The optimization of in vitro culture of Lithospermum erythrorhizon and shikonin production

Background: Lithospermum erythrorhizon is a medicinally valuable plant with diverse biological activities. Objective: This research focused on optimizing the in-vitro cultivation of L. erythrorhizon and establishing an efficient protocol for extracting shikonin from callus tissue. Methods: leaf explants were subjected to various concentrations of Kinetin (Kin), 1-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), Indole-3-acetic acid (IAA), and 6-Benzylaminopurine (BAP) in the LS culture medium to study callus induction. Different concentrations of LS, M9, and MS media, along with varying amounts of BAP, NAA, Kin, and IAA, were employed to investigate callus regeneration. The rooting was examined in concentrations of Indole-3-butyric acid (IBA). For shikonin production, callus was cultivated in LS, MS, and M9 media containing 0.6 mg.l-1 Kin and 2 mg.l-1 NAA. Results: The findings shown that the highest callus induction rate occurred in LS medium with 0.6 mg.l-1 Kin and 2 mg.l-1 NAA (93.3%). full-strength LS medium containing 2 mg.l-1 BAP and 1 mg.l-1 NAA led to the highest shoot regeneration. Using 1 mg.l-1 IBA in the LS medium let to improve rooting percentage (> 70%). The suitable substrate for the acclimatization was vermiculite. The shikonin content analysis in callus indicated that the M9 medium was more effective than MS and LS media in producing shikonin-containing calli. The results demonstrated that the concentration of shikonin in calli grown in M9 medium was increased with the number of day’s post-explant cultivation. Conclusion: This research can serve as a model for future investigations into optimizing the cultivation conditions of L. erythrorhizon.