小麦规模化转基因技术体系构建及其应用

doi:10.3864/j.issn.0578-1752.2014.21.003

摘要/Abstract

摘要： 在主要农作物中，小麦属于遗传转化比较困难的作物，转化效率较低，重复性较差，转化规模较小，优良转基因材料较少，基因工程育种进程明显落后于大豆、玉米、棉花、水稻等作物。目前，应用于小麦中的转基因技术主要包括基因枪介导法和农杆菌介导法，有些实验室也采用花粉管通道、离子束注入、激光微束穿刺、PEG、花粉介导和农杆菌浸花等方法。在外植体利用方面，多数研究主要利用小麦幼胚及其愈伤组织作为起始转化材料，以成熟胚、幼穗、花药愈伤组织为材料转化成功的报道还比较少，需要进一步探索。在转化效率方面，基因枪报道为0.1%—16.7%，农杆菌报道为0.7%—44.8%，变化幅度较大。在目标基因转化方面，除了nptⅡ、bar、hpt、GUS、GOX、pmi、ALS等筛选基因和报告基因外，转化的功能基因主要涉及小麦品质、抗病性、耐旱性、抗蚜虫和抗除草剂等性状改良。农杆菌介导和基因枪介导转化小麦幼胚的转化效率除与受体基因型有关外，还与受体材料的生理状态有关，供体植株生长期间的温度条件、光照条件、营养条件和水分条件对转化效率有至关重要的影响，开花到幼胚取样期间适宜的昼夜温度有利于转化后胚性愈伤组织诱导和候选转基因植株的获得。从整体水平看，中国小麦转基因技术研究虽然取得了较大进展，如建立了小麦成熟胚高频率再生体系并应用于小麦转化，改进了小麦幼胚再生体系和转化体系，将一批抗病、耐旱和品质改良相关基因转入小麦，初步建立了小麦规模化转基因技术体系，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。认为转化效率较低、基因型依赖性强、人工气候条件不够先进、转化队伍不稳定是限制中国小麦规模化转基因技术发展的瓶颈；建立主栽品种转化体系、提高转化效率、开展多基因转化、开发安全型转化技术、避免载体骨架序列插入、减少基因沉默、实现定点整合等是小麦转基因技术研究的发展趋势；通过对组织培养技术、植株再生和转化相关基因的研究，以及优良受体基因型筛选、培养基改良和各个转化影响因素的优化、集成等，克服农杆菌转化小麦的瓶颈，提高小麦转化效率，扩大转化规模。文章重点综述了基因枪和农杆菌转化技术在小麦中的应用和发展，回顾了近5年中国小麦规模化转基因技术研究进展，对于促进转基因小麦新品种培育和小麦功能基因组学研究具有一定参考价值。

关键词: 普通小麦, 转基因技术, 转化效率, 规模化

Abstract: 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 nptⅡ, 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.

Key words: common wheat, transgenic system, transformation efficiency, large-scale

叶兴国，徐惠君，杜丽璞，何光源，王轲，林志珊. 小麦规模化转基因技术体系构建及其应用[J]. 中国农业科学, 2014, 47(21): 4155-4171.

YE Xing-guo, XU Hui-jun, DU Li-pu, HE Guang-yuan, WANG Ke, LIN Zhi-shan. Establishment and Application of Large-Scale Transformation Systems in Wheat[J]. Scientia Agricultura Sinica, 2014, 47(21): 4155-4171.

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