• 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.

• 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.25 no.4
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• pp.274-281
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• 1987

The effects of bovine serum albumin, myoinositol and ergosterol on protoplast formation, regeneration and fusion from auxotrophic mutants of Candida pseudotropicalis were examined. Frequency of protoplast formation ranged from 48 to 98% depending on auxotrophic types. When myoinositol (0.5mg/ml) and ergosterol (0.1mg/ml) were supplemented in the medium of cell growth, and bovine serum albumin (4mg/ml)was added to protoplasting buffer, 50-100% of cells were converted to protoplasts. Such a treatment of three additives improved 2.2-3.0 fold of regeneration rate of protoplasts. The fusion frequencies between complementary auxotrophs ranged from $7.0\times 10^{-4}$ to $1.5\times 10^{-3}$ in the optimal conditions. These values showed 1.9-2.3 fold increase when compared with fusion frequencies obtained without the treatment of additives. These results suggested that these comsion frequencies obtained without the treatment of additives. These results suggested that these xompounds may improve protoplast regeneration and fusion between complementary auxotrophs used in this study.

• 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.

• The Korean Journal of Mycology
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• v.14 no.2
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• pp.165-174
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• 1986

Conditions for production, fusion and reversion of protoplasts of Aspergillus niger were investigated, and an attempt was made to enhance fusion frequency. Auxotrophic mutants and morphological mutants were induced by U.V. irradiation $(9.9\;erg/mm^2,\;13min)$ on Aspergillus niger. Maximum yield of protoplasts was obtained from 21 hr cultured mycelia by using 1% driselase in 0.6 M KCl or 0.6 M $NH_4Cl$ as osmotic stabilizer. The optimal temperature for mycelium digestion was $30^{\circ}C$, and the optimal pH was 6.0. Protoplasts produced at different digestion period showed heterogeneity in size and vacuole content. Maximal frequency of protoplasts reversion was obtained on 0.6 M KCl stabilized agar medium at pH 5.0. Reversion frequencies of protoplasts produced for 3 hr and 1 hr mycelial digestion were 8.0% and 15.3%, respectively. The optimal concentration of PEG(m.w. 6000) for protoplast fusion was 30%, and that of $CaCl_2$ was $1{\sim}50\;mM$. The optimal pH and period for the reaction of PEG solution were 8.0 and 10 minutes, respectively. Fusion frequencies between auxotrophic protoplasts produced for 3 hr-mycelial digestion were $0.06{\sim}0.42%$, and those for 1 hr-mycelial digestion were $0.09{\sim}0.54%$.

• 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.

• 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.

• Korean Journal of Microbiology
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• v.22 no.1
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• pp.49-56
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• 1984

Formation and regeneration of protoplasts by Novozym 234 from Kluyveromyces fragilis N100 and Candida pseudotropicalis CBS607 were studied. This enzyme was more effective on cells grown at exponential phase than those at stationary one to convert intact cells into protoplasts. As osmotic stabilizer, ammonium sulphate was suitable for not only protoplast formation but also regeneration in K. fragilis as well as in C. pseudotropicalis. Optimal enzyme concentration was 3mg per ml in K. fragilis and 1~3mg per ml in C. pseudotropicalis, respectively. After the exposure of K. fragilis cells to 3mg per ml of enzyme for 3hr at 30$^{\circ}C$ , approximately 95% of protoplast formation of all observed cells was obgained, while about 100% from C. pseudotropicalis under the same condition was produced. The regeneration frequency of protoplasts by this enzyme was much lower than that by snail enzyme(Glusulase) although Novozym 234 converted cells from above two species into protoplasts free of cell debris effectively, compared with Glusulase. Novozym 234 appears to be suitable for subcellular fractionation to obtain nuclei or other organelles rather than protoplast regeneration.

• Korean Journal of Microbiology
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• v.24 no.2
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• pp.154-160
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• 1986

In order to develope interspecific fusion of the genus Cellulomonas capable of assimilation cellulose, the optimun conditions for the protoplast formation was investigated to examine the susceptibility of cell wall, between different species of the same genus using scanning electron microscope. The variation in the susceptibilities of Cellulomonas sp. CS 1-1 and C. flavigena to lysozyme treatment were considerably remarkable, although they belong to the same genus. The rate of protoplast formation of CS1-1 was 99.9% being treated with lysozyme $(100{\mu}g/ml)$ for 30 minute and that of C. flavigena was about 80% being treated at the concentration of $600{\mu}g/ml$ of lysozyme for 6 hours. The susceptibility of cell wall to the lysozyme treatment on protoplast formation of the strain, CS1-1 seems not to be depend on the cultural periods of cells. On the contrary, that of C. flavigena was considerably depend on the periods. Cells of C. flavigena at mid exponential phase could be more efficiently converted to protoplast cells than those at late exponential phase be done. The rate of the protoplast formation was 95%, when cells of C. flavigena at mid exponential phase were treated with lysozyme $600{\mu}g/ml$ for 6 hours and observed by SEM. In the evalution of protoplast formation of the CS1-1 results of counting method in plate after osmotic shock treatment were similar to the results of the direct observation method by means of SEM. But in the case of C. flavigena the latter method was much more reliable than the former, because the differences between the number of spheroplasts and protoplasts were not able to figure out on conuting the number of protoplast after osmotic shock tretment.