Sucrose treatment and explant water content: Critical factors to consider in development of successful cryopreservation protocols for shoot tip explants of the tropical species dioscorea rotundata (YAM)

Abstract

A study was conducted to determine the optimum methods for conditioning explants to be used in the development of a simple protocol for long-term conservation of the germplasm of Dioscorea rotundata via cryopreservation. Shoot tips from cultures maintained in vitro were exposed to high concentrations of sucrose prior to silica gel-based dehydration and vitrification solution-based cryopreservation protocols. Explant water contents were determined, and ultrastructural studies were also carried out. Initially, culturing explants on medium supplemented with 0.3 M sucrose for 3-5 d considerably reduced tissue water content from about 12.2 g g-1 dry mass to between 4.8 and 5.5 g g-1 dry mass before cryoprotection with modified PVS2 (MPVS2) or silica gel dehydration. Ultrastructural studies indicated that cells had deposits of starch in plastids following sucrose treatments. Survival for D. rotundata shoot tips treated with MPVS2 vitrification solution, unloaded with 1.0 M sucrose medium and cooled to -70°C, was 16% for 15 min treatment and 44% for 40 min. After the 40 min MPVS2 treatment the TTZ test indicated 88% viability retention of explants cooled to -70°C, and 44% at -196°C. Plantlet development was obtained for -70°C-cooled shoot tips, whereas only callus development occurred from tissues exposed to liquid nitrogen. Explant regeneration was not obtained with air-dehydration techniques. It was concluded that vitrification-solution based cryopreservation presently offers the best option for conservation of this Dioscorea species. © CryoLetters.