Optimization of an in vitro protocol for the production of ascorbic acid in Hibiscus cannabinus leaf-derived normal root cultures

Background: Hibiscus cannabinus (kenaf) is cultivated in many parts of the world for its bast fibers that find extensive industrial applications. In the southern states of India, kenaf is a popular leafy vegetable. The leaves are rich in ascorbic acid. Material and Methods: The present study reports an efficiently optimized protocol for in vitro normal root cultures for the biomass establishment of kenaf and the production of substantial amounts of ascorbic acid. The explants prepared from cotyledonary leaves of 15 day old in vitro seedlings of kenaf were inoculated onto Murashige and Skoog (MS) basal medium supplemented with 0.5-3 mg/L indole-3-acetic acid (IAA) or indole-3-butyric acid (IBA) or naphthalene acetic acid (NAA) individually for root initiation. Results: MS medium containing 2 mg/L NAA provided the best response (100% of explants) with a root length of 12-14 cm. A light greenish friable callus was also obtained in most of the explants. Efficient root biomass and ascorbic acid production was evident upon sub-culturing ~1 cm length root segments onto MS liquid medium containing the same growth regulators regimen used for the initiation of roots. Root cultures showed maximum biomass by the eighth week (5.51 g/100 g fresh weight). Ascorbic acid production was maximal in the seventh week (48.43 mg/100 g fresh weight). Conclusions: This protocol will facilitate scale up studies for obtaining higher yields of ascorbic acid in vitro using kenaf root cultures on a commercial scale; it could also be used to investigate the kinetics of the ascorbic acid biosynthetic pathway.