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Optimization of protoplast formation, regeneration, and viability in Microsporum gypseum

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Abstract

Factors affecting high yields, regeneration frequencies, and viability of protoplasts from clonal cultures of Microsporum gypseum were investigated. Maximum yields of protoplasts were obtained after 6 hrs digestion of 2–4 days old mycelium with Novozyme 234 using CaCl2 (0.4 M) as an osmotic stabilizer and glycine + HCl (pH 4.5) as the buffer system. Mercaptoethanol + dithiothreitol (0.01 M) proved to be the best pretreatment of mycelium prior to digestion with enzyme. A regeneration frequency of 94.4% was obtained using the top agar method with complete medium (pH 6.5) containing 0.5% agar and 0.4 M CaCl2 as an osmoticum. Colonies from regenerated protoplasts on medium containing CaCl2 were pigmented and completely powdery with high sporulation. Protoplast viability was studied in osmotic stabilizer supplemented with glucose or glutamine. After 24 hrs, glucose (2%) and glutamine (2%) enhanced protoplast viability by 22% and 23%, respectively. Protein synthesis, as measured by 3H-lysine uptake, matched the viability profile determined by fluorescence microscopy.

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References

  1. Adriana M, Klooster Ziel D, Sietsma JH, Wouters JTM. Formation and regeneration of Geotrichum candidum protoplasts. J Gen Microbiol 1973; 74: 205–209.

    Google Scholar 

  2. Ahmadjian V. The Lichen Symbiosis. Waltham, Massachusetts: Blaisdell Publishing Co, 1967.

    Google Scholar 

  3. Ahmadjian V, Chadegani M, Koriem AM, Paracer S. DNA and protoplast isolation from lichen and lichen symbionts. Lichen Physiol Biochem 1987; 2: 1–11.

    Google Scholar 

  4. Anne J. Protoplasts of filamentous fungi in genetics and metabolite production. Experientia 1983; 46: 167–178.

    Google Scholar 

  5. Anne J, Eyssen H, DeSomer P. Formation and regeneration of Penicillium chrysogenum protoplasts. Arch Microbiol 1974; 98: 159–166.

    Google Scholar 

  6. Anne J, Eyssen H, DeSomer P. Somatic hybridization of Penicillium roquefortii and Penicillium chrysogenum after protoplast fusion. Nature 1978; 719–721.

  7. Anne J, Peberdy JF. Conditions for induced fusion of fungal protoplasts in polyethylene glycol solutions. Arch Microbiol 1975; 105: 201–205.

    Google Scholar 

  8. Anne J, Peberdy JF. Induced fusion and fungal protoplasts following treatment with polyethylene glycol. J Gen Microbiol 1976; 92: 413–417.

    Google Scholar 

  9. Arnold WN, Garrison RG. Isolation and characterization of protoplasts from Saccharomyces rouxii. J Bact 1979; 137: 1386–1394.

    Google Scholar 

  10. Benitez T, Ramos S, Garcia Acha I. Protoplasts from Trichoderma viride. Arch Microbiol 1975; 103: 199–203.

    Google Scholar 

  11. Berliner MD, Reca ME. Protoplasts of systemic dimorphic fungal pathogens: Histoplasma capsulatum and Blastomyces dermatitidis. Mycopathol 1969; 37: 81–85.

    Google Scholar 

  12. Billich A, Keller U, Kleinkauf H, Zocher R. Production of protoplasts from Fusarium scirpi by lytic enzymes from Streptomyces tsusimaensis. Appl Microbiol Biotechnol 1988; 28: 442–444.

    Google Scholar 

  13. Campbell MC, Stewart JL. The medical mycology handbook. New York: John Wiley and Sons, 1980.

    Google Scholar 

  14. Chaffin WL. Toward gene manipulation with selected human fungal pathogens. In: Bennett JW, Lasure LL, eds. Gene Manipulation in Fungi. New York: Academic Press 1985; 469–487.

    Google Scholar 

  15. Cordy RA, Brink JJ. The effect of nitrous oxide on protein methylation in the developing rat optic nerve. Ann NY Acad Sci 1984; 435: 396–398.

    Google Scholar 

  16. Crowe HM, Levitz SM, Sugar AM. Rapid method for the production of Blastomyces dermatitidis protoplasts. Exp Mycology 1987; 11: 159–161.

    Google Scholar 

  17. Davis B. Factors influencing protoplast isolation. In: Peberdy JF, Ferenczy L. Fungal Protoplasts: Application in Biochemistry and Genetics. New York: Marcel Dekker 1985; 45–71.

    Google Scholar 

  18. DeVries OMH, Wessels JGH. Release of protoplasts from Schizophyllum commune by a lytic preparation from Trichoderma viride. J Gen Microbiol 1972; 73: 13–22.

    Google Scholar 

  19. Eyssen H. Formation of protoplasts of Penicillium chrysogenum and some other filamentous fungi. Agricultura 1977; 25: 21–44.

    Google Scholar 

  20. Ferenczy L. Current questions of gene transfer via protoplast fusion in microorganisms. Experientia 1983; 46: 137–142.

    Google Scholar 

  21. Ferenczy L, Kevei F, Szegedi M. Increased fusion frequency of Aspergillus nidulans protoplasts. Experientia 1975; 31: 50–52.

    Google Scholar 

  22. Ferenczy L, Kevei F, Szegedi M, Franko A, Rohik I. Factors affecting high frequency fungal protoplast fusion. Experientia 1976; 32: 1156–1158.

    Google Scholar 

  23. Ferenczy L, Kevei F, Zsolt J. Fusion of fungal protoplasts. Nature 1974; 248: 793–794.

    Google Scholar 

  24. Ferrer S, Ramon D, Vicente E. An improved method for the production of protoplasts of Podospora anserina. Experientia 1983; 45: 304–305.

    Google Scholar 

  25. Fukui K, Sagara Y, Yoshida N, Matsuoka T. Analytical studies on regeneration of protoplasts of Geotrichum candidum by quantitative thin-layer-agar plating. J Bact 1969; 98: 256–263.

    Google Scholar 

  26. Garcia Acha I, Lopez-Belmonte F, Villanueva JR. Regeneration of mycelial protoplasts of Fusarium culmorum. J Gen Microbiol 1966; 45: 515–523.

    Google Scholar 

  27. Gascon S, Ochoa AG, Novaes M, Villanueva JR. Lytic action of strepzymes from Micromonospora AS Arch Mikrobiol 1965; 51: 156–167.

    Google Scholar 

  28. Gaugy D, Fevre M. Protoplast production from Saprolegnia monoica. Microbios 1982; 34: 89–98.

    Google Scholar 

  29. Gaugy D, Fevre M. Regeneration and reversion of protoplasts from different species of Penicillium. Microbios 1985; 44: 285–293.

    Google Scholar 

  30. Gillian T, Garber RC, Yoder OC. Transformation of the fungal maize pathogen Cochliobolus heterostrophus using the Aspergillus nidulans amdS gene. Mol Gen Genetics 1985; 201: 450–453.

    Google Scholar 

  31. Hamlyn PF, Bradshaw RE, Mellon FM, Santiago CM, Wilson JM, Peberdy JF. Efficient protoplast isolation from fungi using commercial enzymes. Enzyme Microb Technol 1981; 3: 321–325.

    Google Scholar 

  32. Hebraud M, Fevre M. Protoplast production and regeneration from mycorrhizal fungi and their use for isolation of mutants. Can J Microbiol 1988; 34: 157–161.

    Google Scholar 

  33. Homolka L, Vyskocil P, Pilat P. Use of Protoplasts in the improvement of filamentous fungi: Mutagenization of protoplasts of Oudemansiella mucida. Appl Microbiol Biotechnol 1988; 26: 166–169.

    Google Scholar 

  34. Ishikawa F, Oishi K. Formation and regeneration of protoplasts from Conidiobolus lamprauges. J Gen Microbiol 1985; 131: 3311–3316.

    Google Scholar 

  35. Jacobsen T, Jensen B, Olsen J, Allermann K. Preparation of protoplasts from mycelium and arthroconidia of Geotrichum candidum. Can J Microbiol 1985; 31: 93–96.

    Google Scholar 

  36. Kevei F, Peberdy JF. Further studies on protoplast fusion and interspecific hybridization within the Aspergillus nidulans group. J Gen Microbiol 1984; 130: 2229–2236.

    Google Scholar 

  37. Lenhart K, Hejtmanek M, Hejtmankova N, Kunert J. Biochemical mutants of Microsporum gypseum. Acta Univ Olomuc Fact Med 1972; 63: 115–130.

    Google Scholar 

  38. Mellon FM, Peberdy JF, MacDonald KD. Hybridization of Penicillium chrysogenum and Penicillium baarnense by protoplast fusion: genetics and biochemical analysis. Experientia 1983; 45: 310–311.

    Google Scholar 

  39. Musilkova M, Fencl Z. Formation of protoplasts in Aspergillus niger. Folia Microbiol 1966; 11: 472–474.

    Google Scholar 

  40. Musilkova M, Fencl Z. Some factors affecting the formation of protoplasts in Aspergillus niger. Folia Microbiologica 1968; 13: 235–239.

    Google Scholar 

  41. Necas O. The mechanism of regeneration of yeast protoplasts. I. Physical conditions. Folia Biol 1962; 8: 256–261.

    Google Scholar 

  42. Nombela C, Pomes R, Gil C. Protoplasts fusion hybrids from Candida albicans morphological mutants. CRC Crit Rev Microbiol 1987; 15: 79–85.

    Google Scholar 

  43. Ogawa K, Ohara H, Toyama N. Intraspecific hybridization of Aspergillus awamori var. Kawachi by protoplast fusion. Agric Biol Chem 1988; 52: 337–342.

    Google Scholar 

  44. Peberdy JF. Fungal protoplasts: Isolation, reversion, and fusion. Ann Rev Microbiol 1979; 33: 21–39.

    Google Scholar 

  45. Peberdy JF. Wall biogenesis by protoplasts. In: Burnett JH, Trinci APJ, eds. Fungal Walls and Hyphal Growth. London: Cambridge University Press 1979; 49–70.

    Google Scholar 

  46. Peberdy JF. Genetic recombination in fungi following protoplast fusion and transformation. In: Smith JE, eds. Fungal Differentiation. New York: Marcel Dekker 1983; 559–581.

    Google Scholar 

  47. Peberdy JF, Buckley CE, Daltrey DC, Moore PM. Factors affecting protoplast release in some filamentous fungi. Trans Br Mycol 1976; 67: 23–26.

    Google Scholar 

  48. Peberdy JF, Gibson RK. Regeneration of Aspergillus nidulans protoplasts. J Gen Microbiol 1971; 69: 325–330.

    Google Scholar 

  49. Pfeifer TA, Khachatourians GG. The formation of protoplasts from Beauveria bassiana. Appl Microbiol Biotechnol 1987; 26: 248–253.

    Google Scholar 

  50. Picataggio SK, Schamhart DHJ, Montenecourt BS, Eveleigh DE. Sphaeroplast formation and regeneration in Trichoderma reesei. Eur J Appl Microbiol Biotechnol 1983; 17: 121–128.

    Google Scholar 

  51. Price JS, Storck R. Production, purification and characterization of an extracellular chitosanase from Streptomyces. J Bact 1975; 124: 1574–1585.

    Google Scholar 

  52. Quigley DR, Taft CS, Strak T, Selitrennikoff CP. Optimal conditions for the release of protoplasts of Neurospora using novozyme 234. Exp Mycology 1987; 11: 236–240.

    Google Scholar 

  53. Ramirez-Leon IF, Ruiz-Herrera. Hydrolysis of walls and formation of Sphaeroplasts in Mucor rouxii. J Gen Microbiol 1972; 72: 281–290.

    Google Scholar 

  54. Rhodes JC, Kwon-Chung KJ. A new efficient method for protoplast formation in Cryptococcus neoformans. Abstr Annu Meet Am Soc Microbiol 1982, p. 336.

  55. Rhodes JC, Kwon-Chung K. Production and regeneration of protoplasts from Cryptococcus. Sabouraudia 1985; 23: 77–89.

    Google Scholar 

  56. Sallen E, Gay L, Jouffre E. Formation and regeneration of protoplasts from Fusarium oxysporum and other carnation pathogen. Microbios 1988; 53: 71–81.

    Google Scholar 

  57. Scott EM, Gorman SP, Wright LR. Ultrastructure of protoplasts from mycelium and microconidia of Trichophyton mentagrophytes. J Med Veter Mycology 1985; 23: 31–36.

    Google Scholar 

  58. Sietsma JH, de Boer WR. Formation and regeneration of protoplasts from Pythium 2142. J Gen Microbiol 1973; 74: 211–217.

    Google Scholar 

  59. Smith PK, Krohu RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC. Measurement of protein using bicinchoninic acid. Anal Biochem 1985; 150: 76–85.

    Google Scholar 

  60. Srikantha J, Ramananda Rao G. A method for isolation of protoplasts from dermatophytes. J Gen Microbiol 1984; 130: 1503–1506.

    Google Scholar 

  61. Thomas KR, Davis B. The effect of calcium on protoplast release from species of Aspergillus. Microbios 1980; 28: 69–80.

    Google Scholar 

  62. Van Heeswijck R. The formation of protoplasts from Mucor species. Carlsberg Res Commun 1984; 49: 597–609.

    Google Scholar 

  63. Villanueva JR, Garcia Acha I. Production and use of fungal protoplasts. In: Booth C, ed. Methods in Microbiology. New York: Academic Press, 1971; 4: 665–718.

    Google Scholar 

  64. Wessels JGH, Van der Valk P, de Vries OMH. Wall synthesis by fungal protoplasts. In: Peberdy JF, Rose AH, Rogers HJ, Cocking EC, eds. Microbial and Plant Protoplasts. New York: Academic Press 1976; 267–281.

    Google Scholar 

  65. Widholm JM. The use of fluorescein diacetate and phenosafranine for determining viability of cultured plant cell. Stain Technology 1972; 47: 189–194.

    Google Scholar 

  66. Yabuki M, Kasai Y, Ando A, Fujii T. Rapid method for converting fungal cells into protoplasts with a high regeneration frequency. Exp Mycology 1984; 8: 386–390.

    Google Scholar 

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  1. Department of Biology, Clark University, 01610-1477, Worcester, MA, USA

    Mostafa Chadegani, John J. Brink, Afaf Shehata & Vernon Ahmadjian

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  1. Mostafa Chadegani
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Chadegani, M., Brink, J.J., Shehata, A. et al. Optimization of protoplast formation, regeneration, and viability in Microsporum gypseum . Mycopathologia 107, 33–50 (1989). https://doi.org/10.1007/BF00437588

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