Optimization of Callus Induction and Cell Suspension Culture of Betula pendula Roth for Improved Production of Betulin, Betulinic Acid, and Antioxidant Activity

This study aimed to establish an in vitro protocol for callus induction and cell suspension cultures of silver birch (Betula pendula Roth) and to assess the antioxidant activity of cell extract and the accumulation of betulin (B) and betulinic acid (BA). Callus was induced from the inner bark of stems on Gamborg (B5) and Nagata-Takebe (NT) media, and 23 different concentrations of growth regulators were tested. NT medium with 2.5 mg L-1 2,4-dichlorophenoxyacetic acid and 0.5 mg L-1 6-benzylaminopurine was optimal for callus induction. Cell suspension cultures were developed to improve cell growth and metabolite production under different concentrations of NT vitamins. Methanolic cell extracts indicated that the BA content of callus increased from 1 to 9 mo, with a maximum 2.01 mg g(-1) (dry weight [DW]) at 9 mo. The highest level of B observed in cell suspension cultures (0.96 mg g(-1) DW) was obtained on medium supplemented with 4x NT vitamins. The highest antioxidant activities of the 1,1-diphenyl-2-picrylhydrazyl free radical, 77 and 76%, were found in callus extract at 9 mo and cell extract in the 4x NT vitamin treatment, respectively. In conclusion, the use of NT medium supplemented with 4x NT vitamins effectively increased cell growth and the accumulation of B, BA, and antioxidant activity in cell suspension cultures of B. pendula.