Protoplast-Based Transient Expression and Gene Editing in Shrub Willow (Salix purpurea L.)

Abstract

Shrub willows (Salix section Vetrix) are grown as a bioenergy crop in multiple countries and as ornamentals across the northern hemisphere. To facilitate the breeding and genetic advancement of shrub willow, there is a strong interest in the characterization and functional validation of genes involved in plant growth and biomass production. While protocols for shoot regeneration in tissue culture and production of stably transformed lines have greatly advanced this research in the closely related genus Populus, a lack of efficient methods for regeneration and transformation has stymied similar advancements in willow functional genomics. Moreover, transient expression assays in willow have been limited to callus tissue and hairy root systems. Here we report an efficient method for protoplast isolation from S. purpurea leaf tissue, along with transient overexpression and CRISPR-Cas9 mediated mutations. This is the first such report of transient gene expression in Salix protoplasts as well as the first application of CRISPR technology in this genus. These new capabilities pave the way for future functional genomics studies in this important bioenergy and ornamental crop.

Keywords: Salix; gene editing; protoplasts.

Figure 1

(A) sfGFP overexpression; (B) eGFP overexpression; (C) eYGFPuv overexpression; (D) mCherry overexpression; (E) percent transformation efficiency of each fluorescent protein tested shown as mean ± standard deviation of three independent scopes.

Figure 2

(A) Schematic showing the T-DNA design for CRIPSR/Cas9 mediated knockout, exons are shown in blue and gRNA binding sites in red, relative gene lengths are not shown to scale; (B) Alignments of CRISPR mutations with read counts (left) and mutation type (right) for each of the six gRNAs. For brevity, alternative haplotypes with the same mutation were combined, PAM sequences are highlighted in red, and substitutions are highlighted in green.

Figure 3

Image of two-week-old cuttings of Salix purpurea clone 94006, arrows point to leaves used for protoplast extraction.