• Journal of Life Science
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• v.9 no.5
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• pp.531-536
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• 1999

As an attempt to preserve resources in marine biological organisms, we first constructed a cDNA library from the red alga Porphyra yezoensis. The library construction method from P. yezoensis consists of three steps; those include protoplast presparation, RNA isolation, and phage library construction. Protoplast was prepared in order to remove much of the carbohydrate compounds which are characteristics of algal cell walls. Carbohydrate contamination in the purified RNA may inhibit further enzyme reactions, those carbohydrates should be removed. RNA samples prepared from protoplast still seemed to contain residual amount of carbohydrate because mRNA isolation with conventional method failed. We therefore developed a method with Poly ATtract mRNA isolation system. The constructed phage library was tested by analyzing cDNA insert in phage vector from randomly picked ten independent white plagues. All of the phages contained cDNA inserts with sizes ranging 0.5kb and 2.0kb.

• Korean Journal of Microbiology
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• v.27 no.2
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• pp.98-107
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• 1989

Intraspecific fusants, produced by protoplast fusion of auxotrophic mutants from Trichoderma koningii ATCC 26113, were segregated into various strains including parental types, non-parental auxotrophic hybrids, and prototrophic hybrids on complete plate. Interestingly, some of non-parental prototrophic hybrids revealed to have enhanced cellulolytic activity incomparison with other strains of parents or hybrids derived thereafter. It was also evident that prototrophic hybrids of aneuploid could be constructed after the spontaneous segregation of complementing fusants produced through the protoplast fusion.

• Microbiology and Biotechnology Letters
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• v.21 no.2
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• pp.101-106
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• 1993

The optimal conditions for the production and regeneration of L. helveticus protoplasts were examined. The protoplast formation of L. helveticus was most efficient obtained when the cells grown to mid and late logarithmic phase in MRS medium were used. The maximum number of protoplasts was obtained when lysozyme and mutanolysin were used to lysis the cell wall in 20mM HEPES buffer (pH 7.0) containing 1M sucrose. Regeneration was accomplished with a complex medium containing 10% sucrose, 10mM MaCl2, 20mM CaCl2, 5% gelatin and 0.5% bovine serum albumin. The regeneration frequency of the protoplasts was 10-20% after 5 days of incubation at 30C.

• Microbiology and Biotechnology Letters
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• v.15 no.2
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• pp.95-97
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• 1987

The protoplast formation of Streptomyces mitakaensis was monitored with scanning electron microscopy and transmission electron microscopy. The normal cells formed regular mycelium and spore, and their cell wall and cell membrane appeared to be normal, but the cell wall of the lysozyme treated cells (1 mg/$m\ell$) was damaged, which was finally disappeared from cells to become protoplast in 30 to 60 minutes.

• Proceedings of the Microbiological Society of Korea Conference
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• 2000.10a
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• pp.85-91
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• 2000

Aspergillus oryzae is a filamentous fungus classified in the group Aspergillaceae Ascomycetes. It is an important microorganism for industrial production of enzymes and fermented food productions. It secrets large quantities of proteins or enzymes into the culture medium which makes this organism appealing for the production of heterologous proteins. Recently Electric field-mediated transformation method, electroporation, has been applied to fungal transformation. In this study, fungal transformation was carried out by bypassing the protoplast isolation step, decreasing the culturing time and non-protoplast transformation for the increment of transformation efficiency. Transformants were obtained with electroporation in optimal condition 2,500 voltage, 1,540 ohm and 0.50 capacitance. More than 1,000 transform ants were obtained with 6-10 hrs cultured mycelia without enzyme treatment, called non-protoplast transformation.

• The Korean Journal of Mycology
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• v.23 no.4 s.75
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• pp.305-309
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• 1995

To investigate optimal conditions for the protoplast formation and regeneration of Phanerochaete chrysosporium, preparations of three enzymes were used to liberate protoplasts from its 20 hrs-old mycelium on cellophan membrane covered agar media. Novozym 234 alone with 0.6M sucrose was the most effective for isolation of protoplasts from the mycelium with 3hrs incubation time at $39^{\circ}C$ in shaking condition of 120 rpm. The poly-R medium stabilized with 0.6M mannitol was the best for regeneration of the protoplasts.

• Journal of the Korean Society of Tobacco Science
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• v.1 no.2
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• pp.138-149
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• 1979

For the preliminary study on tobacco cell fusion as one of new breeding techniques, the conditions that would be most effective in isolation, fusion, and culture of tobacco protoplasts were examined ; 1. The enzyme solution of 0.5% macerozyme and 2% cellulase( or meicellase) was the most economic and efficient in isolating protoplasts from tobacco leaves. 2. The proper incubation period of tobacco leaves in cell wall digesting solution was 4 hours. 3. As an osmotic stabilizer, sorbitol or mannitol solutions were employed. The concentration of 0.5~0.7 M of either hexitol gave satisfying results as the osmotic stabilizer. 4. The calcium concentration appeared to be an important factor in protoplast fusion. The adhesion of protoplasts was enhanced by enrichment of calcium ion in PEG solution. The highest frequency of protoplast fusion was obtained when tobacco protoplasts were incubated in PEG solution. containing 9mM CaCl2. 5. Cell divisions of the isolated protoplasts were continued and have generated colonies when they were grown on B-5 medium at 28$^{\circ}C$.

• The Korean Journal of Mycology
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• v.12 no.1
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• pp.9-14
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• 1984

Some factors affecting the protoplast release from mycelia of Plurotus ostreatus using commercial lytic enzymes were investigated. The highest yields of the protoplast were obtained from four days old mycelia grown in mushroom complete medium. The solution of 0.8M $MgSO_4$, or KCI showed good results as the osmotic stabilizer for releasing the protoplast. Novozym 234 was the most effective among commercials tested. The concentration of the enzyme and pH of the enzyme solution were optimal at 15mg/ml and $5.5{\sim}6.0$ for the protoplast release, respectively. Mycelial digestion was optimal at about $28^{\circ}C$ and was better in the reciprocal shaking bath (75 oscillations/min) than the stationary culture.

• ALGAE
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• v.21 no.1
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• pp.109-124
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• 2006

Responses to various types of mechanically induced wounding were followed in the giant-celled Caulerpalean species, Derbesia tenuissima, using time-lapse video-microscopy. Gametophyte vesicle cells. Puncture wounding: the gametophyte cell seals the puncture in 5 min. This is followed by cycles of ruptures and sealing, ending with full recovery in 24 hrs. Cut wounding: the protoplast immediately retracts away from the wall and reforms an intact, deflated protoplast that expands to fill the original cell within 21 hrs. Crush wounding (internal). When retained within the cell wall many protoplast fragments condense, round up, and coalesce; the reconstituted protoplast expands until it attains complete recovery, filling the original cell shape in 12 hrs. Crush wounding (external). Protoplast fragments extruded from the crushed cell are more numerous and smaller taking longer to recover. Most fragments become spherical, transforming into small viable cells capable of reproduction in several days. Sporophyte filaments. Crush wounding creates many small fragments that initially condense, coalesce and then expand within the wall to restore a complete filament with normal cytoplasmic streaming within 5 hrs. Reproduction: gametophyte. Our culture isolates produce more females than males (30:1). Gametangia develop one day before discharge that occurs explosively (1/6 sec) at first morning light. The vesicle cell forms successive gametangia every 14 days. Sporophyte. Each sporangium develops on a lateral branch that becomes isolated by the creation of successive basal plugs. After cytoplasmic cleavage and differentiation the stephanokont spores are discharged. The spores settle quickly and germinate forming gametophyte cells.

• Microbiology and Biotechnology Letters
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• v.32 no.2
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• pp.101-109
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• 2004

In order to enhance the productivity of AVM $B_{la}$ produced by Streptomyces avermitilis as a secondary metabolite, we established a basic protocol necessary for protoplast fusion with high-producing strains as a fusion partner, and then obtained various kinds offusants by adopting a massive strain-development procedure (a miniaturized strain screening system). An alternative fusion method using UV and/or NTG mutation of protoplasts was developed to screen genetic recombinants without specific selectable markers. In this method, the mutants obtained by protoplast fusion after UV and/or NTG treatment (95% death rate) of the respective fusion partner (protoplasts of the respective mutants resistant against L-isoleucine antimetabolites such as O-methylthreonine and/or azaleucine) were regarded as DNA-recombined protoplast fusants. Notably it was demonstrated that most of the protoplast recombinants obtained by the UV mutation method were able to biosynthesize higher amount of AVM $B_{la}$ , reaching almost three times higher level (almost equal to the industrial productivity), compared to the average AVM Bla amount of the parallel mother strains.