Fusion of Avena sativa mesophyll cell protoplasts by electrical breakdown

Studies with the light microscope were carried out on mesophyll cell protoplasts of Avena sativa which had been made to undergo fusion by reversible electrical breakdown of the cell membrane. In order to establish close membrane contact between the cells, an important prerequisite for fusion, a method known as dielectrophoresis was used. In an inhomogeneous alternating electrical field the protoplasts adhere to the electrodes and to each other in the direction of the field lines. The cells which were thus brought into close contact with each other could be made to fuse by the application of a field pulse of high amplitude (about 750 V/cm) and short duration (20–50 μs). The field strength required for fusion exceeds the value necessary for the electrical breakdown of the cell membrane. Fusion took place within some minutes and led to a high yield of fused protoplasts. The fusion of cells being in the electric field occured in a synchronous manner. In some of the fusion experiments part of the protoplasts of A. sativa were stained with neutral red. When these cells were fused with unstained protoplasts, the vacuoles from the different cells within the fused aggregate could be shown to remain separate for quite some time.     

Electrically Stimulated Fusion of Different Plant Cell Protoplasts : MESOPHYLL CELL AND GUARD CELL PROTOPLASTS OF VICIA FABA

Cell fusion is induced between guard cell and mesophyll cell protoplasts of Vicia faba by electrical field application. The process of fusion is initiated by electrical breakdown of the cell membrane. Prior to the application of an external electrical field pulse which brings about reversible breakdown of the membrane, the cells (suspended in a low-conducting medium) are brought into close contact with one another by exposing them to an external alternating, nonuniform field (5 volts, electrode distance, 200 micrometers; 500 kiloHertz). During this process, they form “pearl chains” which may become sufficiently long to form bridges between the electrodes. The process is reversible as long as this voltage is not exceeded. Cell fusion is initiated as a result of an electrical field pulse of 50 microseconds duration and of sufficiently high intensity to induce reversible electrical breakdown of the membranes. The process of fusion is completed within 40 minutes or less in the case of guard cell protoplasts, as well as in the case of fusion between guard cell and mesophyll cell protoplasts. The fused cells are spherical in shape, if the fusion product consists only of two or three cells.     

Fusion characteristics of plant protoplasts in electric fields

The electrical parameters important in the fusion of plant protoplasts aligned dielectrophoretically in high-frequency alternating electric fields have been established. Protoplasts were aligned in an alternating electric field between two relatively distant (1 mm) electrodes, by dielectrophoresis induced by field inhomogeneities caused by the protoplasts themselves. This arrangement allowed ease of manipulations, large throughput and low loss of protoplasts. In analytical experiments, sufficiently large samples could be used to study pulse duration-fusion response relations at different pulse voltages for protoplasts of different species, tissues and size (mesophyll protoplasts of Solanum brevidens, Triticum aestivum, Hordeum vulgare; suspension-culture protoplasts of Nicotiana sylvestris, N. rustica, Datura innoxia and S. brevidens; root-tip protoplasts of Vicia faba, hypocotyl protoplasts of Brassica napus). The percentage of aligned protoplasts that fused increased with increasing pulse parameters (pulse duration; voltage) above a threshold that was dependant on pulse voltage. The maximum fusion values obtained depended on a number of factors including protoplast origin, size and chain length. Leaf mesophyll protoplasts fused much more readily than suspension-culture protoplasts. For both types, there was a correlation of size with fusion yield: large protoplasts tended to fuse more readily than small protoplasts. In short chains (five protoplasts), fusion frequency was lower, but the proportion of one-to-one products was greater than in long chains (ten protoplasts). In formation by electrofusion of heterokaryons between mesophyll and suspension-culture protoplasts, the fusion-frequency response curves reflected those of homofusion of mesophyll protoplasts rather than suspension-culture protoplasts. There was no apparent limitation to the fusion of the smallest mesophyll protoplast with the largest suspension-culture protoplasts. Based on these observations, it is possible to direct fusion towards a higher frequency of one-to-one (mesophyll/suspension) products by incorporating low densities of mesophyll protoplasts in high densities of suspensionculture protoplasts and by using a short fusion pulse. The viability of fusion products, assessed by staining with fluorescein diacetate, was not impaired by standard fusion conditions. On a preparative scale, heterokaryons (S. brevidens mesophyll-N. sylvestris or D. innoxia suspension-culture) were produced by electrofusion and cultured in liquid or embedded in agar, and were capable of wall formation, division and growth. It is concluded that the electrode arrangement described is more suitable for carrying out directed fusions of plant protoplasts than that employing closer electrodes.     

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.

     

Effects of Microgravitation on Electrofusion of Plant Cell Protoplasts

Electrofusion of evacuolated with vacuolated mesophyll protoplasts of Nicotiana tabacum was performed as part of the German Sounding Rocket Program (TEXUS 17, 1988). The results indicate a significant increase not only in the yield of 1:1 hybrids, but also in homo- and multifusion products. Hybrids obtained under microgravity have been shown to be viable to a higher degree with respect to their ability for light-dependent O₂-evolution (independent of other substrates than bicarbonate). This finding is of interest for fusion experiments were only limited numbers of fusion partners are available (e.g. protoplasts from embryogenic tissues) or where fusion yields are extemely low under 1 × gravity (e.g. protoplasts of different specific density).     

Isolation of Intact and Functional Chloroplasts from Mesophyll and Bundle Sheath Protoplasts of the C(4) Plant Panicum miliaceum

A procedure is described for isolating and purifying mesophyll protoplasts and bundle sheath protoplasts of the C₄ plant Panicum miliaceum. Following enzymic digestion of leaf tissue, mesophyll protoplasts and bundle sheath protoplasts are released and purified by density centrifugation. The lower density of mesophyll protoplasts allowed rapid separation of the two protoplast types. Evidence for separation of mesophyll protoplasts and bundle sheath protoplasts (up to 95% purity) is provided from light microscopy (based on size difference in both chloroplasts and protoplasts), levels of marker enzymes in the preparations (i.e. pyruvate, Pi dikinase and phosphoenolpyruvate carboxylase for mesophyll and ribulose-1,5-bisphosphate carboxylase for bundle sheath), and differences in substrate-dependent O₂ evolution by chloroplasts isolated from protoplasts.     

Culture of plant somatic hybrids following electrical fusion

Summary Electrically-induced protoplast fusion has been used to produce somatic hybrids between Nicotiana plumbaginifolia and Nicotiana tabacum. Following fusion of suspension culture protoplasts (N. plumbaginifolia) with mesophyll protoplasts (N. tabacum) heterokaryons were identified visually and their development was followed in culture. Because electrical fusion is a microtechnique, procedures were developed for culturing the heterokaryons in small numbers and at low density. The fusion and culture procedures described are rapid, uncomplicated and repeatable. Good cell viabilities indicate that the fusion procedure is not cytotoxic. Fused material was cultured 1–2 days at high density in modified K3 medium (Nagy and Maliga 1976). The heterokaryons were isolated manually and grown, at low density in conditioned media. Calli have been regenerated. Esterase isozyme patterns confirm the hybrid character of calli and clonally-derived plantlets recovered from these fusions.     

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     

三倍体‘银中杨’叶肉原生质体制备的优化

以三倍体杨树品种‘银中杨’（Populus alba×P.berolinensis Yinzhong）无菌苗叶片为材料,对其原生质体分离及纯化条件进行研究,为进一步通过细胞融合、基因工程等进行品种改良探索新的途径。结果表明：酶的种类及浓度、渗透压、酶解时间对‘银中杨’叶肉原生质体分离效果有显著影响,适宜的分离条件为CPW+3% Cellulase RS+0.5% Macerozyme R-10+0.3% Pectinse Y-23+0.6 mol/L甘露醇+0.6 g/L MES+1 g/L BAS,酶解时间为8 h,原生质体产量和活力分别为2.13×10⁷个/g和80.18%;‘银中杨’叶肉原生质体纯化最佳方法为上浮法蔗糖等密度离心,且蔗糖浓度为40%时原生质体产量最高（1.06×10⁷个/g）,可满足进一步的原生质体培养等技术的要求。     

A method for production of interspecific hybrids within Brassiceae via somatic hybridization,using resynthesis of Brassica napus as a model

A general method for the production of somatic hybrids within Brassiceae has been developed using mesophyll protoplasts from one parent and hypocotyl protoplasts from the other. Fusion products were easily identified by their intermediate phenotype between the parental protoplasts. They could be isolated 24 h after fusion with micropipettes using a micromanipulator. At that time their frequency was about 15%. They were cultured in small volumes, 10 μl, and a plating efficiency of 14% was obtained. Hybrid calli were obtained from the fusion products, which was confirmed by isozyme analysis. Ploidy level of one hybrid shoot was determined by flow cytometric DNA analysis.     

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 CaCl₂ up to 5 mm, and protoplast fusion in the presence of 3 mm CaCl₂ 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.     

Culture and fusion of pollen protoplasts of Brassica oleracea L. var. italica with haploid mesophyll protoplasts of B. rapa L. ssp. pekinensis

Hybrid callus was formed from the successful protoplast fusion between pollen protoplasts of Brassica oleracea var. italica and haploid mesophyll protoplasts of Brassica rapa. The pollen protoplast isolation frequency in broccoli was highly related to the ratio of trinucleate pollens in the male gametophyte population. Large quantities of pollen protoplasts with high vigor could be isolated, and the isolation frequency reached up to 90% in 6.0-7.0 mm long flower buds with about 94.7% trinucleate-stage pollens. Pollen protoplasts could be collected and purified by discontinuous gradient centrifugation. In 1% Na-alginate embedding culture, cell divisions were observed but no further development was found. The haploid mesophyll protoplasts were isolated from in vitro haploid plants of B. rapa. Results strongly showed the variability in culturability of mesophyll protoplasts from different haploid lines. Both pollen protoplasts and haploid mesophyll protoplasts retained a stable round shape in the designed prefusion solution with an osmotic pressure of 0.74 osmol/kg. Polyethylene glycol was used for the protoplast fusion, and 40% polyethylene glycol 4000 enabled the highest fusion frequency of about 20%. Some postfusion protoplasts showed cell divisions up to callus proliferation. Calli were screened by random amplified polymorphic DNA analysis for their hybrid character. Results revealed the existence of the hybrid calli. Some of the hybrid calli grew well with green color and shoot primordia. According to our knowledge, this is the first report about a hybrid formation between two haploid protoplasts. Potential comprehensive applications, as well as problems of this technique, are discussed.     

Separation of mesophyll protoplasts and bundle sheath cells from maize leaves for photosynthetic studies

Mesophyll protoplasts and bundle sheath strands of maize (Zea mays L.) leaves have been isolated by enzymatic digestion with cellulase. Mesophyll protoplasts, enzymatically released from maize leaf segments, were further purified by use of a polyethylene glycol-dextran liquid-liquid two phase system. Bundle sheath strands released from the leaf segments were isolated using filtration techniques. Light and electron microscopy show separation of the mesophyll cell protoplasts from bundle sheath strands. Two varieties of maize isolated mesophyll protoplasts had chlorophyll a/b ratios of 3.1 and 3.3, whereas isolated bundle sheath strands had chlorophyll a/b ratios of 6.2 and 6.6. Based on the chlorophyll a/b ratios in mesophyll protoplasts, bundle sheath cells, and whole leaf extracts, approximately 60% of the chlorophyll in the maize leaves would be in mesophyll cells and 40% in bundle sheath cells. The purity of the preparations was also evident from the exclusive localization of phosphopyruvate carboxylase (EC 4.1.1.31) and NADP-dependent malate dehydrogenase (EC 1.1.1) in mesophyll cells and ribulose 1,5-diphosphate carboxylase (EC 4.1.1.39), phosphoribulokinase (EC 2.7.1.19), and “malic enzyme” (EC 1.1.1.40) in bundle sheath cells. NADP-glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.13) was found in both mesophyll and bundle sheath cells, while ribose 5-phosphate isomerase (EC 5.3.1.6) was primarily found in bundle sheath cells. In comparison to the enzyme activities in the whole leaf extract, there was about 90% recovery of the mesophyll enzymes and 65% recovery of the bundle sheath enzymes in the cellular preparations.     

Acceleration of Chloroplast Division in Somatic Cell Hybrids between Mesophyll and Cell Culture Protoplasts

Tobacco mesophyll protoplasts were fused with protoplasts fromcultured cells by electric fusion. When the fusion productswere cultured for 2 d, chloroplast division was observed inthe heterokaryocytes under fluorescence microscopy after stainingwith DAPI, while such chloroplast division was not observedwhen mesophyll protoplasts alone were cultured in the same condition. ³Permanent address: Research Institute of House Food IndustryCo. Ltd., Mikuriya Sakaemachi, Higashi-Osaka-shi, Osaka, 577Japan. (Received June 21, 1988; Accepted November 22, 1988)     

Ribulosebisphosphate carboxylase activity and photosynthetic o(2) evolution rate in vicia guard-cell protoplasts

Activities of ribulose-1,5-bisphosphate carboxylase and rates of photosynthetic O₂ evolution were measured in guard-cell and mesophyll protoplasts from Vicia faba. The ribulose-1,5-bisphosphate carboxylase activity of guard-cell protoplasts was 30% of that of mesophyll protoplasts; however, the O₂ evolution rate was 3 times higher in guard-cell protoplasts than in mesophyll protoplasts on a chlorophyll basis. When the dark-adapted, guard-cell protoplasts were illuminated by red light, O₂ was evolved with an induction period, which became shorter when the protoplasts were reilluminated. High activity of irreversible NADP-glyceraldehyde-3-phosphate dehyrogenase was found in guard-cell protoplasts. Several lines of evidence revealed that there was virtually no contamination by mesophyll cells in guard-cell preparations. These results indicate that guard cells fix CO₂ photosynthetically and imply that the cells utilize a considerable proportion of reducing equivalents from water for reactions other than CO₂ fixation.     

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     

Electric field-induced fusion of isolated vacuoles and protoplasts of different developmental and metabolic provenience

Using an electric field pulse technique, we induced fusion between vacuoles and protoplasts of Kalanchoë daigremontiana , between protoplasts from etiolated and green leaf mesophyll, and between mesophyll protoplasts from plants of different physiological properties ( Avena sativa : C₃ mechanism of photosynthesis, Kalanchoë daigremontiana : crassulacean acid metabolism). Close membrane contact amongst protoplasts or between protoplasts and vacuoles (as required for fusion) was achieved by the application of an alternating, non-uniform electric field to the suspension. Due to the dielectrophoresis effect the cells attach to each other along the field lines. The fusion process is initiated by the injection of an electric field pulse of high intensity and short duration (μs range). The field intensity has to be sufficiently high to induce reversible breakdown in the area of close membrane contact. After the application of the field pulse, the fusion process is initiated and completed within seconds to a few minutes, depending on the material investigated.
Fusion occurs between protoplasts and vacuoles as well as between protoplasts of different species. Both tonoplast and plasma membranes completely intermingled, indicating that in contrast to suggestions in the literature these membranes are compatible. Furthermore the cytoplasms of etiolated and green protoplasts obviously do not mix after fusion is completed, as etioplasts and chloroplasts kept separated from each other. In all experiments the volume of the fusion product equalled the sum of the compartments that underwent fusion. The wide spectrum of possible applications resulting from these fusion experiments in relation to metabolic problems is discussed.     

Preparation of leaf mesophyll protoplasts for transient gene expression in Brachypodium distachyon

Transient gene expression systems using protoplasts have been widely used for rapid functional characterization of genes in many plant species. Brachypodium distachyon has recently been employed as a model plant for studies on biofuel grass species and grass crops because of its small genome size, short life-span, and availability of efficient transformation systems. Here, we report the an efficient protocol for the preparation of leaf mesophyll protoplasts from Brachypodium seedlings. We also modified the polyethylene glycol (PEG)-mediated transformation procedure to optimize experimental conditions, such as duration of enzyme digestion, PEG incubation time, and plasmid DNA concentration and size. The green fluorescence protein (GFP)- and ??-glucuronidase (GUS)-coding genes were used as reporters to evaluate the feasibility of this transient expression system. We found that the yield of viable protoplasts was highest after 3 h of enzymatic digestion. Viability of enzyme-digested protoplasts was moderately maintained up to 24 h in Mmg preincubation solution. In addition, the highest transient expression of reporter genes was obtained when protoplasts were transformed with 20 ??g of plasmid DNA and incubated for 16 h. Together with the recent completion of the Brachypodium genome sequence, the Brachypodium transient expression system using leaf mesophyll protoplasts can be widely used for cellular, molecular, and biochemical studies of genes involved in carbon metabolism and signaling pathways mediating intrinsic and environmental cues.     

Polyamine Metabolism and Osmotic Stress : I. Relation to Protoplast Viability

Cereal leaves subjected to the osmotica routinely used for protoplast isolation show a rapid increase in arginine decarboxylase activity, a massive accumulation of putrescine, and slow conversion of putrescine to the higher polyamines, spermidine, and spermine (HE Flores, AW Galston 1984 Plant Physiol 75: 102). Mesophyll protoplasts from these leaves, which have a high putrescine:polyamine ratio, do not undergo sustained division. By contrast, in Nicotiana, Capsicum, Datura, Trigonella, andVigna, dicot genera that readily regenerate plants from mesophyll protoplasts, the response of leaves to osmotic stress is opposite to that in cereals. Putrescine titer as well as arginine and ornithine decarboxylase activities decline in these osmotically stressed dicot leaves, while spermidine and spermine titers increase. Thus, the putrescine:polyamine ratio in Vigna protoplasts, which divide readily, is 4-fold lower than in oat protoplasts, which divide poorly. We suggest that this differing response of polyamine metabolism to osmotic stress may account in part for the failure of cereal mesophyll protoplasts to develop readily in vitro.     

Selection and enrichment of plant protoplast heterokaryons of Brassicaceae by flow sorting

The experimental conditions for an efficient and reproducible enrichment of fusion products by flow cytometry, using protoplasts of different Brassica species as hybridization material, have been investigated. The heterokaryons were identified by the endogenous chlorophyll autofluorescencence of mesophyll protoplasts of one parent and the fluorescense of exogenously supplied carboxyfluorescin to the hypocotyl protoplasts of the other parent. By using a low head drive frequency (11 kHz), a large nozzle (110 μm) and a low nozzle pressure (30–35 kPa) good survival of the protoplasts was obtained after sorting. Heterokaryons were sorted using these parameters and on average 80% of the protoplasts were fusion products as judged by microscopy. They were cultured in small volumes, 150 μl, and started to divide after 3–5 days and regenerated calli easily. Isozyme analysis of the calli confirmed that 81% had the pattern typical for a hybrrid. Differentiation into shoots have been obtained from some of the hybrid calli; these shoots were also confirmed to be true hybrids.