Agroinfiltration of grapevine leaves for fast transient assays of gene expression and for long-term production of stable transformed cells

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 (b::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.

Agrobacterium-mediated transient MaFT expression in mulberry (Morus alba L.) leaves

“…[6][7][8][9][10] Agro-infiltration is a simple transformation process that exhibits highly efficient transformation and easy operation in several plant species including Arabidopsis, tobacco, grapevine, potato, switchgrass, and tomato. [9][10][11][12][13][14] In woody plants, several studies of transient gene expression by biolistic bombardment, protoplast transfection, and Agrobacterium co-cultivation in Populus have been reported, 15,16) while there is limited report on transient gene expression in mulberry.…”

Development and Use of Biotechnology Tools for Grape Functional Analysis

“…In grapevines, this technology has been recently applied to analyze the function of several genes such as VviAdh [75], VviPIP2 [76], VviCCD1 [77], and VviWRKY33 [78]. Likewise, this method has been proved to be very useful for transgenic complementation [79][80][81], technical studies [82][83][84][85][86][87], promoter analysis [88][89][90][91], subcellular localization analysis [92], and protein production [69,93] in grapevine. Nonetheless, the genetic background and plant growth conditions are key factors in performing successful Agrobacterium-mediated transformation.…”

“…The bacteria enter the intercellular air spaces within the leaf and transform a very large percentage of the internal mesophyll cells. The agroinjection method involves a needleless syringe that can be filled with the bacterial suspension and then pressed against the underside of a leaf to infiltrate the suspension by active pushing through the stomata [92]. This method can be used to rapidly and simply generate a chimeric plant, where a large number of leaf cells contain the gene of interest.…”

“…Nowadays, this method represents an easy and noninvasive technique that allows gene expression in the whole leaf [67]. Agroinfiltration is usually performed on tissues of young plantlets grown in vitro, as greenhouse grown plants have often been described as recalcitrant to this technique [67,92]. Santos-Rosa et al [69] transiently overexpressed stilbene synthase genes in detached grapevine leaves in order to study the influence of stilbenes on downy mildew infection.…”

Cloning of SjCA gene and its expression analysis on upland cottons

“…The transgenic plants were mainly acquired by Agrobacterium tumefaciens-mediated transformation and bombardment of embryo or callus via a desktop gene gun. In the process of Agrobacterium-mediated in planta transformation, the genotype is generally found to influence T-DNA transfer and transgene expression in Arabidopsis and grapevine [51][52][53][54] . Studies have reported in other plants species, the choice of the Agrobacterium strain appeared critical and specific because of specific host-bacteria inactions [52,55] .…”