• The Korean Journal of Mycology
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• v.22 no.2
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• pp.130-137
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• 1994

This experiment was carried out to investigate proper conditions for protoplast isolation and regeneration from mycelia of Lyophyllum decastes. Novozym 234(10 mg/ml) with 0.6 M $MgSO_4$ in phosphate buffer(pH 4.0) was proper for protoplast isolation. The optimal reaction time of the mycelium with the lytic enzyme was four hours in shaking condition at 120 strokes per min. When the mycelium of L. decastes was cultured at $24^{\circ}C$ for 5 days, the formation of protoplasts was effective. The liquid medium was more effective for protoplast isolation than the solid medium. In the liquid medium, high yields of protoplasts were obtained from 0.6 M $MgSO_4$ osmotic stabilizer. Protoplasts of L. decastes were regenerated to normal hyphal growth and the regeneration frequency of the protoplasts in the complete agar medium containing Triton X-100(0.0025%) was $5.94{\sim}8.32%$. The regeneration medium stabilized with 0.6 M sucrose was the best for regeneration of the protoplasts. In contrast to protoplast formation, regeneration was inhibited by the inorganic salts used as osmotic stabilizer.

• The Korean Journal of Mycology
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• v.17 no.3
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• pp.137-144
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• 1989

Effects of incompatibility existing between intra-and interspecies in Pleurotus spp. on protoplast fusion, clamp formation of their fusants and fruitbody production were investigated. Protoplast fusion between intra-and interspecies of the fungus was achieved by Poly ethylene glycol treatment. The fusion frequency between intraspecies was a little higher than that of interspecies. Fusion frequency between interspecies was not correlated with their similarities based on isozyme patterns. In case of protoplast fusion between intra-and interspecies, the fusants from the compatible isolates produced normal fruit bodies, while those from the incompatible isolates did not produce clamp connections and fruit bodies except those of a few isolates presumed mutants.

• The Korean Journal of Mycology
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• v.16 no.2
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• pp.70-78
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• 1988

To obtain basic information for the genetic analysis and breeding of Flammulina velutipes, some factors affecting the release of protoplasts from the fungus were studied. Potato Dextrose peptone Agar medium was suitable for the growth of the mycelium and the protoplast formation of F. velutipes. The culture age for the high yields of protoplast was 5 days on PDPA. Few protoplasts were formed from the mycelium cultured on Mushroom minimum Media. The highest yield of protoplasts was obtained in enzyme solution containing Novozyme 234 plus cellulase CP at 10 mg $ml^{-1}$ concentration, while a half amount of protoplasts was obtained in enzyme solution containing Novozyme 234 only. The optimal reaction time of the mycelium in the Iytic enzyme mixtures was 3 hours. The best osmotic stabilizer for the protoplast formation of the mycelium was 0.6M sucrose without buffer at pH 6.2.

• Korean Journal of Agricultural Science
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• v.12 no.1
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• pp.22-30
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• 1985

Protoplasts of Pisum sativum L. were isolated and cultured from leaf mesophyll tissue. The successful yield of protoplast was obtained in an enzyme solution of 2% 'Onozuka R-10' and 2 % 'Macerozyme R-10' contained 6mM $CaCl_2{\cdot}2H_2O$ within 4 hours. They were divided in B5 culture medium supplemented with 2mg/l kinetin, 1mg/l 2, 4-D and 0.2% Difcobacto agar. Divisions of the protoplasts were continued and led to colony formation for 1 months. The colony from protoplasts of pea mesophyll tissue was formed to callus after subculture in a medium contained macronutrients and amino acids of BII medium and micronutrients and vitamins of B5 medium, and also supplemented with 2mg/l kinetin 2mg/l NAA.

• Journal of Korean Society of Forest Science
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• v.71 no.1
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• pp.9-13
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• 1985

Protoplasts were isolated from cotyledons of germinating Pinus densiflora S. et Z. seeds. The seeds were germinated for nine days, and excised embryonic cotyledons about 3-4mm long were incubated with Cellulase Onozuka R-10 and Macerozyme. After 8 hours of incubation, large number of viable protoplasts were isolated. Isolated protoplasts were cultured in a medium containing basal salts of $B_5$ medium, vitamines, amino acids, organic acid, sugars, and growth hormones. The first evidence of protoplast budding was observed after twelve hours in culture, and it suggested that high potential of the embryonic cotyledons for rapid cell division affected the early budding, rather than effect of culture medium was shown in twelve hours. The three- to four-cell stage was reached after three to four days of culture. Most cell divisions were achieved by additional buddings rather than equal binary cell division. No further cell division was observed beyond the four-cell stage. Protoplasts isolated from fully expanded cotyledons (germinated for 17 to 24 days) seldom initiated or failed to initiate cell division.

• Current Research on Agriculture and Life Sciences
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• v.2
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• pp.15-22
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• 1984

This study was conducted to identify the enzyme treatment time, enzyme concentration and plant growth condition for isolation of potato mesopyll, it was also performed to determine the adquate sucrose molarity on purification of protoplasts and to investigate the incubation time, PEG concentration and DMSO effect for potato-petunia protoplast fusion. The results were summarized as follows: The optimal time of incubation in enzyme solution was 3 - 4 hours and high humidity and low light intensity made plants more effective to protoplast releasing enzymes. Our experimental results showed that the pectolyase Y-23 was an ideal agent for isolation from mesophyll cultured in vitro compared with macerozyme. The enzyme solution with 0.5 % macerozyme and 2 % cellulase was very effective and the purity of healthy protoplast was better in 0.4 and 0.5 M sucrose than in others. It was revealed that the rate of potato-petunia fusion according to the incubation time with PEG was effective at 30 min incubation and percentage of protoplast aggregation was increased by high molecular weight and concentration of PEG. Percentage of potato-petunia protoplast heteroplasmic aggregation was increased by 4 to 16 % in PEG 6000 compared with PEG 4000 and PEG 1500. Addition of 5 to 10 % DMSO to the PEG solution increased to the heteroplasmic aggregation of potato-petunia from 2 to 4 %.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.42 no.5
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• pp.615-625
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• 1997

An efficient protocol for plant regeneration from protoplasts of the indica rice variety IR43 has been developed. The procedure involved plating of embryogenic suspension-derived protoplasts on the surface of a filter membrane overlaying agarose-embedded feeder cells. Lolium multiflorum cell suspensions were preferable to these of Oryza ridleyi as feeder cells and Lolium suspensions supported colony formation from up to 0.68％ of the protoplasts, depending on the age of cell suspensions. Plant regeneration frequency was significantly improved by using maltose alone or in a 1:1(w/w) combination with sucrose as carbohydrate source and a simple dehydration treatment using a high concentration of agarose in the regeneration medium. Medium containing maltose or maltose mixed with sucrose increased the plant regeneration frequency compared with medium containing sucrose alone. The plant regeneration frequency was increased to 30.7 to 70.7％ following dehydration treatment, while the non-treated controls showed a regeneration frequency of 3.1 to 30.6％. Protoplast-derived plants were transferred to the glasshouse, flowered with morphologically normal.

• Korean Journal of Microbiology
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• v.25 no.3
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• pp.198-204
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• 1987

Biparental clones and recombinant clones were obtained by spheroplast fusion of Pseudomonas putida KU218R-3 and P.putida KU428. Formation of the fusion product was the most effective when the Pseudomonas spheroplast mixture were treated with 40% plyethyleneglycol(PEG) 6000 for 10min at room temperature, The fusants which selected by indirect method were obtained at an average frequency of 10.8%. Most of the fusants were biparental clones (10.4%) and the recombinant clones were produced in low yield (0.42%). Fusants, at the frequency of 4% were obtained without PEG 6000, which shows that fusion is not strictly dependant on PEG. The stability of fusants were examined. Most of the biparental clones were segregated to parental form amd late recombinants were formed on further propagation of biparental clone but the recombinant clones were nery stable.

• Korean Journal of Plant Tissue Culture
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• v.25 no.1
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• pp.7-11
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• 1998

Protoplasts were isolated from the parenchyma supension cultured cells of Streptanthus tortus using hydrolytic enzymes, 0.03% cellulase + 0.02% pectinase. Phloem cells and companion protoplasts were isolated from differentiated suspension cultured cells using hydrolytic enzymes, 0.2% macerase + 0.03% cellulase + 0.02% pectinase + 0.025% rohamet PC. Isolated parenchyma -and companion- protoplasts transported glucose into the cells, but not transported sucrose at all. On the other hand, isolated phloem cells transported sucrose into the cells actively, but not transported glucose. These results show for the first time that loading of sucrose into the phloem cells without nucleus was possible without contributing of companion cells and companion cells had not the ability to transport sucrose directly because of lack of sucrose carriers in the membrane. The sucrose transport into the isolated phloem cells depend on metabolic energy.

• 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.