Fungal Protoplasts

  • J. F. Peberdy
Part of the The Mycota book series (MYCOTA, volume 2)


In the 40 years since the first reported isolation of protoplasts from filamentous or mycelial fungi (Peberdy 1971), these structures have become central to research in several key areas of fungal biology. The switch in focus in genetics from the classical to a molecular approach is the major factor. Two genetic techniques, protoplast fusion and transformation, together with electrophoretic techniques to determine the molecular karyotype of fungi are all dependent on the effective use of protoplasts. As a consequence, other aspects of research that employ protoplasts have benefited more generally. At the beginning, protoplasts held high curiosity value but their scientific potential was not fully realized. However, as the desire to extend the protoplast isolation technique to more and more fungi has occurred, correspondingly more attention has been given to improving this essential process. One outcome has been an overall improvement in protoplast yields for many fungi, which in turn has reawakened the interest of researchers addressing cell biological, physiological, and biochemical questions and led to the adoption of protoplasts as experimental tools wherever relevant.


Mycelial Fungus Protoplast Fusion Trichoderma Reesei Neurospora Crassa Aspergillus Nidulans 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Andrade CM, Oliviera FM, Linardi VR (1992) Candida fennica: enhancement of protoplast formation and fusion. Can J Microbiol 8: 897–810Google Scholar
  2. Anné J, Peberdy JF (1985) Protopast fusion and inter-species hybridization in Penicillium. In: Peberdy JF, Ferenczy L (eds) Fungal protoplasts. Marcel Dekker, New York, pp 259–278Google Scholar
  3. Avram D, Petcu I, Radu M, Dan F, Stan R (1992) Electrically induced protoplast fusion for ergosterol-producing yeast strain improvement. J Basic Microbiol 32: 369–372PubMedCrossRefGoogle Scholar
  4. Barrett V, Lemke PA, Dixon RK (1989) Protoplast formation from selected species of ectomycorrhizal fungi. Appl Microbiol Biotechnol 30: 381–387CrossRefGoogle Scholar
  5. Bennett JW (1985) Protoplasts and secondary metabolism. In: Peberdy JF, Ferenczy L (eds) Fungal protoplasts. Marcel Dekker, New York, pp 189–203Google Scholar
  6. Bigger CH, White MR, Braymer HD (1972) Ultrastructure and invertase secretion of the cot-2 mutant of Neurospora crassa. J Gen Microbiol 71: 159–166Google Scholar
  7. Bos CJ (1985) Protoplasts from fungal spores. In: Peberdy JF, Ferenczy L (eds) Fungal protoplasts. Marcel Dekker, New York, pp 73–86Google Scholar
  8. Bradshaw RE, Peberdy JF (1984) Protoplast fusion in Aspergillus: selection of interspecific heterokaryons using antifungal inhibitors. J Microbiol Methods 3: 27–32CrossRefGoogle Scholar
  9. Brigidi P, Matteuzzi D, Fava F (1988) Use of protoplast fusion to introduce methionine overproduction into Saccharomyces cerevisiae. Appl Microbiol Biotechnol 28: 268–271CrossRefGoogle Scholar
  10. Bulone V, Chanzy H, Gay L, Girard V, Fèvre M (1992) Characterization of chitin and chitin synthase from the cellulosic cell wall fungus Saprolegnia monoïca. Exp Mycol 16: 8–21CrossRefGoogle Scholar
  11. Chun SB, Chin JE, Bai S, An G-H (1992) Strain improvement of Phaffia rhodozyma by protoplast fusion. FEMS Microbiol Lett 93: 221–226CrossRefGoogle Scholar
  12. Daboussi MJ, Gerlinger G (1992) Parasexual cycle and genetic analysis following protoplast fusion in Nectria haematococca. Curr Genet 21: 385–392CrossRefGoogle Scholar
  13. Davis B (1985) Factors influencing protoplast isolation. In: Peberdy JF, Ferenczy L (eds) Fungal protopasts. Marcel Dekker, New York, pp 45–73Google Scholar
  14. Deed A, Seviour RJ, Swift IE (1990) Production of carotenoids by protoplasts of Acremonium diospyri. Enzyme Microb Technol 12: 940–944CrossRefGoogle Scholar
  15. Didek-Brumec M, Gaberc-Porekar V, Alacevié M, Sociè H (1993) Strain improvement of Claviceps Purpurea by protoplast fusion without introducing auxotrophic markers. Appl Microbiol Biotechnol 38: 746–749CrossRefGoogle Scholar
  16. Eggerding C, Randall JA, Sargent ML (1975) An altered invertase in the cot-2 mutant of Neurospora crassa. J Gen Microbiol 89: 102–112PubMedGoogle Scholar
  17. Ferenczy L (1984) Fungal protoplast fusion: basic and applied aspects. In: Beers RE, Bassett EG (eds) Cell fusion: gene transfer and transformation. Raven Press, New York, pp 145–169Google Scholar
  18. Font de Mora J, Herrero E, Sentendreu R (1993) A kinetic study on the regeneration of Candida albicans protoplasts in the presence of cell wall synthesis inhibitors. FEMS Microbiol Lett 111: 43–48Google Scholar
  19. Fungaro MHP, de Azevedo JL, Pizzirani-Kleiner AA (1992) Genetic recombination after protoplast fusion in Candida sp. Rev Bras Genet 15: 499–507Google Scholar
  20. Furlaneto MC, Pizzirani-Kleiner AA (1992) Intraspecific hybridisation of Trichoderma pseudokoningii by anastomosis and protoplast fusion. FEMS Microbiol Lett 90: 191–196CrossRefGoogle Scholar
  21. Gabriel M, Kopeckâ M, Svoboda A (1992) Structural rearrangement of the actin cytoskeleton in regenerating protoplasts of budding yeasts. J Gen Microbiol 138: 2229–2234Google Scholar
  22. Gadau ME, Ling AJ (1992) Protoplast fusion in fungi. In: Arora DK, Elander RP, Mukerji KG (eds) Handbook of applied mycology. Vol 4: Fungal biotechnology. Marcel Dekker, New York, pp 101–128Google Scholar
  23. Gautam SP, Gupta AK, Pandey S (1993) Hybrid construction by fusion of protoplasts of Endomycopsis fibuligera and Saccharomyces cerevisiae. Cytobios 73: 183–188PubMedGoogle Scholar
  24. Hamlyn PF, Bradshaw RE, Mellon FM, Santiago CM, Wilson JM, Peberdy JF (1981) Efficient protoplast isolation form fungi. Enzyme Microb Technol 3: 321–325CrossRefGoogle Scholar
  25. Hinnen A (1985) Some aspects of yeast transformation. In: Peberdy JF, Ferenczy L (eds) Fungal protoplasts. Marcel Dekker, New York, pp 323–336Google Scholar
  26. Hocart MJ, Peberdy JF (1990) Protoplasts and the improvement of fungi used in biological control. In: Whipps JM, Lumsden RD (eds) Biotechnology of fungi for improving plant growth. Cambridge University Press, Cambridge, pp 235–258Google Scholar
  27. Hoh YK, Tan TK, Yeoh H-H (1992) Protoplast fusion of ß-glucosidase producing Aspergillus niger strains. Appl Biochem Biotechnol 37: 81–88PubMedCrossRefGoogle Scholar
  28. Jang J-C, McElreath SD, Tainter FH (1993) A membrane technique for producing protoplasts of Cryphonectria parasitica. Curr Microbiol 26: 113–116CrossRefGoogle Scholar
  29. Jochovâ J, Rupes I, Streiblovâ E (1991) F-actin contractile rings in protoplasts of the yeast Schizosaccharomyces. Cell Biol Int Rep 15: 607–610PubMedCrossRefGoogle Scholar
  30. Jochovâ J, Rupes I, Peberdy JF (1993) The effect of the microtubule inhibitor benomyl on protein secretion in Aspergillus nidulans. Mycol Res 97: 23–27CrossRefGoogle Scholar
  31. Kaul W, Rossow U, Emeis C-C (1993) Screening for microorganisms with cell wall lytic activity to produce protoplast-forming enzymes. Appl Microbiol Biotechnol 39: 574–576CrossRefGoogle Scholar
  32. Kelkar HS, Shankar VS, Deshpande MV (1990) Rapid isolation and regeneration of Sclerotium rolfsii protoplasts and their potential application for starch hydrolysis. Enzyme Microb Technol 12: 510–514CrossRefGoogle Scholar
  33. Kevei F, Peberdy JF (1985) Interspecies hybridization after protoplast fusion in Aspergillus. In: Peberdy JF, Ferenczy L (eds) Fungal protoplasts. Marcel Dekker, New York, pp 241–258Google Scholar
  34. Kida K, Kume K, Morimuar S, Sonoda Y (1992) Repeated-batch fermentation process using a thermotolerant flocculating yeast constructed by protoplast fusion. J Ferment Bioeng 74: 169–173CrossRefGoogle Scholar
  35. Kinoshita Y, Hayase S, Higuchi M, Yamamoto Y, Ahmadjian V, Yoshimura I, Yamada Y (1991) Improvement of protoplast isolation from lichen mycobionts. Agric Biol Chem 55: 1891–1892CrossRefGoogle Scholar
  36. Kiramura K, Imura M, Lee SP, Kato Y, Usami S (1989) Intergeneric hybridization between Aspergillus niger and Trichoderma viride by protoplast fusion. Agric Biol Chem 53: 1589–1596CrossRefGoogle Scholar
  37. Kitamoto Y, Mori N, Yamamoto M, Phiwa T, Ichikawa Y (1988) A simple method for protoplast formation and improvement of regeneration from various fungi using an enzyme from Trichoderma harzianum. Appl Microbiol Biotechnol 28: 445–450CrossRefGoogle Scholar
  38. Kolar H, Mischak H, Kammel WP, Kubicek CP (1985) Carboxymethylcellulase and ß-glucanase secretion by protoplasts of Trichoderma reesei. J Gen Microbiol 131: 1339–1347Google Scholar
  39. Kumari JA, Panda T (1992) Studies on critical analysis of factors influencing improved production of protoplasts from Trichoderma mycelium. Enzyme Microb Technol 14: 241–248CrossRefGoogle Scholar
  40. Kunkel W, Groth I, Jacob H-E, Risch S, Harnisch M, May R, Berg H, Katenkamp U (1987) Electrofusion of protoplasts of Penicillium chrysogenum. Stud Biophys 119: 1–3Google Scholar
  41. Lodder S, Wood D, Gull K (1993) A protoplasting technique with general applicability to molecular karyotyping of hymenomycetes. J Gen Microbiol 139: 1063–1067Google Scholar
  42. Layton AC, Kuhn DN (1988) Heterokaryon formation by protoplast fusion of drug-resistant mutants of Phytopthora megasperrna f.sp. glycinea. Exp Mycol 12: 180–194CrossRefGoogle Scholar
  43. Lein J (1986) The Panlabs penicillin strain improvement programme. In: Vanek Z, Hostalek Z (eds) Overproduction of microbial metabolites: strain improvement and process control strategies. Butterworths, Boston, pp 105–139Google Scholar
  44. May G (1992) Fungal technology. In: Kinghorn JR, Turner G (eds) Applied molecular genetics of filamentous fungi. Blackie Academic & Professional, London, pp 1–27CrossRefGoogle Scholar
  45. Obraztsova IN, Nutsubidze NN, Prabakaran K, Demina VA, Gernet MV (1991) Production and fusion of pro 60 J.F. Peberdy: Fungal Protoplasts toplasts of fungus producers of lignolytic and cellulolytic enzymes, biochemical characteristics of the fusant enzyme complex. Microbiology (Eng! Trans!)Google Scholar
  46. Ogawa K, Brown JA, Wood TM (1987) Interspecific hybridization of Trichoderma reesei QM 9414 by protoplast fusion using colour mutants. Enzyme Microb Technol 9: 229–232CrossRefGoogle Scholar
  47. Peberdy JF (1971) Protoplasts from fungi. Sci Prog (Oxford) 60: 73–86Google Scholar
  48. Peberdy JF (1979a) Fungal protoplasts: isolation, reversion and fusion. Annu Rev Microbio! 33: 21–39CrossRefGoogle Scholar
  49. Peberdy JF (1979b) Wall biogenesis by protoplasts. In: Burnett JH, Trinci APJ (eds) Wall and hyphal growth. Cambridge University Press, Cambridge, pp 49–70Google Scholar
  50. Peberdy JF (1983) Genetic recombination in fungi by protoplast fusion and transformation. In: Smith JE (ed) Marcel Dekker, New YorkGoogle Scholar
  51. Peberdy JF (1985) Mycolytic enzymes. In: Peberdy JF, Ferenczy L (eds) Fungal protoplasts. Marcel Dekker, New York, pp 31–44Google Scholar
  52. Peberdy JF (1987a) Developments in protoplast fusion in fungi. Microbio! Sci 4: 108–111Google Scholar
  53. Peberdy JF (1987b) Genetics of Acremonium. In: Peberdy JF (ed) Penicillium and Acremonium: biotechnology handbooks. Plenum Press, New York, pp 93–111Google Scholar
  54. Peberdy JF (1989) Fungi without coats — protoplasts as tools for mycological research. Mycol Res 93: 1–20CrossRefGoogle Scholar
  55. Peberdy JF (1994) Protein secretion in fungi: trying to understand a highly productive black box. Trends Biotechnol 12: 50–57PubMedCrossRefGoogle Scholar
  56. Peberdy JF, Ferenczy L (1985) Fungal protoplasts. Marcel Dekker, New YorkGoogle Scholar
  57. Peberdy JF, Hocart MJ (1987) Protoplasts as tools in the genetics of plant pathogenic fungi. In: Day PR, Jellis GJ (eds) Genetics and plant pathogenesis. Blackwell, Oxford, pp 127–141Google Scholar
  58. Ridout CJ, Coley-Smith JR, Lynch JM (1993) Fractionation of extracellular enzymes from a mycoparasitic strain of Trichoderma harzianum. Enzyme Microb Technol 10: 180–187CrossRefGoogle Scholar
  59. Roncal T, Ugalde UO, Barnes J, Pitt D (1991) Production of protoplasts of Penicillium cyclopium with improved viability and functional properties. J Gen Microbiol 137: 1647–1651Google Scholar
  60. Sanchez A, Larriba G, Villanueva JR, Villa TG (1980) Glycosylation is not necessary for the secretion of exo-1,3-ß-glucanase by Saccharomyces cerevisiae protoplasts. FEBS Lett 121: 283–286CrossRefGoogle Scholar
  61. Selitrennikoff CP, Bloomfield EC (1984) Formation and regeneration of protoplasts of wild-type Neurospora crassa. Curr Microbiol 11: 113–118CrossRefGoogle Scholar
  62. Sharma VK, Goel R, Johari BN (1988) Isolation, purification and regeneration of protoplasts from Sporotrichium thermophile conidiospores. Biochem Int 17: 895–906PubMedGoogle Scholar
  63. Sienko M, Stepien P, Paszewski A (1992) Generation of genetic recombinants in Trichosporon cutaneum by spontaneous segregation of protoplast fusants. J Gen Microbiol 138: 1409–1412PubMedGoogle Scholar
  64. Sonnenberg ASM, Wessels JGH (1987) Heterokaryon formation in the basidiomycete Schizophyllum commune by electrofusion of protoplasts. Theor Appl Genet 74: 654–658CrossRefGoogle Scholar
  65. Stovall ME (1992) Interstrain spheroplast fusion of Alternaria alternata. Appl Microbiol Biotechnol 37: 480–484CrossRefGoogle Scholar
  66. Tahoun MK (1993) Gene manipulation by protoplast fusion and penicillin production by Penicillium chrysogenum. Appl Biochem Biotechnol 39 /40: 445–453PubMedCrossRefGoogle Scholar
  67. Tamova G, Betina V, Farkas V (1993) An efficient method for producing protoplasts from Trichoderma viride. Folia Microbiol 38: 214–218CrossRefGoogle Scholar
  68. Tashpulatov Zh, Shul’man TS, Baibaev BG, Mirzarakhimova M (1991) Formation and regeneration of protoplasts of the cellulolytically active fungus Trichoderma harzianum 19. Microbiology (Eng! Trans) 60: 377–380Google Scholar
  69. Torres KB, Bruckner B, Meier B (1992) Obtaining mutants for protoplast fusion of gibberellin-forming Gibberella fujikuroi strains. Appl Biochem Biotechnol 33: 83–95PubMedCrossRefGoogle Scholar
  70. Toyama H, Toyama N (1990) Protoplast fusion between Aspergillus oryzae and Aspergillus niger in relation to their multinuclear nature. J Ferment Bioeng 69: 186–188CrossRefGoogle Scholar
  71. Toyama H, Yokoyama T, Shinmyo A, Okada H (1984) Interspecific protoplast fusion of Trichoderma. J Biotechnol 1: 25–35CrossRefGoogle Scholar
  72. Trevithick JR, Metzenberg RL (1966) Molecular sieving by Neurospora cell walls during secretion of invertase isoenzymes. J Bacteriol 92: 1010–1015PubMedGoogle Scholar
  73. Tsukamoto C, Magae Y, Taniguchi H, Sasaki T (1990) Changes in the number of nuclei during formation of giant protoplasts in Pleurotus cornucopiae. J Gen Appl Microbio! 36: 451–454CrossRefGoogle Scholar
  74. Vagvölgyi Cs, Ferenczy L (1991) Isolation of nuclei from Aspergillus nidulans protoplasts. FEMS Microbio! Lett 82: 247–252Google Scholar
  75. Vagvölgyi Cs, Ferenczy L (1992) Transfer of isolated nuclei into protoplasts of Aspergillus nidulans. Experientia 48: 271–272CrossRefGoogle Scholar
  76. Vainstein MH, Peberdy JF (1991) Location of invertase in Aspergillus nidulans: release during hyphal wall digestion and secretion by protoplasts. Mycol Res 97: 1270–1274CrossRefGoogle Scholar
  77. van den Hondel CAMJJ, Punt PJ (1991) Gene transfer systems and vector development. In: Peberdy JF, Caten CE, Ogden JE, Bennett JW (eds) Applied molecular genetics of fungi. Cambridge University Press, Cambridge, pp 1–28Google Scholar
  78. Virk S, Johri BN, Singh SP (1992) Protoplasts from Malbranchea pulchella var. sulfurea: isolation and regeneration. J Gen Appl Microbiol 38: 75–78CrossRefGoogle Scholar
  79. Wangemann M, Schauz K (1989) Release of protoplasts from Ustilago maydis using Novozym 234. Angew Bot 63: 95–101Google Scholar
  80. Wellmann H, Shauz K (1993) DMI-resistance in Ustilago maydis. II. Effect of Triadimeton on regenerating protoplasts and analysis of fungicide uptake. Pestic Biochem Physiol 46: 55–64Google Scholar
  81. Yanagi SO, Monma M, Kawasumi T, Hino A, Kito M, Takebe I (1985) Conditions for isolation of and colony formation by mycelial protoplasts of Coprinus macrorhizus. Agric Biol Chem 49: 171–179CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 1995

Authors and Affiliations

  • J. F. Peberdy
    • 1
  1. 1.Department of BiologyChinese University of Hong KongShatin, New TerritoriesHong Kong

Personalised recommendations