Organogenic callus-mediated regeneration coupled with optimized elicitors for hypericin production in Hypericum perforatum

Hypericum perforatum L., popularly termed St. John's Wort, is extensively utilized as a contemporary antidepressant formulation. Nevertheless, biosynthesis of its pharmacologically active constituents, notably hypericin (Hyp), is hampered by vulnerability to abiotic stress, inherent genetic variability, and protracted cultivation timeline. To date, a comprehensive tissue-culture-based system enabling whole-plant regeneration alongside target metabolite production in H. perforatum remains to be fully realized. In this study, the callus induction, plant regeneration, target metabolite accumulation, and key gene expression under treatments of exogenous hormones and elicitors were investigated. For the whole-plant regeneration, a 1.50-fold greater of organogenic callus induction rate (98.50%) and a 5.02-fold lower of subculture browning rate (5.27%) of stems compared with anthers, the differentiation rate reached ca. 99.0% under combination of 6-BA (0.5 mg/L) and NAA (1.0 mg/L) with 52 seedlings generated from the buds, IBA (0.5 mg/L) was more conducive to root induction than GA 3 (0.5 mg/L), and the seedling survival rates reached over 97% in peatmoss. For the target metabolite accumulation, the callus relative growth rate and target metabolite contents ( i.e. , Hyp, total flavonoids, and phenolic) generally exhibited a trend of first increasing and then decreasing with increasing concentrations of elicitors ( i.e. , SA, MeJA, and CuSO 4 ), with 50 μmol/L MeJA showing the optimal elicitor; meanwhile, the expression levels of key genes ( i.e. , CCL2 , PKS , and Hyp-1 ) were upregulated in response to the elicitors. Finally, a comprehensive and efficient in vitro approach for whole-plant regeneration and target metabolite production is proposed. These findings provide useful references for rapid propagation and large-scale suspension culture of H. perforatum .