from goosegrass conferring resistance to dinitroaniline herbicides were created for transformation of monocotyledonous and dicotyledonous plants. These constructs contained beta- and/or mutant alpha-tubulin genes driven either by ubiquitin or CaMV 35S promoter. The constructs were used for biolistic transformation of finger millet and soybean or for Agrobacterium-mediated transformation of flax and tobacco. Trifluralin, the main representative of dinitroaniline herbicides, was used as a selective agent in experiments to select transgenic cells, tissues and plantlets. Selective concentrations of trifluralin estimated for each species were as follows: 10 microM for Eleusine coracana, Glycine max, Nicotiana plumbaginifolia and Nicotiana sylvestris; 3 microM for Linum usitatissimum. PCR and Southern blotting analyses of transformed lines with a specific probe to nptII, alpha-tubulin or beta-tubulin genes were performed to confirm the transgenic nature of regenerated plants. Band specific for the mutant alpha-tubulin gene was identified in transformed plant lines. Results confirmed the stable integration of the mutant tubulin gene into the plant genomes. The present study clearly demonstrates the use of a plant mutant tubulin as a selective gene for plant transformation.     

Unintended effects of genetic modifications and methods of their analysis in plants

The problem of unintended effects, which may be caused by genetic modification of plants, is analyzed. The factors that provoke the occurrence of unintended effects in genetically engineered plants, the consequences caused by the influence of these factors, and the possibility to minimize such effects by the use of improved methods of genetic engineering are discussed. Modern methodological approaches that can be applied to the analysis of unintended effects considered as a stage of safety assessment of transgenic plants, especially the methods of molecular profiling with the use of different “-omic” technologies, are described.     

A comparative proteomics approach to detect unintended effects in transgenic Arabidopsis

Recently, as part of biosafety assessments, unintended effects have been given much attention. In this study, we applied a proteomics approach to elucidate the unintended effects of random T-DNA insertion in transgenic plants. Separated proteins extracted from 12 transgenic Arabidopsis thaliana with different T-DNA insertion sites and from wild-type （ecotype Col-o） were analyzed. In the transgenic plants, 102 significantly altered protein spots were detected, in which 59 were up-regulated and 43 down-regulated. MALDI-TOF MS analysis showed that most of these expression level-altered proteins were involved in energy transfer, oxidative respiration and photosynthesis. However, none of these proteins was a toxic protein or allergen. Using plants with or without cold treatment, a natural environmental stress, as controls, we found that the number of the altered proteins was even less in those transgenic plants than those triggered by the cold treatment, suggesting that the transgenic events had a weaker impact on the plants than the environmental stresses. Interestingly, the phosphinothricin acetyl transferase （PAT）, the BAR-encoded protein, was detected in nine out of twelve different T-DNA insertion lines at five different insertion sites. These data suggest that the most significant impact of transgenic events on the host plants is from the transgene itself, i.e., from the predictable intended effects, rather than unintended effects. This study also suggests that the proteomics approach has the potential to detect the unintended effects in transgenic plants.     

转基因植物中外源非目的基因片段的生物安全研究进展

生物安全（ｂｉｏｓａｆｅｔｙ）是指与以人类和环境为对象的生物学研究所产生的效应相关的安全性［1］,或对生物危害的检测、评价、监测、防范和治理的科学技术体系 ① 。遗传修饰生物体（ＧＭＯ）,特别是转基因植物的安全性是生物安全的重要内容,近几年已经引起政府、社会和科学界的广泛关注。转基因植物对人体健康的负面影响的考虑要点主要包括对人体的毒性、过敏原性、营养成分的改变与抗营养因子以及抗生素抗性等。对生态环境的负面影响即生态学风险的考虑要点包括转基因植物导致杂草问题,产生新病毒或加重病害,对非目标生物的影…     

A comparative proteomics approach to detect unintended effects in transgenic Arabidopsis

Recently, as part of biosafety assessments, unintended effects have been given much attention. In this study, we applied a proteomics approach to elucidate the unintended effects of random T-DNA insertion in transgenic plants. Separated proteins extracted from 12 transgenic Arabidopsis thaliana with different T-DNA insertion sites and from wild-type (ecotype Col-o) were analyzed. In the transgenic plants, 102 significantly altered protein spots were detected, in which 59 were up-regulated and 43 down-regulated. MALDI-TOF MS analysis showed that most of these expression level-altered proteins were involved in energy transfer, oxidative respiration and photosynthesis. However, none of these proteins was a toxic protein or allergen. Using plants with or without cold treatment, a natural environmental stress, as controls, we found that the number of the altered proteins was even less in those transgenic plants than those triggered by the cold treatment, suggesting that the transgenic events had a weaker impact on the plants than the environmental stresses. Interestingly,the phosphinothricin acetyl transferase (PAT), the BAR-encoded protein, was detected in nine out of twelve different T-DNA insertion lines at five different insertion sites. These data suggest that the most significant impact of transgenic events on the host plants is from the transgene itself, i.e., from the predictable intended effects, rather than unintended effects. This study also suggests that the proteomics approach has the potential to detect the unintended effects in transgenic plants.     

An efficient method for the production of marker-free transgenic plants of peanut (Arachis hypogaea L.)

Recombinant genes conferring resistance to antibiotics or herbicides are widely used as selectable markers in plant transformation for selecting the primary transgenic events. However, these become redundant once the transgenic plants have been developed and identified. Although, there is no evidence that the selectable marker genes are unsafe for consumers and the environment, it would be desirable if the marker genes can be eliminated from the final transgenic events. The availability of efficient transformation methods can enable the possibility of developing transgenic events that are devoid of the marker gene/s upfront. Taking advantage of the high and consistent transformation potential of peanut, we report a technique for developing its transgenics without the use of any selectable marker gene. Marker-free binary vectors harboring either the phytoene synthase gene from maize (Zmpsy1) or the chitinase gene from rice (Rchit) were constructed and used for Agrobacterium tumefaciens-mediated transformation of peanut. The putative transgenic events growing in vitro were initially identified by PCR and further confirmed for gene integration and expression by dot blots assays, Southern blots, and RT-PCR where they showed a transformation frequency of over 75%. This system is simple, efficient, rapid, and does not require the complex segregation steps and analysis for selection of the transgenic events. This approach for generation of marker-free transgenic plants minimizes the risk of introducing unwanted genetic changes, allows stacking of multiple genes and can be applicable to other plant species that have high shoot regeneration efficiencies.     

Possible consequences of the overlap between the CaMV 35S promoter regions in plant transformation vectors used and the viral gene VI in transgenic plants

Multiple variants of the Cauliflower mosaic virus 35S promoter (P35S) are used to drive the expression of transgenes in genetically modified plants, for both research purposes and commercial applications. The genetic organization of the densely packed genome of this virus results in sequence overlap between P35S and viral gene VI, encoding the multifunctional P6 protein. The present paper investigates whether introduction of P35S variants by genetic transformation is likely to result in the expression of functional domains of the P6 protein and in potential impacts in transgenic plants. A bioinformatic analysis was performed to assess the safety for human and animal health of putative translation products of gene VI overlapping P35S. No relevant similarity was identified between the putative peptides and known allergens and toxins, using different databases. From a literature study it became clear that long variants of the P35S do contain an open reading frame, when expressed, might result in unintended phenotypic changes. A flowchart is proposed to evaluate possible unintended effects in plant transformants, based on the DNA sequence actually introduced and on the plant phenotype, taking into account the known effects of ectopically expressed P6 domains in model plants.     

Non-coding RNAs in Crop Genetic Modification: Considerations and Predictable Environmental Risk Assessments (ERA)

Of late non-coding RNAs (ncRNAs)-mediated gene silencing is an influential tool deliberately deployed to negatively regulate the expression of targeted genes. In addition to the widely employed small interfering RNA (siRNA)-mediated gene silencing approach, other variants like artificial miRNA (amiRNA), miRNA mimics, and artificial transacting siRNAs (tasiRNAs) are being explored and successfully deployed in developing non-coding RNA-based genetically modified plants. The ncRNA-based gene manipulations are typified with mobile nature of silencing signals, interference from viral genome-derived suppressor proteins, and an obligation for meticulous computational analysis to prevaricate any inadvertent effects. In a broad sense, risk assessment inquiries for genetically modified plants based on the expression of ncRNAs are competently addressed by the environmental risk assessment (ERA) models, currently in vogue, designed for the first generation transgenic plants which are based on the expression of heterologous proteins. Nevertheless, transgenic plants functioning on the foundation of ncRNAs warrant due attention with respect to their unique attributes like off-target or non-target gene silencing effects, small RNAs (sRNAs) persistence, food and feed safety assessments, problems in detection and tracking of sRNAs in food, impact of ncRNAs in plant protection measures, effect of mutations etc. The role of recent developments in sequencing techniques like next generation sequencing (NGS) and the ERA paradigm of the different countries in vogue are also discussed in the context of ncRNA-based gene manipulations.     

Field performance of transgenic citrus trees: Assessment of the long-term expression of uidA and nptII transgenes and its impact on relevant agronomic and phenotypic characteristics

ABSTRACT: BACKGROUND: The future of genetic transformation as a tool for the improvement of fruit trees depends on the development of proper systems for the assessment of unintended effects in field-grown GM lines. In this study, we used eight transgenic lines of two different citrus types (sweet orange and citrange) transformed with the marker genes beta-glucuronidase (uidA) and neomycin phosphotransferase II (nptII) as model systems to study for the first time in citrus the long-term stability of transgene expression and whether transgene-derived pleiotropic effects occur with regard to the morphology, development and fruit quality of orchard-grown GM citrus trees. RESULTS: The stability of the integration and expression of the transgenes was confirmed in 7-year-old, orchard-grown transgenic lines by Southern blot analysis and enzymatic assays (GUS and ELISA NPTII), respectively. Little seasonal variation was detected in the expression levels between plants of the same transgenic line in different organs and over the 3 years of analysis, confirming the absence of rearrangements and/or silencing of the transgenes after transferring the plants to field conditions. Comparisons between the GM citrus lines with their non-GM counterparts across the study years showed that the expression of these transgenes did not cause alterations of the main phenotypic and agronomic plant and fruit characteristics. However, when comparisons were performed between diploid and tetraploid transgenic citrange trees and/or between juvenile and mature transgenic sweet orange trees, significant and consistent differences were detected, indicating that factors other than their transgenic nature induced a much higher phenotypic variability. CONCLUSIONS: Our results indicate that transgene expression in GM citrus remains stable during long-term agricultural cultivation, without causing unexpected effects on crop characteristics. This study also shows that the transgenic citrus trees expressing the selectable marker genes that are most commonly used in citrus transformation were substantially equivalent to the non-transformed controls with regard to their overall agronomic performance, as based on the use of robust and powerful assessment techniques. Therefore, future studies of the possible pleiotropic effects induced by the integration and expression of transgenes in field-grown GM citrus may focus on the newly inserted trait(s) of biotechnological interest.     

Altered GAI activity of hybrid aspen has minimal effects on the performance of a polyphagous weevil,Polydrusus sericeus

Genetically modified plants can have unintended effects on herbivores, even when the transgene is not explicitly devised for pest protection. These effects may lead to altered ecological relationships in areas where transgenic plants are deployed. We tested several genetically‐modified hybrid aspen clone INRA 717‐IB4 [Populus tremula L. ×P. alba L. (Salicaceae)] lines expressing transgenic constructs with altered gibberellic acid expression for their effects on a polyphagous insect, Polydrusus sericeus (Schaller) (Coleoptera: Curculionidae). Longevity, egg production, and leaf consumption were measured in laboratory assays. Only male longevity was negatively affected by foliage from genetically modified trees, and in no case was oviposition or leaf consumption significantly altered. The importance of males in population dynamics varies among species, but we do not have evidence it is a major driver in this system. Contrary to our hypothesis of increased performance on modified foliage, adult P. sericeus were rarely affected by GM foliage, suggesting that not all genetic modifications that alter secondary metabolism in plants will necessarily have a strong effect on specific insect‐plant relations, supporting the view that evaluations continue to require case by case analysis.     

Influence of transgenesis on genome variability in cucumber lines with a thaumatin II gene

The development of new plant varieties by genetic modification aims at improving their features or introducing new qualities. However, concerns about the unintended effects of transgenes and negative environmental impact of genetically modified plants are an obstacle for the use of these plants in crops. To analyze the impact of transgenesis on plant genomes, we analyze three cucumber transgenic lines with an introduced thaumatin II gene. After genomes sequencing, we analyzed the transgene insertion site and performed variant prediction. As a result, we obtained similar number of variants for all analyzed lines (average of 4307 polymorphisms), with high abundance in one region of chromosome 4. According to SnpEff analysis, the presence of genomic variants generally does not influence the genome functionality, as less than 2% of polymorphisms have high impact. Moreover, analysis indicates that these changes were more likely induced by in vitro culture than by the transgenesis itself. The insertion site analysis shows that the region of transgene integration could cause changes in gene expression, by gene disruption or loss of promoter region continuity.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12298-021-00990-8.     

Assessment of unanticipated unintended effects of genetically modified plants on non‐target organisms: a controversy worthy of pursuit?

A typical risk hypothesis addressed during the environmental risk assessment (ERA) of genetically modified (GM) plants for cultivation is that the novel traits intentionally introduced into GM plants do not adversely affect non‐target organisms (NTOs). However, genetic modification may potentially also lead to unintended changes in the GM plant which could raise safety concerns. Therefore, the European Food Safety Authority (EFSA) advocates the characterization of ecological interactions between the GM plant and representative NTOs as part of the ERA of GM plants for cultivation in the European Union. Yet, this requirement is not unanimously accepted by stakeholders. Here, we present EFSA's approach to assess potential adverse effects on NTOs and summarize some of the stakeholders' views, mostly opposing EFSA's position on scientific grounds.     

Molecular characterization for food safety assessment of a genetically modified late blight resistant potato: an unusual case

Standard food safety assessments of genetically modified crops require a thorough molecular characterization of the novel DNA as inserted into the plant that is intended for commercialization, as well as a comparison of agronomic and nutritional characteristics of the genetically modified to the non-modified counterpart. These characterization data are used to identify any unintended changes in the inserted DNA or in the modified plant that would require assessment for safety in addition to the assessment of the intended modification. An unusual case of an unintended effect discovered from the molecular characterization of a genetically modified late blight resistant potato developed for growing in Bangladesh and Indonesia is presented here. Not only was a significant portion of the plasmid vector backbone DNA inserted into the plant along with the intended insertion of an R-gene for late blight resistance, but the inserted DNA was split into two separate fragments and inserted into two separate chromosomes. One fragment carries the R-gene and the other fragment carries the NPTII selectable marker gene and the plasmid backbone DNA. The implications of this for the food safety assessment of this late blight resistant potato are considered.