The stimulatory effect of thidiazuron (TDZ) has been investigated in shoot multiplication for a simple, efficient, rapid, and commercially applicable regeneration protocol of an important medicinal plant, Cassia alata. Furthermore, the effects of an increased photosynthetic photon flux density (PPFD) on photosynthesis, the functioning of the photosynthetic apparatus, and the response of the antioxidant enzymatic system were studied during the ex vitro establishment of micropropagated plantlets. Multiple shoots were induced by culturing nodal explants excised from an aseptic seedling on Murashige and Skoog (MS) medium supplemented with various concentrations (1.0, 2.5, 5.0, 7.5, or 10.0 μM) of TDZ for different treatment durations (2, 3, 4, or 6 wk). The highest number of shoots (17.9 ± 0.3) and longest shoot length (4.6 ± 0.1 cm) were achieved when explants were exposed to 5.0 μM TDZ for 4 wk and subsequently subcultured on growth regulator-free MS medium for 8 wk. In vitro rooting of isolated shoots was best achieved on full-strength MS medium containing 0.5 μM indole-3-butyric acid (IBA). The micropropagated shoots with well-developed roots were successfully established in pots containing Soilrite™ followed by garden soil and grown in greenhouse with an 85% survival rate. During the acclimatization period, significant changes in the activity of the antioxidant enzymatic system were observed. An increase in superoxide dismutase (SOD) activity was measured throughout the acclimatization period. Likewise an upregulation of catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) enzyme activities were also observed. Pigment (chlorophyll a and b and carotenoids) content in ex vitro-formed leaves was significantly higher compared with those grown in vitro. These observed changes reflected the ability of plants to develop an antioxidant enzymatic defense system aiding in survival against oxidative stress and in reducing release of free radicals.
