Showing papers on "Protoplast published in 1976"
TL;DR: It is concluded that the observed formation of prototropic bacteria is due to protoplast fusion, a process which does not induce prophage development, and that the only stable products of the resulting diploid state are haploid recombinants.
Abstract: Prototrophic Bacillus subtilis cells can be formed in the presence of DNase as a result of cell fusion occurring in mixed populations of protoplasts derived from two parental strains which are both nutritionally-complementing and polyauxotrophic. No prototrophs ever appear from mixed nonprotoplasted bacteria, or from the auxotrophic parental protoplasts plated separately. The frequency of prototroph formation, which is appreciable only when the mixed protoplasts are exposed to polyethylene glycol treatment, may exceed 1 X 10(-4) of the total protoplast population initially present, which is 1 to 4 X 10(-3) of those protoplasts which reverted to the bacillary form. It is strongly dependent on the number and chromosomal location of the markers used in the selection of the prototrophs, and it is unaffected when either one of the parental strains bears the phage phi105 in the inducible prophage state. No auxotrophic bacteria, parental or otherwise, were found as segregants from repeatedly isolated protrotrophic clones growing in a nonselective medium. Unselected markers segregate among the selected recombinants. It is concluded that the observed formation of prototropic bacteria is due to protoplast fusion, a process which does not induce prophage development, and that the only stable products of the resulting diploid state are haploid recombinants.
182 citations
TL;DR: Heterokaryon formation, at high frequency, after induced intraspecific fusion between protoplasts of auxotrophs of Penicillium chryosogiwim, P .
Abstract: Although protoplast fusion is of current interest because of its possibilities in pure and applied genetics, only a few reports exist on intraspecific fusion between fungal protoplasts and subsequent selection of heterokaryons. Naturally-occurring fusion between protoplasts of Geotrichum candidurn (Ferenczy, Kevei & Zsolt, I 974) and Aspergillz4s nidulans (Ferenczy, Kevei & Szegedi, I 975) has been reported. Binding & Weber (1974) described fusion between protoplasts of Phycomyces blakc~slceanus induced by seawater or calcium ions at high pH, but the frequency of heterokaryon formation was low in all cases. We here report heterokaryon formation, at high frequency, after induced intraspecific fusion between protoplasts of auxotrophs of Penicillium chryosogiwim, P . putulurn, P. roquejortii, A. nidulans, A . niger and Cephalosporium acrernonium, and fusion between protoplasts of P. chrysogenu~?z and P. rzotutunz auxotrophs, using solutions containing polyethylene glycol (PEG) mol.wt. 6000.
157 citations
TL;DR: Haploid microspore-derived plants from amphidiploid Brassica napus give rise in culture to very large numbers of secondary stem embryoids from single epidermal cells, which form larger plantlets which can be grown to maturity.
Abstract: Haploid microspore-derived plants from amphidiploid Brassica napus give rise in culture to very large numbers of secondary stem embryoids from single epidermal cells. The embryoids can be excised, transferred to fresh medium and induced to repeat the process 4–6 times. Eventually the embryoids form larger plantlets which can be grown to maturity. Spontaneous amphihaploid plants or microspore-derived plants growing either in vitro or in vivo have been used as a source of plant protoplasts. Such protoplasts have been induced to produce calluses capable of both root and shoot production. The limited number of plantlets so far obtained from large numbers of calluses have retained their haploidy. The significance of haploid single-cell cultures of such agriculturally important fodder crops is discussed.
123 citations
TL;DR: The unusual chromosome numbers found in the parasexual hybrids may have been due primarily to triple fusions followed by losses during callus growth, accompanied by selection for a particular range of aneuploidy favorable for development of plantlets from calli.
Abstract: Protoplasts of Nicotiana glauca and N. langsdorffi were prepared from leaf tissue by enzymatic digestion and were fused with the aid of polyethylene glycol. The mixed population of protoplasts was grown first on an enriched medium (M3 of Kao, et al.) and was then transferred to a medium lacking phytohormones, which selects against the parental types. A total of 174 calli that grew on the hormoneless medium were obtained. Mature flowering plants were differentiated from 19 different calli, and more than one from three of these, making 23 regenerated plants in all. Each of the plants was shown to be a parasexual hybrid in that it formed tumors; and the corolla, leaf, and plant habit were similar to, but somewhat different from, the amphiploid produced by cross pollination. The parasexual hybrids were examined cytologically and, instead of the amphiploid number 42, they were found to have a range of from 56 to 64 chromosomes, which accounted for the different characteristics observed. At meiosis mostly bivalents were formed and pollen fertility was high, averaging 84 percent. Seeds or progeny have been obtained from 16 of the hybrids. The unusual chromosome numbers found in the parasexual hybrids may have been due primarilymore » to triple fusions (giving 60-66 chromosomes) followed by losses during callus growth, accompanied by selection for a particular range of aneuploidy (2n = 56 to 64) favorable for development of plantlets from calli.« less
114 citations
TL;DR: A procedure for protoplast formation in Escherichia coli is described and removal of the cell wall was confirmed by examination of cells in thin-section preparations.
Abstract: A procedure for protoplast formation in Escherichia coli is described. Removal of the cell wall was confirmed by examination of cells in thin-section preparations.
91 citations
TL;DR: In culture only N. sylvestris had a low plating efficiency, although N. otophora protoplasts were found to be sensitive to light, and plants were recovered from callus tissue originating from isolated protoplast of all the species and hybrids.
87 citations
TL;DR: A rapid method based on fatty acid analysis for the simultaneous quantitative determination of phospholipid and lipopolysaccharide content of whole cells is reported and is interpreted as results of the adaptation of the L-form to life in the state lacking an envelope by formation of a physically more stable but still sufficiently fluid protoplast membrane.
Abstract: Cells of the stable, protoplast L-form of Proteus mirabilis contain 1.5 to 2 times more extractable lipid, mostly phospholipid, per dry weight than cells of the bacterial form.
Under identical conditions of cultivation the qualitative and quantitative composition of the phospholipid is very similar in both cell forms. The range of mole percentages of individual phospholipid species is 76–80 for phosphatidylethanolamine, 10–13 for phosphatidylglycerol, 3.9–5.5 for diphosphatidylglycerol and 1.0–2.1 for lysophospholipid. However, all phospholipid species in the L-form differ from those of the bacterial form by a lower content of long-chain fatty acids and a higher content of short-chain fatty acids.
Growth of the L-form in the presence of growth-stimulating horse serum results in a change of phospholipid composition accompanied by the uptake of phosphotipid and fatty acids from the serum into L-form phospholipid.
L-form protoplasts synthesize the same two types of Lipopolysaccharide, I and II, that were previously identified in the bacterial form of Proteus mirabilis. However, only small amounts of the more hydrophilic lipopolysaccharide II are present in the L-form. Lipopolysaccharides from both cell forms have virtually identical polysaccharide compositions but differ strikingly in the relative content of fatty acids in their lipid-A moieties. Molar ratios of tetradecanoic acid, hexadecanoic acid and 3-hydroxytetradecanoic acid are 5:1:6 in the bacterial form and 5:0.1:6 in the L-form grown in serum-free medium.
The observed differences between the bacterial form and the protoplast L-form are interpreted as results of the adaptation of the L-form to life in the state lacking an envelope by formation of a physically more stable but still sufficiently fluid protoplast membrane.
A rapid method based on fatty acid analysis for the simultaneous quantitative determination of phospholipid and lipopolysaccharide content of whole cells is reported.
67 citations
TL;DR: Protoplasts from mesophyll cells and callus cells of rice (Oryza sativa L.) have been isolated by enzyme treatment and healthy root formation through transfer to suitable medium is up to now the morphogenetic reaction of the calli.
Abstract: Protoplasts from mesophyll cells and callus cells of rice (Oryza sativa L.) have been isolated by enzyme treatment involving 2% pectinase followed by 3% cellulase at pH 5.4 in 0.45 M mannitol (“viable” protoplasts from mesophyll cells in 50–60% yield, 60–70% yield of “viable” protoplasts from callus cells through treatment with the mixture of the above mentioned enzymes at the same concentration). Our completely defined medium is the combination of three established media (Table 1). Culture conditions are: soft agar in petri dishes at 26° C, where they regenerated cell walls after 24 h. The first cell division was observed after 4 days in culture for callus protoplasts and after 5 days in culture for mesophyll protoplasts. Cell division continues thereafter, and after 4 weeks of culture small white calli were visible in the petri dishes. The type of plant material (whitish leaf sheaths) and cell density are important factors for the efficiency of colony formation (30% plating efficiency). Healthy root formation through transfer to suitable medium is up to now the morphogenetic reaction of the calli.
67 citations
TL;DR: Since this wall autolysine is a factor produced by the cells themselves at a particular stage in their life cycle it represents a non-foreign agent in contrast to all other enzymic methods previously employed for protoplast isolation.
Abstract: Prior to fusion, gametes ofChlamydomonas reinhardii discard their cell walls. This naturally occuring phenomenon has provided the basis for a method of protoplast isolation from both gametes and vegetative cells within the genusChlamydomonas. When synchronized cultures of compatibleChlamydomonas gametes are mixed it is possible, after removal of the cells, to obtain a solution having a high cell wall lytic activity. That vegetative and gamete cells after treatment with this “gamete-autolysine” are indeed protoplasts has been proved by various light and electron microscopical methods. The species specifity of this autolysine, its difference to the previously described “sporangialautolysine” (Schlosser 1966) and furthermore its use in the large scale production of protoplasts is also described. Since this wall autolysine is a factor produced by the cells themselves at a particular stage in their life cycle it represents a non-foreign agent in contrast to all other enzymic methods previously employed for protoplast isolation.
63 citations
TL;DR: Electron microscopy confirmed the absence of a cell wall in once-washed pellets of Dunaliella primalecta and protein quality was good.
Abstract: The protein content of once-washed pellets of Dunaliella primalecta was 35 to 48%. Protein quality was good. Electron microscopy confirmed the absence of a cell wall.
58 citations
TL;DR: The formation of interspecific somatic hybrids between Penicillium roquefortii and P. chrysogenum by PEG-induced fusion of their protoplasts is reported.
Abstract: FUSION of fungal protoplasts after treatment with polyethylene glycol (PEG) has been described as a new technique for intra-specific heterokaryon formation at high frequency1–3. We now report the formation of interspecific somatic hybrids between Penicillium roquefortii and P. chrysogenumby PEG-induced fusion of their protoplasts.
TL;DR: The plant culture conditions are described such that protoplasts may be isolated throughout the year using low enzyme concentrations, and a one-step method involving the addition of sodium citrate to pectinase plus cellulase gives high yield of protoplast from hard plants.
Abstract: Factors involved in the isolation of protoplasts from the leaves of tomato plants grown over a wide range of environmental conditions have been studied. Increases in calcium pectate in summer grown (“hard”) plants are suggested as a barrier to cell wall degradation. A one-step method involving the addition of sodium citrate to pectinase plus cellulase gives high yield of protoplasts from hard plants.
Attempts to convert isolated palisade cells to protoplasts have failed.
The plant culture conditions are described such that protoplasts may be isolated throughout the year using low enzyme concentrations.
TL;DR: Carrot cells cultured in vitro as well as protoplasts isolated from these cells formed embryos and grew into mature plants that exhibited a higher frequency of tetraploids than those grown from untreated protoplast and from the original cells.
Abstract: Carrot cells cultured in vitro as well as protoplasts isolated from these cells formed embryos and grew into mature plants. Treatment of the protoplasts with polyethylene glycol (PEG) did not interfere with the capacity of this material to multiply and differentiate. Plants grown from PEG-treated protoplasts exhibited a higher frequency of tetraploids than those grown from untreated protoplasts and from the original cells.
TL;DR: The capacity to divide proved the protoplast combinations tested to be compatible when fused with those isolated from leaves of two Pisum sativum varieties, four Nicotiana species and Colchicum autumnale.
TL;DR: Uptake of exogenous DNA by isolated tobacco mesophyll protoplasts was studied using radioactive single-stranded DNA of bacteriophage fd and was greatly enhanced by the presence of poly-L-ornithine and 2n ++ in the medium.
TL;DR: Maximal yields of protoplasts were affected by the nature and the molarity of the osmotic stabilizer used, the pH of the lytic medium, period of lytic digestion and age and amount of mycelium used in the digestion mixture.
Abstract: Factors affecting the release of protoplasts from mycelium of Aspergillus flavus Link, A. nidulans (Eidam) Wint., Penicillium chrysogenum Thom, Fusarium culmorum (W. G. Smith) Sacc. and Neurospora crassa Shear & Dodge, using a Streptomyces lytic enzyme preparation, were investigated. Maximal yields of protoplasts were affected by the nature and the molarity of the osmotic stabilizer used, the pH of the lytic medium, period of lytic digestion and age and amount of mycelium used in the digestion mixture.
TL;DR: Factors influencing the fusion frequency of protoplasts have been examined with auxotrophic mutants of Aspergillus nidulans and optimum conditions were a total of 5 to 15 million protoplast per ml.
Abstract: Factors influencing the fusion frequency of protoplasts have been examined with auxotrophic mutants ofAspergillus nidulans. The optimum conditions were a total of 5 to 15 million protoplasts per ml, and 25% polyethylene glycol (PEG) 4000 or 6000 as fusogenic agent in 10 to 100 mM CaCl2 solution.
TL;DR: High yields of viable protoplasts were produced from pea leaves provided that only leaves of the same age were used in each preparation and special emphasis has been given in this paper to the choice of leaf material for protoplast isolation.
Abstract: High yields of viable protoplasts were produced from pea leaves provided that only leaves of the same age were used in each preparation. The conditions under which the pea plants were grown and the age of the plants were also important. The protoplasts were cultured in a medium supplemented with 1 mg/1 2iP and 1 mg/1 2,4-D. They were able to regenerate cell walls within two days. After 5 days cell divisions were apparent and sustained divisions led to callus formation. Special emphasis has been given in this paper to the choice of leaf material for protoplast isolation.
TL;DR: Systematic tests resulted in a nutrient solution containing the following, in milligrams per liter, for the culture of protoplasts isolated from Nicotiana tabacum L. callus cells: Murashige and Skoog salts, and regeneration of cell wall has been observed in 85% and resumption of cell division among 35% of the protoplast isolates.
Abstract: Systematic tests resulted in a nutrient solution containing the following, in milligrams per liter, for the culture of protoplasts isolated from Nicotiana tabacum L. callus cells: Murashige and Skoog salts (T. Murashige and F. Skoog, 1962. Physiol. Plant. 15: 473-497); sucrose, 15,000; mannitol, 110,000; alpha-naphthaleneacetic acid, 0.6; kinetin, 0-0.1; thiamine.HCl, 10; pyridoxine.HCl, 10; nicotinic acid, 5; myo-inositol, 100; and glycine, 2. In this medium, regeneration of cell wall has been observed in 85% and resumption of cell division among 35% of the protoplast isolates.
TL;DR: The envelope of intact chloroplasts readily fused with the protoplasts plasmalemma to establish continuity between the chloroplast stroma and the protoplast cytoplasm.
TL;DR: The fusion products were characterized by the presence of soybean leucoplasts and sweet clover chloroplasts, which appeared to be degenerating but other cytoplasmic organelles were typical of actively growing plant cells.
Abstract: Protoplasts from cultured cells of soybean (Glycine max L) and from sweet clover (Melilotus officinalis L) mesophyll cells were fused with polyethylene glycol and subsequently cultured for six days The resulting fusion products as well as unfused protoplasts of each parental species regenerated cell walls and divided The fusion products were characterized by the presence of soybean leucoplasts and sweet clover chloroplasts The chloroplasts appeared to be degenerating but other cytoplasmic organelles were typical of actively growing plant cells The fate of individual nuclei could not be determined
TL;DR: Chloroplasts from the alga, Vaucheria dichotoma (L.) Ag.
Abstract: Chloroplasts from the alga, Vaucheria dichotoma (L.) Ag., are taken up into protoplasts of carrot (Daucus carota L.) during polyethylene-glycol treatment. Since chloroplasts are found with equal frequency in uni- and multinucleate protoplasts, chloroplast uptake does not depend on protoplast fusion. However, higher frequencies of chloroplast uptake are observed when experimental conditions favor greater aggregation of protoplasts. The intracellular localization of chloroplasts is confirmed by electron microscopy, and it is shown that the chloroplasts, once within the protoplasts, are not surrounded by a limiting membrane of carrot origin.
TL;DR: Evidence was obtained that the phosphate-induced enhancement of infection is mediated through an effect on interaction of virus and poly-l-ornithine during pre-inoculation incubation, and mini-aggregates of the kind produced during incubation with phosphate play an important role in infection.
Abstract: Summary
In the presence of poly-l-ornithine at 1 µg/ml, the uptake of [3H]-labelled particles of tobacco rattle virus (TRV) by tobacco mesophyll protoplasts during inoculation depended on the virus and protoplast concentrations in the inoculation mixture. Uptake decreased 15- to 30-fold in the absence of poly-l-ornithine. Substituting phosphate for citrate buffer in the inoculum greatly increased infection but had little effect on virus uptake. For infections detected by staining with fluorescent antibody to virus particles, the ID50 occurred at an uptake of 30 long TRV particles per protoplast using phosphate buffer, and 250 using citrate. Evidence was obtained that the phosphate-induced enhancement of infection is mediated through an effect on interaction of virus and poly-l-ornithine during pre-inoculation incubation. Batches of protoplasts differing in susceptibility to infection did not differ similarly in uptake of inoculum virus.
Electron microscopy of sections of freshly inoculated protoplasts revealed single TRV particles associated end-on with normal-looking plasmalemma, and aggregates that contained densely stained material plus TRV particles, associated with breaks in the plasmalemma, and adjacent to areas of intracytoplasmic vesiculation. Some aggregates entered the cytoplasm and some TRV particles entered the vesicles. The aggregates were on average smaller after inoculation with phosphate-containing than with citrate-containing inocula. It is suggested that mini-aggregates of the kind produced during incubation with phosphate play an important role in infection.
TL;DR: Protoplasts were isolated from leaves of shoot cultures ofNicotiana tabacum var.
Abstract: Protoplasts were isolated from leaves of shoot cultures ofNicotiana tabacum var. Xanthi andPetunia hybrida, nuclei fromPetunia protoplasts and subprotoplasts from the liquid part of tomato pericarp. They were submitted in several combinations to agglutination by polyethyleneglycol (Kao and Michayluk, 1974) and to fusogenic treatment at pH 9 (Keller and Melchers, 1973) in calcium nitrate solution (Binding, 1974a; Schieder, 1974a). The fate of heterospecific symplasms was investigated during subsequent culture in medium NT-A (Nagata-Takebe medium, modification A: Binding, 1975) and in medium V47 (Binding, 1974b). Cell divisions occurred in tobacco +Petunia andPetunia + tomato symplasms which contained only a few tomato chromoplasts. Incompatibility is supposed to be responsible for the failure of divisions in tobacco + tomato symplasms and inPetunia + tomato symplasms containing a large tomato subprotoplast. The advantage of subprotoplasts for hybridization experiments is discussed in comparison to protoplast fusion and organell transplantation.
TL;DR: The distribution and synthesis of nucleic acids and proteins during gibberellic acid-induced germination of spores of Anemia phyllitidis were studied in order to relate biochemical activity with morphogenetic aspects of germination.
Abstract: The distribution and synthesis of nucleic acids and proteins during gibberellic acid-induced germination of spores of Anemia phyllitidis were studied in order to relate biochemical activity with morphogenetic aspects of germination. Germination is accompanied by the hydrolysis of storage protein granules and the localized appearance of cytoplasmic RNA, protein, and insoluble carbohydrates in a small area adjoining the spore wall and surrounding the nucleus. The protoplast of the spore enlarges in this region, the spore wall breaks and a protonemal cell is formed which contains many chloroplasts. A second division in the spore at right angles to the first yields a rhizoid cell. Autoradiography of 3H-thymidine incorporation has shown that DNA is synthesized both in the nucleus and in the immediately surrounding cytoplasm of the germinating spore until some time after the first division, although a strictly nuclear DNA synthesis is observed later. Synthesis of RNA and proteins is limited to the presumptive regions of the germinating spore which become the protonema and rhizoid, shifting to specific sites in these cells as germination proceeds. The nucleus of the spore continues to be biosynthetically
TL;DR: The protoplasts showed extreme sensitivity to the nature and concentration of the exogenous hormone supply, which resulted in high mitotic activity in protoplast cultures with low population densities.
Abstract: Protoplast cultures were prepared from explants of the roots of seedling peas. In defined, synthetic media these cultures were mitotically active. A variety of culture conditions were investigated and the influences of these conditions on the mitotic activity of the protoplasts were observed. Marked inhibition of mitoses were observed after exposure to high light intensities, and in the absence of a proper exogenous supply of hormones. The protoplasts showed extreme sensitivity to the nature and concentration of the exogenous hormone supply. The protoplasts were mitotically active at low population densities (6,000–8,000 protoplasts/ ml of medium). They did not divide in cultures in which glucose was supplied as a carbohydrate source, but divided actively in cultures in which sucrose (2%) was supplied. The responses to temperature and pH were similar to most protoplast systems which have been reported. The definition of a wide variety of optimal culture conditions resulted in high mitotic activity in protoplast cultures with low population densities.
TL;DR: The cell isoperoxidases in sweet potato plants did not reflect the possible differential mRNA synthesis in relation to organ, age, or injury, and were mainly found in the ionically bound and protoplast fractions, respectively.
Abstract: Leaves and storage roots of sweet potato plants (Ipomea batatas) showed the same qualitative isoperoxidase patterns and a similar distribution of distinctive isoperoxidases between the cell protoplast and cell wall free, ionically bound, and covalently bound fractions. No changes in the qualitative isoenzyme spectrum were found in relation to age, mechanical injury, or ethylene action. Thus, as in tobacco plants, the cell isoperoxidases in sweet potato did not reflect the possible differential mRNA synthesis in relation to organ, age, or injury. Transcription does not seem to be a limiting factor in injury- and ethylene-dependent peroxidase enhancement during the first 24 hr.The contribution of the wall ionically bound and protoplast fractions was highest in young and old leaves, respectively. In the protoplast and wall ionically and covalently bound fractions, 14, 6, and 5 isoenzyme bands were found; in addition, 4 bands, not detected in the protoplast, were also revealed in the covalently bound fraction. The distinctive "juvenile" and, developing with age, "mature" isoforms were mainly found in the ionically bound and protoplast fractions, respectively.The injury-enhanced and/or ethylene-enhanced peroxidase development was most pronounced in young leaves. Ethylene suppressed some injury-enhanced, had no effect on some other injury-enhanced, and greatly promoted some of the injury-unaffected or enhanced isoperoxidases. After ethylene removal, an increase in the "mature" isoforms was found in the protoplast of intact leaves.Electron microscopy of leaves revealed peroxidase in membrane-bound vesicles located mainly in the vacuole; a thin layer of reaction products was also found on the wall's outer surface. No Golgi apparatus were seen in the cells of control or ethylene-treated intact leaves. In ethylene-treated intact or injured leaves accumulations of reaction products between the plasmalemma and wall were also found. Numerous Golgi apparatus with dark stained vesicles were seen in injured, and especially in injured and ethylene-treated leaves; the vacuolar bodies seemed to occur in very great number.