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Enhanced tolerance of transgenic grapevines expressing chitinase and β-1,3-glucanase genes to downy mildew

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Abstract

An Agrobacterium-mediated transformation protocol for grapevine cv. Crimson Seedless using sonication and anti-necrotic agents has been optimized, and transgenic lines carrying wheat chitinase and β-1,3-glucanase genes have exhibited enhanced tolerance to downy mildew incited by Plasmopara viticola. cDNA clones encoding chitinase and β-1,3-glucanase have been isolated from a cDNA library, constructed from scab-infected Sumai-3 wheat, and introduced into a plant cloning vector to generate the plasmids pCAMBAR.chi.11 and pCAMBAR.638. Embryogenic cultures, established from in vitro-derived leaves, of Crimson Seedless were used as explants for Agrobacterium tumefaciens-mediated transformation studies. Sonication of somatic embryos in a bacterial suspension of A. tumefaciens and incorporation of anti-necrotic agents in the co-cultivation medium significantly enhanced transformation efficiency. Transformation efficiency of embryos with either chitinase or β-1,3-glucanase gene was highest when embryos were suspended in a bacterial cell suspension at 0.5 OD600 and sonicated for 2 or 3 s at 60 kHz. Transformation efficiency with chitinase was highest on incorporation of 2 or 3 mg l−1 phenylalanine, 1 or 2 mg l−1 silver nitrate or 400 mg l−1 l-cysteine in co-cultivation medium while incorporation of 20 mg l−1 sodium thiosulphate produced highest transformation efficiency with β-1,3-glucanase. Confirmed transgenic grapevine lines harboring anti-fungal genes exhibited higher levels of chitinase and β-1,3-glucanase transcripts as well as enzymatic activities. Moreover, transgenic lines showed enhanced tolerance to P. viticola infection following detached leaf assays.

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Acknowledgments

Financial support in the form of Senior Research Fellowship (SRF) by the Council of Scientific and Industrial Research (CSIR), Govt. of India to Nookaraju and supply of chitinase and glucanase vectors by Dr. Muthukrishnan Subbarat, Professor in Biochemistry, Kansas State University are gratefully acknowledged.

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Authors and Affiliations

  1. Plant Tissue Culture Division, CSIR National Chemical Laboratory, Pune, 411 008, India

    Akula Nookaraju & Dinesh C. Agrawal

  2. Department of Bioenergy Science and Technology, Chonnam National University, Gwangju, 500 757, Republic of Korea

    Akula Nookaraju

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  1. Akula Nookaraju
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  2. Dinesh C. Agrawal
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Correspondence to Dinesh C. Agrawal.

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Nookaraju, A., Agrawal, D.C. Enhanced tolerance of transgenic grapevines expressing chitinase and β-1,3-glucanase genes to downy mildew. Plant Cell Tiss Organ Cult 111, 15–28 (2012). https://doi.org/10.1007/s11240-012-0166-1

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