Suspension Culture Optimization and Transcriptome-Guided Identification of Candidate Regulators for Militarine Biosynthesis in Bletilla striata

Background: Bletilla striata is a medicinal orchid, whose bioactive constituent militarine has therapeutic interest but limited natural availability. Suspension culture coupled with transcriptomics offers a scalable production route and a means to uncover biosynthetic regulators. Methods: Four B. striata landraces were evaluated. Single-factor experiments and response surface methodology optimized sucrose, NH4NO3, and agitation to maximize biomass and militarine yield. Militarine and four related metabolites were quantified by HPLC-UV. For transcriptomics, RNA from high- and low-producing landraces was sequenced on Illumina HiSeq, assembled de novo, and analyzed with RSEM (FPKM) and DESeq2 to identify DEGs. Results: The landrace SMPF-NL achieved the highest militarine yield (33.06 mg/g) under optimized conditions (sucrose, 35 g/L; NH4NO3, 625 mg/L; agitation, 135 rpm; and half-strength MS medium with 1.0 mg/L of 6-BA, 3.0 mg/L of 2,4-D, and 0.5 mg/L of NAA). Transcriptomic profiling highlighted candidate biosynthetic and regulatory genes, including SuSy2, SUS, ALDO, AOC3, Comt, GOT2, MAOB, BGLU20, and BGLU22. Conclusions: We present an optimized suspension culture system and transcriptomic leads that lay the groundwork for the functional validation and scale-up of controlled militarine production.