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Bauhinia forficata Link. is a native South American plant, which possesses volatile compounds with pharmaceutical and medicinal properties such as antidiabetic and anti-inflammatory effects. However, the conservation and propagation of this plant are complicated by its recalcitrant seeds and delayed flowering transition. Hence, tissue culture is employed for the safe and efficient propagation of B. forficata. However, the optimal conditions for the in vitro cultivation of B. forficata remain unknown. Thus, this study aimed to characterize the volatile profile of adult B. forficata field plants and evaluate the effects of different light intensities (43 and 70 μmol m−2 s−1), gas exchange rates (14 and 25 µL L−1 s−1), and exogenous sucrose concentrations (0, 20, and 30 g L−1) on their in vitro development. The results showed that β-caryophyllene is the major volatile compound produced by B. forficata. Moreover, culturing in a medium containing 30 g L−1 of sucrose and flasks with membranes that allow CO2 exchange at the rate of 25 µL L−1 s−1 produced vigorous and hardened plants with high survival rates independent of irradiance. This study is the first to report the optimal in vitro culture conditions for B. forficata as a reference for future studies on micropropagation and secondary metabolite production using this species. [ABSTRACT FROM AUTHOR]