Optimizing disinfection and adventitious shoot propagation in Cyclocarya paliurus stem tissue culture: enzymatic and flavonoid metabolomics insights

Cyclocarya paliurus (Batal.) Iljinskaja is a medicinal and endangered woody plant, but shoot culture is challenged by higher contamination and lower propagation efficiency. This study evaluated the effects of various sterilization and propagation treatments on shoot culture, and further analyzed enzymatic activity and flavonoid metabolomics at 20, 40, 60, and 80 days of culture using UPLC-MS. The optimal disinfection method-cold storage at 4 °C for 8 days, followed by immersion in warm water at 37 °C, and sterilization with 0.1% mercuric chloride for 5 min-resulted in a contamination rate of 12.72%. The highest propagation coefficient (5.91-fold) was achieved on ½ MS medium supplemented with 0.5 mg/L 6-BA, 0.01 mg/L TDZ, 0.3 mg/L GA₃ and 0.02 mg/L IBA at 60 days. Enzymatic activity followed the order: SOD > POD > PPO > PAL. A total of 151 flavonoid-related metabolites were identified and classified into 11 categories, including flavonols (28.48%), flavones (18.54%), flavanones (10.60%), benzoic acid and its derivatives (6.62%), flavanols (5.96%), flavanonols (5.30%), isoflavanones (4.64%) and others. Metabolite content varied with culture duration, with catechin (a flavanol) showing the highest content (> 1000 µg/g) during shoot culture. Pretreatment of “lower-temperature storage and warm-water immersion” efficiently reduced stem contamination, and significant associations existed between enzymatic activity, flavonoid profiles, and the propagation of adventitious shoot. These findings offer a promising strategy to improve shoot propagation, in vitro conservation and utilization in C. species.