Genetic Transformation and Hybridization

Plant Cell Reports

, Volume 28, Issue 3, pp 387-395

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

  • Maria Luiza Peixoto de OliveiraAffiliated withPlant Biology Department, Plant Tissue Culture Laboratory/BIOAGRO, Federal University of Viçosa
  • , Vicente J. FebresAffiliated withHorticultural Sciences Department, Institute of Food and Agricultural Sciences, Plant Molecular and Cellular Biology Program, University of Florida
  • , Marcio Gilberto Cardoso CostaAffiliated withBiological Sciences Department, State University of Santa Cruz (UESC) Email author 
  • , Gloria A. MooreAffiliated withHorticultural Sciences Department, Institute of Food and Agricultural Sciences, Plant Molecular and Cellular Biology Program, University of Florida
  • , Wagner Campos OtoniAffiliated withPlant Biology Department, Plant Tissue Culture Laboratory/BIOAGRO, Federal University of Viçosa

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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.

Keywords

Agrobacterium tumefaciens Orange SAAT Agroinfiltration