• The Korean Journal of Mycology
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• v.22 no.2
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• pp.138-144
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• 1994

Conditions for isolation of protoplasts from spores and mycelia of Rhizopus nigricans were studied. Larger amount of protoplasts was obtained from swollen spores in liquid medium contained with 5% of 2-deoxy-D-glucose for 4 hours than from mycelia. Enzyme mixture of Novozym 234(2%) and ${\beta}-glucuronidase(5000\;unit/ml)$ was most effective for the isolation of protoplasts from swollen spores and from mycelia. The solution of 0.6 M $MgSO_4$ or mannitol and pH 6.0 showed good results as the osmotic stabilizer and the optimal condition of pH of the enzyme solution for the isolation of protoplast from the swollen spores, respectively. At this condition, $8.0{\times}10^6\;cells/ml$ of protoplasts was obtained from swollen spores by digestion with lytic enzyme mixture for 2 hours.

• 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.

• The Korean Journal of Mycology
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• v.13 no.2
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• pp.79-82
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• 1985

The experiment of protoplast regeneration and reversion were undertaken to provide a basic techniques for protoplast manipulation. Protoplasts of Pleurotus florida and Pleurotus ostreatus were reverted to normal hyphal growth and the reversion frequency of both fungal protoplasts were $0.24{\sim}3.19%$. Reversion medium stabilized with 0.6 M potassium chloride and sucrose was better than the other stabilized one. The protoplast reversion frequency was increased when various amino acid and vitamin compounds were added to the hypertonic mushroom complete agar medium.

• Microbiology and Biotechnology Letters
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• v.13 no.4
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• pp.383-389
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• 1985

Optimum conditions of PEG treatment for the intergeneric fusion of yeast protoplasts were investigated. Fusants were selected by nutritional complementation on minimal medium. The intergeneric fusion frequency between pro-toplasts of S. cerevisiae and C. tropicalis was distributed 10$^{-4}$ to 10$^{-6}$, depending on the combination of parental strains. PEG 4000 or 6000 are equally effective. 30%(w/v) PEG 4000 was found to be optimum and below 20% its stabilizing effect was lost, resulting in protoplast lysis, and optimum pH was 8.0. The efficiency of PEG was enhanced by higher temperature of the PEG solution, and by the addition of Ca ions. The stimulating effect of Ca ions in the range of 1 mM to 100 mM proved similar.

• Journal of Plant Biology
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• v.34 no.1
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• pp.19-23
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• 1991

Culture of embryogenic callus and suspension were induced from rice seeds in MS2.5 medium. In hormone free N6 medium, whole plantlets were regenerated from embryogenic callus. We observed cell division and reformation of embryogenic callus on culture of protoplast isolated from embryogenic cell suspensions. In addition, we studied the influencing factors on viability of protoplast treated with electroporation. Viability was decreased according to the increase of voltage and capacitance during electroporation. An optimal level of viability was obtained after treatment with $200-300\;V/1180\;\mu\textrm{F}$ in HEM buffer at $4^{\circ}C$..

• 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.

• KSBB Journal
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• v.6 no.2
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• pp.201-205
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• 1991

We have investigated influencing factors on viability of Bletilla striata protoplasts electroporated in the presence of various electrical conditions. Cultures of embryogenic callus and embryogenic cell suspension were established with immature seeds of Bletilla striata. Viabilty of electroporated protoplasts was decreased according to the increaseing of electroporation voltage and capacitance. An optimal condition of electroporation for viable protoplasts was in HBM buffer at $4^{\circ}C$.

• The Korean Journal of Mycology
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• v.23 no.4 s.75
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• pp.305-309
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• 1995

To investigate optimal conditions for the protoplast formation and regeneration of Phanerochaete chrysosporium, preparations of three enzymes were used to liberate protoplasts from its 20 hrs-old mycelium on cellophan membrane covered agar media. Novozym 234 alone with 0.6M sucrose was the most effective for isolation of protoplasts from the mycelium with 3hrs incubation time at $39^{\circ}C$ in shaking condition of 120 rpm. The poly-R medium stabilized with 0.6M mannitol was the best for regeneration of the protoplasts.

• Journal of Aquaculture
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• v.1 no.1
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• pp.103-108
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• 1988

Protoplasts were isolated enzymatically from thalli of Porphyra tenera in relation to its utilization in breeding. As the concentration of the enzymes and incubation period increased, so did the yield of protoplasts. When a 250 mg fresh weight of the thalli was incubated in the $12\%$ abalone digestive enzyme mixture at $22^{\circ}C$ for 3 hours, about $2.5\times10^6$ protoplasts were released. The size of protoplasts ranged between 9$\mu$m and 25$\mu$m, with an average of 14.5$\mu$m.

• Microbiology and Biotechnology Letters
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• v.32 no.2
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• pp.101-109
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• 2004

In order to enhance the productivity of AVM $B_{la}$ produced by Streptomyces avermitilis as a secondary metabolite, we established a basic protocol necessary for protoplast fusion with high-producing strains as a fusion partner, and then obtained various kinds offusants by adopting a massive strain-development procedure (a miniaturized strain screening system). An alternative fusion method using UV and/or NTG mutation of protoplasts was developed to screen genetic recombinants without specific selectable markers. In this method, the mutants obtained by protoplast fusion after UV and/or NTG treatment (95% death rate) of the respective fusion partner (protoplasts of the respective mutants resistant against L-isoleucine antimetabolites such as O-methylthreonine and/or azaleucine) were regarded as DNA-recombined protoplast fusants. Notably it was demonstrated that most of the protoplast recombinants obtained by the UV mutation method were able to biosynthesize higher amount of AVM $B_{la}$ , reaching almost three times higher level (almost equal to the industrial productivity), compared to the average AVM Bla amount of the parallel mother strains.