Background

The advancement of Ensifer-mediated transformation (EMT) technology to successfully transform dicots viz., Arabidopsis thaliana, Solanum tuberosum, Nicotiana tabacum, Manihot esculenta, Brassica napus, and the monocot; Oryza sativa, has been previously reported (Wendt et al., 2012; Zuniga-Soto et al., 2015; Chavarriaga-Aguirre et al., 2016; Rathore et al., 2016). Additionally, genomic analysis of E. adhaerens OV14 by Rudder et al. (2014) revealed the bacterium possessed a 7.7 Mb genome comprised of two circular chromosomes (3.96 Mb and 2.01 Mb) and two plasmids (1.61 Mb and 125 Kb). A comparative analysis of the genome of E. adhaerens OV14 with Agrobacterium tumefaciens C58 (classic genetic engineer) and Sinorhizobium meliloti 1021 (a rhizobia with a propensity for low rates of genetic transformation; Broothaerts et al., 2005), highlighted that both E. adhaerens OV14 and S. meliloti 1021 contain homologs to several chromosomal-based genes essential for Agrobacterium-mediated transformation (AMT). However, a suite of genes which positively influence the successful transformation of a plant genome were found to be only present in the genome of E. adhaerens OV14 but absent from S. meliloti 1021 (Rudder et al., 2014). Overall, the sequence analysis of the E. adhaerens OV14 genome has significantly expanded the knowledge base describing the genetic systems that regulate the transformation of plant genomes via EMT.

To date several transient transformation systems have been proposed to study gene function in plants in regard to the use of A. tumefaciens (Wroblewski et al., 2005; Gelvin, 2006; Bhaskar et al., 2009; Li et al., 2009; Van Loock et al., 2010; Hwang et al., 2013; Krenek et al., 2015). Whereas, the stable transformations of any plant species are lengthy processes to test the utility of a bacterial transfection system to transform the plants. The advantages of transient transformation methods include rapid production of results, functional genomic studies and recombinant protein production (Van Loock et al., 2010; Krenek et al., 2015). The model plant A. thaliana is a powerful research tool with which to study the molecular, genetic and biochemical processes that support genetic transformation of somatic tissues as well as in planta (Provart et al., 2016). In contrast to the several months typically required for the transformation of primary crop species, these investigations require a rapid, reproducible and easy quantification method to determine the rate of transient transformation. Previously, Gelvin (2006) reported an efficient and reproducible quantitative assay for Agrobacterium-mediated Transformation using A. thaliana roots. This assay has well served the purpose of testing either Agrobacterium strains or A. thaliana ecotypes/mutants in the authors’ laboratory for more than two decades, reflecting the significance and competency of the assay in publications such as Shi et al. (2014). Conversely, a transient transformation method to facilitate comparable studies via EMT is not available. In response, the Ensifer-mediated A. thaliana Root Transformation (E-ART) protocol presented here is designed to address this deficit so that specific genetic and microbiological factors that support/enhance EMT can be identified to support the application of the technology to agronomically important crop species. The protocol is a modified version of an existing AMT based quantitative A. thaliana roots assay (Gelvin, 2006). While developing the E-ART protocol, we learnt that it is possible to improve transient GUS expression in A. thaliana root segments by adjusting several experimental factors (e.g., time of co-cultivation, acetosyringone concentrations, etc.) involved in the early stages of E. adhaerens transfection. E-ART, being the first quantitative method of transient gene expression for EMT will facilitate the rapid evaluation of novel E. adhaerens strains in plant transformation while also providing a platform to assess the genetic response of plants to EMT.