Induction of Embryogenic Callus, Protoplast Isolation, and PEG-Mediated Transformation Protocols in Eucommia ulmoides

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Abstract
Eucommia ulmoides, a tree species native to China, holds considerable medicinal, ecological, and industrial importance. However, the absence of an efficient and stable genetic transformation system poses significant challenges to gene function studies and molecular breeding in E. ulmoides. Protoplasts, which lack cell walls, serve as effective receptors for transient transformation and are thus ideal for genetic engineering research. In this study, we identified the optimal conditions for callus induction using a full-factorial experimental design and accurately identified embryogenic callus through paraffin sectioning. Furthermore, we developed an efficient protoplast isolation and PEG-mediated transient transformation system using embryogenic callus as the starting material. Our findings revealed that the optimal medium for inducing embryogenic callus was B5, supplemented with 1.5 mg/L 6-BA, 0.5 mg/L NAA, 30 g/L sucrose, and 7 g/L agar. This medium achieved a callus induction rate of 97.50% and an embryogenic callus induction rate of 86.30%. For protoplast isolation, the best conditions involved enzymatic digestion with 1.5% cellulase R-10 and 1.0% macerozyme R-10 at an osmotic pressure of 0.6 M for 4 h, resulting in 1.82×10⁶ protoplasts/g FW with 91.13% viability. The highest transfection efficiency (53.23%) was attained when protoplasts were cultured with 10 µg of plasmid and 40% PEG4000 for 20 min. This study successfully established a stable and efficient system for protoplast isolation and transient transformation in E. ulmoides, offering technical support for exploring somatic hybridization and transient gene expression in this species.

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