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

Shrub willows ( 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 , 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 leaf tissue, along with transient overexpression and CRISPR-Cas9 mediated mutations. This is the first such report of transient gene expression in 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.