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Isolation and Regeneration of Protoplasts from Laccaria fraterna - an Ectomycorrhizal Fungus


Affiliations
1 Methodist Hospital Research Institute, Houston, TX 77030, United States
2 Baylor College of Medicine, Houston, TX 77030, United States
3 Morgan State University, Baltimore, MD 21251, United States
 

An efficient method for high yields of protoplast isolation and regeneration and viability was achieved in Laccaria fraterna, an ectomycorrhizal fungus. In this study, we standardized the optimal conditions such as mycelial age, temperature, pH and osmotic stabilizers for the release and regeneration of protoplasts in L. fraterna. Maximum number of protoplasts (5.1 × 108) was isolated from 4 day old mycelia suspended in an osmotically stabilized MMC buffer (pH 5.0) with 0.5 M man- nitol and 10 mg/mL Novozyme 234. Maximum yields of protoplasts were released from the mycelium using Novozyme 234 after 3 h. Protoplasts exhibited two kinds of regeneration patterns in liquid media and one in solid medium. Almost all the protoplasts were nucleated and viable as observed by acridine orange and fluorescein diacetate staining. The regeneration frequency was as high as 36% under optimal conditions. When colonies from regenerated protoplasts were inoculated with Eucalyptus globulus, few plants showed ectomycorrhizal association. Results of this study indicate that this fungus could be potentially used in transformation, protoplast fusion and other genetic studies.

Keywords

Ectomycorrhizal Fungus, Laccaria fraterna, Novozyme 234, Protoplasts, Regeneration Patterns
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  • Mahadevan A, Raman N, Natarajan, editors. Mycorrhizae for green Asia. Proceedings: First Asian Conference on Mycorrhizae. Madras, India: Alamu printing works; 1988 Jan 29–31; p. 336–344.

  • Chellappan P, Christy SAA, Mahadevan A. Multiplication of Arbuscular Mycorrhizal Fungi on ischolar_mains. In: Mukerji KG, Manoharachary C, Chamola BP, editors. Techniques in mycorrhizal studies. Dordrecht: Kluwer; 2002.

  • Peberdy JF. Fungi without coats-protoplasts as tools for mycological research. Mycol Res. 1989; 93:1–20.

  • Chadegani M, Brink JJ, Shehata A, Ahmadjian V. Optimization of protoplast formation, regeneration, and viability in Microsporum gypseum. Mycopathol. 1989; 107:33–50.

  • Gold MH, Cheng TM, Alic M. Formation, fusion, and regeneration of protoplasts from the wild type and auxotrophic strains of the white rot basidiomycete Phanerochaete chrysosporium. Appl. Environ. Microbiol. 1983; 46:260–263.

  • De Vries OMH, Wessels JGH. Effectiveness of a lytic enzyme preparation from Trichoderma viride in increasing spheroplasts from fungi, particularly basidiomycetes. Antoine van leeuwenhoek J Microbiol Serol. 1973; 39:397–400.

  • Kropp BR, Fortin JA. Formation and regeneration of protoplasts from the ectomycorrhizal basidiomycete Laccaria bicolor. Can J Bot. 1986; 64:1224–1226.

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

  • Barrett V, Dixon RK. Protoplast formation from selected species of ectomycorrhizal fungi. Appl Microbiol Biotech. 1989; 30:381–387.

  • Barrett V, Dixon RK, Lemke PA. Genetic transformation from selected species of ectomycorrhizal fungi. Appl Microbiol Biotechnol. 1990; 33:313–316.

  • Tommerup IC, Bougher NL, Malajczuk N. Laccaria fraterna, a common ectomycorrhizal fungus with mono- and bi-sporic basidia and multinucleate spores: comparison with the quadristerigmate, binucleate spored Laccaria laccata and the hypogenous relative Hydnangium carneum. Mycol Res. 1991; 95:689–698.

  • Marx DH. The influence of ectotrophic mycorrhizal fungi on the resistance of pine ischolar_mains to pathogenic infections. In,. antagonism of mycorrhizal fungi ischolar_main pathogenic fungi and soil bacteria. Phytopathol. 1969; 59:53–163.

  • Burpee LL, Sanders PL, Cole H, Kim SH. A staining technique for nuclei of Rhizoctonia solani and related fungi. Mycol. 1978; 70:1281–1283.

  • Barak R, Chet I. Determination by fluoresceindiacetate staining of fungal viability during mycoparasitism. Soil Biol Biochem. 1986; 18:315–319.

  • Kasuya MCM, Muchovej RMC, Bellei MM, Borges AC. In vitro ectomycorrhizal formation in six varieties of pine. Forest Ecol. Manage. 1992; 47:127–134.

  • Zamocky M, Ludwig R Peterbauer C, Hallberg BM, Divne C, Nicholls P et al. Cellobiose dehydrogenase-a flavocytochrome from wood-degrading, phytopathogenic and saprotropic fungi. Curr Prot Pep Sci. 2006; 7:255–280.

  • Cao WG, Crawford DL. Purification and some properties of beta-glucosidase from the ectomycorrhizal fungus Pisoli- thus tinctorius Strain SMF. Can J Microbiol. 1993; 39:125–129.

  • Stulten H, Kong FX, Hampp R. Isolation and regeneration of protoplasts from the ectomycorrhizal ascomycete Cenococcum geophilum Fr. Mycorr. 1995; 5: 259–266.

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

  • Díez J. Invasion biology of Australian ectomycorrhizal fungi introduced with eucalypt plantations into the Iberian Peninsula Biol Invasions. 2005; 7:3–15.

  • Read DJ. Mycorrhizas in ecosystems. Experientia. 1991; 47:376–391.

  • Hashiba T. Isolation of fungal protoplasts. In: Arora DK, Elander RP, Mukerji KG, editors. Handbook of applied Mycology. New York: Dekker; 1991.

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

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

  • Chen XY, Hampp R. Sugar uptake by protoplasts of the ectomycorrhizal fungus Amanita muscaria. New Phytol. 1993; 125:601–608.

  • Davis B. Factors influencing protoplast isolation. In. Peberdy JF, Ferenczy L, editors. Fungal protoplasts application in biochemistry and genetics. New York: Marcel Dekker; 1985.

  • Dìas ES, Araujo EF, Guimãraes WV, Muchovej RMC. Production and regeneration of protoplasts from the mycorrhizal fungus Suillus granulates. W J Microbiol Biotechnol. 1996; 12:625–628.

  • Marx DH. Variability in ectmycorrhizal development and growth among isolates of Pisolithus tinctorius as affected by source, age and reisolation. Can J Forest Res. 1981; 11: 168–174.


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  • Isolation and Regeneration of Protoplasts from Laccaria fraterna - an Ectomycorrhizal Fungus

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Authors

S. A. Anithachristy
Methodist Hospital Research Institute, Houston, TX 77030, United States
M. Arunmani
Baylor College of Medicine, Houston, TX 77030, United States
Viji Sitther
Morgan State University, Baltimore, MD 21251, United States

Abstract


An efficient method for high yields of protoplast isolation and regeneration and viability was achieved in Laccaria fraterna, an ectomycorrhizal fungus. In this study, we standardized the optimal conditions such as mycelial age, temperature, pH and osmotic stabilizers for the release and regeneration of protoplasts in L. fraterna. Maximum number of protoplasts (5.1 × 108) was isolated from 4 day old mycelia suspended in an osmotically stabilized MMC buffer (pH 5.0) with 0.5 M man- nitol and 10 mg/mL Novozyme 234. Maximum yields of protoplasts were released from the mycelium using Novozyme 234 after 3 h. Protoplasts exhibited two kinds of regeneration patterns in liquid media and one in solid medium. Almost all the protoplasts were nucleated and viable as observed by acridine orange and fluorescein diacetate staining. The regeneration frequency was as high as 36% under optimal conditions. When colonies from regenerated protoplasts were inoculated with Eucalyptus globulus, few plants showed ectomycorrhizal association. Results of this study indicate that this fungus could be potentially used in transformation, protoplast fusion and other genetic studies.

Keywords


Ectomycorrhizal Fungus, Laccaria fraterna, Novozyme 234, Protoplasts, Regeneration Patterns

References