Protoplast isolation and transient expression in the precious and economically important tree Toona ciliata

Protoplasts are widely used in the fields of genetic transformation, physiology, and biochemistry, as they can easily absorb exogenous substances. The development and an efficient protoplast isolation and transient transformation system are essential for molecular biology and related research. Toona ciliata, valued for its high-quality and vividly colored wood, represents an economically significant species. In order to promote efficient breeding of the precious fast-growing tree, the establishment of a protoplast isolation and transient transformation system for T. ciliata is particularly important.

The native protoplast isolation system established in this study used 0.3 g of leaves of two-month-old T. ciliata seedlings as the separation material, with an enzyme solution composed of 15 g/L Cellulase R-10 + 15 g/L Macerozyme R-10 + 0.6 M mannitol +10 mM MES +1 mM CaCl₂ + 0.1 % BSA. Protoplasts were isolated in the dark at room temperature with gentle shaking (50 rpm) for 10 h, yielding (89.17 ± 7.21) × 10⁶ protoplasts per gram of fresh weight with a viability of 92.62 ± 0.75 % (n = 3). For transient transformation, the optimal conditions included 40 % PEG, a plasmid concentration of 30 μg/μL, and a 30 min incubation in the dark, resulting in a transformation efficiency of 29.02 ± 6.13 % (n = 3). This highly efficient native protoplast-based transient expression system was successfully applied to determine the subcellular localization of 1-deoxy-D-xylulose 5-phosphate synthase (DXS), a rate-limiting enzyme in the terpenoid biosynthesis pathway in T. ciliata.

The established protoplast isolation and transient expression system provides a foundation for the subsequent identification of gene function and mechanism research, and provides a reliable research platform for the molecular breeding of T. ciliata, supporting future genetic improvement efforts.