Optimization of cytokinin-mediated <i>in vitro</i> shoot induction and multiplication in <i>Bacopa monnieri</i> (L.) Wettst. using nodal explants

Bacopa monnieri (L.) Wettst., commonly known as Brahmi, is a valued medicinal plant in Ayurveda, recognized for its memory-enhancing and stress-relieving properties, largely due to its bioactive compounds, including bacosides, alkaloids, flavonoids, and phenolics. The present study aimed to optimize in vitro shoot induction and multiplication from nodal explants of the species B. monnieri.Explants were cultured on Murashige and Skoog (MS) medium supplemented with varying concentrations of cytokinins, 6-benzylaminopurine (BAP) and kinetin, applied individually or in combination.BAP at 2.5 mg/L induced 58.3% regeneration with 7.0 ± 0.3 shoots per explant and 2.2 ± 0.9 cm shoot length, whereas kinetin at 2.0 mg/L yielded 50.0% regeneration with 6.0 ± 1.8 shoots and 2.1 ± 1.4 cm shoot length. The combined application of 1.0 mg/L BAP + 1.5 mg/L kinetin produced the highest shoot induction (66.6%), generating 8.0 ± 0.9 shoots per explant with 2.6 ± 1.9 cm shoot length, but post-hoc comparison revealed that shoot number did not differ significantly from the best individual BAP treatment (p > 0.05). Thus, the combination may be considered an effective alternative rather than statistically superior to BAP alone. Statistical analyses confirmed normality, homogeneity of variance, and significant differences among treatments (p < 0.05). A hormone-free MS medium (MS0) served as a negative control and failed to induce any shoot buds, confirming the essential role of cytokinins in axillary bud–mediated organogenesis.The present investigation was limited to the shoot induction and multiplication phase of in vitro culture. These results demonstrated that the optimized combination of BAP and kinetin enhances direct shoot organogenesis and multiplication in B. monnieri. However, it is recommended to examine root induction and acclimatization to establish a complete micropropagation protocol.