Development of an efficient leaf protoplast isolation and transient expression system for Artemisia japonica

Artemisia japonica Thunb is an important perennial herb containing abundant chemical compounds utilized in conventional medicine for the treatment of malaria, hepatitis, hypertension, and inflammation. The protoplasts-based transient transformation system is a versatile and convenient tool for functional gene analysis in several Artemisia species. However, effective protoplast preparation and transformation systems are still lacking for A. japonica. We developed an efficient protoplast-based transformation system by optimizing conditions of protoplasts isolation and polyethylene glycol (PEG)-mediated transformation in A. japonica. The optimum conditions for the protoplast preparation were: the enzyme solution containing 1.75% (m/v) cellulase R10, 0.5% (m/v) macerozyme R-10, and 0.4 M D-mannitol with proper leaves treatment and pre-plasmolysis treatment. The maximum protoplast yield was 1.93 × 106 protoplasts g-1(FM) and the viability of protoplasts was approximately 87.5% under optimized conditions using an orthogonal experiment. Furthermore, the transient protoplast transformation efficiency was 47.86% in A. japonica protoplast under the conditions of 40% (m/v) PEG 4000 for 20 min. The establishment of A. japonica protoplasts isolation and transient transformation system can accelerate the gene function studies of A. japonica and provide a fast and simple gene expression platform for molecular, biochemical, and functional gene characterization for other Artemisia species.