In vitro polyploidy induction: changes in morphological, anatomical and phytochemical characteristics of Thymus persicus (Lamiaceae)

Thymus persicus, which grows in the northwest area of Iran, is a valuable natural source of medicinal pentacyclic triterpenoids (PTs), i.e. betulinic acid, oleanolic acid and ursolic acid. An in vitro approach to polyploidy induction in the plant was examined for the first time. Polyploidization was induced using in vitro-grown T. persicus shoot-tips immersed in colchicine (0, 0.05, 0.1, 0.3 and 0.5 %) for 12-48 h. Ploidy levels of regenerates were determined by either flow cytometry or chromosome counting at the metaphase. The results confirmed that the mother diploid plant contained a chromosome number of 2n = 2x = 28, 2C DNA = 1.20 pg, whereas 2n = 4x = 56, 2C DNA = 2.39 pg was detected in the induced autotetraploids (CV% < 4 %). Out of 960 shoot tip segments exposed to colchicine, 7.80 and 1.04 % were tetraploid and mixoploid, respectively. The most efficient conditions for inducing polyploidy were the treatment with 0.3 % colchicine for 12 h, followed by 0.3 % for 24 h. The effect of polyploidization on growth and anatomical characteristics as well as PTs production was also measured. Tetraploids differed markedly from diploids, showing lower plantlet height, shorter roots, thicker stems and darker leaves, as well as longer and wider stomata and reduced stomatal density on the abaxial and adaxial leaf surfaces. Furthermore, a positive trend in triterpenoid production was obtained with the tetraploid and mixoploid T. persicus plants. These findings indicate that tetraploids can potentially be used in further breeding efforts to obtain a wide range of triterpenoids with improved medicinal properties.