Direct shoot regeneration and gene expression profiling for optimized picroside-I biosynthesis in Picrorhiza Kurroa

Picrorhiza kurroa, an endangered medicinal herb, is known for its bioactive iridoid glycosides, the Picrosides, which possess a range of pharmacological properties, including hepatoprotective, anti-inflammatory, and antioxidant activities. As demand for this valuable plant increases, in vitro tissue culture conservation efforts have become critical. We attempted in vitro tissue engineering-assisted direct shoot regeneration from leaf explants of P. kurroa using various growth hormone combinations, for enhanced metabolite production. MS media, admixed with 0.5 mg/L thidiazuron and 1.5 mg/L kinetin, bypassed the callogenesis phase while offering 83% shoot regeneration efficiency. High-performance liquid chromatography analyses with shoots elucidated a high picroside-I content of up to 9.7 µg/mg. Gene expression analysis via qRT-PCR revealed a marked increase in the expression levels of critical genes associated with the Picroside-I biosynthesis pathway-namely HMGR, PMK, DXPS, G10H, DAHPS, and PAL-when compared to shoots generated through the callus-mediated process. Notably, a marked increase in the expression of geraniol synthase, a key gene in the iridoid pathway, was directly correlated with enhanced Picroside-I levels. This in vitro tissue engineering strategy enhances Picroside-I biosynthesis and sets the stage for future biotechnological and pharmaceutical applications in the conservation, genetic improvement, and commercial exploitation of P. kurroa.