Factors affecting protoplast formation by Rhizoctonia solani

Area of Science:

• Mycology
• Enzymology
• Biotechnology

Background:

• Novozym 234 was the standard enzyme for Rhizoctonia solani protoplast production.
• Discontinuation of Novozym 234 necessitated development of alternative methods.

Purpose of the Study:

• To develop a new, effective procedure for Rhizoctonia solani protoplast isolation.
• To identify optimal enzymatic and environmental conditions for protoplast formation and regeneration.

Main Methods:

• Screening of lytic enzymes from Sigma, focusing on Driselase and lysing enzyme combinations.
• Optimization of incubation temperature and time for mycelial treatment.
• Analysis of factors influencing protoplast yield, including growth rate and mycelial density.
• Evaluation of different solid and liquid media for protoplast regeneration.

Main Results:

• A combination of 20 mg/mL Driselase and 10 mg/mL lysing enzyme proved effective for protoplast release.
• Optimal incubation involved 37°C for 15 minutes followed by 34°C for 105 minutes.
• Protoplast yield (1.68 x 10^6 protoplasts/mL) was comparable to Novozym 234 under favorable conditions.
• 1% V-8 juice agar and 10% potato dextrose broth were identified as optimal regeneration media.

Conclusions:

• A novel and effective method for Rhizoctonia solani protoplast production has been established using Driselase and lysing enzyme.
• The findings provide crucial protocols for fungal protoplast isolation and regeneration, supporting further research in R. solani.
• Optimized conditions ensure high yields of viable protoplasts for genetic and cellular studies.