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At least 217 records · Page 12Linked to original sources
PubMed1993

Transient gene expression of foreign genes in preheated protoplasts: stimulation of expression of transfected genes lacking heat shock elements.

Transfection of preheated petunia protoplasts with several biologically active DNA constructs resulted in a significantly higher gene expression than that observed in transfected unheated protoplasts. It was observed with supercoiled, linearized and single-stranded DNA structures that stimulation of transient gene expression in preheated protoplasts was neither dependent on the reporter gene nor on the regulatory elements used. Heat treatment at 42 degrees C also increased expression in protoplasts transfected with a plasmid bearing the tobacco mosaic virus (TMV) translational enhancer, omega. Northern blot analysis revealed that heat treatment of protoplasts before the transfection event greatly increased the amount of the newly synthesized transcripts. Preheating of protoplasts did not affect the transfection efficiency, namely the number of transfected cells in the population, nor the amount of DNA in transfected nuclei, as was inferred from histochemical staining and Southern blot analysis, respectively. The possible mechanism by which heat treatment stimulates transient gene expression of genes lacking obvious heat shock elements is offered. The relevance of the present findings to transient gene expression in plants in general and to viral gene expression in particular is discussed.

Cell Division↗
PubMed1993

Hodgkin-Huxley analysis of whole-cell outward rectifying K(+)-currents in protoplasts from tobacco cell suspension cultures.

The voltage and time dependence of outward-rectifying K+ currents (IK,out) measured in protoplasts from tobacco cell suspension cultures in the whole-cell configuration of the patch-clamp technique are quantitatively analyzed. The voltage and time dependence was described according to the Hodgkin and Huxley model for IK,out currents in the squid giant axon, and to allow comparison, in analogy with the quantitative analysis of IK,out currents in Vicia faba guard cell protoplasts as described by Schroeder (J. Membrane Biol., 107:229-235, 1989). The IK,out from tobacco could be described by a similar model as the IK,out from guard cell protoplasts (i.e., sigmoid activation time course, activation variable raised to second power, single exponential deactivating tail currents, absence of inactivation). However, in contrast to guard cells, both the activation and deactivation time constants were strongly voltage dependent in tobacco protoplasts. The voltage dependence of the transition rates for channel opening and channel closing was slightly asymmetrical and inverse to the asymmetry found in guard cells. The data presented show that the voltage-dependent kinetic properties of the IK,out conductance of tobacco protoplasts are different from these properties in guard cell protoplasts. This analysis provides a basis for the study of IK,out conductance function and modulation.

Cells, Cultured↗
PubMed1980

Transfection of protoplasts from Streptomyces lividans 66 with actinophage SH10 DNA.

The slightly modified procedure for the transformation of protoplasts of S. coelicolor A3 (2) with SCP2 plasmid DNA and polyethylenglycol (PEG) (Bibb et al., 1978) was extended to infection of protoplasts of S. lividans 66 with actinophage SH10 DNA (Klaus et al., 1979). Maximal yield of transfected protoplasts was obtained at 20% PEG, 3 MM sodium-citrate and 150 mM NaCl final concentrations. The efficiency of transfection was determined to be about 2 x 10(-8) to 2 x 10(-7). The average value of competent protoplasts was about 1-2 x 10(-4) of regenerating protoplasts. In comparison with outgrowing spores infected with phage particles the average burst size of transfected protoplasts was reduced from 100 to 10 pfu/infected cell, the latent period prolonged from 45 min to 120 min and the rise period was not affected.

Bacteriophages↗
PubMed1990

Optimum conditions for efficient transformation of Streptomyces venezuelae protoplasts.

Optimum conditions for protoplast regeneration and transformation of Streptomyces venezuelae ETH14630 have been established. Protoplasts from mycelium grown to the stationary phase and treated with lysozyme in P medium under mild conditions gave the best regeneration frequency. Transformation of protoplasts with naked DNA was very efficient using either polyethylene glycol of mol. wt. 4000 or 6000, at concentrations of 28.5% or 36% (w/v) respectively. About 10(7) transformants/micrograms DNA could be isolated using protoplasts derived from cells cultivated to the early exponential growth phase in LB medium containing 0.2%-0.6% glycine and subsequently treated at 30 degrees -32 degrees C with 20 mg lysozyme/ml in P medium for 30 min. Selection of the transformants occurred on MRYE plates containing less than 10(5) regenerating protoplasts per plate. Higher protoplasts densities considerably decreased the regeneration frequency of the transformants.

Biotechnology↗
PubMed1984

Papulacandin B: inhibitor of biogenesis of (1----3)-beta-D-glucan fibrillar component of the cell wall of Saccharomyces cerevisiae protoplasts.

The effect of papulacandin B on regenerating protoplasts of Saccharomyces cerevisiae was studied by light and electron microscopy. In liquid media it inhibited the biogenesis of (1----3)-beta-D-glucan fibrillar nets; as a result, the protoplasts did not grow polarly but only spherically. The effect was reversible. Instead of the nets the inhibited protoplasts synthesized only individual microfibrils soluble in hydroxide; these were not joined in the nets and were partially masked by amorphous material. The microfibrils disintegrated after lysis and did not maintain the shape of protoplasts. Protoplasts inhibited in solid media grew spherically up to 25 micron but they did not divide or revert, in spite of forming cell walls. These walls were amorphous and fragile and they disintegrated during preparation. Papulacandin B did not decrease the viability of protoplasts and did not interfere with their growth, biogenesis of alkali-soluble glucan microfibrils or amorphous wall matrix. It inhibited specifically the synthesis of alkali-insoluble branched (1----3)-beta-D-glucan, a necessary building unit required for the formation of the fibrillar component of the cell wall responsible for the cell wall shape, its rigidity and tensile strength.

Aminoglycosides↗
PubMed1975

The isolation of protoplasts of the fission yeast Schizosaccharomyces by Trichoderma viride and snail enzymes.

The formation of protoplasts of the fission yeasts Schizosaccharomyces pombe and Schizosaccharomyces versatilis after the combined application of snail enzymes and Trichoderma viride enzymes in an osmotic stabilizer (0.4M KCl, pH 5.5) was studied by light and electron microscopy. The effect of the enzymes used leads during 30 min to the formation of 100% protoplast population. Using electron microscopy no original walls or wall remnants were detected in the suspension of protoplasts. Protoplasts are viable and in liquid nutrient medium they regenerate cell walls and revert into normal cells. Such a protoplast population may be useful for biochemical study of protoplast metabolism by quantitative methods as well as for the chemical study of regenerating cell walls.

Animals↗
PubMed1985

Absence of glucose-stimulated transport in yeast protoplasts.

Protoplasts of Saccharomyces cerevisiae prepared by snail-gut juice treatment were compared in their transport properties with intact cells. 1. Constitutive monosaccharide transport (D-xylose, 6-deoxy-D-glucose), as well as inducible transport of D-galactose, were unaltered. 2. Phosphorylation-associated transport of 2-deoxy-D-glucose was enhanced in protoplasts, possibly as a consequence of removal of the unstirred layer of the cell wall. 3. Proton-driven transports of trehalose, L-leucine, L-proline and monophosphate could not be activated by preincubation with D-glucose, apparently owing to lack of proton-solute coupling in transport. Utilization of glucose was not depressed but respiration was reduced by about 50% while acidification of the external medium after glucose addition was inhibited by more than 90%. This may be related to the inability of protoplast plasma membrane H-ATPase to be activated by glucose and hence to impaired proton-translocating capacity. Uranyl ions inhibited generally much less in protoplasts than in intact cells although their binding to protoplasts was greater (maximum 0.68 fmol per cell but 3.2 fmol per protoplast).

Biological Transport↗
PubMed2003

Characterization of whole-cell K+ currents across the plasma membrane of pollen grain and tube protoplasts of Lilium longiflorum.

Outward and inward currents, mainly carried by K(+), were detected in protoplasts of pollen grains (PG) and pollen tubes (PT) of Lilium longiflorum Thunb. by using the whole-cell configuration of the patch-clamp technique. The outward K(+) current (I(K+ out)) was similar in both protoplast types, while the inward K(+) current (I(K+ in)) was higher in pollen tube protoplasts. In PT but not in PG protoplasts, inward K(+) currents were already detectable at negative membrane voltages usually monitored in lily pollen. I(K+ in) consisted of a slow and a fast current component, as revealed by fitting a sum of two exponential functions to the time-dependent current. The contribution of the fast component to the total inward current was higher in PT than in PG protoplasts, which was even more evident at acidic pH of the external medium. Therefore, based on the measured characteristics, the I(K+ in) of PT protoplasts may contribute to the endogenous K(+) currents surrounding a growing pollen tube.

Cell Membrane↗
PubMed2003

Somatic embryogenesis and fertile green plant regeneration from suspension cell-derived protoplasts of rye ( Secale cereale L.).

A method for somatic embryogenesis and fertile green plant regeneration from suspension cell-derived protoplasts of rye (Secale cereale L. cv. Auvinen) was developed. Fast-growing and friable embryogenic calli with a high regeneration capacity were induced from immature rye inflorescences using modified MS medium. These friable embryogenic calli were used for suspension culture initiation in liquid AA medium. A high yield of protoplasts was obtained from suspension cell clumps after 3-5 days of subculture. Isolated protoplasts were cultured in KM8p medium. The frequency of protoplast cell divisions and colony formations in liquid culture medium were similar to those on agarose-solidified medium. Compact embryogenic calli were developed from protoplast-derived microcalli in growth medium mMS. Approximately 7% of the transferred embryogenic calli produced green shoots on N6 regeneration medium. Of 33 green plants, 28 were fertile with normal flowering and seed set. The ratio of green and albino plantlets was 1:4. Rye protoplast-derived green plants showed normal diploid characters as determined by flow cytometer analysis and chromosome counting.

Cell Division↗
PubMed1998

Gravity and light control of the developmental polarity of regenerating protoplasts isolated from prothallial cells of the fern Ceratopteris richardii.

A procedure has been developed for isolating protoplasts from prothalli of Ceratopteris richardii which can be cultured and are capable of regeneration. Protoplasts were isolated from 2-week-old gametophytes in a medium containing wall-digesting enzymes in 0.5 M sucrose, followed by purification of the released protoplasts by floating them up into a 0.5 M sorbitol layer. Regeneration occurred over a period of 10-24 days, and, under optimal osmotic conditions, followed the developmental pattern seen during spore germination, in that the first division gave rise to a primary rhizoid. Thus, prothallial protoplasts are comparable to germinating spores as suitable models for studies of developmental polarity in single cells. As in germinating spores, the polarity of development in regenerating protoplasts is influenced by the vectors of gravity and unilateral light. However, the relative influence of light in fixing this polarity is greater in regenerating protoplasts, while in germinating spores, the influence of gravity is greater.

Cell Polarity↗
PubMed1998

Synergism in replication of cymbidium mosaic potexvirus (CymMV) and odontoglossum ringspot tobamovirus (ORSV) RNA in orchid protoplasts.

An in vitro orchid protoplast isolation method to study replication kinetics of CymMV and ORSV was developed. This method allows the isolation of viable and raphid-free petal protoplasts from an orchid hybrid, Dendrobium Sonia (Dendrobium Caesar x Dendrobium Tomie Drake). The optimum field strength for both viral RNA to achieve good efficiency of electroporation was 750 V/cm and the optimum viral RNA concentration required was 1 microgram and 4 micrograms per 2 x 10(6) protoplasts for CymMV and ORSV, respectively. Autoradiographs of Northern blots depicting the viral genomic and subgenomic RNA in the extracts, referred to as the "Replication Footprint Profiles" (RFP) of specific CymMV/ORSV virus were prepared at different time intervals. Viral RNA synthesis reached a maximum at 18 h for CymMV and 24 h for ORSV. When CymMV and ORSV viral RNA were electroporated into the protoplasts simultaneously, detection signals of both the positive and negative strand viral RNA increased as compared to the singly infected protoplasts. Thus, synergism in replication of CymMV and ORSV was observed in orchid protoplasts.

Electroporation↗
PubMed2006

Development of a quantitative method for determination of the optimal conditions for protoplast isolation from cultured plant cells.

An index [kv: average isolation rate of viable protoplast (number/ml min)] was established to evaluate the optimal conditions for protoplast isolation from cultured plant cells. The optimal conditions for protoplasts isolation from Nicotiana tabacum BY2 cultured cells could be determined on the basis of the kv [31.7 x 10(3) (number/ml min)]. The colony-forming efficiency of the protoplasts was about 46%. The optimal conditions for protoplasts isolation from Catharanthus roseus [kv = 38.1 x 10(3) (number/ml min)] and Wasabia japonica [kv = 14.2 x 10(3) (number/ml min)] cultured cells could also be determined. Furthermore, a method for rapid regenerating cell wall of protoplast in liquid culture using alginate gel containing locust bean gum was developed.

Alginates↗
PubMed1987

The plasma membrane of yeast protoplasts exposed to hypotonicity becomes porous but does not disintegrate in the presence of protons or polyvalent cations.

Protoplasts of Saccharomyces cerevisiae swelled, lysed and disintegrated when exposed to hypotonic solutions at neutral pH. At pH 4.5 or lower the hypotonically treated protoplasts did not disintegrate and they retained their intracellular proteins, nucleic acids and nucleotides. However, they became leaky for K+ and Ca2+, indicating that pores had been created in the surface membrane, relaxing the osmotic stress. Upon readjustment of pH to neutral, the hypotonically treated protoplasts released the intracellular content and disintegrated. Also, at low pH, protoplasts did not swell in isotonic ammonium acetate and were refractory to the permeabilizing effect of nystatin and to lysis with low concentrations of detergents. Protoplasts were similarly protected against lysis and disintegration by hypotonic treatment or by detergents, even at neutral pH, if the incubation media contained polyvalent cations, especially Zn2+, La3+, spermine, and Ca2+ chelated with EDTA. The protoplasts exposed to hypotonic stress at low pH did not respire and could not regenerate into viable cells. Effects of H+ and polyvalent cations on intramembrane forces acting between molecules of membrane phospholipids are considered along with possible changes in interactions between membrane proteins.

Cell Membrane↗
PubMed2005

Conditioned culture for protoplasts isolated from chrysanthemum: an efficient approach.

An efficient approach was described for the culture of protoplasts isolated from chrysanthemum, and factors affecting the plating efficiency, defined as the number of protoplasts developing microcolonies divided by the number of cultured protoplasts, were investigated here. A yield of 4.86+/-0.22x10(5) mesophyll protoplasts per gram fresh weight were achieved by an enzymolysis procedure. The viability of fresh isolated protoplasts was 64.5+/-8.2% as confirmed by Evans blue staining. Sustained cell division and microcolony formation from the protoplasts were supported by modified Murashige and Skoog medium, complemented with 1:4 diluted conditioned medium harvested from three different callus suspension cultures. Results showed that the plating efficiency was obviously influenced by various concentrations of agarose and antibiotic, and significantly increased by conditioned medium harvested from chrysanthemum suspensions, while the other two species had a positive effect generally. Results also indicated that the growth-stimulative effect was reduced when the conditioned medium was stored for 6-14 days prior to use, suggesting the unstable characteristic of conditioning factors.

Cell Culture Techniques↗
PubMed2005

Tuber borchii mycelial protoplasts isolation, characterization and functional delivery of liposome content, a new step towards truffles biotechnology.

The filametous ascomycete Tuber borchii is a plant-symbiotic ectomycorrhizal microrganism with an high value due to the production of hypogeous fruitbodies (truffles). The present work was undertaken to develop a procedure for the release of T. borchii viable protoplasts from Tuber mycelium, isolate ATTC 96540; several factors which affect the isolation, morphology and viability were examined and developed in order to improve applications of T. borchii protoplasts in morphological, biochemical and genetic investigations (protoplast fusion or transformation). Functional delivery of liposome content into T. borchii protoplasts has also been examined with a cytotoxic ribosome inactivator as saporin. T. borchii protoplasts incubation/fusion with saporin containing liposomes were made to demonstrate the absence of cell wall of 16 days cultured protoplasts.

Ascomycota↗
PubMed2026

An effective method for isolation and regeneration of Solanum tuberosum mesophyll protoplasts for transgene-free genome editing.

An effective system for isolating and regenerating protoplasts is crucial for research in genome engineering. This study focused on refining a protocol for the isolation and regeneration of mesophyll protoplasts from the leaves of Solanum tuberosum cv. Kufri Jyoti. Key factors influencing protoplast yield and viability, such as dark pretreatment, pre-plasmolysis, enzyme concentrations, and osmoticum levels, were thoroughly assessed and optimized. The highest protoplast yield and viability were achieved with an enzyme mixture of 1.0% cellulase R-10 and 0.5% macerozyme R-10 after 16 h of incubation. Furthermore, culturing on a Murashige and Skoog-based medium (MSPI) without ammonium nitrate, enriched with an osmoticum concentration of 0.4 M and a carefully adjusted auxin-to-cytokinin ratio, successfully facilitated protoplast division, microcalli proliferation, and minicalli formation. Callus proliferation and shoot induction were accomplished on MS13K medium supplemented with naphthaleneacetic acid (NAA) and zeatin riboside. Root initiation and elongation were promoted on MS basal medium supplemented with indole-3-butyric acid (IBA) at 1 mg/L. The regenerated plantlets were subsequently acclimatized and hardened under controlled greenhouse conditions. This robust protoplast-to-plant protocol serves as a crucial resource for the introduction of ribonucleoprotein complexes into plant cells, facilitating accurate, transgene-free genome editing.

Callus induction↗
PubMed2006

Transfection of Arabidopsis protoplasts with a Plum pox virus (PPV) infectious clone for studying early molecular events associated with PPV infection.

The development of novel strategies against plant viral diseases relies on a better understanding of molecular virus-host interactions. Here, we report an easy, efficient and reproducible protocol for Arabidopsis protoplast isolation and transfection to study the infection and replication of a potyvirus, Plum pox virus (PPV). Macerozyme and cellulose were used to release protoplasts from Arabidopsis leaf tissues, and polyethylene glycol-mediated DNA uptake was employed for transfection of a PPV infectious clone. Protoplast viability was monitored by fluorescein diacetate staining, and transfection efficiency was estimated by transient expression of the green fluorescent protein. The protocol allowed production of 95% viable mesophyll protoplasts and a successful transfection rate of 35%. The system was used further in a time-course experiment to investigate PPV viral RNA accumulation. It was found that 3 h post-transfection (hpt) in the transfected protoplasts viral RNA increased by about 150-fold and progressively accumulated to reach the maximum at 12 hpt. Viral RNA then decreased dramatically at 24 hpt reaching 40% of its peak level. Considering the availability of the whole genome microarrays, and other genomic resources of Arabidopsis, the synchronized single-cell (protoplast) infection system will be useful for elucidating early molecular events associated with PPV infection.

Arabidopsis↗
PubMed2001

Cucurbit protoplast isolation for the study of plant virus replication.

A cucurbit protoplast isolation protocol was established for the study of plant virus replication in vivo. This protocol is applicable to both cucumber and squash leaf tissue with significant increases in yields of viable protoplasts suitable for electroporation, compared to other published methods. A combination of Cellulase RS, Macerozyme R10 and mannitol was used as digestion enzymes and osmoticum. An average of 1.7x10(7) protoplasts per gram of fresh leaf tissue were obtained from cucumber cultivar Bet-alpha. Both cucumber cultivar Shimson and squash cultivar First Taste produced an average yield of 6.0x10(6) protoplasts per g of fresh leaf tissue. Electroporation of 10 microg of Zucchini yellow mosaic potyvirus (ZYMV-S) RNA into the protoplasts resulted in virus replication and synthesis of coat protein (CP). SDS-PAGE and immunoblotting were used to detect the CP 48 h post-electroporation. This protocol is highly reproducible and will assist researchers who require cucurbit protoplasts to study virus replication.

Cell Separation↗