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

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 viti...

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Bibliographic Details
Published in:Plant cell, tissue and organ culture tissue and organ culture, 2012-10, Vol.111 (1), p.15-28
Main Authors: Nookaraju, Akula, Agrawal, Dinesh C.
Format: Article
Language:English
Subjects:
Airborne microorganisms
Biomedical and Life Sciences
Chitinase
Cloning
Cloning vectors
Cultivation
Downy mildew
Efficiency
Embryos
Enzymatic activity
Explants
Fungicides
Genes
Genetic transformation
Incorporation
Leaves
Life Sciences
Original Paper
Phenylalanine
Plant Genetics and Genomics
Plant Pathology
Plant Physiology
Plant Sciences
Plasmids
Scab
Silver
Silver nitrate
Sodium
Sodium thiosulfate
Somatic embryos
Sonication
Thiosulfates
Wheat
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Summary: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 OD 600 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.
ISSN:0167-6857
1573-5044
DOI:10.1007/s11240-012-0166-1