• Journal of Life Science
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• v.2 no.3
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• pp.180-188
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• 1992

Protoplast의 분리는 기계적 방법, 효소처리에 의한 방법 등을 들 수 있다. 효소처리에 의한 방법으로는 적합한 효소의 선정, 조합 및 농도가 중요하고, 융합에는 고 pH-고 Ca법, FEG법 등 여러가지 융합방법이 있으나 최근에는 전기융합에 의한 방법이 개발되어 실용화되고 있다. 본고에서는 식물 protoplast의 전기융합의 개요와 필자의 실혐결과를 중심으로 설명하였다.

• Applied Biological Chemistry
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• v.30 no.4
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• pp.300-304
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• 1987

Effects of benzyladenine (BA) on viability, cell wall regeneration and division of soybean (Glycine max, Var. Acme) protoplasts isolated from suspension cells of cotyledonary callus were investigated. The uptake of BA by the protoplasts was also studied. BA increased protoplast viability, and promoted cell wall regeneration and cell division. The level of BA in protoplasts was increased to a maximum at about 20 hour incubation and 2/3 of the total amount of BA accumulated in protoplast was absorbed within 6 hours.

• ALGAE
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• v.20 no.3
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• pp.239-249
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• 2005

Some marine coenocytic green algae could form protoplasts from the extruded protoplasm in seawater. The dissociated cell components of the coenocytic protoplasm could be reunited into live cells and, hence, the formation of new species by mixing protoplasms from different coenocytic cells has been predicted. Our results showed that an incompatibility barrier was present during protoplast formation in coenocytic algae to exclude foreign inorganic particles or alien cell components. No inorganic particles or alien cell components were incorporated into protoplast formed spontaneously in seawater. Even when the inorganic particles or alien cell and/or cell component were incorporated into protoplast in some experimental condition, they were expelled from the protoplast or degenerated within several days. A species-specific cytotoxicity was observed during protoplast hybridization between the protoplasms of Bryopsis spp. and Microdictyon umbilicatum. The cell sap of M. umbilicatum could destroy the cell components of Bryopsis spp., but had no effect on Chaetomorpha moniligera. Species C. moniligera and Bryopsis did not affect protoplast generation of either species. The wound-induced protoplast formation in vitro might have evolved in some coenocytic algae as a dispersal method, and the incompatibility barrier to alien particles or cell and/or cell component could serve as a protective mechanism for successful propagation.

• 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.

• Applied Microscopy
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• v.13 no.1
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• pp.49-61
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• 1983

Protoplast releasing mechanisms from Trichoderma koningii, fine structures of the released protoplsts, and their regeneration mode were studied by scanning and transmission electron microscopy. Two types of protoplast releasing mechanisms were observed. In one mechanism, cytoplasm emerged through a cell wall pore developed by cell lytic enzymes and formed a spherical protoplast. In the other mechanism, as the cell wall became progressively thinner, the inner cytoplasm partially rounded to form nonspherical bodies which became spherical protoplasts after being released into the enzyme solution. But, these two types of protoplast releasing mechanisms did not seem to be. mutually exclusive but could occur on the same mycelium simultaneously. And it appeared that cytoplasm which did not become a protoplast by the first mechanism could from a protoplast by the second mechanism. The preparations contained two types of protoplasts, released from different sites of the mycelia. Those released from younger mycelia had dense cytoplasm and small vesicles. Those released from the older mycelia had less dense cytoplasm and larger vacuoles. In the case of regeneration, before producing normal mycelia, most of the protoplasts assumed aberrant tube and yeast-like-forms. Normal mycelia were produced at the end of the yeastlike-forms and sometimes in the middle of the aberrant tube.

• Microbiology and Biotechnology Letters
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• v.14 no.2
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• pp.175-179
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• 1986

In order to develope a protoplast fusion of the genus Cellulomonas having high assimilibility of cellulose, the optimum conditions for the protoplast formation of Cellulomonas flavigena NCIB 12901 was investigated and observed by means of Scanning Electron Microscope. The results suggested that the susceptibility of the cell wall by lysozyme treatment on protoplast formation was considerably depend on the cultural periods of the cells. Cells of C. flavigena at mid exponential phase could more efficiently convert to protoplast cells than those at late exponential phase did. The rate of the protoplast formation was 95%, even though the rate was over 99.9% on counting by indirect method after osmotic shock treatment, when cells of the organism at mid exponential phase were treated with lysozyme (400$\mu\textrm{g}$/$m{\ell}$) for 6 hours and observed by SEM. In the evaluation of protoplast formation of the genus Cellulomonas, direct method of the observation under Scanning Electron Microscope was much more reliable than the counting method of protplasts after osmotic shock treatment. Because defferences between the number of spheroplast and protoplast were not able to be figured out on counting the number of protoplast after osmotic shock treatment.

• 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.23 no.3
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• pp.359-364
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• 1995

Two lactic strains, Leuconostoc mesenteroides Lu5 and Pediococcus pentosaceus P1 isolated from Kimchi, were used to determine the optimum conditions for protoplast formation and regeneration. The maximum protoplast formation rate was obtained with both strains at early exponential growth phase and decreased rapidly during growth phase. For P. pentosaceus P1, 30 $\mu$g/ml of lysozyme treatment was sufficient to obtain over 90% of protoplast formation and 300 $\mu$g/ml for L. mesenteroides Lu5, showing great difference in sensitivity of these strains to lysozyme. For both strains, best results were obtained at pH 7, using 0.5 M sucrose as osmotic stabilizer. For regeneration of protoplast, the highest regeneration rate was obtained after 15 minutes of lysozyme treatment and declined drastically with prolonged digestion.

• Korean Journal of Food Science and Technology
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• v.16 no.3
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• pp.363-367
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• 1984

Conditions for efficient formation and regeneration of protoplasts of Streptococcus lactis ATCC 11454 were investigated. Addition of 20mM DL-threonine into growth medium, growth phase and lysozyme concentration had significant effects on protoplast formation. Approximately, 20% regeneration efficiency was obtained by optimizing the medium composition and modifying the plating procedure.

• 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.