Effects of explant type on callogenesis and the amount of betulin and betulinic acid produced under in vitro conditions in two birch species (Betula spp.)

Betulin and betulinic acid are from the most important anticancer and anti-HIV metabolites, and the birch species (Betula spp.) bark is considered as the primary source of these metabolites. Due to the extinction of these tree species in Iran, it is necessary to replace the metabolites extraction from the birch bark with modern methods such as cell and tissue culture to produce the metabolites. The aim of this study was to determine the effect of explant type on the amount of betulin and betulinic acid produced in calli of two birch species B. pendula and B. litwinowii under in vitro conditions compared to the amount of metabolites extracted from the tree bark. Bark and leaf explants of two mentioned species were cultured in WPM medium containing 1 mg/L BAP and 0.1 mg/L 2,4-D for callogenesis. The amount of betulin and betulinic acid in three-month calli was measured using the HPLC technique and compared with the amount of these metabolites in one-centimeter stem bark samples taken from nature. The bark explant was more successful in callogenesis, and calli derived from this explant had more active ingredients. The amount of betulin and betulinic acid from the extract of bark sample taken from nature was respectively obtained to be 5.23 and 2.91 percent for B. pendula, and 5.65 and 2.52 percent for B. litwinowii. Moreover, calli derived from the bark explant of B. pendula and B. litwinowii contained 0.023 and 0.016 percent of betulin and 0.053 and 0.057 percent of betulinic acid, respectively. Generally, the results indicated that the bark explant was more capable of callogenesis and secondary metabolite induction than the leaf explant in both birch species under in vitro conditions.