Release of transgenic plants: biodiversity and population-level considerations

Many transgenic plant gene products introduced into soil are likely to degrade so quickly that they will have little impact upon the ecosystem. Some products, however, will be more recalcitrant and thus may exhibit potentially adverse effects upon the soil ecosystem. Potential changes in specific groups of organisms, functional groups of organisms and biodiversity are discussed. Methods to detect changes and the impact of changes are considered.     

释放后的转抗病虫基因作物对土壤生物群落的影响

土壤生物,尤其是土壤微生物多样性与活性的保持是农业生态系统健康稳定的基础,农业活动尤其是农作物植被类型的改变对土壤生物的群落结构和活性具有显著的影响。释放后的转基因作物作为生态系统的一种新的生物组分,被引入农田生态系统之间后所引发的农田生物群落（包括土壤微生物群落）的变化及其对农业生态系统的健康与稳定产生的影响,已成为研究热点,本文对转抗虫Bt基因作物、转T4-溶菌酶基因作物,转蛋白酶抑制剂I基因作物的基因产物、作物残体在土壤中的行为（如降解产物的存留形态与生物活性）及其对根际或残体周围土壤中各类生物,尤其是微生物群落结构与功能的影响进行了简要综合评述,指出基因表达产物的后效肯定是存在的且长远的,由其引发的土壤生物群落结构的变化是复杂的,因而有必要对不同类型的转基因作物释放后的生态效应做长期的跟踪研究,建议未来的研究工作应集中在以下3个方面：（1）不同的转基因表达产物在环境中的迁移、结构变化、消长动态及其对生物保持毒杀性的时间;（2）不同类型转基因的植物对土壤生物群落结构的影响趋势;（3）在实验条件下,研究分离纯化的各种转基因表达产物对土壤各生物功能类群的影响。     

转基因植物对传粉蜂类影响的研究进展

随着转基因作物的大规模商业种植,其对非靶生物的安全性问题已经成为转基因生物风险评估的重要内容。有些转基因植物是需要蜂类传粉的显花植物．有些则可作为蜂类的食物来源。抗性转基因植物可能对蜂类产生直接和间接的影响。转基因植物的花粉或者花蜜可能对蜂类产生直接影响,测试化学杀虫剂安全性的相同方法被用来评估这种影响。很多研究测试了纯化的转基因蛋白质(如Bt,蛋白酶抑制剂)对蜜蜂和熊蜂的影响。间接影响来自于遗传转化可能使转基因植物的表型产生的异常变化,特别是花的改变。这种影响是用转基因植物进行评估的。转基因植物对蜂类的影响研究所测定的参数包括蜂类的肠道生理、取食和嗅觉学习行为、经口毒性和寿命等。对在实验室中以幼蜂或者成年蜂为对象、在封闭条件下针对蜂群的研究以及蜂类在转基因植物上的取食行为方面的研究进行了总结。结果表明,转基因植物是否对传粉蜂类产生影响以及影响的大小主要取决于转基因植物的生物学特征、转基因蛋白的性质和表达量。一般来说,Bt毒素蛋白和葡聚糖酶等蛋白质对蜂类没有影响;而某些蛋白酶抑制剂和几丁质酶则可能对蜂类产生不利影响,蜂类所摄取到这类蛋白质的剂量将决定影响的程度。然而,由于采用的研究方法各不相同,难以对不同的研究结果进行分析和比较,并得出明确的结论。     

Ecological Risk Assessment and Regulation for Genetically-Modified Ornamental Plants

Classic plant breeding has increased the beauty and utility of ornamental plants, but biotechnology can offer completely new traits for plants used in homes and gardens. The creation of blue petal color in carnations and roses are examples where biotechnology has created novelty that conventional hybridization cannot match. However, all innovations have benefits and risks, and future commercialization of transgenic ornamental plants raises complex questions about potential negative impacts to managed landscapes and natural ecosystems. Predictive ecological risk assessment is a process that uses current knowledge to estimate future environmental harms or benefits arising from direct or indirect exposure to a genetically-modified (GM) plant, its genes, or gene products. This article considers GM ornamental plants in the context of current ecological risk assessment principles, research results, and current regulatory frameworks. The use of ecological risk assessment by government agencies to support decision-making is reviewed in the context of ornamental plants. Government risk assessments have usually emphasized the potential for pollen-mediated gene flow, weediness in managed areas, invasion of natural areas, and direct harm to nontarget organisms. Some of the major challenges for predictive risk assessment include characterizing gene flow over time and space, plant fitness in changing environments, and impacts to nontarget organisms, communities and ecosystems. The lack of baseline information about the ecology and biodiversity of urban areas, gardens, and natural ecosystems limits the ability to predict potential hazards, identify exposure pathways, and design hypothesis-driven research. The legacy of introduced ornamental plants as invasive species generates special concern about future invasions, especially for GM plants that exhibit increased stress tolerance or adaptability. While ecological risk assessments are a valuable tool and have helped harmonize regulation of GM plants, they do not define the acceptable level of risk or uncertainty. That responsibility belongs to regulators, stakeholders and citizens.     

Genetic transformation of lignin degrading fungi facilitated by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

Background  

White-rot fungi are primarily the major degraders of lignin, a major obstacle for commercial exploitation of plant byproducts to produce bioethanol and other industrially important products. However, to improve their efficacy for lignin degradation, it has become necessary to genetically modify these organisms using appropriate vectors. Agrobacterium tumefaciens, a soil phytopathogenic bacterium, generally transforms plants by delivering a portion of the resident Ti- plasmid, the T-DNA (transfer DNA). The trans-Kingdom gene transfer is initiated by the activity of Ti-plasmid encoded vir (virulence) genes in response to low-molecular-mass phenolic compounds such as acetosyringone. A. tumefaciens played a major role in plant genetic engineering and basic research in molecular biology, accounting for nearly 80% of the transgenic plants produced so far. Initially, it was believed that only dicotyledons, gymnosperms and a few monocotyledonous species could be transformed by this bacterium; but recent reports have totally changed this scenario by demonstrating that many 'recalcitrant' species not included in its natural host range can also be transformed, especially filamentous fungi.     

Degradation of the Cry1Ab protein within transgenic Bacillus thuringiensis corn tissue in the field

Large quantities of Bacillus thuringiensis (Bt) corn plant residue are left in the field after harvest, which may have implications for the soil ecosystem. Potential impacts on soil organisms will also depend on the persistence of the Bt toxin in plant residues. Therefore, it is important to know how long the toxin persists in plant residues. In two field studies in the temperate corn-growing region of Switzerland we investigated degradation of the Cry1Ab toxin in transgenic Bt corn leaves during autumn, winter and spring using an enzyme-linked immunosorbent assay (ELISA). In the first field trial, representing a tillage system, no degradation of the Cry1Ab toxin was observed during the first month. During the second month, Cry1Ab toxin concentrations decreased to approximately 20% of their initial values. During winter, there was no further degradation. When temperatures again increased in spring, the toxin continued to degrade slowly, but could still be detected in June. In the second field trial, representing a no-tillage system, Cry1Ab toxin concentrations decreased without initial delay as for soil-incorporated Bt plants, to 38% of the initial concentration during the first 40 days. They then continued to decrease until the end of the trial after 200 days in June, when 0.3% of the initial amount of Cry1Ab toxin was detected. Our results suggest that extended pre- and post-commercial monitoring are necessary to assess the long-term impact of Bt toxin in transgenic plant residues on soil organisms.     

转转基因植物对根际土壤生态系统的影响

随着全世界转基因植物种植的普及,转基因植物对生态环境的影响也受到人们的广泛关注。本文针对转基因植物对土壤生态系统带来的潜在风险做了较全面的探讨,概述了转基因植物在土壤中的残留、外源基因的水平转移及其表达产物对土壤生物、土壤理化性质的影响,为今后更安全利用转基因植物提供借鉴。     

Test systems to determine the ecological risks posed by toxin release from Bacillus thuringiensis genes in crop plants

Procedures for the selection of species for ecotoxicological risk assessment of Bacillus thuringiensis (Bt) gene products in the epigeal and hypogeal environments are proposed. Although species can be selected on the basis of ecological realism and functional importance, the number of organisms requiring testing and the nature of the test procedures remain uncertain with such a selectively toxic material. The heterogeneity of the soil environment, the stratification of plant material at different stages of breakdown and decomposition and the aggregation and patterns of movement of the soil fauna and flora impose problems for the design of ecologically relevant test methods. Similarly, the impact upon beneficial invertebrates, if toxic effects are detected, will be mediated by the scale and pattern of transgenic plant release in the fragmented agricultural landscape. To properly assess the ecological risks posed by a widely released toxin with a narrow spectrum of effects, a combination of laboratory tests, field experiments and longer-term monitoring will be required.     

提高植物磷营养效率（候选）基因研究进展

低磷限制植物产量提高和品质改良是全球亟待解决的土壤养分问题之一,利用现代转基因技术结合常规育种手段培育磷高效新品种是解决这一问题的有效途径。目前,已有百余个磷营养效率候选基因被克隆,但真正用于植物转基因育种实践的却寥寥无几,且进展缓慢。究其原因可能是由于现有候选基因种类繁杂且未系统分类,有些基因的功能尚未明确,缺少克隆与育种交流平台等问题。鉴于此,本文将目前已克隆的植物磷营养效率候选基因按照其功能不同进行分析,对基因的生物学功能及其潜在应用价值进行归纳总结。这不仅为分子生物学家选择目标基因,解析植物磷高效分子机制提供参考,同时为育种学家进行基因转化,培育磷高效新品种搭建平台。     

花青素调节基因VlmybA2对番茄遗传转化

以小型番茄 Micro-Tom 为材料,利用农杆菌介导法导入花青素调节基因VlmybA2。对抗性筛选出的再生植株进行 GUS 组织染色和 PCR 检测,证明外源基因已经整合到 Micro-Tom 中,转基因番茄根、茎、叶脉、果皮均呈紫色,花色为黄紫嵌合。而野生型的根为白色,茎、叶脉呈绿色,果皮为红色,花为黄色。对转基因番茄的花青素含量、叶片叶绿素含量和光合速率等生理指标进行测定,花青素含量有显著增加,叶绿素含量降低,VlmybA2基因过量表达会降低植株的光合效率,但对植株正常生长影响并不显著。VlmybA2 基因既可增加抗衰老物质花青素含量,又可作为转基因植株的报告基因。     

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.     

Enhancement of plant-microbe interactions using a rhizosphere metabolomics-driven approach and its application in the removal of polychlorinated biphenyls

Persistent organic pollutants, such as polychlorinated biphenyls (PCBs), are a global problem. We demonstrate enhanced depletion of PCBs using root-associated microbes, which can use plant secondary metabolites, such as phenylpropanoids. Using a "rhizosphere metabolomics" approach, we show that phenylpropanoids constitute 84% of the secondary metabolites exuded from Arabidopsis roots. Phenylpropanoid-utilizing microbes are more competitive and are able to grow at least 100-fold better than their auxotrophic mutants on roots of plants that are able to synthesize or overproduce phenylpropanoids, such as flavonoids. Better colonization of the phenylpropanoid-utilizing strain in a gnotobiotic system on the roots of flavonoid-producing plants leads to almost 90% removal of PCBs in a 28-d period. Our work complements previous approaches to engineer soil microbial populations based on opines produced by transgenic plants and used by microbes carrying opine metabolism genes. The current approach based on plant natural products can be applied to contaminated soils with pre-existing vegetation. This strategy is also likely to be applicable to improving the competitive abilities of biocontrol and biofertilization strains.     

High‐throughput detection and screening of plants modified by gene editing using quantitative real‐time polymerase chain reaction

Gene editing techniques are becoming powerful tools for modifying target genes in organisms. Although several methods have been developed to detect gene‐edited organisms, these techniques are time and labour intensive. Meanwhile, few studies have investigated high‐throughput detection and screening strategies for plants modified by gene editing. In this study, we developed a simple, sensitive and high‐throughput quantitative real‐time (qPCR)‐based method. The qPCR‐based method exploits two differently labelled probes that are placed within one amplicon at the gene editing target site to simultaneously detect the wild‐type and a gene‐edited mutant. We showed that the qPCR‐based method can accurately distinguish CRISPR/Cas9‐induced mutants from the wild‐type in several different plant species, such as Oryza sativa, Arabidopsis thaliana, Sorghum bicolor, and Zea mays. Moreover, the method can subsequently determine the mutation type by direct sequencing of the qPCR products of mutations due to gene editing. The qPCR‐based method is also sufficiently sensitive to distinguish between heterozygous and homozygous mutations in T₀ transgenic plants. In a 384‐well plate format, the method enabled the simultaneous analysis of up to 128 samples in three replicates without handling the post‐polymerase chain reaction (PCR) products. Thus, we propose that our method is an ideal choice for screening plants modified by gene editing from many candidates in T₀ transgenic plants, which will be widely used in the area of plant gene editing.     

Extracellular release of a heterologous phytase from roots of transgenic plants: does manipulation of rhizosphere biochemistry impact microbial community structure?

To maintain the sustainability of agriculture, it is imperative that the reliance of crops on inorganic phosphorus (P) fertilizers is reduced. One approach is to improve the ability of crop plants to acquire P from organic sources. Transgenic plants that produce microbial phytases have been suggested as a possible means to achieve this goal. However, neither the impact of heterologous expression of phytase on the ecology of microorganisms in the rhizosphere nor the impact of rhizosphere microorganisms on the efficacy of phytases in the rhizosphere of transgenic plants has been tested. In this paper, we demonstrate that the presence of rhizosphere microorganisms reduced the dependence of plants on extracellular secretion of phytase from roots when grown in a P-deficient soil. Despite this, the expression of phytase in transgenic plants had little or no impact on the microbial community structure as compared with control plant lines, whereas soil treatments, such as the addition of inorganic P, had large effects. The results demonstrate that soil microorganisms are explicitly involved in the availability of P to plants and that the microbial community in the rhizosphere appears to be resistant to the impacts of single-gene changes in plants designed to alter rhizosphere biochemistry and nutrient cycling.     

Implications of transgenic, insecticidal plants for insect and plant biodiversity

Genetically modified plants are widely grown predominantly in North America and to a lesser extent in Australia, Argentina and China but their regions of production are expected to spread soon beyond these limited areas also reaching Europe where great controversy over the application of gene technology in agriculture persists. Currently, several cultivars of eight major crop plants are commercially available including canola, corn, cotton, potato, soybean, sugar beet, tobacco and tomato, but many more plants with new and combined multiple traits are close to registration. While currently agronomic traits (herbicide resistance, insect resistance) dominate, traits conferring “quality” traits (altered oil compositions, protein and starch contents) will begin to dominate within the next years. However, economically the most promising future lies in the development and marketing of crop plants expressing pharmaceutical or “nutraceuticals” (functional foods), and plants that express a number of different genes. From this it is clear that future agricultural and, ultimately, also natural ecosystems will be challenged by the large-scale introduction of entirely novel genes and gene products in new combinations at high frequencies all of which will have unknown impacts on their associated complex of non-target organisms, i.e. all organisms that are not targeted by the insecticidal protein. In times of severe global decline of biodiversity, pro-active precaution is necessary and careful consideration of the likely expected effects of transgenic plants on biodiversity of plants and insects is mandatory.In this paper possible implications of non-target effects for insect and plant biodiversity are discussed and a case example of such non-target effects is presented. In a multiple year research project, tritrophic and bitrophic effects of transgenic corn, expressing the gene from Bacillus thuringiensis (Bt-corn) that codes for the high expression of an insecticidal toxin (Cry1Ab), on the natural enemy species, Chrysoperla carnea (the green lacewing), was investigated. In these laboratory trials, we found prey-mediated effects of transgenic Bt-corn causing significantly higher mortality of C. carnea larvae. In further laboratory trials, we confirmed that the route of exposure (fed directly or via a herbivorous prey) and the origin of the Bt (from transgenic plants or incorporated into artificial diet) strongly influenced the degree of mortality. In choice feeding trials where C. carnea could choose between Spodoptera littoralis fed transgenic Bt-corn and S. littoralis fed non-transgenic corn, larger instars showed a significant preference for S. littoralis fed non-transgenic corn while this was not the case when the choice was between Bt- and isogenic corn fed aphids. Field implications of these findings could be multifold but will be difficult to assess because they interfere in very intricate ways with complex ecosystem processes that we still know only very little about. The future challenge in pest management will be to explore how transgenic plants can be incorporated as safe and effective components of IPM systems and what gene technology can contribute to the needs of a modern sustainable agriculture that avoids or reduces adverse impacts on biodiversity? For mainly economically motivated resistance management purposes, constitutive high expression of Bt-toxins in transgenic plants is promoted seeking to kill almost 100% of all susceptible (and if possible heterozygote resistant) target pest insects. However, for pest management this is usually not necessary. Control at or below an established economic injury level is sufficient for most pests and cropping systems. It is proposed that partially or moderately resistant plants expressing quantitative rather than single gene traits and affecting the target pest sub-lethally may provide a more meaningful contribution of agricultural biotechnology to modern sustainable agriculture. Some examples of such plants produced through conventional breeding are presented. Non-target effects may be less severe allowing for better incorporation of these plants into IPM or biological control programs using multiple control strategies, thereby, also reducing selection pressure for pest resistance development.     

Marker Gene Controversy in Transgenic Plants

Biosafety implications of selectable marker genes that are integrated into the transgenic plants are discussed. In the laboratory, selectable marker genes are used at two stages to distinguish transformed cells out of a large population of nontransformed cells: 1) initial assembly of gene cassettes is generally done in E. coli on easily manipulatable plasmid vectors that contain the selectable marker genes which often code for antibiotic inactivating enzymes, and 2) Then the gene cassettes are inserted into the plant genome by various transformation methods. For selection of transformed plant cells, antibiotic and herbicide resistance genes are widely used. Consequently, transgenic plants can end up with DNA sequences of selectable markers that are functional in E. coli and plants. The potential for horizontal gene transfer of selectable markers from transgenic plants to other organisms both in the environment and in the intestine of humans and animals is evaluated. Mechanisms and consequences of the transfer of marker genes from plants to other organisms is examined. Strategies to avoid marker genes in plants are discussed. It is possible to avoid the use of controversial selectable markers in the construction of transgenic plants.     

Seasonal changes in the rhizosphere microbial communities associated with field-grown genetically modified canola (Brassica napus)

The introduction of transgenic plants into agricultural ecosystems has raised the question of the ecological impact of these plants on nontarget organisms, such as soil bacteria. Although differences in both the genetic structure and the metabolic function of the microbial communities associated with some transgenic plant lines have been established, it remains to be seen whether these differences have an ecological impact on the soil microbial communities. We conducted a 2-year, multiple-site field study in which rhizosphere samples associated with a transgenic canola variety and a conventional canola variety were sampled at six times throughout the growing season. The objectives of this study were to identify differences between the rhizosphere microbial community associated with the transgenic plants and the rhizosphere microbial community associated with the conventional canola plants and to determine whether the differences were permanent or depended on the presence of the plant. Community-level physiological profiles, fatty acid methyl ester profiles, and terminal amplified ribosomal DNA restriction analysis profiles of rhizosphere microbial communities were compared to the profiles of the microbial community associated with an unplanted, fallow field plot. Principal-component analysis showed that there was variation in the microbial community associated with both canola variety and growth season. Importantly, while differences between the microbial communities associated with the transgenic plant variety were observed at several times throughout the growing season, all analyses indicated that when the microbial communities were assessed after winter, there were no differences between microbial communities from field plots that contained harvested transgenic canola plants and microbial communities from field plots that did not contain plants during the field season. Hence, the changes in the microbial community structure associated with genetically modified plants were temporary and did not persist into the next field season.     

Seasonal Changes in the Rhizosphere Microbial Communities Associated with Field-Grown Genetically Modified Canola (Brassica napus)

The introduction of transgenic plants into agricultural ecosystems has raised the question of the ecological impact of these plants on nontarget organisms, such as soil bacteria. Although differences in both the genetic structure and the metabolic function of the microbial communities associated with some transgenic plant lines have been established, it remains to be seen whether these differences have an ecological impact on the soil microbial communities. We conducted a 2-year, multiple-site field study in which rhizosphere samples associated with a transgenic canola variety and a conventional canola variety were sampled at six times throughout the growing season. The objectives of this study were to identify differences between the rhizosphere microbial community associated with the transgenic plants and the rhizosphere microbial community associated with the conventional canola plants and to determine whether the differences were permanent or depended on the presence of the plant. Community-level physiological profiles, fatty acid methyl ester profiles, and terminal amplified ribosomal DNA restriction analysis profiles of rhizosphere microbial communities were compared to the profiles of the microbial community associated with an unplanted, fallow field plot. Principal-component analysis showed that there was variation in the microbial community associated with both canola variety and growth season. Importantly, while differences between the microbial communities associated with the transgenic plant variety were observed at several times throughout the growing season, all analyses indicated that when the microbial communities were assessed after winter, there were no differences between microbial communities from field plots that contained harvested transgenic canola plants and microbial communities from field plots that did not contain plants during the field season. Hence, the changes in the microbial community structure associated with genetically modified plants were temporary and did not persist into the next field season.