Abstract
Willow (Salix spp. L.) species are fast-growing trees and shrubs that have attracted emergent attention for their potential as feedstocks for bioenergy and biofuel production, as well as for pharmaceutical and phytoremediation applications. This economic and environmental potential has propelled the creation of several genetic and genomic resources for Salix spp. Furthermore, the recent availability of an annotated genome for Salix purpurea has pinpointed novel candidate genes underlying economically relevant traits. However, functional studies have been stalled by the lack of rapid and efficient coupled regeneration-transformation systems for Salix purpurea and Salix spp. in general. In this report, we describe a fast and highly efficient hairy root transformation protocol for S. purpurea. It was effective for different explant sources and S. purpurea genotypes, with efficiencies between 63.4% and 98.7%, and the screening of the transformed hairy roots was easily carried out using the fluorescent marker DsRed. To test the applicability of this hairy root transformation system for gene functional analysis, we transformed hairy roots with the vector pGWAY-SpDRM2, where the gene SpDRM2 encoding a putative Domain Rearranged Methyltransferase (DRM) was placed under the control of the CaMV 35S constitutive promoter. Indeed, the transgenic hairy roots obtained exhibited significantly increased expression of SpDRM2 as compared to controls, demonstrating that this protocol is suitable for the medium/high-throughput functional characterization of candidate genes in S. purpurea and other recalcitrant Salix spp.
Highlights
The use of A. rhizogenes to induce transformed hairy roots represents a novel approach in Salix, as these species are recalcitrant to regeneration and transformation with A. tumefaciens
Previous reports suggest that hairy roots can be induced on a wide range of woody species, using in vitro (Alpizar et al, 2006; Bosselut et al, 2011; Yoshida et al, 2015; Plasencia et al, 2016) and in planta strategies (Alagarsamy et al, 2018), and from different explant types, e.g. seedlings, roots, stems, and leaves
The fluorescent marker DsRed facilitates the identification of transgenic roots, allowing the non-destructive and precocious identification of transgenic roots (Limpens et al, 2004; Chabaud et al, 2006; Meng et al, 2019)
Summary
There is still a need to develop a hairy root transformation protocol that would allow the rapid characterization of gene function in Salix spp. We propose a reproducible, rapid and highly efficient A. rhizogenes-mediated hairy root transformation system for S. purpurea (Figure 1; details in Supplementary Material S1) In this method, the transformed hairy roots are detectable by fluorescent markers, allowing an easy and fast selection of transgenic roots. To validate the results of the first experiment and to check if the developed protocol could be applicable to different S. purpurea genotypes, a second transformation experiment (Supplementary Table S2.1—Experiment B) was performed comparing two nonrelated genotypes, ELB3/6 and ELB2/5, using two-week-old in vitro clonal lines of S. purpurea grown in MS30, as this was shown to be the best condition to maximize the transformation efficiency. Based on the results obtained, we expect that this transformation method will be a valuable tool for the medium-/high-throughput functional characterization of candidate genes in S. purpurea
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