Volume 12 Part 1 Article 15
Title: Isolation and Reversion of Protoplasts from Homokaryotic Mycelium of Agrocybe aegerita
Authors: T. Noel and J. Labarere
Conditions affecting the release of protoplasts and their reversion into hyphal form were examined in the edible Basidicntycete Agrocybe aegerita. Protoplasts will be useful in order to work out gene transfer through cell fusion and transformation procedures.
In Basidicmycetes, protoplasts are obtained by the enzymatic digestion of the cell wall in an appropriate osmotic stabilizer. Under these conditions, protoplasts have been previously isolated from Schizophyllum commune (de Vries O.M.H., Wessels J.G.H. 1972), Coprinus cinereus (Moore D. 1975; Yanagi et al. 1985), Lentiinus edodes (Ushiyama R., Nakai Y. 1977; Soon Woo H., Yoon Y. 1985), Tricholona matsutake (Abe et al. 1982), Volvariella volvacea (Santiago C M . 1982), Volvariella banbycina (Stille B. 1984), Collybia velutipes and Pleurotus ostjreatus (Yamada et al. 1983), Pleurotus ostreatus and Pleurotus sp. cfr. Florida (Yoo et al. 1984), Pleurotus comucopiae (Wakabayashi et al. 1985).
Comparison between the methods used and the results obtained by the different authors shows that no standard conditions are existing in fungi for isolating protoplasts suited for reversion. As isolation of protoplasts has never been described in Agrocybe aegerita, a method was established for ob1:aining protoplasts frcm vegetative mycelium.
The ccranercial enzymatic preparation Novozym 234, produced by submerged fermentatJ-on of Trichoderma harzianum, was used in this study. The effect of several parameters such as enzymatic concentration, digestion period and the pH of incubation buffer on protoplast yield were first determined. Then, sctne factors influencing reversion of protoplasts were studied. We particularly investigated the effect of the conditions in which protxjplasts were released, mainly enzymatic concentration and incubatJ.on buffer pH, on the protoplast reversion capacity. Finally, several culture media and plating methods were used to irrprove the reversion percentage of protoplasts.Please login to download the PDF for this proceeding.