Environmental fate and behaviour of antibiotic resistance genes and small interference RNAs released from genetically modified crops

Rising global populations have amplified food scarcity across the world and ushered in the development of genetically modified (GM) crops to overcome these challenges. Cultivation of major crops such as corn and soy has favoured GM crops over conventional varieties to meet crop production and resilience needs. Modern GM crops containing small interference RNA molecules and antibiotic resistance genes have become increasingly common in the United States. However, the use of these crops remains controversial due to the uncertainty regarding the unintended release of its genetic material into the environment and possible downstream effects on human and environmental health. DNA or RNA transgenes may be exuded from crop tissues during cultivation or released during plant decomposition and adsorbed by soil. This can contribute to the persistence and bioavailability in soil or water environment and possible uptake by soil microbial communities and further passing of this information to neighbouring bacteria, disrupting microbial ecosystem services such as nutrient cycling and soil fertility. In this review, transgene mechanisms of action, uses in crops, and knowledge regarding their environmental fate and impact to microbes are evaluated. This aims to encapsulate the current knowledge and promote further research regarding unintended effects transgenes may cause.     

The impact of genetically modified crops on soil microbial communities.

Genetically modified (GM) plants represent a potential benefit for environmentally friendly agriculture and human health. Though, poor knowledge is available on potential hazards posed by unintended modifications occurring during genetic manipulation. The increasing amount of reports on ecological risks and benefits of GM plants stresses the need for experimental works aimed at evaluating the impact of GM crops on natural and agro-ecosystems. Major environmental risks associated with GM crops include their potential impact on non-target soil microorganisms playing a fundamental role in crop residues degradation and in biogeochemical cycles. Recent works assessed the effects of GM crops on soil microbial communities on the basis of case-by-case studies, using multimodal experimental approaches involving different target and non-target organisms. Experimental evidences discussed in this review confirm that a precautionary approach should be adopted, by taking into account the risks associated with the unpredictability of transformation events, of their pleiotropic effects and of the fate of transgenes in natural and agro-ecosystems, weighing benefits against costs.     

Letting the gene out of the bottle: the population genetics of genetically modified crops

Genetically modified (GM) plants are rapidly becoming a common feature of modern agriculture. This transition to engineered crops has been driven by a variety of potential benefits, both economic and ecological. The increase in the use of GM crops has, however, been accompanied by growing concerns regarding their potential impact on the environment. Here, we focus on the escape of transgenes from cultivation via crop x wild hybridization. We begin by reviewing the literature on natural hybridization, with particular reference to gene flow between crop plants and their wild relatives. We further show that natural selection, and not the overall rate of gene flow, is the most important factor governing the spread of favorable alleles. Hence, much of this review focuses on the likely effects of transgenes once they escape. Finally, we consider strategies for transgene containment.     

提高转基因作物生物安全性的分子策略

随着各类转基因作物的问世及其农产品的不断上市,转基因作物的安全性问题已成为公众关心的焦点。综述了提高转基因作物生物安全性的几种分子策略,其中包括选择标记的去除,转基因的组织特异表达和诱导性表达以及转基因逃逸的控制等。     

The impact of hybrids between genetically modified crop plants and their related species: biological models and theoretical perspectives

Forces affecting the rate of spread and increase of hybrids between genetically modified crop plants and their related species remain qualitatively similar, irrespective of whether genetic modification was achieved using traditional methods, those of biotechnology or as a result of the natural evolutionary process. However, the precise magnitude of the forces and, consequently, the likely environmental impact of such hybrids, may depend strongly on the nature of the gene or genes introduced into the native species. While many classes of transgenes are similar to those manipulated by conventional breeding techniques or evolution, biotechnology offers the potential to introduce genes into crops which are novel both from the point of view of function and origin. The qualitative similarity between transgenes and the products of conventional or evolutionary modification suggests that a historical view of the environmental impact of hybrids between traditionally produced crops or exotic species and their relatives would be of use in estimating the probable fate of hybrids containing transgenes in the environment. However, with certain classes of transgenes for which there are no existing analogues, there will need to be greater care in assessing the possible risks associated with release into the environment.     

The ecological risks of transgenic plants.

Biotechnologies have been utilized "ante litteram" for thousands of years to produce food and drink and genetic engineering techniques have been widely applied to produce many compounds for human use, from insulin to other medicines. The debate on genetically modified (GM) organisms broke out all over the world only when GM crops were released into the field. Plant ecologists, microbiologists and population geneticists carried out experiments aimed at evaluating the environmental impact of GM crops. The most significant findings concern: the spread of transgenes through GM pollen diffusion and its environmental impact after hybridisation with closely related wild species or subspecies; horizontal gene transfer from transgenic plants to soil microbes; the impact of insecticide proteins released into the soil by transformed plants on non-target microbial soil communities. Recent developments in genetic engineering produced a technology, dubbed "Terminator", which protects patented genes introduced in transgenic plants by killing the seeds in the second generation. This genetic construct, which interferes so heavily with fundamental life processes, is considered dangerous and should be ex-ante evaluated taking into account the data on "unexpected events", as here discussed, instead of relying on the "safe until proven otherwise" claim. Awareness that scientists, biotechnologists and genetic engineers cannot answer the fundamental question "how likely is that transgenes will be transferred from cultivated plants into the natural environment?" should foster long-term studies on the ecological risks and benefits of transgenic crops.     

Molecular strategies for gene containment in transgenic crops

The potential of genetically modified (GM) crops to transfer foreign genes through pollen to related plant species has been cited as an environmental concern. Until more is known concerning the environmental impact of novel genes on indigenous crops and weeds, practical and regulatory considerations will likely require the adoption of gene-containment approaches for future generations of GM crops. Most molecular approaches with potential for controlling gene flow among crops and weeds have thus far focused on maternal inheritance, male sterility, and seed sterility. Several other containment strategies may also prove useful in restricting gene flow, including apomixis (vegetative propagation and asexual seed formation), cleistogamy (self-fertilization without opening of the flower), genome incompatibility, chemical induction/deletion of transgenes, fruit-specific excision of transgenes, and transgenic mitigation (transgenes that compromise fitness in the hybrid). As yet, however, no strategy has proved broadly applicable to all crop species, and a combination of approaches may prove most effective for engineering the next generation of GM crops.     

The impact of hybrids between genetically modified crop plants and their related species: general considerations

Factors influencing the fate and impact of hybrids between crop plants and their related species operate from the early zygote, through to plant establishment in different habitats, to their ability to form self-sustaining populations. Many of the classes of genes being introduced by modern methods of genetic modification are similar to those manipulated by conventional plant breeding. In assessing the impact of transgenes in hybrids between crops and related species, therefore, it is important to be informed about the consequences of hybridization between conventionally bred varieties and their relatives. Some transgenes will have novel effects (e.g. production of pharmaceutical substances or certain fatty acids) on plants, and are likely to need specific assessment studies to determine their impact on hybrids. This will be particularly important if there is the possibility of these transgenes becoming established in wild populations. Some recommendations for further research are outlined.     

转基因植物中外源基因及其表达产物转移的途径

随着转基因植物商品化应用的增多,全面了解转基因植物潜在的生态风险性尤为重要。国内外对“转基因植物中外源基因向野生亲缘物种漂移的可能性”、“昆虫对抗虫转基因植物的耐受性”以及“转基因植物对生物多样性的潜在影响”等问题已进行了广泛研究。对转基因植物中外源基因及其表达产物的几种可能转移途径作了概述。着重介绍了“经花粉散布或与野生亲缘物种杂交等途径引起的外源基因转移”以及“转基因植物对土壤生态系统的影响”等方面的研究情况。此外,还对“鉴定外源基因及其表达产物存在的方法”进行了简要探讨。     

Recent advances in development of marker-free transgenic plants: Regulation and biosafety concern

During the efficient genetic transformation of plants with the gene of interest, some selectable marker genes are also used in order to identify the transgenic plant cells or tissues. Usually, antibiotic- or herbicide-selective agents and their corresponding resistance genes are used to introduce economically valuable genes into crop plants. From the biosafety authority and consumer viewpoints, the presence of selectable marker genes in released transgenic crops may be transferred to weeds or pathogenic microorganisms in the gastrointestinal tract or soil, making them resistant to treatment with herbicides or antibiotics, respectively. Sexual crossing also raises the problem of transgene expression because redundancy of transgenes in the genome may trigger homology-dependent gene silencing. The future potential of transgenic technologies for crop improvement depends greatly on our abilities to engineer stable expression of multiple transgenic traits in a predictable fashion and to prevent the transfer of undesirable transgenic material to non-transgenic crops and related species. Therefore, it is now essential to develop an efficient marker-free transgenic system. These considerations underline the development of various approaches designed to facilitate timely elimination of transgenes when their function is no longer needed. Due to the limiting number of available selectable marker genes, in future the stacking of transgenes will be increasingly desirable. The production of marker-free transgenic plants is now a critical requisite for their commercial deployment and also for engineering multiple and complex trait. Here we describe the current technologies to eliminate the selectable marker genes (SMG) in order to develop marker-free transgenic plants and also discuss the regulation and biosafety concern of genetically modified (GM) crops.     

Challenges for transgene detection in landraces and wild relatives: learning from 15 years of debate over GM maize in Mexico

Maize is one of the world’s five staple cereals and its traditional varieties constitute a global resource critical to future agricultural development. Fifteen years ago, claims that transgenes had spread into traditional landrace maize in Mexico started an international discussion on the scale and significance of transgene flow from genetically modified (GM) crops to centres of crop origin and genetic diversity. The initial discovery of transgenes in landrace maize sparked an intense environmental dispute in which the culture and traditions of indigenous people were seen as threatened by the unchecked spread of biotechnological inventions from multinational corporations. This dispute was reflected in a political and legal battle over the regulatory status of GM crops in Mexico, which continues today as approvals of GM maize for cultivation remain subject to contestation in the courts. These legal, political and environmental disputes have been fanned by the existence of a significant scientific controversy over the methods for GM detection. The use of various approaches and a lack of harmonized methods specific for monitoring and detection of transgenes in landraces has generated both positive and negative results for GM contamination in Mexico over the years. In this paper, we review the peer-reviewed literature on transgene detection in Mexican maize and highlight the challenges associated with transgene detection in landraces. In doing so, we identify the key methodological aspects under dispute and pinpoint the research bottlenecks and needs for building the capacity to effectively monitor transgene escape from GM crops to wild relatives or landraces.     

The case for genetically modified crops with a poverty focus

Recently seven National Academies of Science produced a report on transgenic plants and world agriculture. The report provides scientific perspectives to the ongoing public debate about the potential role for transgenic technology in world agriculture. In this article, we develop the themes of the report and emphasize the potential for future genetically modified (GM) crops with a poverty focus, emphasizing the potential of GM resistance to plant parasitic nematodes for subsistence potato farmers in Bolivia. We judge that a range of incremental gains to crop yields from many transgenes are valuable for future world security. We advocate the establishment of a standard that GM crops must achieve before they are both biosafe and appropriate for resource-poor farmers and we believe that the best interests of the poor require biotechnologists to work towards that objective.     

The impact of genetic modification of human foods in the 21st century: a review

Genetic engineering of food is the science which involves deliberate modification of the genetic material of plants or animals. It is an old agricultural practice carried on by farmers since early historical times, but recently it has been improved by technology. Many foods consumed today are either genetically modified (GM) whole foods, or contain ingredients derived from gene modification technology. Billions of dollars in U.S. food exports are realized from sales of GM seeds and crops. Despite the potential benefits of genetic engineering of foods, the technology is surrounded by controversy. Critics of GM technology include consumer and health groups, grain importers from European Union (EU) countries, organic farmers, environmentalists, concerned scientists, ethicists, religious rights groups, food advocacy groups, some politicians and trade protectionists. Some of the specific fears expressed by opponents of GM technology include alteration in nutritional quality of foods, potential toxicity, possible antibiotic resistance from GM crops, potential allergenicity and carcinogenicity from consuming GM foods. In addition, some more general concerns include environmental pollution, unintentional gene transfer to wild plants, possible creation of new viruses and toxins, limited access to seeds due to patenting of GM food plants, threat to crop genetic diversity, religious, cultural and ethical concerns, as well as fear of the unknown. Supporters of GM technology include private industries, research scientists, some consumers, U.S. farmers and regulatory agencies. Benefits presented by proponents of GM technology include improvement in fruit and vegetable shelf-life and organoleptic quality, improved nutritional quality and health benefits in foods, improved protein and carbohydrate content of foods, improved fat quality, improved quality and quantity of meat, milk and livestock. Other potential benefits are: the use of GM livestock to grow organs for transplant into humans, increased crop yield, improvement in agriculture through breeding insect, pest, disease, and weather resistant crops and herbicide tolerant crops, use of GM plants as bio-factories to yield raw materials for industrial uses, use of GM organisms in drug manufacture, in recycling and/or removal of toxic industrial wastes. The potential risks and benefits of the new technology to man and the environment are reviewed. Ways of minimizing potential risks and maximizing the benefits of GM foods are suggested. Because the benefits of GM foods apparently far outweigh the risks, regulatory agencies and industries involved in GM food business should increase public awareness in this technology to enhance worldwide acceptability of GM foods. This can be achieved through openness, education, and research.     

Crop seed spillage along roads: a factor of uncertainty in the containment of GMO

Feral populations of crop species along roadsides contribute to the uncertainty regarding the containment of genetically modified (GM) crops, as the feral populations could promote the persistence of transgenes outside of cultivated fields. Roadside populations of several common crop species are known to occur far from arable fields, and the dispersal pathways that promote their recruitment in road verges are unclear. Human-aided dispersal, in particular adhesive dispersal by vehicles, has been suggested as a possible vector, but this has not yet been proven experimentally. We sampled the seed rain from vehicles inside two motorway tunnels in an urban environment to reveal the contribution of crop species to seeds unintentionally dispersed by traffic beyond agricultural production areas. Three species of arable crops, wheat Triticum aestivum , rye Secale cereale and oilseed rape Brassica napus , were among the most frequent species deposited by vehicles inside the motorway tunnels. Each of the three species was clearly more predominant in one direction of traffic. While seeds of Triticum aestivum and Secale cereale were primarily transported into the city, Brassica napus was significantly more abundant in samples from lanes leading out of the city. Seed sources in the local surroundings of the tunnels were virtually nonexistent, and the high magnitude of seed deposition combined with high seed weights suggests a dispersal mechanism different from other species in the sample, at least for Triticum aestivum and Secale cereale . This provides evidence that spillage during transport is a major driver for long-distance dispersal of crops. Our results suggest that seed dispersal by vehicles is the major driver in the recruitment of roadside populations of arable crops, providing a possible escape route for GM crops. Risk management should thus aim at curbing transport losses of GM crops.     

Probability functions to build composite indicators: A methodology to measure environmental impacts of genetically modified crops

There is an on-going debate on the environmental effects of genetically modified crops to which this paper aims to contribute. First, data on environmental impacts of genetically modified (GM) and conventional crops are collected from peer-reviewed journals, and secondly an analysis is conducted in order to examine which crop type is less harmful for the environment. Published data on environmental impacts are measured using an array of indicators, and their analysis requires their normalisation and aggregation. Taking advantage of composite indicators literature, this paper builds composite indicators to measure the impact of GM and conventional crops in three dimensions: (1) non-target key species richness, (2) pesticide use, and (3) aggregated environmental impact. The comparison between the three composite indicators for both crop types allows us to establish not only a ranking to elucidate which crop is more convenient for the environment but the probability that one crop type outperforms the other from an environmental perspective. Results show that GM crops tend to cause lower environmental impacts than conventional crops for the analysed indicators.     

Transgenic plants and biosafety: science, misconceptions and public perceptions

One usually thinks of plant biology as a non-controversial topic, but the concerns raised over the biosafety of genetically modified (GM) plants have reached disproportionate levels relative to the actual risks. While the technology of changing the genome of plants has been gradually refined and increasingly implemented, the commercialization of GM crops has exploded. Today's commercialized transgenic plants have been produced using Agrobacterium tumefaciens-mediated transformation or gene gun-mediated transformation. Recently, incremental improvements of biotechnologies, such as the use of green fluorescent protein (GFP) as a selectable marker, have been developed. Non-transformation genetic modification technologies such as chimeraplasty will be increasingly used to more precisely modify germplasm. In spite of the increasing knowledge about genetic modification of plants, concerns over ecological and food biosafety have escalated beyond scientific rationality. While several risks associated with GM crops and foods have been identified, the popular press, spurred by colorful protest groups, has left the general public with a sense of imminent danger. Reviewed here are the risks that are currently under research. Ecological biosafety research has identified potential risks associated with certain crop/transgene combinations, such as intra- and interspecific transgene flow, persistence and the consequences of transgenes in unintended hosts. Resistance management strategies for insect resistance transgenes and non-target effects of these genes have also been studied. Food biosafety research has focused on transgenic product toxicity and allergenicity. However, an estimated 3.5 x 10(12) transgenic plants have been grown in the U.S. in the past 12 years, with over two trillion being grown in 1999 and 2000 alone. These large numbers and the absence of any negative reports of compromised biosafety indicate that genetic modification by biotechnology poses no immediate or significant risks and that resulting food products from GM crops are as safe as foods from conventional varieties. We are increasingly convinced that scientists have a duty to conduct objective research and to effectively communicate the results--especially those pertaining to the relative risks and potential benefits--to scientists first and then to the public. All stakeholders in the technology need more effective dialogues to better understand risks and benefits of adopting or not adopting agricultural biotechnologies.     

Evaluating genetic containment strategies for transgenic plants

One of the primary concerns about genetically engineered crop plants is that they will hybridize with wild relatives, permitting the transgene to escape into the environment. The likelihood that a transgene will spread in the environment depends on its potential fitness impact. The fitness conferred by various transgenes to crop and/or wild-type hybrids has been evaluated in several species. Different strategies have been developed for reducing the probability and impact of gene flow, including physical separation from wild relatives and genetic engineering. Mathematical models and empirical experimental evidence suggest that genetic approaches have the potential to effectively prevent transgenes from incorporating into wild relatives and becoming established in wild populations that are not reproductively isolated from genetically engineered crops.     

Design for controllability

The safety of genetically modified crops remains a contested issue, given the potential risk for human health and the environment. To further reduce any risks and alleviate public concerns, terminator technology could be used both to tag and control genetically modified plants.Efforts to design genetically modified (GM) crops have focused on minimizing the amount of foreign DNA present in the genome. One reason for this development is to address consumer concerns about unforeseeable effects of either the transgenes or the technology used to introduce them into plant genomes. The latter risk has not been completely assessed, but the former can be dealt with both by minimizing the amount of the foreign DNA inserted and by taking precautionary steps in the selection and adaptation of the transgene itself. In fact, first-generation GM crops contain many unnecessary DNA sequences, such as antibiotic-resistance genes and T-DNA border sequences.These efforts, however, are not only in response to consumer concerns; they will also be helpful to GM engineers. To make maize tolerant to high sodium concentrations, for example, it only requires the introduction of a salt-tolerance gene. Other DNA sequences, such as antibiotic-resistance genes, are only needed to select transgenic cells. Afterwards, they become unnecessary at best and detrimental at worst, as they preclude the use of the same antibiotic to select for the introduction of further foreign DNA sequences. New methods are already being used to transfer transgenes and cisgenes, and to introduce specific mutations that do not require any marker genes [1]. Along with these efforts, discussions have begun about establishing a precise definition of GM [2,3]. The main argument is that if the plant does not contain any transgenes, it is not subject to GM regulation [3,4].But are these new endeavours sufficient to prevent potential harm to humans or the environment? The accident at the nuclear reactor in Fukushima, Japan, in March 2011, demonstrated that a disastrous event can overcome even supposedly safe design—the power plant was not built to withstand the double impact of an earthquake and a tsunami. Thus, rather than simply minimizing risk, we need to develop an emergency control that can shut down everything if the system gets out of control. In the case of GM organisms, no matter how they have been created, we need to be able to trace every individual plant and control its biological activity.In short, we need a tag that identifies a GM crop as such. It is usually possible to identify a GM plant or plant product by using PCR-based analysis to detect the transgene or selection markers. However, new techniques that enable site-specific mutagenesis or the introduction of cisgenes without selection markers would generate ‘stealth'' GM products that are unidentifiable. Furthermore, private companies do not necessarily share information about the nature of the transgene, the selection markers and the exact technique used to generate a certain plant line, which would also make detection impossible. Thus, an easily identifiable tag would help to identify GM crops no matter how and where they have been created.This should be a germination control or ‘terminator'' gene, such as that developed by Monsanto under the moniker ‘genetic use restriction technology'' (GURT), but which was abandoned for commercial use after severe protests [5,6]. One variety of GURT would make the viability of the transgene dependent on treatment with a specific chemical; it would be easy to design GM plants based on GURT that would stop the production of viable seeds if not regularly treated with the activator compound. As transgenic plants with the terminator gene would not grow without the reagent, the escaping of transgenes into the wild would be highly unlikely. If a particular GM line were found to be harmful to human health or the environment after release, the only necessary action to eliminate them would be to withhold treatment.Natural organisms cannot be controlled as easily. Invasive species, such as Caulerpa taxifolia, have disastrous effects on the ecosystems into which they are introduced, and human efforts to keep them under control have largely failed. Thus, the most important aspect of artificial products is that they must be controllable. The nuclear disasters at Fukushima and Chernobyl happened because humans lost control over the reactors. As with reactors, GM crops are artificial constructs over which we must maintain control.Thus, the international community, including plant breeding companies, should discuss the possibility of tagging all GM crops with the terminator system. As the tag and the introduced gene must be inseparable, GM engineers must insert any designed transgene with a terminator tag in tandem. Such GM organisms could then be considered as the ‘same in kind'' as non-GM plants with a similar phenotype after appropriate risk and safety assessment. Of course, possibilities other than the terminator system should be investigated; however, at the moment it is the best option. If the idea of a general tag for all GM plants were accepted, researchers could then improve the tag to make it more compact and safer than Monsanto''s terminator technology and give us even stricter control over GM organisms. The terminator is not a terminus, but a start.     

Impact of interspecific hybridization between crops and weedy relatives on the evolution of flowering time in weedy phenotypes

Background

Like conventional crops, some GM cultivars may readily hybridize with their wild or weedy relatives. The progressive introgression of transgenes into wild or weedy populations thus appears inevitable, and we are now faced with the challenge of determining the possible evolutionary effects of these transgenes. The aim of this study was to gain insight into the impact of interspecific hybridization between transgenic plants and weedy relatives on the evolution of the weedy phenotype.

Methodology/Principal Findings

Experimental populations of weedy birdseed rape (Brassica rapa) and transgenic rapeseed (B. napus) were grown under glasshouse conditions. Hybridization opportunities with transgenic plants and phenotypic traits (including phenological, morphological and reproductive traits) were measured for each weedy individual. We show that weedy individuals that flowered later and for longer periods were more likely to receive transgenic pollen from crops and weed×crop hybrids. Because stem diameter is correlated with flowering time, plants with wider stems were also more likely to be pollinated by transgenic plants. We also show that the weedy plants with the highest probability of hybridization had the lowest fecundity.

Conclusion/Significance

Our results suggest that weeds flowering late and for long periods are less fit because they have a higher probability of hybridizing with crops or weed×crop hybrids. This may result in counter-selection against this subset of weed phenotypes, and a shorter earlier flowering period. It is noteworthy that this potential evolution in flowering time does not depend on the presence of the transgene in the crop. Evolution in flowering time may even be counter-balanced by positive selection acting on the transgene if the latter was positively associated with maternal genes promoting late flowering and long flowering periods. Unfortunately, we could not verify this association in the present experiment.     

The release of genetically modified crops into the environment. Part I. Overview of current status and regulations

In the past 6 years, the global area of commercially grown, genetically modified (GM) crops has increased more than 30-fold to over 52 million hectares. The number of countries involved has more than doubled. Especially in developing countries, the GM crop area is anticipated to increase rapidly in the coming years. Despite this high adoption rate and future promises, there is a multitude of concerns about the impact of GM crops on the environment. Regulatory approaches in Europe and North America are essentially different. In the EU, it is based on the process of making GM crops; in the US, on the characteristics of the GM product. Many other countries are in the process of establishing regulation based on either system or a mixture. Despite these differences, the information required for risk assessment tends to be similar. Each risk assessment considers the possibility, probability and consequence of harm on a case-by-case basis. For GM crops, the impact of non-use should be added to this evaluation. It is important that the regulation of risk should not turn into the risk of regulation. The best and most appropriate baseline for comparison when performing risk assessment on GM crops is the impact of plants developed by traditional breeding. The latter is an integral and accepted part of agriculture.