Factors affecting protoplast formation by Rhizoctonia solani

Abstract

Novozym 234 was the most frequently used enzyme for production of Rhizoctonia solani protoplasts. Since manufacture of this enzyme was discontinued in the late 1990s, a new procedure was developed by testing lytic enzymes from Sigma and by examining factors affecting protoplast formation. The combination of 20 mg/mL Driselase and 10mg/mL lysing enzyme was effective in releasing protoplasts from R. solani. The optimal condition for enzyme treatment of mycelium was incubation at 37 degrees C for 15 min followed by 34 degrees C for 105 min. The amount of protoplasts produced was positively correlated with growth rate and negatively correlated with mycelial density. Under favorable conditions, R. solani mycelia released 1.68 x 10(6) protoplasts/mL that is comparable with that produced with Novozym 234. Among various media tested, the best solid medium for protoplast regeneration was 1% V-8 juice agar, while the best liquid medium was 10% potato dextrose broth.

Protoplast Transformation for Genome Manipulation in Fungi

“…This preparation contains β-glucanase, cellulase, protease, and chitinase activities. Lysing enzymes from Rhizoctonia solani , apart from β-(1-3)-glucanases, also contain protease, pectinase, and amylase activities (Liu et al 2010 ). Also Driselase from Basidiomycetes sp.…”

“…Also Driselase from Basidiomycetes sp. has been used for digestion of arabinoxylans by fungal glycanases (Liu et al 2010 ). This commercial preparation contains laminarinase, xylanase, and cellulase (Product from Sigma-Aldrich).…”

“…PDA (Potato Dextrose Agar) was the medium used for Lentinus lepideus protoplast preparation (Kim et al 2000 ). Rhizoctonia solani was grown in V8 juice agar (Liu et al 2010 ), P. brasiliensis in PYG (peptone, yeast extract, and glucose) medium (De Borba et al 1994 ), and Pseudozyma fl occulosa in YMPD (yeast extract, malt extract, peptone, and dextrose) (Cheng and Belanger 2000 ) for protoplast preparation. S. nodorum , C. sativus , P. teres , and C. beticola are grown in Fries medium (Liu and Friesen 2012 ).…”

“…For U. maydis cellulases and the commercial product Driselase (enzyme mixture containing cellulases, pectinase, laminarinase, xylanase, and amylase) were used for cell wall digestion (Waard 1976 ). Likewise, for cell wall digestion of R. solani (Liu et al 2010 ) and F. graminearum (Goswami 2012 ), a combination of lysing enzymes and Driselase were used. For S. nodorum , C. sativus , P. teres , and C. beticola a combination of β-1,3 glucanase and Driselase (from Sigma-Aldrich) is used for cell wall digestion (Liu and Friesen 2012 ).…”

“…Other alternatives are 0.5 M mannitol which was applied for protoplasting of C. cinerea (Binninger et al 1987 ) and sucrose for Podospora sp. (Brygoo and Debuchy 1985 ), L. lepideus (Kim et al 2000 ), P. fl occulosa (Cheng and Belanger 2000 ), and R. solani (Liu et al 2010 ) as osmotic stabilizers. For T. virens , sorbitol is the osmotic stabilizer of choice for protoplast preparation (Catalano et al 2011 ).…”