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    Efficient, Safe and Large-Scale Transgenic Technology: Opportunities and Challenges
    WAN Jan-min, LI Yu
    Scientia Agricultura Sinica    2014, 47 (21): 4139-4140.   DOI: 10.3864/j.issn.0578-1752.2014.21.001
    Abstract538)   HTML3)    PDF (216KB)(1030)       Save
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    Establishment and Application of Large-Scale Transformation Systems for Rice
    ZHANG Xin, FU Ya-ping, ZHOU Jun-li, GUO Xiu-ping, LIU Wen-zhen, WU Jie-fang, WU Chuan-yin, WAN Jian-min
    Scientia Agricultura Sinica    2014, 47 (21): 4141-4154.   DOI: 10.3864/j.issn.0578-1752.2014.21.002
    Abstract777)   HTML7)    PDF (523KB)(1331)       Save
    Rice, one of the most important food crops and a model monocot plant, its genetic transformation has attracted widespread attention. Since the world’s first case of transgenic rice got success in 1988, the rice genetic transformation technology has been developed rapidly. In 1994, in a milestone work of Agrobacterium-mediated transformation of a number of japonica rice cultivars, rice genetic transformation system has been more advanced after nearly 20 years of development. Presently, Agrobacterium-mediated transformation and microparticle bombardment are the two most widely used methods for rice genetic transformation, while pollen tube pathway, electroporation and polyethylene glycol (PEG) medium are also employed in some laboratories. Agrobacterium transformation is the method of choice because it is of easy and low cost, the high transformation efficiency, the low copy number of transgenes inserted into the host genome and the stability of expression over generations. Nowadays more than 80% of the transgenic rice is transformed by the Agrobacterium method. Although there are a lot of reports in improving the methods and technology of rice transformation, difficulties and limitations still exist in rice genetic transformation. The transformation efficiency of several elite japonica and many indica varieties is still unsatisfactory as it is much lower. Some transformation protocols are laborious, time consuming, and highly genotype-dependent. Besides, there is a problem of somaclonal variations or somatic mutation frequently occurs in plant cells during in vitro culture. Therefore, it is very important to establish the high efficient, safety, large-scale and standard transformation systems for diverse varieties of rice. This paper briefly reviewed the history and development of rice genetic transformation system. Especially, the recent progress of rice transgenic technology for large scale performance in China was described. Many factors are known to affect the efficiency of genetic transformation, such as the genotype of the recipient plants, the type and age of the tissue being inoculated, the strain of Agrobacterium, the expression vector, the composition of the culture medium, as well as various conditions of tissue culture. Extensive research has been conducted to optimize the high-efficiency transformation system for different genotypes of rice. Various factors affecting the transformation efficiencies were explored. Modification in culture conditions for embryogenic calli induction and regeneration from mature seed and also the compatibility of the Agrobacterium strain were successful in examined transformation protocols. The highly efficient Agrobacterium-mediated transformation system for japonica and indica rice using immature embryos or mature embryo-derived calli as the explant has been established for the large-scale production of transgenic rice by integrating new technology method. Also the Agrobacterium-mediated co-transformation system has been preliminary developed and some experimental parameters for marker-free rice transformation have been optimized. However, a big gap between the update status in China and the advanced level in some renowned international biotechnology companies is still existed in either transformation efficiency or transformation scale of rice. It was concluded that bio-safety, high efficiency and large scale were the bottlenecks for production GM rice products and its industrialization. Future studies in this field should be focused on the use of commercial rice varieties, improvement of transformation efficiency, development of marker-free rice plants, introduction of multiple target genes, integration of foreign genes at specific site, etc. The review also analyzed the problems existing in large-scale rice transformation system and provided strategies to improve it. It may provide some useful information for efficient production of transgenic rice for practical use as well as for studying gene function.
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    Establishment and Application of Large-Scale Transformation Systems in Wheat
    YE Xing-guo, XU Hui-jun, DU Li-pu, HE Guang-yuan, WANG Ke, LIN Zhi-shan
    Scientia Agricultura Sinica    2014, 47 (21): 4155-4171.   DOI: 10.3864/j.issn.0578-1752.2014.21.003
    Abstract628)   HTML4)    PDF (1087KB)(1366)       Save
    Wheat is a plant that is relatively difficult to be genetically modified through traditional transgenic approaches among several major field crops. Factors restricting the transformation of wheat include small scale and low efficiency of transformation, poor repeatability, and fewer availability of transgenic materials with satisfied appearances, and thus leading to the lagged-behind of wheat in transgenic breeding compared to soybean, maize, cotton, and rice. Presently, transgenic techniques mainly used in wheat transformation are biolistic particle and Agrobacterium-mediated transformation based on in vitro tissue culture, although methods of pollen tube pathway, ion beam implantation, laser microbeam puncture, polyethylene glycol (PEG) medium, pollen-mediated, and Agrobacterium dipping floral have also been employed in some laboratories. In the aspect of recipient tissues, wheat immature embryo and its derived callus were mostly used as initiating materials for the transformation of target genes, but transgenic wheat plants were also reported to be obtained by using mature embryo, inflorescence, and anther derived callus, which still need to be investigated and confirmed. As for the transformation efficiency reported in wheat, 0.1%-16.7% is for particle bombardment and 0.7%-44.8% for Agrobacterium. A big variation still existed. In the matter of alien transferring genes for wheat, the target genes are mainly involved in wheat enhancement of flour quality, disease resistance, drought tolerance, aphids resistance, and herbicide tolerance except some selection or reporter genes such as npt, bar, hpt, GUS, GOX, pmi, and ALS. The transformation efficiency of wheat immature embryos mediated by Agrobacterium and particle gun is dramatically affected not only by genotypes, but also by the physiological status of mother plants which is determined by temperature, light, nutrition, and water supply during the growth period. Especially, the suitable day/night temperature during the period between anthesis and sampling of the donor plants is mostly beneficial to the embryonic callus induction and putative transgenic regeneration after transformation. Generally, the development of wheat transgenic technology in China has been largely progressed, for examples, the establishment of high regeneration system of wheat mature embryos and its successful application, optimization of regeneration and transformation system of wheat immature embryos, introduction of a group of interested genes related to wheat improvement in disease control, drought resistance and quality enhancement, preliminary setting up of wheat transformation system with larger scale, etc. However, there is still a big gap between the current status in China and the top-level international biotechnology companies regarding to the efficiency or scale in wheat transformation. It is thought that low transformation rate, strong genotype-dependence, and imperfect artificial climate conditions, as well as unstable manipulating team are the bottleneck to limit the application of wheat transformation technology by large scale in China. Future studies in this field should include the use of commercial wheat varieties, improvement of transformation efficiency, development of marker-free wheat plants, introduction of multiple target genes, integration of foreign genes at specific sites, avoidance of backbone insertion and transgene silencing, etc. Wheat transformation efficiency and scale will be improved and expanded by further modifications of plant regeneration system, exploration of regeneration and transformation-associated genes. Other aspects involve the screening of ideal genotypes, medium and technique improvement of co-culture, optimization and combination of various factors in transformation process. Development and application of biolistic particle and Agrobacterium mediated wheat transformation was reviewed in this article. Particularly, the progress of wheat transgenic technology for large scale performance in the latest five years was studied, which may provide useful information for the transformation of candidate genes for functional analysis and the breeding of transgenic wheat varieties.
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    Establishment and Application of Large-Scale Transformation Systems for Maize
    LIU Yun-jun, JIA Zhi-wei, LIU Yan, ZHANG Deng-feng, LI Yu
    Scientia Agricultura Sinica    2014, 47 (21): 4172-4182.   DOI: 10.3864/j.issn.0578-1752.2014.21.004
    Abstract489)   HTML2)    PDF (418KB)(811)       Save
    Maize has the most widely planted scope and the largest output in the world, and makes important contributions to food security. However, pests, weeds, drought, salinity and other biotic and abiotic stress seriously affect maize production. The application of transgenic maize varieties with insect-resistance, herbicide-tolerance, disease-resistance and other traits could reduce the maize yield losses. Maize large scale transformation system has developed rapidly, and the transgenic insect-resistant and herbicide-tolerant maize varieties have also been commercially applied for 18 years, bringing huge economic, social and ecological benefits. Global biotech crop acreage increased from 1.7 million hectares in 1996 to 175 million hectares in 2013. The commercially used transgenic maize events were selected from a large number of independent transgenic events, and had several good characteristics, i.e. single copy of foreign genes, genetic stability, no vector backbone insertion, no effect on maize own traits. Foreign big companies and some public research institutions have established high efficient maize transformation system. In China, the study on maize transformation system started late, and currently the maize transformation system has been preliminarily established. However, it is necessary to further improve the efficiency and the scale of maize transformation system in China. Particle bombardment and Agrobacterium-mediated method are two main methods for maize transformation system. The article describes the principle, the development process and the application of these two methods. Agrobacterium-mediated transformation is the most mainstream of plant genetic transformation methods with the characteristics of low cost, low foreign gene copy number and stable gene expression, so Agrobacterium-mediated transformation method is more suitable for large scale maize transformation system. Over the last decade in China, some achievements have been made in maize transformation method by selecting good explants, optimizing Agrobacterium infection method. The established maize transformation system has also been used to develop a lot of valuable transgenic maize events with insect-resistance or herbicide-tolerance. The perspectives of the development of maize transformation were also discussed. The authors believe that the development trend of maize transformation system include: 1) screening explants from commercial maize inbred lines, 2) raising single-copy transformation event rates without enhancing vector backbone insertion, 3) multi-gene transformation technology, 4) gene targeting technology, 5) safety transformation technology. China should closely follow the development trend of maize transformation system, focus on the development of multi-gene transformation technology, gene targeting technology and safety transformation technology, to better serve the gene function research and the development of transgenic maize products. In China, if the transgenic maize events which were obtained by the large scale maize transformation system are commercially grown in the future, it will bring enormous economic, social and environmental benefits, and will promote the further development of maize transformation system. This review article might provide some useful information for the research on maize transformation system.
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    Establishment and Application of Efficient Transformation System for Cotton
    LIU Chuan-liang, TIAN Rui-ping, KONG De-pei, LI Feng-lian, SHANG Hai-hong, CHEN Xiu-jun
    Scientia Agricultura Sinica    2014, 47 (21): 4183-4197.   DOI: 10.3864/j.issn.0578-1752.2014.21.005
    Abstract482)   HTML7)    PDF (1592KB)(1075)       Save
    This assay is a summary of the application of transgenic technology in cotton all over the world, including the establishment and research progress of cotton transgenic technology for large-scale performance in China in recent years, the main problems and development trend of transgenic technology in cotton, which gives an insight into the transgenic cotton for researchers and makes a great significance to help them working efficiently. Transgenic technology has achieved great progress in cotton bollworm resistance by GMOs, and will gradually get important progresses in disease and adverse resistance in cotton, etc. Early transformation events are transformed by Coker cultivars, and nowadays rapid progresses have been made in transgenic technology for the success of different cotton species of tissue culture in Gossypium raimondii, G. arboretum, G. barbadense, G. hirsutum, etc. and three major transformation methods containing biolistic particle, pollen tube pathway and Agrobacterium-mediated transformation. Cotton transgenic technology for large-scale performance in China is established mainly by Cotton Research Institute of Chinese Academy of Agricultural Sciences and other institutes in China. It forms a trinity system for cotton large-scale transformation by focusing on the optimization of Agrobacterium-mediated method through the selection of efficient transformation vectors, transformation system establishment of major cotton varieties or lines, optimization of tissue culture conditions, in addition to the improvement of biolistic particle and pollen tube pathway methods. This transformation system involves in establishment of Agrobacterium mediated transformation system selecting CRI24 as the transgenic receptor, getting new materials such as W12 whose differentiation rate is up to 100% by petiole tissue culture screening and transformation efficiency increased by 2.88 times compared with before, development of embryonic callus transformation system by biolistic particle method, improvement of the transformation efficiency by pollen tube pathway. Using the transform system, many transgenic materials or lines are obtained and some of them are bred to insect-resistant cotton varieties, more than 200 genes for 41 scientific research institutes in China have been identified their gene functions, and provides a large number of new materials for cotton breeding. It is concluded that strong genotype-dependence is the bottleneck for cotton transformation, expanding the genotype of transgenic receptor, improving the transformation efficiency, and scaling up the system are the subjects of cotton transformation for a long time. At the same time, concerning the efficiency and the public anxiety, further studies in this field should be focused on exploring and discovering safer and more effective transformation system such as multi-gene transformation, plastid transformation, fixed-point conversion or gene stacking, developing safe or maker-free transgenic technique. At last, the strict requirement for safety evaluation needs to be further researched on the genetically modified materials. With the publication of cotton genome sequences, it will be a new direction for cotton basic and applied research to clone cotton genes, which will provide valuable information for the transformation of candidate genes and breeding of transgenic cotton varieties.
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    Establishment and Prospect of Efficient Transformation Systems for Soybean
    HOU Wen-sheng, LIN Kang-xue, CHEN Pu, JIA Zhi-wei, ZHOU Yang, YU Yang, LIU Yan-hua
    Scientia Agricultura Sinica    2014, 47 (21): 4198-4210.   DOI: 10.3864/j.issn.0578-1752.2014.21.006
    Abstract502)   HTML2)    PDF (443KB)(776)       Save
    Soybean [Glycine max (L.) Merr.] is the genetically modified (GM) crops that is earliest commercially planted on large scale. GM soybean is retaining its position as the GM crop occupied the largest scale globally with food, nutritional, industrial, and pharmaceutical uses. From 1996 to 2012, total 763.1 million hectares GM soybean had been planted and by growing GM soybean, almost 37 billion US dollars income was brought to farmers. In 2013, about 84.5 million hectares of GM soybean were planted around the world in 11 countries which accounted for 48% of all the GM crops hectarage and 79% for soybean grown all over the world. Though herbicide-resistant transgenic soybean holds a leading market share in the world, soybean has been regarded as a recalcitrant crop to be transformed for many years. Efficient and stable transformation is a restrictive factor for production of transgenic soybean and gene function research. The most widely and routinely used transformation systems are cotyledonary node-Agrobacterium-mediated transformation and somatic embryo-particle-bombardment-mediated transformation. The first fertile transgenic soybean plants were produced nearly simultaneously by these two methods in 1988. Even after more than two decades, these two methods have continued to be improved and have produced most transgenic soybean plants. The transformation efficiencies of these improved protocols have been extended and the system was successfully adapted to embryogenic suspension cultures for the regeneration of fertile transgenic soybean plants. These ready systems enable us to improve agronomic characteristics or to analyze gene function in soybean by transgenic approaches. In this review, recent advances and problems in research of soybean transformation system were described, with a focus on the characteristics of soybean regeneration systems such as organogenesis, somatic embryogenesis and protoplasts system. The representative soybean transformation systems mediated by Agrobacterium tumefaciens and particle bombardment have been summarized, and described their experimental parameters such as soybean genotype, explant, regeneration system, screening system, transformation frequencies. Analysis suggests that the genotype, explant type and so on can be used in the transformation of soybean has been developed and the transformation frequencies has been improved significantly. Some reports showed higher transformation efficiency over 10% and even some over 30% in some cases. However, the date of transformation frequencies had been obtained in some research reports that involved small sample size. The transformation frequencies between different experimental replications often repeated differences greatly. Sometimes, obtained high transformation frequencies in the single factor experiment, often doesn’t get well again in multi-factor integrated experiment. It indicated that the transformation efficiency still is affected by some unknown factors that is hard to control. On the other hand, the transformation efficiency still relies on the skill of the technicians and on the tissue culture conditions of the laboratory. That leads to some problems such as the same transformation process is poor in reproducibility between laboratories, the transformation frequencies are greatly different between operators, the transformation frequencies are not stable in the same laboratory. And those problems hindered the establishment of high-throughput transformation systems for soybean. Meanwhile, the research result of soybean transformation and transgenic lines was reviewed, and the current transgenic lines for soybean breeding in China were surveyed. Further, some new genetically modified technologies used in soybean were prospected, such as genome editing techniques and precise genome modification techniques.
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    Construction and Application of a Scale Transgenic Technology System for Pigs
    MU Yu-lian, RUAN Jin-xue, WU Tian-wen, CHENG Ying, WEI Jing-liang, FAN Jun-hua, LI Kui
    Scientia Agricultura Sinica    2014, 47 (21): 4211-4223.   DOI: 10.3864/j.issn.0578-1752.2014.21.007
    Abstract385)   HTML2)    PDF (474KB)(574)       Save
    Swine is an important economic animal and pig breeding plays an important role in the development of agriculture in China. At the same time, as a kind of commonly used experimental animals, pigs are similar in anatomy, physiology and genetics to humans and important for life science research. To meet the specific needs of human beings, animal genomes can be modified by transgenic technology using genetic engineering or other experimental techniques, and the target gene can be passed on to the offspring steadily. Therefore, establishment of a scale swine transgenic technology system is essential for animal breeding, gene function and human disease model research. This paper reviewed the research progress of transgenic swine technology system both in China and foreign countries, summarized the principle of the selection of target genes, described the relative merits and applicable scope of specific gene transfer method such as pronuclear microinjection, somatic cell nuclear transfer, zinc-?nger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/ Cas9), sperm vector and lentiviral vector and so on, expounded the relevant regulatory element and system of gene expression regulation strategy. In recent years, China has made great progress in scale transgenic technology system research since the commencement of National Transgenic Major Projects: including explored a large number of functional genes; built ZFN, TALEN, CRISPR/Cas9 mediated genome modification technology platform; optimized the isolation, culture and screening process of donor cells; improved the somatic cell nuclear transfer and pronuclear microinjection system, and combined them with the novel genome editing techniques for site-specific, safe and efficient integration of exogenous gene; through a further cloning, embryo transfer, artificial insemination and other conventional breeding techniques integrated innovation, finally established the transgenic pig propagation technology system, so that the number of transgenic pigs can be expanded in a short time; provided technical service and trained technical staff for many domestic institutions, made transgenic technology to get a wider range of applications. But a series of problems still exist in the scale swine transgenic technology system of China: the lack of domain genes controlling the transgenic pigs related traits and with independent intellectual property; the transgenic technology supporting system is still not perfect; the potential risks of marker genes; the low integration efficiency and instability of exogenous gene; the inefficiency of gene targeting technology; the low piglets survival rate and so on. All of these are considered to be the bottlenecks of development of scale swine transgenic technology. Therefore, in order to realize the technological breakthrough of scale swine transgenic system, the important functional genes and regulatory element which have independent intellectual property must be explored by related omics tools, construct more efficient, safe and multi-gene transgenic vectors to achieve coordinated and efficient expression of exogenous genes, and improve the related technology in the preparation process of transgenic pigs. In conclusion, the transgenic swine breeding of China will enter a new high-speed development period under the support of scale swine transgenic technology system.
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    Establishment and Prospect of Efficient Transgenic System for Cattle
    MENG Qing-yong, LIU Chun-cheng, WANG Meng, ZHANG Kuo, DAI Yun-ping, GUO Ying, FEI Jing, LI Ning
    Scientia Agricultura Sinica    2014, 47 (21): 4224-4233.   DOI: 10.3864/j.issn.0578-1752.2014.21.008
    Abstract338)   HTML3)    PDF (2054KB)(669)       Save
    With the continuous development of life science and the expanding needs of industry application, transgenic technology, a basic technology of life science research, is expanding to life science industry application, which includes big domestic animal transgenic technology industrialization application. GM technology is discussed in this paper about the application and the development in transgenic cattle breeding, combining with the history of transgenic cattle in the world and the bovine transgenic breeding system construction in China. Through introduction, integration and innovation of the modern animal biotechnology, a safe, efficient and large-scale cattle transgenic technology system was established, which should improve the implement in the production of cattle and more extensive application in breeding practice. The purpose of transgenic technology for cattle breeding is improvement of the ability of disease resistance, the milk quality, the milk or meat output and the bioreactor of producing some functional proteins. At present, the scale GM technology system platform of cattle is nearly mature, which level has attained world class performance. More in-depth work will be focused on the regulation of functional gene and security control of import methods. In the future, the bovine transgenic breeding work should be led to “safe, efficient, large-scale”, which includes tissue specific promoters, optimizing codon, adjusting the genetic modification, point knock-in genes, marker free and so on. Disease-resistant and high-yielding dairy breeding is the essential direction of the development of the livestock industry. Some active functional proteins could be produced by cattle gland reactor, so establishment of a perfective efficient transgenic system for cattle is one of the prominent development direction in the future.
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    Approaches of Establishment of Technological System of Transgenic Sheep
    LIU Ming-jun, ZHANG Xue-mei, LI Wen-rong, HUANG Jun-cheng, WANG Li-qin
    Scientia Agricultura Sinica    2014, 47 (21): 4234-4245.   DOI: 10.3864/j.issn.0578-1752.2014.21.009
    Abstract346)   HTML2)    PDF (502KB)(895)       Save
    Transgenic approach can break-through the barrier of breeds, integrate multiple gene effects and modify the genome based on the requirement of mankind, therefore it exhibits great potentials. Since the first transgenic animal born in 1982, more than ten species of transgenic animals have been reported. The methods to generate transgenic animals were developed from original pronuclear microinjection to nuclear transfer, viral vector transgenesis, and concurrent genome editing. The aims of the transgenic animal are also expanded from establishment of transgenic animal technique to generation of transgenic model, improvement of the productive performance or product quality, as bioreactor to produce high value pharmaceutical proteins, and breeding new animal breeds. Hereby the worldwide approaches, the faced problems, and the tendency to produce transgenic sheep were summarized. The proceedings of generation of transgenic novel breeds or animal models, and the characteristics of transgenic techniques were reviewed. The barrier and bottleneck of transgenic techniques were also addressed. Particularly, the feature, tendency and potential of the concurrently highlighted genome editing technique were comprehensively documented. Moreover, the necessity to establish a technological system of transgenic sheep was elucidated, and the present status, gain of innovation and breakthrough, highlighted target of transgenic sheep in domestic in future were reviewed. Meanwhile, the outlook of the economic, ecological and social effects resulted from transgenic sheep were further analyzed and previewed as well.
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