Showing papers on "Protoplast published in 1981"
TL;DR: In this article, the chloroplast type (N. tabacum = NT or N. knightiana = NK) in the plants was determined on the basis of the species specific EcoRI restriction pattern of the chloropslast DNA.
Abstract: Protoplasts of Nicotiana tabacum SRI (streptomycin resistant) and of Nicotiana knightiana (streptomycin sensitive) were fused using polyethylene glycol treatment. From three heterokaryons 500 clones were obtained. From the 43 which were further investigated, 6 resistant, 3 sensitive, and 34 chimeric (consisting of resistant and sensitive sectors) calli were found. From eight clones, a total of 39 plants were regenerated and identified as somatic hybrids. Chloroplast type (N. tabacum = NT or N. knightiana = NK) in the plants was determined on the basis of the species specific EcoRI restriction pattern of the chloroplast DNA. Regenerates contained NT (13 plants) or NK (15 plants) plastids but only the plants with the NT chloroplasts were resistant to streptomycin. This finding and our earlier data on uniparental inheritance points to the chloroplasts as the carriers of the streptomycin resistance factor.
364 citations
TL;DR: Fusions Involving Nonviable Protoplasts, Genetic Mapping by Protoplast Fusion, and Detection of extrachromosomal inheritance are studied.
Abstract: Gram-poSlllve bacteria . Gram-negative bacteria . Protoplast Regeneration . GENETIC INTERACTIONS IN INTRASPECIFIC PROTOPLAST FUSIONS ..... ... . . . . . . . . ... . . ... . . . . ... ......... . . ... ... ... . . . . . . . . . . . . ....... . . . . . The Measurement of Fusion . Optim!!l Conditions for Protoplast F.usio� : .. : : .. GenetIc Events After Protoplast FUSIOn In Gram-Poslflve BacteTla .. Streptomyces . Bacillus . ComparisollS between Streptomyces and Bacillus . Genetic Mapping by Protoplast Fusion .. Chromosomal linkage . Detection of extrachromosomal inheritance . Fusions Involving Nonviable Protoplasts .. Protoplast Fusion in Gram-Negative Bacteria . INTERSPECIFIC PROTOPLAST FUSION . PLASMID TRANSFER BY PROTOPLAST FUSION ... . . . . . . . . . . . .. LOSS OF PLASMIDS BY PROTOPLASTING .. . . . . . . . . . . . . . . . .. . . . ........ .. PLASMID TRANSFORMATION AND TRANSFECTION OF PROTOPLASTS .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Techniques .
158 citations
TL;DR: Fusion transfer of a chimeric plasmid consisting of the herpes simplex virus type 1 (strain KOS) EcoRI fragment F in pBR325 resulted in expression of some viral genomic sequences in about 5% of the mammalian cells, as detected by indirect immunofluorescence.
Abstract: The protoplast fusion technique of Schaffner (W. Schaffner, Proc. Natl. Acad. Sci. U.S.A. 77:2163-2167, 1980) has been adapted to introduce cloned herpes simplex virus genes into cultured mammalian cells. The technique involves digesting bacterial cell walls with lysozyme to produce protoplasts and then fusing the protoplasts to mammalian cells by treatment with polyethylene glycol. For monitoring transfer, protoplasts were labeled with the fluorescent dye fluorescein isothiocyanate before fusion. After fusion, greater than 50% of the mammalian cells were fluorescent, demonstrating that bacterial material was transferred with high frequency. Transfer of plasmid pBR325 occurred at frequencies of 1 to 2%, as measured by in situ hybridization. Fusion transfer of a chimeric plasmid consisting of the herpes simplex virus type 1 (strain KOS) EcoRI fragment F in pBR325 resulted in expression of some viral genomic sequences in about 5% of the mammalian cells, as detected by indirect immunofluorescence. One Ltk- cell in 300 to 500 was transformed to the TK+ phenotype after fusion with protoplasts carrying the chimeric plasmid pX1, which consists of pBR322 and the BamHI fragment coding for the herpes simplex virus type 1 thymidine kinase gene.
144 citations
TL;DR: A finely dispersed cell suspension culture from the friable callus of the ‘Black Mexican Sweet’ line of maize was obtained and showed sustained cell divisions and gave rise to callus.
Abstract: A finely dispersed cell suspension culture from the friable callus of the ‘Black Mexican Sweet’ line of maize was obtained. Protoplasts from this cell culture, when grown in a simplified medium described here, showed sustained cell divisions and gave rise to callus.
132 citations
119 citations
TL;DR: This chapter discusses the transition from cell to cell, and the role of RNA and protein in this transition.
Abstract: INTRODUCTION .. TRANSITION FROM CELL TO PROTOPLAST .. Ultrastructure . Hydrolases . RNA. Protein, and DNA Synthesis ..
114 citations
Book•
01 Jan 1981
TL;DR: Reading this book with the PDF genetics of higher plants applications of cell culture will let you know more things about cell culture.
Abstract: Excellent book is always being the best friend for spending little time in your office, night time, bus, and everywhere. It will be a good way to just look, open, and read the book while in that time. As known, experience and skill don't always come with the much money to acquire them. Reading this book with the PDF genetics of higher plants applications of cell culture will let you know more things.
100 citations
TL;DR: Regeneration capacities of isolated protoplasts of 77 dicotyledonous species have been checked and it emerged that the following parameters were appropriate in various taxa: shoot tip regions of axenic shoot cultures, V-KM as protoplast culture medium, high plating densities, repeated dilutions of the regenerated cell clusters, and prolonged culture on V-M agar media.
99 citations
TL;DR: The results confirm the ability of alfalfa to regenerate plants from protoplasts, but with genotypes and media sequences different from those previously published, and illustrate both the usefulness and the problems encountered in applying media developed for tissue-derived calli to regeneration of Plants from protoplast- derived calli.
97 citations
TL;DR: A method was developed for isolation of recombinants after fusion between mutants of S. areus carrying unselectable markers and appears to have simple applications in the construction of strains with multiple mutant characters.
Abstract: The experimental conditions for plasmid transfer and genetic recombination in Staphylococcus aureus and some coagulase-negative staphylococci by protoplast fusion are described. Protoplasts were prepared by treatment with lysostaphin and lysozyme in a buffered medium with 0.7 to 0.8 M sucrose. Regeneration of cell walls was accomplished on a hypertonic agar medium containing succinate and bovine serum albumin. Transfer of plasmids occurred after treatment of the protoplast mixtures with polyethylene glycol (molecular weight, 6,000) not only between strains of the same species but also between parents of different species, although at approximately 100 times lower frequency in the latter case. Recombination of the chromosomal genes in fused protoplasts required simultaneous treatment of the mixed protoplasts with polyethylene glycol and CaCl2. A method was developed for isolation of recombinants after fusion between mutants of S. areus carrying unselectable markers. Antibiotic resistance plasmids were introduced into the parental strains and used as primary markers to detect protoplast fusion. Chromosomal recombinants were found among the clones with both parental plasmids at a high frequency. The method appears to have simple applications in the construction of strains with multiple mutant characters.
92 citations
TL;DR: With the modified procedure described, maximum frequencies of genetic recombinants were obtained by treating parental protoplasts with 40 to 60% polyethylene glycol 1000 and highest recombinant frequencies were obtained under conditions which favoured efficient protoplast regeneration.
Abstract: Protoplasts from four different species of Streptomyces regenerated cells efficiently in hypertonic soft agar medium overlaid on partially dehydrated regeneration medium. The efficiencies of regeneration were strongly dependent upon the incubation temperatures for cell growth and for protoplast regeneration. Cell growth temperatures (before protoplast formation) required for efficient protoplast regeneration varied from species to species, and did not necessarily correlate with the optimum temperatures for protoplast regeneration. Under the best conditions, protoplasts from all four species were able to regenerate viable cells at nearly 100% efficiency and also formed confluent lawns of mycelia when plated in high concentrations. The temperatures for cell growth and protoplast regeneration also affected the frequencies of genetic recombinants obtained by protoplast fusion in S. fradiae, and highest recombinant frequencies were obtained under conditions which favoured efficient protoplast regeneration. With the modified procedure described, maximum frequencies of genetic recombinants were obtained by treating parental protoplasts with 40 to 60% polyethylene glycol 1000.
TL;DR: In this article, 2μm DNA plasmids are present in all brewing strains examined but the growth temperature of some strains is critical before significant numbers of plasmid can be detected.
Abstract: Hybridization, spheroplast (protoplast) fusion and transformation have been studied as means to genetically manipulate brewing and related yeast strains. Hybridization is a secondary technique for verifying the gene composition of recombinants produced by fusion and transformation. Further, it can be used to study gene dosage and gene suppression effects. Fusion is more empirical than manipulative since little control can be exerted over the genotypic makeup of the fusion product. Transformation with native DNA is a more subtle technique than fusion since the introduction of single traits into strains can be controlled. 2μm DNA plasmids are present in all brewing strains examined but the growth temperature of some strains is critical before significant numbers of plasmid can be detected. KEYWORDS Saccharomyces cerevisiae;, Sacch. uvarum (carls bergensis);, Kluyveromyces lactis;, hybridization, fusion, transformation, plasmid.
TL;DR: Results suggest that, in general, the light-induced conversion of NAD to NADP occurs in the chloroplast and is catalyzed by photoactivated NAD kinase using photochemically produced ATP.
Abstract: Light-induced conversion of NAD to NADP was investigated in higher plants. Upon illumination, conversion of NAD to NADP was observed in intact leaves of wheat and pea following incubation in the dark. This conversion was also observed in mesophyll protoplasts of wheat leaves when they were isolated in the dark or isolated in light and then preincubated in the dark. Chloroplasts isolated from wheat protoplasts prepared in the dark carried out the conversion. The conversion in the mechanically isolated spinach chloroplasts was observed only when they were isolated in the dark from leaves preincubated in darkness.Sucrose density gradient centrifugation of wheat protoplast extracts and differential centrifugation of protoplast extracts from various plants showed that most of the NAD kinase was localized in the chloroplasts. Therefore, the conversion of NAD to NADP is considered to occur in the chloroplasts. However, with extracts of maize mesophyll protoplasts, the enzyme was localized in the extrachloroplast fraction. The NAD kinase was activated some 30% by illumination of leaves or protoplasts of pea and wheat after preincubation in the dark.These results suggest that, in general, the light-induced conversion of NAD to NADP occurs in the chloroplast and is catalyzed by photoactivated NAD kinase using photochemically produced ATP.
TL;DR: The results demonstrate that remote somatic hybridization may have applications although true amphidiploids may not be obtainable and the transfer of small units of genetic material between distantly related species by protoplast fusion seems to be a more realistic approach than the combination of complete, highly diverse genomes.
Abstract: A somatic hybrid cell line, cloned from an individual protoplast-fusion product between Arabidopsis thaliana and Brassica campestris, gave rise to formation of numerous plants differing drastically in morphology. Analysis of these various regenerants, all of which originated from one and the same heterokaryon derived from the fusion of two cells, shows the unspecific elimination of chromosomes of both parental species during the callus growth phase. Whereas the parental cells have so far not been sucessfully regenerated into plants, several of their different asymmetric hybrids are capable of morphogenesis. Furthermore, chromosomal analysis indicates extensive recombination. Most of the plants are predoinantly morphologically regular. Abnormalities are mostly limited to the flowers which tend to undergo phyllody. The results demonstrate that remote somatic hybridization may have applications although true amphidiploids may not be obtainable. The transfer of small units of genetic material between distantly related species by protoplast fusion seems to be a more realistic approach than the combination of complete, highly diverse genomes.
TL;DR: Plants were regenerated following intraspecific fusion of leaf protoplasts from two naturally occurring genotypes of Nicotiana debneyi, which differed in the EcoRl fragmentation pattern of chloroplast DNA and in the nuclear-coded phosphoglucomutase isozymes.
Abstract: Plants were regenerated following intraspecific fusion of leaf protoplasts from two naturally occurring genotypes of Nicotiana debneyi. The two genotypes differed in the EcoRl fragmentation pattern of chloroplast DNA and in the nuclear-coded phosphoglucomutase (Pgm) isozymes. There was no conscious selection for hybrid genotypes during protoplast culture or plant regeneration. Among 225 plants screened for Pgm, six were identified as nuclear hybrids. Restriction endonuclease and filter hybridisation analysis revealed that the cytoplasms of the hybrids contained one or other but never both parental chloroplast DNAs. The sorting out of chloroplasts was random and complete; the limit of detecting a rare chloroplast-DNA type in a mixture was 0.1%.
TL;DR: Although the isolated chloroplasts always retained about 13% of the proteolytic activity of the protoplasts, this could be accounted for by that which became associated with the chloroplast during their isolation.
Abstract: Mesophyll protoplasts were isolated from the leaves of wheat and corn seedlings. After purification the protoplasts were judged to be free of contaminating proteases in the isolation enzymes based on specific activity of the proteases in comparison to leaf tissue and their response to inhibitors that "differentiated" between leaf and isolation enzyme proteases. Wheat protoplasts showed rates of photosynthesis of 95 to 100 micromoles O(2) per milligram chlorophyll per hour, while corn exhibited rates of 35 to 85 micromoles O(2) per milligram chlorophyll per hour, indicating the intactness of the chloroplasts within the protoplasts. These chloroplasts were isolated from the protoplasts using the procedure of Robinson and Walker (1979 Arch Biochem Biophys 196: 319-323). Yields of 91 and 82% intact chloroplasts were obtained from wheat and corn, respectively, based on the distribution of ribulose bisphosphate carboxylase in wheat and NADP-malate dehydrogenase in corn. Vacuoles were obtained from the protoplasts using a modification of the techniques of Wagner and Siegelman (1975 Science 190: 1298-1299) and Saunders (1979 Plant Physiol 64: 74-78). The vacuoles were at least 98% free of protoplast contamination as determined by assaying for "marker" enzymes of chloroplasts, mitochondria, and endoplasmic reticulum. Assuming one vacuole per protoplast, the vacuoles contained 4% of the soluble protein of the protoplasts in wheat and 8% in corn. All the proteolytic activity associated with the degradation of ribulose bisphosphate carboxylase in the protoplasts could be accounted for by that localized within the vacuoles. Although the isolated chloroplasts always retained about 13% of the proteolytic activity of the protoplasts, this could be accounted for by that which became associated with the chloroplasts during their isolation.
TL;DR: Somatic hybrid plants of Nicotiana nesophila and N. stocktonii with N. tabacum were produced by protoplast fusion, and local lesion-type resistance to tobacco mosaic virus has been observed in leaves of these somatic hybrid plants.
Abstract: Somatic hybrid plants of Nicotiana nesophila and N. stocktonii with N. tabacum (cultivated tobacco) were produced by protoplast fusion. These combinations cannot be achieved with conventional sexual hybridization, yet are important in that the wild Nicotiana species are resistant to numerous diseases. Hybridity was verified by chromosome number, isoenzyme analysis, morphological characteristics, and genetic behavior. Local lesion-type resistance to tobacco mosaic virus has been observed in leaves of these somatic hybrid plants.
TL;DR: Measurement of total cell protein and cell volumes during culture indicated that a relationship existed between these parameters and the initiation of mitosis, and the significance of these observations is discussed.
Abstract: A microfluorimetric procedure, employing the fluorescent stain 33258 Hoechst, has been developed for the investigation of the process of DNA synthesis during the initial stages of culture of tobacco (N. tabacum cv. Xanthi) leaf protoplasts.
In this system, the freshly-isolated protoplasts exhibited a unimodal distribution of nuclear DNA content characteristic of the diploid state. The almost immediate onset of DNA synthesis during culture resulted in a doubling of nuclear DNA levels prior to the first mitoses. Although the majority of the protoplasts subsequently entered into synchronous mitosis and cell division, a proportion of the remainder developed into large polyploid cells. Upon further culture, the polyploid cells became subdivided into clusters of small diploid cells. Measurement of total cell protein and cell volumes during culture indicated that a relationship existed between these parameters and the initiation of mitosis. The significance of these observations is discussed.
TL;DR: Examination of other strains produced by similar fusions showed that they also contained reiterated DNA sequences, and isolated from a Streptomyces strain produced by interspecific protoplast fusion contained a tandemly repeated six kilobase pair sequence which constituted 15%–18% of the total DNA.
Abstract: DNA isolated from a Streptomyces strain produced by interspecific protoplast fusion of Streptomyces jumonjinensis and Streptomyces lipmanii contained a tandemly repeated six kilobase pair sequence which constituted 15%–18% of the total DNA. Examination of other strains produced by similar fusions showed that they also contained reiterated DNA sequences.
TL;DR: The results demonstrate that amino acid analog resistance and plant regeneration ability can be used as complementing markers to select for fusion hybrids since both can be expressed in the hybrids.
TL;DR: Calculations showed that the efficiency of infection by this method compares favorably with those by the existing methods for inoculating protoplasts from mesophyll cells with TMV-RNA, indicating that the significance of using protoplast from cultured cells for infection with plant viruses and their nucleic acids is discussed.
TL;DR: Fusion of protoplasts from two cell lines of Daucus carota L. resistant to the amino acid analogues S(2-aminoethyl)-L-cysteine (AEC) or 5-methyltryptophan (5MT) produced large numbers of double-resistant clones on a selective medium containing AEC and 5MT.
TL;DR: A simple enzymatic method is described for the isolation of protoplasts from radicles of 24 hours germinated Phaseolus aureus (Mung Bean) seeds, with liquid over agar cultures being superior to liquid medium alone.
TL;DR: It is demonstrated that protoplast fusion is the only possibility for obtaining hybrids between these two species, and no embryos, seeds, or plantlets were obtained, thus demonstrating the feasibility of producing sexual hybrids through in vivo and in vitro methods by cross pollination.
Abstract: After protoplast fusion somatic hybrid calli were obtained by complementation selection between an albino mutant of Datura innoxia and the wildtype of Atropa belladonna (Krumbiegel and Schieder, 1979. Planta 145, 371–375). In the present study experiments are described concerning leaf and shoot induction on several media supplemented with different combinations and concentrations of hormones. Except for fleshy leaves and embryos, no well-formed shoot could be obtained. However, under standard culture conditions after one and a half years, one line produced numerous green shoots, showing a reduced number of chromosomes from Atropa belladonna. The loss of some chromosomes decreased the degree of somatic incompatibility. The additional appearance of shoots with albino sectors, of total albino shoots, and of green shoots showing a different phenotype, demonstrated that the elimination of the chromosomes occurred not only once, but several times. At least one shoot nearly stable in chromosome content and green subline could be obtained possessing only 6 chromosomes of Atropa belladonna and the original chromosome number of Datura innoxia. Experiments were carried out to test the feasibility of producing sexual hybrids through in vivo and in vitro methods by cross pollination. However, no embryos, seeds, or plantlets were obtained, thus demonstrating that protoplast fusion is the only possibility for obtaining hybrids between these two species.
TL;DR: Intact protoplasts show no oxygen evolution with 3-phosphoglycerate or with oxalacetate plus pyruvate as substrates, even when these substances are provided at high concentrations, while protoplast extracts and chloroplasts display rates of oxygen evolution of 2-3 µmol min-1 (mg Chl)-1 with the same substances.
Abstract: A procedure is described for the preparation of metabolically active mesophyll protoplasts from maize, and of functional, intact chloroplasts from these protoplasts. Intact protoplasts show no oxygen evolution with 3-phosphoglycerate or with oxalacetate plus pyruvate as substrates, even when these substances are provided at high concentrations. On the other hand, protoplast extracts and chloroplasts display rates of oxygen evolution of 2-3 µmol min-1 (mg Chl)-1 with the same substances. Pyruvate stimulates oxalacetate-dependent oxygen evolution substantially, indicating good coupling between non-cyclic electron flow and phosphorylation. Low PI concentrations stimulate 3-phosphoglycerate-dependent oxygen evolution; high PI concentrations, and pyridoxal phosphate, inhibit this activity, suggesting a common carrier for 3-phosphoglycerate and PI.
TL;DR: The application of an electric field pulse of proper strength and duration to a yeast protoplast suspension turned out to be a more effective tool in production of fusion products that conventional methods.
Abstract: Prototrophic colonies could be selected on minimal medium after mixing of protoplasts from diauxotrophic mutants of the yeasts Saccharomycopsis lipolytica and/or Lodderomyces elongisporus and treatment with polyethylene glycol (PEG) in the presence of calcium chloride. This is the result of protoplast fusion and complementation of auxotrophic deficiencies. Under identical conditions an electric field pulse in the mus-range applied via an electric discharge to the protoplast-PEG mixture resulted in a drastic enhancement of the protoplast fusion rate. The presence of polyethylene glycol was demonstrated to be a prerequisite for fusion in this case, too. The frequency of hybrid formation detected a prototrophic colonies could be increased in the case of intraspecific fusion at initial electric field strengths between 2.5 and 5 kV . cm-1. The application of an electric field pulse of proper strength and duration to a yeast protoplast suspension turned out to be a more effective tool in production of fusion products that conventional methods. Large numbers of parasexual hybrids for different selection programmes in yeast genetics and for industrial purpose may be delivered by this technique.
TL;DR: Tobacco mesophyll protoplasts cultivated in vitro do not synthesize a measurable quantity of chloroplastic ribosomal RNA, but actively synthesize cytoplasmic ribosome RNA, polyadenylated RNA, and proteins, which are related to the ageing phenomenon induced by isolation from the plant and in-vitro culture.
Abstract: Tobacco mesophyll protoplasts cultivated in vitro do not synthesize a measurable quantity of chloroplastic ribosomal RNA, but actively synthesize cytoplasmic ribosomal RNA, polyadenylated RNA, and proteins. These syntheses are essentially independent of the presence of hormones in the culture medium and are thus related to the ageing phenomenon induced by isolation from the plant and in-vitro culture. At all stages of culture and in all culture media, protoplasts incorporate low levels of thymidine into their DNA. However, the incorporation of considerable quantities of thymidine, indicative of the S phase, only takes place after 25–30 h and requires the presence of auxin and cytokinin.
TL;DR: The myo-inositol pathway is operative in cell wall regenerating tobacco protoplasts in comparison with that derived from tobacco mesophyll and tobacco tissue cultured on solid agar or in suspension, indicating the secretion of pectic material into the culture medium.
TL;DR: The authors' method allows the production of a large number of parasexual hybrids for different purposes and genetic evidence for fusion has been obtained by isolating parasexually produced hybrids resulting from combination of the genomes of both mutants.
Abstract: Under the action of a high electric field pulse a pronounced stimulation of yeast protoplast fusion initiated by polyethylene glycol and Ca (2+) ions was found. The fusion rate was enhanced by a factor of 200 for different genetically marked strains of the yeast Saccharomyces cerevisiae as compared with fusion without electric field treatment. Genetic evidence for fusion has been obtained by isolating parasexually produced hybrids resulting from combination of the genomes of both mutants.Our method allows the production of a large number of parasexual hybrids for different purposes.
TL;DR: Direct measurements of [(14)C]maltose uptake indicated transport across the chloroplast envelope at rates similar to the transport of glucose, and rapid separation of cytoplasm and chloroplasts following exposure to (14)CO(2) showed that maltose was present in both fractions.
Abstract: When mannose was included in the enzyme incubation medium during the preparation of protoplasts from leaves of spinach, maltose was an early product of protoplast photosynthesis and, after 12 minutes, accounted for up to 15% of the 14C incorporated from 14CO2. Maltose was not detected in protoplasts prepared in the normal enzyme medium. Rapid separation of cytoplasm and chloroplasts following exposure to 14CO2 showed that maltose was present in both fractions. Direct measurements of [14C]maltose uptake indicated transport across the chloroplast envelope at rates similar to the transport of glucose. The source of maltose and site of its initial formation are discussed.