共查询到20条相似文献,搜索用时 15 毫秒
1.
Summary Mesophyll protoplasts isolated from Petunia hybrida were subjected to UV radiation (280–360 nm) in an attempt to assess whether (a) UV radiation has an effect on cortical microtubule organization, (b) UV radiation affects the progression of protoplasts through the cell cycle, and (c) there is a connection between the effect of UV radiation on cell division and the polymerization state of the microtubules. The proto plasts were irradiated with the following UV doses: 4, 8, 12, and 24mmol photons/m 2, 30 min after isolation. Cell cycle analysis and immuno-localization of microtubules were carried out 0, 24, 48, and 72 h after irradiation. The length of cortical microtubules was determined after irradiation and in corresponding controls. We found that UV radiation induced breaks in cortical microtubules resulting in shorter fragments with increasing dose. Also, the protoplasts were delayed in their progression through the cell cycle, with G1 and G2 phases being affected as well as the S phase. The commencement of DNA synthesis in the irradiated protoplasts followed the re-establishment of a microtubule network. At 48 h after irradiation the protoplasts in all treatments, except for the 24 mmol/m 2, had cortical microtubules of similar length, and at 72 h after irradiation only the protoplasts that had received 24 mmol photons/m 2 had not started dividing.Abbreviations BSA
bovine serum albumin
- DMSO
dimethyl sulfoxide
- FDA
fluorescein diacetate
- MT
microtubules
- MTSB
microtubule stabilizing buffer
- PAR
photosynthetically active radiation (400–700 nm)
- PBS
phosphate buffered saline
- UV
ultraviolet 相似文献
2.
Protoplasts were isolated enzymatically from meristematic tissues of the brown algae, Fucus serratus, using a combination of 2% cellulase R-10 Onozuka, 0.5% macerozyme and 1% crude extract of gland gut of Aplysia
vaccaria. The main factors affecting protoplast yield were identified. Protoplasts were produced in large quantities from apical region of thallus and from plantlets compared to mature explants. Yields were greatly improved by the addition of sodium citrate and bovine serum albumin in the enzymatic solution and could reach 5.8 × 10 6 protoplasts per gram of fresh wt. The applicability of these optimal parameters to other species Fucus vesiculosus was shown. 相似文献
3.
Protoplasts were isolated enzymatically from the carrageenophyte red alga Grateloupia turuturu (Halymeniales, Rhodophyta) that occurs along the coast of the French Channel in Normandy. Effects of the main factors on
the protoplast yield were identified to improve the isolation protocol. The optimal enzyme composition for cell wall digestion
and protoplast viability consisted of 2% cellulase Onozuka R-10, 0.5% macerozyme R-10, 2% crude extract from viscera of Haliotis tuberculata, 0.8 M mannitol, 20 mM sodium citrate, 0.3% bovine serum albumin at 25°C, and 4-h incubation period. The protoplasts were
approximately 5–15 μm in diameter, liberated mainly from the surface cell layers. Maximum yield was 1.5 × 10 7 protoplasts g -1 fresh tissue. The protoplasts underwent initial division after 14 days with a high density level of 1 × 10 6 cells mL -1 in culture medium and developed into microthalli of a line of two to six cells. 相似文献
4.
A technique is described for the reversible, lectin-mediated immobilization of plant protoplasts on agarose beads. Cyanogen-bromide-activated agarose beads were coated with protein (gelatine or bovine serum albumin) and lectins were subsequently linked to the protein layer using glutaraldehyde. The technique has possible applications in protoplast fusion-product isolation, cellrecognition studies, and membrane isolation.Abbreviations BSA
bovine serum albumin
- Con A
concanavalin A
- FDA
fluorescein diacetate
- PNA
peanut agglutinin
- WGA
wheat-germ agglutinin 相似文献
5.
A procedure was developed for the enzymic isolation of large quantities of protoplasts from the cortex of Zea mays L. WF9 × MO 17 roots. Cortex was separated from the primary root, sectioned, and the cell walls digested for 3.5 hours in 2% (w/v) Cellulysin, 0.1% Pectolyase Y-23, 1 millimolar CaCl 2, 0.05% bovine serum albumin, 0.5 millimolar dithiothreitol in 0.6 molar mannitol (pH 5.6). Cortical cell protoplasts were collected by centrifugation and purified by flotation in a Ficoll step gradient. The yield of protoplasts was approximately 650 × 10 3/gram fresh tissue. To obtain maximum yield it was essential to include an effective pectinase (Pectolyase Y-23) and protectants (bovine serum albumin and dithiothreitol) in the digestion medium. Cortical cell protoplasts exhibited energy-dependent uptake of K+ (86Rb), H232PO4−, and 36Cl− as well as net H+ extrusion. Ion fluxes were sustained for at least 3 hours. Influx of K+ was highest between pH 7.5 and 8.0, whereas the influx of H2PO4− was greatest between pH 4.0 and 5.0. K+ and H2PO4− influx and net H+ efflux were inhibited by respiratory poisons such as cyanide (0.1 millimolar) and oligomycin (5 micrograms per milliliter), and by inhibitors of plasma membrane ATPase such as diethylstilbestrol (50 micromolar). Calculated flux for Cl− was low, but not greatly different from that observed for other plant cells. K+ flux was somewhat high, probably because the K+ concentration in the cortical cells was below steady-state. The results indicate that isolated cortical cell protoplasts retain transport properties which are similar to those of root tissue. 相似文献
6.
Summary A convenient and rapid isolation procedure for root cell protoplasts suitable for patch clamp experiments, was developed for root cells of tomato ( Lycopersicon esculentum) and Plantago species, grown on hydroculture. The procedure is based on a minimal exposure of cells to cell wall degrading enzyme mixtures. After an incubation period of 30 min in a cell wall degrading enzyme mixture all free floating cells were discarded. Subsequently the root material was rinsed and a second group of cells, still present inside the tissue, was freed by application of mechanical pressure. The newly released protoplasts were filtered and collected on the glass bottom of a patch clamp dish. The bathing medium was rinsed extensively removing cellulose fibrils and protoplasts not attached to the glass. Removal of these cellulose fibrils significantly improved the seal success ratio. The isolated protoplasts were suitable for patch clamp experiments in the cell-attached patch, the whole cell and the isolated patch configuration.Abbreviations BSA
bovine serum albumin
- BTP
bis-tris propane
- CAP
cell-attached patch
- OOP
outside out patch
- PEG
polyethylene glycol
- WC
whole cell 相似文献
7.
Yields of 10 6–10 8 peach mesophyll cells and protoplasts · gfw -1 were obtained depending on factors such as digesting enzymes, and leaf size. Onozuka R-10 (2%) in combination with Macerase (0.5%) was found best for protoplast isolation and mediocre for cell isolation among several enzyme combinations tested. Viability was 90% for protoplasts and 60% for cells. Pectolyase Y23 was found to be ineffective in our investigation. Small leaves, 4–10 mm in length, were a superior source for protoplast isolation than medium or big expanded leaves, 22–30 mm in length. The high yields of protoplasts could be obtained only when keeping the ratio of leaf biomass to volume of digesting enzyme solution under 20 mg ml -1. Purification of protoplasts on a sucrose gradient yielded about 10 7 protoplasts · gfw -1, however, the preparation was still contaminated by intact cells. Protoplasts were cultured under different growth regulators and physical conditions. Limited growth and division of protoplasts embedded in agarose drops were observed.Abbreviations BA
6-benzyladenine
- IBA
indolebutyric acid
- FDA
fluorescein diacetate
- MES
2-M-morpholinoethane sulphonic acid
- MS
Murashige and Skoog
- NAA
-naphthaleneacetic acid
- PVP
polyvinylpyrrolidone 相似文献
8.
The subcellular distribution of soluble protease in anthesis-stage, anthocyanin-containing Hippeastrum cv. Dutch Red Hybrid petal protoplasts has been reevaluated and that of Triticum aestivum L. var. Red Coat leaf protoplasts determined using 125I-fibrin as a protease substrate and improved methods for protoplast and vacuole volume estimation. Results indicate that about 20% of the Hippeastrum petal-soluble protease and about 90% of the wheat leaf-soluble protease can be assigned to the vacuole. Protoplast isolation enzyme labeled with 125I has been used to assess the efficiency of removing isolation enzyme from protoplasts by repeated washing and by separation of protoplasts from debris using density centrifugation. Results of these studies suggest that protoplasts prepared by both methods retain low levels of isolation enzyme. However, when protoplasts prepared by either method were lysed with washing medium lacking osmoticum, little isolation enzyme contaminated the lysates. 相似文献
9.
Summary Protoplasts of the moss Anoectangium thomsonii Mitt. were isolated from preplasmolyzed protonemal filaments, grown in suspension cultures, after the digestion of the cell wall by the enzymes cellulase and macerozyme or driselase. Driselase was more effective than cellulase and macerozyme. After purification these protoplasts were plated in the form of small agar drops in modified Kofler's medium without hormones and incubated in the dark at 26 ± 2 °C. Cell walls regenerated within three days and cell divisions started seven days after the initiation of the cultures. When the regenerants were transferred to normal protonemal culture medium and illuminated by 3,000 lux continuous light, a multi-cellular protonema developed which formed leafy gametophores on salicylic acid supplemented medium. 相似文献
10.
The sub-cellular distribution of chitin synthetase was studied in homogenates of Saccharomyces cerevisiae protoplasts. Use of a mild disruption method minimized rupture of vacuoles and ensuing contamination of subcellular fractions by vacoular proteinases. After fractionation of whole or partially purified homogenates through an isopycnic sucrose gradient chitin synthetase activity was found to be distributed between two distinct particulate fractions with different buoyant density and particle diameter. When whole homogenates were used, about 52% of the chitin synthetase loaded was localized in a microvesicular population identified as chitosomes (diameter 40–110 nm; bouyant density ( d) = 1.146 g/cm 3). Another vesicular population containing 26% of the activity was identified as plasma membrane vesicles because of its large mean diameter (260 nm), its high buoyant density ( d = 1.203 g/cm 3) and by the presence of the vanadate-sensitive ATPase activity. Moreover, after surface labeling of protoplasts with 3H-concanavalin A, the label cosedimented with the presumed plasma membrane vesicles. There was a negligible cross-contamination of the chitosome fraction by yeast plasma membrane markers. In both the plasma membrane and the chitosome fractions, the chitin synthetase was stable and essentially zymogenic. Activation of the chitosome fraction produces microfibrils 100–250 nm in length. Our results support the idea that chitosomes do not originate by plasma membrane vesiculation but are defined sub-cellular organelles containing most of the chitin synthetase in protoplasts of Saccharomyces cerevisiae. 相似文献
11.
An index [ kv: average isolation rate of viable protoplast (number/ml min)] was established to evaluate the optimal conditions for protoplast isolation from cultured plant cells. The optimal conditions for protoplasts isolation from Nicotiana tabacum BY2 cultured cells could be determined on the basis of the kv [31.7 × 10 3 (number/ml min)]. The colony-forming efficiency of the protoplasts was about 46%. The optimal conditions for protoplasts isolation from Catharanthus roseus [ kv = 38.1 × 10 3 (number/ml min)] and Wasabia japonica [ kv = 14.2 × 10 3 (number/ml min)] cultured cells could also be determined. Furthermore, a method for rapid regenerating cell wall of protoplast in liquid culture using alginate gel containing locust bean gum was developed. 相似文献
12.
Cultured protoplasts of young, unexpanded leaves of the wild lettuce, Lactuca perennis, divided to produce cell colonies in an agarose-solidified, modified MS medium with reduced levels of inorganic salts, together with 2,4- d, NAA and zeatin at 0.2, 0.1 and 0.5 mg 1 -1 respectively. Organogenesis followed the initial transfer of protoplastderived colonies to modified MS medium with 2,4- d, NAA and zeatin (0.1, 1.0 and 0.2 mg 1 -1 respectively) and then to full-strength MS medium with 6-BA and NAA (0.4 and 0.05 mg 1 -1). Shoots were rooted on agar-solidified MS medium lacking growth regulators. Regenerated shoots were established ex vitro, 21 weeks after protoplast isolation.Abbreviations 6-BA
6-benzyladenine
- BSA
bovine serum albumin
- d
days
- 2.4- d
2,4-dichlorophenoxyacetic acid
- f. wt.
fresh weight
- IAA
indoleacetic acid
- MES
2 [N-morpholino]ethane sulphonic acid
- MS
Murashige & Skoog (1962) 相似文献
13.
A method of isolating grape mesophyll protoplasts was developed to facilitate the eventual use of genetic engineering techniques in this species. The effects of several factors influencing protoplast isolation could be evaluated quickly by using leaf disks 1 cm in diameter and known volumes of maceration and wash media. The best yields of mesophyll protoplasts were obtained using medium sized leaves of grapevines kept in the dark for 24 hours prior to maceration in 1% Cellulysin, 0.5% Macerase, 0.7 M mannitol, 5 ppm 2, 4 D, 0.1 ppm BAP, 1/10 strength Murashige and Skoog medium, and incubated at 22°C in cool-white fluorescent light (70–100 E m -2 s -1) for 24 hours. Over 30×10 6 protoplasts per cm 2 of leaf were produced using these conditions. This method of screening factors affecting protoplast isolation could be applicable to other species. 相似文献
14.
Average specific density of individual cells of pure cultures of Chromatium warmingii and Chromatium vinosum were measured by isopicnic gradient centrifugation with Percoll during growth at constant illumination as a function of the increasing content of intracellular sulfur. Cell number and volume, bacteriochlorophyll a, sulfide, and sulfur were followed in the cultures along with cellular buoyant density. Poly--hydroxybutyrate was monitored at several points during growth of the cultures. The density of C. warmingii changed from 1.071 to 1.108 g cm -3 (sulfur content per cell varied from 0 to 1.71pg). C. vinosum changed its density from 1.096 to 1.160 g cm -3 (sulfur content per cell varied from 0 to 0.43 pg). Maximum sulfur content in pg of sulfur per m 3 of cell volume were 0.178 for C. warmingii and 0.294 for C. vinosum. Measurement of the differences in buoyant density, volume and sulfur content before and after ethanol extraction of cells with and without intracellular sulfur, allowed tentatively to estimate the density of sulfur inside the cells as 1.219 g cm -3. Isolation of sulfur globules and centrifugation in density gradients gave a density higher than 1.143 g cm -3 for these intracellular inclusions.Non-common abbreviations Bchl
Bacteriochlorophyll
- DMB
Density Marker Beads
- PHB
poly--hydroxybutyrate 相似文献
15.
Following a geometrically asymmetrical cell division during germination of spores of the fern Onoclea sensibilis L., the small cell differentiates into a rhizoid and the large cell divides to form the protonema. Using silver-staining of two-dimensional gels, we have examined the soluble proteins of spores during germination and of separated rhizoid protoplasts and protonemal cells. Of over 500 polypeptides followed, nearly 25% increased or decreased in prominence during spore germination and the initial phases of rhizoid elongation. Soluble proteins from purified protoplasts of young rhizoids were quantitatively different from those of protonemal cells and germinated spores. Nine polypeptides which appeared after cell division were substantially more prominent in rhizoid protoplasts than in whole germinated spores and have been putatively designated rhizoid-specific polypeptides. The differences in the soluble protein composition of young rhizoids and protonemal cells probably reflect the differential organelle distribution between the two cells as well as differential net protein synthesis in the cytoplasms of the two cells. 相似文献
16.
Methods are described for the isolation of functional protoplasts from secondary pulvinus tissue (flexor and extensor) and from leaf mesophyll tissue of primary leaves of Phaseolus coccineus L. Integrity of the protoplasts was shown by vital staining and their ability to evolve oxygen in the light. Extensor-cell protoplasts increased their volume for up to 60% upon addition of 10 mM KCl. This K +-induced swelling was accompanied by increased rates of proton extrusion. 相似文献
17.
Summary A method is described for isolating mesophyll protoplasts from leaves and secretory cell protoplasts from salt glands of the facultative halophyte, Ceratostigma plumbaginoides (L.). Rates of ATP hydrolysis in both cell types were determined, and levels in secretory cell protoplast preparations were fourfold higher than those in mesophyll protoplast preparations, based on total protein. The rate of ATP hydrolysis was sensitive to azide and vanadate, and stimulated by Triton-X-100. Additionally, immunoblot procedures using an antibody to the plasma membrane H +/ATPase was used to compare ATPase levels of the mesophyll and secretory cell protoplasts. Results indicate that secretory cells have a higher concentration of H +/ATPase than mesophyll cells, consistent with their putative function in salt glands.Abbreviations ATP
adenosine triphosphate
- BSA
bovine serum albumin
- DIDS
diisothiocyano-2,2'-disulfonic acid stilbene
- DNP
dinitrophenol
- DTT
dithiothreitol
- FITC
fluorescein isothiocyanate
- NAD +/NADH
nicotinamide adenine dinucleotide
- SDS
sodium dodecylsulfate 相似文献
18.
Mesophyll protoplasts isolated from in vitro-grown Nicotiana tabacum L. shoots were subjected to electrofusion.Dielectrophoresis was induced by an AC field of 50 V cm -1 inter-electrode distance and 0.5 MHz oscillation frequency. Fusion was effected by two 0.7 kV cm -1 DC pulses, each of 50 s duration, applied within one second of each other. Various chemical treatments were tested for their effects on dielectrophoresis efficiencies (percentages of protoplasts that made contact with at least one other protoplast under the AC field), fusion efficiencies (percentages of protoplasts participating in fusion events), cell lysis (percentages of protoplasts bursting during the electrofusion processes), overall viabilities of fusion products 24 h post-fusion and overall plating efficiencies 7 d post-fusion (percentages of fusion-derived cells that had undergone division). The various attributes assessed on the electrofusion of protoplasts in the control treatment, 10% mannitol, differed considerably for experiments carried out on different days. Relative to the control treatment, only the Ca 2+ treatments, and to a lesser extent lipase treatment reduced dielectrophoresis efficiencies. Polyamines, cytochalasins and Ca 2+ treatments significantly reduced cell lysis percentages. All electrofusion facilitators tested (except for spermine at 150 mg l -1, the cytochalasins B and D, and Ca 2+ treatments) increased fusion efficiencies to more than 1.5 times those obtained with the standard 10% mannitol electrofusion medium. Ca 2+ treatments increased overall viabilities of fusion products by more than 1.5 times. With the exception of the prostaglandins, lecithin and CaCl 2 treatments, overall plating efficiencies were reduced by treatment of protoplasts with fusion facilitators. Substantial increases in overall plating efficiencies over those observed in the control treatment were obtained using prostaglandin F 2a, lecithin and CaCl 2.2H 2O treatments. The implications of the results are discussed.Abbreviations AC
alternating current, approx.-approximately
- BA
benzylaminopurine, cv.-cultivar
- DC
direct current, diam.-diameter
- FDA
fluorescein diacetate
- MS
Murashige & Skoog (1962)
- NAA
napthaleneacetic acid
- PCM
protoplast culture medium
- PIM
protoplast isolation medium
- PPM
protoplast purification medium
- rpm
revolutions per minute
- SD(n)
standard deviation of a variate
- SEM
standard error of the mean 相似文献
19.
Axenic shoot cultures of virus-free Vitis vinifera L. cv. Soultanina were a highly efficient source for isolation of viable protoplasts. Optimum results were obtained with leaves of 50–100 mg fresh weight, leaf discs of 0.7 cm in diameter, 100 and 15 U ml -1 Cellulase R-10 and Macerozyme R-10, respectively, and 18 h reaction time in either light or in darkness. Protoplast yield was approx. 25×10 6 viable protoplasts per g fresh weight and their size ranged from 12 to 44 m. During a 20-day culture period, the maximum survival rate obtained was approx. 40%. A plating density of 10×10 5 protoplasts per ml resulted in increased survival rates. Various growth regulators and glutamine did not significantly improve survival rates of protoplasts, whereas extract from coconut added to the culture medium caused an increase in the survival rates of protoplasts. Cell elongation at a significant rate and divisions were observed. [ 14C]glucose uptake was studied as an index of cell membrane integrity and functioning. Uptake rate of glucose by protoplasts was linear for up to 60 min, fully inhibited by NaN 3, with an optimum pH of 4.8. Protoplasts 24 h old exhibited significantly lower rates of glucose uptake. 相似文献
20.
Summary Mesophyll protoplasts from Nicotiana glauca were fused with epidermal protoplasts from N. langsdorffii by an electric pulse. After the fusion products were centrifuged on stepwise density gradient centrifugation using Percoll and sea water, somatic hybrids were observed at 70%–80% in the fraction recovered from the intermediate specific gravity fraction between epidermis and mesophyll protoplasts. From offsprings of these somatic hybrids, teratomatous plants were regenerated. Since the difference of specific gravity between mesophyll and epidermis protoplasts is inherent, this procedure can be essentially applied to obtain somatic hybrids between any combination of plants. The significance of this study is discussed in relation to obtaining somatic hybrids between plant materials without any appropriate genetic markers. 相似文献
|