Passivation effect of iodacetamide and ultraviolet light on tobacco protoplasts

【Objective】 This study aimed to explore the critical dose of tobacco protoplast passivation for improvement and innovation of tobacco germplasm by asymmetric somatic hybridization.【Method】 Tobacco protoplasts were treated with iodacetamide(IOA) of different concentrations(0.1,0.25,0.5,0.75,and 1.0 mmol/L),three centrifuging methods(centrifuging for 5 min at 700 r/min without standing,standing for 5 min and then centrifuging for 5 min at 700 r/min,and standing for 3 min and then centrifuging for 2 min at 700 r/min),and irradiation of ultraviolet light(UV)(exposure by 0.5,1,4,5,7,10,and 15 min in two ultraviolet lamp of 15 W)to investigate the critical dose for passivation of tobacco protoplast.【Result】 Tobacco protoplast was very sensitive to IOA.The treatment with 0.1 mmol/L IOA before standing for 3 min and centrifuging for 2 min at 700 r/min had the highest percentage of the protoplast under the critical condition(92.9%),which became the best to tobacco protoplast passivation.Continuous irradiation 15 min was the critical treatment to tobacco mesophyll protoplasts,and the protoplast ratio under the critical condition was 91.4%.The fused protoplast can divide and form cell colonies.【Conclusion】 The treatment with 0.1 mmol/L IOA,standing for 3 min and then centrifuging for 2 min at 700 r/min and continuous irradiation for 15 min was the critical dose of tobacco protoplast passivation,two kinds of critical dose of two tobacco protoplasts after being treated with critical dose restored ability of division due to complementation after fusing and forming hybrid.