Investigation of morphological, physiological, histological characteristics and hypericine content of 20 populations of Hypericum perforatum L. in vitro

St. John's Wort, known as Hypericum perforatum L., is a valuable medicinal plant in the Hypericaceae family. Its most common use is for its antidepressant properties. The active compounds hypericin and hyperforin in St. John's Wort and their proven therapeutic effects have made this plant one of the most valuable medicinal plants worldwide. Hypericin, a major plant metabolite, has antiviral and anticancer effects. Hypericin belongs to the naphthodianthrones family and is light-sensitive. It mainly accumulates in the dark-colored glands on the leaves and flowers of the plant, and its concentration ranges from 0.3% to 3.0% in the leaves and 1% to 14% in the flower buds, depending on the plant variety, height, light conditions, and season. Hyperforin is another important compound of the phloroglucinols group found abundantly in St. John's Wort flowers, especially in the stamens and the fruit. However, it is also present in large amounts in the leaves. Hyperforin content of 6.9% in flower buds, 8.5% in unripe fruit, and 5.1% in leaves has been reported. Unlike hypericin, hyperforin accumulates in transparent glands. Since hypericin and hyperforin depend on environmental conditions, asexual reproduction is preferred over sexual reproduction. In this study, 20 populations from 20 different regions of Iran were collected and examined for micropropagation optimization. In addition to identifying the most suitable medium for micropropagation of these populations, the morphological, physiological, biochemical, and histological characteristics of them were also investigated. To prepare the modified culture medium, ½ MS salts, FeEDDHA, B5 vitamins, and two g.l-1 Glycine was used. The MS basal medium was used as a control after adjusting the pH to 8.5 and adding 8.6 g.l-1 Agar, the desired medium, was distributed into culture vessels and autoclaved at 121°C and 2 bar pressure for 20 minutes. Seven explants containing 1-2 buds were placed in each culture vessel. All cultures were incubated in a growth room with 8 hours of darkness and 16 hours of light at an intensity of 1500-2000 lux, a temperature of 25°C, and a relative humidity of 60-70% for four weeks. After the emergence of new shoots, data were collected on the number of branches, length of branches, fresh weight, and dry weight. High-performance liquid chromatography (HPLC) was used to measure the hypericin content in the leaves of the plantlets. This study shows that changing the culture medium composition in most studied populations increases branch length and number. Reducing the amount of mineral salts in the culture medium has affected the plant's osmotic relationships to water and nutrient uptake. This and increased glycine concentration have created a better fine growth trend. This study showed that among twenty native es of Iran, the North Khorasan population had the highest morphological (shoot number, shoot length), physiological (fresh weight, dry weight), and biochemical (hypericin content) indices in the modified medium. Also, histological studies in this study showed a larger growth of dark glands in the modified medium than in the basal medium. Based on the findings for twenty native populations of Hypericum perforatum in Iran, the North Khorasan population had the highest morphological (shoot number, shoot length), physiological (fresh weight, dry weight), and biochemical (hypericin content) indices in the modified medium. Also, histological studies showed a larger growth of dark glands in the modified medium than in the basal medium.