Large-scale production of in vitro plantlets of sweet flag (Acorus calamus L.) and quantitative analysis of α- and β-asarone contents using high pressure liquid chromatography

Acorus calamus is a perennial; an endangered and potential medicinal plant that belongs to the Acoraceae family has a range of medicinal properties and fragrant oils, which are being used in various industries. In the current investigation, the highly proficient and reproducible method for comprehensive plantlet regeneration was established and compared the α- and β-asarone contents in between the in vivo as well as in vitro raised plantlets. Amongst the combinations of plant growth regulators (PGRs), maximum shoots per explant (7.23) were observed with KIN. Later, the shoots were subcultured on MS medium supplemented with BAP, IAA and GA₃ showed a maximum percentage of responses in shoot multiplication and elongation. The elongated shoots were subcultured on MS medium fortified with IBA for root initiation and development, and successively the plantlets were acclimatized in plastic pots in the culture room and greenhouse. Quantitative analysis of α- and β-asarone was performed using HPLC from in vitro and in vivo raised plantlets. A significant difference was noted in α-asarone content of in vitro plantlets than in vivo plantlets whereas the least difference was noted in β-asarone content. The results of the present study revealed the potential reproducible protocol for largescale multiplication of A. calamus in 130 days. Further, the regeneration protocol will be a very useful platform for in vitro breeding with different PGRs to analyze the impact on improvement of bioactive molecules such as α- and β-asarone content and fragrant oils, which have huge demand in pharmaceutical and cosmetic trades and owing to their high industrial values.