Agroinfiltration of grapevine leaves for fast transient assays of gene expression and for long-term production of stable transformed cells
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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.
Keywords:Vitis vinifera Plant cell cultures Agrobacterium Transformation Subcellular localization
We thank Professor Roger Tsien for providing us the pRSET-mRFP1 vector and Prof. Albrecht von Arnim for providing the pAVA554-YFP vector. We thank Dr. Jim Haseloff for providing the pBIN-m-gfp5-ER vector. We thank Prof. Paolo Trost for providing the GAPA1 cDNA clone. This research was supported by the “Progetto di Ateneo” by the University of Padova to M. Z.
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