Development of Medium and Bioreactor System for the Cultivation of Hairy Roots of Datura Metel and Scopolamine Production

The effects of phytohormones (auxin and cytokinin), sucrose and the ratios of ammonium to nitrate upon root branching and elongation of Datura metel hairy roots were studied on modified B5O medium. Addition of phytohormones in the medium is fatal to Datura metel hairy root cultures. However, root branching and root elongation were enhanced with addition of nitrate at a concentration of 9mM-36mM without any ammonium in the medium. The effects of ammonium, nitrate, phosphate and l-ascorbic acid on the growth of Datura metel hairy root cultures were investigated under three different light conditions. The combination effects of ammonium and media strength at half and full strength of B5O media on the growth of D. metel hairy root cultures have also been studied. The root growth and morphology were significantly (p=0.05) influenced by the light conditions. Light-grown cultures have ticker roots and less branching, thus had slower growth than dark-grown cultures. Root cultures grown in half strength media had a lower growth rate as compared to root cultures grown in full strength media at all concentrations of ammonium. High concentration of ammonium had an inhibitory effect on the roots. However, root cultures grown in full strength media received less impact from ammonium toxicity suggested that the presence of certain chemical at higher initial concentration suppressed the toxicity of ammonium on the root tissue. The effect of nitrate on the growth of root cultures was not significantly (p=0.05) influenced by light conditions. On the contrary, the effect of phosphate was greatly influenced by light conditions. The root growth was enhanced remarkably, up to 12.4 g/L at 4.4 mM of initial phosphate concentration in the presence of light. The induction of lateral roots was also enhanced dramatically at the concentration of initial phosphate higher than 4.4 mM. This suggested that the branching capability of the hairy root cultures could be improved by supplying phosphate in media in abundant. Scopolamine level in hairy root cultures was increased dramatically to 1.6 mg/g cell dry weight at 4.4 mM of initial phosphate concentration in the dark. The increment of scopolamine level in the root cultures may relate to the root branching. A 1 liter aerated tank without agitation comprised of a polycarbonate cylinder with dimensions of 10 cm in internal diameter and 16.5 cm in height, containing a stainless steel mesh with a mesh size of 2 mm was designed for the cultivation of hairy roots of Datura metel. The effect of hydrodynamic stress that was caused by air flow to the root culture was determined by conductometry. The result of this study shows that root cultures were easily injured by the air flow rate higher than 1.7 vvm. After 16 days of incubation, the root culture filled up almost 95% of the tank space, which gave the final biomass of 175 g fresh weight per liter (8.9 g dry weight per liter), 1.75 mg/g cell dry weight of scopolamine and 0.80 mg/L cell dry weight of hyoscyamine. The productivity of scopolamine in D. metel root cultures grown in the bioreactor is comparable to those cultures in the shake flask.

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