• KSBB Journal
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• v.5 no.3
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• pp.229-234
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• 1990

The isolation and regeneration of protoplasts are necessary for protoplast fusion of edible mushrooms. In this study, over 5$\times$107 ml-1 protoplasts of Agrocybe cylindracea were isolated using the method described by Yanagi. Enzyme mixture of cellulase Onozuka R10(2%), chitinase (0.2%) and Novozym 234(0.1%) was most effective for the isolation of protoplasts and the yield of protoplasts was 4.85$\times$107 ml-1. 0.6M sucrose was the most effective osmotic stabilizer. The maximum amount of mycelia and yield of protoplasts were obtained from 5~7 days cultured mycelia. In the case of 5~7% days cultured mycelia, the digestion time with lytic enzyme was 4~6 hours. ACM and MCM medium were most effective for the regeneration and reversion of protoplasts, and reversion frequency was 6.9~7.0%. 0.6M sucrose was most stable osmotic stabilizer.

• Korean Journal of Plant Tissue Culture
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• v.28 no.3
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• pp.153-157
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• 2001

Protoplasts were isolated from hypocotyls, cotyledons, and young leaves of Chinese cabbage grown under in vitro environmental condition. An enzyme mixture of 1% Cellulysin and 0.5% Macerozyme in combination with 0.4 M mannitol was most effective condition for protoplast isolation. The highest yield of protoplasts, 7.6$\times$10$^{5}$ protoplast/g of fresh weight, was obtained from the treatment of leaves for 12~16 hours at 27~28$^{\circ}C$ with shaking at 30 rpm. The most suitable medium for an initial cell division was K8p basal medium supplemented with 5 mg/L 2,4-D and 2 mg/L kinetin. Within 7~10 days, protoplasts derived from hypocotyl and cotyledon tissues formed cell colonies. When the cells were grown at the size of 8~10 cells, they were embedded into semi-solid medium containing 0.2% agarose. Calli derived from protoplast culture were transferred to the 100 different types of plant regeneration media, but no completely regenerated plants from inbred lines of Chinese cabbage used for this study wore obtained, though frequent rooting took place in several media tested.

• Microbiology and Biotechnology Letters
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• v.14 no.4
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• pp.305-310
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• 1986

To improve the starch fermentation ability of yeast, hybrids were introduced by protoplast fusion of S. cerevisiae and S. diastaticus. The protoplasts of parental auxotrophic cells were fused in the presence of 10 mM CaCl$_2$and 30% of polyethyleneglycol (M.W 4, 000). The frequencies of fusant formation varied depending upon the strains used and were 3.51$\times$10$^{-4}$ to 5.04$\times$10$^{-4}$ for the regenerated protoplasts. The strains capable of extensive starch hydrolysis produce only 10% to total fusants. The 4 strains were finally selected by the results of starch fermentation and genetic stability test. The DNA content and cell volume of the fusants were greater than those of the parental strains.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.16 no.2
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• pp.68-74
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• 1983

A moderately halophilic bacterium, Listeria denitrificans HB-38, isolated from mud on the seashore in Sooyoung bay, Pusan, showed the requirement of $4\%$ sodium chloride for cell growth in a medium with salts typical of a marine environment, and showed that of $10\%$ in a medium with salts typical of a terrestrial environment. The optimum temperature and pH for growth were $40^{circ}C$ and pH 7.5 in the medium containing $10\%$ sodium chloride and ions typical of a terrestrial environment. Sodium chloride as a protoplast stabilizer gave more stability than sorbitol or sucrose, meanwhile the protoplast did not require higher concentration of stabilizer than that of E. coli protoplast. Succinic dehydrogenase of HB-38 had a halophilic property showing maximal activity in the presence of $9\%$ sodium chloride. The strain HB-38 did not harbor an extrach-romosomal DNA.

• Journal of Plant Biology
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• v.26 no.4
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• pp.191-196
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• 1983

The isolatin and culture of protoplasts from hypocotyl originated callus of Phaseolus vulgaris cv. Damyang were carried out. The maximum protoplast yield of 4.6$\times$105 per gram fresh callus, using the 13-day-old callus, was obtained by digeston for 6 hours in the enzyme solution. After 10 day-culture of the isolated callus protoplsts, plating efficiency was 50%. Thereafter, cell cluster medium, and followed by leading to callus formation on an agar medium after 3 weeks of the liquid culture.

• Journal of Plant Biology
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• v.23 no.2
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• pp.49-54
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• 1980

The optimal conditions for the protoplast isolation from the leaves of pea (Pisum sativum L. cv. Sparkle) and barley (Hordeum vulgare L. cv. Baecdong) were determined in order to achieve a somatic hybridization between two species. It was revealed that the use of 0.5M sorbitol as an osmoticum was appropriate for pea. The yield of intact protoplasts was the highest (40%) when pea leaves were incubated in the enzyme solution for 4 hours. In case of barley, the optimal concentrations of cellulase, pectinase and mannitol as the enzyme solution were 2%, 1% and 0.35M, respectively. And the yield of barley protoplasts was the highest(87%) when leaves were incubated in this enzyme solution for 3.5 hours. A fusion of protoplasts from pea and barley was induced by PEG treatment enriched with calcium salts within 60 minutes.

• Korean Journal of Microbiology
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• v.21 no.4
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• pp.213-220
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• 1983

Conditions for isolation of protoplasts from conidiospores of Trichoderma koningii ATCC 26113 were tested. Maximum production of conidial protoplasts was obtained by preincubation of conidiospores on liquid minimal medium for 8 1/2 hrs. and by reaction with cell wall lytic enzyme for 3 hrs. Among effective cell wall lytic enzymes (Driselase, p-Glucuronidase, Novozyme and Zymolyase), Driselase was the most effective one on the production of conidial protoplasts. The production of conidial protoplasts was also enhanced by addition of 2-Deoxy-D-Glucose $(25{\mu}g/ml)$ into liquid minimal medium. Over 70% of the initial swollen conidia, preincubated in liquid minimal medium supplemented with 2-Deoxy-D-Glucose $(25{\mu}g/ml)$, were converted to protoplasts by incubation with 2% (w/v) commercial lytic enzyme Driselase at $28^{\circ}C$ for 3 hrs. The reversion frequency of the conidial protoplasts was about 30 times (25-50%) higher than that of mycelial protoplasts (0.6-1.3%).

• Current Research on Agriculture and Life Sciences
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• v.5
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• pp.19-26
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• 1987

The experiments were conducted to identify several factors affecting isolation and culture of mesophyll protoplasts in Solanum melongena var. fructualbo. Higher viable plotoplasts were obtained, when isolated in 1.5% macerozyme, 0.2% macerozyme, 0.6M mannitol, 0.01M MES, 0.2% BSA containing solution adjusted to pH 6.3 for 4 hours. One hour plasmolysis of the material before digestion of leaf tissue was effective for protoplast yield and viability. The method of washing and purification of crude protoplasts, ESS process with 0.7M mannitol and 0.6M sucrose solution. was the best way to get purified protoplasts with viability. As isolated protoplasts were cultured in 8P-KM medium at a density of $2.5{\times}10^4/ml$, the cells were enlarged after 3 to 5 days from culture, subsequently the cells were divided and resulted in colonies.

• Journal of Korean Society of Forest Science
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• v.71 no.1
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• pp.45-49
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• 1985

Protoplast-source meterial and enzyme strength had a significant influence on protoplast yield from hybrid poplar, Populus alba ${\times}$ P. grandidentata. The yield of protoplasts from in vitro culture of 1 month-old plantlets was more than that from greenhouse grown 4 month-old stock plant. In vitro cultured plantlets regulary produced more viable protoplasts with E-I enzyme solution (0.5% cellulase and 0.1% macerase) than those with E-II enzyme solution (1.0% cellulase and 0.2% macerase) after overnight incubation. The mean yield of protoplasts from in vitro cultured plantlets was $4{\times}10^6$ with E-I enzyme solution. Cell division was observed in these protoplast cultures after 7-10 days. Protoplast-derived hybrid poplar cells survived over 3 weeks in culture and some continuous cell divisions were evident. Other aspects associated with protoplasts from in vitro cultured plantlet are also discussed.

• Journal of Mushroom
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• v.2 no.1
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• pp.33-37
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• 2004

To develop neohaplonts for Ganoderma breeding, protoplasts were isolated from dikaryotic mycelium and regenerated. Selection rate of neohaplonts varied between ASI7074, ASI7091, ASI7094, ASI7100 and ASI7115, showing 5.24% on the average. Auxotropic mutants from Ganoderma monokarions were recovered by UV irradiation on protoplasts. Protoplast survival rates were 1.9% ASI 7074, 0.17% ASI 7091, and zero percent ASI 7100 using 300 second irradiation. Four auxotrophic strains were recovered from 1,536 colonies screened that will be further utilized for protoplast fusion and transformation.