Cryopreservation of Dendrobium Orchid Varieties and the Potential for the Elimination of Cymbidium Mosaic Virus

Cryopreservation offers an effective means of maintaining orchid germplasm with minimal space and time requirements once materials are prepared and frozen. These protocols have also been successfully used to eliminate crop viruses. Here, droplet-vitrification techniques were applied to Dendrobium orchid varieties to assess regeneration rates and the potential for the elimination of Cymbidium mosaic virus.  Shoot tips 2–3 millimeters long were excised from in vitro orchid plantlets and precultured on a semi-solid 0.3 M sucrose media for three days. Excised materials were then placed in a loading solution (0.4 M sucrose + 2 M glycerol) for 20 minutes and then subjected to plant vitrification solution 2 (PVS2) (0.4 M sucrose + 30% glycerol + 15% ethylene glycol + 15% DMSO) for an additional 20 minutes at room temperature.  Shoot tips were then suspended in PVS2 droplets on foil strips, plunged directly into liquid nitrogen, and held for one hour. After exposure to liquid nitrogen, foil strips containing shoot tips were transferred to an unloading solution (1.2 M sucrose) for 40 minutes, transferred to semi-solid media in petri dishes (2% sucrose) and placed in the dark for 1 week, moved to dim lighting for one week, and finally returned to normal lighting conditions.   Droplet-vitrification was successfully applied to three Dendrobium varieties and assessed for use in virus elimination.