Factors affecting the electrofusion efficiency ofPorphyra protoplasts

The effects of various factors on the electrofusion efficiencies ofPorphyra protoplasts were investigated. These factors were protoplast stabilizing reagents, divalent cations, membrane digestive enzymes and cold storage of the protoplasts. Fusion efficiencies were dependent on the concentrations of reagents used to adjust the osmotic pressure of the medium. With mannitol or sorbitol the maximum fusion efficiency (approximately 16%) was observed at concentrations of 0.6 to 0.7 M; glucose was less effective. Brief treatment of the protoplasts with pronase stimulated electrofusion, whereas treatment with proteinase K, trypsin, phospholipase C or lipase repressed fusion. The addition of Ca²⁺ at 10^-5 to 10^-4 M in the protoplast medium enhanced the fusion efficiency to approximately four times that of the non-treated control. Sr²⁺ and Co²⁺ also stimulated electrofusion, but less effectively than Ca²⁺. The fusion capacity of the protoplasts remained stable for about 3 h when kept on ice, but decreased gradually when left at room temperate.     

Electrofusion of protoplasts from celery (Apium graveolens L.) with protoplasts from the filamentous fungus Aspergillus nidulans

A method was developed for electrofusion of higher-plant protoplasts from celery and protoplasts from the filamentous fungus Aspergillus nidulans. Initially, methods for the fusion of protoplasts from ecch species were determined individually and, subsequently, electrical parameters for fusion between the species were determined. Pronase-E treatment and the presence of calcium ions markedly increased celery protoplast stability under the electrical conditions required and increased fusion frequency with A. nidulans protoplasts. A reduction in protoplast viability was observed after electrofusion but the majority of the protoplasts remained viable over a 24-h incubation period. A small decline in protoplast respiration rate occurred during incubation but those celery protoplasts fused with A. nidulans protoplasts showed elevated respiration rates for 3 h after electrofusion.Abbreviations AC alternating current - DC direct current     

Factors affecting protoplast electrofusion efficiency

The electrofusion efficiency of protoplasts isolated from a carrot (Daucus carota) suspension culture was increased by treatment with 0.1 mg/ml lysolecithin, 2.5% dimethylsulfoxide (DMSO), or 0.5 mM Ca²⁺. The lysolecithin and DMSO treatments substantially increased protoplast lysis, whereas calcium treatment did not. The enzymes used for protoplast isolation were also found to have a dramatic effect on the efficiency of fusion. A mixture of Cellulysin and Driselase led to a two-fold enhancement of fusion as compared with Driselase alone. The stimulation by Cellulysin appears to be due to enzymatic modification of the cell surface. However, comparison of the time course for wall digestion with the development of susceptibility to electrofusion suggests that the effect of Cellulysin is not simply due to removal of the cell wall. Brief treatment of the cells with pronase or proteinase K also doubled the efficiency of fusion. Taken together, these results indicate that electrofusion efficiency can be enhanced by the method used for protoplast isolation; they also suggest that modification of membrane/cell-surface proteins during protoplast isolation may be particularly important in determining electrofusion efficiencies.Abbreviations a.c. alternating current - d.c. direct current - DMSO dimethylsulfoxide - NAA naphthaleneacetic acid - PEG polyethylene glycol     

Effects of la on surface charges, dielectrophoresis, and electrofusion of barley protoplasts

When dielectrophoresis and electrofusion of barley (Hordeum vulgare var Moor) leaf protoplasts were assayed in the presence of 0.1 to 1 millimolar lanthanum ion (La³⁺) in the basal medium (0.7 molar mannitol, 1 millimolar piperazine-N, N-bis[2-ethanesulfonic acid]-Na [pH 6.7], 0.1 millimolar CaCl₂), dielectrophoresis and induction of electrofusion were strongly inhibited. The latter remained inhibited and the former recovered by about 60% after washing the La³⁺ -treated protoplasts without EDTA. These inhibitions were almost completely abolished by washing the La³⁺ -treated protoplasts with 1 millimolar EDTA. Inductively coupled plasma atomic emission spectroscopic analysis revealed that protoplasts retained a considerable amount of La³⁺ after washing without EDTA and released most of the bound La³⁺ by washing with 1 millimolar EDTA. This tightly bound La³⁺ seemed responsible for the inhibition of electrofusion and dielectrophoresis that was observed in the La³⁺ -treated protoplasts after washing. ζ-potentials of protoplasts were -39.0±3.2 millivolts, -16.7 ± 2.6 millivolts, and virtually zero in media containing 0, 0.1, and 0.3 millimolar La³⁺ (I = 7.2 millimolar), respectively, and had a positive value (+ 14.2 ± 2.2 millivolts) in the presence of 1 millimolar La³⁺. These effects of La³⁺ on ζ-potentials were easily abolished by washing without EDTA. This indicates that charged species located at the surface of plasma membrane of protoplasts cannot account for the sites at which La³⁺ exerts its inhibition of dielectrophoresis and electrofusion. In contrast, the promotion of spherical fusion and the reduction of broken fusion products observed in the presence of La³⁺ were almost completely abolished by washing without EDTA. Our results also indicate that the initial induction and development of electrofusion can be studied independently.     

High plating efficiency and plant regeneration frequency in low density protoplast cultures derived from an embryogenic Brassica napus cell suspension

Protoplasts were isolated from an embryogenic cell suspension culture derived from microspores of Brassica napus cv. Jet Neuf. Protoplast yield varied with the cell suspension growth medium. Optimization of protoplast plating density, manipulation of culture medium, carbon source and medium matrix, and inclusion of Ficoll resulted in protoplast plating efficiencies close to 30%. Placement of the protoplasts close to the gas interface contributed greatly to the elevated plating efficiency. Low density cultures could be induced to regenerate calli at optimum plating efficiencies if grown in the presence of nurse culture. This is of great advantage for manipulation of individual protoplasts or for microinjection. Plants were regenerated directly from the cell suspension or from the protoplast cultures.Abbreviations BA N⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA naphthaleneacetic acid     

Behavior and viability of tobacco protoplasts in response to electrofusion parameters

This investigation examines responses of protoplasts in a systematic and quantitative way to the various electrical treatments used to achieve electrofusion and their individual and cumulative effect on protoplast viability. Mesophyll and cell suspension protoplasts from two species of the same genera, Nicotiana tabacum and N. rustica var brasilia were used in these experiments. Optimal frequencies for alignment of tobacco protoplasts were between 500 kilohertz and 2 megahertz at 100 volts per centimeter. Variations in frequency and voltage of the alternating current (AC) field caused predictable movements of protoplasts within an electrofusion chamber. AC frequencies below 10 hertz or above 5 megahertz significantly decreased the viability of protoplasts in the fusion chamber as estimated by fluorescein diacetate staining 1 hour after treatment. Although the direct current (DC) pulse appeared to have a slight detrimental effect on protoplast viability, this effect was not significantly different from untreated control preparations.

Protoplasts from both leaf mesophyll cells and suspension cells were induced to fuse with one or more 10 to 30 microseconds DC square wave pulses of approximately 1 kilovolt per centimeter after the protoplasts had been closely appressed with an AC field.

     

Importance of myo-inositol,calcium, and ammonium for the viability and division of tomato (Lycopersicon esculentum) protoplasts

Plants from four cultivars of Lycopersicon esculentum were grown under different conditions, in controlled environment chambers. Low light intensity, long photoperiod (16 h), 25° C/17°C temperature alternance (day/night) were found to be the most convenient conditions for obtaining viable protoplasts. The use of myo-inositol as an osmoticum in the digestion medium and the adjustment of the pH to 6.5, instead of the usual 5.8, for this medium increased the yield of viable protoplasts and enhanced their stability. Under these conditions neither pretreatment (dark and cold treatments), nor preplasmolysis of leaf tissues, were required before protoplast isolation. The concentrations of ammonium nitrate, calcium chloride, myo-inositol, and sucrose were found to be critical for the success of protoplast culture. A medium containing 5 mM ammonium nitrate, 40 mM calcium chloride, 10 mg l^-1 adenine sulfate, 0.5% myo-inositol and 6% sucrose gave sustained protoplast divisions. Under these conditions, plating efficiency ranged from 5% for the cultivar Lukulus to 15% for the cultivar Golden Sunrise.Abbreviations BA benzylaminopurine - CaCl₂ calcium chloride, 2,4,-D-2,4-dichlorophenoxyacetic acid - EDTA ethylene diamine tetraacetic acid - KCl potassium chloride - MES-2-N morpholino ethane sulfonic acid - MgCl₂ magnesium chloride - NH₄NO₃ ammonium nitrate - NAA naphthalene acetic acid, p-protoplasts     

Electrofusion of giant protoplasts of Pleurotus cornucopiae

Summary Giant protoplasts of Pleurotus cornucopiae were fused, using the glass microelectrode electrofusion technque; the percentage fusion achieved was 70%. To induce fusion, Ca²⁺ was necessary, a 10 mM concentration giving the best result. Polyethylene glycol 4000 (PEG) promoted fusion but also increased the adhesion of protoplasts, which caused them to be irreversibly attached to the electrodes. Fusion was always completed within 1 min after a single electrical pulse had been applied. The fused protoplast was isolated with a glass micropipette and was found to regenerate into a colony.     

Fusion of plant protoplasts: a study using auxotrophic mutants of Nicotiana plumbaginifolia,Viviani

Summary Protoplast fusion studies between various auxotrophic mutants of Nicotiana plumbaginifolia were performed to optimize conditions for PEG-mediated fusion and to identify factors influencing the plant protoplast fusion process. Numerous parameters in the isolation, culture, and fusion of protoplasts were tested, and established fusion protocols were compared. Fusion rates, calculated on the basis of colony growth on selection medium (genetic complementation), ranged from 10^–4 to 10^–2. Conditions that allow rapid and reproducible fusions at the highest rates were established. Particular emphasis was given to fusion of mesophyll-derived protoplasts, for which the ability to regenerate fertile plants from fusion products was shown to be particularly high. Preliminary experiments using electric-field mediated fusion suggest that electrofusion may offer significant advantages over the traditional chemical fusion.     

Induction of fast-growing and morphologically different strains through intergeneric protoplast fusions of Ulva and Enteromorpha (Ulvales,Chlorophyta)

Isolated protoplasts of Ulva pertusa and Enteromorpha prolifera were electrically fused. Treatment of protoplasts in 1% protease for 15–20 min prior to fusion enhanced fusion ability. Protoplasts from each fusion partner were mixed together in 1:1 ratio in low conductivity electrofusion solution at a density of 1 × 10⁵ cells ml⁻¹ before subjecting them to electrofusion. The protoplasts were aligned in AC field (1MHz, 25 V for 10–15 s) and subsequently fused by a high intensity single DC pulse of 250 V for 25 μs duration. Fusion buffer supplemented with 1 mM calcium and 1 mM magnesium yielded optimum fusion frequencies (about 18–24%). Entrapment of fusion treated cells inside agarose/agar plate facilitated marking and regeneration of fusion products. The regeneration patterns of fused protoplasts were similar to normal (unfused) protoplast development. Most of the regenerated plants from fusion products had a thallus similar to either U. pertusa type or E. prolifera type. Although some of the plants of the former were morphologically similar to U. pertusa, but most had a higher growth rate (1.9 to 1.5 times) than U. pertusa. Furthermore the thallus of some plants had a characteristic irregular and dentate margin, which was never observed in the parental type.     

Colony formation and plant regeneration from mesophyll protoplasts of Solanum etuberosum

A procedure for the culture of Solanum etuberosum mesophyll protoplasts with subsequent shoot regeneration is described. Several factors affected protoplast yield, colony formation, and shoot regeneration from in vitro plants. A protoplast isolation medium with 0.6 M sucrose produced twice the yield as one with 0.3 M sucrose. uowever, a higher concentration of osmoticum was inhibitory to colony development unless it was diluted into a lower osmoticum medium in a bilayer system. A 16 hour light/8 hour dark photoperiod for stock plants allowed twice the protoplast yield compared to plants grown under continuous light but no effect was found on subsequent colony formation or shoot regeneration. The concentrations of four major salts in the protoplast plating medium were critical for a high frequency of colony formation from protoplasts. Levels of 0.25 × or 1 × were considerably better than 4 ×. Fast colony formation, but at a lower efficiency, was obtained with a monolayer plating method. A bilayer plating system allowed a higher efficiency but colonies developed more slowly. For the best treatments, the frequency of colony formation from protoplasts ranged from 2.4 to 3.6 × 10^-3 with 37% to 66% of the colonies producing shoots ten weeks after protoplast isolation.Cooperative investigation of the USDA-ARS and the Wisconsin Agric. Exp. Stn.     

Effects of prostaglandins e(2) and f(2alpha) on the electrofusion of pea mesophyll protoplasts

The effects of prostaglandins E₂ and F_2α on the electrofusion of pea (Pisum sativum cv Ran 1) mesophyll protoplasts were examined. Prostaglandins E₂ and F_2α influenced electrofusion by lowering the threshold voltage necessary for fusion of dielectrophoretically arranged pairs of protoplasts. The direct current voltage threshold decreased with increasing Ca²⁺ concentration up to 0.1 millimolar CaCl₂ and the effects of prostaglandins E₂ and F_2α were more pronounced when CaCl₂ was present in the medium. Treatment with calcium channel blocker methoxy verapamil did not change the prostaglandin effects, while the addition of ethyleneglycol-bis (β-aminoethyl either)-N,N,N′,N′-tetraacetic acid, which binds free Ca²⁺, increased the threshold voltage. Influence of prostaglandins E₂ and F_2α and Ca²⁺ on the membrane fluidity was investigated by analysis of pyrene fluorescence spectra. The values of the ratio between the maximum fluorescence emission intensities of the excimer and the monomer forms (I_ex/I_mon) indicated that prostaglandins and Ca²⁺ decrease the membrane fluidity. It is proposed that electrically evoked displacement of plasmalemma components takes part in the fusion process (U Zimmermann 1982 Biochim Biophys Acta 694: 227-277). We suggest that prostaglandins E₂ and F_2α facilitate the electrofusion of pea mesophyll protoplasts by changing the fluidity of plasmalemma.     

An improved procedure for plant regeneration from indica and japonica rice protoplasts

Plant regeneration from protoplasts of two commercially cultivated Indian indica rice varieties, Pusa Basmati 1 and Java, has been accomplished by plating embryogenic cell suspension-derived protoplasts on the surface of filter membranes overlying agarose-embedded feeder cells of Lolium multltiflorum and Oryza ridleyi, combined with the use of a maltose-containing shoot regeneration medium. Embryogenic cell suspension cultures of Pusa Basmati 1 and Jaya were initiated from mature seed scutellum-derived calli in liquid R₂ medium modified by the addition of 560 mg l^–1 of proline and 1.0 % (w/v) maltose. In both varieties, protoplast plating efficiencies up to 0.4 % were obtained, depending on the nature of the feeder cells. L. multiflorum feeder cells induced a 6-fold higher plating efficiency than feeder cells of O. ridleyi. In combination, O. ridleyi and L. multiflorum feedercells further enhanced protoplast plating efficiency. Protoplast-derived cell colonies were not obtained from protoplasts of either indica varieties in the absence of feeder cells. MS-based medium containing kinetin (2.0 mg l^–1) and -naphthaleneacetic acid (0.5 mg 1^–1), together with sucrose and maltose both at 1.5 % (w/v), induced green shoot regeneration in 44 % of protoplast-derived tissues, depending on the feeder cells used for protoplast culture. In both varieties, tissues obtained using O. ridleyi feeder cells were more morphogenic than tissues obtained using L. multiflorum feeder cells, either alone or in combination with cells of O. ridleyi. In the japonica rice variety Taipei 309, this new procedure resulted in a 30-fold increase in plant regeneration from protoplasts compared to previous published procedures.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - GPFs growth promoting factors - NAA -naphthaleneacetic acid On leave from Department of Genetics, Haryana Agricultural University, Hisar, IndiaOn leave from Biotechnology Centre, Punjab Agricultural University, Ludhiana, India     

An efficient and rapid procedure for plantlet regeneration from chicory mesophyll protoplasts

An efficient procedure for plantlet regeneration from chicory mesophyll protoplasts has been developed in order to perform protoplast fusion experiments. Protoplasts were isolated from a genotype of Italian red chicory (CH 363) and purified by centrifugation in a solution containing 13% (w/v) sucrose to collect uniform protoplasts in size. After 2 days culture at a density of 2×10⁴ protoplasts ml⁻¹ of liquid medium, protoplasts were cultured following three different procedures: in liquid medium, stratified in semi-solid medium, and embedded in Ca-alginate droplets. Four different media were used and culture procedures were evaluated recording the protoplast viability, protoplast division frequency and plating efficiency for each experiment. The embedding of protoplasts in Ca-alginate droplets enhanced both division frequency and plating efficiency for chicory mesophyll cells. Furthermore, this procedure shortened the cycle of plant regeneration from protoplasts, which could be completed in eight weeks. This revised version was published online in June 2006 with corrections to the Cover Date.     

Immunological evidence for transfer of the barley nitrate reductase structural gene to Nicotiana tabacum by protoplast fusion

Summary A protoplast fusion experiment was designed in which the selectable marker, nitrate reductase (NR), also served as a biochemical marker to provide direct evidence for intergeneric specific gene transfer. NR-deficient tobacco (Nicotiana tabacum) mutant Nia30 protoplasts were the recipients for the attempted transfer of the NR structural gene from 50 krad -irradiated barley (Hordeum vulgare L.) protoplasts. Barley protoplasts did not form colonies and Nia30 protoplasts could not grow on nitrate medium; therefore, selection was for correction of NR deficiency allowing tobacco colonies to grow on nitrate medium. Colonies were selected from protoplast fusion treatments at an approximate frequency of 10^-5. This frequency was similar to the Nia30 reversion frequency, and thus provided little evidence for transfer of the barley NR gene to tobacco. Plants regenerated from colonies had NR activity and were analyzed by western blotting using barley NR antiserum to determine the characteristics of the NR conferring growth on nitrate. Ten plants exhibited tobacco NR indicating reversion of a Nia30 mutant NR locus. Twelve of 26 regenerated tobacco plants analyzed had NR subunits with the electrophoretic mobility and antigenic properties of barley NR. These included plants regenerated from colonies selected from 1) co-culturing a mixture of Nia30 protoplasts with irradiated barley protoplasts without a fusion treatment, 2) a protoplast fusion treatment of Nia30 and barley protoplasts, and 3) a fusion treatment of Nia30 protoplasts with irradiated barley protoplasts. No barley-like NR was detected in plants regenerated from a colony that grew on nitrate following selfed fusion of Nia30 protoplasts. Because tobacco plants expressing barley-like NR were recovered from mixture controls as well as fusion treatments, explanations for these results other than protoplast fusionmediated gene transfer are discussed.     

The hydraulic conductivity as a criterion for the membrane integrity of protoplasts fused by an electric field pulse

The hydraulic conductivity of the membrane, Lp, of fused plant protoplasts was measured and compared to that for unfused cells, in order to identify possible changes in membrane properties resulting from the fusion process. Fusion was achieved by an electric field pulse which induced breakdown in the membranes of protoplasts in close contact. Close membrane contact was established by dielectrophoresis. In some experiments pronase was added during field application; pronase stabilizes protoplasts against high field pulses and long exposure times to the field. The Lp-values were obtained from the shrinking and swelling kinetics in response to osmotic stress. The Lp-values of fused mesophyll cell protoplasts of Avena sativa L. and of mesophyll and guard cell protoplasts of Vicia faba L. were found to be 1.9±0.9·10^-6, 3.2±2.2·10^-6, and 0.8±0.7·10^-6 cm·bar^-1·s^-1, respectively. Within the limits of error, no changes in the Lp-values of fused protoplasts could be detected in comparison to unfused protoplasts. The Lp-values are in the range of those reported for walled cells of higher plants, as revealed by the pressure probe.Abbreviations GCP guard cell protoplast - Lp hydraulic conductivity - MCP mesophyll cell protoplast     

Plant regeneration of protoplasts isolated from suspension cultures of Solanum lycopersicoides

A protocol was developed for the isolation, culture and plant regeneration of protoplasts isolated from suspension cultures of Solanum lycopersicoides Dun. (LA 1990). Protoplasts were isolated by an overnight enzyme digestion, further purified by washing in W5 salts solution, and plated in two modified MS protoplast culture media with and without type VII agarose. The addition of agarose to the two culture media did not enhance plating efficiencies and shoot regeneration percentages and in some cases was even inhibitory. Unlike the experience with some other solanaceous species, the deletion of ammonium from the protoplast culture medium was not found to be beneficial. Protoplasts sustained continuous division in the modified MS media and up to 70% of the protoplast-derived calli readily regenerated shoots on MS salts and vitamins medium containing zeatin and GA.     

Possible involvement of calmodulin and the cytoskeleton in electrofusion of plant protoplasts

Calmodulin antagonists, trifluoperazine, chlorpromazine, calmidazolium, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), strongly inhibited the electrofusion of barley (Hordeum vulgare L. cv Moor) protoplasts with a marked increase of broken fusion products, after 60 minutes of incubation. W-5, a dechlorinated analog of W-7, was found less effective for the inhibition than W-7. Ethyleneglycol-bis(β- aminoethylether)-N,N′-tetraacetic acid a Ca²⁺ chelator, La³⁺, a surface Ca²⁺ antagonist, and verapamil, a Ca²⁺ channel blocker, also inhibited electrofusion. Dielectrophoresis was inhibited by La³⁺. A microtubule inhibitor, vinblastine, inhibited electrofusion strongly while colchicine, slightly. A microfilament inhibitor, cytochalasin B, promoted fused cells to become spherical while phalloidin did not affect electrofusion.     

Electrical fusion for optimal formation of protoplast heterokaryons in Nicotiana

The electrical fusion of protoplasts has been studied in order to maximize the formation of heterokaryons for culture. Heterokaryons of Nicotiana tabacum L. mesophyll protoplasts and N. plumbaginifolia Viviani supension-cell protoplasts were identified in fixed and stained as well as living material; a quantitative fusion index was thereby developed. With this index the efficiencies of various electric fields and fusion-chamber designs have been determined. Optimal fusion was obtained with an alternating-current (AC) field of 150 V/cm and direct-current (DC) square-wave pulses of 1000 V/cm. A new, simple-to-use, largescale fusion chamber is described in which batches of up to 5·10⁵ protoplasts (0.5 ml of cells at 10⁶/ml) can be fused in 5–7 min with efficiencies approaching 40%. Half of the fusion products are heterokaryons, thus fusion is random. Of the fusion products, 60% are bi- or trinucleate. Using fusion procedures similar to those described here Bates and C. Hasenkampf (1985, Theor. Appl. Genet., in press) have recovered viable somatic hybrids which have been regenerated.Abbreviations AC alternating current - DC direct current - PEG polyethylene glycol     

Influence of pH and sucrose concentration on nonenzymatic and enzymatic isolation of protoplasts from mature pollen of Tulbaghia violacea

Studies on protoplast isolation were carried out with mature pollen grains of Tulbaghia violacea Harv. (Liliaceae). Pollen grains drifted from surface sterilized crushed anthers were incubated either in a nonenzymatic solution composed of Nitsch medium and sucrose, or in the same solution supplemented with 1% cellulase Onozuka R-10 and 1% Macerozyme R-10. The process of protoplast release was studied as a function of pH and sucrose concentration of nonenzymatic and enzymatic solutions. For nonenzymatic isolation, the tested range of pH and sucrose concentration was from 3.3 to 13.1 and from 0.015 to 1.12 M (final solution osmolality from 200 to 1,300 mOs kg^-1 H₂O), respectively. In the former case, the release of protoplasts occurred only at nonphysiological pH (12.2 to 13.1) and could be observed after several seconds to 120 min, depending on pH and sucrose concentration of medium. Under enzymatic incubation, viable protoplasts were released more rapidly (3 to 35 min) and in more physiological conditions, the optimum being pH 5.8 and final medium osmolality 652 mOs kg^-1 H₂O. Speed, manner of protoplast release, number and quality of protoplasts were dependent on interactions of pH and sucrose concentration.