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Biolistic transformation of Carrizo citrange (Citrus sinensis Osb. × Poncirus trifoliata L. Raf.)

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Abstract

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The development of transgenic citrus plants by the biolistic method.

Abstract

A protocol for the biolistic transformation of epicotyl explants and transgenic shoot regeneration of immature citrange rootstock, cv. Carrizo (Citrus sinensis Osb. × Poncirus trifoliata L. Raf.) and plant regeneration is described. Immature epicotyl explants were bombarded with a vector containing the nptII selectable marker and the gfp reporter. The number of independent, stably transformed tissues/total number of explants, recorded by monitoring GFP fluorescence 4 weeks after bombardment was substantial at 18.4 %, and some fluorescing tissues regenerated into shoots. Fluorescing GFP, putative transgenic shoots were micro-grafted onto immature Carrizo rootstocks in vitro, confirmed by PCR amplification of nptII and gfp coding regions, followed by secondary grafting onto older rootstocks grown in soil. Southern blot analysis indicated that all the fluorescing shoots were transgenic. Multiple and single copies of nptII integrations were confirmed in five regenerated transgenic lines. There is potential to develop a higher throughput biolistics transformation system by optimizing the tissue culture medium to improve shoot regeneration and narrowing the window for plant sampling. This system will be appropriate for transformation with minimal cassettes.

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Acknowledgments

This research was supported by the Citrus Research and Development Foundation (CRDF), an arm of the Florida Citrus Growers, awards 583 and 15-045C to JZ. Facilities were provided by the CRDF and the University of Florida, IFAS, Citrus Research and Education Center, in Lake Alfred, FL.

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Authors and Affiliations

  1. Plant Pathology Department, Institute of Food and Agricultural Sciences, Citrus Research and Education Center, University of Florida, Lake Alfred, FL, 33850, USA

    Hao Wu, Yosvanis Acanda, Hongge Jia, Nian Wang & Janice Zale

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  1. Hao Wu
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  2. Yosvanis Acanda
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  3. Hongge Jia
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  4. Nian Wang
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  5. Janice Zale
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Correspondence to Hao Wu or Janice Zale.

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Communicated by V. Srivastava.

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Wu, H., Acanda, Y., Jia, H. et al. Biolistic transformation of Carrizo citrange (Citrus sinensis Osb. × Poncirus trifoliata L. Raf.). Plant Cell Rep 35, 1955–1962 (2016). https://doi.org/10.1007/s00299-016-2010-2

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  • DOI: https://doi.org/10.1007/s00299-016-2010-2

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