Agrobacterium-mediated genetic transformation and regeneration of transgenic plants using leaf segments as explants in Valencia sweet orange
Agrobacterium-mediated genetic transformation and regeneration of transgenic plants using leaf segments as explants in Valencia sweet orange
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巴伦西亚甜橙叶段作为外植体的农杆菌介导的遗传转化和转基因植物再生
DOI：
10.1007/s11240-011-0092-7
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发表时间：
2012-05-01
影响因子：
3
通讯作者：
Liu, Ji-Hong
中科院分区：
文献类型：
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作者：
Khan, Ehsan Ullah;Fu, Xing-Zheng;Liu, Ji-Hong
本研究以绿色荧光蛋白（gfp）为重要标记物，通过农杆菌介导的瓦伦西亚甜橙（Citrus sinensis L. Osbeck）叶片片段的转化，试图建立转基因植株再生方案。结果表明，卡那霉素浓度为50 mg l（-1）时，叶片再生对卡那霉素的敏感性最高。此外，还优化了影响瞬时gfp表达频率的因素，包括叶龄、农杆菌浓度、侵染时间和共培养时间。不定芽在含有Murashige和Tucker基础培养基加0.1 mg l(-1) α -萘乙酸（NAA）、0.5 mg l(-1) 6-苄基腺苷（BA）和0.5 mg l（-1）动蛋白（KT）的培养基上再生。3月龄离体苗叶片片段，浓度为OD600 0.6，浸泡10 min，共培养3 d，瞬时gfp表达频率、芽再生响应和转化效率最高。利用优化后的参数，在选择性培养基(MT盐中添加0.5 mg l(-1) BA、0.5 mg l(-1) KT、0.1 mg l（-1) NAA、50 mg l（-1）卡那霉素和250 mg l（-1）头孢噻肟）上恢复了独立转化植株，转化效率为23.33%。荧光显微镜下证实了gfp在叶片和再生芽中的表达。利用gfp和nptII基因特异性引物进行聚合酶链反应（PCR）分析，进一步证实了该转基因基因在独立转基因植株中的整合。本文描述的转化方法可能为利用叶段作为外植体产生转基因植物铺平道路。
In this study, attempts were made to develop a protocol for regeneration of transgenic plants via Agrobacterium tumefaciens-mediated transformation of leaf segments from 'Valencia' sweet orange (Citrus sinensis L. Osbeck) using gfp (green fluorescence protein) as a vital marker. Sensitivity of the leaf segments regeneration to kanamycin was evaluated, which showed that 50 mg l(-1) was the best among the tested concentrations. In addition, factors affecting the frequency of transient gfp expression were optimized, including leaf age, Agrobacterium concentration, infection time, and co-cultivation period. Adventitious shoots regenerated on medium containing Murashige and Tucker basal medium plus 0.1 mg l(-1) alpha-naphthaleneacetic acid (NAA), 0.5 mg l(-1) 6-benzyladenine (BA) and 0.5 mg l(-1) kinetin (KT). The leaf segments from 3-month-old in vitro seedlings, Agrobacterium concentration at OD600 of 0.6, 10-min immersion, and co-cultivation for 3 days yielded the highest frequency of transient gfp expression, shoots regeneration response and transformation efficiency. By applying these optimized parameters we recovered independent transformed plants at the transformation efficiency of 23.33% on selection medium (MT salts augmented with 0.5 mg l(-1) BA, 0.5 mg l(-1) KT, 0.1 mg l(-1) NAA, 50 mg l(-1) kanamycin and 250 mg l(-1) cefotaxime). Expression of gfp in the leaf segments and regenerated shoots was confirmed using fluorescence microscope. Polymerase chain reaction (PCR) analysis using gfp and nptII gene-specific primers further confirmed the integration of the transgene in the independent transgenic plants. The transformation methodology described here may pave the way for generating transgenic plants using leaf segments as explants.