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Agrobacterium-mediated transformation of Phalaenopsis by targeting protocorms at an early stage after germination

  • Genetic Transformation and Hybridization
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Abstract

A transformation procedure for phalaenopsis orchid established by using immature protocorms for Agrobacterium infection was aimed at the introduction of target genes into individuals with divergent genetic backgrounds. Protocorms obtained after 21 days of culture on liquid New Dogashima medium were inoculated with Agrobacterium strain EHA101(pIG121Hm) harboring both β-glucuronidase (GUS) and hygromycin resistance genes. Subculture of the protocorms on acetosyringone-containing medium 2 days before Agrobacterium inoculation gave the highest transformation efficiencies (1.3–1.9%) based on the frequency of hygromycin-resistant plants produced. Surviving protocorms obtained 2 months after Agrobacterium infection on selection medium containing 20 mg l−1 hygromycin were cut transversely into two pieces before transferring to recovery medium without hygromycin. Protocorm-like bodies (PLBs) proliferated from pieces of protocorms during a 1-month culture on recovery medium followed by transfer to selection medium. Hygromycin-resistant phalaenopsis plants that regenerated after the re-selection culture of PLBs showed histochemical blue staining due to GUS. Transgene integration of the hygromycin-resistant plants was confirmed by Southern blot analysis. A total of 88 transgenic plants, each derived from an independent protocorm, was obtained from ca. 12,500 mature seeds 6 months after infection with Agrobacterium. Due to the convenient protocol for Agrobacterium infection and rapid production of transgenic plants, the present procedure could be utilized to assess expression of transgenes under different genetic backgrounds, and for the molecular breeding of phalaenopsis.

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Abbreviations

ND:

New Dogashima

PLB:

Protocorm-like body

GUS:

β-Glucuronidase

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Author notes

  1. Kei-ichiro Mishiba

    Present address: Iwate Biotechnology Research Center, 22-174-4 Narita, Kitakami City, Iwate, 024-0003, Japan

Authors and Affiliations

  1. Laboratory of Plant Cell Technology, Faculty of Horticulture, Chiba University, 648 Matsudo, Matsudo City, Chiba, 271-8510, Japan

    Kei-ichiro Mishiba, Dong Poh Chin & Masahiro Mii

Authors
  1. Kei-ichiro Mishiba
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  2. Dong Poh Chin
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  3. Masahiro Mii
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Correspondence to Masahiro Mii.

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Communicated by K.K. Kamo

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Mishiba, Ki., Chin, D.P. & Mii, M. Agrobacterium-mediated transformation of Phalaenopsis by targeting protocorms at an early stage after germination. Plant Cell Rep 24, 297–303 (2005). https://doi.org/10.1007/s00299-005-0938-8

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  • DOI: https://doi.org/10.1007/s00299-005-0938-8

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