Protoplasts Isolation and Transient Transformation System Optimization for Poplar 84K (Populus alba × Populus...

In poplar, the protracted stable genetic transformation procedure constrains rapid gene functional analyses. To address this limitation, we optimized a protocol for the high-yield isolation and efficient transient transformation of protoplasts from leaves of tissue-cultured poplar 84K (Populus alba × Populus glandulosa). Through systematic refinement, we determined that an enzymatic digestion solution containing 3% cellulase R-10, 0.3% macerozyme R-10, 0.8% pectolyase R-10, and 0.4 M mannitol was optimal. This formulation, applied over a 3 h digestion period, yielded 12.9 × 10⁶ protoplasts per gram fresh weight, with 93.45% viability. Furthermore, we optimized the parameters for polyethylene glycol -mediated transformation. Using 60 µg of plasmid DNA, 40% polyethylene glycol 4000, and a 20 min incubation, we achieved a high transfection efficiency of 68.67%. The established transient expression system thus provides a reliable, rapid, and effective platform for functional characterization-related studies, such as subcellular localization, protein-protein interactions, and gene expression analyses in poplar, thereby supporting molecular breeding applications.