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; )     

Constitution of Mitochondrial and Chloroplast Genomes in Male Sterile Tobacco Obtained by Protoplast Fusion of Nicotiana tabacum and N. debneyi

Chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA) of malesterile tobacco plants obtained by fusion of Nicotiana tabacumprotoplasts and X-irradiated N. debneyi protoplasts were analyzed.Digestion of cpDNA isolated from ten male sterile lines withfour restriction endonucleases (EcoRI, XhoI, SmaI and HindIII)indicated that these lines possessed either one or the otherparental chloroplast genome. Neither mixture of two types ofcpDNA nor unique restriction fragments were detected in anyof the cases examined. The genetic constitution of chloroplastgenomes identified by restriction analysis of cpDNA showed goodagreement with that based on isoelectric focusing of the largesubunit of the Fraction I protein. The mtDNA from five fusion-derivedmale sterile plants showed banding patterns quite differentfrom each other and from the parental plants. Each plant exhibitednew restriction fragments not found in the parental species.These findings indicate that recombinational events in the mitochondrialgenomes take place rather frequently in the mixed cytoplasmsafter protoplast fusion, whereas the mixed chloroplasts becomesegregated to homogeneity. (Received June 19, 1987; Accepted October 5, 1987)     

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)     

A Comparison of Methods for Plasmid Delivery into Plant Protoplasts

Three methods of plasmid delivery to mesophyll protoplasts ofNicotiana tabacum cv. Xanthi have been evaluated. Specifically;a) chemically stimulated uptake of isolated plasmid, b) deliveryof plasmid encapsulated in liposomes, and c) fusion of plasmid-containingspheroplasts, were combined with divalent cation (Ca²⁺ and Mg²⁺)or polyalcohol [polyethylene glycol (PEG) and polyvinyl alcohol(PVA)] treatments. The quantity and quality of plasmid associatedwith intact protoplasts, was assessed by DNA-DNA blot hybridisationanalysis, following stringent washing to separate intact protoplastsfrom non-viable protoplasts and debris. Treatments which increasedassociation of plasmid with protoplasts decreased protoplastviability. Optimum association of plasmid with protoplasts,in the context of acceptable loss of viability, was achievedwhen protoplasts were interacted with either naked plasmid orliposomeencapsulated DNA in the presence of 15% w/v PEG 6000,or with Escherichia coli spheroplasts containing chloramphenicol-amplifiedplasmid in the presence of 25% w/v PEG 6000. Divalent cationsdid not stimulate significant plasmid delivery without unacceptableloss of protoplast viability. Strategies to further increasethe efficiency of plasmid delivery are discussed. (Received June 21, 1984; Accepted August 20, 1984)     

Effects of Physical Parameters upon Protoplast Electrofusion

The effects of three physical parameters upon protoplast electrofusionwere studied using protoplasts from cultured cells of Coptisjaponica and Euphorbia millii. The osmotic potential of themedium did not appreciably affect the AC-field-induced protoplast-pairformation, but significantly influenced the fusion process ofthe paired protoplasts in response to DC pulses. The optimumosmotic potential was 0.55 to 0.60 Osm/kg H₂O in our system.The density of the medium markedly influenced both pair formationand fusion process. The optimum density was 1.13 to 1.14 g/cm₃,and at this density the yield of the fused protoplasts increasedto more than twice that of the control (1.10 g/cm₃). Hydrophiliccoating of the bottom surface of the chamber with Gellan gumor polyacrylamide gel was also effective for both pair formationand the fusion process, while coating with hydrophobic siliconewas entirely inhibitory. Possible interpretations of the effectsof these physical parameters upon protoplast electrofusion arepresented. ¹Permanent address: Biochemical Research Laboratories, KanegafuchiChemical Industry Co., Ltd., Takasago, Hyogo 676, Japan. (Received December 21, 1987; Accepted March 18, 1988)     

In vitro double fertilization in Nicotiana tabacum (L.): polygamy compared with selected single pair somatic protoplast and chloroplast fusions

In vitro polygamy was studied mainly by using isolated sperm and central cells of tobacco in order to elucidate the mechanism that might be involved in preventing in vivo polygamy. In 17.5% 4000 M.W. polyethylene glycol, only when two sperm cells were made close enough to each other and adhered to a female cell simultaneously was polygamy possible. If one sperm cell fused with the egg or central cell, within 30 min another sperm cell could not fuse with the same egg or central cell. Similar phenomena were found in selected single somatic cell fusion. When more than two protoplasts adhered to each other simultaneously, fusion was always successful; after two protoplasts fused, within 30 min the fusion products could not fuse with another protoplast under the same conditions. This comparative study revealed this characteristic to be shared by both sexual and somatic cell fusion. However, after cytoplasm reorganization was complete in the fusion product, it was possible for the fusion product to fuse with the third protoplast. This indicates that the obstruction to additional fusion was present only during a certain period after the preceding fusion under certain condition. The possible reason for the effect is discussed. Received: 7 March 2000 / Revision accepted: 15 June 2000     

Fusion between interphase and mitotic plant protoplasts : Induction of premature chromosome condensation

Morphological changes in interphase nuclei were cytologically studied in heterophasic dinucleate cells formed by the fusion of mitotic and interphase plant protoplasts. Mitotic protoplasts were isolated from a partially synchronized suspension culture of wheat (Triticum monococcum). The mitotic cells were accumulated by colchicine after release of hydroxyurea block. Treatment of protoplast populations with polyethylene glycol-dimethyl sulphoxide solution resulted in metaphase-interphase fusion. Three hours after fusion, the appearance of chromosomes with single chromatid as well as of fragmented, pulverized chromatin in heterophasic cells indicated the induction of premature chromosome condensation (PCC) in somatic wheat cells. Condensation in interphase nuclei of mitotically inactive rice protoplasts was also detected after fusion with mitotic wheat protoplasts.     

Rhizobium Attachment and Curling in Asparagus, Rice and Oat Plants

Observations by scanning electron microscopy revealed that rhizobiaattach to the surface of rice protoplasts with regenerated cellwalls, isolated mesophyll cells of asparagus, and root hairsof rice and oat seedlings. Those strains of rhizobia, namelyRhizobium leguminosarum biovar trifolii, Bradyrhizobium japonicumand Bradyrhizobium sp., attach to the cells of these monocotsin the same manner as they attach to the host dicots tested.Escherichia coli did not attach. These results suggest thatthe attachment of rhizobia is not a host-specific process. Whenoat seedlings were infected by R. l. trifolii, hair curlingwas observed. The interactions between monocot plants and rhizobiais discussed in this paper. (Received June 12, 1989; Accepted November 9, 1989)     

Direct Transfer of Plasmid DNA from Intact Yeast Spheroplasts into Plant Protoplasts

We developed a polyethylene glycol (PEG)-mediated direct DNAtransfer method from intact Saccharomyces cerevisiae spheroplastsinto Arabidopsis thaliana protoplasts. To monitor the DNA transferfrom yeast to plant cells, ß-glucuronidase (GUS) reportergene in which a plant intron was inserted was used as a reporter.This intron-GUS reporter gene on a 2µm-based plasmid vectorwas not expressed in yeast transformants, while it expressedGUS activity when the plasmid DNA was introduced into plantcells. When a mixture of 1 x 10⁸ of S. cerevisiae spheroplastsharboring the plasmid and 2 x 10⁶ of A. thaliana protoplastswas treated with PEG and high pH-high Ca²⁺ solution (0.4 M mannitol,50 mM CaCl₂, 50 mM glycine-NaOH pH 10.5), GUS activity was detectedin the extract of the plant cells after a three-day culture.The GUS activity was higher than that of a reconstitution experimentin which the mixture of 1 x 10⁸ of S. cerevisiae spheroplastswhich did not carry the reporter gene, 2 x 10⁶ of A. thalianaprotoplasts and the same amount of the reporter plasmid DNAas that contained in 1 x 10⁸ of S. cerevisiae spheroplasts,was treated with PEG and high pH-high Ca²⁺ solution. Moreover,the GUS gene expression was resistant to micrococcal nucleasetreatment before and during PEG treatment. From these results,we concluded that plasmid DNA can be directly transferred fromintact yeast spheroplasts to plant protoplasts by a nuclease-resistantprocess, possibly by the cell fusion. ²Deceased on September 15, 1992.     

The Membrane Potential of Enzymatically Isolated Nitella expansa Protoplasts as Compared with their Intact Cells

With the enzymatically isolated Nitella protoplasts, sufficientinsertions of micro-electrodes to make a stable measurementof the membrane potential by the conventional method could notbe made because of an ‘elasticity’ of the outermembrane. We developed an effective method in which a micro-electrodecould be inserted after the outer membrane was punctured bypassing an electrical impulse through the micro-electrode. Inthis method, Ca ions play a crucial role in the ‘punching’and ‘healing’ processes of the protoplast membrane. The effects of the cations K⁺, Na⁺, Ca²⁺ and the anions Cl^–,, , on the membrane potentials of Nitella expansa protoplasts were compared with those of intactcells. The membrane potential of protoplasts was less negativethan that of intact cells when concentrations of Na or K, inthe presence of Ca, were below certain levels which increasedwith increasing Ca concentration; and it tended to become identicalto that of intact cells when Na or K concentrations were beyondthose levels. Beyond those levels for K the membrane potentialsof both protoplasts and intact cells typically seemed to bethe Nernst potentials in the presence of 0•1 to 30 molm^–3 Ca²⁺. However, for Na, the difference in potentialsbetween intact cells and protoplasts decreased at much higherconcentrations than for K. Increase of Ca always gave less negativeprotoplast potentials than those in intact cells. Replacementof Ca by Mg did not change the membrane potential of intactcells, although it was deleterious to protoplasts. The cellwall potential of intact cells was also measured by the micro-electrodetechnique and was revealed as a simple Donnan potential, assumingthe fixed negative charge density of 0•8 equivalent perdm³. The membrane potential of intact cells seems to be a significantreflection of the plasmalemma potential which is thought tobe measured directly in their protoplasts in terms of ionicselectivity and concentration dependency of the ion speciesexamined. In addition, increased sensitivity to calcium in protoplastpotentials compared to intact cells is suggested, though themembrane potential of intact cells seems to be largely preservedin their enzymatically isolated protoplasts. Key words: Membrane potential, protoplasts, Nitella expansa, cell wall potential     

Protoplast fusion between Monostroma nitidum and Porphyra yezoensis and subsequent growth of hybrid plants

Protoplasts from Monostroma nitidum (Chlorophyta) and Porphyra yezoensis (Rhodophyta) were fused utilizing the PEG method. The heterofusants were easily identified by the color of the hybrid cells. The frequency of fusion between the different protoplasts was 1.4%. The heterofusants were transferred into ES medium and cultured. The survival rate of heterofusants after 30 days cultivation was 2–7%. Two plants exhibited hybridizable DNA fragments as compared to parental DNA fragments. Additionally, these two plus a third hybrid plant had characteristic fatty acid components of both the Chlorophyta and Rhodophyta. The results indicate that genetic recombination is possible between these two genera. The field for breeding by protoplasts fusion should be extended across the divisions. This revised version was published online in June 2006 with corrections to the Cover Date.     

Direct transfer of a cold-tolerant Ogura male-sterile cytoplasm into cabbage (Brassica oleracea ssp. capitata) via protoplast fusion

Cold tolerant cytoplasmic male-sterile (CMS) cabbage (Brassica oleracea var. capitata) was produced by the fusion of leaf protoplasts from fertile cabbage and cold-tolerant Ogura CMS broccoli lines. The cabbage lines tested showed great variation in plant regeneration from unfused protoplasts; three with high regenerability were selected as the fusion partners. Several procedures for eliminating the nuclear DNA of the broccoli fusion partner were tested. Diploid cabbage plants were identified by flow cytometry and morphological characters. Gamma-irradiation (30 krad) was the most successful procedure; isolation of cytoplasts from broccoli leaf protoplasts, followed by gamma-irradiation of the cytoplast fraction, also produced diploids. UV-irradiation of the broccoli protoplasts was less effective. PCR using primers for an Ogura CMS-specific mitochondrial DNA sequence permitted the identification of cybrids likely to be CMS. Over 200 diploid plants with the CMS-specific sequence were obtained from 66 independent fusion products and three cabbage lines. Plants were ready for transfer into soil within 8 months after fusion. The plants identified as CMS by PCR produced male-sterile flowers. Our procedures permit the transfer of a desirable male-sterile cytoplasm into cabbage much more rapidly than conventional backcrossing procedures. Received: 4 June 1996 / Accepted: 2 August 1996     

Surface Charge Density Estimation of Vigna mungo Protoplasts Using a Fluorescent Dye, 9-Aminoacridine

We showed that the surface charge density of protoplasts canbe estimated by the 9-aminoacridine method. The estimated surfacecharge density of the protoplasts isolated from elongating regionsof Vigna mungo root was – 39 ? 8 mC/m². The negative surfacecharge density increased when protoplasts were treated withglutaraldehyde or when EDTA was added to the protoplast suspensionmedium. These results support the validity of our estimationof the surface charge density of protoplasts by the 9-aminoacridinemethod. The concentration of amino groups at the surface ofthe protoplasts was estimated to be 34 mC/m². (Received June 19, 1987; Accepted April 11, 1988)     

Production of hybrid cells from single protoplasts of sunflower hypocotyl and broad bean guard cells by electrical fusion

The C4-directed enzymatic apparatus in guard cells is known to be coupled with a reduction in photorespiration resulting in a higher survival rate and yield potential of crops. This has led to searches for guard-cell-specific genes and promoters and methods for transferring them into C3-plants. To explore this possibility we performed somatic fusions between guard cell protoplasts from Vicia faba and hypocotyl protoplasts from Helianthus annuus, using a technique which allows fusion of a single pair of individually selected protoplasts. We obtained fusions in 30–70% of attempts. Culture of the hybrid fusion products in a liquid nutrient medium, without conditioning or feeder cells, produced microcolonies consisting of 8–9 cells within 9 days of culture. This revised version was published online in June 2006 with corrections to the Cover Date.     

Isolation of Hair Cells from Azolla filiculoides var. japonica Leaves

A procedure has been developed to isolate hair cells from Anabaenacontaining packets of the aquatic fern Azolla. Unbroken algalpackets, isolated by enzyme treatment and flotation on 12.5%Percoll solution, contained hair cells, Anabaena filaments andthe envelope membrane. When the packet suspension was gentlypipetted, hair cells attached to the envelope membrane becamedetached from the Anabaena filaments. Hair cells were separatedby flotation on 34% Percoll solution and further purified bysifting through nylon mesh. Both branched and unbranched cellswere isolated and about 70% remained unstained by 0.1% trypanblue. Electron microscopic observation showed that these cellswere protoplasts enclosed by a thin envelope and had well preservedinternal structures. The activities of ammonia-assimilatingenzymes in the hair cells were much higher than those in Azollaleaves, while the activities in the endophytes were repressedto very low levels. These results suggest that hair cells playan important role in the assimilation of the nitrogen whichthe endophytes fix and release into the cavity. (Received March 24, 1986; Accepted June 28, 1986)     

Role of Coccoliths in the Utilization of Inorganic Carbon by a Marine Unicellular Coccolithophorid, Emiliania huxleyi: a Survey Using Intact Cells and Protoplasts

The utilization of inorganic carbon and role of the coccolithswere investigated in intact cells and protoplasts of a marineunicellular calcareous alga, Emiliania huxleyi. Protoplastswith high photosynthetic activity were obtained by artificialdecalcification with 50 mM MES-NaOH (pH5.5). (1) The kineticsof the photosynthetic evolution of O₂ at various concentrationsof externally added NaHCO₃ were the same for intact cells andprotoplasts, indicating that the kinetic properties with respectto dissolved inorganic carbon (DIC) were not affected by thepresence or absence of the coccoliths on the cell surface. Double-reciprocalplots and plots of the concentration of substrate divided byvelocity (s/v) against the concentration of substrate (s) werebiphasic in the case of both intact cells and protoplasts. TheCO₂-utilization reaction was, therefore, considered to involvetwo processes with different values of K_m and V_max. From thekinetic analyses, K_m and V_max [µmoles O₂ (ml PCV)^–1h^–1] were deduced to be 92 µM and 76.3 for a "low-K_m"reaction and 4.1 mM and 252 for a "high-K_m" reaction, respectively.(2) In short-term (40-min) experiments, time courses of thetotal uptake of ¹⁴C-DIC and the incorporation of ¹⁴C into acid-stableproducts of photosynthesis and the internal pool of DIC, determinedas acid-labile compounds, under CO₂-limiting conditions (80µM) were very similar for intact cells and protoplasts.However, incorporation of ¹⁴C into CaCO₃ apparently occurredmore slowly in protoplasts than in intact cells. (3) In longterm (24-h) experiments, patterns of incorporation of ¹⁴C werealmost same for intact cells and protoplasts, with the exceptionthat the amount of ¹⁴C incorporated into CaCO₃ was much smallerin the former than the latter. The production of Ca¹⁴CO₃ increasedduring the course of 10 h after a 4-h lag. However, after 10h the level of Ca¹⁴CCO₃ started to decrease. The decrease wasaccompanied by an increase in ¹⁴C in the products of photosynthesis,suggesting that CaCO₃ was reutilized for the photosyntheticfixation of CO₂ and, therefore, that the coccoliths functionas sites of storage of DIC. However, the internal level of DICremained at the same level even after the supply of externalDIC has been almost completely depleted. (Received July 25, 1995; Accepted December 11, 1995)     

Involvement of Calyculin A- and Okadaic Acid-sensitive Protein Phosphatase in the Blue Light Response of Stomatal Guard Cells

Calyculin A (CA) and okadaic acid (OA), inhibitors of proteinphosphatases, inhibited blue light (BL)-dependent H⁺pumpingin Vicia guard cell protoplasts at half-inhibitory concentrationsof 4.5 nM and 400 nM, respectively. Light-induced stomatal openingin Viciaepidermis was completely suppressed by CA at 100 nMand by OA at 1 µM. These results suggest that CA- andOA-sensitive protein phosphatase is involved in the BL responseof stomatal guard cells. (Received June 27, 1997; Accepted September 2, 1997)     

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.     

Somatic hybridization in higher plants

Summary Somatic hybridization in higher plants has come into focus since methods have been established for protoplast fusion and uptake of foreign DNA and organelles by protoplasts. Polyethylene glycol (PEG) was an effective agent for inducing fusion. Treatment of protoplasts with PEG resulted in 5 to 30% heterospecific fusion products. Protoplasts of different species, genera and even families were compatible when fused. A number of protoplast combinations (soybean + corn, soybean + pea, soybean + tobacco, carrot + barley, etc.) provided fusion products which underwent cell division and callus formation. Fusion products initially were heterokaryocytes. In dividing heterokaryocytes, random distribution of mitotic nuclei was observed to be accompanied by multiple wall formation and to result in chimeral callus. Juxtaposition of mitotic nuclei suggested nuclear fusion and hybrid formation. Fusion of heterospecific interphase nuclei was demonstrated in soybean + pea and carrot + barley heterokaryons. Provided parental protoplasts carry suitable markers, the fusion products can be recognized. For the isolation and cloning of hybrid cells, fusion experiments must be supplemented with a selective system. Complementation of two nonallelic genes that prevent or inhibit growth under special culture conditions appears as the principle on which to base the selection of somatic hybrids. As protoplasts of some species have been induced to regenerate entire plants, the development of hybrid plants from protoplast fusion products is feasible and has already been demonstrated for tobacco. Presented in the formal symposium on Somatic Cell Genetics at the 27th Annual Meeting of the Tissue Culture Association, Philadelphia, Pennsylvania, June 7–10, 1976.     

Carbon Dioxide Fixation by Guard Cell Protoplasts of Commelina communis

Biochemical studies of epidermal tissue may not reflect metabolismof the guard cells which represent less than 5% of the tissuevolume. Pure samples of guard cell protoplasts of Commelinacommunis were therefore used to investigate CO₂ fixation ratesand ¹⁴C-labelling patterns of metabolites in the light and thedark. Qualitatively, results were similar in most respects tothose obtained in a previous study (Schnabl, 1980) for guardcell protoplasts of Vicia faba. CO₂ fixation rates by guardcell protoplasts of C. communis were the same in the light andthe dark but about 50 times lower than the values Schnabl obtainedfor V.faba. The ¹⁴C-labelling pattern of metabolites in C. communiswas also similar in the light and the dark: over 60% of thetotal fixed was in malate with only 1% in sugar phosphates.Label was also detected in starch, aspartate, glutamate andcitrate but not in glycollate as previously recorded in V. fabaguard cell protoplasts. The results confirm the view that the reductive pentose phosphatepathway does not occur in guard cells of C. communis. Key words: CO₂ fixation, Guard cell protoplasts, Stomata