Study on cryopreservation and genetic stability of embryogenic calli induced from Dioscorea bulbifera microtuber / 中草药

ABSTRACT

Objective: To discuss the influence of several factors on the cryopreservation of embryogenic calli induced from Dioscorea bulbifera microtuber by droplet-vitrification and to test the genetic stability of the regenerated plantlets after freezing from the aspects of morphology, physiology, DNA content, as well as the photosynthetic characteristics and chlorophyll fluorescence parameters in this paper. Methods: Plant tissue culture (including microtuber induction and embryogenic callus induction), plant physiology index detection (including total chlorophyll, soluble protein, soluble sugar and superoxide dismutase enzyme and peroxide enzyme activity), and cell flow cytometry were applied. Results: The best cryopreservation conditions of embryogenic callus of D. bulbifera microtuber were as following Embryogenic calli were precultured in liquid media of MS+KT 2 mg/L+NAA 0.5 mg/L+2, 4-D 0.5 mg/L+0.3 mol/L sucrose for 1 d and then treated in loading liquid (MS+2 mol/L glycerol+0.4 mol/L sucrose, pH 5.8) for 20 min. In order to dehydrate, embryogenic calli were transferred in 100% PVS2 at 0℃ for 40 min. After dehydration, the embryogenic calli were inoculated to PVS2 small drops in the aluminum foil strips and then dipped in liquid nitrogen (LN). Finally the aluminum foil strips were quickly transferred to freezing tube that filled with LN and then put into LN tank. After conserving for 1 d in LN, the aluminum foil strips were removed and the embryogenic calli were immersed into liquid washing media (MS+KT 2 mg/L+NAA 0.5 mg/L+2, 4-D 0.5 mg/L+1.2 mol/L sucrose, pH 5.8) preheated in 37℃ warm water. After separated from the aluminum foil strips, the embryogenic calli were washed with fresh liquid washing media at room temperature for there times, 10 min each time. After washing, the embryogenic calli were transferred onto differentiation medium (MS+KT 2 mg/L+NAA 0.5 mg/L+30 g/L sucrose+5 g/L agar), and cultured in dark for 2 d and then cultured in 12 h/d photoperiod, the cell survival rate reaches above 89%. The morphological and physiological indexes and the content of DNA of two kinds of plantlets, which regenerated from cryopreserved and non-cryopreserved embryogenic calli induced from D. bulbifera microtuber by droplet-vitrification, showed no significant difference (P>0.05). Conclusion: Cryopreservation technology system of embryogenic calli induced from D. bulbifera microtuber by droplet-vitrification is established and the regeneration plants have no genetic variation, which provides the theoretical basis and technical basis for the long-term preservation of germplasm resources in the plants of Dioscorea L.