Development of an improved and simple shoot tip cryoconservation protocol for cryobanking of Bacopa monnieri (L.) Wettst. germplasm

Abstract

Bacopa monnieri (L.) Wettst. is a high value medicinally important plant used as a memory enhancer, the germplasm of which requires to be conserved long-term due to its over-exploitation. Access to an efficient shoot tips cryoconservation protocol that results in high post-thaw regrowth and is applicable to many genotypes is a prerequisite for long-term conservation of valuable germplasm. Experiments were conducted to assess the effect of shoot tip size and cryoconservation techniques, including vitrification (V) and droplet-vitrification (DV), to improve post-thaw regrowth in two accessions of B. monnieri. Optimal results after cryopreservation were obtained when small size (SS – 0.5 × 0.5 mm) shoot tips were excised from cultures pretreated on MS basal medium supplemented with 0.3 M sucrose (SM) for 4 to 16 wk, precultured on SM medium for 2 d and dehydrated with Plant Vitrification Solution-2 (PVS2) at 0 °C for 35 min. High post-thaw regrowth of > 50% was obtained using V technique, which was further enhanced by using DV technique (approximately 80%). Genetic integrity of regenerated plantlets after cryoconservation using V and DV showed no significant variation among the control and cryoconserved plants using 39 Inter Simple Sequence Repeats (ISSR) markers. Further, the developed protocol was evaluated in other four accessions and four-fold enhanced post-thaw regrowth (approximately 80%) and reduction of preparation time by approximately 20 wk was achieved. These results demonstrated that this improved protocol has potential to be implemented for cryobanking of in vitro germplasm of Bacopa monnieri.