Preparation of protoplasts from Chlorella protothecoides

Area of Science:

• * Biotechnology and Algal Research
• * Microbiology and Cell Biology

Background:

• * Efficient isolation of viable protoplasts is crucial for genetic manipulation of microalgae.
• * Chlorella protothecoides is a significant algal species with potential applications in various fields.

Purpose of the Study:

• * To develop and optimize an enzymatic method for protoplast isolation from Chlorella protothecoides.
• * To assess the viability and regeneration capacity of the isolated protoplasts.

Main Methods:

• * Screening of four commercially available enzymes for protoplast yield.
• * Optimization of enzyme concentrations (cellulase R-10, snailase), buffer conditions (Tris buffer, pH 6.0), osmotic stabilizer (D-mannitol), temperature (30°C), and incubation time (16 h).
• * Assessment of protoplast viability using phenosafranin staining and regeneration rates in a D-mannitol-containing medium.

Main Results:

• * Optimal enzymatic digestion achieved with 2% cellulase R-10 and 1% snailase in 25 mM Tris buffer (pH 6.0) with 0.6 M D-mannitol.
• * Peak protoplast density obtained after 16 hours of treatment at 30°C.
• * High protoplast viability (nearly all green) confirmed by phenosafranin staining.
• * Approximately 70% regeneration rate achieved using 0.6 M D-mannitol as an osmotic stabilizer.

Conclusions:

• * A robust and efficient enzymatic protocol for Chlorella protothecoides protoplast isolation has been established.
• * The developed method ensures high protoplast viability and regeneration potential.
• * This protocol is valuable for advancing genetic studies and applications of Chlorella protothecoides.