Optimization of protoplast formation, regeneration, and viability in Microsporum gypseum

Area of Science:

• Mycology
• Cell Biology
• Biotechnology

Background:

• Protoplast isolation and regeneration are essential for genetic manipulation of fungi.
• Microsporum gypseum is a dermatophyte with clinical and ecological significance.
• Optimizing protoplast protocols is key to advancing research on this species.

Purpose of the Study:

• To investigate factors influencing high yields, regeneration frequencies, and viability of Microsporum gypseum protoplasts.
• To establish an efficient protocol for protoplast isolation and regeneration.
• To assess the impact of different pretreatment and media conditions on protoplast quality.

Main Methods:

• Mycelial digestion using Novozyme 234 with CaCl2 as an osmotic stabilizer and glycine-HCl buffer.
• Pretreatment of mycelium with mercaptoethanol and dithiothreitol.
• Regeneration using the top agar method with complete medium, agar, and CaCl2.
• Protoplast viability assessment using fluorescence microscopy and protein synthesis measurement (3H-lysine uptake).

Main Results:

• Maximum protoplast yields achieved after 6 hours digestion of 2-4 day old mycelium.
• Optimal pretreatment involved mercaptoethanol + dithiothreitol (0.01 M).
• A regeneration frequency of 94.4% was obtained with specific medium composition and osmoticum.
• Glucose (2%) and glutamine (2%) significantly enhanced protoplast viability after 24 hours.

Conclusions:

• The study successfully optimized conditions for high-yield protoplast isolation and regeneration in Microsporum gypseum.
• The established protocol ensures high regeneration frequencies and protoplast viability.
• Enhanced viability was observed with glucose or glutamine supplementation, correlating with protein synthesis.