In vitro propagation from seeds and enhanced synthesis of podophyllotoxin from root callus of SinoPodophyllum hexandrum Royle T.S. Ying (Himalayan Mayapple) − An endangered medicinal plant

In vitro propagation and secondary metabolite production are the vital biotechnological approaches for the multiplication, conservation, and phytochemicals production from rare, endangered, and industrially important medicinal plants. Himalayan Mayapple (SinoPodophyllum hexandrum Royle T.S. Ying) is a perennial venerate herb of the alpine Himalayas, rhizomes of which yield podophyllotoxin (PTOX), widely used in the pharmaceutical industry for the synthesis of anti-cancer drugs. The low regeneration rate and high demand for this plant emphasized the need for the development of a non-conventional method for its propagation and PTOX production using biotechnological interventions. In the present study, a single-step protocol for in vitro propagation of S. hexandrum through seeds and PTOX production from seedlings-induced leaf and root calli of three geographical locations in Himachal Pradesh (India) is reported. Maximum in vitro seed germination (56.23%) was obtained after 77 days followed by the highest shoot multiplication on MS medium containing BA (2.0 mg/L), NAA (0.2 mg/L), and calcium chloride (0.7 mg/L) after the fourth sub-culture. Rooting was induced at the base of shoots during in vitro shoot multiplication hence, the shoots were directly transferred for hardening without adopting additional rooting passage. For PTOX production, callus was induced from in vitro leaves and roots on B5 medium supplemented with BA (0.1 mg/L) + NAA (1.0 mg/L) and BA (0.5 mg/L) + 2,4-D (0.5 mg/L) under dark incubation. High-performance liquid chromatography (HPLC) analysis revealed maximum PTOX content (0.27 mg/g) from in vitro root callus of district Kinnaur followed by Lahaul-Spiti (0.26 mg/g), and Kullu (0.25 mg/g), thus signifying the role of high altitudes in enhanced PTOX accumulation. The current study offers an insight into the high-frequency regeneration and PTOX production of S. hexandrum, which may serve as significant baseline information for the production of high-quality plant material in the pharmaceutical industry.