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1.
Summary The cell wall regeneration on protoplasts derived from maize mesophyll cells was compared with wall regeneration on protoplasts
derived from suspension cultured cells using light microscopy, transmission electron microscopy, and mass spectrometry. The
time course of cell wall regeneration has shown that the mesophyll protoplasts regenerated walls much slower than the protoplasts
derived from cultured cells. Moreover, cell wall materials on the mesophyll protoplasts were often unevenly distributed. Electron
microscopy has further demonstrated that the mesophyll protoplasts have less organized and compact walls than the protoplasts
from cultured cells. Chemical analysis revealed that the mesophyll protoplasts had a lower ratio of β-(1–3)-glucan to β-(1–4)-glucan than protoplasts from cultured cells. The significance of these results for the viability and development of
protoplasts in culture is discussed.
National Research Council of Canada paper no. 32458. 相似文献
2.
A method is described for regenerating callus from mesophyll protoplasts of a winter variety of Brassica napus. The method combines the use of Ficoll in an initial liquid medium, enhancing early protoplast division and cell colony formation, with a transfer to an agarose system after 10 days culture to give rapid microcalli formation. Further transfers resulted in callus regeneration and the initiation of organogenesis. 相似文献
3.
Expression of totipotency and regeneration potentiality of plant protoplasts is a complex developmental phenomenon. The isolation per se is a stress-inducing procedure, during which, among others, active oxygen species (AOS) are generated. Thereafter, protoplasts undergo cell wall reconstitution, cell elongation and re-enter the cell cycle. AOS are known to participate in cell wall cross-linking and recently hydroxyl radicals were proposed to participate in cell wall loosening. On the other hand, if the antioxidant genes and the overall AOS scavenging machinery is not induced, protoplasts may suffer from oxidative stress and peroxidation of membrane lipids. In an effort to identify potential factors contributing to recalcitrance of plant protoplasts, we present the available information, which correlates AOS and oxidative stress with cell wall reconstitution, dedifferentiation, cell cycle progression, and cell death. Reduced antioxidant machinery and altered redox homeostasis seem to affect the regenerating potential of plant protoplasts and inevitably the protoplast fate (re-entry into cell cycle or cell death). 相似文献
4.
Summary Tobacco mesophyll protoplasts have been examined by electron microscopy during isolation procedures and after 24 hours culture in a medium known to support cell wall regeneration. During isolation the plasmalemma shows little structural differentiation apart from the formation of small vacuoles in the cytoplasm. After 24 hours of culture, several types of activity are seen at the plasmalemma surface. Microtubules, profiles of endoplasmic reticulum, electron dense granules and coated vesicles are associated with the inner surface of the membrane. External to the plasmalemma fibrillar structures occur, both as extensive networks and as individual fibrils apparently associated with the membrane itself. Techniques and criteria for electron microscopy are presented, and the results discussed in terms of plasmalemma function and the regeneration of the cell wall. 相似文献
5.
Protoplasts from Olea europaea L. have been compared in terms of their yield, viability, cell division and callus differentiation. Viable protoplasts were isolated from in vitro cultured leaves and cotyledons by an overnight incubation in an enzyme solution containing 1–1.5% driselase and 0.5M sucrose. This method allowed high yield of purified protoplasts, which floated and formed a dark green band at the meniscus, after centrifugation. Purified protoplasts were diluted to 3×10 4 protoplasts·ml –1 in culture medium. After cell wall regeneration, protoplasts gradually increased their volumes under appropriate conditions. The first divisions occurred during the second week in culture. Division efficiency ranged from 5.2 to 9.8% after 20 days in culture. Two weeks later visible microcolonies developed only from cotyledon protoplasts. After 6 weeks in culture, the microcalli were transferred to a solidified culture medium with 0.6% agarose, which induced active callus growth.Abbreviations OM
olive proliferation medium, Rugini 1984
- Omg
OM for the germination of olive embryos
- OMr=OM
for root induction
- OMp=OM
for protoplasts
- OMc=OM
for callus
- BN
Bourgin and Nitsch medium 1967
- IBA
indol-3-butyric acid
- NAA
naphthalene acetic acid
- 2,4-D
dichlorophenoxyacetic acid. 相似文献
6.
Summary A method to remove the exine from mature tobacco pollen and to release numerous intact pollen protoplasts has been developed. Post-anthesis binucleate pollen was treated with water, buffered with MES at pH 5.5, for two hours. Rupture of the exine was caused by the force of pollen hydration exposing the intine to subsequent enzymatic maceration. The high osmotic pressure (1000 mOsm·kg -1 H 2O) of pollen protoplasts required a special maceration medium, 4% KCl (w/v). Action of an enzyme solution containing 1% (w/v) Macerozyme and 1% (w/v) Cellulase gave rise to viable protoplasts within 4 hours. When cultured in a tobacco mesophyll protoplast culture medium, the pollen protoplasts underwent regeneration of a cell wall, formation of various tube-shaped structures, and division of the generative nucleus into two nuclei. Using a PEG/Ca 2+ method pollen protoplasts were fused with diploid mesophyll protoplasts. Evidence of transfer of chloroplasts into the pollen protoplasts was observed after one day of culture.Abbreviations BCP
bromocresol purple
- FDA
fluoresceindiacetate
- MES
2-(N-morpholino) ethanesulfonic acid
- PEG
polyethyleneglycol 相似文献
7.
Oat leaf mesophyll protoplasts were evacuolated and shown to develop acidic
vacuoles when cultured for 3 d. Vacuole development was followed by cell
wall formation. Developing vacuoles, stained with acridine orange, took the
form of a tubular network when viewed by confocal laser scanning
microscopy. The tubules expanded and fused to form a series of
interconnected vacuoles. When thin sectioned material was examined by
transmission electron microscopy, the tubular network appeared as a number
of small, expanding vesicles. The vacuolar
H +-ATPase, H +-PPase and a
membrane integral protein of 23 kDa (VM23) were shown, by Western blotting,
to be removed from protoplasts following evacuolation. After 5 d culture
the H +-ATPase and H +-PPase,
but not VM23, were detectable in microsomal fractions.This study describes,
for the first time, successful vacuole regeneration in a monocotyledenous
plant. This regeneration follows a similar pattern to that seen in
non-cereal protoplasts. 相似文献
8.
Methods are described for the enzymatic release of protoplasts from leaves of Petunia hybrida and for the utilization of protoplasts in studies in plant developmental biology. As a result of spontaneous fusion during cell wall degradation of leaf material, fresh preparations can contain a high proportion of multinucleate protoplasts. This level can be dramatically reduced by a gradual plasmolysis of the material prior to enzyme incubation.Leaf protoplasts maintained in liquid media are seen to undergo cell wall synthesis, “budding,” and limited regenerated cell division sometimes associated with anthocyanin production. Under such conditions, multinucleate cells are formed as a result of mitosis without cytokinesis.Protoplasts, plated out in a fully defined medium, undergo cell wall synthesis followed by sustained progeny cell division with eventual cell colony production. Cell colonies, derived from individual mesophyll protoplasts, grow rapidly upon subculture, to produce callus capable of shoot differentiation and ultimately whole plant formation. Protoplasts isolated from varieties of P. hybrida were found to differ in their cultural requirements. 相似文献
9.
Stomatal movement is an energetic oxygen-requiring process. In the present study, the effect of oxygen concentration on mitochondrial respiratory activity and red-light-dependent photosynthetic oxygen evolution by Vicia faba and Brassica napus guard cell protoplasts was examined. Comparative measurements were made with mesophyll cell protoplasts isolated from the same species. At air saturated levels of dissolved oxygen in the protoplast suspension media, respiration rates by mesophyll protoplasts ranged from 6 to 10μmoles O 2 mg ?1 chl h ?1, while guard cell protoplasts respired at rates of 200–300 μmoles O 2 mg chl ?1 h ?1, depending on the species. Lowering the oxygen concentration below 50–60 mmol m ?3 resulted in a decrease in guard cell respiration rates, while rates by mesophyll cell protoplasts were reduced only at much lower concentrations of dissolved oxygen. Rates of photosynthesis in mesophyll cell protoplasts isolated from both species showed only a minor reduction in activity at low oxygen concentrations. In contrast, photosynthesis by guard cell protoplasts isolated from V. faba and B. napus decreased concomitantly with respiration. Oligomycin, an inhibitor of oxidative phos-phorylation, reduced photosynthesis in mesophyll cell protoplasts by 27–46% and in guard cell protoplasts by 51–58%. The reduction in both guard cell photosynthesis and respiration following exposure to low oxygen concentrations suggest close metabolic coupling between the two activities, possibly mediated by the availability of substrate for respiration associated with photosynthetic electron transport activity and subsequent export of redox equivalents. 相似文献
10.
Large yields (1.85 × 10 7/g.f.wt.) of viable protoplasts were obtained from leaves of axenic shoot cultures of Malus Xdomestica Borkh. cv. Greensleeves. Protoplasts cultured in liquid or agarose semi-solidified KM8P medium underwent cell wall regeneration and colony formation.Protoplast-derived cell colonies developed to callus on semi-solid KM8 medium. This is the first report of callus formation from mesophyll protoplasts of apple.Abbreviations BAP
6-benzylaminopurine
- K
kinetin
- Z
zeatin
- GA 3
gibberellic acid
- IBA
3-indole butyric acid
- NAA
1-naphthalene acetic acid
- IAA
3-indole acetic acid
- ABA
abscisic acid
- f.wt.
fresh weight
- MS
Murashige and Skoog (1962) 相似文献
11.
A protocol for plant regeneration from mesophyll and callus protoplasts of Robinia pseudoacacia L. was developed. For leaves from in vitro raised shoots, an enzyme combination of 2.0% cellulose and 0.3% macerozyme for
a digestion period of 20 h resulted in the best yield of protoplasts (9.45 × 10 5 protoplast/g fresh weight). Mesophyll-derived protoplasts started cell wall regeneration within 24 h of being embedded in
Nagata and Takebe (NT) medium supplemented with 5 μM NAA and 1 μM BAP followed by the first cell division on day three of
culture and micro-colony (32 cells) formation within day 7–10 in the same medium. However, using callus as the starting material,
a combination of 2.0% cellulose and 1.0% macerozyme for a digestion period of 24 h gave the highest protoplast yield (3.2 × 10 5 protoplast/g fresh weight). Cell wall regeneration in callus-derived protoplasts started within 24 h followed by the first
cell division on the day three (96 h) and the appearance of microcolonies of more than 32 cells by the end of first week (144 h)
of culture on solid WPM medium supplemented with 5 μM NAA and 1 μM BAP. Microcalli were visible to the naked eye after 45 days
on solid WPM medium. Proliferation of macro-calli was successfully accomplished on solid Murashige and Skoog (MS) medium with
5 μM NAA and 5 μM BAP. Both mesophyll and callus protoplast-derived calli produced shoots on MS medium with 0.5 μM NAA and
1 μM BAP within 25–30 days and multiplied on MS medium with 1.25 μM BAP. Excised microshoots were dipped in 1–2 ml of 2.0 μM
IBA for 24 h under dark aseptic conditions and transferred to double sterilized sand for rooting. The flasks containing sand
were inoculated with Rhizobium for in vitro nodulation. Forty-five plants transferred to pots in the glasshouse established well. 相似文献
12.
Summary To identify possible reasons that may contribute to recalcitrance in plant protoplasts, the time course of new cell wall deposition
was studied by scanning electron microscopy in protoplasts of a recalcitrant species, the grapevine. Results showed that microfibrils
were developed after 2 days of culture, that complete cell wall formation occurred on Day 6 to 7 of protoplast culture, and
its ultrastructural appearance was identical to that of grapevine leaf-derived callus cells. In addition, a comparative study
was undertaken on [U- 14C]glucose uptake and incorporation in ethanol-soluble, cellulosic, and noncellulosic polysaccharide fractions in protoplasts
of grapevine and of a readily regenerating species, tobacco, during culture. There was a significantly higher [U- 14C]glucose uptake by tobacco than by grapevine protoplasts. The label distribution in the ethanol-soluble, cellulosic, and
noncellulosic fractions of newly synthesized cell walls differed quantitatively between the two species. In particular, the
labeled glucose incorporated in the noncellulosic cell wall fraction was threefold greater in tobacco than in grapevine protoplasts.
Differences were also revealed in the monosaccharide composition of this fraction between the two species. Addition of dimethyl
sulfoxide to the culture medium resulted in a dramatic increase in [U- 14C]glucose uptake by grapevine protoplasts, whereas it exhibited a limited effect in tobacco protoplasts. It showed no effect
on the ultrastructural characteristics of new cell wall nor on the incorporation rate of labeled glucose in the cellulosic
and noncellulosic cell wall fractions. 相似文献
13.
Isolated mesophyll protoplasts of Brassica juncea (L.) Czern., cv. RLM 514 upon culture in suitable growth medium, regenerated cell wall, underwent cell division and formed cellular colonies. Subsequent induction of embryoid (embryogenesis) and shoot bud (organogenesis) formations in such cell masses resulted in regeneration of 186 and 42 plantlets respectively.Abbreviations NT
Nagata and Takebe, 1971
- B5
Gamborg et al. 1968
- KM
Kao and Michayluk, 1975
- GK 2
Schenck and Hoffmann, 1978
- MS
Murashige and Skoog, 1962
- BAP
6-benzyladenine
- 2, 4-D
2, 4-dichlorophenoxyacetic acid
- NAA
napthaleneacetic acid
- GA 3
gibberellic acid 相似文献
14.
Protoplasts were isolated from leaves of glasshouse-grown plants of Centaurea cyanus and axenic shoot cultures of Senecio x hybridus. Upon culture, using modified MS-based media, protoplasts of both systems entered division to produce callus, followed by plant regeneration. Leaf protoplasts of Callistephus chinensis entered sustained division only following the preconditioning for 24h of peeled leaf tissues on agar-solidified MS-based medium. Protoplasts were also isolated from cell suspensions of C. chinensis and divided in MS-based or KM media. However, only leaf mesophyll protoplasts of Callistephus produced callus, which developed shoots.The establishment of protoplast-to-plant protocols for these ornamental species has provided a basis for broadening their gene pools through somatic hybridisation.Abbreviations BAP
6-benzylaminopurine
- NAA
-naphthaleneacetic acid
- 2,4-D
2,4-dichlorophenoxyacetic acid
- MS
Murashige and Skoog (1962)
- KM
Kao and Michayluk (1975)
- g.f.wt.
gram fresh weight 相似文献
15.
Moricandia arvensis is of interest as it is a dicotyledonous species which has C 3 — C 4 intermediate photosynthesis, a mechanism which results in enhanced recapture of photorespired CO 2. Leaves from cultured shoot tips were used as a source for mesophyll cell protoplasts. Approximately 1% of the protoplasts which survived the first few days of culture produced calli. On a suitable regeneration medium, 30–60% of the calli regenerated one or more shoots. From among the regenerating shoots eight were selected, transferred to soil and grown to flowering in the glasshouse; all were fertile. The development of a protoplast regeneration system provides the opportunity to use transformation and somaclonal variation as tools in the genetic analysis of the C 3–C 4 character in this species.Abbreviations GDC
glycine decarboxylase
- 2,4-D
2,4-dichlorophenoxyacetic acid
- BAP
benzyl aminopurine
- NAA
naphthalene acetic acid
- ABA
abscisic acid 相似文献
16.
An efficient procedure for plantlet regeneration from chicory mesophyll protoplasts has been developed in order to perform
protoplast fusion experiments. Protoplasts were isolated from a genotype of Italian red chicory (CH 363) and purified by centrifugation
in a solution containing 13% (w/v) sucrose to collect uniform protoplasts in size. After 2 days culture at a density of 2×10 4 protoplasts ml −1 of liquid medium, protoplasts were cultured following three different procedures: in liquid medium, stratified in semi-solid
medium, and embedded in Ca-alginate droplets. Four different media were used and culture procedures were evaluated recording
the protoplast viability, protoplast division frequency and plating efficiency for each experiment. The embedding of protoplasts
in Ca-alginate droplets enhanced both division frequency and plating efficiency for chicory mesophyll cells. Furthermore,
this procedure shortened the cycle of plant regeneration from protoplasts, which could be completed in eight weeks.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
17.
We investigated the function of cell wall hydroxyproline-rich glycoproteins by observing the effects of a selective inhibitor of prolyl hydroxylase, 3,4-dehydro-L-proline (Dhp), on wall regeneration by Nicotiana tabacum mesophyll cell protoplasts. Protoplasts treated with micromolar concentrations of Dhp do not develop osmotic stability and do not initiate mitosis. The architecture of regenerated cell walls was examined using deep-etch, freeze-fracture electron microscopy of rapidly frozen tobacco cells. Untreated protoplasts assemble a dense fibrillar cell wall consisting of laterally associating subelementary fibrils. In contrast, treatment of protoplasts with Dhp alters the structure of the regenerated wall fibrils in several ways: first, the microfibrils are coated with globular knobs; second, some larger fiber bundles have an open ribbon-like appearance; and third, the smallest subelementary fibrils were not visible. Tobacco cells develop an abnormal morphology as a consequence of this abnormal cell wall structure. Thus, inhibition of prolyl hydroxylase results in the regeneration of a cell wall with abnormal structural and functional properties. These data provide experimental evidence that hydroxyproline-rich glycoproteins are important for the structural integrity of primary cell walls and for the correct assembly of other wall polymers, and that wall structure is an important regulator of cell division and cell morphology. 相似文献
18.
The presence of auxin (2,4-D), in the culture medium of tobacco ( Nicotiana tabacum var Maryland) mesophyll protoplasts is necessary both for cell wall regeneration and for passage of the cells from phase G 0 to phase G 1 of the cell cycle. Among about 250 proteins synthesized by protoplasts and characterized by their migration in a two-dimensional electrophoresis gel, 2,4-dichlorophenoxyacetic acid affects the synthesis of 11. Nine proteins are synthesized at a reduced level in the presence of the hormone, of which three are rapidly labeled and short-lived, while the others, which are long-lived, become detectable only after 2 hours of radioactive labeling, suggesting that they undergo slow posttranslational maturation. These nine proteins are proline-rich but the proline radicals are not strongly hydroxylated. The synthesis of these proteins is no longer inhibited by auxin if dichlorobenzonitril, a weed-killer which inhibits cell wall reformation of tobacco protoplasts, is added to the culture medium. Two proteins are only synthesized if protoplasts are cultivated in an auxin-containing medium. These polypeptides are rapidly labeled, and are long-lived. The inhibition of cell wall reformation by dichlorobenzonitril does not modify their synthesis. These results suggest that proteins whose synthesis is reduced by auxin are related to cell wall reformation and that they do not play a role in the induction of the cell cycle. In contrast, proteins whose synthesis is stimulated in the presence of auxin are good candidates for a role in the induction of the cell cycle. 相似文献
19.
A technique is described for the routine isolation of protoplasts from storage parenchyma cells of potato tubers grown in vitro. The protoplasts typically contained many starch grains. On culture, most of the starch grains were metabolised during the first 7 days, after which the cells began to divide. Following further culture, protoplast-derived colonies and calli were obtained, from which shoots and intact plants were regenerated. Cytological study of regenerated plants showed that the majority were octaploid or aneuploid at the octaploid level. This aspect is compared with plants regenerated from mesophyll protoplasts of potato. The use of tuber protoplasts for studies on tissue-specific transient gene expression of chimeric gene constructs, following their introduction into the protoplasts by electroporation, is discussed, together with the uses of tuber protoplasts in fundamental physiological and biochemical studies. 相似文献
20.
An efficient technique has been developed for the isolation of barley megasporocyte protoplasts at early meiotic prophase. Ovules were dissected out of ovaries under aseptic conditions, subjected to a brief enzymatic digestion, and then transferred to a modified Kao medium with 90 g/l sucrose and 20 mM CaCl 2. A small incision was made with a scalpel through the softened epidermal cell layer of the nucellus and the megasporocyte could then be liberated into the medium by applying gentle pressure on the nucellus. The megasporocyte appeared to be completely devoid of a wall and changed its in situ pyriform shape to completely spherical when extruded into the medium. Four to nine protoplasts could typically be isolated per spike. Protoplasts cultured in medium degenerated after a few days. Viability was dramatically improved if protoplasts were co-cultivated with barley microspores undergoing microspore embryogenesis. More than half of the protoplasts were still alive after 6 days of culture, and in some cases they survived more than 12 days of culture. Fluorescence microscopy of the cultured protoplasts stained with 4,6-diamidino-2-phenylindole (DAPI) or aniline blue revealed that the protoplasts remained uninuclear and reformed their callose wall. 相似文献
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