Preparation, purification and regeneration optimizing research of protoplasts from Rhizoctonia solani

Abstract

The aim of this work was to study the standardization of conditions to obtain and regenerate protoplasts of Rhizoctonia solani. The optimal way of harvesting 1.67 × 109protoplasts per gram from Rhizoctonia solani was to deal with 12 h aged mycelium in 0.6 M MgSO4 stabilizer (pH 5.2) combining cellulase and driselase at 35°C for 3 h. Meanwhile, the most suitable condition for protoplast regeneration had been attained. The condition was to deal with 16 h aged mycelium in 0.6 M MgSO4 stabilizer (pH 5.2) combining cellulase with driselase at 35°C for 2.5 h, and incubation at 26°C in the regeneration medium. Furthermore, a two-phase system of 0.6 M sucrose and 0.6 M mannitol was developed for separation and purification of intact protoplasts from broken protoplasts, excellent results were obtained. There was a breakthrough for protoplast technology of R. solani and other fungus was recorded in this paper.   Key words: Preparation, protoplast, purification, regeneration, Rhizoctonia solani