• Journal of The Korean Society of Grassland and Forage Science
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• v.9 no.3
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• pp.119-123
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• 1989

레드 클로버의 葉肉細胞로 부터 原形質體의 裸出에 影響을 미치는 要因들을 調査하여 다음과 같은 結果를 얻었다. 原形質體의 裸出에 使用된 酵素는 Cellulase, Macerozyme, Driselase 및 Pectolyase였으며 그 中 1%의 Cellulase ONOZUKA R-10과 0.5%의 Macerozyme R-10의 組合에서 가장 좋은 結果를 얻었다. 裸出된 原形質體의 適正 膨壓을 維持하기 爲하여 Mannitol의 濃度를 0.3~O.8M의 범위에서 6단계로 조정하였을 때, 0.7M에서 가장 높은 收率을 얻을 수 있었다. 原形質體 裸出을 爲한 酵素溶液의 pH는 5.6에서 가장 좋은 結果를 나타내었으며, 材料로 쓰인 레드 클로버 일을 4가지 크기로 區分하여 裸出實驗한 結果, 작은 잎보다 큰 잎에서 收率이 높은 傾向이었다. 酵素處理 後 裸出된 原形質體의 정제에는 0.8M의 Sucrose가 가장 適當하였다.

• Journal of Plant Biology
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• v.29 no.4
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• pp.263-284
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• 1986

Effects of putrescine, spermidine and spermine on the activity of D-glucose-6-phosphate cyclohydrolase in the Daucus carota L. protoplast cultured for 4 days and effects of polyamines on the incorporation of D-[u-14C]-glucose treated to protoplasts in culture-medium were investigated. The activity of D-glucose-6-phosphate cyclohydrolase was increased by polyamines and among them spermine was the most effective. Polyamiens increased protein synthesis and this due to the increasing effect of the polyamines on the synthesis of glycoprotein which is one of cell wall components. The synthesis of cell polysaccharides, such sa pectic substances, hemicelluloses and cellulose was increased by polyamines, which stimulated synthesis of pectin substances, and hemicellulose more greatly than that of cellulose, and spermidine was the most effective. In the light of the above results it seems that the polyamines increase cell wall regeneration by the stimulation of enzyme activities which synthesize cell wall components.

• Journal of Microbiology
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• v.33 no.4
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• pp.334-338
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• 1995

The electrophoretic karyotypes of 6 species in different Fusarium sections were examined by using contour-clamped homogeneous electric field (CHEF) gel electrophoresis. Intact chromosomal DNA was prepared from protoplasts and up to 9 distinct bands were separated on 0.7% or 0.8% agarose gel under several different conditions. Putative chromosome numbers varied from 6 to 9 amd polymorphic karyotypes were observed in different Fusarium sections. Using Schizosaccharomyces pombe and Saccharomyces cerevisiae chromosomes as standards, the sizes of the Fusarium spp. chromosomes were estimated. The electrophoretic karyotypes of F. moniliforme and F. subglutinans (section Liseola) were similar. Unidentified filamentour fungi, F. beomiforme was much closer to F. axysporum (section Elecgans) in karyotype and the karyotypes of F. napiforme were more similar to those of section Liseola than any other sections. F. graminearum (section Discolor) had a distinctive electrophoretic karyotype.

• 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.13 no.3
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• pp.169-177
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• 1985

The studies were carried out to obtain the basic data for maximizing the protoplast yields from the mycelia of P. ostreatus and P. sajor-caju. Some factors affecting the regeneration of the protoplast of both species and the productivity of their reversion were also examined. The maximum yields of protoplasts were obtained from four days cultured mycelia of both species on cellophan membrane placed on the surface of PSA or MCM media in a petri dish. The optimal concentration of lytic enzyme Novozym 234 for protoplast releasing was 5 mg per ml of 0.5 M phosphate buffer solution with 0.6 M sucrose or 0.6 M $MgSO_4$ at pH 6.0. The greatest number of protoplasts was released 3 hours after incubation of the mycelia of P. ostreatus and after 4 hours for the P. sajor-caju in the lytic enzyme solution. Among the osmotic stabilizer solutions tested 0.6 M sucrose and 0.6 M KCl showed the best regeneration rates of the protoplasts of both species. When 0.75 % agar solution was over-layed on the regeneration media immediately after inoculation of the protoplast the regeneration rates were greatly enhanced. The ampicillin added to the agar solution prevented bacteria from infection. The reverted isolates produced the sporophores and basidial spores just like their parents without any mutations when they were cultivated in a broad mouth bottle with sawdust substrates.

• The Korean Journal of Mycology
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• v.25 no.2 s.81
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• pp.143-151
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• 1997

Interspecific hybrids between Aspergillus niger and Penicillium notatum (Tyr-), hyperlipolytic enzyme-producing fungi, were obtained by nuclear transfer technique. Optimal conditions for formation of intergeneric hybrids were investigated. Maximum production of protoplasts was obtained by 1% Novozyme 234 at $30^{\circ}C$ for 3 hrs and the most effective osmotic stabilizers for the isolation of protoplasts were 0.6 M KCl. Frequencies of hybrid formation by nuclear transfer were $3.8{\times}10^{-3}{\sim}1.3{\times}10^{-3}$. From the observation of genetic stability, conidial size, DNA content, and nuclear stain, it was suggested that their karyotypes are aneuploid. The hybrids showed $1.2{\sim}1.7$ fold higher lipase activities than parental strains. It was strongly supported by results of this study that nuclear transfer technique is much more efficient in the formation of intergeneric hybrids than protoplast fusion and is very useful for the improvement of strains.

• Journal of Plant Biotechnology
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• v.35 no.3
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• pp.235-243
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• 2008

Here we describe a procedure for Chinese cabbage protoplast culture and effects of various treatments. Chinese cabbage protoplasts were isolated from different parts of young seedlings as using an enzyme mixture, of which yield was maximized in seven hours around after digestion. The highest rate of initial cell division followed by micro-callus formation was obtained in the medium with 1.0 mg/L 2,4-D, 0.5 mg/L NAA, and 1.0 mg/L BA when the cotyledon-derived protoplasts were cultured. Initiation of cell division and micro-callus proliferation significantly depended upon Chinese cabbage genotype under a same culture circumstances. The micro-calli developed from cotyledon tissue of Norang-Bom cultivar successfully grew toward callus colonies on the solidified medium with 0.2 mg/L zeatin and 0.1 mM spermidine. The callus colonies generated de novo shoots at the maximum frequency of 4.3% on the medium with 5.0 mg/L BA and 1.0 mg/L NM. Our results might be helpful for further studies to enhance the regeneration efficiency in Chinese cabbage protoplast culture.

• Korean Journal of Microbiology
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• v.24 no.3
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• pp.243-250
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• 1986

Protoplast formation and regeneration from wild-type and auxotrophic mutants of Candida pseudotropicalis CBS 607 as well as fusion between complementary mutants were carried out. Frequencies of protoplast formation from wild-type and histidine or adenine requiring mutants ranged from 96 to 100% whereas those from methionine or tryptophan auxotrophs were 52 and 72%, respectively. When bovine serum albumin(4mg/ml, BSA) was added to protoplasting buffer for cells of methionine or tryptophan auxotrophs grown in a medium supplemented with myoinositol(0.5mg/ml), 96-99 % of cells were converted to protoplasts. Protoplasts were regenerated at the frequencies ranging from 18 to 20%. However, the addition of BSA to protoplasting buffer and the supplement of myoinositol to a medium of cell growth doubled the regeneration rate except adenine auxotroph in which such an improvement was not observed. It was found that optimal concentrations of polyethylene glycol and $CaCl_2$ are 20% and 100mM while optimal pH and exposure time are 6.0 and 30min. The fusion frequencies between complementary mutants ranged from $1.5{\times}10^{-3}\;to\;8.8{\times}10^{-3}$ and were enhanced by the improvement in the rate of protoplast regeneration. When histidine auxotroph was fused with tryptophan mutant, several fusion products were obtained which were found to be in the state of aneuploid or diploid, judging from DNA content and the presence of a large nucleus in the products.

• Microbiology and Biotechnology Letters
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• v.17 no.1
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• pp.1-7
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• 1989

To enhance the capability of starch fermentation of the transformant TSD-14, the heat treated protoplasts of TSD-14 were fused with the protoplasts of C. tropicalis (lys$^-$) in the presence of 30% (w/ v) PEG and 20 mM CaC1$_2$. Fusants were selected by nutritional complementation on minium medium and the fusion frequency was 4.4$\times$10$^{-5}$. All fusants tested were possessed of complemented traits concerning carbon compound assimilation, and the cell volumes of the fusants were approximately 1.5 times larger than the parental strains. The fusants were genetically very stable, and were able to hydrolyze alpha 1,4-glucosidic linkage as well as alpha 1,6-linkage of starch contrary to one of parents TSD-14, The most promising fusant FSC-14-75 produced 8.7% (v/v) of ethanol from 15% liquefied potato starch medium, but the result was enhanced to 9.3% (v/v) by addition of 0.3% peptone. The corresponding fermentation efficiency was 86.0%.

• Korean Journal of Food Science and Technology
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• v.38 no.5
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• pp.712-716
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• 2006

Saccharomyces cerevisiae and Zygosaccharomyces rouxii were electrofused and fermented in germaniumfortified nutrients to produce high-yield, organic germanium. The conditions for the preparation of protoplasts from both strains and for electrofusion were studied. The protoplasts of both cells formed long pearl chains and the cell membranes were lysed and fused through cellulase and high frequency voltage $(450{\sim}750V/128{\sim}512\;{\mu}sec)$. The fusants with the fastest growth were selected, and then characterized for their carbohydrate usage and tolerance to glucose and salts. The glucose tolerance of the fusants was better than that of S. cerevisiae and similar to that of Z. rouxii. The fusants appeared to have resistance to 12% NaCl. The cell size of the fusants was greater than that of the parental strains. The fusant cells contained more gemlanium than the parental cells did. The electrofusion of S. cerevisiae and Z. rouxii increased the cell capacity and accumulation of germanium in the yeasts. This method was proved to be effective to produce a high quantity of organic germanium.