A reliable protocol for the stable transformation of non-embryogenic cells cultures of grapevine (Vitis vinifera L.) and Taxus x media

Abstract

One of the major intent of metabolic engineering in cell culture systems is to increase yields of secondary metabolites. Efficient transformation methods are a priority to successfully apply metabolic engineering to cell cultures of plants that produce bioactive or therapeutic compounds, such as Vitis vinifera and Taxus x media. The aim of this study was to establish a reliable method to transform non-embryogenic cell cultures of these species. The V. vinifera cv. Gamay/cv. Monastrell cell lines and Taxus x media were used for Agrobacterium-mediated transformation using the Gateway-compatible Agrobacterium sp. binary vector system for fast reliable DNA cloning. The Taxus x media and Vitis cell lines were maintained in culture for more than 4 and 15 months, respectively, with no loss of reporter gene expression or antibiotic resistance. The introduced genes had no discernible effect on cell growth, or led to extracellular accumulation of phytoalexin trans-Resveratrol (t-R) in response to elicitation with methylated cyclodextrins (MBCD) and methyl jasmonate (MeJA) in the grapevine transgenic cell lines compared to the parental control. The method described herein provides an excellent tool to exploit exponentially growing genomic resources to enhance, optimize or diversify the production of bioactive compounds generated by grapevine and yew cell cultures, and offers a better understanding of many grapevine and yew biology areas.

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.…”

“…For instance, the compatibility of A. tumefaciens strains with the plant species represents an important variable to be considered in this kind of assay. Thus, the strains most efficiently used for gene transfer into grapevine are probably C58C1 (pCH32) [69,78,86] and EHA105 [75,77,82,83,85,88,90], which contain extra copies of vir genes that make them hypervirulent [61].…”

“…Transformation and regeneration of grapevine plants have been achieved via organogenesis [84], embryogenesis [83,93], or from nonembryogenic cell cultures [86,94], demonstrating that grapevine is not recalcitrant to Agrobacterium infection but this approach is time consuming and takes several months to produce transgenic plants suitable for analyses. In some cases, the production of whole transgenic organisms may not be needed if a large number of cells within a plant can be uniformly and consistently transformed.…”

“…Likewise, it is the most common method to obtain stably transformed grapevines [83,86,88,90,93]. The cocultivation of cell suspension cultures of Gamay Red with EHA105 strain of Agrobacterium was used for studying the expression of the grape dihydroflavonol 4-reductase gene (VviDFR) and the analysis of its promoter region [88].…”

“…Thompson Seedless for genetic transformation via Agrobacterium with a gene involved in the defense system. Martínez-Márquez et al [86] applied the same method to stably transform nonembryogenic cell cultures of two V. vinifera cell lines with the GFP gene under the control of pCaMV35S. This study was relevant for those interested in bioactive metabolite production.…”