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Protoplast fusion in Aspergillus: selection of interspecific heterokaryons using antifungal inhibitors
Journal of Microbiologtcal Methods 3 (1984) 27-32
27
Elsevier JMM 00079
Protoplast fusion in Aspergillus" selection of interspecific heterokaryons using antifungal inhibitors R . E . Bradshaw and J.F. Peberdy* Department of Botany, University of Nottingham, Nottmgham NG7 2RD (England) (Received 3 May 1984) (Accepted 28 June 1984)
Summary Wild-type isolates of Aspergillus nidulans and A. rugulosus have been utilized in protoplast fusion crosses using a novel selection method. Interspecific heterokaryons were produced by combining these strains with auxotrophic, inhibitor-resistant mutants and recovering the fusant colonies using minimal medium containing inhibitor as the selective medium.
Key words: Aspergiilus - Protoplast fusion in fungi - Selection using inhibitors
Introduction
In the parasexual cycle in fungi, heterokaryons and diploids are usually selected by the complementation of auxotrophic markers which differ for the two parents [1]. The value of the parasexual cycle for strain improvement in commercially important fungi is consequently limited because the induction of such nutritional markers can depress yields of desired metabolites [2, 3]. In addition, the isolation of such mutants can be time-consuming and, if mutagenic treatments are used, both chromosomal aberrations and deleterious mutations may be induced simultaneously. These considerations are also important in studies of interspecific hybridization, in which nutritional complementation is commonly employed as a selective method [4--6]. This paper outlines successful attempts to select interspecific heterokaryons and allodiploids of Aspergillus nidulans and A. rugulosus following protoplast fusion, using a novel selection regime which enables wild-type *
To whom correspondence should be addressed.
0167-7012/84/$03.00 © 1984 Elsevier Science Publishers B.V.
28
isolates to be used as one of the parents. In each cross, selection against the latter parent was achieved by its sensitivity to an inhibitor whilst selection against an auxotrophic, inhibitor-resistant parent was achieved by plating on nutritionally deficient medium. The principles of this system are similar to those used to obtain somatic hybrids of mammalian cells [7]. Materials and Methods
Strains and culture conditions The strains of A. nidulans and A. rugulosus used in these experiments are listed in Table 1. Cultures were maintained on complete medium (CM) which contained (g • 1-1): oxoid yeast extract, 2.5; oxoid malt extract, 5.0; glucose, 10.0; Sigma agar, 20.0; CuSO4 • 5H20, trace. The minimal medium (MM) was prepared as described by Pontecorvo [8]. Incubation was at 28°C throughout. TABLE 1 STRAINS USED The gene symbols used for A. rugulosus are: ade, requirement for adenine; whi, white conidiospore colour; acr, resistant to acriflavine; malc, resistant to malachite green. The gene symbols used for resistance to inhibitors in A. nidulans are: acr, resistant to acriflavine; ben, benlate; act, actidione (cycloheximide); oli, oligomycin; ima, lmazilil; car, carboxin. Gene symbols for auxotrophic, non-utdization and spore-colour markers are those of Clutterbuck [14]. Species
Nottingham collection strain no.
Genetic markers
Origin
Aspergillus nidulans
2-1 2-3 2-18
wild-type wild-type
University of Nottingham FGSC (strain no. 4) FGSC (strain no. 407)
2-36
2-112
Aspergillus rugulosus
adE20 blA1; AcrA1; phenA2, pyroA4; lysB5; lacA1, choA1; riboB2 chaA1 suAladE20, yA2, adE20; wA3; galA1; pyroA4; facA303; sB3; nicB8; benAlO, rurAl, carB2 galD5, suAladE20, riboA1, anAl, pro-94, lysF88, pabaA1, yA2, adE20; actB2, oliCl3, tmaA4, benC28, choA1, carA1
3---46
adeA3, whiA3, acr-1 adeA3, whiA3, act-l, malc-2
3-97
wild-type
3-98
wild-type
3-110
wild-type
3--15
J. Van Tuyl
Parasexual recombinant from FGSC 466 x FGSC 529
University of Nottingham Sandoz (Strain no. BP 2061/21) Sandoz (Strain no. BP 2056/32) I.M.I. (Strain no. 91020a)
29
Protoplast isolation Protoplasts were isolated using a combination of the lytic enzymes Novozym 234 (Novo Enzyme Products Ltd., Windsor, Berkshire, England) and Cellulase CP (John and E. Sturge Ltd., Selby, England) as previously described for Aspergillus species [9].
Protoplast fusion Fusion of protoplasts was induced as described in a previous publication [10] except that 0.6M KC1, maintained at pH 5.8 using phosphate buffers, was used as the osmotic stabilizer. At least 107 protoplasts of each parent were combined in each fusion. Fusion products were selected on MM containing 0.6M KCI (MMS) and the appropriate inhibitor at concentrations which prevented growth of protoplasts of the sensitive parents. Protoplast viability was checked by plating on CM + 0.6M KC1 (CMS). The fusion frequency was calculated as the ratio of colonies growing on MMS and CMS (%).
Antifungal inhibitors Acriflavine (a mixture of 2,8-diamino-10-methyl acridinium chloride and 2,8diamino acridine) and cycloheximide (3-(2-3,5-dimethyl-2-oxocyclohexyl)-2hydroxyethylglutarimide) were obtained from Sigma Chemical Co., Poole, Dorset, England. Benlate (methyl-l-(butylcarbamoyl)-2-benzimidazolecarbamate) was from E.I. du Pont de Nemours and Co., Geneva, Switzerland. Carboxin 15,6dihydro-2-methyl-l,4-oxathiin-3-carboxanilide) and imazilil (1-13-(alkyloxy)-2,4chlorophenetyl-imidazole phosphate) were gifts from Dr. B. Baldwin of I.C.I. plc Plant Protection Division, Jeallot's Hill, Berkshire, England. Acriflavine and benlate were dissolved in distilled water; carboxin, cycloheximide and imazilil were dissolved in methanol to prepare stock solutions and added to molten agar to give a maximum solvent concentration of 1%. Results and Discussion
Selection using acriflavine Interspecific heterokaryons were obtained by selection on MMS + acriflavine (25 and 50 ~tg/ml) from six fusions involving different combinations of parental strains (Table 2). The following criteria were used as evidence of interspecies hybridization: (i) slow growth of colonies on MMS + acriflavine selection plates and segregation of these colonies to give both parental types when transferred to CM; (ii) the absence of colonies when parental protoplasts were plated separately with or without treatment with the fusogenic agent, polyethylene glycol (PEG), or as a mixture without PEG-treatment, on MMS + acriflavine; (iii) the production of vigorously-growing sectors by fusion colonies which had been subcultured onto MM + acriflavine. These sectors were poorly sporulating and produced brown pigment, and thus were morphologically similar to the A. nidulans x A. rugulosus allodiploid hybrids previously isolated and described [4]. The hybrid nature of the sectors was suggested by their stability on CM and their segregation on CM con-
3O TABLE 2 SELECTION OF INTERSPECIFIC HETEROKARYONS
O N MMS + A C R I F L A V I N E
Fusion frequency a (% × 10 -3)
Parent strains Auxotrophic, acriflavine-res~stant
Prototrophic, acnflavme-sensit~ve
A. nidulans
A. rugulosus
2-18 2-18
3-97 3-98
A. rugulosus 3--15 3-15 3-46 3-46
A. nidulans 2-1 2-3 2-1 2-3
MMS + 25 ~g - m l acriflavme
MMS + 50~tg - ml -t acriflavine
0.056 0.007
0.020 0.002
0.043 0.025 0.004 0.016
0.018 0.006 0.001 0.005
Ratio of colonies on MMS + acriflavme and CMS x 100 (%).
TABLE 3 SELECTION OF INTERSPECIFIC HETEROKARYONS
Parents Fusion frequency (% × 10 -a)
f 2-112 × 3-97 2-112 × 3-110
Resistance mutation in 2-11: % Survival of 2-112 protoplasts on CMS + inhibitor % Dominance of mutation in diploids of A. nidulans a
Carboxin (100 ~tg. ml -~) 0.005 0.035
carA1
O N MMS + V A R I O U S I N H I B I T O R S
Benlate (5 ~tg. m1-1) 0.011 0.088
benC28
4 5
85.6
85.6
45.4
Cycloheximide (1000 ~tg. ml -~) 0.028 0.122
actB2
7.8
100
Imazihl (0.25 ~tg. ml -~) 0.016 0.094
imaA 4 1.8
42.5
Data from Ref. 13.
taining 1.5-2.0 ~tg/ml benlate to give a range of morphological types. A large number of stable segregants were isolated and characterized from one of the hybrids (2-18 × 3--97) grown on medium containing the haploidization agent benlate. 96 different combinations of the known genetic markers contributed by parent 2-18 were expressed amongst the sample of segregants analyzed [11], which showed without doubt that nuclear fusion and genetic recombination had occurred. The fusion frequencies shown in Table 2 are similar to those obtained by conventional selection; protoplast fusion of complementing auxotrophs of A. nidulans and A. rugulosus which are selected on MMS generally yield fusion frequencies of 0.02-0.40% [12]. With concentrations of acriflavine greater than 50 ~tg/ml in the
31 selection medium, fusion frequencies declined; this was directly correlated with a decline in viability of the resistant parent. Conversely, fusion frequencies were elevated when less than 25 ~tg/ml inhibitor were present, but at such low concentrations a proportion of wild-type A. rugulosus protoplasts were able to revert and grow. Selection using other inhibitors Protoplasts of a strain of A. nidulans, 2-112, which carries several mutations conferring resistance to inhibitors (Table 1), were fused with protoplasts of wildtype strains of A. rugulosus, 3-97 and 3-110. Interspecific heterokaryons were isolated by selection on MMS containing benlate, carboxin, cycloheximide or imazilil (Table 3) and were identified according to the criteria outlined for acriflavine selection. Colonies with the morphology of interspecific hybrids were isolated as sectors from fusion products selected on carboxin and on imazilil. Protoplast fusion of A. nidulans strain 2-36, which is resistant to benlate, with A. rugulosus wild-type 3-97, yielded no fusion products on MMS + benlate. This was an interesting contrast to the fusion involving strains 2-112 and 3-97, and the same fungicide, which formed heterokaryons. These A. nidulans strains have different resistance mutations; 2-36 carries ben AIO (linkage group VIII) which is recessive in intraspecific diploids whilst 2-112 contains ben C28 (linkage group VII) which is semi-dominant [13]. Since strain 2-36 is more resistant to benlate than strain 2-112 [12, 13], it seems likely that the inability of the former to contribute to the formation of fusion products is a consequence of its recessivity, and is therefore inadequately expressed in interspecific heterokaryons. All of the inhibitor-resistant strains which successfully yielded fusion products are known to carry resistance markers which show semi-dominant or dominant expression in intraspecific diploids (Table 3). The method of heterokaryon selection outlined in this paper has the distinct advantage over conventional auxotrophic complementation that wild-type strains can be used in parasexual crosses. A large number of wild isolates could be used, for example, in programs of recombination in industrially important fungi by crossing them with a few specially constructed auxotrophic, inhibitor-resistant strains.
Acknowledgements R.E.B. acknowledges the support of a SERC Postgraduate Studentship.
References 1 Roper,J.A. (1952) Productionof heterozygousdiploidsin filamentousfungi. Experientia8, 14-15 2 Fantini,A.A. (1962) Genetics and antibiotic production of Emericellopsis species. Genetics 47, 161-177. 3 Macdonald, K.D.. Hutchinson, J.M. and Gillett, W.A. (1963) Isolation of auxotrophs of Penicilhum chrysogenum and their penicillinyields. J. Gen. Microbiol. 33,365-374.
32 4 5 6 7
8 9
10 11
12 13 14
Kevei, F. and Peberdy, J.F. (1977) Interspecific hybridization between Aspergillus n,dulans and Aspergillus rugulosus by fusion of somatic protoplasts, J. Gen. Microbiol. 102,255-262. Kevel, F. and Peberdy, J.F. (1979) Induced segregation in mterspecific hybrids of Aspergillus ntdulans and Aspergillus rugulosus obtained by protoplast fusion. Mol. Gen. Genet, 170, 213-218 Ferenczy, L. (1981) Microbial protoplast fusion. Symp. Soc. Gen. Microbiol. 31, 1-34 Jha, K.K. and Ozer, H.L. (1976) Expression of transformation in cell hybrids. I. Isolation and apphcation of density-inhibited Balb/3T3 cells deficient in hypoxanthine phosphoribosyl-transferose and resistant to ouabam. Somat. Cell Genet. 2,215-223. Pontecorvo, G., Roper, J.A., Hemmons, D.W., Macdonald, K.D. and Burton, A.W. (1953) The genetics of Aspergillus nidulans. Adv. Genet. 5, 141-238. Hamlyn, P.F., Bradshaw, R.E., Mellon, F.M., Santiago, C.M., Wilson, J.M. and Peberdy, J.F. (1981) Efficient protoplast isolation from fungi using commercial enzymes. Enzyme Microbiol Technol. 3,321-325. Annd, J. and Peberdy, J.F. (1976) Induced fusion of fungal protoplasts following treatment with polyethylene glycol. J. Gen. Microbiol. 92,413--417. Bradshaw, R.E., Lee, K.U. and Peberdy, J.F. (1983) Aspects of genetic interaction in hybrids of Aspergillus nidulans and Aspergillus rugulosus obtained by protoplast fusion. J. Gen. Microbiol. 129, 3525-3533. Bradshaw, R.E. (1983) Hybridization of Aspergillus species. Ph.D. Thesis. University of Nottingham. U.K. Van Tuyl, J.M. (1977) Genetics of fungal resistance to systemic fungicides. Ph.D. Thesis. Agricultural University of Wageningen, Netherlands. Clutterbuck, A.J. (1974) Aspergillus nidulans. In: Handbook of Genetics, Vol. 1, (King, R.C., ed.) pp. 447-510, Plenum Press, New York.
27
Elsevier JMM 00079
Protoplast fusion in Aspergillus" selection of interspecific heterokaryons using antifungal inhibitors R . E . Bradshaw and J.F. Peberdy* Department of Botany, University of Nottingham, Nottmgham NG7 2RD (England) (Received 3 May 1984) (Accepted 28 June 1984)
Summary Wild-type isolates of Aspergillus nidulans and A. rugulosus have been utilized in protoplast fusion crosses using a novel selection method. Interspecific heterokaryons were produced by combining these strains with auxotrophic, inhibitor-resistant mutants and recovering the fusant colonies using minimal medium containing inhibitor as the selective medium.
Key words: Aspergiilus - Protoplast fusion in fungi - Selection using inhibitors
Introduction
In the parasexual cycle in fungi, heterokaryons and diploids are usually selected by the complementation of auxotrophic markers which differ for the two parents [1]. The value of the parasexual cycle for strain improvement in commercially important fungi is consequently limited because the induction of such nutritional markers can depress yields of desired metabolites [2, 3]. In addition, the isolation of such mutants can be time-consuming and, if mutagenic treatments are used, both chromosomal aberrations and deleterious mutations may be induced simultaneously. These considerations are also important in studies of interspecific hybridization, in which nutritional complementation is commonly employed as a selective method [4--6]. This paper outlines successful attempts to select interspecific heterokaryons and allodiploids of Aspergillus nidulans and A. rugulosus following protoplast fusion, using a novel selection regime which enables wild-type *
To whom correspondence should be addressed.
0167-7012/84/$03.00 © 1984 Elsevier Science Publishers B.V.
28
isolates to be used as one of the parents. In each cross, selection against the latter parent was achieved by its sensitivity to an inhibitor whilst selection against an auxotrophic, inhibitor-resistant parent was achieved by plating on nutritionally deficient medium. The principles of this system are similar to those used to obtain somatic hybrids of mammalian cells [7]. Materials and Methods
Strains and culture conditions The strains of A. nidulans and A. rugulosus used in these experiments are listed in Table 1. Cultures were maintained on complete medium (CM) which contained (g • 1-1): oxoid yeast extract, 2.5; oxoid malt extract, 5.0; glucose, 10.0; Sigma agar, 20.0; CuSO4 • 5H20, trace. The minimal medium (MM) was prepared as described by Pontecorvo [8]. Incubation was at 28°C throughout. TABLE 1 STRAINS USED The gene symbols used for A. rugulosus are: ade, requirement for adenine; whi, white conidiospore colour; acr, resistant to acriflavine; malc, resistant to malachite green. The gene symbols used for resistance to inhibitors in A. nidulans are: acr, resistant to acriflavine; ben, benlate; act, actidione (cycloheximide); oli, oligomycin; ima, lmazilil; car, carboxin. Gene symbols for auxotrophic, non-utdization and spore-colour markers are those of Clutterbuck [14]. Species
Nottingham collection strain no.
Genetic markers
Origin
Aspergillus nidulans
2-1 2-3 2-18
wild-type wild-type
University of Nottingham FGSC (strain no. 4) FGSC (strain no. 407)
2-36
2-112
Aspergillus rugulosus
adE20 blA1; AcrA1; phenA2, pyroA4; lysB5; lacA1, choA1; riboB2 chaA1 suAladE20, yA2, adE20; wA3; galA1; pyroA4; facA303; sB3; nicB8; benAlO, rurAl, carB2 galD5, suAladE20, riboA1, anAl, pro-94, lysF88, pabaA1, yA2, adE20; actB2, oliCl3, tmaA4, benC28, choA1, carA1
3---46
adeA3, whiA3, acr-1 adeA3, whiA3, act-l, malc-2
3-97
wild-type
3-98
wild-type
3-110
wild-type
3--15
J. Van Tuyl
Parasexual recombinant from FGSC 466 x FGSC 529
University of Nottingham Sandoz (Strain no. BP 2061/21) Sandoz (Strain no. BP 2056/32) I.M.I. (Strain no. 91020a)
29
Protoplast isolation Protoplasts were isolated using a combination of the lytic enzymes Novozym 234 (Novo Enzyme Products Ltd., Windsor, Berkshire, England) and Cellulase CP (John and E. Sturge Ltd., Selby, England) as previously described for Aspergillus species [9].
Protoplast fusion Fusion of protoplasts was induced as described in a previous publication [10] except that 0.6M KC1, maintained at pH 5.8 using phosphate buffers, was used as the osmotic stabilizer. At least 107 protoplasts of each parent were combined in each fusion. Fusion products were selected on MM containing 0.6M KCI (MMS) and the appropriate inhibitor at concentrations which prevented growth of protoplasts of the sensitive parents. Protoplast viability was checked by plating on CM + 0.6M KC1 (CMS). The fusion frequency was calculated as the ratio of colonies growing on MMS and CMS (%).
Antifungal inhibitors Acriflavine (a mixture of 2,8-diamino-10-methyl acridinium chloride and 2,8diamino acridine) and cycloheximide (3-(2-3,5-dimethyl-2-oxocyclohexyl)-2hydroxyethylglutarimide) were obtained from Sigma Chemical Co., Poole, Dorset, England. Benlate (methyl-l-(butylcarbamoyl)-2-benzimidazolecarbamate) was from E.I. du Pont de Nemours and Co., Geneva, Switzerland. Carboxin 15,6dihydro-2-methyl-l,4-oxathiin-3-carboxanilide) and imazilil (1-13-(alkyloxy)-2,4chlorophenetyl-imidazole phosphate) were gifts from Dr. B. Baldwin of I.C.I. plc Plant Protection Division, Jeallot's Hill, Berkshire, England. Acriflavine and benlate were dissolved in distilled water; carboxin, cycloheximide and imazilil were dissolved in methanol to prepare stock solutions and added to molten agar to give a maximum solvent concentration of 1%. Results and Discussion
Selection using acriflavine Interspecific heterokaryons were obtained by selection on MMS + acriflavine (25 and 50 ~tg/ml) from six fusions involving different combinations of parental strains (Table 2). The following criteria were used as evidence of interspecies hybridization: (i) slow growth of colonies on MMS + acriflavine selection plates and segregation of these colonies to give both parental types when transferred to CM; (ii) the absence of colonies when parental protoplasts were plated separately with or without treatment with the fusogenic agent, polyethylene glycol (PEG), or as a mixture without PEG-treatment, on MMS + acriflavine; (iii) the production of vigorously-growing sectors by fusion colonies which had been subcultured onto MM + acriflavine. These sectors were poorly sporulating and produced brown pigment, and thus were morphologically similar to the A. nidulans x A. rugulosus allodiploid hybrids previously isolated and described [4]. The hybrid nature of the sectors was suggested by their stability on CM and their segregation on CM con-
3O TABLE 2 SELECTION OF INTERSPECIFIC HETEROKARYONS
O N MMS + A C R I F L A V I N E
Fusion frequency a (% × 10 -3)
Parent strains Auxotrophic, acriflavine-res~stant
Prototrophic, acnflavme-sensit~ve
A. nidulans
A. rugulosus
2-18 2-18
3-97 3-98
A. rugulosus 3--15 3-15 3-46 3-46
A. nidulans 2-1 2-3 2-1 2-3
MMS + 25 ~g - m l acriflavme
MMS + 50~tg - ml -t acriflavine
0.056 0.007
0.020 0.002
0.043 0.025 0.004 0.016
0.018 0.006 0.001 0.005
Ratio of colonies on MMS + acriflavme and CMS x 100 (%).
TABLE 3 SELECTION OF INTERSPECIFIC HETEROKARYONS
Parents Fusion frequency (% × 10 -a)
f 2-112 × 3-97 2-112 × 3-110
Resistance mutation in 2-11: % Survival of 2-112 protoplasts on CMS + inhibitor % Dominance of mutation in diploids of A. nidulans a
Carboxin (100 ~tg. ml -~) 0.005 0.035
carA1
O N MMS + V A R I O U S I N H I B I T O R S
Benlate (5 ~tg. m1-1) 0.011 0.088
benC28
4 5
85.6
85.6
45.4
Cycloheximide (1000 ~tg. ml -~) 0.028 0.122
actB2
7.8
100
Imazihl (0.25 ~tg. ml -~) 0.016 0.094
imaA 4 1.8
42.5
Data from Ref. 13.
taining 1.5-2.0 ~tg/ml benlate to give a range of morphological types. A large number of stable segregants were isolated and characterized from one of the hybrids (2-18 × 3--97) grown on medium containing the haploidization agent benlate. 96 different combinations of the known genetic markers contributed by parent 2-18 were expressed amongst the sample of segregants analyzed [11], which showed without doubt that nuclear fusion and genetic recombination had occurred. The fusion frequencies shown in Table 2 are similar to those obtained by conventional selection; protoplast fusion of complementing auxotrophs of A. nidulans and A. rugulosus which are selected on MMS generally yield fusion frequencies of 0.02-0.40% [12]. With concentrations of acriflavine greater than 50 ~tg/ml in the
31 selection medium, fusion frequencies declined; this was directly correlated with a decline in viability of the resistant parent. Conversely, fusion frequencies were elevated when less than 25 ~tg/ml inhibitor were present, but at such low concentrations a proportion of wild-type A. rugulosus protoplasts were able to revert and grow. Selection using other inhibitors Protoplasts of a strain of A. nidulans, 2-112, which carries several mutations conferring resistance to inhibitors (Table 1), were fused with protoplasts of wildtype strains of A. rugulosus, 3-97 and 3-110. Interspecific heterokaryons were isolated by selection on MMS containing benlate, carboxin, cycloheximide or imazilil (Table 3) and were identified according to the criteria outlined for acriflavine selection. Colonies with the morphology of interspecific hybrids were isolated as sectors from fusion products selected on carboxin and on imazilil. Protoplast fusion of A. nidulans strain 2-36, which is resistant to benlate, with A. rugulosus wild-type 3-97, yielded no fusion products on MMS + benlate. This was an interesting contrast to the fusion involving strains 2-112 and 3-97, and the same fungicide, which formed heterokaryons. These A. nidulans strains have different resistance mutations; 2-36 carries ben AIO (linkage group VIII) which is recessive in intraspecific diploids whilst 2-112 contains ben C28 (linkage group VII) which is semi-dominant [13]. Since strain 2-36 is more resistant to benlate than strain 2-112 [12, 13], it seems likely that the inability of the former to contribute to the formation of fusion products is a consequence of its recessivity, and is therefore inadequately expressed in interspecific heterokaryons. All of the inhibitor-resistant strains which successfully yielded fusion products are known to carry resistance markers which show semi-dominant or dominant expression in intraspecific diploids (Table 3). The method of heterokaryon selection outlined in this paper has the distinct advantage over conventional auxotrophic complementation that wild-type strains can be used in parasexual crosses. A large number of wild isolates could be used, for example, in programs of recombination in industrially important fungi by crossing them with a few specially constructed auxotrophic, inhibitor-resistant strains.
Acknowledgements R.E.B. acknowledges the support of a SERC Postgraduate Studentship.
References 1 Roper,J.A. (1952) Productionof heterozygousdiploidsin filamentousfungi. Experientia8, 14-15 2 Fantini,A.A. (1962) Genetics and antibiotic production of Emericellopsis species. Genetics 47, 161-177. 3 Macdonald, K.D.. Hutchinson, J.M. and Gillett, W.A. (1963) Isolation of auxotrophs of Penicilhum chrysogenum and their penicillinyields. J. Gen. Microbiol. 33,365-374.
32 4 5 6 7
8 9
10 11
12 13 14
Kevei, F. and Peberdy, J.F. (1977) Interspecific hybridization between Aspergillus n,dulans and Aspergillus rugulosus by fusion of somatic protoplasts, J. Gen. Microbiol. 102,255-262. Kevel, F. and Peberdy, J.F. (1979) Induced segregation in mterspecific hybrids of Aspergillus ntdulans and Aspergillus rugulosus obtained by protoplast fusion. Mol. Gen. Genet, 170, 213-218 Ferenczy, L. (1981) Microbial protoplast fusion. Symp. Soc. Gen. Microbiol. 31, 1-34 Jha, K.K. and Ozer, H.L. (1976) Expression of transformation in cell hybrids. I. Isolation and apphcation of density-inhibited Balb/3T3 cells deficient in hypoxanthine phosphoribosyl-transferose and resistant to ouabam. Somat. Cell Genet. 2,215-223. Pontecorvo, G., Roper, J.A., Hemmons, D.W., Macdonald, K.D. and Burton, A.W. (1953) The genetics of Aspergillus nidulans. Adv. Genet. 5, 141-238. Hamlyn, P.F., Bradshaw, R.E., Mellon, F.M., Santiago, C.M., Wilson, J.M. and Peberdy, J.F. (1981) Efficient protoplast isolation from fungi using commercial enzymes. Enzyme Microbiol Technol. 3,321-325. Annd, J. and Peberdy, J.F. (1976) Induced fusion of fungal protoplasts following treatment with polyethylene glycol. J. Gen. Microbiol. 92,413--417. Bradshaw, R.E., Lee, K.U. and Peberdy, J.F. (1983) Aspects of genetic interaction in hybrids of Aspergillus nidulans and Aspergillus rugulosus obtained by protoplast fusion. J. Gen. Microbiol. 129, 3525-3533. Bradshaw, R.E. (1983) Hybridization of Aspergillus species. Ph.D. Thesis. University of Nottingham. U.K. Van Tuyl, J.M. (1977) Genetics of fungal resistance to systemic fungicides. Ph.D. Thesis. Agricultural University of Wageningen, Netherlands. Clutterbuck, A.J. (1974) Aspergillus nidulans. In: Handbook of Genetics, Vol. 1, (King, R.C., ed.) pp. 447-510, Plenum Press, New York.