Morphological and Metabolic Changes During Callus-Based Shoot Regeneration in Chamaecyparis obtusa

Chamaecyparis obtusa is a valuable conifer species, prized for its high-quality timber and bioactive essential oils. However, commercial micropropagation is challenging due to its resistance to vegetative propagation. This study aimed to develop an efficient in vitro plant regeneration system using leaf explants and to profile the metabolic changes during organogenesis. Leaf explants from one-year-old in vitro-grown plantlets were cultured on media with various plant growth regulators (PGRs) to optimize callus induction, shoot multiplication, and rooting. Secondary metabolites were systematically analyzed throughout the developmental stages: explant, callus, regenerated shoot, and rooted plantlet. The most effective shoot regeneration, leading to whole plantlets, was achieved on a medium supplemented with 1.0 mg/L 2,4-dichlorophenoxyacetic acid and 2.0 mg/L thidiazuron. Metabolic profiling revealed significant stage-specific biochemical transitions. High-Performance Liquid Chromatography (HPLC) precisely quantified individual phenolics, resolving cross-reactivity issues seen in total flavonoid colorimetric assays. Gas Chromatography-Mass Spectrometry (GC-MS) also identified substantial shifts in volatile terpenoid biosynthesis during shoot morphogenesis. This integrated protocol provides a reliable platform for mass propagation of C. obtusa and offers fundamental insights into the metabolic dynamics of in vitro development, with significant potential for future horticultural and biotechnological applications.