Protoplast formation

Protoplasts have been liberated in vitro from a wide variety of fungi (Davis, 1985; Peberdy and Ferenczy, 1985), including several ectomycor-rhizal basidiomycetes (Kropp and Fortin, 1986; Hebraud and Fevre, 1988; Barrett et al., 1989). The absence of mitotic spores from ecto-mycorrhizal species requires that mycelial fragments serve as starting material for protoplast formation with these fungi.

Commercially available enzyme mixtures, such as Novozyme 234 or Caylase C3, are generally effective for protoplast generation with fungi. These lytic enzyme preparations include principally /J-1,3 and /3-l,4 glucanases, a-1,3 and a-1,4 glucanases, as well as chitinase and glucuronidase activities. Preparations may also contain proteinase activities detrimental to stability of liberated protoplasts. Care must be taken in evaluating incubation in the enzyme mixtures for efficiency in yielding stable and regenerative protoplasts.

Optimal conditions for protoplast formation and maintenance, involving enzyme concentration/exposure time, culture age, pH and osmotic conditions of buffers/media, vary with individual species. Once the cell wall is removed, an osmotic stabilizer, such as 0.6 m KC1, 1.2 m sorbitol or 0.5 m mannitol, must be present both in maintenance buffers and in regeneration media, at least until some resynthesis of cell wall material has occurred (Tilburn et al., 1983; Peberdy and Ferenczy, 1985; Barrett et al., 1989). Regeneration rates also vary and among ectomycorrhizal fungi are low, often only 1-5%, with yields of the order of 107 viable protoplast regenerates per gram fresh weight of mycelia (Kropp and Fortin, 1986; Hebraud and Fevre, 1988; Barrett et al., 1989).

The ability to form and regenerate large numbers (> 106) of protoplasts is an incentive for attempting transformation with any fungus, provided a suitable vector employing a selectable marker gene is available. To date, a few species of the genera Laccaria and Hebeloma are the only ectomycorrhizal fungi among several evaluated for protoplast yield/regeneration with potential for such experimentation (Kropp and Fortin, 1986; Hebraud and Fevre, 1988; Barrett et al., 1989). The only vectors presently available for such experimentation, however, are those developed for studies involving other filamentous fungi, primarily ascomycetous species.

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