In vitro culture of Balkan endemic and rare Pulsatilla species for conservational purposes and secondary metabolites production

Shoot cultures of Pulsatilla montana ssp. balcana, P. halleri ssp. rhodopaea and P. slaviankae (Zimm.) Jordanov & Kozuharov were initiated from surface-sterilized seeds of the three species, collected at their natural habitats. Shoot cultures of the three species in Murashige and Skoog’s culture medium (MS) exhibited a chlorophyll a/b (chl a/b) ratio ranging from 1, 86 to 3, 01. Moreover by using different concentrations of benzyladenine and 3-indole butyric acid the chl a/b ratio diff ered from species to species, showing a diff erent adaptability of species to diff erent media. Th e shoot cultures of the three Pulsatilla species were also analyzed as an in vitro source of polyphenolic antioxidant substances so they were screened for total phenolics, fl avonoids and anthocyanidins content in three media variants, demonstrating the highest content for these substances in P. montana ssp. balcana and P. halleri ssp. rhodopaea in the growth regulator-free and 6-benzyladenine – supplemented medium, while in medium supplemented with auxin and cytokinin all three species produced commensurable levels of the studied metabolites.