• The Korean Journal of Mycology
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• v.26 no.2 s.85
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• pp.275-280
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• 1998

Factors affecting protoplasts isolation and regeneration of Fomitella fraxinea were investigated. Lytic enzyme mixture of Novozym 234, Cellulase onozuka R-10 and ${\beta}-Glucuronidase$ was found to be the best for the protoplasts isolation. Osmotic stabilizer of 0.6 M sucrose was observed as the best for protoplasts isolation. The highest number of protoplasts was obtained from the F. fraxinea mycelium with lytic enzyme mixture and osmotic stabilizer that had been cultured for 3 hours. The highest regeneration rate of 0.02 % was achieved when the 0.6 M sorbitol was employed as osmotic stabilizer.

• Food Science and Biotechnology
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• v.14 no.4
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• pp.543-546
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• 2005

Optimized conditions for protoplast preparation and regeneration from young hyphae of Monascus ruber were established. Heat shock treatment of spores gave rapid and synchronized germination. Spores collected from cultures grown for 7-8 days at $30^{\circ}C$ were germinated until over 70% germ tubes reached to 3-5 spore length. Enzymatic digestion of young hyphae was optimal with 50 mg/mL Glucanex in 0.1 M sodium citrate buffer containing 0.8 M mannitol as an osmotic stabilizer. Regeneration rate was around 10% when 0.8 M sorbitol was used as an osmotic stabilizer in regeneration medium. These conditions will be applied in genetic study of M. ruber that produces citrinin at high level and thus is good model strain for molecular genetic dissection of citrinin biosynthesis.

• Journal of Life Science
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• v.13 no.2
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• pp.143-149
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• 2003

This experiment was undertaken to investigate proper conditions for protoplast isolation and regeneration from the mycelia of Lyophyllum ulmnrium. Protoplast isolation and regeneration are influenced by a variety of factors such as enzyme, osmotic stabilizer, reaction time and age of mycelia. A combination of Novozyme 234 (10mg/ml) and cellulase Onozuka R-10 (10 mg/ml) with 0.6 M $MgSO_4$ was most effective for isolation of the protoplasts. The optimum reaction time of the mycelia with the lytic enzymes was 3.5~4 hours at $28^{\circ}C$ in shaking condition at 120 strokes per min. High yield of the protoplasts were obtained from its 4~5 days old mycelia on complete agar media. Its protoplasts were regenerated to normal hyphae. Regeneration media with 0.6 M sucrose were proper for regeneration of the protoplasts. Their regeneration frequency on complete agar media was 2.3~2.7%.

• The Korean Journal of Mycology
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• v.14 no.4
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• pp.273-280
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• 1986

The degree of autolysis and lytic enzyme production in the culture filtrate of Rhizopus oryzae was investigated. The formation of protoplast by using autolytic enzymes from Rh. oryzae was also attempted. Protoplasts were liberated from Rh. oryzae mycelium by lytic enzymes present in autolytic-phase culture filtrate. Maximum release of chitosanase and proteolytic enzyme into culture filtrate during autolysis was corresponded to maximum protoplast-liberating activity. High yields of protoplasts were obtained from 10 hr-age of Rh. oryzae mycelium with 0.5 M mannitol as osmotic stabilizer. The optimum temperature and pH for mycelium digestion were $25{\sim}30^{\circ}C$ and $6.0{\sim}6.5$ respectively. The mycelium of the 18 hours cultures were treated with autolytic enzyme in same volume of osmotic stabilizer at $30^{\circ}C$ for 5 hours and then it was confirmed by scanning electoron microscope that protoplast were produced beside the digesting cell wall of the fungi.

• Microbiology and Biotechnology Letters
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• v.16 no.4
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• pp.303-309
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• 1988

In order to establish the process of interspecific protoplast fusion of the genus Cellulomonas capable of utilizing of cellulose, C. flavigena NCIB 12901 and Cellulomonas sp. CSI-1, the optimum conditions for the regeneration and fusion were examined. The condition of suitable osmotic stabilizer for the protoplast regeneration of C. flavigena was established by using 0.4M sorbitol. And then, by addition of 3% po]yvinyl pyrrolidone (PVP) to cell wall regeneration medium, regeneration frequency was increased 3 times higher than that without PVP addition. The optimum conditions for the interspecific protoplast fusion between auxotrophic and antibiotics resistant mutants were obtained with 40%(W/V) of PEG (polyethylene glycol) 6000 as the fusogenic agent and 25mM of CaCl$_2$on treating time for 15 min. The fusion frequency between mutants was from 2.0$\times$10$^{-4}$ to 4.0$\times$10$^{-4}$ under the optimum conditions. The fusants were confirmed to revert from protoplast to cells of rod type during regeneration process and the aggregation of protoplast by PEG was observed. Also the progress of fusion was observed by scanning electron microscopy, Many isolated fusants were shown to be complement clones of both parents which occured at a high frequency among the isolated clones.

• Archives of Pharmacal Research
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• v.10 no.3
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• pp.158-164
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• 1987

To obtain a new strain of Ganoderma lucidum by protoplast fusion technique, its protoplast formation and regeneration were studied. Several factors affecting the protoplast formation and regeneration were investigated to find their optimum conditions. The mycelium was grown for four days on the cellophane membrane placed on G. Incidum complete medium (GCM). When various commercial lytic enzymes were examined for protoplast isolation, the combination of Novozym 234 and $\beta$glucuronidase was found to be effective. An osmotic stabilizer, 0.6 M sucrose in 20 mM phosphate buffer pH 5.8, gave the highest yield of protoplasts. Three-hour incubation in shaking incubator was most suitable for releasing protoplasts. To increase the protoplast yield, pretreatment with 2-mercaptoethanol was carried out. The regeneration frequency in GCM containing 0.6M MgSO$_4$ 7$H_2O$ was shown to be 0.66%.

• Journal of the Korean Society of Tobacco Science
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• v.1 no.2
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• pp.138-149
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• 1979

For the preliminary study on tobacco cell fusion as one of new breeding techniques, the conditions that would be most effective in isolation, fusion, and culture of tobacco protoplasts were examined ; 1. The enzyme solution of 0.5% macerozyme and 2% cellulase( or meicellase) was the most economic and efficient in isolating protoplasts from tobacco leaves. 2. The proper incubation period of tobacco leaves in cell wall digesting solution was 4 hours. 3. As an osmotic stabilizer, sorbitol or mannitol solutions were employed. The concentration of 0.5~0.7 M of either hexitol gave satisfying results as the osmotic stabilizer. 4. The calcium concentration appeared to be an important factor in protoplast fusion. The adhesion of protoplasts was enhanced by enrichment of calcium ion in PEG solution. The highest frequency of protoplast fusion was obtained when tobacco protoplasts were incubated in PEG solution. containing 9mM CaCl2. 5. Cell divisions of the isolated protoplasts were continued and have generated colonies when they were grown on B-5 medium at 28$^{\circ}C$.

• The Korean Journal of Mycology
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• v.21 no.3
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• pp.195-199
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• 1993

This experiments was designed for development of Fusarium oxysporum strains with enhanced activity of polygalacturonase by means of mutants and protoplasts fusion. Six auxotrophic mutants of F. oxysporum were induced by treatment of MNNG. Protoplasts from mutants were formed from early exoponential mycelium after treatment with driselase(10 mg/ml) using 0.6 M KCl as osmotic stabilizer. Fusion experiments between protoplasts of several auxotrophic mutants were done using polyethyleneglycol 8,000 and $CaCl_2$. The frequency of fusion was $5.0{\times}10^{-3}$ as determined from several experiments. Activity of polygalacturonase was determined by the methods of modified DNS. Consequently, the polygalacturonase activities of mutants and fusant derived F. oxysporum were 1.4 to 3.5 times greater than that of the parental strain, and mutant Fx-2 seemed to be the best strain. Thus, the method we used seemed to be favorable for the improvement of strains.

• KSBB Journal
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• v.5 no.3
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• pp.229-234
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• 1990

The isolation and regeneration of protoplasts are necessary for protoplast fusion of edible mushrooms. In this study, over 5$\times$107 ml-1 protoplasts of Agrocybe cylindracea were isolated using the method described by Yanagi. Enzyme mixture of cellulase Onozuka R10(2%), chitinase (0.2%) and Novozym 234(0.1%) was most effective for the isolation of protoplasts and the yield of protoplasts was 4.85$\times$107 ml-1. 0.6M sucrose was the most effective osmotic stabilizer. The maximum amount of mycelia and yield of protoplasts were obtained from 5~7 days cultured mycelia. In the case of 5~7% days cultured mycelia, the digestion time with lytic enzyme was 4~6 hours. ACM and MCM medium were most effective for the regeneration and reversion of protoplasts, and reversion frequency was 6.9~7.0%. 0.6M sucrose was most stable osmotic stabilizer.

• Microbiology and Biotechnology Letters
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• v.16 no.2
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• pp.156-162
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• 1988

Optimal conditions for the formation and regeneration of protoplasts of the cellulolytic fungus Penicillium verruculosum were investigated. Among the various commercial cell wall lytic enzymes tested, 0.5%(w/ v) Novozym 234 was the most effective for protoplast formation. The highest yield of protoplast exceeding 4.5$\times$10$^6$/m$\ell$ obtained when 400mg of 20 hr-old mycelia was incubated with 0.5%(w/v) Novozym 234 at 3$0^{\circ}C$ for 1 hr. The best osmotic stabilizer for the isolation and re-generation of protoplasts was 0.7M sorbital (pH 5.6) and 0.6M MgSO$_4$(pH 5.6), respectively. When 0.6M MgSO$_4$was added as osmotic stabilizer to the complete medium, the maximum regeneration frequency obtained was 4.6-27.8%. Micromorphological change of giant protoplasts into hyphae was observed during incubation in the regeneration liquid medium.