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High-efficiency Agrobacterium-mediated transformation of citrus via sonication and vacuum infiltration

  • Genetic Transformation and Hybridization
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Abstract

An improved method for the Agrobacterium infiltration of epicotyl segments of ‘Pineapple’ sweet orange [Citrus sinensis (L.) Osbeck] and ‘Swingle’ citrumelo [Citrus paradisi Macf. X Poncirus trifoliata (L.) Raf.] was developed in order to increase transformation frequency. Sonication-assisted Agrobacterium-mediated transformation (SAAT), vacuum infiltration, and a combination of the two procedures were compared with conventional Agrobacterium-mediated inoculation method (‘dipping’ method). It was observed that the morphogenic potential of the epicotyl segments decreased as the duration of SAAT and vacuum treatments increased. Transient GUS expression was not affected by the different SAAT treatments, but it was significantly enhanced by the vacuum infiltration treatments. The highest transformation efficiencies were obtained when the explants were subjected to a combination of SAAT for 2 s followed by 10 min of vacuum infiltration. PCR and Southern blot analysis of the uidA gene were used to confirm the integration of the transgenes. The transformation frequencies achieved in this study (8.4% for ‘Pineapple’ sweet orange and 11.2% for ‘Swingle’ citrumelo) are the highest ones reported for both cultivars.

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Abbreviations

BAP:

6-Benzylaminopurine

NAA:

Naphtaleneacetic acid

GUS:

β-Glucuronidase

nptII:

Neomycin phosphotransferase II

SAAT:

Sonication-assisted Agrobacterium-mediated transformation

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Acknowledgments

The authors thank CNPq, FAPEMIG and FAPESB for financial support. M.L.P.O was recipient of scholarships from CAPES and CNPq (Grant number SWE 210112/2006-9) for her sandwich program training at the University of Florida.

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Authors and Affiliations

  1. Plant Biology Department, Plant Tissue Culture Laboratory/BIOAGRO, Federal University of Viçosa, Viçosa, MG, 36570-000, Brazil

    Maria Luiza Peixoto de Oliveira & Wagner Campos Otoni

  2. Horticultural Sciences Department, Institute of Food and Agricultural Sciences, Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL, 32611-0690, USA

    Vicente J. Febres & Gloria A. Moore

  3. Biological Sciences Department, State University of Santa Cruz (UESC), Ilhéus, BA, 45662-000, Brazil

    Marcio Gilberto Cardoso Costa

Authors
  1. Maria Luiza Peixoto de Oliveira
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  2. Vicente J. Febres
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  3. Marcio Gilberto Cardoso Costa
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  4. Gloria A. Moore
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  5. Wagner Campos Otoni
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Correspondence to Marcio Gilberto Cardoso Costa.

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Communicated by G. Phillips.

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de Oliveira, M.L.P., Febres, V.J., Costa, M.G.C. et al. High-efficiency Agrobacterium-mediated transformation of citrus via sonication and vacuum infiltration. Plant Cell Rep 28, 387–395 (2009). https://doi.org/10.1007/s00299-008-0646-2

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  • DOI: https://doi.org/10.1007/s00299-008-0646-2

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