• Title/Summary/Keyword: protoplast isolation

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Optimized Methods for the Isolation of Arabidopsis Female Central Cells and Their Nuclei

  • Park, Kyunghyuk;Frost, Jennifer M.;Adair, Adam James;Kim, Dong Min;Yun, Hyein;Brooks, Janie S.;Fischer, Robert L.;Choi, Yeonhee
    • Molecules and Cells
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    • v.39 no.10
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    • pp.768-775
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    • 2016
  • The Arabidopsis female gametophyte contains seven cells with eight haploid nuclei buried within layers of sporophytic tissue. Following double fertilization, the egg and central cells of the gametophyte develop into the embryo and endosperm of the seed, respectively. The epigenetic status of the central cell has long presented an enigma due both to its inaccessibility, and the fascinating epigenome of the endosperm, thought to have been inherited from the central cell following activity of the DEMETER demethylase enzyme, prior to fertilization. Here, we present for the first time, a method to isolate pure populations of Arabidopsis central cell nuclei. Utilizing a protocol designed to isolate leaf mesophyll protoplasts, we systematically optimized each step in order to efficiently separate central cells from the female gametophyte. We use initial manual pistil dissection followed by the derivation of central cell protoplasts, during which process the central cell emerges from the micropylar pole of the embryo sac. Then, we use a modified version of the Isolation of Nuclei TAgged in specific Cell Types (INTACT) protocol to purify central cell nuclei, resulting in a purity of 75-90% and a yield sufficient to undertake downstream molecular analyses. We find that the process is highly dependent on the health of the original plant tissue used, and the efficiency of protoplasting solution infiltration into the gametophyte. By isolating pure central cell populations, we have enabled elucidation of the physiology of this rare cell type, which in the future will provide novel insights into Arabidopsis reproduction.

Protoplast Isolation and reversion from Pleurotus spodoleucus (참느타리버섯의 원형질체(原形質體) 분리(分離) 및 환원(還元))

  • Yoo, Young-Bok;Byun, Myung-Ok;Go, Seung-Joo;You, Chang-Hyun;Chang, Kwon-Yawl
    • The Korean Journal of Mycology
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    • v.15 no.1
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    • pp.19-22
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    • 1987
  • This experiment was undertaken to investigate proper conditions for protoplast formation from Pleurotus spodoleucus. Novozym 234 was the most effective enzyme and a high yield of protoplasts was obtained. Combination of enzymes did not improve this result. Sucrose gave the best result to support the release and maintain the stability of protoplasts. The optimal reaction time of mycelium with the lytic mixture was 3 hrs. in shaking condition at 120 strokes $min-^1$. When mycelium of P. spodoleucus was cultured for 3 days on mushroom complete agar medium at $27^{\circ}C$, the formation of protoplasts was effective. The mushroom complete agar medium stabilized with 0.6 M sucrose was the most effective for reversion of protoplasts.

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Selection of High Efficient Enzyme for Protoplasts Isolation from Mushrooms (버섯류의 원형질체 나출을 위한 고효율 효소 선발)

  • Kim, Jong-Kun;Kim, Jin-Hee;Kong, Won-Sik;Kang, Hee-Wan
    • The Korean Journal of Mycology
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    • v.38 no.1
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    • pp.21-24
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    • 2010
  • This study was carried out to select cell wall degrading enzymes for maximizing protoplast yield from Basidiomycetes. The protoplasts were released from spore suspension, mycelia cultured on cellophane membrane, and homogenized mycelia of Flammulina velutipes using commercial cell wall degrading enzymes. The highest yield of protoplasts was obtained from the homogenized mycelia treated with the enzyme combination of $Glucanex^R$ 200G and cellulase onozuka R-10. The protocol was also available for Pleurotus ostreatus, P. eryngii, and Hypsizygus marmoreus.

STUDIES ON THE TISSUE CULTURE OF PANAX GINSENG

  • Harn C
    • Proceedings of the Ginseng society Conference
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    • 1974.09a
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    • pp.9-22
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    • 1974
  • Unlike the tissue culture in animals and human being, in higher plants various parts of the plant are cultured for varied purposes, and they are named variously depending on which parts are used as explants or what purposes they are cultured for. Followings are some of the names of culture used frequently: organ culture, tissue culture, callus culture, single cell culture, meristem culture, mericlone culture, ovary culture, ovule culture, embryo culture, endosperm culture, anther culture, pollen culture, protoplast culture, etc.. As the names of the culture indicate, in some kinds of culture the explants used for culture are actually not tissues, but organs, single cells, or protoplasts. It seems, however, convenient to call all of the above-mentioned cultures grossly as tissue culture. Several kinds of tissue culture were attempted using Panax ginseng as material and some of the results were summarized below. 1. Callus culture After dormancy of the sed was broken, whole embryo or parts (hypocotyl, cotyledon and epicotyl) of partly grown embryo were cultured in the media supplemented with growth regulators. Rapid swelling occurred in a few weeks, but most of the swelling was observed only in the basal part of epicotyl, changes in the other parts of embryo appearing in much later stages. The swelling or increase in size, however, was resulted not from the divisions of cells, but from the mere expansion of cell. Real calli were formed about two months after inoculation of explants. Callus tissues developed from cortex, pith, and vascular bundle in the cases of hypo- and epicotyl, from mesophyl tissue in the case of cotyledon. Shoots developed more easily from cotyledons regardless of whether they are detached from or attached to the embryo proper. 2. Culture in the Knudson C medium When cotyledons, detached from or attached to the embryo proper, were cultured in the growth regulator-free Knudson C medium comprision only several kinds of mineral compounds and sucrose, shoot primordium or callus developed profusely and finally plantlets were produced directly from shoot primordium or indirectly through callus. In this medium epidermal cells as well as mesophyl cells of the cotyledon became meristematic and divided, changing into multinucleate cells or multicellular bodies, developing eventually into either shoot primordia or calli. 3. Anther culture Anthers were cultured in the media supplemented with various growth regulators applied singly or in combinations. Callus was formed mostly in the connective tissue of anther. Cells of anther wall layers changed in appearance, but no division occurred. Microspores of all stages in development were not changed, ruling out the possibility that microspore-originated callus might be formed. 4. Isolation of protoplast Protoplasts were isolated from young root, leaf, and epicotyl, using 0.7M D-mannitols as osmoticum and using macerozyme and cellulase respectively for maceration and digestion of the cell wall. Production in large number of naked intact protoplast was rather difficult as compared with other plant species. Fusion of protoplasts occurred infrequently mainly due to the fewer number of naked protoplasts in the solution.

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Development of Cell Lines for Application of Recombinant DNA Techniques in Crops (작물의 유전자 재조합을 위한 세포주의 개발 연구)

  • Chae, Young-Am;Choi, Kyu-Whan
    • KOREAN JOURNAL OF CROP SCIENCE
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    • v.30 no.2
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    • pp.195-200
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    • 1985
  • This experiment was carried out to know the processes of protoplast isolation, culture and plant regeneration in aims of introducing foreign genes into plant cells through plant gene vector, and cellular selection for plant improvement. The main results indicated that 2% cellulase plus 0.5% macerozyme is proper for isolation of protoplasts from leaf mesophyll cells of N. plumbaginifolia, plating efficiency was higher in 1.4-2.0 x 10$^4$ cells/ml, complete cell wall was regenerated after 2 days culture, cell division and cell mass were observed after 4 days and 2 weeks, respectively, colony was developed after 3 weeks culture, addition of 1-2mg/l BA promoted shoot differentiation while root differentiation did not required hormone and seeds were harvested from more than 100 cell lines for further investigation and study.

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Isolation of Auxotrophic Mutants from Basidiospores of Pleurotus cornucopiae (자외선(紫外線) 조사(照射)에 의한 노랑느타리버섯 담자포자(擔子胞子)의 영양요구성(營養要求性) 균주(菌株) 선발(選拔)에 관한 연구(硏究))

  • Lee, Yeon-Hee;Park, Yong-Hwan;Yoo, Young-Bok;Min, Kyung-Hee
    • The Korean Journal of Mycology
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    • v.14 no.2
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    • pp.185-188
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    • 1986
  • After treatment of basidiospores of P. cornucopiae with ultraviolet light, 84 putative mutants from 4671 isolates were obtained. The highest proportion of auxotrophic mutants was obtained from the isolates irradiated to give $0.4{\sim}1.0%$ survival. Fourteen auxotrophs were selected for protoplast fusion and each genetic marker was identified.

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Present Status and Prospects of in vitro Production of Secondary Metabolites from Plant sin China

  • Chen, Xian-Ya;Xu, Zhi-Hong
    • Proceedings of the Botanical Society of Korea Conference
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    • 1995.06a
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    • pp.40-56
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    • 1995
  • During the past two decades, China has seen her great progress in plant biotechnology. Since the Chinese market of herb medicine is huge, while the plant resources are shrinking, particular emphasis has been placed in plant tissue and cell cultures of medicinal plants, this includes fast propagation, protoplast isolation and regeneration, cell suspension cultures and large scale fermentation. To optimize culture conditions for producing secondary compounds in vitro, various media, additives and elicitors have been tested. Successful examples of large scale culture for the secondary metabolite biosynthesis are quite limited : Lithospermum ery throrhizon and Arnebia euchroma for shikonin derivatives, Panax ginseng, P. notoginseng, P. quinquefolium for saponins, and a few other medicinal plants. Recent development of genetic transformation systems of plant cells offered a new approach to in vitro production of secondary compounds. Hairy root induction and cultures, by using Ri-plasmid, have been reported from a number of medicinal plant species, such as Artemisia annua that produces little artemisinin in normal cultured cells, and from Glycyrrhiza uralensis. In the coming five years, Chinese scientists will continue their work on large scale cell cultures of a few of selected plant species, including Taxus spp. and A. annua, for the production of secondary metabolites with medicinal interests, one or two groups of scientists will be engaged in molecular cloning of the key enzymes in plant secondary metabolism.

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Isolation and Characterization of Intraspecific Complementing Fusants of Penicillium verruculosum (Penicillium verruculosum의 종내원형질 융합체의 분리 및 특성)

  • Chung, Ki-Chul;Park, Chang-Ryeol;Suk Bai;Chun, Soon-Bai;Kim, Ki-Chung
    • Microbiology and Biotechnology Letters
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    • v.16 no.3
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    • pp.182-186
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    • 1988
  • The possibility of strain improvement of cellulolytic fungus, Penicillium verruculosum via protoplast fusion was investigated. The cellulolytic activities of the six fusants, finally selected for their hyper-cellulolytics were 2 times of those of wild type and 1.2 to 4.4 times of those parental auxotrophs. It was confirmed that the nuclear fusion occurred in fusants by their DNA contents and nuclear staining with Giemsa. It was also found that the fusants were aneuploids, and their genetic stability was demonstrated from the subculture for four months.

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Chromatin Structural Rearrangement during Dedifferentiation of Protoplasts of Cucumis sativus L.

  • Ondrej, Vladan;Kitner, Miloslav;Dolezalova, Ivana;Nadvornik, Petr;Navratilova, Bozena;Lebeda, Ales
    • Molecules and Cells
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    • v.27 no.4
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    • pp.443-447
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    • 2009
  • This paper reports on the structural rearrangement of satellite DNA type I repeats and heterochromatin during the dedifferentiation and cell cycling of mesophyll protoplasts of cucumber (Cucumis sativus). These repeats were localized in the telomeric heterochromatin of cucumber chromosomes and in the chromocenters of interphase nuclei. The dramatic reduction of heterochromatin involves decondensation of subtelomeric repeats in freshly isolated protoplasts; however, there are not a great many remarkable changes in the expression profile. In spite of that, reformation of the chromocenters, occurring 48 h after protoplast isolation, is accompanied by recondensation of satellite DNA type I; however, only partial reassembly of these repeats was revealed. In this study, FISH and a flow cytometry assay show a correlation between the partial chromocenter and the repeats reassembly, and with the reentry of cultivated protoplasts into the cell cycle and first cell division. After that, divided cells displayed a higher variability in the expression profile than did leaves' mesophyll cells and protoplasts.

Selection of parental monokaryons from Korean Hypsizigus marmoreus by protoplast regeneration (원형질체 재생을 통한 느티만가닥버섯 단핵균주 선발)

  • Oh, Youn-Lee;Kong, Won-Sik;Jang, Kab-Yeul;Shin, Pyung-Gyun;Kim, Eun-Sun;Oh, Min ji;Choi, In-Geol
    • Journal of Mushroom
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    • v.13 no.3
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    • pp.270-273
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    • 2015
  • Hypsizigus marmoreus is commercially the most important edible mushroom in Japan. This mushroom is usually cultivated for a longer period (about 85~120 days) than other mushroom. In order to develop a new cultivar that has a shortened cultivation period, the genome analysis of this strain has been considered. This study aims to obtain parental monokaryotic strains reproducing 'Haemi' cultivar in Hypsizigus marmoreus for reference genome sequencing. The mycelia were cultured in MCM and MYG media for various incubation periods. Homogenized mycelia were treated with commercial cell wall degrading enzymes to maximize protoplasts production yield from Hypsizigus marmoreus. The greatest number of protoplasts was obtained from mycelia cultured in MCM media for 3 days using Novozyme enzyme. The isolated protoplasts were grown in regeneration agar media after two weeks. Regenerated colonies were picked and moved on separated dishes for microscopic observation. Neohaplonts regenerated from dikayotic strains were identified by the absence of clamp connections. We confirmed that one of monokaryotic strains is a parental strain by crossing with an original compatible strain of 'Haemi' cultivar. This parental strain will be used for reference genome sequence analysis.