Agrobacterium-mediated genetic transformation and regeneration of transgenic plants using leaf segments as explants in Valencia sweet orange

Research Note


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 α-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.


Citrus sinensis L. Osbeck Green fluorescence protein Leaf segments Genetic transformation 





Cauliflower mosaic virus


Cetyltrimethylammonium bromide


Green fluorescence protein








α-Naphthaleneacetic acid


Neomycin phosphotransferase II gene




Murashige and Tucker basal medium


Indole-3-acetic acid


Indole-3-butyric acid


Polymerase chain reaction



This work was supported by National Natural Science Foundation of China, the Research Fund for the Doctoral Program of Higher Education (20090146110010), Fok Ying Tong Education Foundation (114034), Wuhan Municipal Project for Academic Leaders (201150530148) and Hubei Provincial Natural Science Foundation (2009CDA080). Khan EU was supported financially by Islamic Development Bank (IDB), Saudi Arabia under PhD Merit Scholarship Program for High Technology.


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Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  1. 1.Key Laboratory of Horticultural Plant Biology (MOE), National Key Laboratory of Crop Genetic Improvement, College of Horticulture and Forestry SciencesHuazhong Agricultural UniversityWuhanChina

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