Establishment of Transient and Stable Gene Transformation Systems in Medicinal Woody Plant Acanthopanax senticosus

Abstract

Background: Transient and stable gene transformation systems play a crucial role in elucidating gene functions and driving genetic improvement in plants. However, their application in medicinal woody plants has been hampered by inefficient procedures for isolating protoplasts and regenerating plant in vitro. Results: Embryogenic callus protoplast isolation and transient transformation system were successfully established, the highest yield of protoplasts was achieved at approximately 1.88×10⁶ cells per gram with a viability of 90% under the combination of 1.5% cellulase and 0.2% lysozyme, with enzymatic digestion for 6 h in darkness followed by centrifugation at 400×g for 5 min. The transient transfection rate of protoplast reached 45.56% at a PEG 4000 concentration of 40%, a transfection time of 40 min, 16 h of dark incubation, a plasmid concentration of 1.5 ng·μL^-1, and a heat shock of 25 min at 45°C. Additionally, 15 Agrobacterium tumefaciens-mediated GUS-positive seedlings were obtained through the somatic embryogenetic pathway under the optimized conditions. Conclusion: This study successfully established both transient and stable genetic transformation systems, paving the way for future molecular biology research in A. senticosus.