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.     

耐草甘膦基因克隆和作物转化研究新进展

评述了耐草甘膦基因克隆和作物转化研究的新进展。生物耐草甘膦的机理主要在于1）过量表达EPSPS（磷酸烯醇式丙酮酰莽草酸合成酶）基因;2）EPSPS基因发生突变而对草甘膦的亲和力减弱。或3）解毒酶作用。现已从大肠杆菌、鼠伤寒沙门氏菌、矮牵牛、拟南芥、烟草、胡萝卜等中克隆了aroA等EPSPS基因并进行了分子生物学研究。作物转基因抗草甘膦育种主要通过三条途径;1）转入经过修饰的或突变的耐草甘膦EPSPS基因,2）转入过表达的EPSPS基因;3）导入草甘膦代谢酶基因。     

Progress in the evaluation of transgenic fish for possible ecological risk and its containment strategies

Genetically improved transgenic fish possess many beneficial economic traits; however, the commercial aquaculture of transgenic fish has not been performed till date. One of the major reasons for this is the possible ecological risk associated with the escape or release of the transgenic fish. Using a growth hormone transgenic fish with rapid growth characteristics as a subject, this paper analyzes the following: the essence of the potential ecological risks posed by transgenic fish; ecological risk in the current situation due to transgenic fish via one-factor phenotypic and fitness analysis, and mathematical model deduction. Then, it expounds new ideas and the latest findings using an artificially simulated ecosystem for the evaluation of the ecological risks posed by transgenic fish. Further, the study comments on the strategies and principles of controlling these ecological risks by using a triploid approach. Based on these results, we propose that ecological risk evaluation and prevention strategies are indispensable important components and should be accompanied with breeding research in order to provide enlightments for transgenic fish breeding, evaluation of the ecological risks posed by transgenic fish, and development of containment strategies against the risks.     

Progress in the evaluation of transgenic fish for possi-ble ecological risk and its containment strategies

Genetically improved transgenic fish possess many beneficial economic traits; however, the commercial aquaculture of transgenic fish has not been performed till date. One of the major reasons for this is the possible ecological risk associated with the escape or release of the transgenic fish. Using a growth hormone transgenic fish with rapid growth characteristics as a subject, this paper analyzes the following: the essence of the potential ecological risks posed by transgenic fish; ecological risk in the current situation due to transgenic fish via one-factor phenotypic and fitness analysis, and mathematical model deduction. Then, it expounds new ideas and the latest findings using an artificially simulated ecosystem for the evaluation of the ecological risks posed by transgenic fish. Further, the study comments on the strategies and principles of controlling these ecological risks by using a triploid approach. Based on these results, we propose that ecological risk evaluation and prevention strategies are indispensable important components and should be accompanied with breeding research in order to provide enlightments for transgenic fish breeding, evaluation of the ecological risks posed by transgenic fish, and development of containment strategies against the risks. Supported by the Development Plan of the State Key Fundamental Research of China (Grant Nos. 2007CB109205 and 2007CB109206), the National Natural Science Foundation of China (Grant No. 30430540), and the ‘863’ High Technology Project (Grant No. 2006AA10Z141)     

The effect of gene drive on containment of transgenic mosquitoes

Mosquito-borne diseases such as malaria and dengue fever continue to be a major health problem through much of the world. Several new potential approaches to disease control utilize gene drive to spread anti-pathogen genes into the mosquito population. Prior to a release, these projects will require trials in outdoor cages from which transgenic mosquitoes may escape, albeit in small numbers. Most genes introduced in small numbers are very likely to be lost from the environment; however, gene drive mechanisms enhance the invasiveness of introduced genes. Consequently, introduced transgenes may be more likely to persist than ordinary genes following an accidental release. Here, we develop stochastic models to analyze the loss probabilities for several gene drive mechanisms, including homing endonuclease genes, transposable elements, Medea elements, the intracellular bacterium Wolbachia, engineered underdominance genes, and meiotic drive. We find that Medea and Wolbachia present the best compromise between invasiveness and containment for the six gene drive systems currently being considered for the control of mosquito-borne disease.     

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.     

Molecular characterization of genetically-modified crops: Challenges and strategies

Molecular characterization lays a foundation for safety assessment and subsequent monitoring of genetically modified (GM) crops. Due to the target-specific nature, conventional polymerase chain reaction (PCR)-based methods cannot comprehensively detect unintended gene insertions, let alone unknown GM events. As more and more new developed GM crops including new plant breeding technology (NPBT) generated crops are in the pipeline for commercialization, alternative -omics approaches, particularly next generation sequencing, have been developed for molecular characterization of authorized or unauthorized GM (UGM) crops. This review summarizes first those methods, addresses their challenges, and discusses possible strategies for molecular characterization of engineered crops generated by NPBT, highlighting needs for a global information-sharing database and cost-effective, accurate and comprehensive molecular characterization approaches.     

Improving containment strategies in biopharming

This review examines the challenges of segregating biopharmed crops expressing pharmaceutical or veterinary agents from mainstream crops, particularly those destined for food or feed use. The strategy of using major food crops as production vehicles for the expression of pharmaceutical or veterinary agents is critically analysed in the light of several recent episodes of contamination of the human food chain by non-approved crop varieties. Commercially viable strategies to limit or avoid biopharming intrusion into the human food chain require the more rigorous segregation of food and non-food varieties of the same crop species via a range of either physical or biological methods. Even more secure segregation is possible by the use of non-food crops, non-crop plants or in vitro plant cultures as production platforms for biopharming. Such platforms already under development range from outdoor-grown Nicotiana spp. to glasshouse-grown Arabidopsis , lotus and moss. Amongst the more effective methods for biocontainment are the plastid expression of transgenes, inducible and transient expression systems, and physical containment of plants or cell cultures. In the current atmosphere of heightened concerns over food safety and biosecurity, the future of biopharming may be largely determined by the extent to which the sector is able to maintain public confidence via a more considered approach to containment and security of its plant production systems.     

Bar基因和转Bar基因作物的研究进展

概述了草丁膦的毒害机理和 Bar基因的抗性机理与表达情况。总结了转 Bar基因作物的来源、抗性遗传特点和其对环境安全性评价等方面的研究进展。展望了 Bar基因和转 Bar基因作物的应用前景。     

Advancing environmental risk assessment for transgenic biofeedstock crops

Transgenic modification of plants is a key enabling technology for developing sustainable biofeedstocks for biofuels production. Regulatory decisions and the wider acceptance and development of transgenic biofeedstock crops are considered from the context of science-based risk assessment. The risk assessment paradigm for transgenic biofeedstock crops is fundamentally no different from that of current generation transgenic crops, except that the focus of the assessment must consider the unique attributes of a given biofeedstock crop and its environmental release. For currently envisioned biofeedstock crops, particular emphasis in risk assessment will be given to characterization of altered metabolic profiles and their implications relative to non-target environmental effects and food safety; weediness and invasiveness when plants are modified for abiotic stress tolerance or are domesticated; and aggregate risk when plants are platforms for multi-product production. Robust risk assessments for transgenic biofeedstock crops are case-specific, initiated through problem formulation, and use tiered approaches for risk characterization.     

Potential for the environmental impact of transgenic crops

In recent years, there has been increasing interest in how changes in agricultural practice associated with the introduction of particular genetically modified (GM) crops might indirectly impact the environment. There is also interest in any effects that might be associated with recombinant and novel combinations of DNA passing into the environment, and the possibility that they may be taken up by microorganisms or other live biological material. From the current state of knowledge, the impact of free DNA of transgenic origin is likely to be negligible compared with the large amount of total free DNA. We can find no compelling scientific arguments to demonstrate that GM crops are innately different from non-GM crops. The kinds of potential impacts of GM crops fall into classes familiar from the cultivation of non-GM crops (e.g., invasiveness, weediness, toxicity, or biodiversity). It is likely, however, that the novelty of some of the products of GM crop improvement will present new challenges and perhaps opportunities to manage particular crops in creative ways.     

Gene dispersal from transgenic crops

The risk of release of genetically modified oilseed rape (Brassica napus) was investigated in relation to interspecific gene flow with hoary mustard (Hirschfeldia incana). Microscopic studies showed polymorphism within the population of hoary mustard for pollen germination on oilseed rape flowers. The transgenic herbicide-resistant and a commercial cultivar of oilseed rape were not different for pollen behaviour and ovule fertilization. Pollen tube growth was slow and erratic in interspecific crosses. Fertilization efficiency of oilseed rape and hoary mustard pollen in interspecific crosses was 15% and 1.3%, respectively, of that in intraspecific crosses. This unequal efficiency in reciprocal crosses was confirmed by hybrid seed set in pods. There was no post-zygotic barrier to the development of hybrid embryos in hoary mustard pods. Up to 26 spontaneous hybrids per male sterile oilseed rape plant, and one per hoary mustard plant, were obtained in field experiments. Hybrids were identified by isozyme electrophoresis, morphology and cytology. All hybrids were triploid with 26 chromosomes, and had low fertility. They produced 0.5 seeds per plant after spontaneous backcrossing with hoary mustard. Some of these descendants were produced from unreduced gametes. Our results suggest that gene flow is likely to occur, but its actual frequency under crop growing conditions remains to be estimated.     

A built-in strategy for containment of transgenic plants: creation of selectively terminable transgenic rice

Plant transgenic technology has been widely utilized for engineering crops for trait improvements and for production of high value proteins such as pharmaceuticals. However, the unintended spreading of commercial transgenic crops by pollination and seed dispersal is a major concern for environmental and food safety. Simple and reliable containment strategies for transgenes are highly desirable. Here we report a novel method for creating selectively terminable transgenic rice. In this method, the gene(s) of interest is tagged with a RNA interference cassette, which specifically suppresses the expression of the bentazon detoxification enzyme CYP81A6 and thus renders transgenic rice to be sensitive to bentazon, a herbicide used for rice weed control. We generated transgenic rice plants by this method using a new glyphosate resistant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene from Pesudomonas putida as the gene of interest, and demonstrated that these transgenic rice plants were highly sensitive to bentazon but tolerant to glyphosate, which is exactly the opposite of conventional rice. Field trial of these transgenic rice plants further confirmed that they can be selectively killed at 100% by one spray of bentazon at a regular dose used for conventional rice weed control. Furthermore, we found that the terminable transgenic rice created in this study shows no difference in growth, development and yield compared to its non-transgenic control. Therefore, this method of creating transgenic rice constitutes a novel strategy of transgene containment, which appears simple, reliable and inexpensive for implementation.     

Advances in development of transgenic pulse crops

It is three decades since the first transgenic pulse crop has been developed. Todate, genetic transformation has been reported in all the major pulse crops like Vigna species, Cicer arietinum, Cajanus cajan, Phaseolus spp, Lupinus spp, Vicia spp and Pisum sativum, but transgenic pulse crops have not yet been commercially released. Despite the crucial role played by pulse crops in tropical agriculture, transgenic pulse crops have not moved out from laboratories to large farm lands compared to their counterparts - 'cereals' and the closely related leguminous oil crop - 'soybean'. The reason for lack of commercialization of transgenic pulse crops can be attributed to the difficulty in developing transgenics with reproducibility, which in turn is due to lack of competent totipotent cells for transformation, long periods required for developing transgenics and lack of coordinated research efforts by the scientific community and long term funding. With optimization of various factors which influence genetic transformation of pulse crops, it will be possible to develop transgenic plants in this important group of crop species with more precision and reproducibility. A translation of knowledge from information available in genomics and functional genomics in model legumes like Medicago truncatula and Lotus japonicus relating to factors which contribute to enhancing crop yield and ameliorate the negative consequences of biotic and abiotic stress factors may provide novel insights for genetic manipulation to improve the productivity of pulse crops.     

Molecular analysis of gene conversion in spermatids from transgenic mice

Investigations into the mechanisms and properties of gene conversion in mammals are greatly restricted by the inability to recover all the products of a meiosis. Additionally, the study of this process has been hampered by the lack of visible markers to detect gene conversion, especially when the events are rare. In previous work, we developed a transgenic system for detection and quantitation of gene conversion events in the germline of mice (Murti, J.R., Bumbulis, M., Schimenti, J.C., 1992. High frequency germline gene conversion in transgenic mice. Mol. Cell. Biol. 12, 2545–2552) that could be exploited as an assay for recombinogenic chemicals (Murti, J.R, Schimenti, K.J., Schimenti, J.C., 1994. A recombination-based transgenic mouse system for genotoxicity testing. Mutat. Res. 307, 583–595). A specific intrachromosomal gene conversion event between two complementarily defective lacZ genes resulted in the production of β-galactosidase in spermatids, enabling a measurement of conversion frequency. Here, we report that the anticancer drug, cisplatin, increased gene conversion in meiotic stage cells in these transgenic mice. Furthermore, a method was developed for direct molecular analysis of transgene conversion events in single or pooled lacZ-positive spermatids. The ability to identify gametes that have undergone a rare gene conversion event, followed by molecular amplification of the recombinant gene, should make it possible to investigate the mechanisms of genetic recombination in mammals in greater detail than previously possible.