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Agrobacterium-mediated transformation of protocorm-like bodies in Cymbidium

  • Genetic Transformation and Hybridization
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Abstract

Genetically transformed plants of Cymbidium were regenerated after cocultivating protocorm-like bodies (PLB) with Agrobacterium tumefaciens strain EHA101 (pIG121Hm) that harbored genes for β-glucuronidase (gus), hygromycin phosphotransferase (hpt) and neomycin phosphotransferase II (nptII). PLB of three genotypes maintained in liquid new Dogashima medium (NDM), were subjected to transformation experiments. The PLB inoculated with Agrobacterium produced secondary PLB, 4 weeks after transfer onto 2.5 g L−1 gellan gum-solidified NDM containing 10 g L−1 sucrose, 20 mg L−1 hygromycin and 40 mg L−1 meropenem. Transformation efficiency was affected by genotype and the presence of acetosyringone during cocultivation. The highest transformation efficiency was obtained when PLB from the genotype L4 were infected and cocultivated with Agrobacterium on medium containing 100 μM acetosyringone. Transformation of the hygromycin-resistant plantlets regenerated from different sites of inoculated PLB was confirmed by histochemical GUS assay, PCR analysis and Southern blot hybridization.

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Abbreviations

AS:

Acetosyringone

GUS:

β-Glucuronidase

Hm:

Hygromycin

Km:

Kanamycin

ND:

New Dogashima

PLB:

Protocorm-like body

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Acknowledgements

We Thank Hitaka Orchid Co., Ltd. for providing Cymbidium seeds and Mukoyama Orchids Co., Ltd. for providing PLB of RY.

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Authors and Affiliations

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

    Dong Poh Chin & Masahiro Mii

  2. Plant Molecular Biology Group, Graduate School of Life and Environmental Science, Osaka Prefecture University, 1-1 Gakuen Sakai, Osaka, 599-8531, Japan

    Kei-ichiro Mishiba

Authors
  1. Dong Poh Chin
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  2. Kei-ichiro Mishiba
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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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Chin, D.P., Mishiba, Ki. & Mii, M. Agrobacterium-mediated transformation of protocorm-like bodies in Cymbidium . Plant Cell Rep 26, 735–743 (2007). https://doi.org/10.1007/s00299-006-0284-5

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  • DOI: https://doi.org/10.1007/s00299-006-0284-5

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