Cryopreservation of plantlet nodes of Dioscorea opposita Thunb. using a vitrification method

A cryopreservation method by vitrification was developed for long-term storage of Dioscorea opposita Thunb., a valuable native medicinal plant species in Henan Province of China. The cryopreservation protocol was established with cultivar B and evaluated with another four cultivars, Tiegun, 47, Taigu and Huaiqing 1. The results showed that nodes with a bud excised from 60 d plantlets were desirable for the cryopreservation. The optimum procedure was established as: 1) the plantlets were cultured on the Murashige and Skoog (MS) medium supplemented with 2 mg L(-1) KT and 0.02 mg L(-1) NAA at 4 degree C for 7 d before nodes with length of 1-1.5 cm were excised; 2) the nodes were precultured at 4 degree C for 7 d on the MS supplemented with 10 percent dimethyl sulfoxide (DMSO) followed by loading with 60 percent Dioscorea vitrification solution 1 (DVS1): 22 percent (w/v) glycerol + 13 percent (w/v) ethylene glycol + 13 percent (w/v) polyethylene glycol + 10 percent (w/v) DMSO for 60 min at 0 degree C and dehydrated with 100 percent DVS1 for 60 min at 0 degree C; 3) the nodes were then immersed into liquid nitrogen (LN) directly and conserved for 180 d; 4) after rapid thawing in a water-bath at 37 degree C, the nodes were rinsed four times with MS medium supplemented with 5 percent sucrose, then transferred to the MS medium supplemented with 2 mg L(-1) kinetin (KT) and 0.02 mg L(-1) NAA for regeneration. In the present research the regeneration rate of cv. B was about 77.1 percent, those of cvs. Tiegun and Huaiqing 1 were 67.2 percent and 54.0 percent respectively, while cvs. Taigu and 47 were about 40 percent. There were no visual changes observed between the plantlets regenerated from nodes with and without cryopreservation in terms of the morphology indices, indicating that the method established could be applicable to D. opposita with optimized protocol.