meta-Topolin-induced enhanced biomass production via direct and indirect regeneration, synthetic seed production, and genetic fidelity assessment of Bacopa monnieri (L.) Pennell, a memory-booster plant

The present study reports enhanced in vitro mass propagation, synthetic seed production and its regeneration, acclimatization, and genetic fidelity assessment of in vitro regenerants of Bacopa monnieri (L.) Pennell; a medicinal plant renowned for its memory-enhancing property. For the first time, meta-Topolin (mT) as well as N6-benzyladenine (BA) and kinetin at their variable concentrations (0.5, 1. 1.5, 2 and 2.5 mg/l) were supplemented individually in Murashige and Skoog (Physiol Plant 15:473–497, 1962) (MS) basal medium to induce multiple shoots from shoot tip explants, initially collected from two-month-old vegetatively propagated plants. The best result in terms of multiplication (~ 7 shoots/explant) and biomass accumulation (~ 451.3 mg fresh weight, ~ 104.3 mg dry weight) was obtained in MS medium supplemented with 1 mg/l mT. For rooting of in vitro shoots, supplementation of 1.5 mg/l IAA and 0.5 mg/l IBA in MS media resulted in induction of the maximum number (~ 20) and highest length (~ 5.5 cm) of roots, respectively. Most efficient indirect regeneration and biomass production from green and hard basal calli (induced spontaneously in BA-supplemented media) was recorded in a simple MS medium. Synthetic seeds were produced from in vitro shoot tips using 2.5% sodium alginate and 75 mM calcium chloride solutions and were successfully regenerated in half-strength MS medium. Six-week-old in vitro regenerants were transferred to different substrates for acclimatization, among which cocopeat was found to be the best exhibiting the highest survival and growth in external conditions. Finally, molecular marker-(ISSR) and phenology-based genetic fidelity assessment of the in vitro regenerants exhibited that the in vitro regenerants were true-to-type in nature within themselves as well as with the mother plant.