Establishment of regeneration system of and molecular association analysis of coumarin secondary metabolism.

INTRODUCTION

is a medicinally critical species whose sustainable utilization is hindered by declining wild populations and insufficient cultivation systems. This study aimed to establish an optimized regeneration system for and elucidate the molecular mechanisms underlying coumarin biosynthesis, focusing on the roles of key phenylpropanoid pathway genes.

METHODS

Leaf, stem, and root explants were cultured on MS media supplemented with varying concentrations of 2,4-D, 6-BA, and IBA to induce callus, proliferation, differentiation, and rooting. Coumarin content (praeruptorins A, B, E) was quantified via HPLC, while RT-qPCR analyzed expression levels of , and genes across tissues.

RESULTS

Optimal media were identified: MS + 0.5 mg/L 2,4-D + 0.5 mg/L 6-BA (callus induction, 85.71% efficiency), MS + 1.0 mg/L 6-BA + 0.5 mg/L IBA (proliferation), and MS + 0.5 mg/L IBA (rooting, 69.44% success). Adventitious buds exhibited the highest total coumarin content (3.67× callus), with roots of seedlings accumulating 1.67× more coumarin than leaves. Expression of PpC2'H dominated across materials (), correlating strongly with coumarin levels.

DISCUSSION

The study demonstrates that hormonal crosstalk (auxin-cytokinin balance) critically regulates morphogenesis, while acts as a bottleneck gene for coumarin synthesis. These findings enable targeted metabolic engineering (e.g., overexpression) to enhance yields, offering a sustainable alternative to wild harvesting.