Thidiazuron regulated growth, secondary metabolism and essential oil profiles in shoot cultures of Ajuga bracteosa

Ajuga bracteosa a highly demanding pharmaceutical plant, is getting endangered in several regions of the world due to ruthless and destructive harvesting. Research efforts need to be done for its sustainable production and utilization. In this study, effects of Thidiazuron (TDZ) in comparison to conventional cytokinins were tested in vitro on shoot morphogenesis, antioxidant system and biosynthesis of volatile organic compounds in A. bracteosa. Thidiazuron alone acted as the best growth regulator for direct shoot morphogenesis in explants by promoting highest induction of shoots (88%) in stem explants cultivated on MS (Murashige and Skoog) medium supplemented with 1.2 ppm (TDZ). At lower doses (0.5 ppm), TDZ resulted in highest multiplication (18 shoots) and elongation (5.8 cm) of shoots. Further, shoot cultures raised in vitro at TDZ (1.2 ppm) displayed higher activities of superoxide dismutase (SOD: 4.2 U/mg protein), peroxidase (POD: 2.8 U/mg protein) and ascorbate peroxidase (APx: 1.8 U/mg protein), respectively. Maximum total phenolic content (TPC; 4.5 mg GAE/g DW) and total flavonoid content (TFC; 3.5 mg QE/g DW), respectively were detected in shoot cultures grown over MS medium combined with 1.2 ppm TDZ plus 2.0 ppm benzylaminopurine (BA). Analysis through gas chromatography-mass spectrometry (GC MS) showed, biosynthesis of 34 volatile compounds in the in vitro raised shoots. Wherein, TDZ (1.2 ppm) resulted in production of substantial amounts of bioactive monoterpene hydrocarbons including a-pinene (5.3%), camphene (4.45%), limonene (3.4%), 1,8 cineole (14.3%), alpha-thujone (9.4%) and oxygenated monoterpenes such as camphor (12.2%), borneol (11.4%) and nerol (9.2%). Thus, besides providing an ex-situ conservation strategy for a high valued medicinal herb, this study has the potential for commercial production of important bioactive volatile compounds.