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 Betulin is a pentacyclic lupane-type triterpene, mainly obtained from Betula species plants with a variety of biological actions such as anti-HIV and anticancer properties. This study aimed to enhance the production of betulin in cultures of Betula litwinowii calli under the influence of concentrations and duration time of elicitors and precursors. Collected leaves from Sangdeh habitat in summer, were cultured in WPM medium containing 2,4-D and BAP hormones. Four month calli were sub-cultured in medium containing elicitors such as salicylic acid, chlorocholine chloride and cobalt chloride and precursors such as sucrose, vitamin and glucose. . Calli were harvested from new culture media after two, three and four weeks and their wet and dry weights were calculated. Data analysis was performed based on two-factor factorial experiment (the first factor, elicitors and precursors each in four levels and the second factor, time with three levels) in a completely randomized design with three replications with SPSS software. Duncan's multiple range test were performed to compare the mean (p≤0.01). The results showed that two week elicitation with chlorocholine chloride (1.5 mg/l for one week) was the best with a more than 3-fold increasing in betulin induction compared to the control treatment (0.068 mg / g). The use of vitamin precursor (ten-fold the normal amount in WPM culture medium) for three weeks, causes the highest increase in betulin induction compared to the control (0.1 mg / g, respectively). In general, in the present study, this treatment is introduced as the best. 

Keywords: betulin, birch, chloro-choline chloride, vitamins

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