摘要
西瓜是我国重要的经济作物,其面积和产量居世界水果前列。由于西瓜资源的遗传背景相对狭窄,对于一些重要病害缺乏可利用的抗性材料,采用转基因手段有助于创制新种质。转基因技术是生物遗传改良和基因功能验证的重要方法,而组织培养和遗传转化是植物转基因成功与否的2个前提条件。西瓜遗传转化采用的方法有多种,而农杆菌介导法应用最广泛。大量的研究表明,西瓜是被公认比较难于转化的作物,目前为止转化效率低仍然是阻碍西瓜转基因的主要瓶颈。本文分析了西瓜组织培养的影响因素,如西瓜的种子贮存时间、基因型、苗龄、外植体类型、激素组合、培养条件;同时分析了农杆菌介导法影响西瓜遗传转化效率的主要因素,包括筛选标记基因、农杆菌菌株类型、预培养时间、农杆菌侵染时间和浓度、共培养时间;探讨了目前西瓜组织培养和遗传转化体系所存在的问题,提出了今后的研究方向。
Watermelon is one of the economically significant cucurbits in China for its production yield and areas rank the first in the world. There are many agricultural problems in the cultivation of watermelon for which traditional breeding method often does not have already explained, such as lacking of resistance cuhivar against viral diseases. However, there are inadequate germplasm resources to meet the needs of watermelon breeders. Other important issues requiring improvement are fruit quality, nutrition, flavour, tolerance of storage of fruits and resistance to abiotic stresses. The above problems are difficult to solve through time-consuming conventional breeding methods, but biotechnology shed more light on solving this issue. Transgenic technology is a powerful tool for gene functional validation and genetic improvement of plants. Tissue culture and genetic transformation are the two basic components for that. Tissue cul- ture is a prerequisite to successful genetic transformation which introduce significantly interest genes into the plant genome while preserving genetic identity of plants. Recent advances in this technology have resulted in successful development of commercially disease and herbicide resistant plants which enhanced tolerance to environment stresses, increased crop productivity and reduced the usage of harmful pesti- cides. The plants have been engineered for safe and inexpensive production in large quantities produced in transgenic plants, as well as plants which possess enhanced nutritional traits to date. In this paper, we analyzed the major factors affecting watermelon tissue culture which is aimed to regenerate explants of cotyledons, hypocotyls, apical buds, anthers, ovaries, protoplasts and leaves. But most watermelon tissue culture used cotyledons as the explants, induction of adventitious shoots was obtained from proximal parts of the coty- ledon incubated on MS medium containing different concentrations of benzylaminpurine 1-7 mg-L-~and low- er concentrations of auxin 0-3 mg· L^-1 or 2 mg· L^-1 of 2,4-D. Regeneration of watermelon in tissue culture has been achieved for various watermelon cultivars. The various factors that influenced tissue culture of watermelon including the seeds storage time, genotypes, age of seedlings, type of explants, composition of culture medium and environmental conditions. However, an efficient system for plant regeneration is essential for in vitro techniques which is useful in procedure of transgenic genes. Recently most transgenic wa- termelon researches focused on improving regeneration of plantlets from callus and adventitious shoots directly, in addition to pursuing efficient transformation methods of foreign genes into watermelon. Agrobacte- rium-mediated genetic transformation currently was the common method in the watermelon transgenic technology. In 1994, Choi in Korea reported the first transgenic watermelon plants regenerated from cotyle- dons explants by Agrobacterium inoculation. Since then, by using modified Agrobacterium-mediated genetic transformation, resistance to various plant diseases has been achieved in transgenic watermelon through the incorporation of genes coding for the coat protein gene of various plant viruses, including Zucchini yellow mosaic virus （ZYMV）, Watermelon mosaic virus （WMV）, Cucumber mosaic virus （CMV） and Cucumber green mottle mosaic virus （CGMMV）. Other researchers have reported the development of genetically engineered watermelon plants through transformation with ACC oxidase antisense gene, antifungal proteins and chitinases etc. Agrobacterium-mediated genetic transformation technology is a highly complex and evolved process involving genetic determinants of both the bacterium and the host plant cell. The watermel- on is still acknowledged recalcitrant crop for transformation. Its transformation efficiency is still very low so far. Here we summarized key factors influencing Agrobacterium-mediated watermelon genetic transformation, including genotypes of the plant, types of explants, plasmid vectors, bacterial strains, the selection markers genes, the Agrobacterium strains, the pre-culture time, the infection concentrations and immersing time of Agrobacterium, the co-culture time. A number of researches indicated that three to ten days cotyledons are used as suitable explants for watermelon transformation, kanamycin is the usual selection marker, and herbicide resistance Bar gene and hygromycin B resistance gene are also used. Some Agrobacterium strains are applied to produce genetic transformation watermelon, such as EHA105, LBA4404, EHA101 and GV3101. There is no evidence to prove which strain is more effective. One to five days pre- culture or without can produce adventitious shoots, the co-cultivation time of watermelon explants with Agrobacterium from one to five days also produce adventitious shoots. The efficiency of plant transforma- tion can depend also on the Agrobacterium density, ranging from 1 × 10^6 to 1 × 10 mg· L^-1 can increase transcient transformation, however, it is not always correlated with higher stable transformation. It also discussed that some questions about tissue culture and genetic transformation of watermelon. It has not yet been solved that vitrification, yellowing, the top necrosis phenomenon in the process of subculture. Cotyle- dons is the mainly used explants affects the ploidy level of watermelon regenerated from tissue culture that high frequency of tetraploid regenerants is a common phenomenon. Some fruit characters are significantly different in morphology, and there is gene escape in transgenic watermelon plants system. The disorganization of meristematic structures following the exposure of explants to Agrobacterium remains the major obstacle to develop efficient transformation technology. These problems seriously restrict the usage of watermelon transgenic technology. In 2013, a powerful tool CRISPR （clustered regulatory interspersed short pal- indromic repeat）/Cas9 （CRISPR associated proteins） gene editing technology in plants was developed.Since then, the CRISPR/Cas9 system has been used in various plant species for targeted genome editing, such as arabidopsis, tobacco, wheat, rice, maize, cucumber and watermelon. It is able to achieve highly flexible and specific targeting. This system can edit multiple target genes simultaneously. We also expect new watermelon germplasm with novel desired traits will be created by this tool in coming years.
出处
《果树学报》
CAS
CSCD
北大核心
2017年第7期905-916,共12页
Journal of Fruit Science
基金
国家现代产业技术体系(CARS-26-13)
中国农业科学院创新工程(CAAS-ASTIP-2016-ZFRI)
国家自然科学基金(31572147)
中央级科研院所基本科研业务费专项(1610192017502)
关键词
西瓜
组织培养
遗传转化
Citrullus lanatus
Tissue culture
Genetic transformation
