The use of long-term Scutellaria altissima callus cultures for shoot regeneration, production of bioactive metabolites and micropropagation

Abstract

Different types of seedling explants were used to induce Scutellaria altissima callus formation. The explants were cultured on Murashige and Skoog (MS) agar medium supplemented with 0.1 mg l-1 indole-3-acetic acid (IAA), 0.2 mg l-1 thidiazuron (TDZ) and Schenk and Hildebrandt (SH) agar medium containing 0.1 mg l-1 naphthalene acetic acid (NAA), 0.2 mg l-1 6-benzylaminopurine (BAP), 0.5 mg l-1 2,4-dichlorophenoxyacetic acid (2,4-D). Hypocotyl explants had the highest frequency of callus formation and the calli had the best shoot regeneration potential. Two types of hypocotyl-derived calli named SH-type and MS-type were maintained for two years under in vitro conditions and characterized in terms of shoot proliferation and ability to produce bioactive metabolites (baicalin, wogonoside, verbascoside). The results indicate that the SH-type callus grown for two years under dark conditions was characterized by high regeneration efficiency: 13 shoots per culture with a length of about 4 cm. The callus also accumulated a high concentration of flavone glycosides: baicalin (32.4 mg g-1 dry weight) and wogonoside (7.5 mg g-1 dry weight). On the other hand, MS-type calli of the same age cultured under photoperiod light conditions produced the highest amount of verbascoside (10.6 mg g-1 dry weight). Inter simple sequence repeat (ISSR) analysis of S. altissima shoots developed via callus organogenesis showed their genetic similarity to shoots originated from the seeds. The regenerated plantlets from calli S. altissima shoots were direct rooted ex vitro in pots and acclimatized in the greenhouse with a survival rate of 90 to 95% after 12 weeks. No morphological abnormalities were observed in the micropropagated plants. Key words: Callus culture, inter simple sequence repeat (ISSR) analysis, flavonoids, Scutellaria altissima, shoot regeneration, verbascoside.