Factors Analysis In Protoplast Isolation And Regeneration From A Chalkbrood Fungus, Ascosphaera Apis

Current need for genetic engineering for Ascosphaera apis and absence of reports have initiated us to target development of an efficient and reproducible protocol to make this fungus amenable to genetic studies and transformation. The fungus was isolated from chalkbrood mummies and checked for its identity. Different enzymolysis and osmotic pressure stabilizing agents along with different growth mediums, incubation periods, pH and temperature have been utilized for isolation and regeneration of protoplasts. The fungus demonstrated varying responses in terms of yield and regeneration rates to different factors tested. Liquid growth medium and shorter incubation periods has yielded the highest isolated protoplast number (34.00x10(5) mL(-1)) while use of 50mg mL(-1) driselase was the best enzymolysis agent, yielded 98.36 x 10(5) mL(-1) of protoplasts at 5.8 pH and 28 degrees C. Exponentially growing mycelial culture provided the highest viability (90%). Citric acid-monohydrate with NaCl (0.8 mole L-1) as osmotic stabilizer and 240 min of enzymolysis time have supported 53.06% protoplast regeneration, which is the first and highest to be reported for a fungus. With this first time reported protocol, viable protoplasts were obtained and regenerated successfully from A. apis. Thus, we believe, an important foundation has been set for efficient genetic manipulation of this fungus. (C) 2014 Friends Science Publishers