Agroinfiltration of grapevine leaves for fast transient assays of gene expression and for long-term production of stable transformed cells |
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Authors: | Michela Zottini Elisabetta Barizza Alex Costa Elide Formentin Cristina Ruberti Francesco Carimi Fiorella Lo Schiavo |
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Institution: | (1) Dipartimento di Biologia, Università Degli Studi di Padova, Via U. Bassi 58/B, 35131 Padua, Italy;(2) Istituto di Genetica Vegetale, Palermo (CNR), Corso Calatafimi 414, 90128 Palermo, Italy |
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Abstract: | Agrobacterium-mediated transient assays for the analysis of gene function are used as alternatives to genetic complementation and stable
plant transformation. Although such assays are routinely performed in several plant species, they have not yet been successfully
applied to grapevines. We explored genetic background diversity of grapevine cultivars and performed agroinfiltration into
in vitro cultured plants. By combining different genotypes and physiological conditions, we developed a protocol for efficient
transient transformations of selected grapevine cultivars. Among the four cultivars analyzed, Sugraone and Aleatico exhibited
high levels of transient transformation. Transient expression occurred in the majority of cells within the infiltrated tissue
several days after agroinfiltration and, in a few cases, it later spread to a larger portion of the leaf. Three laboratory
strains of Agrobacterium tumefaciens with different virulence levels were used for agroinfiltration assays on grapevine plants. This method promises to be a powerful
tool to perform subcellular localization analyses. Grapevine leaf tissues were transformed with fluorescent markers targeted
to cytoplasm (free GFP and mRFP1), endoplasmatic reticulum (GFP::HDEL), chloroplast (GAPA1::YFP) and mitochondria (β::GFP).
Confocal microscope analyses demonstrated that these subcellular compartments could be easily visualized in grapevine leaf
cells. In addition, from leaves of the Sugraone cultivar agroinfiltrated with endoplasmic reticulum-targeted GFP-construct,
stable transformed cells were obtained that show the opportunity to convert a transiently transformed leaf tissue into a stably
transformed cell line.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. |
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Keywords: | Vitis vinifera Plant cell cultures Agrobacterium Transformation Subcellular localization |
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