Deciphering the efficiency of meta-Topolin in de novo shoot regeneration, L-Dopa biosynthesis, survival rate, and the evaluation of genetic stability in Hybanthus enneaspermus (L.) F. Muell.

In this experimental investigation, an indirect organogenesis approach was employed to propagate Hybanthus enneaspermus (L.) F. Muell. The comparative effectiveness of 6-Benzyladenine (BA) and meta-Topolin (mT) in de novo shoot regeneration was examined. Nodal segments generated callus in the Murashige & Skoog (MS) media infused employing diverse auxins. A deep green, densely compacted, and six weeks nourished calli raised at a concentration of 2 mg L⁻¹ 2, 4-Dichlorophenoxyacetic acid (2, 4-D) was optimized for de novo shoot regeneration. The best shoot regenerating frequency per segment of nodal callus was recorded with 2.0 mg L⁻¹ mT treatment of twelve weeks, which was higher than BA. Robust mT-derived microshoots cultured in 1.5 mg L⁻¹ of Indole-3-butyric acid (IBA) triggered the maximum quantity of roots per shootlet than the BA-derived microshoots. HPLC quantification demonstrated that the mT-derived microshoots yield 1.27-fold higher content of L-Dopa and a 35% increased survival rate than that of the BA-derived microshoots. The homogeneity of micropropagated plantlets was analyzed through RAPD, ISSR, and SCoT molecular markers, which ultimately confirmed the genetic uniformity among the in vitro cloned plantlets of H. enneaspermus. This is the first study that attempts to bring forth the potentiality of mT in de novo shoot regeneration and the creation of L-Dopa in H. enneaspermus. The consequences of the mT prove to be outperforming BA in terms of shoot regeneration, L-Dopa biosynthesis, and survival rate.