• 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.

• Microbiology and Biotechnology Letters
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• v.13 no.2
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• pp.137-144
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• 1985

Studies were conducted on the conditions for preparation of yeast protoplasts utilizing Hansenula anomala var. anomala FRI YO-32 as well as Saccharomyces cerevisiae KFCC 32356 and a lytic enzyme from the snail Helix pomatia. The cell wails of the strain FRI YO-32 and S cerevisiae were found to be resistant to activity of the snail lytic enzyme if they were not treated with thiol compounds. Dithiothreitol was found to be more effective than 2-mercaptoethanol, but the latter was considered to be practical. As factors influencing the formation of yeast protoplast, it was considered to be concentration and incubation time of 2-mercaptoethanol or the lytic enzyme, growth stages in yeast cultivation, initial number of yeast cells, and concentration of osmotic stabilizer (KCI). Optimum conditions for the preparation of yeast protoplasts were determined.

• Archives of Pharmacal Research
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• v.20 no.3
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• pp.206-211
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• 1997

The optimal conditions for the production and regeneration of the protoplasts from Lentinula edodes were studied. Protoplast formation from the mycelia of L. edodes which were cultured in liquid medium showed a significantly high yield compared with that of the mycelia which were cultured on cellophane covered agar media. A mixture of Novozyme 234 (15 mg/ml) and Cellulase Onozuka R10 (10 mg/ml) in 0.6 M mannitol (pH 4) was optimal lytic enzyme for the protoplast release. The optimal incubation time and mycelia age were 3.5-4 hours at $30^{\circ}C$ and 6-8 days, respectively. Regeneration frequency was 0.18% plated onto a medium containing 0.6 M sucrose, and 0.08% plated onto a medium containing mannitol. But hardly any regeneration was observed in the media containing NaCl, KCl, or $MgSO_{4}$ More than 90% of the protoplasts contained nuclei and the nucleus number per protoplast was 1.1. The DNA content per nucleus was 5.1 pg. The diameter of the protoplast was $3-5{\mu}m$ and it had a well defined cell structure.

• Korean Journal of Agricultural Science
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• v.13 no.2
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• pp.185-192
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• 1986

Some factors affecting the protoplast release from mycelia of Ganoderma lucidum and regeneration of the protoplast were investigated and the results obtained are summarized as follows; Novozym 234 as a lytic enzyme was the most effective for the protoplast release from mycelia of Ganoderma lucidu m and its optimal concentration was 10mg per ml of osmotic stabilizer. The highest number of protoplasts were released after 3 hours incubation in the reciprocal shaking bath at 120 oscillations a minute. Among six osmotic stabilizers tested, 0.6M sucrose showed the best result. SCM medium showed good mycelial growth and high yields of protoplasts. The protoplasts released from the mycelium of G. lucidum were regenerated at 0.20 to 0.27 percent on MCM, MMM and SCM. Of the cultures obtained from protoplasts regenerated, 13 to 29 percent were monokaryon.

• Preventive Nutrition and Food Science
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• v.3 no.3
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• pp.211-215
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• 1998

Preparative isolation of lipid granules from Fhodotorula glutinis, which has been studied for long time to produce edible lipids, was carried out by flotation method in Ficoll-Linear density gradient. When the isolated lipid granules were suspended in a series of solutions containing varying concentration of osmotic stabilizer (sorbitoal and mannitol) ranging from 0.8M to 0M, the lipid granules appeared to be disrupted at a concentration between 0.8M and 0.7, and again at a concentration below 0.1M, suggesting that lipid granules have a membraneous structure and that at least two types of lipid granules are present. Compositional analysis of lipids from lipid granules revealed that lipids are composed mainly of neutral lipids (87.8% of total lipids), predominantly as triacylglycerols (71.89%). Marked differences were observed inphospholipids between lipids of lipid granules and those of whole cells . The major components of phospholipids in lipid granules and inwhole cells are phosphatidylcholine(38.6%) and phosphatidylserine(42.8%), respectively. In addition, significant differences were also observed in the fatty acid composition of phospholipids. As phospholipids are important structural components of membranes, these differences lead to the suggesting that the membrane of lipid granules may be distinct functionally and structurally from other membranes of yeast cells. The major fatty acid components of neutral lipidss of whole cells and lipid granules are palmitic , oleic and linoleic acid. However , degreeof fatty acid unsaturation of neutal lipids of lipid granules was much lower than that of neutral lipids of whole cells.

• Korean Journal of Plant Tissue Culture
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• v.22 no.1
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• pp.7-11
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• 1995

Protoplasts were isolated from hypocotyl tissues of 5-day-old Brassica oleracea var capitata Green Challenger seedlings. Several media were used for protoplast culture and shoot regeneration. The shoot-regeneration rapacity of protoplast derived callus depended on the initial culture medium. Protoplasts were cultured in liquid medium (B5 medium supplemented with CaCl2, 2H2O 600mg/L, g1ucose 20g/L, D-mannito1 70g/L, NAA lmg/L, BA lmg/L, 2.4-D 0.25 mg/L)at 27$^{\circ}C$ under the dark After 5 to 10 days, cultlues were diluted with medium with a reduced osmotic stabilizer and then transferred to illuminated conditions. The culture medium was changed with the fresh medium at 7- to 10-day-intervals until the formation of microcallus. Hypocotyl protoplast-derived callus proliferated when transferred to MS medium supplemented with NAA lmg/L, BA 1mg/L and GA$_3$ 0.02mg/L. Upon transfer to MS basal medium without growth regulators, roots were produced. In an attempt to increase the regeneration frequency, 10g/L polyvinylpyrrolidone was added to the regeneration medium, but the shoot regeneration was mot improved. The regenerated whole plants were acclimated in a sterized soilless mixture(vermiculite 2;perlite 2;peat moss1) in a culture room.

• Korean Journal of Microbiology
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• v.28 no.1
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• pp.47-54
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• 1990

In order to investigate physiological characteristics of fusants by interspecific protoplast fusion of the genus Cellulomonas, protoplasts of Cellulomonas flavigena NCIB 12901 and Cellulomonas bibula NCIB 8142 were fused and cell wall regenerated. To give gene maker, C. bibula was treated with 500 ug/ml NTG for 1 hr and arginine requiring auxotrophic mutants were isolated. Protoplasts of the genus Cellulomonas were obtained by treatment with $600{\mu}{\textrm{g}}$/ml lysozyme, and 0.5M sorbitol was optimal for osmotic stabilizer on protoplast fromation. Protoplast fusion was enhanced by 40% PEG)M.W.6,000) containing 25 mM $CaCl_{2}$ at $30^{\circ}C$ for 30 min and fusion frequency between C. bibula and C. flavigena was $5\times 10^{-4}$. Processes of protoplast formation, cell wall regeneration and protoplast fusion were obsdrved by scanning electron microscope. By comparing enzyme activities of cellulase, exocellobiohydrolase, .betha.-glucosidase of the parent strains of Cellulomonas with those of thier mutants and fusants, fusants with increased enzyme activity were obtained. By the studies on nutritional requirement, antibiotic resistance, cellulolytic enzyme activities, type of peptidoglycan and motility of two mutants and fusants, fusants were proved to be recombinant of both mutant strains.

• The Korean Journal of Mycology
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• v.27 no.6 s.93
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• pp.399-405
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• 1999

Intergeneric hybrids between Saccharomyccopsis fiburigera KCTC 7393 and Saccharomyces cerevisiae KCTC 7049 (tyr-, ura-) were obtained by nuclear transfer technique. Nuclei isolated from the wild type S. fiburigera strain were transfered into auxotrophic S. cerevisiae mutants and new strains showing an increased starch degrading capability were selected. Maximum production of protoplasts was obtained from the treatment with 0.1 % Novozym 234 at $30^{\circ}C$ for 90 min, and most effective osmotic stabilizer for the isolation of protoplasts was 0.6 M KCl at pH 5.8. The frequency of protoplast regeneration was 14.64% under the conditions. Genectic stability, conidial size, DNA content, and nuclear stain suggested that the fusants were aneuploidy. The specific activity of ${\alpha}-amylase$ was observed to increase about $1.2{\sim}1.9$ folds.

• The Korean Journal of Mycology
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• v.16 no.4
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• pp.214-219
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• 1988

Factors affecting protoplast formation and reversion were investigated in Pleurotus sapidus kalchbr. For release of protoplast, enzyme mixture of Novozyme 234, ${\beta}-D-glucanase$ and ${\beta}-glucuronidase$ was most effective, when mycelium of 0.6 M sucrose solution as osmotic stabilizer without addition of buffer solution. The yield of protoplast was highest with mycelium cultured for 4 days on mushroom complete agar medium at ${30}^{\circ}C$. Protoplasts of Pleurotus sapidus were reverted to normal hyphal growth with maximum reversion frequency of 2% on Mushroom complete agar medium stabilized with 0.6 M sucrose solution and covered by 0.75% agar layer.

• Microbiology and Biotechnology Letters
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• v.20 no.1
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• pp.6-13
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• 1992

- Protoplast fusion between lincomycin resistant Lactobacillus casei KCTC 1121 and rifarnpicin resistant Lactobacillus delbrueckii JK-414 was attempted to obtain the improved strains. Protoplasts of L. casei and L. delbrueckii were produced by mutanolysin digestion at $42^{\circ}C$ for 15 min. L. casei cells were converted to protoplasts by treating with 5 $\mu g$ / m l of mutanolysin in 20 mM HEPES buffer (pH 7.0) containing 0.75 M sucrose at the middle logarithmic growth phase. In case of L. delbrueckii 1.0 M sucrose was used osmotic stabilizer. Regeneration of protoplast in both strains was efficiently accomplished on the regeneration medium containing 10% sucrose, 6 mM $MgC1_2, 6 mM CaC1_2$, and 2.5% gelatin. Protoplast fusion between L. casei and L. delbrueckii was carried out in the presence of 40% of PEG 4,000. The frequency of protoplast fusion was found to be about $3.2\times 10^4$. Acid production of L. casei was better than that of L. delbrueckii. Among fusants, F23 and F35 exhibited excellent lactic acid production. F23 and F24 exhibited the improved proteolysis compared to that of the parent strains and they had twice as much as DNA content of the parents.