Diospyros anisandra is a small tree that contains metabolites with antitubercular, antibacterial, larvicidal, antioxidant, antihistaminic, and antiviral properties in its leaves, stem bark, and roots. However, this plant is slowly growing and takes about 10 years to reach maturity in situ. Therefore, it makes it an ideal model to apply tissue cultures and thus be able to have the metabolites of interest as required. In the present work we report for the first time, the establishment and comparison of three different protocols of in vitro culture of D. anisandra. This work aimed to establish stable in vitro cultivation conditions with seeds, leaves and nodal segments. They were cultured in Murashige and Skoog (MS) medium supplemented with plant growth regulators such as Gibberellic acid (GA3), zeatin, 6-benzylaminopurine (BAP), and 2,4-dichlorophenoxyacetic acid (2,4-D). The results showed that a germination efficiency of 73% was achieved in seeds with 57.8 µM GA3. In leaf regeneration, 80% regeneration of embryonic mass was achieved with 28.9 µM of BAP and zeatin. Finally, in nodal segments, an 85% efficiency in axillary shoot regeneration was achieved with 28.9 µM zeatin. On the other hand, the hexane extract of the plant segments of all treatments was analyzed by thin layer chromatography and gas chromatography-mass spectrometry (GC/MS). The main compounds detected are plumbagin, stigmasterol, β-sitosterol, taraxasterol, vitamin E, betulinic acid among others in the three systems studied, therefore, we can conclude that success was obtained in generating a cultivation method that allows us to have these compounds in less than 1 year of growth.
