• Title/Summary/Keyword: Hypocotyl protoplast

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The effects of cytokinin and plating density on protoplast culture of sunflower

  • Chitpan Kativat;Witsarut Chueakhunthod;Piyada Alisha Tantasawat
    • Journal of Plant Biotechnology
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    • v.49 no.4
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    • pp.331-338
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    • 2022
  • Sunflower (Helianthus annuus L.) protoplasts were isolated from seven-day-old etiolated hypocotyls of 10 A line and four-week-old fully expanded young leaves of PI 441983 line in vitro seedlings using an enzymatic method. Purified protoplasts were collected by filtration and floatation in sucrose solution. Semi-solid protoplast culture was performed using the L4 regeneration protocol with various culture media and plating densities to achieve the highest efficiencies for protoplast culture of hypocotyl and mesophyll protoplasts of 10 A and PI 441983 lines, respectively. The concentrations in liquid L'4M medium and different plating densities were evaluated in two types of cytokinins, the adenine-type 6-benzyladenine (BA) and the phenylurea-type thidiazuron (TDZ). The highest colony formation was achieved in both sunflower lines when 0.5 mgL-1 BA and 0.5 mgL-1 TDZ were applied with a high plating density (3 × 105 protoplasts mL-1). These conditions led to 38.45% and 39.40% colony formation for hypocotyl protoplasts of the 10 A line and mesophyll protoplasts of the PI 441983 line, respectively. Moreover, many hypocotyl protoplast-derived colonies developed into micro-calli. In addition, superior development of both sunflower protoplasts was observed with all plating densities when BA was used in combination with TDZ. This finding will be applicable to future sunflower hybrid production via somatic hybridization.

Plant Regeneration from Hypocotyl-Derived Protoplasts of Brassica oleracea var. capitata (양배추 배축 원형질체로부터 식물체 재분화)

  • 이연희;조현석;서석철;김호일
    • Korean Journal of Plant Tissue Culture
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    • v.22 no.1
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    • pp.7-11
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    • 1995
  • Protoplasts were isolated from hypocotyl tissues of 5-day-old Brassica oleracea var capitata Green Challenger seedlings. Several media were used for protoplast culture and shoot regeneration. The shoot-regeneration rapacity of protoplast derived callus depended on the initial culture medium. Protoplasts were cultured in liquid medium (B5 medium supplemented with CaCl2, 2H2O 600mg/L, g1ucose 20g/L, D-mannito1 70g/L, NAA lmg/L, BA lmg/L, 2.4-D 0.25 mg/L)at 27$^{\circ}C$ under the dark After 5 to 10 days, cultlues were diluted with medium with a reduced osmotic stabilizer and then transferred to illuminated conditions. The culture medium was changed with the fresh medium at 7- to 10-day-intervals until the formation of microcallus. Hypocotyl protoplast-derived callus proliferated when transferred to MS medium supplemented with NAA lmg/L, BA 1mg/L and GA$_3$ 0.02mg/L. Upon transfer to MS basal medium without growth regulators, roots were produced. In an attempt to increase the regeneration frequency, 10g/L polyvinylpyrrolidone was added to the regeneration medium, but the shoot regeneration was mot improved. The regenerated whole plants were acclimated in a sterized soilless mixture(vermiculite 2;perlite 2;peat moss1) in a culture room.

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Isolation and Culture of Phaseolus vulgaris L. Callus Protoplasts (강남콩(Phaseolus vulgaris L.) Callus의 원형질체 유이 및 배양)

  • 김상구
    • 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.

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Callus Formation and Rooting of Inbred Lines of Chinese Cabbage (Brassica campestris ssp. perkinensis) Though Protopalst Culture (원형질체 배양을 통한 배추 〔Brassica campestris ssp. perkinensis〕캘러스 형성 및 뿌리분화)

  • 염옥희;전익조;김혜진;백남권;임학태
    • 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.

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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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Protoclonal variation in cabbage (Brassica oleracea ssp. capitata) (양배추 protoclone의 변이)

  • 이연희;조현석;김호일;나종현;이인원
    • Korean Journal of Plant Tissue Culture
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    • v.24 no.3
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    • pp.175-181
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    • 1997
  • Plants were regenerated from hypocotyl protoplast culture of cabbage (F$_1$hybrid Green Challenger) and were transplanted on fields. The ploidy level of regenerated and seed-grown plants was measured by flow cytometry. In total 125 regenerated plant, diploid (2n), tetraploid(4n), and mixoploid (2n+4n) were 72.8%, 25.6%, and 1.6%, respectively. Most of the regenerated plants had normal phenotype, whereas several plane showed abnormal phenotype such as heavy leaf incision, savoy, and wave. The regenerated plants with abnormal phenotype and different ploidy level were analysed by isozyme and RAPD, but no significant difference was found.

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