• Title/Summary/Keyword: Protoplast isolation

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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%.

Protoplast Production from Sphacelaria fusca (Sphacelariales, Phaeophyceae) Using Commercial Enzymes

  • Avila-Peltroche, Jose;Won, Boo Yeon
    • Journal of Marine Bioscience and Biotechnology
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    • v.12 no.1
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    • pp.50-58
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    • 2020
  • Sphacelaria is a filamentous brown algal genus that can be epibiotic on macroalgae, marine plants, and sea turtles. Its important role in benthic ecosystems, exposure to different stressors (e.g., grazing), and use as a model organism make Sphacelaria ideal for assessing physiological responses of organisms to environmental inputs. Single-cell RNA sequencing is a powerful new probe for understanding environmental responses of organisms at the molecular (transcriptome) level, capable of delineating gene regulation in different cell types. In the case of plants, this technique requires protoplasts ("naked" plant cells). The existing protoplast isolation protocols for Sphacelaria use non-commercial enzymes and are low-yielding. This study is the first to report the production of protoplasts from Sphacelaria fusca (Hudson) S.F. Gray, using a combination of commercial enzymes, chelation, and osmolarity treatment. A simple combination of commercial enzymes (cellulase Onozuka RS, alginate lyase, and driselase) with chelation pretreatment and an increased osmolarity (2512 mOsm/L H2O) gave a protoplast yield of 15.08 ± 5.31 × 104 protoplasts/g fresh weight, with all the Sphacelaria cell types represented. Driselase had no crucial effect on the protoplast isolation. However, the increased osmolarity had a highly significant and positive effect on the protoplast isolation, and chelation pretreatment was essential for optimal protoplast yield. The protocol represents a significant step forward for studies on Sphacelaria by efficiently generating protoplasts suitable for cellular studies, including single-cell RNA sequencing and expression profiling.

Effect of Incubation Time, Concentration of Enzyme, and 2,4-D on Isolation and Callus Formation of Protoplast from Callus of Citrus junos (遊離시간 , 酵素處理 및 2,4-D 농도가 재래 유자(Citrus junos)의 캘러스由來 原形質體 遊離 및 培養에 미치는 영향)

  • 오성도;김영숙
    • Korean Journal of Plant Tissue Culture
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    • v.25 no.5
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    • pp.335-339
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    • 1998
  • The factors affecting the isolation and culture of the protoplast of embryogenic callus, derived from immature ovule in Citrus junos, were examined. An incubation time in enzyme solution of 16 hrs was preferable for protoplast isolation. Efficient protoplast yields were obtained from the treatment of equal concentration of 0.7 M $\textrm{BH}_{3}$ to the enzyme solution containing 1.0% cellulase, 1.0% macerozyme and 0.2% pectolyase. Protoplast cultured in MT medium with 0.1 mg/L 2,4-D showed vigorous division and some of them formed callus. Induced callus was subcultured on solid MT medium but the callus showed very slow growth. The above results show the possibility to culture from protoplast fusion in Citrus genera.

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Efficient isolation, culture and regeneration of Lotus corniculatus protoplasts

  • Raikar, S.V.;Braun, R.H.;Bryant, C.;Conner, A.J.;Christey, M.C.
    • Plant Biotechnology Reports
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    • v.2 no.3
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    • pp.171-177
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    • 2008
  • This paper reports an improved protocol for isolation, culture and regeneration of Lotus corniculatus protoplasts. A range of parameters which influence the isolation of L. corniculatus protoplasts were investigated, i.e., enzyme combination, tissue type, incubation period and osmolarity level. Of three enzyme combinations tested, the highest yield of viable protoplasts was achieved with the combination of 2% Cellulase Onozuka RS, 1% Macerozyme R-10, 0.5% Driselase and 0.2% Pectolyase. The use of etiolated cotyledon tissue as a source for protoplast isolation proved vital in obtaining substantially higher protoplast yields than previously reported. Culture of the protoplasts on a nitrocellulose membrane with a Lolium perenne feeder-layer on the sequential series of PEL medium was highly successful in the formation of microcolonies with plating efficiencies 3-10 times greater than previous studies. Shoot regeneration and intact plants were achieved from 46% of protoplast-derived cell colonies.

The Conidial Protoplast Fusion of Cellulolytic Fungus Trichoderma koningii (섬유소 분해균인 trichoderma koningii의 분생자 원형질체 융합에 관하여)

  • 홍순우;하영칠;박희문
    • Korean Journal of Microbiology
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    • v.22 no.4
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    • pp.207-214
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    • 1984
  • Improved methods for the isolation and purification of conidial protoplast were investigated and conidial proplast from auxotrophic mutants were fused. The reaction time for isolation of protoplasts from the swollen condiospores preincubated in liquid minimal medium supplimented with 2-deoxy-D-glucose was shorten by reaction with mixture of 2% driselase and 2% ${\beta}-glucuronidase$ (1:1). The conidial protoplast could be highly purified by using 5% Ficoll 400 as a centrifugation medium. Nucleus of the conidial protoplast was stained with Giemsa stain and the conidial protoplast had one nucleus. It was also confirmed that the conidial protoplast was true protoplast with no cell wall remnant at the outside of plasma menbrane. Fusion frequencies of the conidial protoplast from auxotrophic mutants ranged from $3.4{\times}10^{-1}\;to\;4.9{\times}10^{-1}$. These values were higher than those of mycelial protoplast by a factor of 5 to 28.

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Construction and Analysis of cDNA Library from Porphyra yezoensis (방사무늬 김의 cDNA Library 제조 및 분석)

  • 서수분;이은경;김영진
    • 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.

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Studies on the Isolation, Culture and Fusion of Protoplasts from Plant Mesophyll and Cells Cultured in vitro (식물원형질체의 분리, 배양 및 융합에 관한 연구)

  • Choi, S.J.;Son, S.H.;Chang, W.C.
    • KOREAN JOURNAL OF CROP SCIENCE
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    • v.27 no.2
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    • pp.147-154
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    • 1982
  • This study was conducted to investigate an effective method of protoplast isolation, the plating efficiency for cell division, and fusion of plant protoplasts by polyethylene glycol for somatic hybridzation. The effectiveness of protoplast isolation was different with the various enzyme concentrations, but, in the protoplast isolation from tobacco mesophyll, the enzyme solution with 0.5% macerozyme and 2.0% cellulase was very effective. The protoplast isolation from callus cultured in vitro for a long period was not obtained in any of the enzyme solution used. Protoplasts divided actively at cell densities above $10^44/ml and at $25^{\circ}C$ under 12hr illumination by inflorecient light (l50 Lux), regardless of presence of agar. The highest frequency of protoplast fusion was obtained after treatment with a solution of 0.33 M polyethylene glycol 1500.

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Isolation of Protoplasts from Cultured Cells of Potato (Solanum tubersoum L.) Tuber Tissue (감자(Solanum tuberosum L.) 괴경의 배양세포로부터 원형질체의 분리)

  • 정상호
    • Journal of Plant Biology
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    • v.29 no.1
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    • pp.11-18
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    • 1986
  • Protopasts were isolated from cultured cells of potato (Solanum tuberosum L.) tuber tissue. The ability of callus formation from the culture cells was higher in cultivars Dejima and Superior than in Shimabara and Irish Cobbler on Lam's medium. Therefore, the former was used as sources for protoplast isolation. Friable calli were transferred to liquid media and cells in exponential phase were used for protoplast isolation. In both of Dejima and Superior, the yield of protoplasts was high in the enzyme solution of 2% Onozuka cellulase and 1% macerozyme. Also, viability of isolated protoplasts was very good. Thus, it seems that these protoplasts would be applicable to various aims of research.

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Protoplast Isolation and Reversion from Ganoderma lucidum and Ganoderma sp. (Ganoderma lucidum과 Ganoderma sp.의 원형질체(原形質體) 나출(裸出) 및 환원(還元))

  • Um, Seung-Duk;Chae, Young-Am;Yoo, Young-Bok;You, Chang-Hyun;Cha, Dong-Yeul
    • The Korean Journal of Mycology
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    • v.16 no.1
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    • pp.21-25
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    • 1988
  • This experiment was carried out to investigate proper conditions for protoplast isolation and reversion from Ganoderma lucidum and Gctnoderma sp.. In G. lucidum, 10 mg. $ml^{-1}$ Novozyme 234 with 0.6 M sucrose was proper for protoplast isolation. The optimal reaction time of mycelium with lytic enzyme was five hrs. Protoplast isolation from four-day-old mycelium was the most effective. Protoplast isolation from four-day-old mycelium in G. sp. was optimum in the combination of N ovozyme 234 and ${\beta}-glucuronidase$ with 0.6 M sucrose. MCM was suitable for reversion in G. lucidum while SCM was good for G. sp.. The most effective osmoticum stabilizer for protoplast reversion in G. lucidum and G. sp. was 0.6 M sucrose.

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Isolation and Culture of Mesophyll Protoplasts from in vitro Cultured Populus alba × P. glandulosa (현사시(Populus alba × P. glandulosa) 기내배양엽육(器內培養葉肉) 조직(組織)에서의 원형질체(原形質體)의 분리(分離) 및 배양(培養))

  • Park, Young Goo;Han, Kyung Hwan
    • Journal of Korean Society of Forest Science
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    • v.73 no.1
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    • pp.33-42
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    • 1986
  • This study was carried out to investigate the optimum conditions for isolation and culture of mesophyll protoplasts from Populus alba ${\times}$ P. glandulosa. The results obtained from the experiments are as follows; 1) The suitable concentration of BAP for shoot multiplication was 0.4 mg/l. 2) High yield and viability of isolated protoplasts were obtained by our high enzyme-short time incubation method. 3) Optimum enzyme concentrations for mesophyll protoplast isolation were Cellulase 2%, Macerozyme 0.8%, Hemicellulase 1.2%, Driselase 2%, and Pectolyase Y-23 0.05%. 4) 0.6M mannitol in enzyme solution was the most effective for protoplast isolation and viability. 5) The most adequate pH level of enzyme solution was pH 5.6. 6) The effect of DTT and MES buffer was significant. 7) For protoplast purification, 0.6M sucrose was the most proper concentration. 8) The adding effect of Dextran T40 in floating solution was important. 9) The mesophyll protoplasts isolated through our high enzyme-short time incubation method revealed successful response to culture condition over 3 weeks of culture.

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