Gene Flow,Risk Assessment and the Environmental Release of Transgenic Plants

The release of genetically modified plants is governed by regulations that aim to provide an assessment of potential impact on the environment. One of the most important components of this risk assessment is an evaluation of the probability of gene flow. In this review, we provide an overview of the current literature on gene flow from transgenic plants, providing a framework of issues for those considering the release of a transgenic plant into the environment. For some plants gene flow from transgenic crops is well documented, and this information is discussed in detail in this review. Mechanisms of gene flow vary from plant species to plant species and range from the possibility of asexual propagation, short- or long-distance pollen dispersal mediated by insects or wind and seed dispersal. Volunteer populations of transgenic plants may occur where seed is inadvertently spread during harvest or commercial distribution. If there are wild populations related to the transgenic crop then hybridization and eventually introgression in the wild may occur, as it has for herbicide resistant transgenic oilseed rape (Brassica napus). Tools to measure the amount of gene flow, experimental data measuring the distance of pollen dispersal, and experiments measuring hybridization and seed survivability are discussed in this review. The various methods that have been proposed to prevent gene flow from genetically modified plants are also described. The current “transgenic traits” in the major crops confer resistance to herbicides and certain insects. Such traits could confer a selective advantage (an increase in fitness) in wild plant populations in some circumstances, were gene flow to occur. However, there is ample evidence that gene flow from crops to related wild species occurred before the development of transgenic crops and this should be taken into account in the risk assessment process.     

Future prospects for transgenic crops

Transgenic (genetically modified) crops are grown at present on more than 40 million hectares in 13 countries around the world. These `first generation' products principally comprise soybean, maize (corn) and cotton resistant to herbicides and/or insects. This review considers the wide range of `second generation' products under development and testing in many commercial and academic laboratories. Such products include examples for a variety of food, medical, veterinary and industrial purposes. In addition the review assesses the present state of public acceptance of transgenic products.     

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.     

Plant genetic engineering in Monsanto company: from the first laboratory experiments to worldwide practical use

The history of modern biotechnology of agricultural plants is briefly considered in the article. Methods of genetic transformation and regeneration of transgenic plants as well as the mechanisms of resistance of genetically modified plants to herbicides and pests are discussed. By the example of genetically modified varieties and hybrids there are shown the ways of solving the problem of weeds and pests. The questions of biosafety legislation in different countries are considered.     

Field testing of virus resistant transgenic plants

A number of crop plants have been genetically modified for the purpose of resisting virus infection. Different resistance types have been observed in transgenic crops. The practical value of genetically modified, virus resistant, economically important crops can be evaluated only by field testing. The criteria for effective field resistance to viral disease can vary significantly depending on the crop and the virus. Furthermore, field testing is required to determine whether important agronomic properties of modified crops were changed by plant transformation and to confirm that the resistance observed under controlled environment is effective also under natural field conditions and to demonstrate the economical value of virus resistant, transgenic plants.     

Overexpression of Rice Ferrochelatase I and II Leads to Increased Susceptibility to Oxyfluorfen Herbicide in Transgenic Rice

Protoporphyrin IX is a photosensitizer and a causative agent of rice membrane lipid peroxidation in plant cells. Protoporphyrinogen IX oxidase (PPO) is the molecular target of PPO-inhibiting herbicides, which trigger a massive increase in protoporphyrin IX. Thus, any possible method to decrease the levels of protoporphyrin IX upon challenge with PPO-inhibiting herbicides could be employed to generate plants resistant to such herbicides. We generated transgenic rice plants overexpressing rice ferrochelatase isogenes encoding ferrochelatase enzymes, which convert protoporphyrin IX into protoheme, to see whether the transgenic plants have phenotypes resistant to PPO-inhibiting herbicides. The resulting transgenic rice plants were all susceptible to oxyfluorfen (a diphenyl-ether-type PPO-inhibiting herbicide), as judged by cellular damage with respect to cellular leakage, chlorophyll loss, and lipid peroxidation. In particular, the transgenic plants expressing rice ferrochelatase II without its plastid targeting sequence showed higher transgene expression and oxyfluorfen susceptibility than lines expressing the intact ferrochelatase II. Possible susceptibility mechanisms to oxyfluorfen herbicide in the transgenic rice plants are discussed.     

Transgenic rice plants expressing a Bacillus subtilis protoporphyrinogen oxidase gene are resistant to diphenyl ether herbicide oxyfluorfen

Protoporphyrinogen oxidase (Protox), the penultimate step enzyme of the branch point for the biosynthetic pathway of Chl and hemes, is the target site of action of diphenyl ether (DPE) herbicides. However, Bacillus subtilis Protox is known to be resistant to the herbicides. In order to develop the herbicide-resistant plants, the transgenic rice plants were generated via expression of B. subtilis Protox gene under ubiquitin promoter targeted to the cytoplasm or to the plastid using Agrobacterium-mediated gene transformation. The integration and expression of the transgene were investigated at T0 generation by DNA and RNA blots. Most transgenic rice plants revealed one copy transgene insertion into the rice genome, but some with 3 copies. The expression levels of B. subtilis Protox mRNA appeared to correlate with the copy number. Furthermore, the plastidal transgenic lines exhibited much higher expression of the Protox mRNA than the cytoplasmic transgenic lines. The transgenic plants expressing the B. subtilis Protox gene at T0 generation were found to be resistant to oxyfluorfen when judged by cellular damage with respect to cellular leakage, Chl loss, and lipid peroxidation. The transgenic rice plants targeted to the plastid exhibited higher resistance to the herbicide than the transgenic plants targeted to the cytoplasm. In addition, possible resistance mechanisms in the transgenic plants to DPE herbicides are discussed.     

Costs of transgenic herbicide resistance introgressed from Brassica napus into weedy B. rapa

Wild relatives of genetically engineered crops can acquire transgenic traits such as herbicide resistance via spontaneous crop–wild hybridization. In agricultural weeds, resistance to herbicides is often a beneficial trait, but little is known about possible costs that could affect the persistence of this trait when herbicides are not used. We tested for costs associated with transgenic resistance to glufosinate when introgressed into weedy Brassica rapa . Crosses were made between transgenic B. napus and wild B. rapa from Denmark. F₁ progeny were backcrossed to B. rapa and BC₁ plants were selected for chromosome numbers similar to B. rapa . Further backcrossing resulted in a BC₂ generation that was hemizygous for herbicide resistance. We quantified the reproductive success of 457 BC₃ progeny representing six full-sib families raised in growth rooms (plants were pollinated by captive bumblebees). Pollen fertility and seed production of BC₃ plants were as great as those of B. rapa raised in the same growth rooms. Segregation for herbicide resistance in BC₃ plants was 1:1 overall, but the frequency of resistant progeny was lower than expected in one family and higher than expected in another. There were no significant differences between transgenic and nontransgenic plants in survival or the number of seeds per plant, indicating that costs associated with the transgene are probably negligible. Results from this growth-chamber study suggest that transgenic resistance to glufosinate is capable of introgressing into populations of B. rapa and persisting, even in the absence of selection due to herbicide application.     

转基因番茄口服疫苗的现状,问题及对策

日趋成熟的植物基因工程技术为转基因植物口服疫苗的研究提供了基础并带来了广阔的发展前景.番茄是最常用的转基因植物受体材料之一,可以直接生食避免了加热过程对外源蛋白的损伤.随着转基因植物免疫保护机制的进一步阐明以及国际番茄基因组测序计划的完成,转基因番茄口服疫苗的发展也迎来了新的机遇.但与此同时,我们也应看到转基因番茄口服疫苗仍存在诸多不确定的因素,如外源蛋白表达量不高、口服时可能被消化以及转基因植物安全性问题等.如何成功解决这些问题将成为转基因番茄口服疫苗发展过程中的重大考验.综述了植物口服疫苗这一概念出现后的近二十年中,番茄作为受体材料研制口服疫苗的现状,问题及对策,旨在为转基因番茄口服疫苗的进一步研究提供思路.     

转基因抗虫抗除草剂棉花对棉铃虫抗性及对草甘膦耐受性研究

本论文研究了抗虫又抗除草剂棉花对草甘膦的耐受程度,比较了这一双抗性状的棉花与单抗虫棉的抗虫效果。结果表明,抗虫又抗除草剂棉花对草甘膦有较好的耐受性,四叶期喷施草甘膦后抗虫抗除草剂棉花可以安全生长,蕾期喷施草甘膦对棉花的开花率和结铃率有影响。抗虫又抗除草剂棉花和单抗虫棉对棉铃虫Helicoverpa armigera(Hübner)均具有较好的防治效果,苗期棉花叶片对棉铃虫防治效果最好,后期防治效果下降到49.2%和46.6%,吐絮期防治效果又上升到57.0%和53.1%。     

植物遗传转化新技术和新方法

目前通过遗传转化技术获得了许多植物的转基因植株,一些重要农作物转基因新品种已进入产业化阶段,展现出极好的应用前景。但随着研究的不断深入,在如何提高遗传转化效率和转基因安全性等方面,一些新的技术和方法不断出现并得到应用,如胚状体再生系统、叶绿体转化系统、超声波辅助农杆菌介导法、位点特异重组MATvector系统、正选择系统以及新的转基因分子检测方法,使遗传转化技术向高效、安全方向发展,新一代的转基因植物也将会更适应人们和生态环境的需求。     

棉铃虫对Bt杀虫剂的抗性遗传方式

随着转基因棉花种植面积的日益增加,棉铃虫Helicoverpa armigera（Hübner)对Bt的抗性已经成为一个不容忽视的问题。发展转多价基因作物是当前缓解害虫对Bt抗性的最有效措施。本研究以经室内多年筛选的、抗性倍数达2 000多倍的Bt杀虫剂（含多种蛋白）抗性品系为材料,通过生物测定和不同的杂交试验,测定棉铃虫对Bt杀虫剂的抗性遗传方式,以期为Bt生物农药的抗性治理提供一定的依据,同时为制定棉铃虫对转多基因作物的抗性治理策略提供一定的参考。对敏感亲本和抗性亲本杂交产生的F1代的研究结果表明,杂交品系的抗性倍数分别为22.2倍和24.6倍;抗性显性度D值均小于0,分别为-0.20和-0.17,抗性为常染色体不完全隐性遗传。对4种回交后代和2种自交后代F2的研究结果表明,实际死亡率与期望死亡率差异较大,说明抗性是由单基因多个位点或多基因控制。     

Assessment of effects of Bt‐oilseed rape on large white butterfly (Pieris brassicae) in natural habitats

Much effort has been allocated to the definition of risk, relevant for the assessment of genetically modified plants. However, few studies have emphasised the limitations in testing methods. In this study, tests for and effects on non‐target herbivores were exemplified and evaluated for Pieris brassicae (L.) (Lepidoptera: Pieridae) and a genetically modified Brassica napus L. (Brassicaceae) expressing the Bacillus thuringiensis (Bt)‐toxin Cry1Ac. It was established that this herbivore recognises and accepts the transgenic plant as a host. It was found that ovipositing females of P. brassicae preferred the transgenic variety for egg‐laying. Therefore, effects of the transgenic host plant on the herbivore were determined. Larvae feeding on the Bt‐plants experienced 100% mortality for all larval stages. Based on these observations, a population model was established. The model showed that larval survival is increased with amount of food (number of plants) and reduced with the frequency of transgenic specimens, number of host plants needed for completing larval development, and number of egg‐laying butterflies. Such models may both aid the design of further tests for effects and support the assessment whether population effects are likely to occur due to the presence of insect‐resistant plants outside the agricultural area.     

转基因玉米的研究进展和食用安全性评价

玉米是我国重要的粮食作物和饲料作物。目前转基因技术作为生物育种技术的代表,已成为国际上育种的前沿和核心技术。世界各国利用转基因技术相继研发了具有多种优良性状的转基因玉米,创造了巨大的经济效益;同时统一、有效的监管措施是转基因玉米研发、推广和商业化的重要基础。食用安全性评价是有效监管的前提。针对转基因玉米的商业化和控制玉米重要性状基因的研究进展进行了综述,并对转基因玉米的食用安全性评价进行归纳分析,以期为我国转基因玉米的研发、管理和推广提供理论参考。     

Natural glufosinate resistance of soil microorganisms and GMO safety

Bacteria and fungi from pristine soil, never exposed to glufosinate herbicide, were isolated and analyzed for glufosinate tolerance. Seven of the 15 tested isolates were sensitive to 1 mM glufosinate (an active ingredient of many nonselective contact herbicides), 5 were resistant to 4 mM glufosinate and 3 even to 8 mM glufosinate in liquid medium. None of the isolated microorganisms carried the gene for glufosinate resistance bar (bialaphos resistance) in its genome and at least in some of glufosinate-resistant isolates the increased glutamine synthetase level was detected as a possible resistance mechanism. The transfer of the bar glufosinate resistance gene from transgenic maize Bt 176 into glufosinate-sensitive soil bacterium Bacillus pumilus S1 was not detected under the laboratory conditions by a classical plate count method and PCR. The ecological risk of potential bar gene transfer from genetically modified plants into soil microcosms under natural circumstances is discussed.     

Metagenomic‐based impact study of transgenic grapevine rootstock on its associated virome and soil bacteriome

For some crops, the only possible approach to gain a specific trait requires genome modification. The development of virus‐resistant transgenic plants based on the pathogen‐derived resistance strategy has been a success story for over three decades. However, potential risks associated with the technology, such as horizontal gene transfer (HGT) of any part of the transgene to an existing gene pool, have been raised. Here, we report no evidence of any undesirable impacts of genetically modified (GM) grapevine rootstock on its biotic environment. Using state of the art metagenomics, we analysed two compartments in depth, the targeted Grapevine fanleaf virus (GFLV) populations and nontargeted root‐associated microbiota. Our results reveal no statistically significant differences in the genetic diversity of bacteria that can be linked to the GM trait. In addition, no novel virus or bacteria recombinants of biosafety concern can be associated with transgenic grapevine rootstocks cultivated in commercial vineyard soil under greenhouse conditions for over 6 years.     

Use of a Mutated Protoporphyrinogen Oxidase Gene as an Effective In Vitro Selectable Marker System that Also Conveys <Emphasis Type="Italic">in planta</Emphasis> Herbicide Resistance in Sugarcane

Genetic engineering can be used to introduce economically important traits in sugarcane cultivars. Part of any transformation process involves the selection of genetically transformed cells. In this study, an efficient sugarcane in vitro selection system was developed using mutated protophorhyrinogen oxidase (PPO) genes as selectable markers. Two PPO genes, that encode proteins targeted either to the mitochondria or plastid, were isolated from tobacco and maize. Site-directed mutagenesis was used to alter the nucleotide sequence of these genes so that the resulting proteins are less sensitive to diphenylether type herbicides. Sugarcane callus was genetically transformed through particle bombardment with constructs allowing expression of either transgene, and putative transgenic calli were selected on fomesafen. It took approximately 4 weeks to select herbicide resistant calli clones on 10 mg/l fomesafen in the presence of light, which increased the selection pressure, and a further 8 weeks to regenerate resistant plantlets. PCR analysis confirmed that all regenerated putative transgenic sugarcane plants contained the transgene. All transgenic plants showed levels of herbicide resistance when planted in soil.     

Evaluation of three herbicide resistance genes for use in genetic transformations and for potential crop protection in algae production

Genes conferring resistance to the herbicides glyphosate, oxyfluorfen and norflurazon were developed and tested for use as dominant selectable markers in genetic transformation of Chlamydomonas reinhardtii and as potential tools for the protection of commercial‐scale algal production facilities against contamination by organisms sensitive to these broad‐spectrum herbicides. A synthetic glyphosate acetyltransferase (GAT) gene, when fitted with a strong Chlamydomonas promoter, conferred a 2.7×‐fold increase in tolerance to the EPSPS inhibitor, glyphosate, in transgenic cells compared with progenitor WT cells. A mutant Chlamydomonas protoporphyrinogen oxidase (protox, PPO) gene previously shown to produce an enzyme insensitive to PPO‐inhibiting herbicides, when genetically engineered, generated transgenic cells able to tolerate up to 136× higher levels of the PPO inhibitor, oxyfluorfen, than nontransformed cells. Genetic modification of the Chlamydomonas phytoene desaturase (PDS) gene‐based gene sequences found in various norflurazon‐resistant organisms allowed production of transgenic cells tolerant to 40× higher levels of norflurazon than nontransgenic cells. The high efficiency of all three herbicide resistance genes in producing transgenic cells demonstrated their suitability as dominant selectable markers for genetic transformation of Chlamydomonas and, potentially, other eukaryotic algae. However, the requirement for high concentrations of glyphosate and its associated negative effects on cell growth rates preclude its consideration for use in large‐scale production facilities. In contrast, only low doses of norflurazon and oxyfluorfen (~1.5 μm and ~0.1 μm , respectively) are required for inhibition of cell growth, suggesting that these two herbicides may prove effective in large‐scale algal production facilities in suppressing growth of organisms sensitive to these herbicides.     

Cantaloupe line CZW-30 containing coat protein genes of cucumber mosaic virus,zucchini yellow mosaic virus,and watermelon mosaic virus-2 is resistant to these three viruses in the field

Cantaloupe line CZW-30 containing coat protein gene constructs of cucumber mosaic cucumovirus (CMV), zucchini yellow mosaic potyvirus (ZYMV), and watermelon mosaic virus 2 potyvirus (WMV-2) was investigated in the field over two consecutive years for resistance to infections by CMV, ZYMV, and/or WMV-2. Resistance was evaluated under high disease pressure achieved by mechanical inoculations and/or natural challenge inoculations by indigenous aphid vectors. Across five different trials, homozygous plants were highly resistant in that they never developed systemic symptoms as did the nontransformed plants but showed few symptomatic leaves confined close to the vine tips. Hemizygous plants exhibited a significant delay (2–3 weeks) in the onset of disease compared to control plants but had systemic symptoms 9–10 weeks after transplanting to the field. Importantly, ELISA data revealed that transgenic plants reduced the incidence of mixed infections. Only 8% of the homozygous and 33% of the hemizygous plants were infected by two or three viruses while 99% of the nontransformed plants were mixed infected. This performance is of epidemiological significance. In addition, control plants were severely stunted (44% reduction in shoot length) and had poor fruit yield (62% loss) compared to transgenic plants, and most of their fruits (60%) were unmarketable. Remarkably, hemizygous plants yielded 7.4 times more marketable fruits than control plants, thus suggesting a potential commercial performance. This is the first report on extensive field trials designed to assess the resistance to mixed infection by CMV, ZYMV, and WMV-2, and to evaluate the yield of commercial quality cantaloupes that are genetically engineered.