Transgenic, transplastomic and transient approaches for foreign gene expression in plants

The review represents the latest results of the experiments carried out at the Department of genetic engineering of the Institute of cell biology and genetic engineering of the National Academy of Sciences of Ukraine in the field of creation oftransgenic and transplastomic plants as well as the use of transient expression for production of recombinant proteins. The new approaches of promoterless gene expression in transgenic plants and construction of transplastomic plants using "clipboard" species are discussed.     

Transgenic, transplastomic, and transient approaches to alien gene expression in plants

Recent experiments are reviewed, which aimed at developing the transgenic and transplastomic plants, as well as at accumulating recombinant proteins due to transient expression. These experiments were performed in the Department of Genetic Engineering of the Institute of Cell Biology and Genetic Engineering of the National Academy of Science, Ukraine. The new approaches have been developed to induce promoterless gene expression in transgenic plants and to obtain transplastomic plants with the use of “clipboard” species.     

Ex-post assessment of genetically modified,low level presence in Canadian flax

Canada is the world’s largest producer and exporter of flaxseed. In 2009, DNA from deregistered genetically modified (GM) CDC Triffid was detected in a shipment of Canadian flaxseed exported to Europe, causing a large decrease in the amount of flax planted in Canada and a major shift in export markets. The flax industry in Canada undertook major changes to ensure the removal of transgenic flax from the supply chain. To demonstrate compliance, Canada adopted a protocol involving testing grain samples (post-harvest) using an RT-PCR test for the construct found in CDC Triffid. Efforts to remove the presence of GM flax from the value chain included reconstituting major flax varieties from GM-free plants. The reconstituted varieties represented the majority of planting seed in 2014. This study re-evaluates GM flax presence in Canadian grain stocks for an updated dataset (2009–2015) using a previously described simulation model to estimate low-level GM presence. Additionally, losses to the Canadian economy resulting from the reduction in flax production and export opportunities, costs associated with reconstituting major flax varieties, and testing for the presence of GM flax along the flax value chain are estimated.     

Opinion building on a socio-scientific issue: the case of genetically modified plants

This paper presents results from a study with the following research questions: (a) are pupils' opinions on genetically modified organisms (GMOs) influenced by biology teaching; and (b) what is important for the opinion pupils hold and how does knowledge work together with other parameters such as values? 64 pupils in an upper secondary school answered questionnaires, in which they expressed opinions and gave arguments on applications of GMOs, before and after biology courses. The pupils' knowledge of genetics and GMOs was also investigated. Eleven pupils were then in-terviewed to examine their reasoning in more depth. More pupils were positive about genetically modified tomatoes after the courses. Males were more positive than females. No correlation was found between knowledge of basic genetics and opinion. Most of the pupils could express arguments for and against the applications but they built their personal opinion on different arguments. An important concern was potential risks. Depending on risk judgement and/or how they trusted scientists, the pupils came to different conclusions. Few had any idea of how the different applications are risk assessed or how scientists work. Other important factors for decision-making were the purpose of the application, the time perspective and feelings.     

Argonic performance and field resistance of genetically modified,virus-resistant potato plants

Time consuming potato breeding programmes for virus resistances may be shortened by engineering virus resistance in existing cultivars or advanced breeding lines. Under field conditions genetically modified potato plants expressing viral coat protein genes of potato virus X, Y and potato leaf roll virus showed improved resistance up to near immunity. Despite the occurence of variation in the level of virus resistance and in phenotypic identity, in all cases true to type transgenic clones with improved virus resistance could be selected. These results indicate that improving potato cultivars or advanced breeding lines, by selectively adding new traits while preserving intrinsic properties, is commercially feasible.     

Edible genetically modified microorganisms and plants for improved health

The development of new strategies for the delivery of vaccine antigens or immune modulators to the mucosal tissue includes innovative approaches such as the use of genetically modified food microorganisms and plants. Even though the 'proof-of-concept' has recently been established for these two systems, key questions mainly related to efficacy and risk of breaking oral tolerance remain to be critically addressed in the immediate future.     

Animal nutrition with feeds from genetically modified plants

Plant breeders have made and will continue to make important contributions toward meeting the need for more and better feed and food. The use of new techniques to modify the genetic makeup of plants to improve their properties has led to a new generation of crops, grains and their by-products for feed. The use of ingredients and products from genetically modified plants (GMP) in animal nutrition properly raises many questions and issues, such as the role of a nutritional assessment of the modified feed or feed additive as part of safety assessment, the possible influence of genetically modified (GM) products on animal health and product quality and the persistence of the recombinant DNA and of the 'novel' protein in the digestive tract and tissues of food-producing animals. During the last few years many studies have determined the nutrient value of GM feeds compared to their conventional counterparts and some have additionally followed the fate of DNA and novel protein. The results available to date are reassuring and reveal no significant differences in the safety and nutritional value of feedstuffs containing material derived from the so-called 1st generation of genetically modified plants (those with unchanged gross composition) in comparison with non-GM varieties. In addition, no residues of recombinant DNA or novel proteins have been found in any organ or tissue samples obtained from animals fed with GMP. These results indicate that for compositionally equivalent GMP routine-feeding studies with target species generally add little to nutritional and safety assessment. However, the strategies devised for the nutritional and safety assessment of the 1st generation products will be much more difficult to apply to 2nd generation GMP in which significant changes in constituents have been deliberately introduced (e.g., increased fatty acids or amino acids content or a reduced concentration of undesirable constituents). It is suggested that studies made with animals will play a much more important role in insuring the safety of these 2nd generation constructs.     

Tolerance to aluminum in genetically modified tobacco plants

Genetically modified tobacco plants tolerant to high aluminum concentrations were developed by integration of constructs containing rhamnolipid genes (rhlA and rhlB). At an aluminum concentration of 200 mM in ionite soil, the control plants perished, whereas the transgenic plants, although they were inhibited, continued to grow and produced seeds.     

Quantification of cuticular permeability in genetically modified plants

More and more studies on genetically modified plants are identifying parts of the genetic code with putative involvement in creating the cuticular barrier. Unfortunately, many of these studies suffer from the inadequacy of the chosen methods to quantify, in a reasonably unambiguous way, if and how the efficacy of the cuticular barrier is affected by the genetic change. A short overview of relevant findings is given and a more stringent experimental approach to quantifying effects on cuticular permeability in genetically modified plants proposed.     

Bioavailability of transgenic microRNAs in genetically modified plants

Background

Transgenic expression of small RNAs is a prevalent approach in agrobiotechnology for the global enhancement of plant foods. Meanwhile, emerging studies have, on the one hand, emphasized the potential of transgenic microRNAs (miRNAs) as novel dietary therapeutics and, on the other, suggested potential food safety issues if harmful miRNAs are absorbed and bioactive. For these reasons, it is necessary to evaluate the bioavailability of transgenic miRNAs in genetically modified crops.

Results

As a pilot study, two transgenic Arabidopsis lines ectopically expressing unique miRNAs were compared and contrasted with the plant bioavailable small RNA MIR2911 for digestive stability and serum bioavailability. The expression levels of these transgenic miRNAs in Arabidopsis were found to be comparable to that of MIR2911 in fresh tissues. Assays of digestive stability in vitro and in vivo suggested the transgenic miRNAs and MIR2911 had comparable resistance to degradation. Healthy mice consuming diets rich in Arabidopsis lines expressing these miRNAs displayed MIR2911 in the bloodstream but no detectable levels of the transgenic miRNAs.

Conclusions

These preliminary results imply digestive stability and high expression levels of miRNAs in plants do not readily equate to bioavailability. This initial work suggests novel engineering strategies be employed to enhance miRNA bioavailability when attempting to use transgenic foods as a delivery platform.

     

Review: genetically modified plants for the promotion of human health

Plants are attractive biological resources because of their ability to produce a huge variety of chemical compounds, and the familiarity of production in even the most rural settings. Genetic engineering gives plants additional characteristics and value for cultivation and post-harvest. Genetically modified (GM) plants of the “first generation” were conferred with traits beneficial to producers, whereas GM plants in subsequent “generations” are intended to provide beneficial traits for consumers. Golden Rice is a promising example of a GM plant in the second generation, and has overcome a number of obstacles for practical use. Furthermore, consumer-acceptable plants with health-promoting properties that are genetically modified using native genes are being developed. The emerging technology of metabolomics will also support the commercial realization of GM plants by providing comprehensive analyzes of plant biochemical components.     

Evolution of a regulatory framework for pharmaceuticals derived from genetically modified plants

The use of genetically modified (GM) plants to synthesize proteins that are subsequently processed, regulated and sold as pharmaceuticals challenges two very different established regulatory frameworks, one concerning GM plants and the other covering the development of biotechnology-derived drugs. Within these regulatory systems, specific regulations and guidelines for plant-made pharmaceuticals (PMPs) - also referred to as plant-derived pharmaceuticals (PDPs) - are still evolving. The products nearing commercial viability will ultimately help to road test and fine-tune these regulations, and might help to reduce regulatory uncertainties. In this review, we summarize the current state of regulations in different countries, discuss recent changes and highlight the need for further regulatory development in this burgeoning, new industry. We also make the case for the harmonization of international regulations.     

Novel roles for genetically modified plants in environmental protection

Transgenic plants of environmental benefit typically consist of plants that either reduce the input of agrochemicals into the environment or make the biological remediation of contaminated areas more efficient. Examples include the construction of species that result in reduced pesticide use and of species that contain genes for either the degradation of organics or the increased accumulation of inorganics. Cutting-edge approaches, illustrated by our own work, focus on the applicability of genetically modified (GM) plants that produce insect pheromones or that are specifically tailored to the phytoremediation of cadmium or PCBs. This paper discusses the role that the next generation of GM plants might play in preventing and reducing chemical contamination and in converting contaminated sites into safe agricultural or recreational land.     

The tolerance of tobacco genetically modified plants to aluminium

Introduction of the vector constructions with rhamnolipid genes (rhlA and rhlB) into tobacco plants has resulted in development of genetically modified plants which were tolerant to high concentrations of aluminium. While under aluminium concentration of 200 mM in a soil (ionit resin) all control plants perished, transgenic tobacco plants continued to grow and were fertile.     

Transgenic plants: performance,release and containment

This review focuses on transgenic plants, from the initial stages of the genetic modification process in the laboratory to their release stage in the field and indicates possible areas of concern and strategies for dealing with them. The classes of marker genes and issues about their safety, the gene flow and strategies that are used to isolate transgenic plants genetically are specifically examined. In addition, an assessment is provided of the phenomena which affect the performance of transgenic plants, such as gene disruption, the pleiotropic effect on plant phenotype and genetic variation. Finally, strategies are suggested for preventing unexpected consequences of transgenic plant production.The author is with the Department of Genetics, University of Leeds, Leeds LS2 9JT, UK     

Risk mitigation of genetically modified bacteria and plants designed for bioremediation

While the possible advantages of bioremediation and phytoremediation, by both recombinant microbes and plants, have been extensively reviewed, the biosafety concerns have been less extensively treated. This article reviews the possible risks associated with the use of recombinant bacteria and plants for bioremediation, with particular emphasis on ways in which molecular genetics could contribute to risk mitigation. For example, genetic techniques exist that permit the site-specific excision of unnecessary DNA, so that only the transgenes of interest remain. Other mechanisms exist whereby the recombinant plants or bacteria contain conditional suicide genes that may be activated under certain conditions. These methods act to prevent the spread and survival of the transgenic bacteria or plants in the environment, and to prevent horizontal gene flow to wild or cultivated relatives. Ways in which these genetic technologies may be applied to risk mitigation in bioremediation and phytoremediation are discussed.

     

Risk mitigation of genetically modified bacteria and plants designed for bioremediation

While the possible advantages of bioremediation and phytoremediation, by both recombinant microbes and plants, have been extensively reviewed, the biosafety concerns have been less extensively treated. This article reviews the possible risks associated with the use of recombinant bacteria and plants for bioremediation, with particular emphasis on ways in which molecular genetics could contribute to risk mitigation. For example, genetic techniques exist that permit the site-specific excision of unnecessary DNA, so that only the transgenes of interest remain. Other mechanisms exist whereby the recombinant plants or bacteria contain conditional suicide genes that may be activated under certain conditions. These methods act to prevent the spread and survival of the transgenic bacteria or plants in the environment, and to prevent horizontal gene flow to wild or cultivated relatives. Ways in which these genetic technologies may be applied to risk mitigation in bioremediation and phytoremediation are discussed.