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PubMed1982

Studies on the labelling of terpenoids in shoots and cells or protoplasts from Calendula officinalis leaves.

1. From the leaves of Calendula officinalis, homogeneous preparations of isolated cells and protoplasts have been obtained which effectively synthesized terpenoid compounds from [1-14C]acetate. 2. The quantitative determinations have shown that the level of sterols, beta-amyrin and oleanolic acid was similar in shoots, isolated cells and protoplasts but the level of polyprenols was the highest in shoots, lower in cells and the lowest in protoplasts. 3. The determination of the dynamics of labelling with radioactive acetate has shown that sterols are labelled at a similar rate in shoots, cells and protoplasts, whereas the labelling of beta-amyrin, oleanolic acid and polyprenols is delayed in cells and protoplasts as compared with shoots. The labelling of beta-amyrin and oleanolic acid reached eventually the level observed in the shoots, but the labelling of polyprenols at this time was twice as high in the cells and protoplasts as in the shoots.

Acetates↗
PubMed1994

Hypocotyl protoplast culture in Brassica napus L.

Protoplasts were isolated from the hypocotyls of two oilseed rape (Brassica napus L.) cultivars, Yunbei 2 and Ningyou 7. Light treatment during seed germination could apparently increase their cell division frequency. The purified protoplasts were cultured with three different methods. The method called "agarose island" was found superior to two others ("thin liquid layer" and "agarose solid layer"). The protoplasts cultured in the "agarose island" divided more rapidly and developed into calli more frequently. Shoot regeneration occurred soon after the transfer of protoplast-derived calli onto the differentiation medium. Individual shoots were rooted on the rooting medium. Whole plants were transplanted into pots, and grew well in the phytotron. The "agarose island" method might be suitable for genetic transformation of protoplasts because the liquid medium surrounding the "island" could be conveniently replaced without strong disturbing the microenvironment of protoplasts plated.

Biotechnology↗
PubMed1993

[Use of the protoplast fusion and regeneration method for screening antibiotic producers among inactive strains of Streptomyces].

Intraspecies fusion of protoplasts of two strains of Streptomyces fradiae, i.e native protoplasts of an inactive strain INA 00708 and heat inactivated protoplasts of a neomycin-producing strain ATCC 10745, and regeneration of the protoplasts of the inactive strain INA 00708 resulted in formation of clones producing neomycin and clones synthesizing antibiotics of an unknown nature differing from neomycin. All the active clones were unstable and lost their antibiotic activity in subcultures. Regeneration of the protoplasts of 4 different inactive strains of Streptomyces sp. also resulted in formation of active clones which were unstable and lost their capacity for the antibiotic synthesis after the first subculture. The data in principal indicate to the possible use of protoplast fusion and regeneration in screening of cultures producing new antibiotics among inactive strains of streptomycetes. However, the efficiency of such procedures is low since the experiments are labor-consuming and the resulting active clones are genetically unstable.

Anti-Bacterial Agents↗
PubMed1995

Ultrahigh-resolution low-voltage SEM reveals ultrastructure of the glucan network formation from fission yeast protoplast.

The refined field emission SEM, S-900 LV which gives better resolution especially at low voltages below 5 kV was developed for ultrahigh resolution scanning electron microscopy. A visualization test at x 300,000 was made using a gold-evaporated magnetic tape, and the resolution was found to be about 1 nm at 2.5 kV. The ultrastructure of the cell wall, especially the reverting glucan network, from the protoplast of Schizosaccharomyces pombe was studied using this improved ultrahigh-resolution low-voltage SEM (UHR-LVSEM). The results with uncoated reverting protoplasts observed with this microscope revealed that the network was originally formed as secreted particles scattered on the protoplast surface and these were subsequently stretched to microfibrils about 2 nm thick. The microfibrils were twisted around each other and joined together so that they developed into 8-nm-thick fibrils, forming a ribbon-shaped network of glucans about 16-nm-thick which covered the entire protoplast surface. The UHR-LVSEM images of reverting protoplasts treated with glucanase confirmed that the particles scattered on the protoplast surface in the initial stage of regeneration were glucan in nature.

Animals↗
PubMed1998

The use of cryopreserved apical protoplasts from Curvularia lunata for electrotransformation.

An electroporation method, utilizing cryopreserved protoplasts, has been developed for the steroid 11-hydroxylating fungus Curvularia lunata strain IM 2901. Protoplasts released from the apical parts of 24- and 48-h-old mycelia were suspended in cryopreservation buffer and stored at -75 degrees C for several weeks. The thawed and freshly prepared (control) protoplasts were electroporated with pAN 7-1 plasmid carrying the Escherichia coli hygromycin B resistance gene (hph) under the control of Aspergillus nidulans sequences. The electroporation efficiency of the control protoplasts with plasmid pAN 7-1 was 7.5 and 12.0 transformants per microgram DNA (protoplasts liberated from 24- and 48-h-old mycelia, respectively). Protoplasts released from the younger mycelium were more stable according to their reversion ability to mycelial form and transformation efficiency. After 16 weeks of cryopreservation the yield of electroporation was 61.3% of the control value. All isolated electrotransformants proved to be stable for a period of > 4 months even without selective pressure.

Cryopreservation↗
PubMed1987

Parsley protoplasts retain differential responsiveness to u.v. light and fungal elicitor.

The differential response of cultured parsley cells to u.v. irradiation and elicitor treatment is a paradigm for analysis of specific plant defense responses. We demonstrate that freshly isolated parsley protoplasts, in the absence of detectable cell wall, maintain fully the ability to be activated by these important environmental factors. Stimulated protoplasts synthesize typical qualitative patterns and amounts of potentially protective flavonoid glycosides and coumarin phytoalexins following either u.v. irradiation or treatment with fungal elicitor, respectively. Induced accumulation of mRNAs and enzymes of the phenylpropanoid biosynthetic pathways is nearly identical in protoplasts and cells. Stimulation of protoplasts with elicitor requires only a short period of contact, which is not sufficient for cell wall regeneration. Importantly, there is no activation of these pathways during protoplast preparation. These results establish that parsley protoplasts respond appropriately to two physically distinct stimuli and might serve as an especially suitable system for the analysis of signal transduction and gene activation.

Journal Article↗
PubMed1981

Fate of plasmid DNA in transformation of Bacillus subtilis protoplasts.

Polyethylene glycol-treated protoplasts of B. subtilis can be transformed by plasmid DNA at very high frequencies (Chang and Cohen 1979). From analysis of plasmid mediated transformation of transformation-deficient mutants it appeared that mutants, reduced in the transformation by plasmid DNA in the competent state, were plasmid transformation-proficient when transformed as protoplasts. By means of CsCl-gradient centrifugation of re-extracted plasmid DNA it could be demonstrated that plasmid DNA enters the protoplasts in the double-stranded form. In addition, sucrose gradient centrifugation of the re-extracted plasmid DNA showed that the entered DNA is predominantly present as covalently closed circular DNA. The efficiency of plasmid transformation in protoplasts was found to be close to one (each plasmid molecule having entered into the protoplasts gives rise to a transformed cell). This is in good agreement with the observation that little, if any, damage is done to this DNA during or after entry into protoplasts.

Bacillus subtilis↗
PubMed1976

Fusion of bacterial protoplasts.

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.

Bacillus subtilis↗
PubMed1984

Dimethyl sulfoxide can initiate cell divisions of arrested callus protoplasts by promoting cortical microtubule assembly.

A serious problem in the technology of plant cell culture is that isolated protoplasts from many species are reluctant to divide. We have succeeded in inducing consecutive divisions in a "naturally" arrested system-i.e., protoplasts from a hibiscus cell line, which do not divide under standard conditions-and in an artificially arrested system-i.e., colchicine-inhibited callus protoplasts of Nicotiana glutinosa, which do readily divide in the absence of colchicine. In both cases, the reinstallation of a net of cortical microtubules, which had been affected either by colchicine or by the protoplast isolation procedure, resulted in continuous divisions of the formerly arrested protoplasts. Several compounds known to support microtubule assembly in vitro were tested for their ability to promote microtubule assembly in vivo. Best results were obtained by addition of dimethyl sulfoxide to the culture medium. Unlimited amounts of callus could be produced with the dimethyl sulfoxide method from protoplasts which never developed a single callus in control experiments.

Journal Article↗
PubMed1992

Electrically induced protoplast fusion using pulse electric fields for dielectrophoresis.

The formation of protoplast chains in suspensions of isolated pea (Pisum sativum cv Ran 1) mesophyll protoplasts induced by electric fields was studied. The chain formation induced by a sine-wave field (2 V, peak to peak; 500-0.1 kHz) was compared to that induced by an alternating pulse field (1 V, amplitude; 0.1-0.4 kHz). An increased number of dielectrophoretically paired protoplasts, formation of protoplast chains in the presence of CaCl(2) up to 5 mm, and protoplast fusion in the presence of 3 mm CaCl(2) were found when the pulse field was applied. The present results suggest the possibility of electrically induced protoplast fusion at cation concentrations that prevent fusion when sine-wave fields are applied.

Journal Article↗
PubMed1997

Blue-Light-Induced Shrinking of Protoplasts from Maize Coleoptiles and Its Relationship to Coleoptile Growth.

Protoplasts isolated from red-light-grown maize (Zea mays L.) coleoptiles shrank transiently upon brief exposure (e.g. 30 s) to blue light under background irradiation with red light. The maximal volume reduction (about 4% at a saturating fluence) occurred about 5 min after blue-light stimulation. The response was prevented by the anion-channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid. Red light and far-red light did not induce any comparable response. Protoplasts of different sizes and those isolated from different coleoptile positions showed similar responses. After treatment with a saturating blue-light pulse, the protoplasts became responsive to a second pulse and gained full responsiveness within 5 min, suggesting that the photoreceptor system involves a dark-reversible component. The response to continuous blue light was also found to be transient. The protoplast volume was reduced during about 6 to 9 min of irradiation and returned within the next 30 min to the control level. The response to continuous blue light was saturated at 30 [mu]mol m-2 s-1. However, when the fluence rate was enhanced 10-fold after a period of irradiation at 30 [mu]mol m-2 s-1, the protoplasts showed another shrinking response. These and other kinetic results indicate that the photoreceptor system undergoes a photosensory adaptation. Growth in different zones of the coleoptile was inhibited by blue light transiently after pulse stimulation, as well as during continuous stimulation. It was concluded that the observed protoplast shrinking is related to the blue-light-induced inhibition of coleoptile growth.

Journal Article↗
PubMed1973

Response of Nicotiana mesophyll protoplasts of normal and tumorous origin to indoleacetic Acid in vitro.

Enzymatically isolated mesophyll protoplasts of the two normal, nontumor-forming parent species Nicotiana glauca and N. langsdorffii and two of their tumor-prone interspecific hybrids were maintained in a 0.5 m mannitol solution supplemented with various concentrations of auxin (indoleacetic acid) and the growth inhibitor abscisic acid. The bursting response of protoplasts in medium containing indoleacetic acid in physiological concentrations showed that protoplasts from the tumorous hybrids tolerate auxin in up to 30 times higher concentrations than protoplasts from parent plants. The "survival" of all protoplast preparations in comparable abscisic acid containing media was significantly greater than that in the indoleacetic acid supplemented solutions. Protoplasts in vitro respond with bursting only after the external indoleacetic acid concentrations reach levels comparable to those of endogenous auxins present in these cells. The data are discussed in conjunction with previous observations on uptake and maintenance of indoleacetic acid levels in tumorous Nicotiana tissues.

Journal Article↗
PubMed1978

RNA synthesis in whole cells and protoplasts of centaurea: a comparison.

Protoplasts enzymically isolated from suspension cultures of Centaurea cyanus L. incorporate radioactive precursors into RNA with kinetics similar to that of whole cells. There are differences, however, in several other aspects of RNA metabolism. The proportion of total RNA that contains poly(A) sequences (25 to 30%) is similar in both freshly isolated protoplasts and whole cells after a 20-minute pulse with [(3)H]adenosine. After a 4-hour pulse, however, poly(A)-containing RNA makes up 30% of the total RNA in protoplasts whereas it drops to 8% in whole cells. There appears to be a faulty processing of ribosomal precursor into the mature ribosomal species, as the precursor seems to accumulate to higher levels relative to the mature 18S and 25S rRNAs in protoplasts as compared to whole cells. Additional differences are seen in the size distributions of poly(A)-containing RNA, although the length of the poly(A) segment is similar in both protoplasts and whole cells. Within 24 hours protoplasts appear to have resumed a pattern of RNA synthesis similar to that of whole cells.

Journal Article↗
PubMed1979

Protoplasts surviving freezing to -196 C and osmotic dehydration in 5 molar salt solutions prepared from the bark of winter black locust trees.

Free protoplasts were prepared from the living bark tissue of the trunk of summer and winter black locust trees by enzymic digestion of thin slices of the tissue for 3 hours in a medium containing 2% Onozuka cellulase, 2% Rhozyme pectinase, and 2% Driselase in mannitol solutions using 0.4 molar mannitol for summer tissue and 1.0 molar mannitol for winter tissues. Cleaned suspensions of protoplasts and also thin slices of tissue with cells intact were frozen to temperatures of -10 C, -20 C, -30 C, -40 C and liquid nitrogen in sucrose and balanced salt solutions. Similar suspensions of protoplasts were also subjected to strong osmotic dehydration (plasmorrhysis) in a series of balanced salt solutions of increasing molarity. Tests for survival showed that protoplasts retain the same properties of either extreme susceptibility or extreme resistance to injury by freezing or osmotic dehydration as the cells from which they are prepared. Winter protoplasts showed capability for tolerating freezing to -196 C and plasmorrhysis in 5 molar salt solutions. These results indicate that protoplasts are a valid and useful system for investigating the properties of the protoplasm and surface membranes associated with the seasonal development of extreme hardiness in the cells of woody plants.

Journal Article↗
PubMed1980

Photosynthetic and Photorespiratory Carbon Metabolism in Mesophyll Protoplasts and Chloroplasts Isolated from Isogenic Diploid and Tetraploid Cultivars of Ryegrass (Lolium perenne L.).

Photosynthetic (14)CO(2) fixation, [(14)C]glycolate formation, and the decarboxylation of [1-(14)C]glycolate and [1-(14)C]glycine by leaf mesophyll protoplasts isolated from isogenic diploid and tetraploid cultivars of ryegrass (Lolium perenne L.) were examined. The per cent O(2) inhibition of photosynthesis in protoplasts from the tetraploid cultivar was less than that of the diploid line at both 21 and 49% O(2). Kinetic studies revealed that the K(m) (CO(2)) for photosynthesis by the diploid protoplasts was about twice that of the tetraploid line. In contrast, the K(i) (O(2)) for protoplast photosynthesis was similar in both cultivars, as was the potential for oxidizing glycolate and glycine to CO(2) via the photorespiratory carbon oxidation cycle. Although the maximal rates of glycolate accumulation by the isolated protoplasts in the presence of 21% O(2) and a glycolate oxidase inhibitor were similar in the two cultivars, the percentage of total fixed (14)C entering the [(14)C]glycolate pool and the ratio of the rate of [(14)C]glycolate formation to (14)CO(2) fixation at 21% O(2) and low pCO(2) were about two times greater in protoplasts and intact chloroplasts isolated from the diploid line compared to the tetraploid. These results fully support the recent observation that a doubling of ploidy in various ryegrass cultivars reduced the K(m) (CO(2)) of purified ribulose bisphosphate carboxylase-oxygenase by about one-half without affecting the K(i) (O(2)) (Garrett 1978 Nature 274: 913-915).

Journal Article↗
PubMed1980

Enzymic determination of metabolites in the subcellular compartments of spinach protoplasts.

A method for determining the subcellular metabolite levels in spinach protoplasts is described. The protoplasts are disrupted by centrifugation through a nylon net, releasing intact chloroplasts which pass through a layer of silicone oil into perchloric acid while the remaining cytoplasmic components remain over the oil and are simultaneously quenched as acid is centrifuged into them. Cross-contamination is measured and corrected for using ribulose 1,5-bisphosphate as a chloroplastic marker and phosphoenolpyruvate carboxylase as a cytoplasmic marker. A method for separation of intact protoplasts from the medium by silicone oil centrifugation is described, which allows a correction to be made for the effect of free chloroplasts and broken protoplasts. Methods for inhibiting chloroplast photosynthesis, without inhibiting protoplasts, are presented. It is demonstrated that ribulose 1,5-bisphosphate, ATP, ADP, AMP, inorganic phosphate, hexose phosphate, triose phosphate, fructose 1,6-bisphosphate, and 3-phosphoglycerate can be reliably recovered in the subcellular fractions isolated from protoplasts, and measured by enzymic substrate analysis.

Journal Article↗
PubMed1980

Corn Root Protoplasts: ISOLATION AND GENERAL CHARACTERIZATION OF ION TRANSPORT .

A method was developed for the large scale and rapid isolation of intact viable corn root protoplasts. Pure and metabolically active protoplasts were collected using a flotation technique. Vital staining tests, light and electron microscopy, and measurements of basic metabolic processes indicated that the isolated protoplasts were metabolically active, and that the plasmalemma and other organelles were well preserved. The isolated protoplasts performed normal, active ion transport functions. Time course of K(+) and inorganic phosphate (H(2)PO(4) (-)) influx and the effects of external pH, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, fusicoccin, and diethylstilbestrol on K(+) and inorganic phosphate influx and net H(+) efflux in isolated protoplasts correlated well with data obtained on root segments. Data presented indicated that isolated protoplasts from roots can be used to gain additional insights into the mechanism of ion transport in plant cells.

Journal Article↗
PubMed1982

Aggregation of plant protoplasts by artificial lipid vesicles.

Sonicated unilamellar lipid vesicles, consisting of egg lecithin, stearylamine, and cholesterol in 7:2:1 molar ratios, promoted the aggregation of tobacco (Nicotiana glutinosa) protoplasts with the aid of mono- or divalent cations.A reaction mixture containing liposomes (0.4 micromoles lipid per milliliter), 50 millimolar CaCl(2), 0.5 molar mannitol, and 5 x 10(5) protoplasts per milliliter resulted in approximately 25% protoplast aggregation. To achieve the maximum protoplast aggregation, 1.6 x 10(8) liposomes per protoplast per hour would be required.The kind of liposomes effective in protoplast aggregation were positively charged, small-size vesicles which were obtained either by 60-minute sonication or by membrane filtration in conjunction with sonication.

Journal Article↗