Establishment of a system for tissue culture regeneration and isolation of Camellia yubsienensis, and PEG-mediated transient expression of mesophyll protoplasts

Camellia yubsienensis has several distinctive characteristics, including thin fruiting shells, high seed yield, and a notable degree of resilience to adverse conditions. In the present study, C. yubsienensis stem segments with buds were used as explants. Single-factor experiments and orthogonal experimental designs were employed to identify the optimal conditions for establishing a tissue culture regeneration system for C. yubsienensis and optimal conditions for the isolation and PEG-mediated transient expression of its mesophyll protoplasts. The results showed that the optimal disinfection method involved 75 % C₂H₅OH for 60 s, followed by 0.1 % HgCl for 20 min, resulting in a low contamination rate of 9.3 %. The best culture medium for bud induction of stem segments was WPM supplemented with 2 mg/L 6-BA, 1.5 mg/L IBA, 1 mg/L GA₃, 7 g/L agar, and 30 g/L sucrose, which achieved a high induction rate of 88.73 %. The optimal sampling time was April 20th, with a high induction rate of 85.56 %. The most favorable genotype was YZ₂, which exhibited faster bud formation and robust growth, with an induction rate of 87.69 %. The best proliferation culture medium consisted of WPM supplemented with 4 mg/L 6-BA, 0.1 mg/L IAA, 2 mg/L GA₃, 7 g/L agar, and 30 g/L sucrose, resulting in a proliferation coefficient of approximately 3.56. The preferred rooting method involved dipping the stem segments in 10 mg/mL IBA for 10 s and 1 mg/mL salicylic acid for 10 s, followed by planting in the optimal culture medium: 1/2 MS supplemented with 0.01 mM GABA, 5 g/L agar, and 30 g/L sucrose, achieving a rooting rate of 83 % after approximately 20 days. The optimum conditions for mesophyll protoplast isolation were 2.5 % Cellulase R-10 + 1.5 % Macerozyme R-10 + 0.45 % snailase with 8 h of enzymatic digestion at an osmotic pressure of 0.4 M, and the production and viability of protoplasts were 9.95×10⁶/g·FW and 93.52 %. The maximum transfection efficiency (68.1 %) was obtained with the incubation of the protoplasts with 25 µg plasmid and 40 % PEG4000 for 30 min. This study established a regeneration system for C. yubsienensis and explored the optimal conditions for the isolation and PEG-mediated transient expression of mesophyll protoplasts, laying the foundation for factory-scale breeding of superior varieties of C. yubsienensis. It also provides technical support for research on somatic cell hybridization and the establishment of genetic transformation systems for C. yubsienensis.