• The Korean Journal of Mycology
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• v.16 no.1
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• pp.21-25
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• 1988

This experiment was carried out to investigate proper conditions for protoplast isolation and reversion from Ganoderma lucidum and Gctnoderma sp.. In G. lucidum, 10 mg. $ml^{-1}$ Novozyme 234 with 0.6 M sucrose was proper for protoplast isolation. The optimal reaction time of mycelium with lytic enzyme was five hrs. Protoplast isolation from four-day-old mycelium was the most effective. Protoplast isolation from four-day-old mycelium in G. sp. was optimum in the combination of N ovozyme 234 and ${\beta}-glucuronidase$ with 0.6 M sucrose. MCM was suitable for reversion in G. lucidum while SCM was good for G. sp.. The most effective osmoticum stabilizer for protoplast reversion in G. lucidum and G. sp. was 0.6 M sucrose.

• The Korean Journal of Mycology
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• v.15 no.1
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• pp.14-18
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• 1987

This experiment was undertaken to investigate proper conditions for protoplast formation from Lyophyllum ulmarium. Combination of Novozym 234, ${\beta}-Glucuronidase$ and ${\beta}-D-Glucanase$ with 0.6 M Sucrose was the most effective for isolation of protoplasts. The optimal reaction time of mycelium with the lytic mixture was 3 hrs in shaking condition at 120 strokes $min-^1$. When the mycelium of L. ulmarium was cultured for 6 days on yeast glucose agar medium at $25^{\circ}C$, the formation of protoplasts was effective. The yeast glucose agar medium stabilized with 0.6 M sucrose was the most effective for reversion of protoplasts.

• Korean Journal of Microbiology
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• v.30 no.6
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• pp.514-518
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• 1992

The present study has been perromed to investigate the optimal conditions for protoplast formation and regeneration of oleandomycin-producing Streptomyces antibioticus (S. antibioticus) KCTC 1081. Mycelia were grown in YME medium containing 0.2% (w/v) glycine and converted into the protoplast by incubating at 35.deg.C for 60 minutes in protoplast buffer (P buffer) containing 4 mg/ml lysozyme. The reversion of protoplasts to the normal filamentous state was examined by the growth on various synthetic agar media. A high reversion rate was obtained by incubating the protoplasts on a hypertonic agar medium containing 20 mM $Mg^{++}$, 5 mM $Ca^{++}$ and 0.3 M sucrose at 28.deg.C for 5 days. From these experiments, we established the improved regeneration medium and a protocol which supports higher and more consistent levels of regeneration of S. antibioticus protoplasts. The regenerant showed an increased antimicrobial activity compared with that of the initial strain.n.n.

• 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.

• The Korean Journal of Mycology
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• v.15 no.1
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• pp.19-22
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• 1987

This experiment was undertaken to investigate proper conditions for protoplast formation from Pleurotus spodoleucus. Novozym 234 was the most effective enzyme and a high yield of protoplasts was obtained. Combination of enzymes did not improve this result. Sucrose gave the best result to support the release and maintain the stability of protoplasts. The optimal reaction time of mycelium with the lytic mixture was 3 hrs. in shaking condition at 120 strokes $min-^1$. When mycelium of P. spodoleucus was cultured for 3 days on mushroom complete agar medium at $27^{\circ}C$, the formation of protoplasts was effective. The mushroom complete agar medium stabilized with 0.6 M sucrose was the most effective for reversion of protoplasts.

• Korean Journal of Microbiology
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• v.21 no.4
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• pp.213-220
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• 1983

Conditions for isolation of protoplasts from conidiospores of Trichoderma koningii ATCC 26113 were tested. Maximum production of conidial protoplasts was obtained by preincubation of conidiospores on liquid minimal medium for 8 1/2 hrs. and by reaction with cell wall lytic enzyme for 3 hrs. Among effective cell wall lytic enzymes (Driselase, p-Glucuronidase, Novozyme and Zymolyase), Driselase was the most effective one on the production of conidial protoplasts. The production of conidial protoplasts was also enhanced by addition of 2-Deoxy-D-Glucose $(25{\mu}g/ml)$ into liquid minimal medium. Over 70% of the initial swollen conidia, preincubated in liquid minimal medium supplemented with 2-Deoxy-D-Glucose $(25{\mu}g/ml)$, were converted to protoplasts by incubation with 2% (w/v) commercial lytic enzyme Driselase at $28^{\circ}C$ for 3 hrs. The reversion frequency of the conidial protoplasts was about 30 times (25-50%) higher than that of mycelial protoplasts (0.6-1.3%).

• Korean Journal of Agricultural Science
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• v.15 no.1
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• pp.15-22
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• 1988

Factors affecting the regeneration, reversion of protoplasts from mycelium of F. velutipes were investigated and the selection of auxotrophic mutants from protoplasts of F. velutipes was performed. PDP medium stabilized with 0.6M sucrose was suitable for the regeneration of protoplasts, and regeneration frequency was 0.47-1.32. The regeneration frequency of protoplasts was increased when nutrients were added to the regeneration medium. Especially, yeast extract was the most effective to regeneration of protoplasts. Regeneration pattern of protoplasts was formation of germ tubes from bud-like cells. 13-18% of monokaryotic strains was appeared from reverted protoplasts. Five of auxotrophic mutants were isolated from strains showed survival frequency of 1.9-16.