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Highly efficient transformation of the GFP and MAC12.2 genes into precocious trifoliate orange (Poncirus trifoliata [L.] Raf), a potential model genotype for functional genomics studies in Citrus

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Abstract

Precocious trifoliate orange (Poncirus trifoliata [L.] Raf), an extremely early flowering mutant of P. trifoliata, is an attractive model for functional genomics research in Citrus. A procedure for efficient regeneration and transformation of this genotype was developed by using green fluorescent protein (GFP) gene as visual marker and etiolated stem segments as explants. In vivo monitoring of GFP expression permitted a rapid and easy discrimination of transgenic shoots and escapes. Transformation efficiency was 20.7% and the transformants were identified by polymerase chain reaction (PCR) and Southern blot analysis. Moreover, the transgenic lines expressed variable amounts of the GFP gene as revealed by real-time PCR analysis. Fifteen transgenic plants flowered 18 months after transfer to the greenhouse and six of them set fruits. GFP expression was also observed in the transgenic flowers and fruits. To test the utility of this system for functional genomics studies, an Arabidopsis thaliana MAC12.2 gene with the potential to produce seedless fruits was introduced into this genotype, and the traits of the transgenic fruits were characterized. The successful transformation of this perennial woody genotype with extremely short juvenility will allow us to test the function of cloned genes in citrus, the improvement of which is hindered by a long juvenility period.

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Abbreviations

AS:

acetosyringone

BA:

6-benzyladenine

GA3 :

gibberellic acid

GFP:

green fluorescent protein

IBA:

indole butyric acid

KT:

kinetin

MT medium:

Murashige and Tucker (1969)

NAA:

α-napthaleneacetic acid

NPT II:

neomycin phosphotransferase II

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Acknowledgements

This research was financially supported by the National Natural Science Foundation of China, the National 863 Project (2006AA100108, 2007AA10Z182), and the Key Project of Hubei Provincial Natural Science Foundation (no. 2008CDA069).

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Authors and Affiliations

  1. National Key Laboratory of Crop Genetic Improvement, National Center of Crop Molecular Breeding, Huazhong Agricultural University, Wuhan, 430070, China

    Bin Tan, Ding-Li Li, Shi-Xiao Xu, Gai-En Fan, Jing Fan & Wen-Wu Guo

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  1. Bin Tan
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  2. Ding-Li Li
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  3. Shi-Xiao Xu
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  4. Gai-En Fan
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  5. Jing Fan
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Correspondence to Wen-Wu Guo.

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Communicated by F. Gmitter

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Tan, B., Li, DL., Xu, SX. et al. Highly efficient transformation of the GFP and MAC12.2 genes into precocious trifoliate orange (Poncirus trifoliata [L.] Raf), a potential model genotype for functional genomics studies in Citrus . Tree Genetics & Genomes 5, 529–537 (2009). https://doi.org/10.1007/s11295-009-0206-0

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