Polyethylene glycol and electric field treatment of plant protoplasts: characterization of some membrane properties

Petiolar protoplasts of a dihaploid line of winter oilseed rape Brassica napus L. ssp. oleifera were exposed to fusogenic polyethylene glycol (PEG) and electric field treatments. The surface properties and stability of membrane components of the treated protoplasts were investigated by contact angle measurements in aqueous two-phase systems and differential scanning calorimetry, respectively. The leakage of intracellular components was estimated with respect to amino acids, proteins and DNA. Both fusogenic treatments resulted in the same apparent changes in membrane surface hydrophobicity and the same destabilization of membrane components. However, the PEG-treated protoplasts were more leaky than both the control and the electric field-treated protoplasts. The results indicate that the molecular mechanisms of PEG- and electrical field-induced fusion are similar. However, the effects of the latter appear to be less harmful presumably because the parameters for electric field treatment are more easily controlled.     

Induction of cell fusion of plant protoplasts by electrical stimulation

When an electric impulse of a few milliseconds was applied topoint-adherence protoplasts isolated from cultured cells ofRauwolfia serpentina through glass capillary microelectrodes,fusion of the protoplasts was immediately induced. This phenomenonseems to be related to transient changes in the membrane state,such as membrane excitation, induced by electrical stimulation. (Received February 22, 1979; )     

Quantitative studies on the viability of yeast protoplasts following dielectrophoresis

Abstract Protoplasts from Saccharomyces cerevisiae and Saccharomyces diastaticus were collected in a non-homogeneous alternating electric field. The dependence of the viability of the protoplasts on different conditions of collection was tested by determining the regeneration rates in each case. The parameters varied in collection were the field strength (0.33 kV/cm–6.67 kV/cm), the frequency of the alternating field (1–2 MHz) and the collection time (2–10 min). The introduction of a new type of fusion chamber (meander chamber) permitted, for the first time, quantitative exposure of protoplasts to the electric field as well as their complete transference into the regeneration medium. The regeneration rates of yeast protoplasts collected under those conditions employed for electrofusion did not differ from those of protoplasts which had been maintained under the same experimental conditions but were not subject to the influence of an alternating electric field. The two yeast strains were fused together (collection 1 kV/cm; pulse 15 kV/cm; duration of pulse 40 μs) and the fusion products were introduced into a selection medium for regeneration. The fusion rate was about 4.8 × 10⁻⁴; on average 272 colonies grew on the selection medium for each chamber filling.     

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.     

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.     

Possible role of transient electric fields in freezing-induced membrane destabilization

Summary Evidence is presented to support the hypothesis that electrical potentials generated during the freezing of aqueous solutions (the Workman-Reynolds effect) may contribute to the destabilization of the plasma membrane and cryoinjury of isolated protoplasts. Specifically. (1) electric potential diffrences of sufficient magnitude to cause lysis of the plasma membrane occur during the rapid freezing of isolated protoplasts suspended in sorbitol: (2) survival of protoplasts is inversely correlated with the magnitude of the potential difference and (3) cold acclimation increases the stability of the plasma membrane to applied electric fields. A discussion is given of the different physical phenomena though to be involved in the Workman-Reynolds effect. The basis equations for these phenomena are outlined.     

Co-electroporation of rice protoplasts with RNAs of cucumber mosaic and tobacco mosaic viruses

Cucumber mosaic virus (CMV) RNA was used to study electroporation conditions suitable for protoplasts from rice suspension cultures. Rice protoplasts required a stronger and shorter electric pulse than tobacco protoplasts for introduction of viral RNA. Under optimized conditions, CMV infection was established in 65 % of electroporated protoplasts. In contrast, electroporation with tobacco mosaic virus (TMV) RNA did not result in infection of rice protoplasts. However, when TMV RNA was electroporated into rice protoplasts together with CMV RNA, TMV production was demonstrated in 15 % of protoplasts. Differential staining with fluorescent antibodies against the two viruses showed that the protoplasts producing TMV were without exception also infected by CMV. The results show that CMV replicates in rice protoplasts by itself, whereas TMV does so only with the aid of CMV.Abbreviations CMV cucumber mosaiv virus - PBS phosphate buffered saline - TMV tobacco mosaic virus.     

Inhibition of Tobacco Mosaic Virus Reproduction in Isolated Tobacco Protoplasts by Means of Pancreatic Ribonuclease

Treatment of protoplasts with RNase solutions (1 — 100 μg/ml) sharply reduced their ability to support TMV reproduction. Treatment of protoplasts with RNase both prior to and after inoculation with the virus, and also treatments of varying duration, had approximately the same effect on inhibiting TMV reproduction; however, the effect became sharply intensified with higher RNase concentrations. The authors assume that the RNase inhibitive effect in protoplasts is ensured by the specific structure of this relatively small positively-charged macromolecule, not by its enzymic properties. The RNase molecules apparently have a damaging effect on the plasmalemma and possibly on other cell membranes with which the initial stages of TMV reproduction are associated. The development of the RNase damaging effect can be prevented by bivalent metal ions in the incubation medium, especially by calcium and manganese. The protective effect of cations is presumably caused not only by the charge, but by their steric configuration since K⁺ and Mg⁺⁺ are ineffective in protecting the protoplasts.     

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.

     

Cell relaxation after electrodeformation: effect of latrunculin A on cytoskeletal actin

Precise measurement of the mechanical properties of a cell provides useful information about its structural organization and physiological state. It is interesting to understand the effect of individual components on the mechanical properties of the entire cell. In this study, we investigate the influence of the cytoskeletal actin on the viscoelastic properties of a cell. Actin-specific agents, including latrunculin A and jasplakinolide, are used to alter the organization of the cytoskeletal actin. Brassica oleracea protoplasts are treated with the drugs and deformed under an external electric potential. The relaxation processes of single protoplasts after electrodeformation are measured. The data are analyzed by a model-independent spectrum recovery algorithm. Two distinct characteristic time constants are obtained from the relaxation spectra. Treatment with latrunculin A increases both of the relaxation time constants. The longest relaxation times for control, latrunculin A treated, and jasplakinolide treated cells are determined to be 0.28, 1.0, and 0.21 s, respectively.     

Wirkung von Virushemmstoffen auf die Infektion von Tabakprotoplasten mit dem Tabakmosaik-Virus

Effects of virus inhibitors on the infection of tobacco protoplasts with tobacco mosaic virus Yeast extract inhibits the infection of Nicotiana glutinosa plants with tobacco mosaic virus (TMV), whereas in N. sandérae yeast extract is not effective. This phenomena was compared with the effect of yeast extract on protoplasts, and on the infection of protoplasts of both tobacco species with TMV. Additionally, skim milk and ribonuclease were included in the experiments as further inhibitors of early stages of virus infection. It was examined whether these inhibitors damage non-inoculated protoplasts (a), and whether they affect virus infections in protoplasts as they do in cells of intact plants (b). To investigate protoplast damage by the inhibitors, conductivity measurements of protoplast suspensions containing inhibitors, and the ability of protoplasts for cell wall regeneration after treatment with the inhibitors, were used. Inhibitor concentrations which prevent virus infections in plants did not damage the protoplasts. The inhibitor effect on the course of infection was investigated by protoplast treatments before, during and after inoculation with TMV, and by addition of the substances to the culture medium. Measurements of virus content in protoplasts after cultivation revealed different results for the three inhibitors, however, there was no difference in the response of protoplasts from the two tobacco species to yeast extract. It is concluded that there are principal differences between the inhibition of plant and protoplast infections. Therefore, it is unlikely that protoplasts are a useful system for the mode of action studies on inhibitors of early stages of virus infection in plants.     

The effect of vibration noise in space relevant experiments

The experiments performed were initiated as a part of the post-flight investigations after the "PROTO" experiment performed on IML-1. The present experiments were performed with protoplasts prepared using the same standard isolation procedures as for the IML-1. The protoplasts were vibrated for 24 h with and without air bubbles in the protoplast cultivation bags and in the range of 1 to 20 Hz with 4 mm amplitude. The vibrations were found to have a negative effect on the viability of the protoplasts in bags without air bubbles and the vibration threshold seemed to lie around 20 Hz. Air bubbles are likely to cause cavitation-like conditions, thus increasing the mechanical strain on the free-floating protoplasts. During the 30 days microgravity mode on the ISS, mechanical vibrations would not be expected to have a significant influence on potential protoplast experiments. Experiments with durations overlapping the rendezvous and reboost mode may be exposed to critical vibration levels.     

外源胆固醇对离体原生质体在冰冻—化冻过程中的保护作用

在适宜浓度(0.1～0.4m mol/L)的胆固醇存在下,经冰冻处理的小麦叶肉原生质体在化冻后存活率均高于对照,其中以0.4m mol/L胆固醇对原生质体的保护效果最佳。胆固醇对卷心菜叶肉原生质体的抗冰冻能力具有类似效果。从而表明外源胆固醇与质膜结合后,对稳定质膜结构和保持正常功能具有一定的作用。     

Electro-enhancement of division of plant protoplast-derived cells

Summary Electric field pulses, ranging from 250 to 2000 V and of 10 to 50 sec duration, were assessed for their effect on the growth in culture of isolated protoplasts ofGlycine canescens, Prunus avium × pseudocerasus, Pyrus communis, Solanum dulcamara andSolanum viarum. Three successive voltage pulses between 250 and 1000 V caused a small decrease in protoplast viability, but promoted cell division and enhanced significantly the plating efficiency. A higher percentage of electro-pulsed protoplasts showed sustained growth in culture to the microcallus stage compared to untreated protoplasts. The rate of cell division was also stimulated in electro-treated protoplasts. These observations are discussed in relation to present knowledge of the effects of electrical treatments on plant and animal cells.     

Induction of a long-lived fusogenic state in viable plant protoplasts permeabilized by electric fields

Electropermeabilized tobacco mesophyll protoplasts are shown to fuse by creating cell contact several minutes after electropulsation. Electropermeabilization was analysed by measuring calcein uptake. Experiments were performed at low temperature to avoid resealing of protoplast transient permeation structures. These results confirm that the long-lived permeabilized state induced by the electric field is associated to a fusogenic state, under viability conditions. This is indicative that as for mammalian cells, the electric field-induced membrane modifications, which give the permeable state, are such as to decrease the magnitude of the intercellular repulsive forces between plant protoplasts. Such a fusion method may be useful for somatic hybrids production with protoplasts showing morphological and physiological differences.     

Production of somatic hybrids of moss by electrofusion

Summary Mixtures of protoplasts of two auxotrophic mutants of Physcomitrella patens, one requiring thiamine (aneurine), the other p-aminobenzoic acid, have been subjected to electrofusion. The protoplasts were aligned in an alternating electric field (500 KHz, 20 V RMS/cm) and induced to fuse by a brief DC pulse (800 V/cm, time constant lms). After culture, first on complete medium and then on selective medium, hybrid plants were obtained at a frequency of 3%. One hybrid with a morphology typical of polyploidy was also observed.Dedicated to Professor Georg Melchers to celebrate his 50-year association with the journal     

The permeability of electroporated cells and protoplasts of sugar beet

A simple method has been developed to determine the changes in permeability of protoplasts and intact cells when electroporated. Cells and protoplasts of sugar beet, Beta vulgaris L., were subjected to electric pulse treatments of different field strengths, pulse number and pulse duration, and the ability to accumulate and retain the hydrophilic dye phenosafranine was determined spectrophotometrically. Results of timecourse studies of phenosafranine accumulation and retention indicated that pores are formed or enlarged rapidly in the plasmamembrane and remain permeable to phenosafranine for relatively long periods; the half-life of the pores was temperaturedependent. Both cells and protoplasts retained the highest levels of phenosafranine when supplied with a series of five rectangular pulses of 50 s duration and of field strength 2500 V·cm^-1. If these parameters were exceeded, The phenosafranine content was reduced, concomitant with a decline in viability as indicated by fluorescein-diacetate staining, indicating the loss of the integrity of the plasmamembrane. The pattern of accumulation and retention by protoplasts of radioactivity from [³H]pABD1, a modified bacterial plasmid, was similar to that of phenosafranine, but uptake of the plasmid by cells was not demonstrated. The mothod can be used to determine conditions for the optimum permeabilization of protoplasts for direct gene transfer.     

External electric fields stimulate the electrogenic calcium/sodium exchange in plant protoplasts

External electric fields of low intensity stimulated calcium influx in protoplasts isolated from carrot cell suspension cultures in field intensity dependent and frequency-dependent ways. The field-induced calcium uptake involved a temperature-dependent system that was saturable by external calcium. The induction process appeared mainly cumulative as long as the morphology of the protoplasts did not change (up to 10 min). The stimulation elicited by the electric fields was effective even after switching the field off; the influx increased for 5 min and then slowed down to its initial value 15 min later. During electrostimulation, an additional amount of ATP was accumulated; on removal of the stimulatory field, the extra amount of ATP was consumed, whereas the plasma membrane was hyperpolarized and sodium ions were expelled from the protoplasts. Inhibition of either ATP accumulation or consumption results in the inhibition of both calcium influx and sodium efflux, demonstrating that these processes are coupled. From the data obtained in this work, it may be concluded that the electric field stimulates an ATP synthase like activity; the consumption of the ATP thus formed elicits an electric potential (probably due to the efflux of cations and more specifically sodium) that drives the influx of calcium.     

Interspecies protoplast fusion inLarix: Comparison of electric and chemical methods

Summary Larix was chosen for the study on interspecies protoplast fusion due to its ability to regenerate plants from protoplasts derived from embryogenic cultures.L. laricina line L2 was used in fusion experiments with eitherL. × eurolepis line L6 orL. × leptoeuropaea line L5. A method of unambiguous labeling of parental protoplasts prior to fusion was developed using vital fluorescent dyes. Of a number of dyes tested, only rhodamine B hexyl ester chloride (R6) and 3,3′-dihexylox-carbocyanine iodide (DiOC₆) stained the protoplasts in a consistent and uniform fashion. The fusion of mixed parental protoplasts that were internally labeled was carried out either in the presence of a 20% polyethylene glycol (PEG) solution or in an electric field. The progress of fusion was readily observed, taking only minutes under the experimental conditions. The fusion products could be identified by dual fluorescence several h after the onset of fusion. Heterofusion frequencies of approximately 18% and 6% in the presence of PEG and an electric field, respectively, were attained. Postfusion cultures betweenL. × laricina protoplasts and protoplasts ofL. × leptoeuropaea gave rise to cell colonies and betweenL. laricina andL. × eurolepis, to mature somatic embryos.     

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.