Two Advanced Cryogenic Procedures for Improving Stevia rebaudiana (Bertoni) Cryopreservation

Abstract

Cryopreservation is a useful tool for the long-term storage of plant genetic resources, and different cryogenic procedures have recently been developed. The present study focused on the use of the Droplet-vitrification (DV) and V cryo-plate protocol for the cryopreservation of Stevia rebaudiana in vitro-derived apical shoot tips and axillary shoot tips. A preliminary test showed that 90 and 120 min PVS2 (Plant Vitrification Solution 2) treatment significantly reduced the regrowth of the explants before immersion in liquid nitrogen (LN). For both procedures tested, the best osmoprotective condition for obtaining a higher regrowth of cryopreserved explants occurred when explants were PVS2 treated for 60 min. After direct immersion in LN, thawing and plating, the highest regrowth recorded was 80% with DV and 93% with V cryo-plate. Moreover, shoot tips proved to be a more suitable material for Stevia cryopreservation. A satisfactory vegetative regrowth was observed in the subcultures following cryopreservation by DV and V cryo-plate cryogenic procedures.

Comparative Study of Three Vitrification Solutions for an Effective Droplet-vitrification Cryopreservation Procedure for Pfaffia Glomerata (Spreng.) Pedersen

“…In the case of the present study, shoot tip regrowth rates were higher and plantlet regeneration and growth was faster than when the vitrification procedure was used [17]. The droplet-vitrification been developed for many other plant species [7,8,12,13,14,15,16,21,22,23,24,25,26,27]. It is a practical, user-friendly technique and is now routinely applied for the cryopreservation of shoot tips of a wide range of plant species and genotypes and could be an alternative for implementing long-term storage of germplasm.…”

“…In other studies, it was reported that the best exposure times to PVS2 were 60 min for Stevia. [8], 20 min. for Rosa hybrida L. [14], 60 min for Malus domestica Borkh.…”

“…Therefore, one of the key factors in the design of a cryopreservation protocol is to select and optimize a dehydration strategy for the explants prior to freezing them in liquid nitrogen, to avoid the formation of ice crystals inside the cells and tissues Treatment of explants with vitrification solutions containing combinations of different cryoprotectants, such as DMSO (dimethyl sulfoxide), ethylene glycol, glycerol, and sucrose, added in specific concentrations has become a common strategy in the design of cryopreservation protocols. nt solutions must be used with caution since they might have a toxic effect or cause osmotic stress to cells of some plant species, compromising viability of the cells in the explant, resulting in cell death, especially if immersion in them occurs at temperatures above 0 °C or for prolonged periods of time [8,31]. Toxic effects can be prevented by selecting adequate cryoprotectants, adjusting the concentration of cryoprotectants in the solution, and the temperature and duration of the The aim of this study to develop a cryopreservation procedure based on the vitrification technique for shoot tips of (Spreng.)…”

“…is a cryopreservation technique that has been successfully applied to many plant species [7,8]. This technique consists of dripping vitrification solution containing one single explant on aluminum foil strips, which are immersed quickly in liquid nitrogen (LN) -196°C.…”

Vitrification Solutions for Plant Cryopreservation: Modification and Properties

“…The temperature during the incubation of plants in PVSs is important for both toxicity and dehydration. The temperature close to 0 • C for plants treated in PVS2 is crucial and had significantly lower lethality than at 22 • C [65,[141][142][143]. When the temperature is subsequently lowered, the penetrating components of PVS2 cryoprotect the cells by restricting the molecular mobility of water molecules and preventing them from nucleating ice crystals [11].…”