Objective  The effects of temperature stress on the accumulation of triterpenoids, flavonoids and defense enzymes in multiple shoots of birch (Betula platyphylla) were investigated so as to lay the foundation for increasing the accumulation and factory production of secondary metabolites in birch.

  Method  Different concentrations (0.5, 1.0, 2.0 and 5.0 mg/L) of 6-benzylaminoadenine (6-BA) were used to induce multiple shoots and establish suspension culture system of birch. Furthermore, multiple shoots were treated by temperature stress so that the synthesis of secondary metabolites in birch was induced.

  Result  The callus of birch could be induced to obtain the multiple shoots by NT medium supplemented with 2.0 mg/L 6-BA. The content of total triterpenoids in birch multiple shoots under temperature stress (45 and 4 ℃) was higher than that of the control (25 ℃), among them the total triterpenoids content increased by 5.03 times after 4 h and then restoration culture for 6 h. Also the content of oleanolic acid in birch multiple shoots treated by 4 ℃ for 1 h and sampled 24 h later was increased by 14.52 times, and even for 2.33 mg/g. And the content of flavonoids was enhanced by 38.37% under 4 ℃ for 4 h and then 25 ℃ for 48 h. Meanwhile, the superoxide dismutase (SOD) activity was highest under 4 ℃ for 4 h and normal culture for 72 h, which was 69.77% higher than that of control, and the catalase (CAT) activity of birch multiple shoots treated by 4 ℃ for 1 h and normal culture for 96 h reached the peak, which was increased to 1.81 times. Then the ascorbate peroxidase (APX) activity of multiple shoots treated at recovery culture for 6 h under 4 ℃ for 1 h was 55.29% higher than that of control. While treated by 45 ℃ for 4 h, the content of flavonoids and oleanolic acid was significantly negatively correlated (P < 0.05) with APX enzyme and SOD activity, respectively. Furthermore, the content of total triterpenes was extremely significantly positively correlated or significantly positively correlated with APX activity (P < 0.01) and CAT activity (P < 0.05), and the content of flavonoids was significantly negatively correlated (P < 0.05) with SOD activity, also CAT activity was significantly correlated with APX activity (P < 0.01) and SOD activity (P < 0.05). These results showed that the three anti-stress enzymes played their collaboration role in the synthesis of secondary products.

  Conclusion  Multiple shoots of birch were successfully induced by 2.0 mg/L 6-BA, and suspension culture system was established. Short time temperature stress could stimulate the significant changes of three defense enzymes (SOD, CAT and APX), which coordinated with each other and promoted the synthesis and accumulation of total triterpenoids and flavonoids in multiple shoots of birch.