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At least 649 records · Page 36Linked to original sources
PubMed1982

Cytoplasmic hybridization in Nicotiana: mitochondrial DNA analysis in progenies resulting from fusion between protoplasts having different organelle constitutions.

Our previous studies indicated that fusion products with one functional nucleus but organelles of the two fusion partners (i.e. heteroplastomic cybrids) could be obtained by fusing X-irradiated (cytoplasmic donor) with non-irradiated (recipient) Nicotiana protoplasts. The present report deals with the analysis of mitochondria in cybrid populations resulting from the fusion of donor Nicotiana tabacum protoplasts with recipient protoplasts having a N. Sylvestris nucleus but chloroplasts of an alien Nicotiana species, and exhibiting cytoplasmic male sterility. The two fusion parents showed significant differences in restriction patterns of their chloroplast and mitochondrial DNA. Four groups of cybrid plants were obtained by this fusion. All had N. sylvestris nuclei but contained either donor or recipient chloroplasts and had either sterile or fertile anthers. There was no correlation between anther fertility and chloroplast type. The mitochondrial DNA restriction patterns of sterile cybrids were similar to the respective patterns of the sterile fusion partner while the mitochondrial DNA restriction patterns of the fertile cybrids were similar to the respective patterns of the fertile fusion partner. The results indicate an independent assortment of chloroplasts and mitochondria from the heteroplastomic fusion products.

Cell Fusion↗
PubMed1976

Localization of mannan at the surface of yeast protoplasts by scanning electron microscopy.

The beta(1-3)glucanase of Basidiomycete QM 806 was used to prepare Saccharomyces cerevisiae and Candida utilis protoplasts. Plasma membranes isolated from S. cerevisiae contained a small amount of mannose and traces of glucose and ribose. Randomly distributed alpha-mannan was detected by scanning electron microscopy at the surface of prefixed protoplasts using colloidal gold labelled with Concanavalin A as a marker. C. utilis protoplasts were also marked with anti-mannan antibodies. Again the distribution of mannan was random. This experiment indicated also that plasma membrane mannan has the same immunochemical determinants as cell wall mannan. It is hypothesized that mannan is mainly located in the outer layer of plasma membranes.

Candida↗
PubMed1975

Chemical analysis of cell wall regeneration and reversion of protoplasts from Schizophyllum commune.

In the presence of MgSo4 as osmotic stabilizer, nucleated protoplasts of Schizophyllum commune developed a large vacuole and could be isolated on the basis of their low buoyant density. All these protoplasts were capable of wall regeneration and about 50 percent reverted to the hyphal mode of growth in liquid medium. The kinetics of the formation of three main cell-wall components, S-glucan (alpha-1,3-glucan), R-glucan (beta-1,3, beta-1,6-glucan) and chitin were studied from the onset of regeneration. S-glucan and chitin accumulation as well as RNA and protein synthesis started simultaneously after a short lag, but R-glucan formation was delayed. The reversion of hyphal tubes only began after several hours of rapid R-glucan synthesis. Cycloheximide (0.5 mug/ml), inhibiting protein synthesis by 98% inhibited the formation of R-glucan and the reversion to hyphal growth but the formation of chitin and S-glucan did start and continued seemingly unimpaired for several hours. This indicates that the enzymes responsible for the synthesis of S-glucan and chitin remained intact during protoplast preparation. Polyoxin D inhibited both the synthesis of chitin and R-glucan and also the reversion to hyphal growth. However, the synthesis of S-glucan was not suppressed. These inhibitor studies as well as the kinetics of R-glucan formation during normal regeneration suggest that the synthesis of R-glucan is required for the initiation of hyphal morphogenesis.

Adenine↗
PubMed1977

Protoplast fusion of Schizosaccharomyces pombe Auxotrophic mutants of identical mating-type.

Protoplasts of methionine- and lysine-requiring h- mutants isolated from the L972 h- strain of Schizosaccharomyces pombe were fused. The protoplasts were obtained from the cells with enzymes produced by Trichoderma viride. When a mixture of the protoplasts was treated with 30% PEG 4000 solution containing 10 mM CaCl2, cell fusion and complementation was attained with a frequency of 0.17%. Both fusion partners were recovered among the spores after crossing of the fusion products with the strain M210 ade6 h+. Cytological and haploidization examinations showed that the fusion cells are not heterokaryons, and that the increased amount of genetic material is situated in one nucleus.

Ascomycota↗
PubMed1984

A simple method for large-scale electrofusion and culture of plant protoplasts.

A simple method is described for the electrofusion of plant protoplasts. Protoplasts were aggregated in a radio-frequency field (10 V RMS, 0.5 MHz) for 15-30 s with an inter-electrode distance of 5 mm. They were then fused with a 300-V DC pulse. The protoplasts were able to divide after this treatment. A transferrable electrode permitted electrofusion of 1-ml volumes of culture in standard tissue-culture dishes in about 20 s.

Cell Fusion↗
PubMed1985

Optimisation of conditions for infection of isolated potato protoplasts with potato virus X.

Conditions for the reproducible infection of potato protoplasts with PVX were determined. Successful virus infection was achieved when potato protoplasts at a concentration of 3 X 10(5) cells/ml were incubated with 20 micrograms/ml PVX and 2 micrograms/ml Poly-L-ornithine (MW 100,000) for 15 minutes at 0 degrees C. Numbers of protoplasts infected varied between 30 and 50 per cent. Preincubation of PVX with PLO for a minimum of 10 minutes was essential for good infection. The type and molecular weight of polycation used had a major effect on virus infection. PLO at 100,000 MW was the most efficient.

Cell Survival↗
PubMed1976

Investigation on the infection of cucumber mesophyll protoplasts with cucumber mosaic virus.

Isolated protoplasts from the first leaf mesophyll of cucumber plants have been successfully infected in vitro with cucumber mosaic virus (CMV). Virus instability before, during and after inoculation of the protoplasts resulted in low infectivities when extracts were assayed on cowpea; however, viral RNA extraction improved the bioassay technique. Attempts to optimize inoculation and incubation of protoplasts are outlined, incorporating the improved assay.

Culture Techniques↗
PubMed1996

Covisualization by computational optical-sectioning microscopy of integrin and associated proteins at the cell membrane of living onion protoplasts.

Using higher-resolution wide-field computational optical-sectioning fluorescence microscopy, the distribution of antigens recognized by antibodies against animal beta 1 integrin, fibronectin, and vitronectin has been visualized at the outer surface of enzymatically protoplasted onion epidermis cells and in depectinated cell wall fragments. On the protoplast all three antigens are colocalized in an array of small spots, as seen in raw images, in Gaussian filtered images, and in images restored by two different algorithms. Fibronectin and vitronectin but not beta 1 integrin antigenicities colocalize as puncta in comparably prepared and processed images of the wall fragments. Several control visualizations suggest considerable specifity of antibody recognition. Affinity purification of onion cell extract with the same anti-integrin used for visualization has yielded protein that separates in SDS-PAGE into two bands of about 105-110 and 115-125 kDa. These bands are again recognized by the visualization antibody, which was raised against the extracellular domain of chicken beta 1 integrin, and are also recognized by an antibody against the intracellular domain of chicken beta 1 integrin. Because beta 1 integrin is a key protein in numerous animal adhesion sites, it appears that the punctate distribution of this protein in the cell membranes of onion epidermis represents the adhesion sites long known to occur in cells of this tissue. Because vitronectin and fibronection are matrix proteins that bind to integrin in animals, the punctate occurrence of antigenically similar proteins both in the wall (matrix) and on enzymatically prepared protoplasts reinforces the concept that onion cells have adhesion sites with some similarity to certain kinds of adhesion sites in animals.

Binding Sites, Antibody↗
PubMed1996

Evidence for genomic changes in transgenic rice (Oryza sativa L.) recovered from protoplasts.

The occurrence of genomic modifications in transgenic rice plants recovered from protoplasts and their transmission to the self-pollination progeny has been verified with the random amplified polymorphic DNA (RAPD) approach. The plant was the Indica-type rice (Oryza sativa L.) cultivar Chinsurah Boro II. The analysed material was: (1) microspore-derived embryogenic rice cells grown in suspension culture, (2) transgenic plants recovered from protoplasts produced from the cultured cells and (3) the self-pollination progeny (two successive generations) of the transgenic plants. DNA purified from samples of these materials was PCR-amplified with different random oligonucleotide primers and the amplification products were analysed by agarose gel electrophoresis. Band polymorphism was scored and used in band-sharing analyses to produce a similarity matrix. Relationships among the analysed genomes were expressed in a dendrogram. The extensive DNA changes evidenced in cultured cells demonstrate the occurrence of somaclonal variation in the material used to produce protoplasts for gene transfer. Quantitatively reduced DNA changes were also found in the resulting transgenic plants and in their self-pollination progenies. While confirming the stability of the foreign gene in transgenic plants, this work gives molecular evidence for the occurrence of stable genomic changes in transgenic plants and points to in vitro cell culture as the causative agent. RAPDs are shown to be a convenient tool to detect and estimate the phenomenon at the molecular level. The methodology is also proposed as a fast tool to select those transgenic individuals that retain the most balanced genomic structure and to control the result of back-crosses planned to restore the original genome.

DNA, Plant↗
PubMed1994

Genetic transformation of plants by protoplast electroporation.

This article describes an optimized protocol for the electroporation of tobacco mesophyll protoplasts together with notes and data on the effects of various parameters and suggestions for work with protoplasts of other species. In this protocol, electroporation is achieved by means of electrical pulses from a high-voltage, capacitive-discharge unit. Procedures are described for measurement of protoplast viability with Evan's blue, the detection of transient expression of CAT and GUS gene plasmid constructs, and for the recovery of stable transformants based on selection for kanamycin resistance.

Cell Survival↗
PubMed1981

Use of ruthenium red for visualization of the regenerating cell wall in yeast protoplasts.

Ruthenium red was used to stain the surface of fresh and regenerating protoplasts in Saccharomyces cerevisiae. The presence of a dense layer, 10--40 nm wide, was demonstrated on the surface of most of the fresh protoplasts. This layer could be removed neither by repeated washings of protoplasts nor ;by their centrifugation. Ruthenium red proved to be a very useful dye for regenerating walls since it stained both the amorphous and the fibrillar components, but to a different degree, thus permitting their easy differentiation.

Cell Wall↗
PubMed2005

Properties of Saccharopolyspora erythraea strains after protoplast regeneration.

Protoplast formation, stabilization and regeneration was improved for 4 strains (erythromycin producers) of Saccharopolyspora erythraea. A modified medium was developed for protoplast regeneration. Parental and protoplast-regenerated strains exhibited changes in morphology, ultrastructure, and antibiotic production.

Culture Media↗
PubMed1992

Transformation of Streptomyces lincolnensis protoplasts with plasmid vectors.

A method for the preparation and regeneration of protoplasts of Streptomyces lincolnensis is described. Mycelium in the early exponential phase appeared to be most suitable for this purpose and yielded up to 25% regenerated intact cells. Transformation of S. lincolnensis protoplasts was achieved using broad-host-range streptomycete plasmid vectors pIJ622, pMP66, pRS410 and pIJ943 constructed from replicons pIJ101, pSLG33 and SCP2. The efficiency of transformation was 3.10(3) transformants per micrograms plasmid DNA when (2-5).10(7) recipient protoplasts were used. Interspecific transformations showed that there is no efficient restriction system in S. lincolnensis that would limit the transfer of genetic information from S. lividans or E. coli.

Blotting, Southern↗
PubMed1998

Occurrence of a parasexual cycle following the transfer of isolated nuclei into protoplasts of Phytophthora parasitica.

Nuclei isolated from metalaxyl-resistant (Mr) protoplasts of Phytophthora parasitica were transferred into chloroneb-resistant (Cnr) protoplasts of the same species, with an average successful rate of 1x10(-4), using a selective medium containing both metalaxyl and chloroneb. No colonies appeared when self-fusion products of donor nuclei or recipient protoplasts were exposed to the selective medium. Similar results were obtained when the reciprocal nuclear transformation was performed. All the zoospore cultures produced by nuclear transformants contained both Mr and Cnr genes, demonstrating the occurrence of karyogamy since most zoospores are uninucleate. An average of 38% of zoospores produced by nuclear transformants were about twice the size of normal zoospores. Each large zoospore contained a single nucleus, also about twice the size of that in normal zoospores, indicating the occurrence of tetraploidy in these zoospores. The other 42% of zoospores were of normal size but contained both Mr and Cnr genes, suggesting the occurrence of mitotic crossing-over following the production of tetraploidy and the subsequent occurrence of diploidization during zoosporogenesis. For the first time, this study demonstrated the completion of events leading to a parasexual cycle following nuclear transfer.

Cell Fusion↗
PubMed2005

Electrochemical protoplast fusion in citrus.

We report here the development of a novel protoplast fusion method for citrus somatic hybridization. This new procedure, which we have named electrochemical protoplast fusion, is based on chemically induced protoplast aggregation, using a low concentration of polyethylene glycol, and DC pulse-promoted membrane fusion. Based on the results of nucleus and mitochondria molecular analyses, we were successful in using this method to regenerate both symmetric somatic hybrids and cybrids. Various parameters, including pulse intensity, pulse length, and composition of the fusion media, were tested, and the optimum fusion condition selected consisted of two 100-micros pulses of 1,500 V cm(-1). Our conclusion is that electrochemical fusion is a reliable and reproducible method that combines the best features of both the chemical and electrical methods, thereby promoting cell division and high embryogenesis rates of the fused cells. It represents a new approach to citrus somatic hybridization. Various interesting features of this new approach are presented and discussed.

Agriculture↗
PubMed2005

Plant regeneration from protoplasts isolated from embryogenic suspension cultured cells of Cinnamomum camphora L.

An efficient and reproducible protocol is described for the regeneration of Cinnamomum camphora protoplasts isolated from cultured embryogenic suspension cells. Maximum protoplast yield (13.1+/-2.1x10(6)/g FW) and viability (91.8+/-3.8%) were achieved using a mixture of 3% (w/v) cellulase Onozuka R10 and 3% (w/v) macerozyme Onozuka R10 in 12.7% (w/v) mannitol solution containing 0.12% (w/v) MES, 0.36% (w/v) CaCl(2).2H(2)O, and 0.011% (w/v) NaH(2)PO(4).2H(2)O. First divisions occurred 7-10 days following culture initiation. The highest division frequency (24.6+/-2.9%) and plating efficiency (6.88+/-0.8%) were obtained in liquid medium (MS) supplemented with 30 g l(-1) sucrose, 0.7M glucose, 0.1 mg l(-1) NAA, 1.0 mg l(-1) BA, and 1.0 mg l(-1) GA(3). After somatic embryo induction and then shoot induction, the protoplast-derived embryos produced plantlets at an efficiency of 17.5%. Somatic embryos developed into well-rooted plants on MS medium supplemented with 1.0 mg l(-1) 3-indole butyric acid (IBA). Regenerated plants that transferred to soil have normal morphology.

Cells, Cultured↗
PubMed2007

Plant regeneration from cell suspension-derived protoplasts of Phalaenopsis.

Protoplasts isolated from cell suspension culture of Phalaenopsis "Wataboushi" were cultured by (a) embedding in gellan gum-solidified hormone-free 1/2 New Dogashima medium (1/2 NDM) containing 0.44 M sorbitol, 0.06 M sucrose and 0.1 g/l L-glutamine (standard method) and (b) beads method using beads of gellan gum or sodium alginate as the gelling agents which were surrounded by liquid NDM. Although, the two beads methods gave less frequency of initial protoplast division than the standard method, the former finally resulted in higher frequency of microcolony formation than the latter. The highest frequency of microcolony formation (23%) was obtained when protoplasts were embedded in 1% Ca-alginate beads and subcultured every two weeks by replacing the surrounding liquid culture medium with a decrease in sorbitol concentration by 0.1 M. Colonies visible to the naked eyes were observed within 2 months of culture and the regenerated calluses were transferred onto hormone-free NDM supplemented with 10 g/l maltose and 0.3% (w/v) gellan gum, on which PLBs were formed and proliferated profusely. The PLBs were regenerated into plantlets after changing the carbon source to 10 g/l sorbitol and successfully acclimatized to greenhouse conditions.

Orchidaceae↗
PubMed2003

Sugarbeet (Beta vulgaris L.): shoot regeneration from callus and callus protoplasts.

The successful application of recombinant DNA technology for crop plants requires efficient regeneration systems. A detailed study on the regeneration potential of callus and callus-derived protoplasts of a recalcitrant species, sugarbeet, was performed. A reproducible and highly efficient method for induction of regenerable friable callus was established from etiolated hypocotyl explants. A reduced sucrose concentration proved beneficial. Successful shoot regeneration could be demonstrated in 10 out of 12 tested lines. Seed germination, followed by callus induction and shoot regeneration required only a single culture medium. Additionally, the regeneration capacity of roots and root-derived callus was demonstrated. Highly efficient plant regeneration was also achieved when using protoplasts isolated from regenerable friable callus induced on etiolated hypocotyls explants. To our knowledge this represents the first report on callus protoplast to plant regeneration in sugarbeet.

Beta vulgaris↗