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Efficient production of marker-free transgenic ‘Tarocco’ blood orange (Citrus sinensis Osbeck) with enhanced resistance to citrus canker using a Cre/loxP site-recombination system

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Abstract

Marker-free transgenic plants alleviate concerns regarding the biosafety of genetically modified organisms and promote their commercialization. In this study, a transformation vector pLI35SAAT, harboring a Cre/loxP-mediated recombination system combined with the isopentenyl transferase (ipt) selectable marker gene and an anti-bacterial peptide gene AATCB, was used to produce marker-free transgenic ‘Tarocco’ blood orange (Citrus sinensis Osbeck) with enhanced resistance to citrus canker. Using ipt positive selection, a transformation efficiency of 21.4 % was achieved. When the DNA between two loxP sites was excised, phenotypically normal shoots gradually appeared from 74.8 % of the transgenic ipt shoots. Their marker-free transgenic nature was confirmed using PCR and sequencing analyses. In vitro evaluations of citrus canker disease resistance revealed that marker-free transgenic plants exhibited an enhanced resistance to Xanthomonas axonopodis pv. citri. The marker-free transgenic plants appeared phenotypically normal under greenhouse conditions. Thus, marker-free transgenic citrus plants with targeted traits can be efficiently produced using a Cre/loxP-mediated recombination system combined with ipt positive selection.

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Acknowledgments

We thank Prof. Xiaochun Zhao from the Citrus Research Institute of Southwest University for critically reading the manuscript. This research was supported by the National Natural Sciences Foundation of China (31272150 to X. Zou), the Fundamental Research Funds for the Central Universities (XDJK2013C160 to H. Peng; XDJK2014A018 to Y. He), the earmarked fund for China Agriculture Research System (CARS-27 to C. Chen) and the Ministry of Agriculture “Introduce International Advanced Agriculture Science and Technology” (2011-G21 to Y. He).

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The authors declare that they have no conflict of interest.

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Correspondence to Shanchun Chen or Xiuping Zou.

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Peng, A., Xu, L., He, Y. et al. Efficient production of marker-free transgenic ‘Tarocco’ blood orange (Citrus sinensis Osbeck) with enhanced resistance to citrus canker using a Cre/loxP site-recombination system. Plant Cell Tiss Organ Cult 123, 1–13 (2015). https://doi.org/10.1007/s11240-015-0799-y

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