• Title/Summary/Keyword: protoplasts

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Effect of the Electric Field on the Plant Protoplasts During Cell Fusion (세포융합시 전계하에서 식물세포가 받는 영향에 관한연구)

  • Lee, Sang-Hoon;Lee, Yon-Min;Cha, Hyeon-Cheol
    • Journal of Biomedical Engineering Research
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    • v.17 no.2
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    • pp.173-178
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    • 1996
  • The objective of this paper is to investigate the effect of AC field on the protoplast of plant cells. The results of investigation will be the basis for the development of etectric cell fusion device. For the experiment, we made the electrode and AC and DC pulse generator and observed the behavior of the protoplasts through the inverted microscope which is connected to the monitor and video recorder by the CCD camera. As a result, the numbers of rotating, moving and destructed protoplasts and viability of the protoplasts have close relation to the amplitude of AC field, while the rotation rate is closely related to the frequency of AC pulse.

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Plant Regeneration from Mesophyll Protoplasts Culture of Solanum sisymbriifolium

  • Kim Hag-Hyun;Shin Un-Dong
    • Journal of Plant Biotechnology
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    • v.7 no.3
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    • pp.169-174
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    • 2005
  • The optimal culture conditions were studied for plant regeneration from mesophyll protoplasts of Solanum sisymbriifolium. Axenic seedlings of S. sisymbriifolium were used as a explant for protoplast culture. Many viable protoplasts were isolated by incubating leaf slices in an enzyme solution containing 0.25% Meicerase and 0.05% Macerozyme for 16 hr at $25^{\circ}C$ without shaking. Protoplast density of $5.0{\times}10^4\;ml^{-1}$ in Kao medium containing 5.0 mg/L NAA, 1.0 mg/L 2,4-D and 1.0 mg/L BA was optimal for colony formation. Most colonies were formed when protoplasts were cultured at $25^{\circ}C$ after initial culture at $30^{\circ}C$ for one week. On the MS agar medium with 1.0 mg/L zeatin, 38.4% of protoplast-derived calli differentiated shoots. These shoots rooted on 1/2MS medium with 5.0 g/L sucrose and 2.5 g/L gellan gum, and developed into whole plants.

The Isolation and Fusion of Pea and Barley Mesophyll Protoplasts (완두와 보리의 엽내세포 원형질체 분이 및 융합)

  • 이광웅
    • 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.

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Studies on Protoplast Isolation and Regeneration of Lyophyllum ulmarium (느티만가닥 버섯의 원형질체 분리와 재생에 관한 연구)

  • 최혜진;김병각;현진원
    • Journal of Life Science
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    • v.13 no.2
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    • pp.143-149
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    • 2003
  • This experiment was undertaken to investigate proper conditions for protoplast isolation and regeneration from the mycelia of Lyophyllum ulmnrium. Protoplast isolation and regeneration are influenced by a variety of factors such as enzyme, osmotic stabilizer, reaction time and age of mycelia. A combination of Novozyme 234 (10mg/ml) and cellulase Onozuka R-10 (10 mg/ml) with 0.6 M $MgSO_4$ was most effective for isolation of the protoplasts. The optimum reaction time of the mycelia with the lytic enzymes was 3.5~4 hours at $28^{\circ}C$ in shaking condition at 120 strokes per min. High yield of the protoplasts were obtained from its 4~5 days old mycelia on complete agar media. Its protoplasts were regenerated to normal hyphae. Regeneration media with 0.6 M sucrose were proper for regeneration of the protoplasts. Their regeneration frequency on complete agar media was 2.3~2.7%.

Optimum Conditions of pH and Ca2+ Concentration for Electrofusion of Tobacco Protoplasts (담배 워형질체의 전기융합을 위하 pH 및 Ca2+ 농도 최적조건 설정)

  • 오인숙;소상섭;김환규
    • KSBB Journal
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    • v.13 no.4
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    • pp.399-403
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    • 1998
  • This study was carried out to optimize the concentration of Ca2+ and pH of fusion medium which affected electrofusion frequency of protoplasts isolated from Nicotiana tabacum L. (cv. BY4) mesophyll cells and callus. The protoplasts were electrofused in the fusion media containing two different Ca2+ concentrations and three different pH regions. Fusion frequency was lower in the fusion medium containing only 13% mannitol as osmotic stabilizer. However, higher degree of fusion frequency (47.3%) was observed in the fusion medium containing 50mM CaCl2 at pH 10.5 than any other conditions. Cell viability was decreased by Ca2+ and high pH treatment in the fusion media, while fusion frequency was increased. It is concluded that Ca2+ is involved in electrofusion of protoplasts.

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Studies on the Induction of Transformation and Mutiplication in Orchid Plants (II) Isolation, Culture and Electroporation of Protoplasts in Bletilla striata (난과 식물의 형질전환 유도 및 다량증식에 관한 연구 (II) 자란의 원형질체 분리, 배양 및 Electroporation)

  • 이정석;김영준황성진황백
    • KSBB Journal
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    • v.6 no.2
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    • pp.201-205
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    • 1991
  • We have investigated influencing factors on viability of Bletilla striata protoplasts electroporated in the presence of various electrical conditions. Cultures of embryogenic callus and embryogenic cell suspension were established with immature seeds of Bletilla striata. Viabilty of electroporated protoplasts was decreased according to the increaseing of electroporation voltage and capacitance. An optimal condition of electroporation for viable protoplasts was in HBM buffer at $4^{\circ}C$.

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Isolation, Culture, and Fusion of Nicotiana Protoplasts (원형질체 분리, 배양 및 Nicotiana 종간 세포융합에 관한 연구)

  • 윤경은;김준철;최상수;손세호
    • 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$^{\circ}C$.

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Protoplasts Isolation and Reversion of Fomitella fraxinea (장수버섯(Fomitella fraxinea)의 원형질체 분리 및 재생)

  • Kim, Kyung-Soo;You, Chang-Hyun;Kong, Won-Sik;Kim, Young-Ho;Cha, Dong-Yeul
    • The Korean Journal of Mycology
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    • v.26 no.2 s.85
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    • pp.275-280
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    • 1998
  • Factors affecting protoplasts isolation and regeneration of Fomitella fraxinea were investigated. Lytic enzyme mixture of Novozym 234, Cellulase onozuka R-10 and ${\beta}-Glucuronidase$ was found to be the best for the protoplasts isolation. Osmotic stabilizer of 0.6 M sucrose was observed as the best for protoplasts isolation. The highest number of protoplasts was obtained from the F. fraxinea mycelium with lytic enzyme mixture and osmotic stabilizer that had been cultured for 3 hours. The highest regeneration rate of 0.02 % was achieved when the 0.6 M sorbitol was employed as osmotic stabilizer.

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Protoplast Isolation and Reversion from Agrocybe cylindracea (Agrocybe cvzindracea의 원형질체 분리 및 환원)

  • Park, Shin;lee, Jae-Sung
    • 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.

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Factors Affecting Electrofusion of Plant Protoplasts (식물 Protoplast의 전기자극 융합에 관여하는 인자)

  • Han, Sung-Kyu;U, Zang-Kual;Kang, Soon-Suon;Riu, Key-Zung;Oh, Sung-Gug
    • Applied Biological Chemistry
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    • v.33 no.1
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    • pp.93-100
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    • 1990
  • The optimum conditions of electric stimulation for electrofusion of protoplasts of petunia, carrot and soybean, and the effects of calcium, magnesium, protease, trypsin, triton X-100, concanavalin A, dimethyl sulfoxide(DMSO), glycerol monooleate and spermine on fusion frequency and/or viability of petunia protoplast were investigated. The optimum frequencies(Hz)-amplitudes(V/cm) of AC Pulse for protoplast pearl-chain formation were 10 kHz-20 V/cm and 1 MHz-60 V/cm for petunia, 100 kHz-40 V/cm and $1\;MHz-40{\sim}60\;V/cm$ for carrot, and $1\;MHz-40{\sim}80\;V/cm$ for soybean, respectively. The optimum condition of DC pulse treatment at the 1 MHz-60 V/cm-15sec treatment of AC for electrofusion of petunia protoplasts was 2.5 kV/cm-40 sec, and under this condition the fusion frequency and viability of protoplasts were 45 % and 10 %, respectively, Both of the protoplasts of carrot and soybean were not fused under the AC and DC conditions tested in this experiment. The electrofusion of petunia protoplasts was stimulated by calcium, and the fusion frequency and the viability of the protoplasts were 43 % and 11 % , respectively at the calcium concentration of 140 mM. Although fusion frequency was not affected by magnesium only, magnesium stimulated fusion frequency in the presence of calcium, and the viability and fusion frequency of petunia protoplasts were 45 % and 13 %, respectively, at 140 mM of magnesium-140 mM of calcium. The relative fusion frequencies of petunia protoplasts to the controls were increased by 2.4, 2.1, 1.6, 1.4, 1.8, 1.5 and 2.2 folds, respectively, by the treatments of protease, trypsin, triton X-100, concanavalin A, DMSO, glycerol monooleate, and spermine. The viabilities of petunia protoplasts were decreased by these substances.

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