Skip to main content
Journal of Wood Science

Official Journal of the Japan Wood Research Society

Download PDF

Regeneration of protoplasts from hyphal strands ofVolvariella volvacea

Journal of Wood Science volume 44, pages 401–407 (1998)Cite this article

Abstract

A series of experiments on the preparation and regeneration of protoplasts from hyphal strands ofVolvariella volvacea (Bull. ex. Fr.) Singer were conducted with the aim of optimizing the conditions for its efficient regeneration. One commercial (Vvcl) and two wild (EAAC-0001 and EAAC-0002) strains ofV. volvacea from the Philippines were used and subjected to varying conditions to determine the most efficient means for regeneration of their protoplasts. The effects of age and type of strain, pH, type and concentration of osmotic stabilizer, enzymatic composition, treatment time, temperature, reciprocal frequency during enzymatic lysis of the cell wall, and centrifugation conditions were investigated. Results showed that the three strains ofV. volvacea had varying responses in terms of yield, size, and ability of their protoplasts to regenerate into the protoplast regeneration medium. Among the three strains, EAAC-0002 had the highest rate of regeneration. The 5-day-old culture ofV. volvacea, when subjected to a combination of 2% Novozyme 234 and 0.2% chitinase in 0.6M mannitol (pH 6.0) for 3 h at 30°C, 90 strokes/min and centrifuged at 1100 g for 10 min; produced an efficient yield of protoplasts with a relatively high regeneration rate.

References

  1. Chang ST, Kwan HS, Kang YN (1995) Collection, characterization and utilization of germplasm ofLentinula edodes. Can J Bet (suppl 1) 73:055–061

    Google Scholar 

  2. Hamlyn PF, Ball C (1979) Recombination studies withCephalosporium acremonium. In: Sebek OK, Laskin AI (eds) Genetics of industrial microorganisms. American Society for Microbiology, Washington, DC, pp 185–191

    Google Scholar 

  3. Birkett JA, Hamlyn PF (1985) Protoplast fusion and genetic analysis inCephalosporium In: Peberdy JF, Ferenczy L (eds) Fungal protoplasts: applications in biochemistry and genetics. Marcel Dekker, New York, pp 207–223

    Google Scholar 

  4. De Vries OMH, Wessels HGH (1972) Release of protoplasts fromSchizophyllum commune by combined action of purified α-1,3glucanase and chitinase derived fromTrichoderma viride. J Gen Microbiol 73:13–22

    Article  Google Scholar 

  5. Moore D (1975) Production ofCoprinus protoplasts by use of chitinase or helicase. Trans Br Mycol Soc 65:134–137

    Article  Google Scholar 

  6. Akamatsu K, Kamada T, Takemaru T (1983) Release and regeneration of protoplasts from oidia ofCoprinus cinereus. Trans Mycol Soc Jpn 24:173–184

    Google Scholar 

  7. Gold MH, Chang TM, Alic M (1983) Formation of fusion and regeneration of protoplasts from wild type and auxotrophic strains of the white rot basidiomycetePhanerochaete chrysosporium. Appl Environ Microbiol 46:260–263

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Yoo YB, Byun MO, Go SJ, You CH, Park YH, Peberdy JF (1984) Characteristics of fusion products between Pleurotus ostreatus and Pleurotus Florida following interspecific protoplast fusion. Korean J Mycol 12:164–169

    Google Scholar 

  9. Chang ST, Li GSF, Peberdy IF (1985) Isolation of protoplasts from edible fungi. MIRCEN J Appl Microbiol Biotechnol 1:185–194

    Article  CAS  Google Scholar 

  10. Hong SW, Yeup Y (1985) Formation and regeneration of protoplasts in Lentinus edodes. Mushroom Newslett Tropics 5(4):4–10

    Google Scholar 

  11. Lau WC, Dhillon EKS, Chang ST (1985) Isolation and reversion of protoplasts ofPleurotus sajor caju. MIRCEN J Appl Microbiol Biotechnol 1:345–353

    Article  Google Scholar 

  12. Magae Y, Kakimoto Y, Kashiwagi Y, Sasaki T (1985) Fruiting body formation from regenerated mycelium ofPleurotus ostreatus protoplasts. Appl Environ Microbiol 49:441–442

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Morinaga T, Kikuchi M, Norm R (1985) Formation and regeneration of protoplasts inCoprinus pellucidus andCoprinus cinereus. Agric Biol Chem 49:523–524

    CAS  Google Scholar 

  14. Wakabayashi S, Magae Y, Kashiwagi Y, Sasaki T (1985) Formation of giant protoplasts fromPleurotus cornucopiae by the cell wall lytic enzymes. Appl Microbiol Biotechnol 21:328–330

    Article  CAS  Google Scholar 

  15. Sudo T, Higaki M (1988) Regeneration and fruiting body formation from protoplasts in Agrocybe cylindracea. Mokuzai Gakkaishi 34:181–183

    Google Scholar 

  16. Eguchi F, Tashiro M, Suzuki T, Higaki M (1990) Preparation and regeneration of protoplasts of edible mushrooms (in Japanese). Mokuzai Gakkaishi 34:181–183

    Google Scholar 

  17. Eguchi F, Fukuzumi T, Higaki M (1993) Production of new species of edible mushrooms by protoplast fusion method. II. Analysis of mycelia and basidiospores of a fusant betweenPleurotus ostreatus andAgrocybe cylindracea. (in Japanese). Mokuzai Gakkaishi 39:825–830

    CAS  Google Scholar 

  18. Eguchi F, Leonowicz A, Higaki M, Fukuzumi T (1994) Laccaseless mutants induced by the regeneration of protoplasts ofPleurotus basidiomycetes. Mokuzai Gakkaishi 40:107–110

    CAS  Google Scholar 

  19. Eguchi F, Higaki M (1991) Cytologic observation of the protoplasts of edible mushrooms. I. Preparing process of protoplasts (in Japanese). Mokuzai Gakkaishi 37:859–864

    Google Scholar 

  20. Eguchi F, Higaki M (1991) Preparation and enlargement of protoplasts (in Japanese) Mokuzai Gakkaishi 37:172–176

    Google Scholar 

  21. Eguchi F, Higaki M (1991) Cytologic observation of the protoplasts of edible mushrooms. II. Regenerating process to mycelia from protoplasts (in Japanese). Mokuzai Gakkaishi 37:865–868

    Google Scholar 

  22. Eguchi F, Higaki M (1992) Production of new species of edibe mushrooms by protoplast fusion method. I. Protoplast fusion and fruiting body formation betweenPleurotus ostreaus andAgrocybe cylindracea (in Japanese). Mokuzai Gakkaishi 38:403–410

    CAS  Google Scholar 

  23. Eguchi F, Higaki M (1993) Preparation and regeneration of protoplasts of edible mushrooms. II. The genusPleurotus (in Japanese). Mokuzai Gakkaishi 39:700–709

    Google Scholar 

  24. Eguchi F, Higaki M (1995) Production of new species of edible mushrooms by protoplasts fusion method. III. Protoplast fusion and analysis of the fusant betweenPleurotus sajor caju andMycoleptodonoides anchisonii. Mokuzai Gakkaishi 41:342–348

    Google Scholar 

  25. Eguchi F, Higaki M (1995) The stability of lactase less mutants induced by the preparation of protoplasts and the cell fusion of basidiomycetes (in Japanese). Mokuzai Gakkaishi 41:598–602

    CAS  Google Scholar 

  26. Eguchi F, Higaki M (1995) Pigment less mutant induced by the regeneration of protoplasts and cell fusion of basidiomycetes (in Japanese). Mokuzai Gakkaishi 41:505–510

    Google Scholar 

  27. Higaki M, Eguchi F (1991) Preparation and enlargement of protoplasts of edible mushrooms (in Japanese). Mokuzai Gakkaishi 37:172–176

    Google Scholar 

  28. Liang ZR, Chang ST (1989) A study on intergeneric hybridization betweenPleuroms sajor-caju andSchizophyllum commune by protoplast fusion. Mushroom Sci 12(Part 1):125–137

    Google Scholar 

  29. Toyoda H, Hirai T, Sumiya H, Kawakami Y, Sakamoto M, Ushiyama R (1984) Preparation and cell wall regeneration of protoplasts of Lentinus edodes. Mem Fac Agr Kinki Univ 17:121–130

    Google Scholar 

  30. Santiago CM Jr (1983) Reversion of mycelial protoplasts ofVolvariella volvacea (Bull. ex. Fr.) Singer. Philippine Agric 66:168–175

    Google Scholar 

  31. Santiago CM Jr (1982) Protoplast isolation in the common tropical mushroom (Volvariella volvacea) using microbial enzyme. Kalikasan Philippine J Biol 11:365–371

    CAS  Google Scholar 

  32. Mukherjee M, Sengupta S (1986) Mutagenesis of protoplasts and regeneration of mycelium in the mushroomVolvariella volvacea. Appl Environ Microbiol 52:1412–1414

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Zhang ZG, Li DP, Fang F (1994) Studies on protoplast technology of filamentous fungi. V. Morphological observations of protoplast fusion. Acta Sci Nat Univ Norm Hum 17(3):41–48

    CAS  Google Scholar 

  34. Wang FD, Ye XE, Ye GZ, Xia ZA (1992) Interspecific protoplast fusion of straw mushroom and behavior of regenerated strains. Acta Agric Shanghai 8(1):14–19

    Google Scholar 

  35. Reyes RG, Eguchi F, Iijima T, Higaki M (1997) Isolation and characterization of the regenerated protoplasts of Volvariella volvacea. In: Proceedings development of agribusiness and its impact on agricultural production in SouthEast Asia. NODAI Center for International Programs, Tokyo, pp 481–490

    Google Scholar 

  36. Reyes RG, Eguchi F, Iijima T, Higaki M (1997) Mycelial growth performance of wild strains ofVolvariella volvacea from the Philippines. In: Nakase T, Takeo K (eds) Proceedings Asian International Mycological Congress, Chiba, Japan, p 87

  37. Tschen JSM, Li IF (1994) Optimization of formation and regeneration of protoplasts from biocontrol agents ofTrichoderma species. Mycoscience 35:257–263

    Article  Google Scholar 

  38. Peberdy JF (1987) Development in protoplast fusion in fungi. Microbiol Sci 4:108–114

    CAS  PubMed  Google Scholar 

  39. Pe'er S, Chet I (1991)Trichoderma protoplast fusion: a tool for improving biocontrol agents. Can J Microbiol 36:6–9

    Article  Google Scholar 

  40. De Vries OMH, Wessels HGH (1973) Release of protoplast fromScizophyllum commune by a lyric enzyme preparation fromTrichoderma viride. J Gen Microbiol 76:319–330

    Article  PubMed  Google Scholar 

  41. Anne J, Eyssen H, de Somer P (1974) Formation and regeneration ofPenicillium chrysogenum protoplasts. Arch Microbiol 98:159–166

    Article  CAS  PubMed  Google Scholar 

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

    Article  Google Scholar 

  43. Peberdy JF (1978) Protoplasts and their development. In: Smith JF, Berry D R (eds) The filamentous fungi (vol 3). Edward Arnold, London, pp 119–131

    Google Scholar 

  44. Hocart MJ, Peberdy JF (1989) Protoplast technology and strain selection. In: Whipps JM, Lumsden RD (eds) Biotechnology of fungi for improving plant growth. Cambridge University Press, Cambridge, UK, pp 235–257

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Faculty of Agriculture, Department of Forestry, Tokyo University of Agriculture, 156-8502, Tokyo, Japan

    Renato G. Reyes, Fumio Eguchi, Tomoaki Iijima & Miyato Higaki

Authors
  1. Renato G. Reyes
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Fumio Eguchi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Tomoaki Iijima
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Miyato Higaki
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Reyes, R.G., Eguchi, F., Iijima, T. et al. Regeneration of protoplasts from hyphal strands ofVolvariella volvacea . J Wood Sci 44, 401–407 (1998). https://doi.org/10.1007/BF01130455

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF01130455

Key words

  • Fungal protoplast
  • Mushroom
  • Protoplast technology
  • Volvariella volvacea