Factors affecting protoplast formation by Rhizoctonia solani

Novozym 234 was the most frequently used enzyme for production of Rhizoctonia solani protoplasts. Since manufacture of this enzyme was discontinued in the late 1990s, a new procedure was developed by testing lytic enzymes from Sigma and by examining factors affecting protoplast formation. The combination of 20 mg/mL Driselase and 10 mg/mL lysing enzyme was effective in releasing protoplasts from R. solani. The optimal condition for enzyme treatment of mycelium was incubation at 37 °C for 15 min followed by 34 °C for 105 min. The amount of protoplasts produced was positively correlated with growth rate and negatively correlated with mycelial density. Under favorable conditions, R. solani mycelia released 1.68 × 106 protoplasts/mL that is comparable with that produced with Novozym 234. Among various media tested, the best solid medium for protoplast regeneration was 1% V-8 juice agar, while the best liquid medium was 10% potato dextrose broth.