Metabolite and expression profiling of steroidal alkaloids in wild tissues compared to bulb derived in vitro cultures of Fritillaria roylei – High value critically endangered Himalayan medicinal herb

Bulbous Fritillaria roylei (known as Jangli lahsan) is an important critically endangered medicinal herb of Astavarga group and widely used in traditional medicinal system. Being a medicinal potential but endangered herb, biotechnological interventions are urgently needed for its restoration and production of bioactive compounds. In the present study, in vitro cultures were established from bulb scales as explant by using optimized sterilization protocol (79.67% survival). From different combinations and concentrations of plant growth regulator tested; high efficiency callus induction (88.89%) and in vitro plantlets regeneration (77.78%) response was obtained in Murashige and Skoog medium supplemented with 0.5 mg/L thidiazuron +2.0 mg/L picloram and 1.0 mg/L kinetin +0.5 mg/L naphthaleneacetic acid, respectively. For the quantification of steroidal alkaloids i.e. sipeimine and peimine; selective, sensitive, and rapid liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) analytical method was developed and validated. Quantification of sipeimine and peimine was done for the first time in established in vitro cultures and naturally grown tissues of F. roylei i.e. bulbs, leaves, stem and flower bud. Among in vitro raised cultures, higher sipeimine content (1.98 μg/g) was detected in callus. Whereas, maximum sipeimine content (599.72 μg/g) was observed in floral bud compared to other wild tissues. Peimine content was detected in bulb (1.13 μg/g), leaf (0.78 μg/g) and stem (0.67 μg/g). Qualitative and quantitative phytochemical screening revealed potentiality of this important medicinal herb for commercial utilization in drug formulation to the industries. Maximum antioxidant activity (IC₅₀) was exhibited in floral buds (0.11 mg/mL), while among in vitro raised cultures; regenerated plantlets (0.80 mg/mL) shown higher scavenging activity than callus (2.71 mg/mL). Higher total phenol content (42.86 μg gallic acid/mg) was observed in bulb whereas, flavonoids content is higher in leaves (68.36 μg quercetin/mg) followed by callus (58.22 μg quercetin /mg). Putative steroidal alkaloids gene expression studies using RT-qPCR showed positive correlation with metabolite content. For the first time, present investigation concluded that floral buds, leaves and stem could also be used as an alternative potential source for steroidal alkaloids and callus culture as sustainable approach for metabolite production to meet industrial standards and demands.