To accelerate the creation of new germplasm of Dioscorea opposita, with the callus of “Minghuai 2” as material, the effects of different concentrations of EMS [0(ck)，0.05%，0.1%，0.2%，0.3%] and different time [0(ck)，6h，12h，24h，48h] on the mortality of callus were studied, so as to determine the semi lethal concentration and time; The effect of callus in different subculture times was compared after semi lethal treatment; And the morphological comparison and molecular identification of regeneration plantlets were carried out; The results show that: The concentration and time of EMS had a significant effect on the death rate of the callus; And the effect of EMS concentration on the death rate of callus was more significant than that of the treatment time; The suitable EMS mutation method was: 0.1%EMS concentration was treated by 48h, 0.2%EMS was treated by 12h; The proliferation coefficient and regeneration rate of callus increased with the increase of subculture times; However, the mutation rate decreased significantly with the increase of subculture; So after mutation, the callus was subcultured once and then transferred to regeneration is the most favorable screening of the mutant; The variation of regenerated plants mainly occurred in leaf color and leaf shape; By RAPD molecular markers, the results showed that the DNA bands of some regeneration plantlets did appear to differ.
