Isolation and Regeneration of Protoplasts from Laccaria fraterna - an Ectomycorrhizal Fungus

An efficient method for high yields of protoplast isolation and regeneration and viability was achieved in Laccaria fraterna, an ectomycorrhizal fungus. In this study, we standardized the optimal conditions such as mycelial age, temperature, pH and osmotic stabilizers for the release and regeneration of protoplasts in L. fraterna. Maximum number of protoplasts (5.1 × 108) was isolated from 4 day old mycelia suspended in an osmotically stabilized MMC buffer (pH 5.0) with 0.5 M man- nitol and 10 mg/mL Novozyme 234. Maximum yields of protoplasts were released from the mycelium using Novozyme 234 after 3 h. Protoplasts exhibited two kinds of regeneration patterns in liquid media and one in solid medium. Almost all the protoplasts were nucleated and viable as observed by acridine orange and fluorescein diacetate staining. The regeneration frequency was as high as 36% under optimal conditions. When colonies from regenerated protoplasts were inoculated with Eucalyptus globulus, few plants showed ectomycorrhizal association. Results of this study indicate that this fungus could be potentially used in transformation, protoplast fusion and other genetic studies.