protoplast formation from selected species of ectomycorrhizal fungi

Appl Microbiol Biotechnol (1989) 30:381-387 Applied AFtcrobiology

Biotechnology © Springer-Verlag 1989

Protoplast formation from selected species of ectomycorrhizal fungi*'**

Victoria Barrett 1, Paul A. Lemke I and Robert K. Dixon 2

1 Department of Botany and Microbiology, Auburn University, Auburn, Alabama, USA 36849 2 School of Forestry, Auburn University, Auburn, Alabama, USA 36849

Summary. Conditions for the formation of protoplasts from selected species of ectomycorrhizal fungi are described. The age of the fungal culture and extent of incubation in a lytic enzyme mixture are critical factors for efficient formation of protoplasts. There is a correlation between the distri- bution of nuclei in hyphal fragments and protoplasts and the frequency of protoplast regeneration. Protoplasts from at least two of the species studied are formed in sufficient numbers and regenerate at suitable frequencies to be useful for development of genetic transformation and cell fusion systems. These fungi can now be consid- ered in experiments designed for the improvement of ectomycorrhizal associations through genetic manipulation of the fungal component.

Introduction

The roots of most vascular plants are colonized by specific root-inhabiting fungi and this relation- ship is termed mycorrhizal. The mycorrhizal asso- ciation is symbiotic and the plant benefits through improved water absorption (Duddridge et al. 1980) and uptake of nutrients (Harley and Smith 1983), resistance to pathogens (Marx 1975) and tolerance to environmental stress (Dixon and Bu- schena 1988). The ectomycorrhizal fungi are associated with many gymnosperms and with some

* Dedicated to Professor Dr. Dr. he. K. Esser on the occasion of his 65th bir thday

** This research was supported in part by the Mclntire-Sten- nis Cooperative Forestry Research Program and is published as Alabama Agriculture Experiment Station Journal No. 6- 881863P

Offprint requests to: P. A. Lemke

woody angiosperms (Harley and Smith 1983). The majority of these fungi belong to the classes Ba- sidiomycotina and Ascomycotina and grow vege- tatively in culture but generally do not sporulate or complete a sexual cycle in the laboratory. These limitations are the principal reasons why ectomycorrhizal fungi have not been studied genetically. A recent report suggests that Hebeloma cylindrosporum will sporulate in combination with Pinus pinaster plantlets grown in laboratory flasks (Debaud and Gay 1987). Homokaryotic mycelia and compatible heterokaryotic (dikaryotic) mycelia of this fungus have been shown to form ectomycorrhizae with comparable efficiencies (De- baud et al. 1988). Similarly, homokaryons and clamped dikaryons of the fungus Laccaria bicolor will establish ectomycorrhizae with Pinus banksiana in vitro (Kropp and Fortin 1986).

Protoplasts can be used for the isolation of cy- toplasmic organelles (Billich et al. 1988), investi- gation of cell wall synthesis and synchronized cell growth (Davis 1985), for cell fusion (Minuth and Esser 1983), and preparation of high molecular weight DNA for cloning and karyotyping. Proto- plasts can also be used as a source material for mutant induction as well as for transformation experiments designed for strain improvement (Ho- molka et al. 1988; Hynes 1986). We have evalu- ated conditions for the generation of protoplasts from selected species of ectomycorrhizal fungi in an effort to identify and develop systems with potential for transformation and other genetic study. While protoplasts have been formed from a vari- ety of fungi including the basidiomycetes Co- prinus cinereus (Yanagi et al. 1985), Tricholoma matsutake (Abe et al. 1982), Collybia veltipes, Pleurotus ostreatus (Yamada et al. 1983) and Usti- la9o maydis (Banks 1983), there are only a few re- ports of protoplast formation from ectomycorrhi-

382 V. Barrett et al. : Protoplast formation from ectomycorrhizal fungi

Table 1. Ectomycorrhizal fungi used in protoplast experiments

Species Source Type"

Cenoeoccum geophilum 155 Hebeloma circinans BR63-10 H. eylindrosporum BR70-60 Laeearia bicolor $447 L. laeeata $443 L. laceata $444 L. laceata $449 Pisolithus tinctorius 285

Suillus luteus VT1616 Thelephora terrestris 223

Quereus alba, Md, USA, isolated in 1955 by E. Hasckaylo A Picea, Haute Savoie, France, isolated in 1963 by G. Bruchet B Pinus pinaster, Le Lands, France, isolated in 1970 by J. C. Debaud BC Tsuga mertensiana, Ore, USA, isolated in 1978 by G. Hunt BC T. mertensiana, Ore, USA, isolated in 1978 by G. Hunt BC Pseudotsuga taxifolia, Ore, USA, isolated in 1977 by G. Hunt BC P. taxifolia Ore, USA, isolated in 1978 by J. Trappe BC Pinus taeda, Ga, USA, isolated in 1987 by D. Marx, passed through pine in BC 1988 by M. Bronson P. densiflora, central South Korea, isolated in 1984 by O. Miller B P. ooearpa, Brazil, isolated in 1977 by T. Krugner BC

a A = ascomycete affinity; B = basidiomycete; BC = basidiomycete with clamp connections

zal fungi including Lacearia bicolor, (Kropp and Fortin 1986) and Hebeloma cylindrosporum, (He- braud and Fevre 1988).

Materials and methods

Culture conditions and protoplast formation. Identification and sources of fungal isolates used in this study are listed in Table 1. Cultures were maintained stationary in 100 ml potato dextrose broth (PDB, Difco, Detroit, Mich, USA) in 250 ml flasks at 22 ° C. Cultures selected for protoplast formation were macerated in a blender (Dynamics Corp. New Haven, Conn, USA) for 10 s to 5 min with the addition of fresh medium. Since Pisolithus tinctorius was reported to be killed by pro- longed maceration (Lapeyrie and Bruchet 1985), it was blended for only 10-15 s. Cenocoeeum 9eophilum can be blended for up to 10 rain with no apparent loss of viability and was usually processed for 3-5 min. Other fungi were routinely macerated for 1 rain. Macerated cultures were incubated in 100 ml PDB in 250-rnl flasks at 22°C without shaking.

Mycelia for protoplast formation were collected on nylon mesh of 60 Ixm porosity (Spectrum Medical Industries, Inc. Los Angeles, Calif, USA), washed once in osmotic buffer (MMC: 0.5 M mannitol, 0.05 M maleic acid, 0.05 M CaC12 pH 5.6 (860 mOsmol) or KMPC: 0 .7M KC1, 0 .8M mannitol, 0.02 M KPO4, 0.05 M CaCl2 pH 6.3 (2500 mOsmol) and col-

lected by centrifugation (1500 x 9, 10 rain). The packed volume of wet mycelia was measured in graduated centrifuge tubes and transferred to 50-ml flasks for incubation with Novozyme 234 (Calbiochem, San Diego, Calif, USA) (5 mg/ml in osmotic buffer) on a shaker (180 rpm) at 31°-32 ° C. Chitinase (1 mg/ ml) (Sigma Chemical Co. St. Louis, Mo, USA) was added to the Novozyme 234 solution for incubation of C. geophilum.

Protoplasts were separated from hyphae by filtration through cotton tamped in the end of a 10-ml disposable sy- ringe and moistened with osmotic buffer. The cotton plug was washed with 15 ml osmotic buffer, the protoplasts were pel- leted by centrifugation (500x9, 15 rain) and washed twice with osmotic buffer and collected each time by centrifugation. The final pellet was resuspended in 1 ml osmotic buffer and the number of protoplasts counted on a haemocytometer. Nu- clei were stained for DNA with Hoechst 33258 (Sigma Chemi- cal Co. St. Louis, Mo, USA) at 100 ~tg/ml and counted by ob- serving fluorescence with an Olympus AH-2 microscope with high pressure mercury fluorescence illumination, a 330-380 nm excitation filter, and a > 420 nm barrier filter,

Media and regeneration of protoplasts. Protoplasts were regen- erated in soft agar or in liquid medium. Osmotically stabilized media tested for regeneration of protoplasts were (1) KCI + M (0.7 M KC1, 0.8 M mannitol, 0.05 M CaCI2, 0.02 M K P O 4 pH 6.3, 0.5% yeast extract, 100 ~tg/1 thiamine HC1, 1.2 rag/1 FeCI3 (Finkelstein et al. 1986), (2) YDS (1.2 M sorbitol, 0.5% yeast extract, 2.0% dextrose, 100 I.tg/1 thiamine HCI, 1.2 rag/1 FeC13

Table 2. Yield and regenerative ability of protoplasts of species and isolates of ectomycorrhizal fungi

Species Yield of Viable Diameter of Regeneration Nucleated protoplasts fragments protoplasts rate (%) cells (%) (per cm 3 wet mycelia) (~m)

C. 9eophilum 155 2.1 × 1 0 6 5 . 0 × 10 7 2-10 0 0 H. eircinans 17.0 × 10 6 7.5 × 10 7 2- 3 1.5 22 H. cylindrosporum 126.0 × 1 0 6 4.7 x 105 2- 7 1.6 24 L. bicolor $447 8 . 6 x 1 0 6 1.4x 105 2- 5 0.1 27 L. laccata $444 179.6 × 10 6 1.1 × 10 6 2- 5 0.7 12 L. laceata $443 60.0 × 106 1.0 x 105 2- 5 0.4 14 L. laccata $449 14.7 × 10 6 - - 2- 5 0.9 13 P. tinctorius 285 2.5 x 106 2.5 X 10 4 2- 4 0 0 S. luteus VT1616 25.0x 106 5.0× 104 2- 5 0 0 T. terrestris 223 3.2 × 10 6 2.0 × 10 4 2- 4 - - - -

V. Barrett et al. : Protoplast formation from ectomycorrhizal fungi 383

E

% % 3.

g-

L.~cc~ria IQccat~ S444 200-

180 ~ 9ka~

/ 160 -

140£

uol 100-

80. 0

60- 40- O"

0--

20 I . / , ~ , ~ , ~

0 5 10 15 Incubation time (hours}

o

~ - - L ,

20 25

18

Y,, 14 E

12

~ 8 %

~ 4

g 2

Lac¢orlo leccatQ $449

5 1C) 1'5

Incubation time (hours)

2O - t

25

E

)

%

3. in

10 T Leccario bicolor 5447 -~- 25

*~ 2o

6- % 15

% 4 - . ~ 10

o J " ' - - ' ~ 0 0 5 10 115 20 - 25

Incubation time (hours)

A

t A

5 10

Suillus luteus VT1616

~'5 ;/o - ' 25

Incubotlon time (hours)

130 120

~, 110 E 100 ~ 9D

8O

~ 50 4O m

"~ 3O o ~- 2O o "6 10

Hebelomo cyllndrosporum A

- - - 0

5 110 ~6 210 25 Incubation time (hours)

~'_ 18 T •

~£ 12

x

0

Hebelama eircinans

5 10 15 20 25 Incubotion time (,hours)

._~ 3.o T Pisolithus tinctorius 2B5 ='~ 2,4

l 2,2 ~>" 2.5 E 2,0.

• >,

2.0 •

1.4 1.5 ~ " 1 , 2

% o "~ 1.0 ,; 1,0

~" 0.8 , A \ ~ o,~ 6 0 . s ~ ~ o.4

"6 ~ 0,2 0.0 ca- 0.o

0 5 10 15 20 25

Incub(~t~on time (ho~,rs)

Cenococcum geophilum 155

zk A

F:U- 5 lO 15 20 25

lncubotlon tlme (hours)

Fig. 1. Kinetics of protop[ast formation illustrating the dependence of protoplast yield on the age of the culture after maceration and the length of the incubation in Novozyme 234; O - - O , 2 days; 0 - - 0 , 4 days; A - - A , 6 days; 4 - - 4 , 10 days

384

(Yelton et al. 1984)), (3) CRM (0.3 M citrate, 0.17% yeast ni- trogen base, 0.5% (NH4)2SO4 pH 5.7, 2.0% dextrose, 100 Ixg/1 thiamine HC1, 1.2 ~tg/1 FeC13 (Tully and Gilbert 1985)), (4) M M N + M (0.6 M mannitol, 0.3% malt extract, 1.0% dextrose, 0.0025% (NH4)zHPO4, 0.05% KH2PO4 pH 5.7, 0.015% MgSO4, 0.005% CaCI2, 0.0025% NaC1, 100 l, tg/l thiamine HC1, 1.2 I-tg/1 FeC13 (Kropp and Fortin 1986)), (5) PRM 0.6 M mannitol, 0.1% bactopeptone, 0.1% bactosoytone, 0.1% KHzPO4 pH 5.6, 0.03% yeast extract, 100 ~g/1 thiamine HC1, 1.2 rag/1 FeC13 (modified from Kitamoto et al. 1988)), (6) PPD (10% polyethy- lene glycol 4000, potato dextrose broth, 0.016 M CaC12, 0.016 M TRIS pH 7.4, 100 ~tg/1 thiamine HC1, 1.2 rag/1 FeC13).

An aliquot of protoplasts was mixed with 10 ml molten soft agar medium (0.7% agar, 430-45 ° C) and poured into a sterile petri dish (100x 15 ram) or over the surface of solid agar medium (2% agar) in a petri dish and incubated at 22°C until colonies appeared. Alternatively, an aliquot of proto-

V. Barrett et al.: Protoplast formation from ectomycorrhizal fungi

plasts was mixed with 10 ml liquid regeneration medium and incubated at 22°C without shaking. After 6-7 days the cells were collected by centrifugation, resuspended in 1 ml regeneration medium, spread on the surface of solid agar medium and incubated at 22 ° C until colonies developed and could be eas- ily counted. Each experiment was controlled for carry-over of hyphal fragments by plating the initial protoplast preparation onto medium without osmotic stabilizer and scoring after 2 weeks for either no growth or negligible numbers of growth foci.

Viable hyphae. Viable fragment counts were made on the macerated fungi. An aliquot of the macerate was taken, diluted in osmotic buffer, and plated onto potato dextrose agar (PDA; Difco, Detroit, Mich, USA). Foci of fungal growth were counted after 7-10 days. Hyphae were examined for nuclei by staining DNA with Hoechst 33258 (Sigma chemical, St. Louis, Mo, USA).

Table 3. Regeneration frequencies of ectomycorrhizal fungal protoplasts in various media

Species Regeneration Medium frequency

C. geophilum 155 0 M M N + M soft agar, liquid 0 CRM soft agar, liquid 0 PPD, PRM, YDS liquid

H. circinans 1.5 x 10 -2 M M N + M soft agar 1.7 × 10 -3 PRM soft agar 0 CRM soft agar 7.0 x 10-4 CRM liquid 1.0 x 10-3 YDS liquid

H. cylindrosporum 4.4 x 10 -4 M M N + M liquid 5.5 × 10 -3 M M N + M soft agar 6.3 x 10 -5 PPD liquid 2.2 x 10 3 CRM liquid 1.6 x 10-2 CRM soft agar 4.4 x 10-4 PRM liquid 1.1 x 10 -3 YDS liquid

L, bicolor $447 1.0 X 10 - 3 PRM soft agar 0 PPD liquid

L, laccata $443 4.0× 10 -3 M M N + M liquid 0 YDS liquid

L. laccata $444 3.8 X 10 - 4 M M N + M soft agar 7.0 x 10-3 M M N + M liquid 0 CRM liquid, soft agar 6.5 x 10-5 PRM liquid 1.2 x 10 - 4 YDS liquid

L, laccata $449 4.5 × 10 - 3 M M N + M liquid 0 CRM, KC1 + M soft agar 5.3 x 10 -4 PPD liquid 4.5 x 10-3 PRM liquid 9.1 x 10 - 3 PRM soft agar 6.4 x 10 -4 YDS liquid

P. tinctorius 285 0 M M N + M soft agar, liquid 0 PRM soft agar, liquid 0 PPD, CRM, YDS liquid

S. luteus VT1616 0 M M N + M soft agar, liquid 0 CRM soft agar, liquid 0 PPD, PRM, YDS liquid

For medium abbreviations see Materials and methods

V. Barrett et al.: Protoplast formation from ectomycorrhizal fungi

Results and discussion

Protoplasts were formed from most of the fungi tested but at dissimilar efficiencies and viabilities (Table 2). Several strains of L. laccata were com- pared and yields of protoplasts varied over an or- der of magnitude. This result suggests strain dif- ferences may significantly influence the isolation of protoplasts from other species of fungi. The kinetics of protoplast formation for cultures aged 2, 4, 6 and 10 days are shown in Fig. 1. Formation of protoplasts was greatly affected by both the age of the culture after maceration and by the length of time of incubation in lytic enzyme solution. Kita- moto et al. (1988) previously suggested that pro- teinases in the lytic enzyme preparation may be detrimental to protoplast survival. Cenococcum geophilum protoplasts formed only in the KMPC osmotic buffer. This buffer has been used success- fully in the formation of Aspergillus protoplasts (Finkelstein et al. 1986) and C. geophilum is re- lated to ascomycetous fungi (Miller 1982). The other ectomycorrhizal fungi are all basidiomycetes and protoplasts are formed in the MMC buffer typically used for basidiomycetes (Kita- moto et al. 1988; Kropp and Fortin 1986; Yanagi et al. 1985).

Regeneration frequencies for all the fungal protoplasts were low (Table 3). Three species (C. geophilum, P. tinctorius, Suillus luteus) did not regenerate from protoplasts in any of the media tested. Protoplasts from these three fungi stained with Hoechst 33258 showed an absence of nuclei. Dilutions of the initial fungal macerates were plated to confirm that viable hyphal fragments were present before incubation in Novozyme 234 (Table 2). Two of the fungal species (P. tinctorius and S. luteus) showed few viable hyphal fragments, and protoplasts from these hyphae failed to regenerate which was obviously due to the lack of nuclei. The mycelia of both fungi and of C. geophilum are composed predominantly of wide hyphae (> 3 Ixm) with thickened and often pig- mented and/or encrusted cell walls. The minority cells of these mycelia are narrow and thin-walled generative hyphae (Ainsworth 1961). Examination of fluorescent-stained hyphae indicated that the nuclei are sparsely distributed and limited to generative hyphae (Fig. 2). Although C. geophilum protoplasts are enucleate, the hyphae exhibit a high degree of viability following maceration.

The best protoplast regeneration (H. cylindrosporum) was 1.6% but rates were typically < 1% (Table 3). Hebeloma cylindrosporum protoplasts were 24% nucleated (872 protoplasts counted).

385

The L. bicolor protoplasts were 27% nucleated (72 protoplasts counted) and those of L. laccata $449 were 13% nucleated (340 protoplasts counted) (Fig. 3) and regeneration frequencies were 0.1% and 0.9%, respectively. These fungi showed abun- dant viable hyphal fragments after maceration.

The potential uses of protoplasts from ectomycorrhizal fungi for experiments in transformation, mutation induction, and cell fusion justify a con- tinued effort to form and regenerate wall-less cells from these fungi. Protoplasts of ectomycorrhizal fungi are potential source material for development of improved strains of fungi which could positively affect the productivity of agronomic and forest crops (Dixon and Marx 1987). While these fungi yield protoplasts at rates equivalent to other fungi (Kitamoto et al. 1988; Yanagi et al. 1985), the low regeneration frequencies make their

Fig. 2. Hyphae of Cenococcum geophilum using (A) visible light and UV illumination and (B) the same field with UV illumination only. Hoechst 33258 stained nuclei of generative hyphae are evident in both photographs

386 V. Barrett et al.: Protoplast formation from ectomycorrhizal fungi

Fig. 3. Protoplasts of Laccaria laceata $449 using (A) visible light and (B) the same field with UV illumination. The majority of protoplasts are enucleate

use in procedures to detect rare events difficult and in some cases impractical. Although transformation of fungi occurs at low frequency, it is typically integrative and stable (Hynes 1986). Mutag- ens which cause mutation at low levels of kill, such as methane sulphonic acid ethyl ester (EMS), are available. Thus, future experiments using protoplasts, despite low regeneration frequencies, are feasible with at least two of the ectomycorrhizal fungi studied, i . e .H, cylindrosporum and L. la¢cata.

References

Abe M, Umetsu H, Nakai T, Sasage D (1982) Regeneration and fusion of mycelial protoplasts of Tricholoma matsutake. Agric Biol Chem 46:1955-1957

Ainsworth GC (1961) Dictionary of the fungi 5th edition. Commonwealth Mycological Institute, Kew, Surrey, UK

Banks GR (1983) Transformation of Ustilago maydis by a plasmid-containing yeast 2-micron DNA. Curr Genet 7:73-77

Billich A, Keller U, Kleinhauf H, Zocher R (1988) Production of protoplasts from Fusarium sciripi by lytic enzymes from Streptomyces tsusimaensis. Appl Microbiol Biotechnol 28:442-444

Davis B (1985) Factors influencing protoplast isolation. In: Peberdy JF, Ferenczy L (eds) Fungal protoplasts. Dekker, New York, pp 45-71

Debaud JC, Gay G (1987) In vitro fruiting under controlled conditions of the ectomycorrhizal fungus Hebeloma cylindrosporum associated with Pinus pinaster. New Phytol 105:429-435

Debaud JC, Gay G, Prevost A, Lei J, Dexheimer J (1988) Ec- tomycorrhizal ability of genetically different homokaryotic and dikaryotic mycelia of Hebeloma cylindrosporum. New Phytol 108:323-328

Dixon RK, Buschena CA (1988) Response of ectomycorrhizal Pinus banksiana and Picea 91auca to heavy metals in soil. Plant Soil 105:265-271

Dixon RK, Marx DH (1987) Mycorrhizae. In: Bonga JM, Dur- zan DJ (eds) Cell and tissue culture in forestry, vol. 2. Mar- tinus Nijhoff, Dordrecht, The Netherlands, pp 336-350

Duddridge JA, Malibari A, Reed DJ (1980) Structure and function of mycorrhizal rhizomorphs with special refer- ence to their role in water transport. Nature 287:834-836

Finkelstein DB, Rambosek JA, Leach J, Wilson RE, Larson AE, McAda PC, Soliday CL, Ball C (1986) Genetic transformation and protein secretion in industrial filamentous fungi. In: Alacevic N, Hranueli D, Ponan Z (eds) Genetics of industrial microorganisms. Proceedings of 5 th Interna- tional Symposium of Genetics of Industrial Microorgan- isms, Split, Yugoslavia 1985. Pliva Press, Zagreb, Yugo- slavia pp 101-110

Harley JL, Smith SE (1983) Mycorrhizal symbiosis. Academic Press, New York

Hebraud M, Fevre M (1988) Protoplast production and regeneration from mycorrhizal fungi and their use for isolation of mutants. Can J Microbiol 34:157--161

Homolka L, Vyskocil P, Pilat P (1988) Use of protoplasts in the improvement of filamentous fungi. I. Mutagenization of protoplasts of Oudemansiella mucida. Appl Microbiol Biotechnol 28:166-169

Hynes MJ (1986) Transformation of filamentous fungi. Exp Mycol 10:1-8

Kitamoto Y, Mori N, Yamamoto M, Ohiwa T, Ichikawa Y (1988) A simple method for protoplast formation and improvement of protoplast regeneration from various fungi using an enzyme from Trichoderma harzianum. Appl Mi- crobiol Biotechnol 28:445-450

Kropp BR, Fortin JA (1986) Formation and regeneration of protoplasts from the ectomycorrhizal basidiomycete Lac- caria bicolor. Can J Bot 64:1224-1226

Lapeyrie FF, Bruchet G (1985) Some factors influencing viability of ectomycorrhizal fungal inoculum. New Phytol 100:585-593

Marx DH (1975) The role of ectomycorrhizae in the protection of pine from root infection by Phytophthora cinnarnomi. In: Bruehl GW (ed) Biology and control of soil-borne plant pathogens. Am Phytopathol Soc, St. Paul, Minn, USA, pp 112-115

Miller OK (1982) Taxonomy of ecto- and ectendomycorrhizal fungi. In: Schenck NC (ed) Methods and principles of mycorrhizal research. Am Phytopathol Soc, St. Paul Min, USA, pp 81-101

Minuth W, Esser K (1983) Intraspecific, interspecific, and in-

V. Barrett et al.: Protoplast formation from ectomycorrhizal fungi 387

tergenic recombination in /~-lactam producing fungi via protoplast fusion. Appl Microbiol Biotechnol 18:38-46

Tully M, Gilbert HJ (1985) Transformation of Rhodosporidium toruloides. Gene 36: 235-240

Yamada O, Magae Y, Kashiwagi Y, Kakimoto Y, Sasaki T (1983) Preparation and regeneration of mycelial protoplasts of Collybia veltipes and Pleurotus ostreatus. Appl Mi- crobiol Biotechnol 17:298-300

Yanagi SO, Monma M, Kawssumi T, Hino A, Kito M, Takebe I (1985) Conditions for isolation of and colony formation

by mycelial protoplasts of Coprinus macrorhizus. Agric Biol Chem 49:171-179

Yelton M, Hamer JE, Timberlake WE (1984) Tranformation of Aspergillus nidulans by using a trpC plasmid. Proc Natl Acad Sci USA 81:1470-1474

Received October 4, 1988/Accepted December 19, 1988

protoplast formation from selected species of ectomycorrhizal fungi

Documents