High-Efficiency Regeneration and Transformation System in Nardostachys jatamansi for Functional Genomics and Bioactive Compounds Enrichment

Abstract

Micropropagation is key for the sustainable conservation of critically endangered plants and often enhances metabolite production. The biochemical fidelity of these regenerated plants is assessed through metabolite profiling. Here, we developed an efficient in vitro regeneration protocol for Nardostachys jatamansi, achieving the highest callus induction rate (93.33% ± 4.08%) using 1.5 mg/L TDZ and 1 mg/L NAA. Combining 1.5 mg/L TDZ and 0.1 mg/L NAA yielded a regeneration efficiency of 68% ± 4.89%, with the highest shoot number (6.4 ± 0.50) under conditions of 15°C ± 2°C. GC-MS analysis revealed elevated primary and secondary metabolites in the regenerated leaves compared to those in the mother plant. In addition, an Agrobacterium tumefaciens-mediated transformation protocol was optimised for subcellular localisation, transient gene expression and virus-induced gene silencing. Results revealed, in comparision to EHA105 (30% ± 5.77%), GV3101 (43.33% ± 3.33%) showed higher transformation efficiency under similar conditions. The highest transformation efficiency for transient expression was achieved with an OD₆₀₀ = 0.6, a 4-h infection, and a 3-day co-cultivation, confirmed through RT-PCR and western blotting. Virus-induced gene silencing using pTRV2:NjPDS effectively silenced the NjPDS gene. This integrated platform, combining micropropagation, transformation and metabolite profiling, provides a scalable tool for enhancing bioactive compounds and genetics in N. jatamansi.