• Mycobiology
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• v.47 no.4
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• pp.483-493
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• 2019

Antrodia cinnamomea is a unique medicinal fungus in Taiwan. It has been found rich in some pharmacologically active compounds for anti-cancer, hangover, and immune regulation etc. With the in-depth study of these components, it would be interesting and important to establish a molecular system for basic studies of A. cinnamomea. Thus, we would like to set up a foundation for this purpose by studying the A. cinnamomea protoplast preparation and regeneration. Firstly, we studied the optimization method of protoplast preparation of A. cinnamomea, and found various factors that may affect the yield during protoplast preparation, such as mycelial ages, pH values, and osmotic stabilizers. Secondly, in the regeneration of protoplasts, we explored the effects of various conditions on the regeneration of protoplasts, including different media and osmotic pressure. In addition, we found that citrate buffer with pH value around 3 dramatically increased the regeneration of protoplasts of A. cinnamomea, and provided a set of regeneration methodology for A. cinnamomea.

• Microbiology and Biotechnology Letters
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• v.13 no.3
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• pp.311-314
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• 1985

Ganodema lucidum protoplasts were formed by the treatment of Novozym 234. The osmotic stabilizers such as mannitol were effective enough to produce protoplasts up to 10$^{6}$ $m\ell$. For regeneration, however, MgSO$_4$.7$H_2O$ was suitable. When inositol and sucrose were employed as osmotic stablizers, the regeneration ratio reached to 0.26％. Overlay of Streptomycin sulfate added agar was required to prevent bacterial contamination.

• Korean Journal of Microbiology
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• v.19 no.4
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• pp.186-191
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• 1981

Protoplast production from Trichoderma koningii ATCC 26113 was investigated for the purpose of doing basic and applied researches by protoplast fusion of the cellulolytic filamentous fungus. High yields of protoplast were obtained by using the 18hr. old mycelia treated with the lytic enzyme Driselase of Kyowa Fermentation Co., Japan, in 0.6M $MgSO_4\;or\;(NH_4)_2SO_4$ as osmotic stabilizers. The optimum temeprature of mycelial digestion was about $28^{\circ}C$ and the number of protoplast increased rapidly after 3hr. digestion. Protoplasts produced at different periods showed distinct morphological heterogeneities in the whole size and the size of vacuole.

• Korean Journal of Microbiology
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• v.23 no.3
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• pp.209-214
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• 1985

The optimum conditions of protoplasts formation from Pyricularia oryzae were investigated with lytic enzymes and osmotic stabilizers. The mycelia were begun to refease the protoplasts in response to the complex enzyme solution after 30-60 minutes and reached to maximum after 2-3hrs. Among the lytic enzymes tested, the mixture solution containing ${\beta}-Glucuronidase$(0.01 ml/ml), Cellulase ONOZUKA-RS(20mg/ml), Driselase (10mg/ml), and Macerozyme R-10 (10mg/ml) resulted in the highest rate of protoplasts releasing of Pyricularia oryzae. The best stabilizer was 0.6M KCl at pH 7.0. Shen the mycelia were digested with enzyme mixture, the stationary culture was better than shaking culture for higher protoplast formation.

• The Korean Journal of Mycology
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• v.20 no.1
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• pp.58-64
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• 1992

This research was focused on investigation of the general condition for protoplast formation of Trichoderma speues. for protoplast formation, the mycelia cultured in YM medium were collected from each growth phase and were treated with the Iytic enzymes. This procedure was carried out by all strains. The most optimal conditions of NOVOZYM 234 and DRISELASE were determined by T. saturnisporum IAM 12535 and T. longibruchiatum IBM 13107, respectively. The effect of osmotic stabilizers appeared ${KCI}>(NH_4)_2{SO_4}>NaCl>mannitol>{MgSO}_4$ and the optimal concentration of each osmotic stabilizer wns determined by 0.6-0.9 M. The optimal condition of DRISELASE for protoplast formation ; optimal pH 5.0, optimal concentration, 2%, optimal reaction time, 4 hours, and optimal temperature, $30^{\circ}C$. The optimal condition of NOVOZYM 234 for protoplast formation ; optimal pH 5.5, optimal concentration 1%, optimal reaction time 3 hours, and optimal temperature $30^{\circ}C$. The optimal culture period of mycelia for protoplast formation was between the initial and the middle exponential phase. Generally, DUSELASE was more effective than NOVOZYM 234 on protoplast formation except for T. longibruchiatum IAM 13107 and T. viride IAM 5141.

• The Korean Journal of Mycology
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• v.21 no.1
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• pp.1-8
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• 1993

ABSTRACT: Factors responsible for protoplast formation and regeneration of Phytophthora capsici were examined. Protoplasts were successfully liberated from the mycelial culture by digestion for 6-9 hrs with Novozym 234 in 0.35 M $CaCl_2$, (pH 5.7) as osmotic stabilizer. Young rapidly-growing mycelium (24 hrs old) showed highest protoplast yields. High concentrations of Novozym 234 were effective in releasing protoplasts from the mycelium. The combination of 0.4 M mannitol and 0.1 M $CaCl_2$ was optimal osmotic stabilizers for protoplast regeneration. The synthetic Henninger media containing all nutritional elements gave the best regeneration rate. The protoplast regeneration was greatly inhibited in the media which were not supplement with amino acids or ${\beta}-sitosterol$. Certain amino acids such as L-aspartic acid and L-glutamic acid remarkably enhanced protoplast regeneration. However, the addition of microelements did not affect protoplast regeneration.

• Microbiology and Biotechnology Letters
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• v.13 no.4
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• pp.377-382
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• 1985

As an essential previous step towards the development of cell fusion to breed a new brewing yearst strain, several factors predicted to affect the protoplast formation of S. cerevisiae, C. tropicalis and E. fibuligera were investigated in order to obtain the protoplasts in high yields. The optimum pH and temperature for the protoplas formation were 7.5 and 35$^{\circ}C$, respectively. Pretreatment of the yeast cells with 2-mercaptoethanol stimulated the protoplast formation and 50mM of the reagent was found as effective. Among several osmotic stabilizers tested for their effect on protoplas formation, 0.6M KCI was comparatively favorable.

• Journal of Life Science
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• v.13 no.4
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• pp.516-521
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• 2003

Optimal conditions of protoplast isolation from the seaweed Prophyra pseudolinearis have been described. The P. pseudolinearis is one of indigenous and dominant Porphyra species in the East Sea region. Protoplasts have been released by enzymatic treatment of 2% agarase and 2% hemicellulase in 25mM MES buffer, pH 6.0 containing 0.5 M sorbitol. The protoplasts could be fused with neutral red-stained protoplasts of P. okamurae by the addition of polyethylene glycol 8000 solution.

• The Korean Journal of Mycology
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• v.22 no.2
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• pp.130-137
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• 1994

This experiment was carried out to investigate proper conditions for protoplast isolation and regeneration from mycelia of Lyophyllum decastes. Novozym 234(10 mg/ml) with 0.6 M $MgSO_4$ in phosphate buffer(pH 4.0) was proper for protoplast isolation. The optimal reaction time of the mycelium with the lytic enzyme was four hours in shaking condition at 120 strokes per min. When the mycelium of L. decastes was cultured at $24^{\circ}C$ for 5 days, the formation of protoplasts was effective. The liquid medium was more effective for protoplast isolation than the solid medium. In the liquid medium, high yields of protoplasts were obtained from 0.6 M $MgSO_4$ osmotic stabilizer. Protoplasts of L. decastes were regenerated to normal hyphal growth and the regeneration frequency of the protoplasts in the complete agar medium containing Triton X-100(0.0025%) was $5.94{\sim}8.32%$. The regeneration medium stabilized with 0.6 M sucrose was the best for regeneration of the protoplasts. In contrast to protoplast formation, regeneration was inhibited by the inorganic salts used as osmotic stabilizer.

• Korean Journal of Agricultural Science
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• v.13 no.2
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• pp.185-192
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• 1986

Some factors affecting the protoplast release from mycelia of Ganoderma lucidum and regeneration of the protoplast were investigated and the results obtained are summarized as follows; Novozym 234 as a lytic enzyme was the most effective for the protoplast release from mycelia of Ganoderma lucidu m and its optimal concentration was 10mg per ml of osmotic stabilizer. The highest number of protoplasts were released after 3 hours incubation in the reciprocal shaking bath at 120 oscillations a minute. Among six osmotic stabilizers tested, 0.6M sucrose showed the best result. SCM medium showed good mycelial growth and high yields of protoplasts. The protoplasts released from the mycelium of G. lucidum were regenerated at 0.20 to 0.27 percent on MCM, MMM and SCM. Of the cultures obtained from protoplasts regenerated, 13 to 29 percent were monokaryon.