Deciphering molecular signaling and metabolite dynamics during in vitro clonal propagation in Ferula assa-foetida
摘要
Ferula assa-foetida L. is a perennial herbaceous plant valued for its oleo-gum resin (asafoetida). Native to Iran and Afghanistan, this plant faces global cultivation challenges due to limited access to high-quality planting material. To address this constraint, a reproducible micropropagation protocol was developed through indirect somatic embryogenesis. Callus was induced from leaf explants using Murashige and Skoog medium supplemented with 1.0 mg L−1 naphthaleneacetic acid (NAA) in combination with 1.5 mg L−1 kinetin (Kn) or 1.0 mg L⁻1 benzylaminopurine (BAP). Somatic embryos developed on callus were cultured with 2.0 mg L⁻1 Kn and 0.05 to 0.1 mg L⁻1 IBA, with various stages of somatic embryo development visualized through scanning electron microscopy (SEM). Shoot proliferation was optimized using 2.0 mg L⁻1 BAP and 0.2 mg L⁻1 NAA, while rooting was effective with 0.05 mg L⁻1 IBA. Acclimatization resulted in 100% survival in sand:vermiculite:soil (1:1:1) and sand:FYM:soil:cocopeat (1:1:1:1). The genetic fidelity of regenerated plantlets was verified using RAPD, ISSR, and SCoT markers, as well as flow cytometry. Differential expression of SERK2, WUS4, CUC3, HMGR1, PAL1, and TPS12 genes highlighted key molecular regulators of organogenesis. Comprehensive metabolic profiling of roots at three stages—in vitro roots, 20-d-old, hardened plants, and 45-d-old, acclimatized plants—using UHPLC-DAD-QTOF-MS led to the identification of 57 metabolites, including coumaric acid, ferulic acid, umbelliferone, and key amino acids. These findings provided a reliable method for clonal propagation and deepened the understanding of cellular reprogramming, aiding future efforts in functional genomics, gene editing, and the improvement of F. assa-foetida.