Isolation of protoplasts from edible fungi - Springer Link

MIRCEN Journal, 1985, 1, 185-194

Isolation of protoplasts from edible fungi S. T. Chang*, G. S. F. Li* & J. F. Peberdy*

*Department of Biology, The Chinese University of Hong Kong, Shatin, N. T., Hong Kong, and tDepartment of Botany, The University of Nottingham, Nottingham Received 20 August 1984; accepted 28 December 1984

Introduction

In the last 15 years, the production and consumption of edible fungi have increased rapidly. The world production of 10 commercially cultivated edible fungi in 1981 was estimated to be 1,357,000 tonnes. The common white mushroom (Agaricus bisporus) accounts for about 70% of the total world production, the Shiitake mushroom (Lentinus edodes) for an additional 14%, the straw mushroom (Folvariella volvacea) 4% and the oyster mushroom (Pleurotus spp.) 2.8%. These four mushrooms together make up the bulk of 90% of the world total production (Chang & Miles 1984). Due to the complex sexual patterns and special growth habits found in the higher basidiomycetes (Raper 1966; Chang & Hayes 1978), our understanding of their genetics and breeding mechanism has been less extensive in comparison with both the lower fungi and higher plants. Similarly the application of protoplast exchanges has not been extensively investigated in the edibte fungi. Neurospora crassa was the first filamentous fungus reported to yield protoplasts (Emerson & Emerson 1958; Bachmann & Bonner 1959). Since then, protoplasts have been obtained from many filamentous fungi (Meinecke 1960; Rodriguez Aguirre & Villanueva 1962; Rodriguez Aguirre et al. 1964; De Vries & Wessels 1972, 1973; Fawcett etal. 1973; Anne et al. 1974; Moore 1975) by the use oflytic enzymes which are mostly prepared from a variety of micro-organisms (Peberdy 1976; Peberdy & Isaac 1976). More recently, some commercial preparations of polysaccharases have been reported to be highly effective for the degradation of fungal cell walls (Hamlyn et al. 1981). The extensive literature relating to techniques for the isolation and culture of fungal protoplasts has been reviewed in detail (Peberdy 1979a, b; Ferenczy 1981). Nevertheless, reports of protoplast isolation from edible fungi are few (Hamlyn et al. 1981; Qiu et al. 1982; Santiago 1982a, b) and so far no information on protoplast reversion in this group of fungi is available. Protoplasts are now regarded as a useful tool for genetic manipulations (Peberdy 1980; Ferenczy 1981) which involve fusion leading to somatic hybridization and gene transformation. Genetic recombinations achieved by these methods are usually impossible when intact cells from different specific or generic origins are used. The success in this interesting area of genetic manipulation depends much on the basic work of protoplast isolation and reversion of the organisms concerned. In this study, mycelium from membrane disc and shake liquid cultures were treated with two mycolytic enzymes for protoplast isolation from

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S.T. Chang, G. S. F. Li and J. F. Peberdy

strains of Agaricus bisporus, Auricular& auricula, Lentinus edodes, Pleurotus safor-caju, Volvariella bombycina and V. volvacea. These are all important cultivated edible fungi with the exception of V. bombycina which has the potential to be cultivated commercially in the near future (Huang & Wu 1982; Chang & Miles 1984). Conditions for reversion of these protoplasts were also investigated. Materials and methods

Organisms Agaricus bisporus Ag-23 was given by Dr T. J. Elliott, Glasshouse Crops Research Institute, UK. Lentinus edodes L-13, a dikaryon (originally TM1 830 from Tomioko, Japan) was acquired from Prof. P. G. Miles, the State University of New York at Buffalo, USA and P. sa]or-caju P1-27, a dikaryon, was obtained from Dr Zakia Bano, Central Food Technological Research Institute, Mysore, India. Pleurotus sajor-caju P1-27-9, is a single basidiospore isolate from P1-27 and V. bombycina Vo-1 was obtained from the Institute of Microbiology, Academia Sinica, China, originally coded AS5.164. Agaricus bisporus Ag-17; Au. auricula Au-2, 11. volvacea V-5 and V-35 are collections from S. T. Chang's Laboratory. Media Volvariella bombycina and 11. volvacea were grown on complete agar medium (CM) (Raper & Miles 1958) containing (g/1): MgSO4.7H20, 0.5; KH2PO4, 0.46; K2HPO4, 1.0; Bacto-peptone (Difco), 2.0; D-glucose, 20.0; Bacto-yeast extract (Difco), 2.0; Bacto-agar (Difco), 20.0; thiamine-HC1, 0.0005. All other cultures were grown on malt extract agar medium (ME) containing (g/1): malt extract (Maltzin trocken, hell, diamalt, Miinchen), 12.0; Bacto-agar (Difco), 20.0. Both media were autoclaved at 121 ~ for 20 min. For the regeneration and reversion of protoplasts, osmotically stabilized agar medium (OSA) was prepared. It consisted of (g/l): Bacto-peptone (Difco), 10.0; Bacto-yeast extract (Difco), 10.0; D-glucose, 10.0; and is supplemented with an appropriate molarity of MgSO4. 7H20, sucrose, or mannitol, as specified in individual experiments. Bottom agar is of 1.5%, while overlaying soft agar is of 0.7% Bacto-agar (Difco). Preparation of myeelium for protoplast isolation Disc mycelium. Discs of cellophane membrane (dialysis tubing, Arthur H. Thomas Co. USA), 5 mm diameter, were laid on a piece of moist filter paper (Whatman No. 1) in a Petri dish and autoclaved at 121 ~ for 20 min. These sterile discs were laid aseptically along the periphery of actively growing mycelial colonies on agar medium. The mycelial discs were ready for protoplast release when mycelia had just grown over the discs. The required incubation period depends on the growth rate of different organisms; 10 h for V. volvacea, 20 h for P. sajor-caju and 30 h for Ag. bisporus and L. edodes. Volvariella bombycina was an exception because mycelia older than 5 h were unsuitable for protoplast production. The amount of mycelium on each disc was determined by weight difference, to 0.1 mg accuracy. Liquid culture. The cultural conditions used to produce mycelium for protoplast isolation were specific for each fungus (Chang & Hayes 1978). Mycelium from a two- to 10-day-old shake flask culture (20 mg fresh wt/ml in a 25 ml flask) was washed two to three times with a stabilizer (0.6 mol/l MgSO4) and by centrifugation (4000 rev/min, 15 min, Sorvall, SS.3). Mycolytic enzymes Novozym 234 (batch PPM 1035) was supplied by Novo Industri A/S Denmark. Lywallzyme

Protoplasts from edible fungi

187

was a gift from the Guangdong Institute of Microbiology, China. Both were in powdered form and were stored at --20~ Each enzyme was dissolved in 0.05 mol/1 Na phosphate buffer (pH 5.8) and sterilized by centrifugation (12,000 rev/min, 4 ~ 1 h). The clear supernatant liquid was used in the digestion mixture. The concentration of enzymes used was 1.33% (w/v).

Osmo tic stabilizers Two organic salts, one sugar and one sugar alcohol were used as osmotic stabilizers. They were: MgSO4.7H20, KC1, sucrose and mannitol. All were prepared in 0.05 mol/1 Na phosphate buffer, pH 5.8 and autoclaved at 121 ~ for 20 min. A range of concentrations, i.e. 0.4-0.8 mol/1 MgSO4 and KC1, 0.2-0.4 mol/1 sucrose and mannitol, were used in protoplast isolation experiments.

Protoplast production Mycelial discs were transferred to a microscope slide. Thirty microlitres of lytic mixture was added immediately to each disc which was then covered with a piece of cover slip. The process of protoplast release was examined under a phase contrast microscope at once, or the assembly was kept in a Petri dish for later observation. All protoplast yields were based on a 30 min digestion period. Direct microscopic observations showed that protoplast release started immediately after the addition of lytic mixture. Protoplast yield was at its maximum after 30 min and prolonged digestion led only to a decrease in protoplast number. To check protoplast yields, digestion was performed in an Eppendorf centrifuge tube (1.5 ml) with one mycelial disc in 30/al lytic mixture. Samples were counted for protoplasts in a haemocytometer (Double Neubauer Ruling). Purification of protoplasts from shake flask cultures essentially followed the method described by Hamlyn et al. (1981). After 4-5 h of incubation with~enzyme mixture, the mycelial debris was removed by filtration through a column of glass wool (ca 4-5 cm long). The f'fltrate was then centrifuged for 25 min at 7000 rev/min. The pellet containing the protoplasts was washed twice with 0.6 mol/1 MgSO4 and suspended in the same stabilizer solution.

Protoplast regeneration and reversion After production and counting of the protoplasts a volume of protoplast suspension (i.e. known number of protoplasts) was added to 5 ml osmotically stabilized soft agar which was kept molten at 40 ~ It was gently mixed and poured to make a thin layer on 8.5 cm plates of solidified osmotically stabilized agar medium (OSA). The plates were incubated at 28 ~ and scored for mycelial colonies for a total period of 7 days. A colony count was made to assess the reversion frequency of the protoplasts. The percentage of reversion refers to the fraction of protoplasts that regenerate a new cell wall and revert to normal hyphal growth. To serve as control, an equal volume of protoplast suspension was added to distilled water before mixing with the soft agar. In this case all the protoplasts should burst and no mycelial colonies should appear on the agar plates unless some mycelial fragments had been included in the sample. The number of mycelial colonies that grew on the OSA plates minus that found on the 'control' plates was used for the calculation of percentage of reversion.

Results

Protoplast production Effect of various osmotic stabilizers. Four osmotic stabilizers were tested for their effect on

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S.T. Chang, G. S. F. Li and J. F. Peberdy

protoplast formation in five fungi. Novozym 234 was used at a concentration of 0.4 mg/30/11 lytic mixture, to digest 0.2 mg mycelium. It was prepared in 0.05 mol/1 Na phosphate buffer (pH 5.8) and in the following ranges of concentrations of osmotic stabilizers (tool/l): 0.2, 0.4, 0.6, 0.8, 1.0 of MgSO4 or KC1; 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, 1.0 of sucrose or mannitol. Table 1 shows the range of molarities of the four stabilizers tested which allowed the protoplasts released to remain intact. Any concentrations above or below were believed to be unsuitable;they would probably be hypotonic or too hypertonic to that of the cytoplasm. Table 1 Range of molarity of four osmotic stabilizers that allowed protoplast production in Ag. bisporus, L. edodes, P. sa]or-ca]u, V. bombycina, and V. volvacea. Organism (strain)

Range of molarity (mol/1) of osmotic stabilizer (in 0.05 mol/1 Na-phosphate buffer, pH 5.8) MgSO4

KC1

Sucrose

Mannitol

0.4-0.8 (0.6)*

0.4-0.8 (0.6)

0.3 (0.3)

0.3 (0.3)

0.4-0.8 (0.4-0.6)

0.4-0.8 (0.4)

0.2-0.3 (0.2-0.3)

0.2-0.3 (0.2)

0.4-0.8 (0.6) 0.4-0.8 (0.6)

O.4-0.8 (0.6) 0.4-0.8 (0.6)

O.2-0.4 (0.3) 0.3 (0.3)

O.2-0.4 (0.3) 0.3 (0.3)

V. bombycina (Vo-1)

0.4-0.6 (0.6)

0.4-0.6 (0.4)

0.3 (0.3)

0.3

V. volvacea (V-5)

0.4-0.8 (0.4)

0.4-0.6 (0.4-0.6)

0.2-0.4 (0.2)

0.2-0.4 (0.2)

Ag. bisporus (Ag-23) L. edodes (L-13) e. sa]or-ca]lg dikaryon (P1-27)

monokaryon (P1-27-9)

* Molarity in parenthesis is the most suitable. In Table 2, the optimum molarity of each individual osmotic stabilizer which was the best for different fungi is given. MgSO4 was the choice for protoplast release from P. sajorcaju (both monokaryon and dikaryon) and K volvacea, KC1 was best for L. edodes and P. sajor-caju, while sucrose was good for L. edodes. In general mannitol was not satisfactory. Protoplasts were released from Ag. bisporus and K bombycina, but the yield was poorer than that of the other fungi tested. Thus, it was difficult to compare the effectiveness of different osmotic stabilizers.

Effect o f mycolytic enzymes. Protoplast yield depends on the amount of enzyme as well as mycelium used in a lyric mixture. According to Hamlyn et al. (1981), 5 mg Novozym 234 was used to digest 20 mg (fresh wt) mycelium of V. volvaeea/ml of lyric mixture. In this study, the following weight ratios of enzyme: mycelium were tested: 4 : 1,2 : 1, 1 : 1, 1 : 2, 1 : 4. Digestions were carried out in 0.4 and 0.6 mol/1 MgSO4 or KC1 and 0.2 and 0.3 tool/1 sucrose or mannitol. In all cases the best protoplast yield was given by the ratio of 2 : 1. At 4: 1, the enzymatic reaction was too drastic; there was evidence that protoplasts had been released because hyphal wall degradation was observed under the microscope


189

Table 2 Comparison of protoplast yield from Ag. bisporus, L. edodes, P. sajor-caju, V. bombycina and V. volvacea at the optimum molarity of different osmotic stabilizers using the enzyme Novozym 234 Molarity (mol/1) of osmotic stabilizer (in 0.05 mol/1 Na phosphate buffer, pH 5.8) Organism (strain)

MgSO4

KC1

0.4

0.4

Ag. bisporus (Ag-23) L. edodes (L-13)

0.6 +

+

+

e. sajor-caju dikaryon (P1-27) monokaryon (P1-27-9)

+++ +++

V. bombycina (Vo-1)

+

V. volvacea (V-5)

0.6

+++

Sucrose

Mannitol

0.2

0.2

+ +++

+ +++ +++

++ +++

++

+

TFTC TFTC

+ +

+ +

0.3 +

++ ++

+ +

0.3

Remarks

1.04 X 106/ml 0.67 • 106/ml TFTC

+

8.32 X 104/ml

+++, indicates best protoplast yield in individual fungus. TFTC, too few to count (either yield is low or protoplasts are unstable). but the protoplast yield was low and this may be attributed to the continuous action of enzyme on the plasma membrane so that protoplasts burst and could not be detected. On the other hand, with the 1 : 4 ratio, only very few protoplasts were released, presumably because insufficient enzyme was used. The effectiveness of Novozym 234 on protoplast release in different fungi varied as shown in Table 2. Using 0.2 mg mycelium and 0.4 mg Novozym 234 in 30/al 0.6 mol/1 MgSO4 (0.05 mol/1 Na phosphate buffer, ph 5.8), the protoplast yield was 1.04 X 106/ml in the dikaryon (P1-27) and 0.67 X 106/ml in the monokaryon (P1-27-9) ofP. sajor-caju. For V. volvacea V5, 0.4 mol/1 MgSO4, which was found to be of the optimum molarity, was used and the yield was 8.32 • 104/ml. The results shown in Table 3 indicate that lywallzyme was of much higher efficiency than Novozym 234 for protoplast production from Ag. bisporus and also from P. sajor-caju but was inferior from V. volvacea. Table 3 Comparison of the effect of two mycolytic enzymes for protoplast release from four edible fungi. Yield of protoplast in MgSO4 (0.6 mol/1) with the concentration of the enzymes 1.33% (w/v). Organism (strain)

Novozym 234

Lywallzyme

Ag. bisporus (Ag-17)

TFTC

2.16 X 106/ml

Au. auricula (Au-2)

-

7.46 X 106/ml

P. sajor-caju (P1-27)

1.14 X 107/ml

3.84 • 107[ml

V. volvacea (V-35)

2.56 X 106/ml

1.92 X 106/ml

TFTC, too few to count. -, not tested.

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Protoplast regeneration and reversion Preparation of protoplasts is of use only if the protoplasts are viable, able to regenerate cell wall and revert to normal hyphal growth. Only two fungi, P. sajor-caju dikaryon P1-27 and V. volvacea V-5, were tested because their protoplast yields were comparatively high. The reversion media were made hypertonic with 0.4 mol/1 MgSO4 or 0.2 or 0.3 tool/1 mannitol. The addition of 0.6 tool/1 MgSO4 to the reversion medium caused precipitation so that only 0.4 mol/1 MgSO4 was used. Protoplast production was carried out with Novozym 234 and osmotically stabilized with MgSO4. Results of preliminary trials were not promising. The reversion frequency of both fungal protoplasts was exceedingly low, never above 1%. Still, it seemed that the MgSO4-stabilized reversion medium was better than the mannitol-stabilized one, and P1-27 protoplasts had a higher reversion frequency. The effect of sucrose as osmotic stabilizer has also been tested. It appeared to be a suitable candidate as well. More investigations are required and they should be carried out using sucrose and MgSO4 as osmotic stabilizers. Discussion The release of protoplasts depends on three major factors: the lytic enzyme used, the osmotic stabilizer and the physiological status of the organisms (Peberdy 1976), and complex interactions exist between them. The mini-scale method reported here is reliable and of great use in rapid screening to determine the proper conditions for protoplast production or to test the effectiveness of different enzyme preparations for protoplast release. Once the appropriate conditions are established, protoplast production can be scaled up by the use of shake cultures. The results show that protoplast production is possible in the edible fungi tested, but that the efficiency of protoplast release varies greatly. Fungal cell walls differ in composition and architecture (Gander 1974) and the mycolytic enzymes used may have different effects in digesting the hyphal walls of different species. Novozym 234 derived from Trichoderma harzianum (previously named T. viride) was less effective in contrast to an extracellular enzyme, lywallzyme, a preparation from T. longibrachiatum (Qiu et al. 1982) on Ag. bisporus hyphal walls (Table 3). De Vries & Wessels (1973) also reported that a lytic enzyme preparation from T. viride was less effective on Ag. bisporus cell wall in comparison with that of other basidiomycetes. However, Novozym 234 gave a better yield of protoplasts on V. volvacea mycelia. By the use of lywallzyme, P. sajor-caju had the highest yield (3.84 • 107/ml), followed by Au. auricula (7.46 • 106/ml), Ag. bisporus (2.16 X 106/ml) and V. volvacea (1.92 • 106]ml). The physiological age of the culture markedly influences protoplast yield and the nature of the culture medium used also has an effect. It has been shown with several organisms that actively growing cultures, preferably in the exponential phase, give the best protoplast yields (Anne et aL 1974; Peberdy et aL 1976; Isaac 1978). The membrane method makes use of mycelia taken from the periphery of actively growing colonies, which are presumably in the early exponential phase of growth. Both effects, culture age and culture medium, suggest that some modification or change in the cell wall might be involved in altering its susceptibility to lysis. Many inorganic salts, sugars and sugar alcohols have been used successfully to provide osmotic support to free protoplasts, with variable effectiveness in different fungi (ViUanueva & Garcia Acha 1971). Inorganic salts seem to be more effective with filamentous fungi; this was so in our studies ofP. sajor-caju and V. volvacea. On the other hand, sucrose appeared to


191

to be a more appropriate osmotic stabilizer in the lytic system of protoplast isolation from L. edodes. In general, protoplasts that emerged in the early period of lytic digestion were smaller in size, mostly homogeneous and not greatly vacuolated. Later, both the size and the number of vacuoles in each protoplast increased. The mycelia and protoplasts ofL. edodes were extensively vacuolated in the presence of sucrose. Although De Vries & Wessels (1972) found that protoplasts produced in the presence of MgSO4 were highly vacuolate and buoyant, our results show that a high degree of vacuolation may not be due to the effect of MgSO4 alone. The effectiveness of different osmotic stabilizers is well understood. Protoplast reversion was studied in solid regeneration media because it has been suggested that wall components, or their precursors, or the enzymes involved in their synthesis, may be lost into the liquid medium whereas they can be retained in a solid medium (Lampen 1968; Necas et al. 1968). In general, there are two patterns of morphological development of protoplast reversion in the mycelial fungi. In one, protoplasts give rise to abnormally shaped germ tubes which ultimately change to normal hyphae at their tips (Peberdy & Gibson 1971; DooijewaardKloosterziel et al. 1973; Anne et al. 1974). In the second, protoplasts first develop a wall to form a primary cell that later produces a normal germ tube (Van der Valk & Wessels 1973). Nevertheless, the distinction in patterns of development is not straightforward and in some fungi both types of development leading to the formation of normal hyphae have been described (Peberdy 1979b). The basic information reported here is considered to be useful for later work on the application of protoplast fusion techniques to the hybridization of fungal species. The technique of protoplast fusion, in by-passing the sexual cycle, is very useful in crossing strains of a particular organism, or even different species, that would not normally interact.

Acknowledgements We are grateful to Ms Lau Yu Mei Lin for her technical aid. We wish to thank NOVO Enzyme Products Ltd. and the Guangdong Institute of Microbiology for providing samples of Novozym 234 and lywallzyme, respectively.

References ANNE, J., EYSSEN, H. & DE SOMER, P. 1974 Formulation and regeneration of Penicillium chrysogenum protoplasts. Archives of Microbiology 98, 159-166. BACHMANN, B.J. & BONNER, D.M. 1959 Protoplasts from Neurospora crassa. Journal o f Bacteriology 78, 550-556. CHANG, S.T. & MILES, P.G. 1984 A new look at cultivated mushrooms. BioScience 34, 358-362. CHANG, S.T. & HAYES, W.A. (eds) 1978 The Biology and Cultivation of Edible Mushrooms. New York & London: Academic Press. DE VRIES, O.M.H. & WESSELS, J.G.H. 1972 Release of protoplasts from Schizophyllum commune by a lytic enzyme preparation from Trichoderma viride. Journal of General Microbiology 73, 13-22. DE VRIES, O.M.H. & WESSELS, J.G.H. 1973 Effectiveness of lytic enzyme preparation from Trichoderma viride in releasing spheroplasts from fungi, particularly basidiomycetes. Antonie van Leeuwenhoek 39,397-400. DOOIJEWAARD-KLOOSTERZIEL, A.M.P., SIETSMA, J.H. & WOUTERS, J.T.M., 1973 Formation and regeneration of Geotrichum candidum protoplasts. Journal of General Microbiology 74, 205-209.

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EMERSON, S. & EMERSON, M.R. 1958 Production, reproduction and reversion of protoplast like structures in the osmotic strain of Neurospora crassa. Proceedings of National Academy of Sciences, U.S. 44,668-671. FAWCETT, P.A., LODER, P.B., DUNCAN, M.J., BEESLEY, T.J. & ABRAHAM, E.P. 1973 Formation and properties of protoplasts from antibiotic-producing strains of Penicillium chrysogenum and Cephalosporium aceremonium. Journal of General Microbiology 79, 293-309. FERENCZY, L. 1981 Microbial protoplast fusion. In Genetics as a Tool in Microbiology, ed. Glover, S.W. & Hopwood, D.A. pp. 1-34. Cambridge University Press. GANDER, J.E. 1974 Fungal cell wall glycoproteins and peptido-polysaccharides. Annual Review of Microbiology 28,103-119. HAMLYN, P.F., BRADSHAW, R.E., MELLON, F.M., SANTIAGO, C.M., WILSON, J.M. & PEBERDY, J.F. 1981 Efficient protoplast isolation from fungi using commercial enzymes. Enzyme Microbiology Technology 3,321-325. HUANG, N.L. & WU, S.Z. 1982 A preliminary study on the silver-silk straw mushroom, Volvariella bombycina (Pers. ex Fr.) Sing. Mushroom Newsletter for the Tropics 3, 6-9. ISAAC, S. 1978 Biochemical properties of protoplasts from Aspergillus nidulans. Ph.D. Thesis, University of Nottingham, England. LAMPEN, J.O. 1968 External enzymes of yeast: their nature and formation. Antonie van Leeuwenhoek 34, 1-18. MEINECKE, G. 1960 Protoplasts from Penicillium glaucum. Nature, London 188,246. MOORE, D. 1975 Production of Coprinus protoplasts by use of chitinase or helicase. Transaction of the British Mycological Society 65, 134-136. NECAS, O., SVOBODA, A., & HAVELKOVA, M. 1968 Mechanism of regeneration of yeast protoplasts. V. Formation of the cell wall in Schizosaccharomyces pombe. Folia biologica, Praha 14, 80-85. PEBERDY, J.F. 1976 Isolation and properties of protoplasts from filamentous fungi. In Microbial and Plant Protoplasts, ed. Peberdy, J.F., Rose, A.H., Rogers, H.J. & Cocking, E.C. pp. 39-50. London: Academic Press. PEBERDY, J.F. 1979a Fungal protoplasts: isolation, reversion and fusion. Annual Review o f Microbiology 33, 21-39. PEBERDY, J.F. (ed) 1979b Protoplasts-Application in Microbial Genetics. Department of Botany, University of Nottingham, England. PEBERDY, J.F. 1980 Protoplast fusion--a tool for genetic manipulation and breeding in industrial microorganisms. Enzyme Microbiology Technology 2, 23-29. PEBERDY, J.F. & GIBSON, R.K. 1971 Regeneration of Aspergillus nidulans protoplasts. Journal of General Microbiology 69,325-330. PEBERDY, J.F. & ISAAC, S. 1976 An improved procedure for protoplast isolation from Aspergillus nidulans. Microbios Letters 3, 7-9. PEBERDY, J.F., BUCKLEY, C.E., DALTREY, D.C. & MOORE, P.M. 1976 Factors affecting protoplast release in some filamentous fungi. Transactions of the British Mycological Society 67, 23-26. QIU, J.Y., LIAO, H.C. & WU, Y.C. 1982 Preparation of an enzyme for lysing the cell wall of higher basidiomycetes. Hereditas, Bei]ing 4, 13-14 (in Chinese). RAPER, J.R. 1966 Genetics of Sexuality in Higher FungL New York: The Ronald Press Company. RAPER, J.R. & MILES, P.G. 1958 The Genetics of Schizophyllum commune. Genetics 43, 530-546. RODRIGUEZ AGUIRRE, M.J. & VILLANUEVA, J.R. 1962 Production of protoplast-like structures from various species of fungi. Nature, London 196, 693-694. RODRIGUEZ AGUIRRE, M.J., GARCIA ACHA, I. & VILLANUEVA,J.R. 1964 Formation of protoplasts of Fusarium culmorum by strepzyme. Antonie van Leeuwenhoek 30, 33--44.


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SANTIAGO, C.M., Jr. 1982a Studies on the Physiology and Genetics of VolvarieUa volvacea (Bull ex Fr.) Sing. Ph.D. Thesis, University of Nottingham, England. SANTIAGO, C.M., Jr. 1982b Production of VolvarieUa protoplasts by use of Trichoderma enzyme. Mushroom Newsletter for the Tropics 3, 3-6. VAN DER VALK, P. & WESSELS, J.G.H. 1973 Mitotic synchrony in multi-nucleate Schizophyllum protoplasts. Protoplasma 78,427--432. VILLANUEVA, J.R. & GARCIA ACHA, I. 1971 Production and use of fungal protoplasts. In Methods in Microbiology, ed. Borth, C., 4 , 6 6 5 - 7 1 8 . New York: Academic Press. Summary Mycelium from the periphery of actively growing colonies on cellophane and from shake flask cultures was used for the isolation of protoplasts from strains of Agaricus bisporus, Auricularia auricula, Lentinus edodes, Pleurotus sajor-ca]u, Volvariella bombyeina and V. volvacea. The mycelial cells were treated with two mycolytic enzymes, Novozym 234 or lywallzyme. Protoplasts were produced from all the edible fungi tested. Pleurotus sa]or-ca]u gave the highest yield (3.84 X 107/ml), followed by Au. auricula (7.46 X 106/ml), Ag. bisporus (2.16 X 106/ml) and V. volvacea (1.92 X 106/ml), when treated with lywallzyme. Agaricus bisporus gave the smallest yield of protoplasts when Novozym 234 was used. The effects of different molarities of osmotic stabilizers were also studied. The cellophane method is simple and quick and can be used as a screening procedure. The yields of protoplasts obtained from liquid cultures were usually higher than those from cellophane cultures.

R~sum~ ]solement de protoplastes d partir de champignons comestibles Du myc6llum pr~lev6 ~ la p~riph~rie de colonies d6volopp~es sur cellophane ou ~ partir de cultures liquides agit~es a ~t~ utillz~ pour la preparation de protoplastes d'Agaricus bisporus, Auricularia auricula, Lentinus edodes, Pleurotus sa]or-ca]u, Volvariella bombycina et V. volvacea. Les cellules myc61iales ont ~t~ trait6es par deux enzymes mycolytiques, Novozym 234 et lywallzyme. Par traitement avec le lywallzyme, des protoplastes ont ~t~ obtenus avec t o u s l e s champignons comestibles ~tudi~s, P. sa]or-ca]u donnant le rendement le plus ~lev~ (3.84 • 107/ml), suivi par Au. auricula (7.46 X 106/ml), Ag. bisporus (2.16 • 106/ml) et V. volvacea 1.92 X 106/ml). Avec le Novozym 234, Ag. bisporus a donn~ le rendement en protoplastes le moins ~lev~. Les effets de diff6rentes molarit~s de stabilisateurs osmotiques ont 6galement 6t~ ~tudi6s. La m~thode de la cellophane est simple et rapide et peut ~tre employee comme proc~d~ de degrossissage. Les rendements en protoplastes obtenus fi partix des cultures llquides sont habituellement plus ~lev~s que ceux ~ partir des cultures sur cellophane. Resumen Aislamiento de protoplastos a partir de setas comestibles Para el aislamiento de protoplastos de cepas de Agaricus bisporus, Auricularia auricula, Lbntinus edodes, Pleurotus sa]or-ca]u, Volvariella bombycina, y V. volvacea se utilizo micelio periferico de colonias que estaban en crecimiento activo en celofan y e n frascos de agitaci6n. Las celulas del micelio se tratar6n con dos enzymas mycollticos: Novozym 234 y lywallzyme. Todas las setas comestibles ensayadas produjer6n protoplastos. Mediante tratamiento con lywallzyme la mejor cosecha de protoplastos la proporciono P. sa]or-ca]u (3.84 • 107/ml) seguido pot Au. auricula (7.46 X 106/ml), Ag. bisporus (2.16 • 106/ml) y V. volvacea (1.92 • 106/ml). Con Novozym 234 Ag. bisporus fu~ la de menor rendimiento. Tambi~n se estudiar6n los efectos de distintos estabilizadores osmoticos a diferentes molaridades.