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Autopolyploid formation of Trichoderma reesei QM9414 by colchicine treatment
JOURNAL or FERMENTATIONAND BIOENGINEERING VO1. 69, No. 1, 51-53. 1990
NOTES Autopolyploid Formation of Trichoderma reesei QM9414 by Colchicine Treatment H I D E O T O Y A M A AND N O B U O T O Y A M A *
Department of Food Technology, Faculty of Horticulture, Minamikyushu University, Takanabe-cho, Hibarigaoka 884, Japan Received 21 August 1989/Accepted 6 November 1989 Conidia of Trichoderma reesei QM9414 were treated with colchicine. Nuclei in coichicine-treated conidia enlarged. When the concentration of colchicine or the treatment time with colchicine increased, the diameter of nuclei became larger. Colchicine-treated conidia generated sectors on a medium containing benomyl. Some sectors formed many conidia or could not produce clear zones on the plate assay medium for cellulase production. According to the D N A assay of conidia, colchicine-treated strains were autopolyploid.
shaker. After 24 h, m a n y small pellets (about 2.0 mm in diameter) appeared in the medium. Ungerminated conidia incubated in the Natick medium containing 8 0 / l g / m l t-arginine, 2.0%o glucose, and 0.1% (w/v) colchicine (pH 10) for 24 h at 30°C by a reciprocal shaker were collected and incubated on the Natick medium containing 8 0 p g / m l L-arginine, 2.0°/oo glucose, 0.1}~ Triton X-100, and 1.5%0 agar (pH 5.0) for 4 d at 30°C to form colonies. Mycelial mats (2 m m × 2 mm) o f these colonies were cut out and put on Natick medium containing 0 . 2 p g / m l benomyl, 8 0 p g / m l L-arginine, 2.0%o glucose, and 1.5~0" agar (pH 5.0) (the haploidizing medium), and incubated for 14 d at 30°C. After incubation, nonsector parts o f colonies that had generated sectors were isolated (Z1) as shown in Fig. 1. W h e n mycelial mats of colonies derived from conidia o f Z1 were put on the haploidizing medium and incubated for 14 d at 30°C, all of colonies formed sectors. Similar experiments were done at p H 3.0, 5.0, and 7.0. A t p H 3.0, the buffer containing 1.0 M HC1 and 1.0 M C H 3 C O O N a was used. For p H 5.0, the buffer containing 0.2 M CH3COOH and 0.2 M CH3COONa was ued. F o r p H 7.0, the buffer containing 0.2 M NaHzPO4 and 0.2 M Na2HPO4 was used. Green mature conidia of Z 1 and no. 83 generated on the Natick medium containing 8 0 p g / m l L-arginine, 2.0°//oo glucose, and 1.5% agar ( p H 5 . 0 ) for 10d of incubation at 30°C were collected in a desiccator and dried for 28 d. After drying, the length of the m a j o r axis of conidia formed by these two types were measured for at least 50 conidia in each strain. D N A of conidia was extracted by the method of Herbert et al. (10) and assayed the D N A contents o f 1 mg o f green mature conidia by the method o f Ceriotti (11). It was possible to compare D N A contents o f 1 mg o f green mature conidia, comparing this with the result of measurement o f length of the conidial m a j o r axis. Table 1 showed that D N A contents of 1 mg of Z1 green mature conidia were apparently larger than those of 1 mg o f no. 83 green mature conidia, consistent with the increased conidial m a j o r axis in Z1. Green mature conidia o f sector parts (Z2) generated from Z1 incubated on the haploidizing medium for 14 d at 30°C were dried in a desiccator for 28 d. After drying, the
Cellulase-producing fungi o f the genus Trichoderma are widely used in industry and there are m a n y reports on its breeding (1). The genus Trichoderma is also used for investigating the action o f cellulase components because the fungus has an suitable cellulase system. However, it is necessary, for m o r e i n f o r m a t i o n on cellulase action, to breed m o r e various mutants, e.g. c h r o m o s o m a l mutants. This fungus is mononucleate (2) and it is easy to grasp the genetic condition o f hybrids, comparing with multinucleate fungi such as Aspergillus oryzae (3). In fact, intraspecific (4) and interspecific p r o t o p l a s t fusion (5) of Trichoderma reesei was done in 1984. In this paper, colchicine treatment was attempted for T. reesei QM9414 to get more various genetic variants without the use o f p r o t o p l a s t fusion or mutation. Colchicine and its derivative colcemid are known to bind tubulin and cause c h r o m o s o m e n o n d i s j u n c t i o n (6). Using these chemicals, a u t o p o l y p l o i d f o r m a t i o n has been done in animals (7) and plants (8). In microbes, colchicine was used for breeding in yeast (Fujii, T. et al., Abstr. A n n u . Meet. Agric. Biol. Chem. Soc. J a p a n , p. 134, 1989) but not used for breeding in T. reesei. In fungi, there are only a few reports o f colchicine treatment, p r o b a b l y because colchicine turns to colchicein, which has no tubulin-binding activity at low pH, the cultivation condition of fungi. Therefore, colchicine treatment o f T. reesei was attempted in high p H cultivation to generate a u t o p o l y p l o i d o f this fungus. Strain no. 83 was an a u x o t r o p h i c mutant ( a r g ) derived from T. reesei QM9414 by UV-irradiation. Conidia o f no. 83 were a d d e d to Natick medium (9) containing 1.4 g o f (NH4)2SO4, 2.0 g of KH2PO4, 0.3 g o f urea, 0.3 g o f CaC12, 0.3 g o f M g S O 4 • H 2 0 , 0.005 g o f FeSO4.7H20, 0.0016 g o f MnSO4. H20, 0.0014 g of ZnSO4. H20, 0.002 g o f CoC12, 0.08 g o f L-arginine, 20.0 g o f glucose, and 1.0 g of colchicine ( W a k o Pure Chem. Co., Ltd.) per liter and the buffer containing 0 . 2 M N a z C O 3 and 0 . 2 M NaHCO3 (pH 10), and incubated on a reciprocal shaker for 24 h at 30°C. After 24 h, no pellet was seen in the medium. As a control, no. 83 were a d d e d into the same medium without colchicine and incubated for 24 h at 30°C by a reciprocal * Corresponding author. 51
52
J. FERMENT. B1OENG.,
TOYAMA AND TOYAMA
FIG. 1. Sector formation of a Z1 colony. No. 83 was an arginine-less mutant of T. reesei QM9414 and Z 1 was 0.1% (w/v) colchicine-treated no. 83 for 24 h at 30°C. ZI generated sectors on the medium containing 0.2pg/ml (w/v) benomyl. The arrow indicates Z2A-E. D N A c o n t e n t s o f these 1 m g o f c o n i d i a were assayed. T h e r e were t w o types in D N A c o n t e n t s o f 1 m g Z2 green m a t u r e c o n i d i a (Table 1), one ( Z 2 A ) had the same D N A c o n t e n t as that o f no. 83 and the o t h e r type (Z2B) had a larger D N A c o n t e n t t h a n that o f no. 83. Z 2 A did not g e n e r a t e new sectors but Z2B g e n e r a t e d new sectors on the h a p l o i d i z i n g m e d i u m i n c u b a t e d for 14 d at 30°C. D r i e d green m a t u r e c o n i d i a o f no. 83 and Z1 were stained by G i e m s a s o l u t i o n . A f t e r t r e a t m e n t with 5 N, 3 N, a n d 1 N HC1 at 6 0 ° C , the d i a m e t e r o f nuclei in c o n i d i a o f no. 83 and Z1 were m e a s u r e d by calipers a m o n g at least 50 c o n i d i a in each strain. T a b l e 2 s h o w e d that Z1 c o n i d i a had larger nuclei t h a n no. 83 c o n i d i a had. D r i e d c o n i d i a o f Z1, after 24 h, 48 h, 96 h, and 120 h o f i n c u b a t i o n at 3 0 ° C with 0 . 1 % ( w / v ) colchicine were stained by G i e m s a s o l u t i o n after p r e t r e a t m e n t . N u c l e a r d i a m e t e r in Z1 c o n i d i a e n l a r g e d as the i n c u b a t i o n t i m e b e c a m e longer. Nuclei in no. 83 c o n i d i a i n c u b a t e d for 72 h e n l a r g e d a b o u t 2 times larger t h a n t h o s e in Z1 c o n i d i a inc u b a t e d for 24 h. This suggests that not only diploids but
TABLE 1.
DNA contents of conidia derived from T. reesei QM9414, no. 83, Z1, Z2A and Z2B
Strains T. reesei QM9414
No. 83 ( a r g ) ZI-I b Z1-2 Z2A-F Z2A-2 Z2B-1 Z2B-2 Z2A-E d Z2A-L
DNA content (,ug/mg conidia) 5.25 5.23 11.45 10.80 5.20 5.22 6.75 7.01 5.30 5.27
Length of major Standard axis of conidia" deviation ~m) (6) 4.72 4.72 7.22 7.54 5.96 6.80 7.09 6.85 6.25 6.76
0.08 0.08 0.23 0.17 0.15 0.13 0.20 0.18 0.15 0.18
The length of major axis of conidia showed average among at least 50 conidia. b ZI-1 and Z1-2 were derived from no. 83 conidia treated with 0.1% (w/v) colchicine for 24h at 30°C with a reciprocal shaker (pH 10). c Z2A and Z2B were sectors generated from Z1 on the haploidizing medium, Z2A did not segregate new sectors on the haploidizing medium but Z2B segregated new sectors. a Z2A-E produced clear zones on the plate assay medium for cellulase production but Z2A-L did not produce clear zones.
also p o l y p l o i d s m a y be g e n e r a t e d by colchicine t r e a t m e n t . Dried c o n i d i a o f Z1 treated with 0 . 0 5 % , 0 . 1 % , 0 . 3 % , and 0.5%o ( w / v ) colchicine for 96 h at 3 0 ° C were stained by G i e m s a s o l u t i o n after t r e a t m e n t . T h e largest nuclei in Z1 c o n i d i a were seen in the case o f 0 . 5 % ( w / v ) colchicine. The nuclear d i a m e t e r o f Z 2 A c o n i d i a was similar to that o f no. 83 and did n o t change d u r i n g cultivation. But the nuclear d i a m e t e r o f Z2B c o n i d i a was i n t e r m e d i a t e between the nuclear d i a m e t e r o f Z1 c o n i d i a and that o f no. 83 conidia. Z 2 A seemed to be m o n o p l o i d and Z2B seemed to be diploid or partial diploid. T h e d i s t r i b u t i o n o f the ability o f c o n i d i a f o r m a t i o n , a u x o t r o p h y , and the ability to f o r m clear zones o n the plate assay m e d i u m for cellulase p r o d u c t i o n c o n t a i n i n g 1 . 0 % ( w / v ) c a r b o x y m e t h y l c e l l u l o s e calcium salt ( C M C Ca) and 0 . 1 % T r i t o n X-100 after 6 d o f i n c u b a t i o n at 3 0 ° C o f Z1, Z 2 A , and Z2B were investigated. T a b l e 3 shows that two types o f strains a p p e a r e d , one ( Z 2 A - E ) p r o duced conidia m u c h m o r e than no. 83 and Z1, and p r o d u c e d a clear z o n e on the plate assay m e d i u m for cellulase p r o d u c t i o n , the o t h e r ( Z 2 A - L ) p r o d u c e d few c o n i d i a and did n o t p r o d u c e clear zones on the plate assay m e d i u m for cellulase p r o d u c t i o n . Z 2 A - E and Z 2 A - L were arginine-less like no. 83 and Z1. A c c o r d i n g to the D N A assay o f conidia, Z 2 A - E and Z 2 A - L seemed to be like m o n o p l o i d s . T h e great a m o u n t o f c o n i d i a in Z 2 A - E was c o n s i d e r e d to be caused by a c h r o m o s o m a l m u t a t i o n , a n e u p l o i d or par-
TABLE 2.
Nuclear number and diameter of nuclei derived from
T. reesei QM9414, no. 83, Z1, Z2A and Z2W
Strains 7". reesei QM9414
No. 83 ( a r g ) ZI-1 Z1-2 Z2A- 1 Z2B-I Z2A-E Z2A-L
Nuclear number a
Diameter of nuclei u (~m)
Standard deviation (8)
1.11 1.11 2.22 2.37 1.39 1.80 1.30 1.25
0.014 0.014 0.057 0.050 0.023 0.049 0.023 0.018
a The nuclear number was an average of at least 50 conidia. b The diameter of nuclei was average among at least 50 conidia. c The nuclear number and the diameter of nuclei were counted on microscopic photos of conidia stained by Giemsa solution.
VoL. 69, 1990
NOTES TABLE 3.
Strains T. reesei QM9414 No. 83 Zl-1 Z2A- 1 Z2B- 1 Z2A-E Z2A-L
53
Distribution of conidia formation, auxotrophy, and clear zone formation among strains No. of colony
Sector formation a
10 10 5 5 5 5 1
+ + -
+ 10 10 0 0 0 0 1
Conidia formation b ~ ~ 0 0 5 5 1 0 0
0 0 0 0 4 5 0
Auxotrophy argarg + 0 10 5 5 5 5 5
10 0 0 0 0 0 0
Clear zone f°rmation~ + + + + + +
a In sector formation, + means the ability to produce new sectors and means the inability to produce new sectors on the haploidizing medium. b In conidia formation, + means a low amount of conidia, 4- means the conidia formation only in the center of colonies, and +- means the large amount of conidia on the Natick medium containing 80/zg/ml L-arginine, 2.00/ooglucose, and 1.5%0 agar (pH 5.0) by 6 d of incubation at 30°C. In clear zone formation, + means the ability to produce clear zones and - means the inability to produce clear zones on the plate assay medium for cellulase production.
tial d i p l o i d . Z 2 A - L m i g h t be u n a b l e to p r o d u c e clear zones o w i n g to a c h r o m o s o m a l m u t a t i o n , such as a deletion. C o l c h i c i n e is k n o w n to b i n d t u b u l i n to cause c h r o m o s o m e n o n d i s j u n c t i o n and increase p l o i d y b e f o r e the division o f cells (12). T h e r e f o r e , c o n i d i a did n o t germ i n a t e d u r i n g colchicine t r e a t m e n t . But small pellets ( a b o u t 1 m m in d i a m e t e r ) a p p e a r e d after 48 h o f i n c u b a t i o n and increased t h o s e a m m o u n t s by 120 h after i n c u b a tion. T h e d i a m e t e r o f nuclei in c o n i d i a derived f r o m these small pellets increased c o m p a r i n g with the d i a m e t e r o f nuclei in no. 83 c o n i d i a . This suggested t h a t e n l a r g e m e n t o f nuclei in c o n i d i a b e g a n to end by 24 h after i n c u b a t i o n and b e g a n to g e r m i n a t e to g e n e r a t e small pellets after 24 h o f i n c u b a t i o n . T h e r e a s o n w h y the e n l a r g e m e n t o f nuclei began to stop was considered to be that colchicine was turned into colchicein by acid p r o d u c t i o n o f the fungi that decreased the p H o f m e d i u m . In fact, the p H o f the m e d i u m was 6.5 after 1 2 0 h i n c u b a t i o n and a l m o s t all c o n i d i a in the m e d i u m g e r m i n a t e d . T h e n u m b e r o f larger nuclei in h y b r i d c o n i d i a o f m u l t i n u c l e a t e f u n g i b e t w e e n A . oryzae and A . k a w a c h i i (13) or b e t w e e n A . oryzae a n d A . niger ( T o y a m a , H. and T o y a m a , N., A b s t r . A n n u . Meet. Agric. Biol. C h e m . Soc. J a p a n , p. 397, 1988) decreased d u r i n g cultivation, p r o b a b l y because o f the influence o f their m u l t i n u c l e a t e n a t u r e but the n u m b e r or the d i a m e t e r o f nuclei in Z 2 A a n d Z2B c o n i d i a did n o t decrease d u r i n g cultivation. This m i g h t suggest the genetic stability o f fungi w h o s e cells are mononucleate. In c o n c l u s i o n , a p o l y p l o i d o f T. reesei Q M 9 4 1 4 was p r o d u c e d by colchicine t r e a t m e n t . This p o l y p l o i d was called A u t o p o l y p l o i d because the nuclei o f this p o l y p l o i d were c o n s t r u c t e d o f the o r i g i n a l c h r o m o s o m e s . It was possible to get genetic v a r i a n t s f r o m this A u t o p o l y p l o i d o n the h a p l o i d i z i n g m e d i u m . This suggested the possibility o f getting v a r i o u s genetic variants, e. g. c h r o m o s o m a l m u t a n t s f r o m A u t o p o l y p l o i d . This m e t h o d o f colchicine t r e a t m e n t was used o n the f o l l o w i n g fungi: A . oryzae IFO5239, A . niger IFO4407 ( T o y a m a , H . and T o y a m a , N . , A b s t r . A n n u . Meet. Agric. Biol. C h e m . Soc. J a p a n , p. 375, 1989) and A . k a w a c h i i IFO4308 ( T o y a m a , H . and T o y a m a , N . , u n p u b l i s h e d data) and to isolate a u t o p o l y p l o i d and genetic v a r i a n t s in each strain.
This report was presented at the annual meeting of the Agricultural Chemical Society of Japan, Niigata, 1989. REFERENCES 1. Manczinger, L. and Ferenezy, L.: Somatic cell fusion of Trichoderma reesei resulting in new genetic combinations. Appl. Microb. Biotechnol., 22, 72-76 (1985). 2. Rosen, D., Edelman, M., Galun, E., and Danon, D.: Biogenesis of mitochondria in Trichoderma viride: structural changes in mitochondria and other spore constituents during conidium maturation and germination. J. Gen. Microbiol., 83, 31-49 (1974). 3. Ishitani, C., Ikeda, Y., and Sakaguchi, K.: Hereditary variation and genetic recombination in koji-molds (Aspergillus oryzae and Asp. sojae). J. Gen. Appl. Microbiol., 2, 401-430 (1956). 4. Toyama, H., Yamaguchi, K., Shinmyo, A., and Okada, H.: Protoplast fusion of Trichoderma reesei, using immature conidia. Appl. Environ. Microbiol., 47, 363-368 (1984). 5. Toyama, H., Yokoyama, T., Shinmyo, A., and Okada, H.: Interspecific protoplast fusion of Trichoderma. J. Biotechnol., 1, 25-35 (1984). 6. Driscall, C. J. and Darvey, N. L.: Chromosome pairing: effect of colchicine on isochromosomes. Science, 169, 290-291 (1970). 7. Cox, D.M.: A quantitative analysis of colcemid-induced chromosomal nondisjunction in chinese hamster cells in vitro. Cytogenet. Cell Genet., 12, 165-174 (1973). 8. Chen, C.H. and Gueden, Y.C.: In vitro production of polyploidy plantlets after colchicine treatment of daylily callus. Amer. J. Bot., 61, 6, Suppl, 1 (1974). 9. Mandels, M.: Microbiological sourse of cellulase. Biotechnol. Bioeng. Symp., 5, 81-105 (1975). 10. Herbert, D., Phipps, P. J., and Strange, R. E.: Chemical analysis of microbial ceils, p. 324-334. In Norris and Ribbons (ed.), Methods in microbiology, 5B. Academic Press, New York (1971). 11. Ceriotti, G.: Determination of nucleic acids in animal tissues. J. Biol. Chem., 214, 59-70 (1955). 12. Fragata, M.: A hypothesis concerning the use of colchicine as a polyploidy inducer. Experientia, 26, 104-106 (1970). 13. Toyama, H. and Toyama, N.: Genetic structure of cycloheximide-nystatin resistant segregant from fusant between Aspergillus oryzae and Aspergillus kawachii. Hakkokogaku, 67, 83-90 (1989).
NOTES Autopolyploid Formation of Trichoderma reesei QM9414 by Colchicine Treatment H I D E O T O Y A M A AND N O B U O T O Y A M A *
Department of Food Technology, Faculty of Horticulture, Minamikyushu University, Takanabe-cho, Hibarigaoka 884, Japan Received 21 August 1989/Accepted 6 November 1989 Conidia of Trichoderma reesei QM9414 were treated with colchicine. Nuclei in coichicine-treated conidia enlarged. When the concentration of colchicine or the treatment time with colchicine increased, the diameter of nuclei became larger. Colchicine-treated conidia generated sectors on a medium containing benomyl. Some sectors formed many conidia or could not produce clear zones on the plate assay medium for cellulase production. According to the D N A assay of conidia, colchicine-treated strains were autopolyploid.
shaker. After 24 h, m a n y small pellets (about 2.0 mm in diameter) appeared in the medium. Ungerminated conidia incubated in the Natick medium containing 8 0 / l g / m l t-arginine, 2.0%o glucose, and 0.1% (w/v) colchicine (pH 10) for 24 h at 30°C by a reciprocal shaker were collected and incubated on the Natick medium containing 8 0 p g / m l L-arginine, 2.0°/oo glucose, 0.1}~ Triton X-100, and 1.5%0 agar (pH 5.0) for 4 d at 30°C to form colonies. Mycelial mats (2 m m × 2 mm) o f these colonies were cut out and put on Natick medium containing 0 . 2 p g / m l benomyl, 8 0 p g / m l L-arginine, 2.0%o glucose, and 1.5~0" agar (pH 5.0) (the haploidizing medium), and incubated for 14 d at 30°C. After incubation, nonsector parts o f colonies that had generated sectors were isolated (Z1) as shown in Fig. 1. W h e n mycelial mats of colonies derived from conidia o f Z1 were put on the haploidizing medium and incubated for 14 d at 30°C, all of colonies formed sectors. Similar experiments were done at p H 3.0, 5.0, and 7.0. A t p H 3.0, the buffer containing 1.0 M HC1 and 1.0 M C H 3 C O O N a was used. For p H 5.0, the buffer containing 0.2 M CH3COOH and 0.2 M CH3COONa was ued. F o r p H 7.0, the buffer containing 0.2 M NaHzPO4 and 0.2 M Na2HPO4 was used. Green mature conidia of Z 1 and no. 83 generated on the Natick medium containing 8 0 p g / m l L-arginine, 2.0°//oo glucose, and 1.5% agar ( p H 5 . 0 ) for 10d of incubation at 30°C were collected in a desiccator and dried for 28 d. After drying, the length of the m a j o r axis of conidia formed by these two types were measured for at least 50 conidia in each strain. D N A of conidia was extracted by the method of Herbert et al. (10) and assayed the D N A contents o f 1 mg o f green mature conidia by the method o f Ceriotti (11). It was possible to compare D N A contents o f 1 mg o f green mature conidia, comparing this with the result of measurement o f length of the conidial m a j o r axis. Table 1 showed that D N A contents of 1 mg of Z1 green mature conidia were apparently larger than those of 1 mg o f no. 83 green mature conidia, consistent with the increased conidial m a j o r axis in Z1. Green mature conidia o f sector parts (Z2) generated from Z1 incubated on the haploidizing medium for 14 d at 30°C were dried in a desiccator for 28 d. After drying, the
Cellulase-producing fungi o f the genus Trichoderma are widely used in industry and there are m a n y reports on its breeding (1). The genus Trichoderma is also used for investigating the action o f cellulase components because the fungus has an suitable cellulase system. However, it is necessary, for m o r e i n f o r m a t i o n on cellulase action, to breed m o r e various mutants, e.g. c h r o m o s o m a l mutants. This fungus is mononucleate (2) and it is easy to grasp the genetic condition o f hybrids, comparing with multinucleate fungi such as Aspergillus oryzae (3). In fact, intraspecific (4) and interspecific p r o t o p l a s t fusion (5) of Trichoderma reesei was done in 1984. In this paper, colchicine treatment was attempted for T. reesei QM9414 to get more various genetic variants without the use o f p r o t o p l a s t fusion or mutation. Colchicine and its derivative colcemid are known to bind tubulin and cause c h r o m o s o m e n o n d i s j u n c t i o n (6). Using these chemicals, a u t o p o l y p l o i d f o r m a t i o n has been done in animals (7) and plants (8). In microbes, colchicine was used for breeding in yeast (Fujii, T. et al., Abstr. A n n u . Meet. Agric. Biol. Chem. Soc. J a p a n , p. 134, 1989) but not used for breeding in T. reesei. In fungi, there are only a few reports o f colchicine treatment, p r o b a b l y because colchicine turns to colchicein, which has no tubulin-binding activity at low pH, the cultivation condition of fungi. Therefore, colchicine treatment o f T. reesei was attempted in high p H cultivation to generate a u t o p o l y p l o i d o f this fungus. Strain no. 83 was an a u x o t r o p h i c mutant ( a r g ) derived from T. reesei QM9414 by UV-irradiation. Conidia o f no. 83 were a d d e d to Natick medium (9) containing 1.4 g o f (NH4)2SO4, 2.0 g of KH2PO4, 0.3 g o f urea, 0.3 g o f CaC12, 0.3 g o f M g S O 4 • H 2 0 , 0.005 g o f FeSO4.7H20, 0.0016 g o f MnSO4. H20, 0.0014 g of ZnSO4. H20, 0.002 g o f CoC12, 0.08 g o f L-arginine, 20.0 g o f glucose, and 1.0 g of colchicine ( W a k o Pure Chem. Co., Ltd.) per liter and the buffer containing 0 . 2 M N a z C O 3 and 0 . 2 M NaHCO3 (pH 10), and incubated on a reciprocal shaker for 24 h at 30°C. After 24 h, no pellet was seen in the medium. As a control, no. 83 were a d d e d into the same medium without colchicine and incubated for 24 h at 30°C by a reciprocal * Corresponding author. 51
52
J. FERMENT. B1OENG.,
TOYAMA AND TOYAMA
FIG. 1. Sector formation of a Z1 colony. No. 83 was an arginine-less mutant of T. reesei QM9414 and Z 1 was 0.1% (w/v) colchicine-treated no. 83 for 24 h at 30°C. ZI generated sectors on the medium containing 0.2pg/ml (w/v) benomyl. The arrow indicates Z2A-E. D N A c o n t e n t s o f these 1 m g o f c o n i d i a were assayed. T h e r e were t w o types in D N A c o n t e n t s o f 1 m g Z2 green m a t u r e c o n i d i a (Table 1), one ( Z 2 A ) had the same D N A c o n t e n t as that o f no. 83 and the o t h e r type (Z2B) had a larger D N A c o n t e n t t h a n that o f no. 83. Z 2 A did not g e n e r a t e new sectors but Z2B g e n e r a t e d new sectors on the h a p l o i d i z i n g m e d i u m i n c u b a t e d for 14 d at 30°C. D r i e d green m a t u r e c o n i d i a o f no. 83 and Z1 were stained by G i e m s a s o l u t i o n . A f t e r t r e a t m e n t with 5 N, 3 N, a n d 1 N HC1 at 6 0 ° C , the d i a m e t e r o f nuclei in c o n i d i a o f no. 83 and Z1 were m e a s u r e d by calipers a m o n g at least 50 c o n i d i a in each strain. T a b l e 2 s h o w e d that Z1 c o n i d i a had larger nuclei t h a n no. 83 c o n i d i a had. D r i e d c o n i d i a o f Z1, after 24 h, 48 h, 96 h, and 120 h o f i n c u b a t i o n at 3 0 ° C with 0 . 1 % ( w / v ) colchicine were stained by G i e m s a s o l u t i o n after p r e t r e a t m e n t . N u c l e a r d i a m e t e r in Z1 c o n i d i a e n l a r g e d as the i n c u b a t i o n t i m e b e c a m e longer. Nuclei in no. 83 c o n i d i a i n c u b a t e d for 72 h e n l a r g e d a b o u t 2 times larger t h a n t h o s e in Z1 c o n i d i a inc u b a t e d for 24 h. This suggests that not only diploids but
TABLE 1.
DNA contents of conidia derived from T. reesei QM9414, no. 83, Z1, Z2A and Z2B
Strains T. reesei QM9414
No. 83 ( a r g ) ZI-I b Z1-2 Z2A-F Z2A-2 Z2B-1 Z2B-2 Z2A-E d Z2A-L
DNA content (,ug/mg conidia) 5.25 5.23 11.45 10.80 5.20 5.22 6.75 7.01 5.30 5.27
Length of major Standard axis of conidia" deviation ~m) (6) 4.72 4.72 7.22 7.54 5.96 6.80 7.09 6.85 6.25 6.76
0.08 0.08 0.23 0.17 0.15 0.13 0.20 0.18 0.15 0.18
The length of major axis of conidia showed average among at least 50 conidia. b ZI-1 and Z1-2 were derived from no. 83 conidia treated with 0.1% (w/v) colchicine for 24h at 30°C with a reciprocal shaker (pH 10). c Z2A and Z2B were sectors generated from Z1 on the haploidizing medium, Z2A did not segregate new sectors on the haploidizing medium but Z2B segregated new sectors. a Z2A-E produced clear zones on the plate assay medium for cellulase production but Z2A-L did not produce clear zones.
also p o l y p l o i d s m a y be g e n e r a t e d by colchicine t r e a t m e n t . Dried c o n i d i a o f Z1 treated with 0 . 0 5 % , 0 . 1 % , 0 . 3 % , and 0.5%o ( w / v ) colchicine for 96 h at 3 0 ° C were stained by G i e m s a s o l u t i o n after t r e a t m e n t . T h e largest nuclei in Z1 c o n i d i a were seen in the case o f 0 . 5 % ( w / v ) colchicine. The nuclear d i a m e t e r o f Z 2 A c o n i d i a was similar to that o f no. 83 and did n o t change d u r i n g cultivation. But the nuclear d i a m e t e r o f Z2B c o n i d i a was i n t e r m e d i a t e between the nuclear d i a m e t e r o f Z1 c o n i d i a and that o f no. 83 conidia. Z 2 A seemed to be m o n o p l o i d and Z2B seemed to be diploid or partial diploid. T h e d i s t r i b u t i o n o f the ability o f c o n i d i a f o r m a t i o n , a u x o t r o p h y , and the ability to f o r m clear zones o n the plate assay m e d i u m for cellulase p r o d u c t i o n c o n t a i n i n g 1 . 0 % ( w / v ) c a r b o x y m e t h y l c e l l u l o s e calcium salt ( C M C Ca) and 0 . 1 % T r i t o n X-100 after 6 d o f i n c u b a t i o n at 3 0 ° C o f Z1, Z 2 A , and Z2B were investigated. T a b l e 3 shows that two types o f strains a p p e a r e d , one ( Z 2 A - E ) p r o duced conidia m u c h m o r e than no. 83 and Z1, and p r o d u c e d a clear z o n e on the plate assay m e d i u m for cellulase p r o d u c t i o n , the o t h e r ( Z 2 A - L ) p r o d u c e d few c o n i d i a and did n o t p r o d u c e clear zones on the plate assay m e d i u m for cellulase p r o d u c t i o n . Z 2 A - E and Z 2 A - L were arginine-less like no. 83 and Z1. A c c o r d i n g to the D N A assay o f conidia, Z 2 A - E and Z 2 A - L seemed to be like m o n o p l o i d s . T h e great a m o u n t o f c o n i d i a in Z 2 A - E was c o n s i d e r e d to be caused by a c h r o m o s o m a l m u t a t i o n , a n e u p l o i d or par-
TABLE 2.
Nuclear number and diameter of nuclei derived from
T. reesei QM9414, no. 83, Z1, Z2A and Z2W
Strains 7". reesei QM9414
No. 83 ( a r g ) ZI-1 Z1-2 Z2A- 1 Z2B-I Z2A-E Z2A-L
Nuclear number a
Diameter of nuclei u (~m)
Standard deviation (8)
1.11 1.11 2.22 2.37 1.39 1.80 1.30 1.25
0.014 0.014 0.057 0.050 0.023 0.049 0.023 0.018
a The nuclear number was an average of at least 50 conidia. b The diameter of nuclei was average among at least 50 conidia. c The nuclear number and the diameter of nuclei were counted on microscopic photos of conidia stained by Giemsa solution.
VoL. 69, 1990
NOTES TABLE 3.
Strains T. reesei QM9414 No. 83 Zl-1 Z2A- 1 Z2B- 1 Z2A-E Z2A-L
53
Distribution of conidia formation, auxotrophy, and clear zone formation among strains No. of colony
Sector formation a
10 10 5 5 5 5 1
+ + -
+ 10 10 0 0 0 0 1
Conidia formation b ~ ~ 0 0 5 5 1 0 0
0 0 0 0 4 5 0
Auxotrophy argarg + 0 10 5 5 5 5 5
10 0 0 0 0 0 0
Clear zone f°rmation~ + + + + + +
a In sector formation, + means the ability to produce new sectors and means the inability to produce new sectors on the haploidizing medium. b In conidia formation, + means a low amount of conidia, 4- means the conidia formation only in the center of colonies, and +- means the large amount of conidia on the Natick medium containing 80/zg/ml L-arginine, 2.00/ooglucose, and 1.5%0 agar (pH 5.0) by 6 d of incubation at 30°C. In clear zone formation, + means the ability to produce clear zones and - means the inability to produce clear zones on the plate assay medium for cellulase production.
tial d i p l o i d . Z 2 A - L m i g h t be u n a b l e to p r o d u c e clear zones o w i n g to a c h r o m o s o m a l m u t a t i o n , such as a deletion. C o l c h i c i n e is k n o w n to b i n d t u b u l i n to cause c h r o m o s o m e n o n d i s j u n c t i o n and increase p l o i d y b e f o r e the division o f cells (12). T h e r e f o r e , c o n i d i a did n o t germ i n a t e d u r i n g colchicine t r e a t m e n t . But small pellets ( a b o u t 1 m m in d i a m e t e r ) a p p e a r e d after 48 h o f i n c u b a t i o n and increased t h o s e a m m o u n t s by 120 h after i n c u b a tion. T h e d i a m e t e r o f nuclei in c o n i d i a derived f r o m these small pellets increased c o m p a r i n g with the d i a m e t e r o f nuclei in no. 83 c o n i d i a . This suggested t h a t e n l a r g e m e n t o f nuclei in c o n i d i a b e g a n to end by 24 h after i n c u b a t i o n and b e g a n to g e r m i n a t e to g e n e r a t e small pellets after 24 h o f i n c u b a t i o n . T h e r e a s o n w h y the e n l a r g e m e n t o f nuclei began to stop was considered to be that colchicine was turned into colchicein by acid p r o d u c t i o n o f the fungi that decreased the p H o f m e d i u m . In fact, the p H o f the m e d i u m was 6.5 after 1 2 0 h i n c u b a t i o n and a l m o s t all c o n i d i a in the m e d i u m g e r m i n a t e d . T h e n u m b e r o f larger nuclei in h y b r i d c o n i d i a o f m u l t i n u c l e a t e f u n g i b e t w e e n A . oryzae and A . k a w a c h i i (13) or b e t w e e n A . oryzae a n d A . niger ( T o y a m a , H. and T o y a m a , N., A b s t r . A n n u . Meet. Agric. Biol. C h e m . Soc. J a p a n , p. 397, 1988) decreased d u r i n g cultivation, p r o b a b l y because o f the influence o f their m u l t i n u c l e a t e n a t u r e but the n u m b e r or the d i a m e t e r o f nuclei in Z 2 A a n d Z2B c o n i d i a did n o t decrease d u r i n g cultivation. This m i g h t suggest the genetic stability o f fungi w h o s e cells are mononucleate. In c o n c l u s i o n , a p o l y p l o i d o f T. reesei Q M 9 4 1 4 was p r o d u c e d by colchicine t r e a t m e n t . This p o l y p l o i d was called A u t o p o l y p l o i d because the nuclei o f this p o l y p l o i d were c o n s t r u c t e d o f the o r i g i n a l c h r o m o s o m e s . It was possible to get genetic v a r i a n t s f r o m this A u t o p o l y p l o i d o n the h a p l o i d i z i n g m e d i u m . This suggested the possibility o f getting v a r i o u s genetic variants, e. g. c h r o m o s o m a l m u t a n t s f r o m A u t o p o l y p l o i d . This m e t h o d o f colchicine t r e a t m e n t was used o n the f o l l o w i n g fungi: A . oryzae IFO5239, A . niger IFO4407 ( T o y a m a , H . and T o y a m a , N . , A b s t r . A n n u . Meet. Agric. Biol. C h e m . Soc. J a p a n , p. 375, 1989) and A . k a w a c h i i IFO4308 ( T o y a m a , H . and T o y a m a , N . , u n p u b l i s h e d data) and to isolate a u t o p o l y p l o i d and genetic v a r i a n t s in each strain.
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