Abstract
Protoplasts of the ectomycorrhizal ascomycete Cenococcum geophilum were isolated from mycelium grown in liquid medium. The method was optimized with regard to culture conditions, preincubation, lytic enzyme system, pH value of the incubation medium, osmotic buffer and incubation temperature for C. geophilum strains SIV and 1448. The yields were 1-3·108 and 7·106 protoplasts per gram fresh weight for C. geophilum SIV and C. geophilum 1448, respectively. Protoplasts from C. geophilum SIV exhibited plasma membrane integrity close to 100% (fluorescein diacetate staining). At least 50% of the protoplasts contained a nucleus (staining with acridine orange). The regeneration of protoplasts from C. geophilum is described for the first time. The regeneration frequency was up to 13%, and, dependent on the conditions of culture (liquid medium, agarose, agar), four types of regeneration patterns could be distinguished Regenerated protoplasts of C. geophilum were capable of forming mycorrhizas with spruce (Picea abies) seedlings.
Similar content being viewed by others
References
Annè J, Eyssen H, De Somer P (1974) Formation and regeneration of Penicillium chrysogenum protoplasts. Arch Microbiol 98:159–166
Anunciacao CE, Guimaraes WV, Araujo EF, Muchovej RMC, e Souza DRN (1990) Production and regeneration of protoplasts of Pisolithus tinctorius. Biotechnol Tech 4:215–220
Barrett V, Lemke PA, Dixon RK (1989) Protoplast formation from selected species of ectomycorrhizal fungi. Appl Microbiol Biotechnol 30:381–387
Benitez T, Villa TG, Notario V, Garcia Acha I (1976) Studies of walls of Trichoderma viride using fluorescent brighteners. Trans Br Mycol Soc 67:485–489
Bull TA (1970) Inhibition of polysaccharases by melanin: enzyme inhibition in relation to mycolysis. Arch Biochem Biophys 137:345–356
Chen XY, Hampp R (1993a) Sugar uptake by protoplasts of the ectomycorrhizal fungus Amanita muscaria. New Phytol 125:601–608
Chen XY, Hampp R (1993b) Isolation and regeneration of protoplasts from gills of Agaricus bisporus. Curr Microbiol 26:307–312
Farquhar ML, Peterson RL (1990) Induction of protoplast formation in the ectomycorrhizal fungus Paxillus involutus by the root rot pathogen Fusarium oxysporum. New Phytol 116:107–113
Fowke LC, Gamborg OL (1980) Applications of protoplasts to the study of plant cells. Int Rev Cytol 68:9–51
Guttenberger M, Hampp R (1992) Ectomycorrhizines — symbiosis-specific or artifactual polypeptides from ectomycorrhizas? Planta 188:129–136
Hacskaylo E, Palmer JG, Vozzo JA (1965) Effect of temperature on growth and respiration of ectotrophic mycorrhizal fungi. Mycologia 57:748–756
Harley FR, Smith SE (1983) Mycorrhizal symbiosis. Academic Press, London
Hashiba T (1991) Isolation of fungal protoplasts. In: Arora DK, Elander RP, Mukerji KG (eds) Handbook of applied mycology, vol 4: Fungal biotechnology. Dekker, New York, pp 129–149
Hébraud M, Fèvre M (1988) Protoplast production and regeneration from mycorrhizal fungi and their use for isolation of mutants. Can J Microbiol 34:157–161
Hutchison LJ (1991) Description and identification of cultures of ectomycorrhizal fungi found in North America. Mycotaxon 42:387–504
Jang JC, McElreath SD, Tainter FH (1993) A membrane technique for producing protoplasts of Cryphonectria parasitica. Curr Microbiol 31:93–96
Kottke I, Oberwinkler F (1986) Mycorrhiza of forest trees — structure and function. Trees 1:1–24
Kottke I, Guttenberger M, Hampp R, Oberwinkler F (1987) An in vitro method for establishing mycorrhizae on coniferous tree seedlings. Trees 1:191–194
Kropp R, Fortin JA (1986) Formation and regeneration of protoplasts from the ectomycorrhizal basidiomycete Laccaria bicolor. Can J Bot 64:1224–1226
Maheshwari SC, Gill R, Maheshwari N, Gharyal PK (1986) Isolation and regeneration of protoplasts from higher plants. In: Reinert J, Bindin H (eds) Differentiation of protoplasts and transformed plant cells. Springer, Berlin Heidelberg New York, pp 3–36
Mikola P (1948) On the physiology and ecology of Cenococcum graniforme. Commun Inst For Fenn 36:1–104
Nagata T, Takebe I (1970) Cell wall regeneration and cell division in isolated tobacco mesophyll protoplasts. Planta 99:12–20
Niederer M (1989) Ektomykorrhiza von Bestandesfichten: die jahreszeitliche Dynamik löslicher Kohlenhydrate und ihre Bedeutung als Vitalitätsindikatoren. PhD thesis, University of Basel
Park JY (1970) A change in color of aging mycorrhizal roots of Tilia americana formed by Cenococcum graniforme. Can J Bot 48:1339–1341
Peberdy JF (1989) Presidential address: Fungi without coats — protoplasts as tools for mycological research. Mycol Res 93:1–20
Roncal T, Ugalde UO, Barnes J, Pitt D (1991) Production of protoplasts of Penicillium cyclopium with improved viability and functional properties. J Gen Microbiol 137:1647–1651
Sipiczki M, Heyer WD, Kohli J (1985) Preparation and regeneration of protoplasts and spheroplasts for fusion and transformation of Schizosaccharomyces pombe. Curr Microbiol 12:169–174
Sonnenberg AS, Wessels JG, Griensven LJ van (1988) An efficient protoplasting/regeneration system for Agaricus bisporus and Agaricus bitorquis. Curr Microbiol 17:285–291
Taylor BE, Wheeler MH, Szaniszlo PJ (1987) Evidence for pentaketide melanin biosynthesis in dermataceous human fungi. Mycologia 79:320–322
Wallenda T, Wingler A, Guttenberger M, Hampp R (1993) Determination of sugars and sugar alcohols in the ectomycorrhizal fungus Amanita muscaria (L. ex Fr.) Hooker. Plant Physiol Suppl 102:177
Wingler A, Guttenberger M, Hampp R (1993) Determination of mannitol in ectomycorrhizal fungi and ectomycorrhizas by enzymatic micro-assays. Mycorrhiza 3:69–73
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Stülten, C.H., Kong, F.X. & Hampp, R. Isolation and regeneration of protoplasts from the ectomycorrhizal ascomycete Cenococcum geophilum Fr.. Mycorrhiza 5, 259–266 (1995). https://doi.org/10.1007/BF00204959
Issue Date:
DOI: https://doi.org/10.1007/BF00204959