• Korean Journal of Microbiology
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• v.31 no.3
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• pp.218-223
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• 1993

Conditions for the release and regeneration of protoplasts form Rhizopus oryzae and intergeneric protoplast fusion between Rhizopus oryzae and Aspergillus oryzae were studied. High yields of protoplast fusion between Rhizopus oryzae and Aspergillus oxyzae were studied. High yield of protoplasts from young germilings of R. oryzae were obtained by using lytic enzymes containing chitosanase (3 mg/ml), chitinase (3 mg/ml) and Novozym 234 (5 mg/ml). 0.5M glucose was used as the osmotic stabilizer and optimum pH of buffer was determined to be pH 7.5-8.0. Under these conditions, protoplasts were formed after about 3-4 hrs incubation. Approximately, 1.0%-4.9% of these protoplasts were formed after about 3-4 hrs incubation. Approximately, 1.0%-4.9% of these protoplasts regenerated on solid medium with a soft agar overlay. We have also carried out protoplasts fusion between R. oryzae and A. oryzae and have succeeded in obtaining three types of intergeneric fusants. In these experiments, 35% PEG-4000 and 10 mM CaCl$_{2}$ were used as fsogenic agents, and auxotrophic properties were used as a genetic marker to select fusants. Complementation frequency be protoplasts fusion of A. oxyzae and R. oryzae was 4.4% * 10$^{-5}$ . The fusant strains of the first type were prototrophs showing an Aspergillus type morphology with dark-yellow sporulation, those of the second type were also Apergillus type morphology but showed no sporulation. And the strains of the third type stopped growing when fusion products grown on regeneration minimal medium were transferred to fresh minimal medium. The formation of fusion products was observed by fluorescent vital stains for complementary labelling of protoplats from R. oryzae and A. oryzae. Rhodamine 6G and fluorescein diacetate wer useful complementary vital stains of Rhizopus and Aspergillus protoplasts for visualization of requency and type (dicell, multicell) of fusion.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.32 no.1
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• pp.117-120
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• 1999

High yields of protoplasts were obtained following enzymatic digestion of the vagetative thalli of marine green alga Monostroma nitidum. The enzyme mixtures containing $4\%$ Cellulase R-10+$3\%$ Macerozyme R-10+$3\%$ Abalone acetone power produced $4.41\times10^6$ protoplasts per 300 mg of fresh tissue. The highest yield of protoplasts was obtained by 270 minutes treatment of the thalli in enzyme solution. Freshly isolated protoplasts were spherical in shape and ranged between $13\~33\mu$m in diameter. The high efficiency of differentiation were obtained by incubating freshly isolated protoplasts in 0.4 M mannitol f/2 medium for 7 days and then transferring to 0.2 M mannitol f/2 medium. Protoplasts began to form new cell walls three days after initial culture and began to germinate after 10 days, and then form a leafy thallus after further culture in f/2 medium. The addition of antibiotics in media inhibited the differentiation of protoplasts in culture.

• Korean Journal Plant Pathology
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• v.11 no.4
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• pp.318-323
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• 1995

The effect of AF-toxin I produced by the strawberry pathotype of Alternaria alternata on the protein synthesis of susceptible strawberry protoplasts was examined by using the radiolabeled amino acids. The incorporation of the radiolabeled amino acids into newly synthesized proteins in the strawberry protoplasts was stimulated by the toxin treatment at relatively low concentrations (2.2$\times$10-11 to 2.2$\times$10-9 M), but not at higher concentrations (2.2$\times$10-8 to 2.2$\times$10-6 M). An one-dimensional SDS-polyacrylamide gel electrophoresis revealed no detectable differences in the proteins synthesized in both the toxintreated and untreated protoplasts. The susceptible strawberry protoplasts were treated with AF-toxin I and stained with Fluostain I to detect the extracellular polysaccharides. The toxin treatment induced the accumulation of extracellular polysccharides in a dose-dependent manner. These results indicate a transient activation of cellular metabolism in the susceptible cells by the toxin exposure.

• Journal of Plant Biology
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• v.28 no.3
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• pp.233-241
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• 1985

The isolation and culture of protoplasts from hypocotyl-derived calluses of Glycine max (L.) Merr. cv. Jangyeop were obtained by digestion for 6 hrs in an enzyme solution containing 3.5% cellulase, 1.5% macerozyme, 10% sorbitol and 0.1% CaCl2.2H2O at pH 5.8. Newly formed cell wall of protoplasts cultured in MS agar medium containing 10 $\mu$M $\alpha$-naphthaleneacetic acid (NAA) and 32 $\mu$M N6-benzylaminopurine (BAP) could be observed after 24 hrs culture. The first cell division of the protoplasts was observed after 3 days of culture; cell clusters after 2 weeks of culture. When transferred to solid media, the protoplasts formed cell clusters gave rise to proliferating calluses.

• Journal of Plant Biology
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• v.31 no.2
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• pp.121-130
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• 1988

In order to clarify effects of phytohormones on the viability and the cell wall regeneration of protoplasts isolated from Nicotiana tobacum L. var. BY4, protoplasts isolated from mesophyll tissue were cultured on the Murashige-Skoog liquid media supplemented with auxin(2, 4-D, NAA, IAA) and/or cytokinin (kinetin, BAP, 2ip). Viability of protopplasts was higher in the culture medium containing auxin and cytokinin, especially in the combination of 2, 4-D and BAP. The effectual cell wall regeneration of protolasts was observed when theprotoplasts were cultrued on the medium supplemented with auxin alone, especially with IAA. Cell wall regernation started from 2-3 days after culture and was not detected at budding regions. When the protoplasts were cultured on the phytohormone-free medium, the viability of protoplasts dramatically decreased 4 days after culture.

• Journal of Plant Biology
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• v.36 no.1
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• pp.105-112
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• 1993

Selection of glyphosate-resistant clones from MNNG-treated mesophyll protoplasts of haploid tobacco and their differentiation were studied. The protoplasts were treated with 0.1 to 100 $\mu\textrm{g}$/mL N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) for 30 min when they expanded to oval shapes. After the treatment, the protoplasts in 4-16 cell stages were transferred to the selective medium containing 1 mM glyphosate for the selection of the glyphosate-resistant colonies. The efficiency of the cell division of the protoplasts in the selective medium decreased as the MNNG concentrations in creased. Optimal MNNG concentration for induction of the glyphosate-resistant clones was 10$\mu\textrm{g}$/mL and mutation frequency was 2.66$\times$10-6. The stability of the glypohsate-resistance of the clones was examined by prolonged subculture in the medium with 1 mM glyphosate, and the resistant clones were survived more than 10 months. Among them one clone has been proliferating and greening and the others were proliferating without greening or greening with slower proliferating.

• Mycobiology
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• v.29 no.1
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• pp.15-18
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• 2001

Protoplast fusion is a useful technique for establishing fungal hybrids to overcome the natural barriers. The ultrastructure of protoplast and its fusion process were observed using a scanning electron microscopy(SEM) and a transmission electron microscopy(TEM). The protoplasts were variable in size from $0.5{\sim}15{\mu}m$ in diameter, and the mean diameter was about $3{\sim}5{\mu}m$. It was impossible to discriminate protoplasts of Lentinula edodes from protoplasts of Coriolus versicolor by size and surface structure. Big aggregates of the dehydrated protoplasts were observed, after polyethylene glycol 4000 treatment. Nucleus, mitochondria, lipid granules and various vesicles having granules were scattered in the cytoplasm. The vesicles were heterogeneous in size and vary from one protoplast to another. The fused membrane layer of the two protoplasts was observed. Time protoplast membrane contact and reorganization of membrane components were essential condition for protoplast fusion. Transmission electron micrograph showed fused protoplasts and flattening of the cells in the area of the membrane contact. We hope that our electron microscopic observations provide some insights into the understanding of the fusion process of protoplast in fungi.

• Mycobiology
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• v.34 no.2
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• pp.73-78
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• 2006

Inter-specific hybridization between Pleurotus pulmonarius and P. florida was attempted through PEG-induced protoplast fusion to select a fusant. The protocol for protoplast release, regeneration and fusion in these two Pleurotus species was standardized using the variables controlling the process. The mixture of mycolytic enzymes, i.e. commercial cellulase, crude chitinase and pectinase, KCl (0.6 M) as osmotic stabilizer, pH 6 of the phosphate buffer and an incubation time of 3 hours resulted in the maximum release of protoplasts from 3-day-old mycelia of P. florida ($5.3{\sim}5.75{\times}10^{7}$ protoplasts/g) and P. pulmonarius ($5.6{\sim}6{\times}10^{7}$ protoplasts/g). The isolated protoplasts of P. florida regenerated mycelium with 3.3% regeneration efficiency while P. pulmonarius showed 4.1% efficiency of regeneration. Polyethyleneglycol (PEG)-induced fusion of protoplasts of these two species resulted in 0.28% fusion frequency. The fusant produced fruiting bodies on paddy straw but required a lower temperature of crop running ($24{\pm}2^{\circ}C$) than its parents which could fruit at $28{\pm}2^{\circ}C$. The stable fusant strain was selected by testing for the selected biochemical markers i.e. Carbendazim tolerance and utilization of the lignin degradation product, vanillin.

• Journal of Plant Biology
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• v.29 no.1
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• pp.1-10
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• 1986

Intra- and inter-specific protoplast fusion of tobacco (Nicotiana tabacum cv. Virginia 115) and pea (Pisum sativum cv. Sparkle) were carried out in highly inhomogeneous alternating electric fields. Under the electric field of alternating current (AC, sine wave), 600 V/cm and 800 kHz for tobacco protoplast, and 600 V/cm and 700 kHz for pea protoplasts, the protoplasts were aggregated in pearl chains. Intra-specific protoplast fusions were most effectively induced within the aggregates of tobacco and pea, respectively, by the additional application of a single high field pulse of direct current (DC, square wave) at 1 kV/cm for 50 $mutextrm{s}$. Inter-specific fusions between protoplasts of the two plants were most effectively induced in the electric field of 600 V/cm and 700 kHz, and square wave pulse at 1 kV/cm for 50 $mutextrm{s}$. The duration of the pulse over the electrical breakdown voltages was simulated from 1 to 100 $mutextrm{s}$ in both tobacco and pea protoplast. The yield of the electrofusion products was significantly high (above 60%), compared with that (20%) of the standard fusion method by polyethylene glycol (PEG) 4,000, and the viability of electrofused protoplasts was above 70%, but that of PEG-fused protoplasts 8~16%, when determined by Evan's blue staining method.

• Korean Journal of Microbiology
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• v.22 no.2
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• pp.103-110
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• 1984

The conditions for the protoplast fusion of auxotrophic mutants of Trichoderma koningii were determined. A preparation of commercial enzyme Driselase was used successfully to isolate protoplasts from the 18 hr old mycelium of T. koningii. The yields of protoplasts production were ranged from $0.3{\times}10^8$ to $2.5{\times}10^8$ protoplasts per mg of damp mycelium of various auxotrophic mutant strains. The regeneration frequencies from $9.3{\times}10^{-3}\;to\;2.0{\times}10^{-1}$ were obtained when the protoplasts from auxotrophic mutants were plated on the malt extract medium containing 0.6M $MgSO_4$, and 2% agar, and the optimal concentration of PEG for protoplst fusion was 30%. Exposure of protoplasts to PEG for 10 min was found to be sufficient to induce high frequency heterokaryon formation. Optimal pH of fusion mixture was determined as 5.5, and 1 mM of calcium chloride in fusion mixture was found to be sufficient to enhance protoplast fusion frequency. Under optimal condition, the fusion frequency of the cross between protoplasts from various auxotrophic mutants were $1.6{\times}10^{-2}\;and\;4.1{\times}10^{-2}$.