To determine the effect of coconut extract on callogenesis of Betula pendula, Roth stem barks were cultured in NT (Nagata and Takebe) basic culture media in two individual experiments: i) cultivation explant in different treatments of coconut extracts combined with 1 mg l−1 2, 4-D (2, 4-Dichlorophenoxyacetic acid) and ii) callogenesis in NT media containing 1.5 mg l−1 2,4-D and 0.5 mg l−1 BAP (6-Benzylaminopurine) and then cultivation under the first experiment treatments. The first experiment demonstrated that not all concentrations of coconut extracts lead to callus induction individually, but callus induction increased 84% in a culture containing 5% coconut extract plus 1 mg l−1 2, 4-D. Based on the results of the second experiment, this treatment also significantly increased the wet and dry weights of the produced calluses. The possibility of increasing the betulinic acid and betulin by ultrasound was also studied. Samples cultivated in the selected culture medium were exposed to ultrasound waves in two forms of 1) one exposure and 2) twice exposure (repetition with 24 hr interval) in steps of 20, 60, 100, and 160 sec, and one treatment as the control. Betulin and the betulinic acid amount were determined using HPLC (High-Performance Liquid Chromatography). The maximum betulinic acid and betulin were obtained in 100 sec in once exposure (2.3 mg g−1 DW) and 160 sec in the twice exposure (0.75 mg g−1 DW) to ultrasound. The results showed that betulinic acid content was more than betulin.
