• Proceedings of the Plant Resources Society of Korea Conference
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• 2003.10b
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• pp.20-20
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• 2003

Isolation and culture of leaf protoplasts from two chilli cultivars (Capsicum annuum var. accumnatum and Bird chilli) were developed to enhance selection process in the somatic hybridization programmes. In order to isolate the protoplasts from leaves of these two chilli cultivars different incubation periods (3, 5 and 10 hours) were tested with combinations of enzyme mixtures containing cellulase and macerozyme. Leaves were incubated on three enzyme mixtures (2% cellulase +0.4% macerozyme, 1% cellulase +0.2% macerozyme and 0.5% cellulase +0.1% macerozyme in 13% mannitol) at 251oC in the dark. Three hours of incubation using 2% cellulase and 0.4% macerozyme was the best for the protoplast isolation of both chilli cultivars tested. The yield was 5${\times}$108protoplasts/m1/g leaf tissue in both chilli varieties. It was found that in the mixed nurse method using Nagata and Takebe (NT) medium supplemented with 1.0mg/12,4-D, NAA and BAP with 0.5M mannitol and 1.2% Sea Plaque agarose is the best medium for protoplast culture. Protoplasts of Capsicum annum var. accumnatum were alive for 14 days forming cell walls and initiating cell division.

• Journal of Microbiology and Biotechnology
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• v.2 no.1
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• pp.46-49
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• 1992

The availability of Phaffia rhodozyma as an astaxanthin sources in the aquaculture industry is limited because of the low carotenoid content of natural isolate. In this study, we have used the protoplast fusion technique to construct cell hybrids with an increased content of astaxanthin from P. rhodozyma. Cell hybrids (F307 and F406) obtained were very stable and produced considerably more astaxanthin (> 1 mg/g yeast) than the wild parent. Karyogamy was confirmed by the isolation of recombinants after mitotic segregation of parental auxotrophic genetic markers, the increased amount of chromosomal DNA/cell and the presence of single nucleus/cell.

• Korean Journal of Microbiology
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• v.28 no.3
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• pp.274-277
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• 1990

The hyphal tip phenoloxidases of Coprinus congregatus were localized by the protoplast-concanavalin A method. Protoplast were generated from cultures grown on a solid medium which was solidified with a new gelling agent, Pluronid Polyol F127, instead of agar. the enzymes were associated with the cell membrane which might work as a transducer in the light recepter complex.

• Journal of Plant Biotechnology
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• v.3 no.1
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• pp.27-31
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• 2001

New types of cytoplasmic male sterility in Brassica species would be very useful for the production of F$_1$, hybrid seeds. Leaves and stems of rapid cycling stock of B.juncea (CrGC4-3) containing Anand CMS were used as experimental materials for plant regeneration from protoplast culture. Very high plant regeneration rate (85%) was found in the Kao & Michayluk medium supplemented with 2 mg/L zeatin, 0.5 mg/L BAP, and 1 mg/L NAA when only leaf, not stem, segments were cultured. Protoplasts were isolated from leaves using mixtures of enzymes (1% Cellulycin, 0.5% Macerozyme) in 0.4 M mannitol and 50 mM $CaCl_2$.$2H_2$O. Mcrocalli induced from protoplasts were transferred to the shoot regeneration medium containing 2 mg/L BAP, 2 mg/L zeatin, and 0.5 mg/L NAA. After 60 days of initial protoplast culture, regenerated plantlets were obtained, acclimatized, transplanted into the pots, and grown up to the flowering stage.

• Journal of the Korean Society of Tobacco Science
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• v.20 no.1
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• pp.40-49
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• 1998

Protoplasts were isolated from mesophyll of tobacco(Nicotiana glauca) transformed with kanamycin-resistant gene (NPT II gene) and potato hairy root callus containing Ri plasmid of Agrobacterium rhiEogenes, and protoplasm fusion was made between the isolated protoplasts. The transgenic tobacco leaf tissue could grow on the media containing high concentrations of kanamycin, but not on the phytohormone-free media. On the other hand, the potato hairy root calli could be cultured on the phytohormone-free media but not on media containing more than 40 ㎍/ml kanamycin. In these conditions, the viability of both protoplasts were above 90%, These selection markers were used for the selection of protoplasts fused between the two, i.e. protoplast fusion was detected using selection media containing 100㎍/ml kanamycin and with no phytohormone. The mixture of 1.0% cellulase, 0.3% macerozyme, and 0.7M mannitol was best for the maximum protoplast production for tobacco, and that of 2.0% cellulase, 2.0% macerozyme, 1.0% dricelase, and 0.5M mannitol for potato. Both tobacco mesophyll and potato callus protoplasts were fused by using PEG solution on the selectable medium. Cell walls were regenerated after 5 days in this medium, and colonies were alive until 4 weeks after cultural, but died after 6 weeks.

• Journal of Korean Society of Forest Science
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• v.71 no.1
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• pp.66-73
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• 1985

Factors affecting isolation and fusion of protoplasts of three Quercus species were investigated and procedures for isolation, purification and fusion of protoplasts of the three species were also established. Unhardened leaves and rapidly growing callus cultures were good source of viable protoplasts. The optimum composition of enzyme mixture for rapid isolation of protoplasts from leaf mesophyll tissues and calli was Cellulase Onozuka R-10 (20g/l, Macerozyme R-10(10g/l), Pectinase(250 units/l, $CaCl_2$, $2H_2O$(14mM), $MgSO_4{\cdot}7H_2O$(1.8mM), $KNO_3$(1.0mM), $H_3BO_3$(1.0mM), $KH_2PO_4$(0.2mM), KI($1.0{\mu}M$), 1,4-dithiothreitol (0.1mM) and mannitol (0.6M). Optimum density of protoplasts for maximum fusion was $2{\times}10^5/ml$ which was the highest protoplast density given in this study. Optimum concentration and duration of PEG 1450 treatment for inducing fusion appeared to be 29%(W/V) final PEG 1450 concentration and 5-10 minutes, respectively.

• Microbiology and Biotechnology Letters
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• v.14 no.5
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• pp.359-363
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• 1986

S. diastaticus hydrolysised $\alpha$-1.4 linkage of the starch and was known fermenting yeast strain, but poorly hydrolized $\alpha$-1.6 linkage of the starch. To improve the starch fermentation ability of yeast, we tried that protoplast fusion between S. diastaticus and C. tropicalis and finally two starins of fusant (FPDC42, FPDC43) were obtained. C. tropicalis well hydrolysis both $\alpha$-1.4 and $\alpha$-1.6 linkanges in the starch. The protoplast of parental auxotrophic cells were fused in the presence of 10mM CaCl$_2$ and 35% of polyethylene glycol (M. W. 4,000). The fusion frequency was 10$^{-5}$ to 10$^{-6}$. Properties of the fusants(genetic stability, assimilation of carbon sources, random spore formation, copper resistance, NaCl tolerance, DNA content, cell size and growth rate) were investigated.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.42 no.3
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• pp.306-316
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• 1997

Embryogenic calli were induced from mature seed scutella of anther culture-derived rice variety Zhonghua 8. Cell suspension cultures were initiated from friable embryogenic calli and utilized as source material for protoplast isolation. Generally, the older and finer cell suspensions gave higher protoplast yields than younger suspension cultures. Protoplasts exhibited sustained cell division and formed microcalli when cultured in KPR medium supplemented with 0.5 mg $l^{-1}$ 2,4-D, 1.0 mg $l^{-1}$ NAA and 0.5 mg $l^{-1}$ zeatin using the agarose embedding procedure without feeder cells. Protoplast plating efficiencies ranged from 0.20 to 0.54％. Microcalli were transferred to MS medium supplemented with 2.0 mg $l^{-1}$ kinetin and 0.5mg $l^{-1}$ NAA for plant regeneration. The regeneration frequencies were 2 to 12％, depending on the cell suspension lines of Zhonghua 8. The plants were transferred to the glasshouse and were fertile.

• Journal of Life Science
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• v.6 no.2
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• pp.94-103
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• 1996

To understand effect of inoculum size, cell density, sucrose concentration and concentrations of MS basal on suspension culture and protoplast isolation of wild viola(Viola patrinii DC.) callus from petiole segments this experiment was conducted. In the lot of 30 mesh inoculum size, two observations were; One was that a considerable increase in the fresh and dry weight of callus was determined. Another was that the callus mass was relatively compact compared with others. A recommendable cell density was 0.4g for 20ml culture medium and the higher sucrose concentration, the higher fresh and dry weight were obtained. The dilution of MS basal salt was differently affected on fresh and dry weight; the highest fresh weight was found in 1x MS salt, while the higest dry weight was in 1/3x dilution.The addition of casein hydrolysate(3g/L) was more effective to increase of both fresh and dry weight. THe contents of protein was great in the inoculum lots with larger inoculum sizeand higher concentration of MS basal salts contenting 3g/L of casein hydrolysate and higher sucrose compared with others. The greatest protoplasts were isolated from the lot of 10 mesh size treated with 1%pectinase SE-150 and 2% cellulase YC. In general, for optimal protoplast isolation the followingconditions were recommended; 1) Use of smaller cell size cultured for 2-5 weeks, and 2) more than 5 hours incubation using the combined mixture of the enzymes with proper concentrations.

• Journal of Aquaculture
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• v.12 no.1
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• pp.7-14
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• 1999

Protoplasts were isolated from the vegetative thalli of Meristotheca papulosa using several commercial and crude enzymes. The suitable enzyme combination for the protoplast isolation was 4% abalone acetone powder, 4% Macerozyme R-10 and 4% Hemicellulase in the filtered seawater buffered with 50mM MES (pH 6.0) containing 0.6M mannitol and 0.5% potassium dextran sulfate. Yield of protoplast was $107.6{\times}10^4$ cells per gram of fresh thallus. Protoplasts were whitish ovoid in shape and ranged between $7{\mu} m$~ $24{\mu} m$ in diameter. Division of protoplasts was first observed 9 days after culture in $ASP_{12}$ medium, and the germination occurred within 25 days. The addition of Guillard's antibiotics in culture media was harmful to the regeneration of protoplasts.