Production of β-β,Dimethylacrylshikonin in Callus Cultures of Onosma echioides Var hispidum Clarke

Callus tissues derived from leaf segments of Onosma echioides var hispidum on three basal media viz. Murashige & Skoog (MS), Gamborg et al (B₅) and White’s containing 3% sucrose produce napthaquinone pigments in presence of and NAA. However, β-β,dimethylacrylshikonin synthesis was triggered in dark in undifferentiated parenchyma cells on B₅ agar medium containing 1 x10⁻⁵ M Kn and 2×10⁻⁶ M IBA when proliferated calli after 16 weeks (4ᵗʰ generation) were transferred to it and incubated at 23 ± 1 °C. The pigment biosynthesis increased linearly from 4ᵗʰ to 6ᵗʰ week after a lag of first 3 weeks. Callus grew exponentially after a lag of 2 weeks and diminished from 6ᵗʰ week onward. During 8 weeks of growth, callus grew from 0.8 to 8.2 g and the β-β, dimethylacrylshikonin showed the highest level of 25.41 μg g⁻¹ of fresh tissue. Light microscopic examination of semi-thin sections of pigmented tissue revealed pigment accumlation between the plasma membrane and cell wall and also in the intercellular spaces. Exposure of cultures to white fluorescent light for more than 2 h resulted in complete repression of pigment bosynthesis. The investigations suggest that the regulatory mechanism for the biosynthesis and accumulation of napthaquinone pigment(s) may be similar to that of Lithospermum erythrorhizon. Pigment producing capability of callus cultures of O. echioides var hispidum can be exploited as an alternative raw source for the production of shikonin derivatives.