水稻规模化转基因技术体系构建与应用

doi:10.3864/j.issn.0578-1752.2014.21.002

摘要/Abstract

摘要： 水稻作为重要的粮食作物和遗传转化的模式植物，其遗传转化一直受到广泛重视。自世界首例转基因水稻于1988年获得成功以来，水稻遗传转化技术体系迅猛发展，尤其是1994年首次通过农杆菌介导实现对粳稻的高频转化，经过近20年的发展，水稻遗传转化技术体系已经比较完善。目前，应用于水稻中的转基因技术主要包括基因枪介导法和农杆菌介导法，一些实验室也采用花粉管通道法、电击法、PEG转化法等。其中，农杆菌介导的转基因方法以其低成本、易操作、转化效率高、单位点插入比例高、后代表达稳定等特点已经成为水稻转化的主流方法，约占水稻转基因报道总数的80%以上。虽然国内外刊物时有转基因方法改进的报道，但是由于种种原因，水稻的转化还受一些因素的限制，例如部分粳稻品种和籼稻受基因型的限制十分明显，转基因效率普遍较低，严重制约了转基因技术在水稻生产中的应用；某些转基因程序过于繁琐，耗时长，成本高，不但导致效率低，而且长时间的组织培养诱发逆转座子转座引起无性系变异干扰了功能研究和育种工作。因此，迫切需要建立高效、安全、规模化和标准化的水稻转基因技术体系。文章综述了国内外水稻转化技术的发展历程，重点回顾了近5年中国水稻规模化转基因技术研究进展，包括围绕不同水稻基因型高效转化体系优化及建立，对影响农杆菌转化效率及植株分化频率等诸多因素如水稻基因型、外植体类型、农杆菌菌株和质粒载体、培养基组分、共培养时间、侵染方式等方面的研究和探索。整合国内外已有研究结果进行技术集成创新，分别以粳稻和籼稻成熟胚、幼胚为外植体，采用农杆菌转化方法，通过优化受体材料和愈伤状态、农杆菌侵染浓度和分化温湿度、工艺流程标准化等多种组分，突破了成熟胚分化难的技术瓶颈，整合了无选择标记等安全转基因技术，实现了粳稻和部分籼稻转化技术的标准化和工厂化，初步建立了安全、高效、规模化水稻转基因技术体系。但与国际先进水平相比，尤其与一些跨国生物技术公司相比，在转化规模和转化效率方面仍然存在较大差距。认为安全、高效、规模化是转基因水稻新品种培育和产业化的重大技术瓶颈。建立水稻主栽品种快速、高效、稳定的转化系统，开发安全型转化技术，开展多基因、大片段基因转化，实现转基因的定点整合和时空控制表达等是水稻转基因技术的发展趋势，针对水稻规模化转基因技术体系存在的问题，提出了相应的对策，对于促进转基因水稻新品种培育和功能基因组学研究具有一定参考价值。

关键词: 水稻, 规模化, 转化体系

Abstract: 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.

Key words: rice, largescale, transformation system

张欣，付亚萍，周君莉，郭秀平，刘文真，吴洁芳，吴传银，万建民. 水稻规模化转基因技术体系构建与应用[J]. 中国农业科学, 2014, 47(21): 4141-4154.

ZHANG Xin, FU Ya-ping, ZHOU Jun-li, GUO Xiu-ping, LIU Wen-zhen, WU Jie-fang, WU Chuan-yin, WAN Jian-min. Establishment and Application of Large-Scale Transformation Systems for Rice[J]. Scientia Agricultura Sinica, 2014, 47(21): 4141-4154.

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