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1.
Changes in the number of protoplasts, viability, protein and chlorophyll content were studied in tobacco mesophyll protoplasts
cultivated either autotrophically in CPW medium with mannitol (MCPW) in the light or heterotrophically in CPW medium with
glucose (GCPW) in the dark. The number and viability of protoplasts in the both cultivation media were unchanged. In MCPW
in the light, the protein and chlorophyll content strongly decreased already after 12 h of cultivation, at 72 h of cultivation,
values dropped to 23.6 % (proteins) and to 3.5 % (chlorophyll) in comparison with the initial content. In GCPW in the dark,
the protein and chlorophyll contents decreased only slightly to 75 % (proteins) and to 57.7 % (chlorophyll). 相似文献
2.
Moges Asmara D. Shibli Rida A. Karam Nabila S. 《In vitro cellular & developmental biology. Plant》2004,40(4):389-395
Summary Cryopreservation of African violet via encapsulation-dehydration, vitrification, and encapsulation-vitrification of shoot
tips was evaluated. Encapsulation-dehydration, pretreatment of shoot tips with 0.3 M sucrose for 2 d followed by air dehydration for 2 and 4 h resulted in complete survival and 75% regrowth, respectively. Dehydration
of encapsulated shoot tips with silica gel for 1 h resulted in 80% survival but only 30% regrowth. Higher viability of shoot
tips was obtained when using a step-wise dehydration of the material rather than direct exposure to 100% plant vitrification
solution (PVS2). Complete survival and 90% regrowth were achieved with a four-step dehydration with PVS2 at 25°C for 20 min
prior to freezing. The use of 2M glycerol plus 0.4M sucrose or 10% dimethyl sulfoxide (DMSO) plus 0.5M sucrose as a cryoprotectant resulted in 55% survival of shoots. The greatest survival (80–100%) and regrowth (80%) was obtained
when shoot tips were cryoprotected with 10% DMSO plus 0.5M sucrose or 5% DMSO plus 0.75M sucrose followed by dehydration with 100% PVS2. Shoot tips cryoprotected with 2M glycerol plus 0.4M sucrose for 20 min exhibited complete survival (100%) and the highest regrowth (55%). In encapsulation-vitrification, dehydration
of encapsulated and cryoprotected shoot tips with 100% PVS2 at 25°C for 5 min resulted in 85% survival and 80% regrowth. 相似文献
3.
Bartolomeo Lercari Franco Tognoni Gianni Anselmo Daniele Chapel 《Physiologia plantarum》1986,67(3):340-344
The regeneration of buds from leaves of Saintpaulia ionantha Wendl cv. Sona and from cotyledons of Lycopersicon esculentum Mill. cv. UC 105 was studied in response to continuous light and daily light pulses. The regeneration of buds from cotyledons of Lycopersicon esculentum is high under continuous red and white light. Trie effect of light may be related to phytochrome. In Saintpaulia ionantha blue light appears to be very effective. The experimental results suggest that the blue light effects involve both a blue light absorbing photoreceptor and phytochrome 相似文献
4.
Intact mesophyll protoplasts from cereal plants were easilyprepared by incubating leaves with the abaxial epidermis peeledoff at 2025?C for 23 hr in 0.6 M mannitol containing1% cellulase at pH 5.6. From one gram (fresh weight) of leaves1.56?106 protoplasts, more than 90% of which were morphologicallyintact, could be obtained. Protoplasts isolated from wheat,oat, corn and barley were efficiently infected with brome mosaicvirus (BMV), and supported viral multiplication. (Received June 21, 1977; ) 相似文献
5.
6.
Adrian D. Butt 《Plant science》1985,42(1):55-59
High yields (1.2–8.0 × 106 g fresh wt.?1) of viable leaf mesophyll protoplasts have been isolated from a range of mature deciduous woody-plant species (Betula pendula, Alnus glutinosa, Salix caprea var. Kuroyanagi, S. alba var. Tristis, Populus Tacatricho (= P. tacamahacca × trichocarpa 32), Ulmus glabra var. camperdown), and juvenile glasshouse-grown material (B. pendula, B. pubescens, A. glutinosa). Protoplasts are only released if chopped leaf tissue is thoroughly washed prior to digestive enzyme addition. The nature of the washing requirement has been investigated and it has been demonstrated that water soluble compounds are released from chopped leaves which modify their cell-wall structure rendering them resistant to enzymic digestion. When analyzed by paper chromatography the leachate from B. pendula leaves was found to contain the hydroxycinnamic acids p-coumaric acid (PCA) and o-coumaric acid (OCA). Pre-incubation of B. pendula tissue (which is normally susceptible to enzymic digestion) in authentic samples of PCA and OCA prior to enzymic incubation, completely suppressed protoplast yields. The relevance of hydroxycinnamic acids to protoplast isolation and plant tissue culture is discussed. 相似文献
7.
Efficient procedures are described for high-yield isolation of mesophyll protoplasts from spring wheat ( Triticum aestivum L. cv. Glenlea), winter wheat ( Triticum aestivum L. cv. Frederick), barley ( Hordeum vulgare L. cv. Bruce) and rye ( Secale cereale L. cv. Puma). Factors such as plant age, composition of the incubation medium during isolation, purification procedures and culture medium affect protoplast yield, viability and metabolic competence, as measured by light-dependent CO2 fixation. Optimal osmolarity of the isolation medium was equivalent to 1.8 times that measured in the leaves of all plant material used. The presence of 2 m M ascorbic acid in the preincubation and isolation medium increased the yield by 50% and conserved viability and metabolic competence. The protoplasts were stable for up to 48 h without loss of either viability or of original activity of CO2 fixation, which was in the order of 100 μmol CO2 (mg chl)−1 h−1 .
In our MC-56 liquid medium these protoplasts regenerated cell walls within 72 h and a few divided. 相似文献
In our MC-56 liquid medium these protoplasts regenerated cell walls within 72 h and a few divided. 相似文献
8.
Leaf discs fromSaintpaulia ionantha xconfusa hybrids (African violet) were transferred between basal medium (BM) containing no hormones and shoot-inducing medium (SIM) containing 2.0 mg 1–1 indole acetic acid and 0.08 mg l–1 6-benzylaminopurine to determine whether there is a window of competence for shoot regeneration. Leaf discs precultured on BM prior to transfer to SIM formed buds 3 days earlier than the controls (leaf discs not precultured) regardless of whether the discs were placed upside down or right side up on the medium. This suggests that cultured leaf cells were not competent for shoot induction during the first 3 days of culture. Leaf discs cultured right side up (abaxial surface to the medium) did not form buds on BM alone, unlike discs cultured upside down. Leaf disc survival was affected by a delay in hormonal exposure, but surviving leaf discs produced as many shoots as control leaf discs. This suggests that in the absence of exogenous plant hormones, cellular competence to regenerate shoots is not lost in excised leaf discs of African violet.Abbreviations BM
Basal medium
- SIMSho
Shoot-inducing medium 相似文献
9.
《Plant science》1987,50(2):133-137
An enzyme mixture of general applicability, containing 1.0% Onozuka R-10, 1.0% Hemicellulase, and 0.1% Pectolyase Y-23 provided the basis for the isolation of large numbers of viable mesophyll protoplasts from woody species within the families Betulaceae, Juglandaceae, Rosaceae and Rutaceae. This is the first report of protoplast isolation from leaves of hazelnut, walnut, sweet cherry, sour cherry and the common pear. 相似文献
10.
In leaf discs ofSaintpaulia ionantha xconfusa hybrid (cv. Virginia) cultured on shoot-inducing medium, periclinal divisions were initiated in epidermal cells 3–5 days after explant isolation. This timing coincided with the time for competence acquisition determined in tissue-transfer experiments. Some of the daughter cells from periclinal divisions formed the target cells which divided both anticlinally and periclinally to form cell division centers (meristemoids), precursors of adventitious shoots. The target cells were not morphologically distinct from other epidermal cells at the light microscope level. It is suggested that the periclinal divisions in epidermal cells represent the dedifferentiation phase during which target (competent) cells are formed. Once the cells have acquired the ability to divide periclinally, both dedifferentiation and shoot induction occur in the presence of exogenous plant hormones.Abbreviations
SIM
Shoot-inducing medium 相似文献
11.
Proteases from mesophyll protoplasts of Melilotus alba were identified by standard proteolytic assays and separated using different chromatographic techniques. Their characterization also included their subcellular location. Besides the evidence for the multiplicity of the proteolytic enzymes, two protease sets were distinguished endopeptidases, which are exclusively vacuolar, and aminopeptidases, which are widely distributed throughout the cell. Cytosol-located enzymes were tested as substrates of the two sets of proteases, by studying comparatively the time-course changes of enzyme activities during incubation in total protoplast extracts, or in cytosol fractions devoid of vacuolar proteases. The degradation of phosphoenolpyruvate-carboxylase protein, a typical cytosolic enzyme, in the presence of purified amino-and endopeptidases, was also estimated by immunoprecipitation studies. Only the vacuolar endopeptidases are effective in the degradation of cytosolic enzymes. Hydrolytic enzyme activities mostly of vacuolar origin were very stable during incubation in total protoplast extracts. These proteins therefore appear to be particularly resistant to proteolytic attack. The results indicate that, in plants, the effective proteolytic system acting on cytosolic enzymes seems to be vacuole-located, and that the selectivity in protein degradation may be imposed by the susceptibility of the protein being degraded and by its transfer into the vacuoles.Abbreviations Leu-pNA
leucine-p-nitroanilide
- lys-p-NA
lysine-p-nitroanilide
-
pCMB
p-chloromercuribenzoic acid
- PEPCase
phosphoenolpyruvate carboxylase
- PMSF
phenylmethylsulfonylfluoride
- SDS-PAGE
sodium dodecyl sulfate-polyacrylamide gel electrophoresis 相似文献
12.
High yields (2.3 × 105 to 1.3 × 106 protoplasts/g.f.wt.) of isolated protoplasts were obtained from cotyledons of Cirus sinensis (L.) Osb. 'Valencia'. Osmotic potential of the medium and enzyme concentrations were important in obtaining high viability of preparations as indicated by FDA fluorescence. Adding malt extract to a Murashige-Tucker basal medium increased plating efficiencies somewhat, but not the rate or duration of cell division. However, modifying the NAA and kinetin concentration optimized plating efficiencies (up to 20%) of protoplasts and also the rate or duration of cell division. The highest plating efficiency and number of cells per colony were obtained on a defined medium containing NAA (15 μ M ). and kinetin (4.6 μ M ). Coincidence of percentage protoplast viability after 13 days (assessed by FDA fluorescence) with plating efficiency after 21 days indicates that FDA fluorescence is an accurate indicator of citrus protoplast viability. 相似文献
13.
Response to chilling of tomato mesophyll protoplasts 总被引:2,自引:0,他引:2
Freshly isolated protoplasts from tomato leaves show two completely different responses to a chilling treatment of 12 h at 7° C prior to culture at 29° C, depending on the presence or absence of glucose in the medium. In the culture medium with glucose as osmoticum, where the rate of cell divisions under optimal culture conditions is relatively high (about 20% plating efficiency), protoplasts were drastically injured by the chilling procedure and died. In the medium with mannitol as the osmoticum instead of glucose, where the plating efficiency even under optimal conditions is rather low (about 8%), protoplasts withstand the chilling procedure. More-over, after the chilling treatment when the protoplasts were transferred to the optimal culture temperature of 29° C, the plating efficiency was raised to about 20%, which is the same level as in the glucose-containing medium without chilling. This effect was not observed when the medium in which the protoplasts were suspended during the chilling period was replaced with fresh medium. This suggests that under these conditions tomato protoplasts produce and excrete a factor in the cold that improves the vitality of the cells or stimulates cell division. The possible relationship between chilling sensitivity of tomato protoplasts and their ability to divide will be discussed. 相似文献
14.
Summary. The ability of plant cells to dedifferentiate represents an important survival strategy invoked in a range of situations from
repair mechanisms following wounding to apomixis. Dedifferentiation requires that somatic cells reprogram and enter the cell
division cycle. This in turn necessitates the accurate partitioning of nuclear content and organelles, such as chloroplasts,
to daughter cells, thereby ensuring continuity of cellular information systems. The distribution of cytoplasm and its organelle
content in mature plant cells is governed by a large, central vacuole, with connections between distant cortical and perinuclear
cytoplasmic domains mediated by transvacuolar strands. Here we examined the changes to vacuolar architecture in Arabidopsis thaliana protoplasts expressing a green-fluorescent protein fusion to a δ-tonoplast-intrinsic protein (δTIP). We found that vacuolar
architecture became increasingly intricate during protoplast culture with the development of numerous transvacuolar strands.
The development of an intricate vacuolar architecture was an actin filament- and not microtubule-dependent process, as is
the case in interphase plant cells. Furthermore, we show that myosin is required for this increased complexity of vacuolar
architecture and the formation of subcortical actin filament arrays. Despite the likelihood that increased vacuolar invagination
would allow better redistribution of cytoplasmic organelles, we found that repositioning of chloroplasts from cortical to
perinuclear cytoplasm was not dependent on transvacuolar strands. Our findings indicate that the vacuole is a dynamic entity
that develops a complex architecture before dedifferentiating plant cells enter cell division.
Supplementary material to this paper is available in electronic form at
Correspondence and reprints: School of Environmental and Life Sciences, University of Newcastle, University Drive, Callaghan,
NSW 2308, Australia. 相似文献
15.
C. L. Webb M. R. Davey J. A. Lucas J. B. Power 《Plant Cell, Tissue and Organ Culture》1994,38(1):77-79
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
16.
Summary A method has been developed for the reuse of cell wall digesting enzymes to isolate protoplasts from actively-growing suspension cultures of plant cells. Protoplasts could be satisfactorily prepared as many as three times using the same enzyme mixture without any loss in yield or viability of the isolated protoplasts. The yields of nuclei isolated from protoplasts prepared with used enzyme solution were comparable to those obtained with fresh enzymes. 相似文献
17.
Chloroplasts prepared from lysed protoplasts of barley mesophyll contain 2–8% of the total acid proteinase activity. This residual activity is not associated with intact chloroplasts isolated by means of density gradient centrifugation. Vacuoles isolated from lysed protoplasts contain 80–85% of the total acid proteinase activity, indicating that the enzyme(s) which is presumably responsible for the degradation of chloroplastic proteins is located largely in the central vacuoles of mesophyll cells. 相似文献
18.
After 1 h plasmolysis in CPW13M solution, highly viable (>75%) protoplasts were isolated from leaves of axenic shoot cultures
of Rosa hybrida L. cv. Abraham Darby using an enzyme mixture containing 1.0% (w/v) Hemicellulase, 0.1% (w/v) Macerozyme, 1.0
(w/v) Cellulase RS, 0.05% (w/v) Pectolyase Y23 and 1.0% (w/v) PVP-10 and from cv. Marie Pavié using an identical mixture but
with Cellulase RS and Pectolyase Y23 at 0.7% (w/v) and 0.1% (w/v), respectively. With both cvs., sustained protoplast division
was achieved after plating in agarose beads with modified KM8p medium containing 1.0% (w/v) polyvinylpyrrolidone (mol. wt.
10 000; PVP-10), 8.91 μM naphthaleneacetic acid (NAA) and 4.44 μM 6-benzyladenine (BA). Protoplast-derived callus gave rise
to roots after transfer to SH medium containing 14 μM 2,4-D.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
19.
A simple method for the isolation of plant protoplasts 总被引:1,自引:0,他引:1
A simple protoplast isolation protocol that was designed to recover totipotent plant protoplasts with relative ease has been
described. The key elements of the protocol are, tissue digestion at slightly elevated temperatures and use of protoplast-releasing
enzymes that are stable and efficient at higher temperatures. Besides enzymes, the protoplast isolation cocktail consisted
of an osmoticum (mannitol or MgSO4), and a protectant (CaCl2 2H2O), all dissolved in distilled water. The protocol has ensured reproducibility, higher yields and is gentle on protoplasts
as the protoplasts obtained were amenable to cell wall regeneration and cell division. Plant regeneration was demonstrated
forNicotiana tabacum cv. Thompson from protoplasts isolated by this method. Wall regeneration and cell division were obtained in other species.
The merits of the protocol are, simple and easy-to-handle procedure, non-requirement of preconditioning of donor plant and
explants, incubation without agitation, satisfactory yields, culturability of the protoplasts isolated and applicability of
the protocol to a large number of species including mucilage-containing plants. 相似文献
20.
Andrea Hoffman Ursula Halfter Peter-Christian Morris 《Plant Cell, Tissue and Organ Culture》1994,36(1):53-58
Conditions for maximising transient expression of GUS in leaf mesophyll protoplasts of Arabidopsis thaliana ecotype C24 were investigated. It was found that the factors most influencing expression levels, with optimum levels in parenthesis, were plasmid DNA quantity (100 g per 5 × 105 protoplasts), inclusion of carrier DNA (50 g), PEG pH and amount (pH above 6, and total PEG concentration at least 9% w/w) and the topological form of the DNA. Linearised plasmid DNA with long flanking sequences 3 and 5 to the marker gene yielded the highest levels of GUS expression.Abbreviations 2,4-d
2,4-dichlorophenoxyacetic acid
- GUS
-glucuronidase
- MU
methylumbelliferone
- PEG
polyethylene glycol
- X-gluc
5-bromo-4-chloro-3-indolyl--glucuronic acid 相似文献