Hairy Root Transformation: A Useful Tool to Explore Gene Function and Expression in Salix spp. Recalcitrant to Transformation

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.

Keywords: Agrobacterium rhizogenes-mediated transformation; Salix purpurea; domains rearranged methyltransferase 2 (DRM2); pGWAY-0; willow.

Figure 1

Workflow of the hairy root transformation of Salix purpurea in vitro plantlets by A4RS harboring a DsRed-based binary vector. Emerging DsRed fluorescent roots are already detected 7 days post inoculation (dpi). (A) 6-week-old S. purpurea in vitro plantlets are sub-cultured to MS medium with full strength macroelements and 30 g L^-1 sucrose (MS30). (B) 14-day-old S. purpurea in vitro plantlets were infected by stabbing the stem with a needle swabbed with Agrobacterium rhizogenes. (C) Infected plants were co-cultivated with A. rhizogenes for 14 days on MS30 supplemented with acetosyringone under dim light. (D) Plants were transferred to MS30 medium supplemented with Augmentin. (E) Generated hairy roots were examined at 21 dpi under a stereo fluorescence microscope. (F) Co-transformed roots were excised and collected for further analysis. (G) In vitro culture of hairy roots and composite plants. Figure adapted from Plasencia et al. (2016), introducing the protocol specificities of hairy roots transformation for S. purpurea.

Figure 2

Relative expression of SpDRM2 in transformed pGWAY-SpDRM2 hairy roots collected in three composite plants, in hairy roots transformed with pGWAY-0 (empty vectors) and wild-type roots. Relative expression is expressed in arbitrary units, by comparing with the expression in wild-type roots. Bars indicate the average of relative expression for pGWAY-DRM2 hairy roots (n = 3; technical replicates) and for pGWAY—0 hairy roots or wild type roots (n = 9 3 biological × 3 technical replicates). Standard error of mean is also indicated above the bars. Different letters above the bars indicate significant differences between conditions (Tukey’s comparison test P-value < 0.05).