• Korean Journal of Agricultural Science
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• v.19 no.1
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• pp.33-39
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• 1992

Effects of buprofezin, a chitin synthesis inhibitor of insects, on mycelial growth, protoplast formation and reversion of Coriolus versicolor, Ganoderma applanatum and G. lucidum were investigated. The mycelial growth of C. versicolor, G, applanatum and G, lucidum was severely inhibited by buprofezin treatment, and the inhibition rate severely as the concentration of the buprofezin increased. Aerial mycelia and oidia formation of the mushrooms were increased by buprofezin treatment, but mycelial morphology was not changed. The rate of protoplast formation and reversion of G, applanatum was greatly increased by the treatment of the buprofezin, while that of G. lucidium was decreased. The rate of protoplast formation of C. versicolor was also increased when buprofezin was added to the medium, but the rate of protoplast reversion was not affected by the treatment, From the results obtained in this experiment, we found that the rate of protoplast. formation and reversion was increased by the treatment of the buprofezin in the mushrooms such as C, versicolor and G, applanatum, whose mycelial growth was fast on the medium, while that of G. lucidum, whose mycelial growth was relatively slow, was decreased by the treatment.

• 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 Agricultural Science
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• v.17 no.2
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• pp.77-81
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• 1990

Effects of buprofezin, an inhibitor of chitin synthesis, on mycelial growth, protoplast formation and reversion of Pleurotus ostreatus and P. sajor-caju were investigated. The mycelial growth of Pleurotus ostreatus and P. sajor-caju was the inhibited by buprofezin treatment, and the inhibition rate was severer as the concentration of the buprofezin increased. Aerial mycelium formation was increased by buprofezin treatment, but mycelial morphology was not changed. Protoplast formation of Pleurotus ostreatus and P. sajor-caju. was significantly increased when buprofezin was added to the culture medium at the concentration of 200~500 ppm and the protoplast reversion of the mushrooms was also increased by the treatment of the buprofezin.

• The Korean Journal of Mycology
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• v.16 no.4
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• pp.214-219
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• 1988

Factors affecting protoplast formation and reversion were investigated in Pleurotus sapidus kalchbr. For release of protoplast, enzyme mixture of Novozyme 234, ${\beta}-D-glucanase$ and ${\beta}-glucuronidase$ was most effective, when mycelium of 0.6 M sucrose solution as osmotic stabilizer without addition of buffer solution. The yield of protoplast was highest with mycelium cultured for 4 days on mushroom complete agar medium at ${30}^{\circ}C$. Protoplasts of Pleurotus sapidus were reverted to normal hyphal growth with maximum reversion frequency of 2% on Mushroom complete agar medium stabilized with 0.6 M sucrose solution and covered by 0.75% agar layer.

• 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.

• The Korean Journal of Mycology
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• v.14 no.3
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• pp.215-223
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• 1986

Protoplasts of P. cornucopiae were reverted to normal hyphal growth and reversion frequency was $0.04{\sim}19%$. The complete medium stabilized with 0.6 M sucrose was most effective for regeneration of protoplasts. When hypertonic mushroom complete medium not containing agar was overlaid, regeneration frequency of protoplasts was the highest rate among the others of topagar. The protoplast reversion frequency and mycelial growth of P. cornucopiae were increased when various amino acids, nucleic acid components and vitamin compound were added to the hypertonic minimal medium. The relation between sources increasing reversion frequency and sources accelerating mycelial growth was similar in amino acids and nucleic acid components but it was different in vitamins. The protoplast reversion frequency showed the highest rate when all sources were added to the regeneration minimal medium. Microscopically, regeneration patterns of protoplasts showed formation of a bud-like structure, direct germination, yeast-like cell chain of the protoplast, and the production of both direct germ tube and yeast-like cell chain from a protoplast.

• The Korean Journal of Pesticide Science
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• v.12 no.1
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• pp.88-96
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• 2008

To obtain protoplasts of Colletotrichum acutatum JC24, conidia were inoculated onto cellophane membrane placed on PDA and incubated at $25^{\circ}C$ for 20 hrs under the dark condition. Cellophane membranes, where mycelia were incubated, were soaked into 2% lysing enzyme solution prepared with 0.02 M phosphate buffer (pH 7.0) including 1.2 M sorbitiol. After treatment in 2% enzyme solution for 2 - 3 hrs, it could be possible to harvest $2-3\;{\times}\;10^6$ protoplasts/mL. The effect of several fungicides on reversion ratio was determined by using the protoplasts obtained from C. acutatum JC24. Any protoplasts could not be reversed to mycelia on reversion PDA amended with $10\;{\mu}\;g\;mL^{-1}$ of propineb. With tebuconazole, inhibition ratio of protoplast reversion was 100 and 0.9% at 0.5 and $0.1\;{\mu}\;g\;mL^{-1}$, respectively, while inhibitory effect on mycelial growth was 85.1 and 75.7%. The inhibitory tendency of carbendazim on protoplast reversion was as same as mycelial growth. In the case of strobilurins, trifloxystrobin and kresoxim-methyl, they only could inhibit protoplast reversion of C. acutatum JC24, when salicylhydroxamic acid (SHAM) was amended into reversion PDA with strobilurins.

• 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.

• Microbiology and Biotechnology Letters
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• v.16 no.2
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• pp.163-167
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• 1988

The conditions for the protoplast fusion of auxotrophic mutants of Penicillium verruculosum were determined. A preparation of commercial enzyme Novozym 234 was used to successfully isolate protoplast from the 20hr old mycelium of P. verruculosum. Under optimal condition, the protoplast yield ranged from 2.4$\times$10$^7$ to 3.0$\times$10$^7$ protoplasts from 400mg of damp mycelia of various auxotrophic mutant strains. The regeneration frequency ranged from 26.6 to 42.4% and the spontaneous reversion frequency of the protoplasts on the regeneration minimal medium was less than 10$^7$. The optimal concentration of PEG 6000 was 20%, and exposure of protoplasts to PEG for 10 min was found to be sufficient for protoplast fusion. Optimal pH of fusion mixture was deter-mined as 5.5 and l0mM of calcium chloride in fusion mixture effectively enhanced the protoplast fusion frequency. Under optimal condition, the fusion frequency between various auxotrophs ranged from 1.8$\times$10$^{-3}$ to 3.5$\times$0$^{-3}$.

• The Korean Journal of Mycology
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• v.17 no.1
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• pp.1-6
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• 1989

The mycelia of Pyricularia oryzae were treated with enzyme solution mixture consisting of Driselase, ${\beta}-Glucuronidase$, Cellulase, and Macerozyme R-10 for the production of protoplasts. More protoplasts were formed from mycelia of race KJ 101 of P. oryzae than that of race KI 315a in the enzyme mixtures. The number of protoplasts was decreased in the untreated control three hrs after the enzyme treatment, whereas the number was increased in the treatments with 10, 50 and 100 mM 2-mercaptoethanol, respectively. The number of protoplasts increased to reach maximum at five hrs after treatment of 10 mM 2-mercaptoethanol, but the least was found in 200 mM. The protoplasts of P. oryzae in a liquid medium containing 2.5% yeast extract, and 2% dextrose reverted to the mycelia after five hrs shaking incubation at $27^{\circ}C$. Some protoplasts produced yeast like buds and the buds were developed to irregularly shaped chains of cell protruded a germ tube like hypha from the distal cell. Once in a while a germ tube like hypha protruded directly from the protoplasts. Except in the first type of reversion, other protoplasts reverted to the normal mycelia. The reversion frequency was highest on PDA with stabilizer of 0.6 M KCl. No reversion of protoplasts occurred on water agar regaardless oftreaatments.