Callus orchis maculata L. as a source of bioactive substances: biotechnology of cultivation

Orchis maculata L. is a medicinal plant that contains alkaloids, flavonoids, carotenoids, anthocyanins, and sterols. However, this plant is listed in the Red Book of Russia. Therefore, domestic pharmacology needs in vitro biotechnological methods for its industrial cultivation. The research objective was to select phytohormones in a nutrient medium that would increase the callus biomass of Orchis maculata L. The study featured callus cultures grown on various nutrient media and such phytohormones as cytokines (kinetin, 6-BAP) and auxins (BCI, 2,4-D). The amount of the phytohormones varied from 0.1 mg to 2.0 mg. The callus cells with the highest dry residue were subjected to extraction with 70 % ethanol at 60 °С for 60 min; the ratio of raw materials : extractant was 1:10. The extract was analyzed for the qualitative and quantitative composition of biologically active substances using HPLC (Shimadzu LC-20 Prominence) and TLC (Sorbfil PTSKh-AF-A). The greatest increase in callus biomass was observed when the ratio of cytokine: auxin was 1:1. On cultivation day 14, the growth index was 7.71 ± 0.01 when using 6-BAP + 2.4-D (1.0 mg + 1.0 mg per 1 L) on nutrient medium No. 29. Medium No. 13, kinetin (1.5 mg) + 2,4-D (1.5 mg), provided the highest amount of dry substances (10.47 ± 0.02). The TLC method (medium No. 13) revealed the content of quercetin and quercetin-3,7-O-β-D-glucopyranosides in trisaccharide and disaccharide forms. The HPLC method detected rutin (73.07 ± 3.65 mg/kg), quercetin (15.08 ± 0.75 mg/kg), apigenin (23.63 ± 1.18 mg/kg), and coleofolide (12.53 ± 0.63 mg / kg). The resulting nutrient medium made it possible to obtain enough callus Orchis maculata L. to extract biologically active substances for medicinal purposes.