• Journal of Plant Biology
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• v.37 no.2
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• pp.183-193
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• 1994

인삼(Panax ginseng C.A. Meyer)의 뇌두 캘러스로부터 분리한 원형질체와 배양된 원형질체의 미세구조 변화와 원형질막 표면을 투과 및 주사전자현미경으로 조사하였다. 분리 직후의 원형질체에서는 캘러스세포에서보다 작은 액포들이 많이 형성되어 있었다. 또한 활면소포체의 수가 증가하였으며 이들은 원형질막과 평행으로 배열하였다. 활면소포체는 가끔 세포질을 둘러싸서 세포질분리체(cytosegresome)를 형성하였고, 이 구조는 세포질을 분해시킨 후 액포로 변화하기도 하였다. 배양된 원형질체에서는 분리 직후의 원형질체와 비교하여 조면소포체, 딕티오좀, 리보좀, 미토콘드리아, proplastid 및 액포 등의 수가 현저하게 증가하였다. 딕티오좀으로부터 많은 소낭들이 형성되었고 이들은 세포질 전반에 걸쳐 존재하였다. 때로는 소낭들이 원형질막 바깥으로 돌출되어 돌기를 형성하기도 하였다. 배양된 원형질체의 표면에는 섬유소로 구성되어 있을 것으로 추정되는 섬유상 구조들이 형성되어 있었다.

• 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.

• Korean Journal of Microbiology
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• v.30 no.3
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• pp.154-159
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• 1992

Forniation ancl ~regcncration oi' conitlial pro1oplast of Pc, ti~i.rlli~in~~~ c~rr~~culo.hryupmel.- - czllulolytic Ihngus. were examined. By using Novozyme 134(1'!/0 w/c) as a cell wall lytic enzyme. the highest yield of protopl;~sts exceeding 501%, war obtained from the qwollen conidiosporcs preincubatrd in the minimal medium containing 2-tleoxy-D-glucose(2-UC;. 75 pglml) for 10-11 11. No protoplast were obtained horn dormant spores. The regeneration frequency of the protoplasts was 49.2'!11. which was higher than that of mycclium originated protoplast (4.6-17.X1X, . in 0.6 M MgS04. pH 5.6). 'l'lie best osmotic stahilizcr ror the isoaltion and regcueration of thc protoplast was 0.6 M iimmonium sulfatc and 0.6 M magnesiuni sulfhte. respectively. 'I'lie process of the protoplast isolaiton l'ro~n swollen cnnirliospore ancl regeneration ha\, ing two pattcrns from protoplast were obsen'etl through light microscope.

• The Korean Journal of Mycology
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• v.13 no.1
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• pp.1-10
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• 1985

This experiment was undertaken to investigate proper conditions for protoplast isolation from strains of Agaricus bisporus, Flammulina velutipes, Lentinus edodes, Pleurotus florida, Pleurotus ostreatus, Pleurotus sajor-caju and Volvariella volvacea. Combination of ${\beta}-D-glucanase$, novozym 234 and snail enzyme with 0.6M $MgSO_4$ in 0.05M Na-maleate buffer, pH 5.8 was the most effective for isolation of protoplasts. High yields of protoplasts were obtained from $4{\sim}5$ days old mycelia of P. florida and P. ostreatus on mushroom complete agar medium. Protoplasts were also obtained in good yield from other fungi but A. bisporus and V. volvacea. In the latter two cases protoplast release was observed; however, the yield was much lower than those of the other fungi.

• Microbiology and Biotechnology Letters
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• v.16 no.4
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• pp.293-296
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• 1988

Stability of spheroplasts prepared from Saccharomyces cerevisiae D-71, a thermophilic strain and Zygosaccharomyces rouxii SR-S, an osmotolerant strain were studied. Stability of spheroplast from Saccharomyces cerevisiae D-71 was highest in 0.8M KCI and 1.0M sorbitol ; that from Zygosaccharomyces rouxii SR-S was highest in 0.4M KCI and mannitol and that from both strains was less than 10% for sonic oscillation at 20Kc for 60 sec. In centrifugation at 10000 x g for 10 min., stability of spheroplast from Saccharomyces cerevisiae D-71 was 93% and that from Zygosaccharomyces rouxii SR-S was 84%. Breakage of spheroplast from Saccharomyces cerevisiae D-71 was 99% and that from Zygosaccharomyces rouxii SR-S was 55% for UV irradiation with 15W UV lamp at a distance of 20 cm for 60 min.

• Korean Journal of Microbiology
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• v.23 no.4
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• pp.265-270
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• 1985

Overall procedure of cell fusion between Brevibacterium flavum and Corynebacterium glutamicum was morphologically observed by transmission electron microscopy. Protoplasts formed by treatment of cells with penicillin G and lysozyme in order were released through the pores generated on a certain region of cell walls to be spherical form. When two different protoplasts were met, cell wall and membrane in the contact zone was disappeared and followed by the mutual exchange of cytoplasmic and/or chromosomal materials. Cell xall regeneration speed of the protoplasts fused was slower than that of the non-fused, whereas the size of the former was confirmed as bigger than that of the latter.

• Korean Journal of Microbiology
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• v.24 no.4
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• pp.364-369
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• 1986

A procedure for the preparation, regeneration and fusion of protoplasts of Streptomyces tubercidicus was confirmed. Also, protoplast releasingprocesses from mycelia were observed by scanning electron microscope. Three types of protoplasts releasing processes-from the hyphal tip, hyphal end regions and lateral regions of the hyphae-were observed. More than 17% regeneration efficiency was obtained by regeneration medium that is composed of tryptone-yeast extract-sodium acetate-$MgCl_2-CaCl_2$-sucrose. Optimal concentrations of $Ca^{++},\;Mg^{++}$ and sucrose in the regeneration medium were 50mM, 0.4-0.5M respectively. Above 30% of fusion frequency of the protoplasts derived from two auxotrophic strains of S. tubercidicus was induced by polyethylene glycol 4000(60% w/v).

• Korean Journal of Plant Tissue Culture
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• v.27 no.6
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• pp.429-434
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• 2000

Isolated protoplasts from ginseng (Panax ginseng C. A. Meyer) callus tissue were cultured in modified MS media supplemented with various concentrations of dimethylsulfoxide (DMSO). The cell wall regeneration rate and cell division efficiency of the protoplasts were increased significantly by 1% DMSO treatment. However, there was no difference in the viability of protoplasts between the DMSO treatment and non-treatment. Transmission electron microscopy revealed that the microtubules were oriented in parallel manner to the plasmalemma after 3 days of culture in medium with 1% DMSO. Further, interconnected cellulose microfibrils were observed on the outer surface of the 3-day-cultured protoplasts by scanning electron microscopy These structures shown by electron microscopy were not observed in protoplasts cultured on DMSO-free media. This studies indicates that DMSO supplemented in culture media seemed to stimulate the cell wall regeneration and cell divisions of protoplasts by forming microtubule organizing centers (MTOC).

• The Korean Journal of Mycology
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• v.18 no.2
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• pp.77-83
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• 1990

Factors influencing the fusion frequency of protoplasts were investigated with auxotrophic mutants of Pleurotus florida and Pleurotus ostreatus. Immediately after the polyethylene glycol (PEG) solution was added, the protoplasts adhered firmly and shrank. During the subsequent dilution with 0.6 M sucrose, the protoplasts regained their normal size and larger bodies were observed. Interspecific heterokaryons were obtained by fusion of the nutritionally complementing protoplasts. Hyphae of the heterokaryotic fusants formed true clamp connections. The optimum conditions were a total of 1 to 15 million protoplasts per ml, 30% polyethylene glycol 8000 solution with adjustment to pH 8.0 and 0.6 M sucrose stabilized regeneration medium. Other parameters such as $CA^{++}$, glycine, exposure time and temperature influenced mainly the viability of the protoplasts.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.27 no.2
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• pp.147-154
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• 1982

This study was conducted to investigate an effective method of protoplast isolation, the plating efficiency for cell division, and fusion of plant protoplasts by polyethylene glycol for somatic hybridzation. The effectiveness of protoplast isolation was different with the various enzyme concentrations, but, in the protoplast isolation from tobacco mesophyll, the enzyme solution with 0.5% macerozyme and 2.0% cellulase was very effective. The protoplast isolation from callus cultured in vitro for a long period was not obtained in any of the enzyme solution used. Protoplasts divided actively at cell densities above $10^44/ml and at $25^{\circ}C$ under 12hr illumination by inflorecient light (l50 Lux), regardless of presence of agar. The highest frequency of protoplast fusion was obtained after treatment with a solution of 0.33 M polyethylene glycol 1500.