Protoplast preparation and regeneration from spores of the biocontrol fungus Pseudozyma flocculosa.
This paper describes a specific protocol for yielding and regenerating protoplasts from spores of the recently described powdery mildew biocontrol agent Pseudozyma flocculosa. With this protocol, a large quantity of protoplasts was obtained from beta-mercaptoethanol-pretreated spores in 3-day-old cultures of P. flocculosa grown in YMPD. Enzymatic digestion was optimal with 0.5% Novozym 234 and 5% Glucanex prepared in 0.6 M KCl in 0.1 M citrate buffer. All liberated protoplasts fluoresced in the presence of fluorescein diacetate indicating that viability was nearly 100%. The regeneration rate was equally outstanding reaching 75% when 0.8 M sucrose was used as osmotic stabilizer in the regeneration medium. This protocol will find useful applications in genetic studies of this poorly characterized and understood biocontrol agent.
Cheng
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Centre de Recherche en Horticulture, Département de Phytologie, Université Laval, Quebec, Canada.
Bélanger
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D001487:Basidiomycota / Q000235:genetics / Q000502:physiology*; D004798:Enzymes / Q000378:metabolism; D009172:Mycology / Q000379:methods; D009997:Osmotic Pressure; D010572:Pest Control, Biological; D011523:Protoplasts / Q000502:physiology*; D013172:Spores, Fungal / Q000502:physiology*
D004798:Enzymes; C045154:novozym 234