Decalepis salicifolia (Bedd. ex. Hook.f.) Venter is a potential natural source of the vanillin isomer, 2-hydroxy-4-methoxybenzaldehyde (2H4MB), an aromatic compound. However, the utilization of the plant is hindered especially due to its critically endangered status and the root-specific accumulation of the compound. The use of in vitro culture techniques offers a sustainable means for the production of valuable metabolites. In this study, an efficient system was established for the production of 2H4MB in the adventitious root cultures of D. salicifolia. Leaf explants of in vitro grown plants produced on an average 4.33 +/- 2.07 number of roots with root initiation frequency of 95.69 +/- 3.74% in woody plant medium supplemented with 0.5 mg/L alpha-naphthalene acetic acid (NAA) and 1.0 mg/L kinetin (Kn). The adventitious root biomass accumulation of 10.61 +/- 0.89 g fresh weight (FW) was obtained in woody plant liquid media containing 0.5 mg/L NAA and 0.3 mg/L indole-3-butyric acid (IBA) in 60 days of inoculation. Field-grown plants of the same age produced 0.30 +/- 0.02 g FW, which was 35-fold lower than the adventitious root culture. The total production of 2H4MB in the same growth period was 4.9-fold higher in adventitious root culture (139.54 mu g) as compared to field-grown plants (28.62 mu g). Furthermore, sucrose concentration of 2% was favorable for biomass accumulation, whereas 5% was favorable for 2H4MB production. On the other hand, media pH 5.0 was suitable for biomass production and pH 7.0 was best suited for accumulation of 2H4MB. The adventitious roots also showed stable production of biomass and 2H4MB over 2 years. The established adventitious root culture system is suitable for further large-scale production of 2H4MB for flavor and fragrance industrial applications.