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Picrorhiza kurroa possesses diverse pharmacological activities due to secondary metabolites, particularly Picroside-I and Picroside-II. Traditional cultivation methods, including tissue culture and open-field farming, face challenges such as high costs, environmental constraints, and long cultivation cycles. Hence, in vitro raised, 3 and 6 month hydroponically cultivated P. kurroa plants were assessed for morpho-physiological traits, secondary metabolites accumulation, and gene expression.

The results showed that 6 month plants achieved maximum morphological growth, including shoot length (17.4 cm), leaf number (18.1), and rootlet length (14.4 cm). While 3 month hydroponically cultivated plants had the highest leaf biomass (1.13 gm FW and 0.11 gm DW), 6 month hydroponically cultivated plants exhibited the greatest root biomass (0.42 gm FW; 0.03 gm DW). Chlorophyll fluorescence parameters and total photosynthetic pigments were higher in 6 month plants. Phytohormone analysis showed higher levels of ABA, GA3, and IAA in both leaf and root tissues, and JA in root tissues. UPLC quantification revealed that PI (1.08%) was found in 3 month hydroponic leaf tissue, whereas PII (2.16%) accumulated in 6 month hydroponic root tissue, suggesting enhanced metabolite production over time. Comparative gene expression profiling demonstrated a positive correlation between pathway genes and metabolite levels. Overall, hydroponic cultivation of P.

kurroa improved morphology, growth, and secondary metabolite production, making it a promising approach for commercial herbal formulations.

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