Comparative Elicitation of Phenolic Accumulation in Salvia officinalis Hairy-Root Cultures Using Salicylic Acid, Silver Nitrate, LED Spectra, and Microbial Preparations

Salvia officinalis L. is a pharmaceutically important species rich in rosmarinic acid, yet strategies to enhance phenolic accumulation in controlled systems remain limited. This study compared three elicitor categories on polyphenol biosynthesis in Agrobacterium rhizogenes-transformed hairy root cultures: chemical (salicylic acid, AgNO3), physical-Light-emitting diode (LED light spectra: white, blue, red, green, darkness), and biological (Bacillus velezensis BPVs2cell lysate and cell-free supernatant, Trichoderma asperellum TdCP biomass lysate). Within the chemical-elicitation series, AgNO3 produced the strongest response, increasing total phenolic content by 39.2% after 7 days compared with the untreated control and inducing an approximately three-fold increase in rosmarinic acid, together with elevated levels of caffeic, syringic, and chlorogenic acids. Salicylic acid exhibited an inhibitory effect, decreasing total phenolic content by 50%. Exposure to blue and red LED light resulted in a two-fold increase in total phenolics compared with white light and darkness, while green light uniquely stimulated the exudation of phenolics into the culture medium. Among biological elicitors, B. velezensis cell-free supernatant (1.5 mL) triggered a 10-fold increase in rosmarinic acid at day 7, whereas T. asperellum produced a transient, non-specific response without targeted rosmarinic acid enhancement. These results demonstrate that elicitor type, dose, and exposure time critically determine phenolic accumulation specificity. AgNO3, blue and red LED illumination, and B. velezensis cell-free supernatant were identified as promising individual treatments in their respective experimental series; however, combined-treatment effects and possible synergism require confirmation in a factorial experiment.