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Increased resistance to crown rust disease in transgenic Italian ryegrass (Lolium multiflorum Lam.) expressing the rice chitinase gene

  • Genetic Transformation and Hybridization
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Abstract

We introduced the rice chitinase (Cht-2; RCC2) gene into calli of Italian ryegrass (Lolium multiflorum Lam.), with a hygromycin phosphotransferase (HPT) gene as a selectable marker, by particle bombardment. Hygromycin-resistant calli were selected and transferred to regeneration medium for shoot formation. Polymerase chain reaction (PCR) analysis revealed regenerants containing the HPT gene. The RCC2 gene was detected in 65.5% of those regenerants. Southern hybridization detected both HPT and RCC2 genes and indicated that the transgenic plants were independently transformed. Expression of the RCC2 gene in the transgenic plants was confirmed by Northern hybridization, reverse transcription–PCR and Western blotting. Bioassay of detached leaves indicated increased resistance to crown rust (Puccinia coronata) in transgenic plants, which exhibited higher chitinase activity than a nontransgenic plant.

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Abbreviations

HPT:

Hygromycin phosphotransferase

MS medium:

Murashige and Skoog medium

PCR:

Polymerase chain reaction

RCC2 :

Rice chitinase Cht-2

RT–PCR:

Reverse transcription–polymerase chain reaction

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Acknowledgements

We thank Dr G. Spangenberg (La Trobe University, Victoria, Australia) for the gift of the plasmid pAcH1, and Dr H. Muylle (Agricultural Research Centre-Gent, Belgium) for kindly teaching us the protocol for in vitro inoculation with crown rust. We also thank Dr K. Sugawara (National Institute of Livestock and Grassland Science, Japan) for helpful discussions, and Miss Y. Nakayama (Japan Grassland Farming and Forage Seed Association) for assistance in producing transgenic plants. This work was funded by a research grant from the Japan Racing Association (JRA).

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Authors and Affiliations

  1. Forage Crop Research Institute, Japan Grassland Farming and Forage Seed Association, 388-5 Higashiakada, Nishinasuno, Tochigi, 329-2742, Japan

    Wataru Takahashi & Yuichi Miura

  2. National Institute of Livestock and Grassland Science, 768 Senbonmatsu, Nishinasuno, Tochigi, 329-2793, Japan

    Masahiro Fujimori, Toshinori Komatsu & Tadashi Takamizo

  3. National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602, Japan

    Yoko Nishizawa

  4. Research Institute, Tamagawa University, 6-1-1 Tamagawa-Gakuen, Machida, Tokyo, 194-8610, Japan

    Tadaaki Hibi

Authors
  1. Wataru Takahashi
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  2. Masahiro Fujimori
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  3. Yuichi Miura
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  4. Toshinori Komatsu
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  5. Yoko Nishizawa
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  6. Tadaaki Hibi
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  7. Tadashi Takamizo
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Corresponding author

Correspondence to Wataru Takahashi.

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Communicated by F. Sato

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Takahashi, W., Fujimori, M., Miura, Y. et al. Increased resistance to crown rust disease in transgenic Italian ryegrass (Lolium multiflorum Lam.) expressing the rice chitinase gene. Plant Cell Rep 23, 811–818 (2005). https://doi.org/10.1007/s00299-004-0900-1

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  • DOI: https://doi.org/10.1007/s00299-004-0900-1

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