Abstract
Optimization of different factors for efficient protoplast release fromMetarhizium anisopliae was investigated. Factors like culture media, age of the mycelium, incubation time, different enzymatic combinations and osmotic stabilizer were studied. Mycelium harvested at 40th h in Sabouraud Dextrose broth showed the best protoplast yield over the other media and age of mycelium tested. An incubation time of 3 h and Lysing enzyme at a concentration of 10 mg/ml was the best among the different enzymatic concentrations and combinations tested and yielded a protoplast release of 7.3×108 protoplasts/ml. The most suitable osmotic stabilizer for efficient protoplast release was 0.7M KCl.
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References
Anjani Kumari J., Panda T. (1992). Studies on critical analysis of factors influencing improved of protoplasts fromTrichoderma reesel mycelium. Enzyme Microb. Technol., 14: 241–248.
Anjani Kumari J., Panda T. (1993). Improved technique on reversion of mycelial protoplast ofTrichoderma reesei into mycelia. Bioprocess Eng., 8: 287–294.
Chawla H.S. (2002). Introduction to Plant Biotechnology. Science Publishers, USA.
Elbouami F. (2001). Isolation and regeneration of protoplasts from mycelium ofFusarium solani. Afr. Crop Sci. J., 9: 351–358.
Hamlyn P.F., Bradshaw R.E., Mellon F.M., Santiago C.M., Wilson J.M., Peberdy J.F. (1981). Efficient protoplast isolation from fungi using commercial enzymes. Enzyme Microb. Technol., 3: 321–325.
Inglis D.G., Goettel M.S., Butt T.M., Strasser H. (2001). Use of Hyphomyecete fungi for managing insect pests. In: Butt T.M., Jackson C.W., Magan N., Eds., Fungi as Biocontrol Agents-Progress Problems and Potential, 1st edn., CAB International Wallingford, UK.
Kawula T.H., Ling A.J. (1984). Production of protoplasts fromBeauveria bassiana blastospores. J. Invert. Pathol., 43: 282–284.
Kim B.K., Kang, J.H., Jin M., Shim M.J., Choi E.C. (2000). Mycelial protoplast isolation and regeneration ofLentinus lepideus. Life Sci., 66: 1359–1367.
Kitamoto Y., Mori N., Yamamoto M., Ohiwa T., Ichikawa Y. (1988). A simple method for protoplast formation and improvement of protoplast regeneration from various fungi using an enzyme fromTrichoderma harzianum. Appl. Microbiol. Biotechnol., 28: 445–450.
Lau W.C., Dhillon E.K.S., Chang S.T. (1985). Isolation and reversion of protoplasts ofPleurotus sajor-caju. World J. Microb. Biotech., 1: 345–353.
Peberdy J.F. (1994). Protein secretion in filamentous fungi — trying to understand a highly productive black box. Trends Biotechnol., 12: 50–57.
Pfeifer T.A., Khachatourians G.G. (1987). The formation of protoplasts fromBeauveria bassiana. Appl. Microbiol. Biotechnol., 26: 248–253.
Robinson H.L., Deacon J.W. (2001). Protoplast preparation and transient transformation ofRhizoctonia solani. Mycol. Res., 105: 1295–1303.
Silveria W.D. (1986). Protoplast fusion and genetic recombiantion inMetarhizium anisopliae. Enzyme Microb. Technol., 9: 149–152.
Thakur R., Sandhu S.S. (2003). Development of transformation system for entomopathogenic fungusMetarhizium anisopliae (ENT 12) based oncnx — gene. Indian J. Microbiol., 43: 187–192.
Yanai K., Horiuchi H., Yano K. (1990). Preparation of protoplasts ofRhizopus niveus and their transformation with plasmid DNA. Agric. Biol. Chem., 54: 2689–2696.
Zhou X., Wei Y., Zhu H., Wang Z., Lin J., Liu L., Tang K. (2008). Protoplast formation, regeneration and transformation from the taxol-producing fungusOzonium sp. Afr. J. Biotechnol., 7: 2017–2024.
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Dhar, P., Kaur, G. Optimization of different factors for efficient protoplast release from entomopathogenic fungusMetarhizium anisopliae . Ann. Microbiol. 59, 183–186 (2009). https://doi.org/10.1007/BF03175619
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DOI: https://doi.org/10.1007/BF03175619