Selection of hybrid plants obtained by electrofusion of vacuolated x evacuolated plant protoplasts in hypo-osmolar solution

Vacuolated and evacuolated tobacco mesophyll protoplasts were electrically fused in hypo-osmolar media by using an alternating field of modulated amplitude for alignment. The vacuolated fusion partner was isolated from Nicotiana tabaccum L. cv Xanthi and the evacuolated one from the streptomycin-resistant strain Nicotiana tabaccum L. cv Petit Havana SR1. The field and osmolarity conditions used ensured relatively high yields of heterologous fusion products despite the differences in density and size of the parental cells. After removal of the evacuolated, streptomycin-resistant fused and unfused protoplasts by flotation of vacuole-containing cells on iso-osmolar sucrose medium, the cybrids and hybrids were cultured in 25 microliters drops of agarose. During the first 5 weeks the non-fused Xanthi-protoplasts were used as a nurse culture. After addition of streptomycin to the growth media, cybrids and hybrids were successfully selected whereas fused and unfused vacuole-containing protoplasts died within 6 days. Only the streptomycin-resistant cybrids and hybrids developed into whole plants. On average a yield of 0.025% of streptomycin-resistant plants (referred to the total number of parental cells) was obtained. Polyacrylamide gel electrophoresis of leaf extracts of these plants showed that at least 50% of the streptomycin-resistant plants had a hybrid-esterase isoenzyme pattern. The protocol can be generalised by fusion of iodoacetamide-inactivated vacuolated protoplasts with meristematic (or evacuolized) protoplasts carrying no genetic marker. Use of evacolated protoplasts for electrofusion with vacuole-containing protoplasts therefore offers a way of overcoming the lack of suitable genetic markers for hybrid selection.     

Determination of physical membrane properties of plant cell protoplasts via the electrofusion technique: prediction of optimal fusion yields and protoplast viability

By variation of physical parameters (field strength, pulse duration) which result in electrofusion and electroporation, properties of the plasma membrane of different types of plant cell protoplasts were analyzed. The lower threshold for that field pulse intensity at which membrane breakdown occurred (recorded as fusion event) depended on pulse duration, protoplast size, and protoplast type (tobacco, oat; vacuolated, evacuolated). This fusion characteristic of plant protoplasts can also be taken as a measure of the charging process of the membrane and allows thus a non-invasive determination of the time constant and the specific membrane capacitance. Although the fusion yield was comparable at pulse duration/field strength couples of, e.g., 10 s/1.5 kV*cm^–1 and 200 s/0.5 kV*cm^–1, hybrid viability was not. Rates of cell wall regeneration and cell division of tobacco mesophyll protoplasts were not affected but may have been increased at short pulse duration/high field strength. Plating efficiency, in contrast, was significantly decreased with longer pulse duration at low field strengths.     

空间细胞电融合关键技术的地基研究——烟草细胞的电融合

本文针对建立空间细胞电融合技术存在的三个主要问题进行了研究。结果表明,用低温(4℃)、融合介质(0.55 mol/L甘露醇)并添加0.1%纤维素酶保存原生质体,72 h内可以使约94%细胞维持无壁状态,同时并未使细胞丧失再生能力,基本满足从地面制备亲本细胞到在微重力条件下进行电融合,对亲本细胞保持无壁状态的要求。为减少剪切力环境对亲本细胞造成的损伤,一方面用超速离心方法对亲本细胞之一去液泡,另一方面用电泳代替蠕动泵混合亲本细胞。而且,由于原生质体壁生长与其膜电位之间存在负相关性,因此利用电泳方法可以有效地富集和优化亲本细胞。根据地面实验结果推测,空间有/无液泡亲本细胞电融合的较适合参数可能为:交流电场强度90V/cm,频率0.8 MHz,排列时间20 s,直流脉冲1.0—1.3 kV/cm,幅宽40μs,两次脉冲。     

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.     

Enhanced hybridoma production by electrofusion in strongly hypo-osmolar solutions

Electrofusion of mammalian cells in strongly hypo-osmolar media containing sorbitol, small amounts of divalent cations and albumin resulted in high yields of hybrids. The number of viable hybrids was higher than any value for chemically- or electrically-mediated fusion reported in the literature. Optimum clone numbers were obtained for fusion of osmotically-stable subclones of murine myeloma cells with DNP-Hy-stimulated lymphocytes provided that the osmolarity of the fusion medium was as low as 75 mosmol/l. Similar results were obtained for fusion of osmotically stable subclones of myeloma cells with the murine hybridoma cell line G8. Due to the dramatic increase in volume the field strength of the breakdown pulse (leading to fusion of the dielectrophoretically aligned cells) has to be reduced, as predicted by theory. The efficacy of hypo-osmolar electrofusion allowed the use of very few cells (about 10(5) lymphocytes or G8 cells per fusion chamber). This figure is considerably smaller than that reported in the literature for iso-osmolar electrofusion. It is significant that, in contrast to iso-osmolar conditions, the fusion yield in hypo-osmolar electrofusion was reproducible over long periods of time and less dependent of variations between cultures. At suspension densities of about 10(6) cells per fusion chamber (normally used in iso-osmolar electrofusion) hypo-osmolar electrofusion of homogeneous cell suspensions resulted in the formation of many giant cells when the appropriate field conditions were applied. Similar high or, at some field strengths, even higher numbers of clones at low cell suspension density were obtained when G8 and myeloma cells were first exposed during the washing procedure to strongly hypo-osmolar media, but then transferred to iso-osmolar solutions for electrofusion. Similar experiments with lymphocytes and myeloma cells failed because of destruction of many lymphocytes by the two osmotic shock steps in rapid succession. Volume distribution measurements of G8 and myeloma cells showed that after re-incubation of the osmotically pre-stressed cells the original volume distribution is largely, but not completely re-established. This and other results indicate that osmotic pressure gradients and associated tensions in the membrane do not play a primary role in the initiation of the electrofusion process. The experiments suggest that due to the osmotic (pre-) stress the membrane permeability is slightly and uniformly increased presumably due to the dissolution of membrane- and cell-skeleton proteins. Obviously, this facilitates electrofusion in hypo-osmolar or subsequently in iso-osmolar solutions.     

Optimization of Electrofusion Parameters and Interspecific Somatic Hybrid Regeneration in Citrus

This study was primarily attempted to optimize the electrofusion parameters using protoplasts isolated from cell suspension cultures of "Page" tangelo ( Citrus reticulata Blanco x C. paradisi Macf) and mesophyll protoplasts of rough lemon ( Citrus jambhiri Lush) as fusion partners. It was shown that the binuclear heterokaryons frequency reached 15% with the following parameters: alternate current (AC) 125 V/cm, AC time 60 s, direct current (DC) 1 250 V/cm, DC pulse width 50 μs, DC pulse interval O. 5 s, No. of DC pulse 3. Considering the fact that different types of protoplasts have different specific weights, higher frequency of the binuclear heterokaryons was obtained by controlling the centrifugation time after fusion. The fusion products regenerated into plantlets after 3 to 4 months of culture. Chromosome counting of the root tips and morphological observation of the regenerants verified that 78% were tetraploids and the rest were diploids with the leaf morphology of mesophyll parent. Peroxidase (POX) isozyme and RAPD analysis indicated that interspecific somatic hybrids were obtained and an autotetraploid plant of mesophyll parent type was also verified.     

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.     

Comparative effects of fusion facilitators on electrofusion attributes of N. tabacum mesophyll protoplasts

Mesophyll protoplasts isolated from in vitro-grown Nicotiana tabacum L. shoots were subjected to electrofusion.Dielectrophoresis was induced by an AC field of 50 V cm^-1 inter-electrode distance and 0.5 MHz oscillation frequency. Fusion was effected by two 0.7 kV cm^-1 DC pulses, each of 50 s duration, applied within one second of each other. Various chemical treatments were tested for their effects on dielectrophoresis efficiencies (percentages of protoplasts that made contact with at least one other protoplast under the AC field), fusion efficiencies (percentages of protoplasts participating in fusion events), cell lysis (percentages of protoplasts bursting during the electrofusion processes), overall viabilities of fusion products 24 h post-fusion and overall plating efficiencies 7 d post-fusion (percentages of fusion-derived cells that had undergone division). The various attributes assessed on the electrofusion of protoplasts in the control treatment, 10% mannitol, differed considerably for experiments carried out on different days. Relative to the control treatment, only the Ca²⁺ treatments, and to a lesser extent lipase treatment reduced dielectrophoresis efficiencies. Polyamines, cytochalasins and Ca²⁺ treatments significantly reduced cell lysis percentages. All electrofusion facilitators tested (except for spermine at 150 mg l^-1, the cytochalasins B and D, and Ca²⁺ treatments) increased fusion efficiencies to more than 1.5 times those obtained with the standard 10% mannitol electrofusion medium. Ca²⁺ treatments increased overall viabilities of fusion products by more than 1.5 times. With the exception of the prostaglandins, lecithin and CaCl₂ treatments, overall plating efficiencies were reduced by treatment of protoplasts with fusion facilitators. Substantial increases in overall plating efficiencies over those observed in the control treatment were obtained using prostaglandin F_2a, lecithin and CaCl₂.2H₂O treatments. The implications of the results are discussed.Abbreviations AC alternating current, approx.-approximately - BA benzylaminopurine, cv.-cultivar - DC direct current, diam.-diameter - FDA fluorescein diacetate - MS Murashige & Skoog (1962) - NAA napthaleneacetic acid - PCM protoplast culture medium - PIM protoplast isolation medium - PPM protoplast purification medium - rpm revolutions per minute - SD(n) standard deviation of a variate - SEM standard error of the mean     

Electrofusion parameters for mouse two-cell embryos

We studied electrofusion of mouse two-cell embryos in order to define parameters which would result in a high yield of fused embryos. Various cell alignment times (from <10 to >60 s) and alternating current percentages (2 to 100%) were examined. The fusion parameters tested were the number of fusion pulses (1-9), pulse length (30-90 mus) and pulse strength (0.50-1.79 kV/cm). Furthermore different combinations of these three parameters were tested. In addition the influence of several embryo culture media on the fusion rates was examined. The results show that the fusion rate of the embryos increases with shorter alignment and higher percentages of the alternating current. The highest fusion rate (95%) was obtained by use of one pulse with a duration of 70 mus and a field strength of 0.60-0.79 kV/cm. The survival rate of the embryos was best if Whitten Medium was used before and after the fusion pulses. The fusion of two-cell stages results in tetraploid embryos which can serve as models for studies in polyploid cells.     

Intensification of lipase biosynthesis as a result of electrofusion of Rhizopus cohnii protoplasts

Our objective was to obtain products of fusion of the filamentous fungus Rhizopus cohnii Rh.c./1 with an increased capacity for lipase biosynthesis in comparison with the original strain. Protoplasts of auxotrophic mutants of the parent strain Rh.c./1 obtained after UV irradiation of the spores were subjected to electrofusion. We found that the largest number of electrofusion products could be obtained with the use of the following process parameters: 1 or 2 impulses immediately following one another with a field intensity of 200 V/cm and an exposition time of 1000 ms at the stage of dielectrophoresis, 1 impulse with a field intensity of 500 V/cm and an exposition time of 10 ms or 20 ms at the stage of fusion, regulated temperature of 4 degrees C before and after the process, rounding time of ca 20 min. Electrofusion of protoplasts of auxotrophic mutants of the Rh.c./1 strain produced 19 fusion products whose lipase biosynthesis capacity in a liquid medium culture was higher than that of the parent strains. The fusion product labelled XIII-21 was selected as the best strain. Lipase activity obtained after its culture in the liquid medium was ca 3.5 times higher than that obtained after the culture of the original strain Rh.c./1.     

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     

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.     

Determination of electric field threshold for electrofusion of erythrocyte ghosts. Comparison of pulse-first and contact-first protocols.

Rabbit erythrocyte ghosts were fused by means of electric pulses to determine the electrofusion thresholds for these membranes. Two protocols were used to investigate fusion events: contact-first, and pulse-first. Electrical capacitance discharge (CD) pulses were used to induce fusion. Plots of fusion yield vs peak field strength yielded curves that intersected the field strength axis at positive values (pseudothresholds) which depended on the protocol and decay half time of the pulses. It was found that plots of pseudothreshold vs reciprocal half time were linear for each protocol; when extrapolated to reciprocal half time = 0 (i.e., t----infinity), these lines intersected the ordinate at values of the field strength considered to be the true electrofusion thresholds. In this fashion, the contact-first protocol gave an electrofusion threshold of 46.5 +/- 11.5 V/mm for hemoglobin-free ghosts (white ghosts) and 40.9 +/- 8.8 V/mm for ghosts with fractional hemoglobin (pink ghosts), while the threshold for the pulse-first protocol applied to pink ghosts was determined to be 93.4 +/- 11.0 V/mm. Although the thresholds depended on the electrofusion protocol, plots of critical field strength vs reciprocal time had the same slopes, i.e., approximately 24 Vs/mm. The results suggest that the fusogenic state induced by an electric pulse in either the contact-first protocol or the pulse-first protocol (long-lived fusogenic state) may in fact share a common mechanism, if the two states are not actually identical.     

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     

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     

一种改进的杂交瘤细胞电融合方法

一种改进的杂交瘤细胞电融合方法邹翔（南京大学医学院南京２１０００８）刘琴芝,裴红英,郁文芳,安节,陈伯权（中国预防医学科学院病毒学研究所北京１０００５２）陈刚,赵南明（清华大学生物科学与技术系生物膜与膜生物工程国家重点实验室北京１０００８４）我们对国...     

Homokaryons from animal and plant cells generated by electrofusion

A new apparatus was constructed which enables the use of the electrofusion method to obtain polynuclear cells of various mammalian cell lines, erythrocytes and plant protoplasts. This technique was applied to both suspensions and monolayers. Electrical and other physical parameters were monitored to find optimal conditions for mutual contact of cells (dielectrophoresis) and subsequent fusion. In the suspension technique, dielectrophoresis of mouse erythrocytes occurred at a field frequency of 20 kHz and a strength of 500 V.cm-1, whereas cultured mammalian cells and plant protoplasts required a frequency of 1-1.4 MHz and a strength of 250-800 V.cm-1. Fusion of cells was induced after the application of 1 to 10 high-voltage pulses of 1-5 kV.cm-1, 10-36 microseconds duration. After these high-voltage pulses were to the monolayer of mouse L cells, about 12% viable homokaryons were obtained.     

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.     

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.

     

Electrofusion of protoplasts from Aspergillus nidulans

Abstract Electrical parameters were determined and quantified for the stimulation of the optimum alignment and fusion of Aspergillus nidulans protoplasts. In a non-homogeneous alternating electrical field A. nidulans protoplasts aligned to form pearl chains associated with the electrodes of the fusion chamber. Most protoplasts were in pearl chains in an alignment field frequency of 3.0 MHz but maximum pair formation occurred at 1.0 MHz. At a field strength between 100 and 1000 V · cm⁻¹ pearl chain formation occurred with minimal protoplast rotation or lysis. The application of DC pulses resulted in protoplast fusion. Most fusion events were observed after two 500 V · cm⁻¹ DC pulses with a 0.5 s interpulse period. Using 1 × 10³ protoplasts · cm⁻³ in a 7 μm fusion chamber a maximum of 17.2 ± 2.0% fusion events were achieved.