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

Research Note

Abstract

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.

Keywords

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

Abbreviations

BA

6-benzyladenine

CaMV

Cauliflower mosaic virus

CTAB

Cetyltrimethylammonium bromide

gfp

Green fluorescence protein

Km

Kanamycin

AS

Acetosyringone

Cef

Cefatoxime

NAA

α-Naphthaleneacetic acid

nptII

Neomycin phosphotransferase II gene

KT

Kinetin

MT

Murashige and Tucker basal medium

IAA

Indole-3-acetic acid

IBA

Indole-3-butyric acid

PCR

Polymerase chain reaction

Notes

Acknowledgments

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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