Abstract: In order to promote the breeding of drought tolerant Bletillagermplasm,the best conditions for EMS and NaN₃ mutagenesis of the Bletillacallus were determined.In this study,the Bletilla′s seeds as materials to induce the callus tissue,and the drought tolerance of callus tissue was studied by PEG stress after combined induction with EMS and NaN₃,respectively.The results showed that the optimal formulation for seed budding was 1/2 MS+1.0mg·L^-1NAA.The optimal formula for inducing callus was 1/2 MS+1.0 mg·L^-16-BA;The optimal formula for inducing callus was 1/2 MS+1.0mg·L^-16-BA;The optimal formulation for callus proliferation was MS+1.0 mg·L^-16-BA+0.5 mg·L^-1NAA.When the concentration of EMS was 0.6%,NaN₃ was 6mmol·L^-1,and the mutagenesis was 6h,the callus tissue reached half death,that was the optimal mutagenesis effect.The Bletilla′s cell membrane induced by EMS are more seriously damaged under drought stress.Both EMS and NaN₃ can improve enzyme activity,but in terms of osmotic regulation,NaN₃ mutation increased greatly,and the mutagenic effect of NaN₃ is better than EMS.