A transient protoplast expression system for functional gene validation in sugarcane: Focus on drought response and immune pathways

Modern sugarcane cultivars are allo‐autopolyploids and aneuploids, which pose great challenges for research and breeding to meet global demand. Recent breakthroughs in gene‐editing technologies offer the potential to expedite the development of high‐yield stress‐resilient sugarcane, a vegetatively propagated crop. However, the limited valuable targets, coupled with complex transgenic methodologies for verifying gene function, hamper this progress. Protoplasts enable rapid gene introduction and function validation, yet an efficient protoplast expression system for elite sugarcane cultivars is currently lacking. Here, we developed a protoplast isolation and transfection method using tissue‐cultured GT42, a primary sugarcane cultivar in China, yielding 1.41 × 10 7 protoplasts/g fresh weight with 78.74% viability and 39.01% transfection efficiency. This system proved effective for various assays, including GCaMP6‐based calcium imaging. Using this system, we showed that abscisic acid biosynthesis gene ScNCED3 acts as an early drought signal, positively regulated by ScPYL4‐12, ScOST1‐12 and ScMPK6‐11, whereas salicylic acid (SA) enhances ScPR1 expression. Notably, the ScNPRs exhibited opposite functionalities compared to those in Arabidopsis , with ScNPR1 negatively and ScNPR3/4 positively activating ScPR1 expression upon SA treatment. Together, we established an efficient protoplast expression system using GT42 sugarcane culm, providing a valuable tool for studying sugarcane gene function under stress.