Transfer of organelles of the alga Chlamydomonas reinhardii into carrot cells by protoplast fusion

A mutant of Chlamydomonas reinhardii which lacks a cell wall was fused with Daucus carota protoplasts using polyethylene glycol and the resulting fusion products were cultured. Fusion involved integration of Chlamydomonas and carrot plasma membranes and the release of algal organelles into the carrot cytoplasm. Chlamydomonas basal bodies, nuclei and chloroplasts were frequently observed in the fusion products. Cultured fusion products regenerated cell walls and divided; most Chlamydomonas organelles degenerated during culture but chloroplasts were still recognizable in the carrot cytoplasm after 10.Abbreviations PEG polyethylene glycol - TEM transmission electron microscopy - SEM scanning electron microscopy This study was undertaken during sabbatical leave in The Research School of Biological Sciences. Australian National University     

Ultrastructure of fusion products from soybean cell culture and sweet clover leaf protoplasts

Summary Protoplasts from cultured cells of soybean (Glycine max L.) and from sweet clover (Melilotus officinalis L.) mesophyll cells were fused with polyethylene glycol and subsequently cultured for six days. The resulting fusion products as well as unfused protoplasts of each parental species regenerated cell walls and divided. The fusion products were characterized by the presence of soybean leucoplasts and sweet clover chloroplasts. The chloroplasts appeared to be degenerating but other cytoplasmic organelles were typical of actively growing plant cells. The fate of individual nuclei could not be determined.Supported by National Research Council of Canada, Grant A6304     

A method for high-frequency intergeneric fusion of plant protoplasts

Summary Protoplasts of Vicia hajastana Grossh. obtained from suspension-culture cells and Pisum sativum L. obtained from leaves adhered tightly to each other in concentrated solutions of high-molecular-weight polyethylene glycol (PEG). The adhesion occurred non-specifically between the free protoplasts from the same species as well as from the different species and genus. It was enhanced by enrichment of the PEG solution with calcium. Very few heteroplasmic fusions occurred during the period when the protoplasts were incubated in the PEG solution. However, many heterokaryons (up to 10%) were formed soon after the PEG solution was diluted out. The same phenomena were also observed in protoplasts from suspension-culture cells of Glycine max L. and from leaves of Hordeum vulgare L. Vicia and soybean protoplasts obtained from cultured cells regenerated cell walls and underwent sustained cell division after such treatment. Some Vicia-pea heterokaryons divided once. Over 10% of the soybean-barley hybrids divided in 7 days. Some divided 4–5 times and formed small clusters of cells in 10 days. The hybrids were recognizable because they contained chloroplasts from the leaf protoplast and exhibited morphological characters typical of the chlorophyll-less cells. None of the protoplasts from pea and barley leaves, either with or without PEG treatment, underwent cell division during the period of observation. The mechanism of adhesion and fusion of the protoplasts has been discussed.National Research Council (Canada) No. 13732.     

Ultrastructure and Isozyme Analysis of Cultured Soybean-Nicotiana Fusion Products

Protoplasts were prepared from 2 days old subcultures of soybean (Glycine max (L.) Merr.) and fragments of young leaves of tobacco (Nicotiana tabacum var. “Xanthi”) according to the methods of Kao. Protoplasts were fused and single fusion products were cultured in Cuprak dishes as previously described. Fusion products were fixed and embedded in plastic by reported methods for electron microscope study. Isoenzyme studies were carried out according to described methods. Proteins were electrophoresed on 5% polyacrylamide gels and stained. Fusion products were easily identified on the basis of the presence of both tobacco chloroplasts and soybean leucoplasts (Fig. 1). The chloroplasts contained typical grana and stroma lamellae; leucoplasts were characterized by numerous starch granules and a paucity of internal lamellae. After 15 hours in culture, thorough mixing of cytoplasm had occurred as evidenced by the distribution of plastids. Fusion of interphase nuclei was not observed in any of the fusion products. Premitotic nuclear fusions which have been reported previously may signify unhealthy fusion products. Fusion products underwent their first cell division within 2–3 days in culture; divi- ding nuclei contained complete sets of both tobacco and soybean chromosomes. During subsequent divisions, hybrids gradually lost some tobacco chromosomes. By 4.5 days, small clusters of hybrid cells were evident. The chloroplasts of such hybrid cells exhibited unusual shapes, possibly as a result of starch accumulation (Fig. 2b, 2c). The leucoplasts remained unchanged. Within 2 weeks, hybrid clusters contained 100–200 cells. Very few chloroplasts were detected in these cells by electron microscopy. The chloroplasts present were highly modified. Typically, these plastids were characterized by enlarged grana and elongated parallel stacks of stroma lamellae. Similar changes in plastid morphology were observed in pea-soybean fusion products cultured for 1 week. It is not possible to determine from the present study whether chloroplasts were being diluted during cell proliferation or whether they were dedifferentiating. Previous ultrastructural research suggests that dedifferentiation of chloroplasts occurs in fusions involving similar species while chloroplasts degeneration is more likely in fusions of widely separated species . Biochemical evidence from studies of the electrophoretic mobility of the plastid-encoded large subunit of ribulose-1, 5-bisphosphate carboxylase and the endonuclease restriction patterns of plastid DNA indicate that plastids may either assort randomly or both plastid types may coexist in cells of regenerated hybrid plants. Chloroplasts were not detected in hybrids cultured for prolonged periods. The leucoplasts in these cells were indistinguishable from leucoplasts of parental soybean cells. Leucoplasts were not diluted during cell division and their numbers were likely maintained by plastid division. Over 20 hybrid cell lines were established and cultured for 7–9 months. Chromosome analysis revealed that many lines including the one illustrated in Fig. 4 retained over one half of the tobacco chromosomes in addition to the full soybean chromosome complement . Zymograms from this same cell line are presented in Fig. 5. The electrophoretic patterns for both dehydrogenases clearly demonstrate that hybridization has been achieved. The shikimate dehydrogenase (SDH) zymogram for the hybrid shows that the broad slow- moving band from soybean and the 2 distinctive fast-moving bands found in Nicotiana are all present in the hybrid. Similarly, for 6-phosphogluconate dehydrogenase (6-PGDH), the hybrid contained the bands from soybean and the 3 slower-moving bands from Nicotiana as well as one of the 2 fast-moving bands found in the latter. This study demonstrates the usefulness of both electron microscopy and isozyme analysis for examining hybrid cells derived from plant protoplast fusion. During the early stages of hybrid culture when small sample size precludes isozyme analysis, ultrastructure studies permit the identification of hybrid cells, after prolonged culture, the isozyme technique is a much more sensitive measure of hybridization than is electron microscopy.     

Asymmetric protoplast fusion between sweet potato and its relatives,and plant regeneration

Experiments were conducted to asymmetrically fuse protoplasts from sweet potato (Ipomoea batatas L. Lam.) and its wild relativesI. trifida Don. andI. lacunosa L. Protoplasts of sweet potato were treated with iodoacetamide, whereas those ofI. trifida Don. andI. lacunosa L. were irradiated with X-rays. The asymmetric protoplast fusion was carried out by the electrofusion method and by polyethylene glycol treatment. Electrically-fused protoplasts initiated cell division, and then formed calli earlier than the polyethylene glycol-fused protoplasts. Plant regeneration occurred only in electrofused calli, suggesting that polyethylene glycol had some toxic effect on plant regeneration ability. Analysis of peroxidase isozymes confirmed the interspecific hybrid characteristics of both the fusion-derived calli and regenerated plants.     

Immunocytochemical localisation of auxin-binding proteins in coleoptiles and embryos ofZea mays L.

Summary The auxin-binding protein ABP-1 was localised immunocytochemically in coleoptiles and immature embryos ofZea mays. Two primary polyclonal antibodies raised against ABP-1 and secondary antibodies were either labelled with FITC or 10 nm gold particles for light microscopy, and with 10 nm gold particles for transmission electron microscopy. Light microscopy revealed that ABP-1 was localised in the epidermal cells of etiolated maize coleoptiles, in subepidermal parenchymatic mesophyll cells of the coleoptile and in the companion cells of the vascular bundles. Most labelling was found in the cytoplasm, less in nuclei and vacuoles and cell walls appeared negative. The region of the plasma membrane exhibited prominent labelling. Embryos showed low labelling throughout their tissues just after excision, but after culture for 7 days intensive labelling was found in the epidermis of the scutellum. Quantitative electron microscopy confirmed that ABP-1 was present in the cytoplasm of epidermal, mesophyll, and companion cells of coleoptiles. Gold particles were neither found in cell walls nor in the cuticle. Areas with ER and dictyosomes within epidermal and mesophyll cells of coleoptiles had a denser labelling with gold particles than elsewhere. Labelling at the plasma membrane, being the site where the auxin binds to the ABP, was observed at low levels in all cells examined, which is due to the method applied. Epidermal cells of embryos cultured for 5 days exhibited high levels of gold particles in ER and nuclei, and lower levels in the cytoplasm. The distribution is only partly in accordance with the model in which ABP is thought to cycle through the plant cell from the ER via the Golgi system towards the plasma membrane.Abbreviations ABP-1 auxin-binding protein 1 - BSA bovine serum albumin - 2,4-D 2,4-dichlorophenoxyacetic acid - EM electron microscopy - LM light microscopy - LR Write London resin white - PBS phosphate-buffered saline - PEG polyethylene glycol - TEM transmission electron microscopy     

Effects of sodium chloride and polyethylene glycol on root-hair infection and nodulation of Vicia faba L. plants by Rhizobium leguminosarum

The effects of sodium chloride and polyethylene glycol (PEG) on the interaction between Rhizobium leguminosarum strain 29d and root hairs of field bean (Vicia faba L. cv. Maris Bead) plants were investigated. Two levels each of NaCl (50 and 100 mol·m^–3) and PEG (100 and 200 mol·m^–3) were given at the time of root-hair formation. Scanning electron microscopy showed rhizobial attachment and colonization on root-hair tips. Adhesion of rhizobia in both lateral and polar orientation, sometimes associated with microfibrils, occurred mainly in crooks at the root-hair tips; most of the infections also occurred here. Bacterial colonization and root-hair curling were both reduced by stress treatments. Polyethylene glycol but not NaCl significantly reduced root-hair diameter. The proportion of root hairs containing infection threads was reduced by 30% under NaCl and by 52% under PEG. The structure of some of the root hairs, epidermal and hypodermal cells, as seen by light microscopy in ultrasections, was distorted as a result of NaCl and PEG treatments; cells showed plasmolysis and folded membranes. After three weeks of treatment, both NaCl and PEG inhibited nodule number by about 50% and nodule weight by more than 60%. It is concluded that the root-hair infection process in Vicia faba is impaired by NaCl and PEG treatments and this in turn results in fewer nodules being produced.Abbreviation PEG polyethylene glycol     

Concanavalin A-mediated agglutination of plant plastids

Cucumber (Cucumis sativus L.) and pear (Pyrus domestica Medik.) fruit proplastids, and pea (Pisum sativum L., cv. Meteor) leaf chloroplasts, extracted by osmotic rupture of protoplasts isolated after degradation of the cell walls by cellulase and pectinase, agglutinated in the presence of Con A. Agglutination of cucumber proplastids was inhibited by anti-Con A and by methyl D-gluco/manno pyranosides but not by methyl D-galactopyranoside. Fluorescein isothiocyanate-conjugated Con A (FITC-Con A) rendered agglutinated clumps fluorescent. If cellulase was omitted from the macerating medium, Con A-mediated agglutination did not occur even if proplatids were subsequently incubated with cellulase. Proplastids and chloroplasts extracted by conventional mechanical disruption methods were not agglutinated by Con A and did not acquire fluorescence with FITC-Con A. However, cucumber proplastids so extracted could be agglutinated by Con A if incubated with cellulase after preparation.Abbreviation Con A Concanavalin A (Jackbean phytohemagglutinin)     

NaCl胁迫对宁夏枸杞叶和幼根显微及超微结构的影响

以宁夏枸杞为材料,采用超薄切片技术制备样品,应用光学显微镜和透射电镜分析了不同浓度NaCl胁迫条件下宁夏枸杞叶和幼根显微及超微结构的变化。结果表明：随着NaCl胁迫的加重,(1)叶片上表皮细胞增厚,栅栏组织细胞出现缩短现象,排列疏松且紊乱;幼根的初生结构无明显变化。(2)叶片栅栏组织中叶绿体不再紧靠在细胞膜上,叶绿体双层膜破坏,基粒片层松散排列,杂乱无章,出现膨胀和空泡现象,淀粉粒和嗜锇颗粒增多,叶肉细胞中线粒体发生轻微变化;幼根中皮层薄壁细胞线粒体形状发生改变,结构破坏,内膜和外膜模糊甚至破裂,大多数嵴模糊,出现空泡现象;细胞核解体,基质外溢。研究表明, 不同浓度的NaCl胁迫对宁夏枸杞叶片和幼根细胞的显微及超微结构影响不同,NaCl浓度大于200 mmol/L时,宁夏枸杞叶片和幼根细胞的显微及超微结构发生了明显变化,且叶肉细胞中线粒体的变化没有叶绿体的变化显著,推测叶肉细胞中线粒体的耐盐性比叶绿体强。     

Dedifferentiation of chloroplasts in interspecific and homospecific protoplast fusion products

Summary Interspecific and homospecific protoplast fusion products (Vicia narbonensis +V. hajastana,V. hajastana+V. hajastana) regenerated cell walls and divided when culturedin vitro for a period of 7 days. While most organelles appeared typical of actively growing plant cells, chloroplasts exhibited structural changes which were interpreted as dedifferentiation.     

Comparison of cell wall regeneration on maize protoplasts isolated from leaf tissue and suspension cultured cells

Summary The cell wall regeneration on protoplasts derived from maize mesophyll cells was compared with wall regeneration on protoplasts derived from suspension cultured cells using light microscopy, transmission electron microscopy, and mass spectrometry. The time course of cell wall regeneration has shown that the mesophyll protoplasts regenerated walls much slower than the protoplasts derived from cultured cells. Moreover, cell wall materials on the mesophyll protoplasts were often unevenly distributed. Electron microscopy has further demonstrated that the mesophyll protoplasts have less organized and compact walls than the protoplasts from cultured cells. Chemical analysis revealed that the mesophyll protoplasts had a lower ratio ofβ-(1–3)-glucan toβ-(1–4)-glucan than protoplasts from cultured cells. The significance of these results for the viability and development of protoplasts in culture is discussed. National Research Council of Canada paper no. 32458.     

Changes in chloroplast number during pea leaf development

Protoplasts were prepared from pea (Pisum sativum L.) leaves throughout development and their contents spread in a monolayer to determine the number of chloroplasts per cell. This approach permitted the rapid analysis of more than 100 cells at each stage of development. The average number of chloroplasts per cell increased from 24±10 to 64±20 during greening and expansion of the first true foliage leaves; all cells containing chloroplasts apparently increase their chloroplast number. A parallel increase in the amount of DNA per nucleus was not observed. As the leaves senesced the chloroplast number gradually decreased to 44±12. We have correlated these changes with our previous results on the percentage of chloroplast DNA per cell. Chloroplast multiplication resulted in a 2.7-fold dilution (from 272 to 102) of the number of copies of the chloroplast DNA molecule per plastid.     

Quantitative ion localization within Suaeda maritima leaf mesophyll cells

Grown under saline conditions, Suaeda maritima accumulates Na⁺ and Cl^- into its leaves, where individual mesophyll cells behave differently in their compartmentation of these ions. Measurements of ion concentrations within selected subcellular compartments show that freeze-substitution with dry sectioning is a valuable preparative technique for analytical electron microscopy of highly vacuolate plant material. Using this approach, absolute estimates were made of Na⁺, K⁺ and Cl^- concentrations in the cytoplasm, cell walls, chloroplasts and vacuoles of leaf mesophyll cells.Abbreviation TAEM transmission analytical electron microscopy     

Intergeneric fusion of Zygnemataceae protoplasts

In intergeneric fusion fromMougeotia andZygnema protoplasts, the fate of fusion products, as well as nuclei and chloroplasts, could be classified according to the number of protoplasts involved from the two algae. Stable elongation growth occurred only in products of groups involving one protoplast from one alga and several protoplasts from the other alga. The features of the elongating products were those of the alga more numerously represented. The different nuclei combined by fusion failed to co-exist. In the groups involving one protoplast from one alga and several from the other, the nucleus from the former degenerated in an early period and only the nuclei from the latter were maintained. Also, the different chloroplasts combined did not co-exist. The genus of the chloroplasts maintained coincided with that of the nuclei maintained. The chloroplasts from the other genus degenerated gradually. An early morphological change in the degenerating chloroplasts was seen in the quantity of starch grains. Later, the chloroplasts generally became rounded, In degeneratingZygnema chloroplasts, thylakoid stacking was prominent. Without collapse of the thylakoid or accumulation of plastoglobules, the degenerating chlorplasts showed rupture of the chloroplast envelope.     

The immuno-histochemical localization of lectin in pea seeds (Pisum sativum L.)

The lectin from the garden pea (Pisum sativum L.) has been localized at the ultrastructural level by the unlabeled peroxidase-antiperoxidase procedure of L.A. Sternberger et al. (1970, J. Histochem. Cytochem 18, 315–333) in 24 h imbibed seeds. Upon examination by light microscopy and transmission electron microscopy, the lectin was only found in the protein bodies of cotyledons and embryo axis. Cell walls as well as membraneous fractions were completely devoid of lectin. These results are discussed in relation to the possible physiological function of seed lectins.Abbreviations PBS phosphate-buffered saline - TBS Tris-buffered saline - PAP-complex horseradish peroxidase-antihorseradish peroxidase soluble complex - NGS normal goat serum - TBS^* Tris-buffered saline containing 0.5 M NaCl, pH 7.6     

Characterization of a connexin homologue in cultured soybean cells and diverse plant organs

Antibodies were prepared against ratliver connexin (27-kDa polypeptide subunit of cell gap junctions found between contacting animal cells) and a putative soybean (Glycine max (L.) Merr.) connexin (29-kDa polypeptide) previously isolated from cultured soybean root cells (SB-1 cell line). The antibodies were utilized to examine the intracellular localization of soybean connexin in these cultured soybean cells and to probe for the presence of a soybean-type connexin in petals, fruits, and leaves from a variety of plants. As judged by specific reactivity on immunoblots, both antibodies against the 27-kDa polypeptide (ratliver connexin) and against the 29-kDa polypeptide (operationally termed soybean connexin) were utilized to demonstrate immunological relatedness of the 27-kDa (rat liver) and the 29-kDa (soybean) polypeptide. Immunofluorescent localization of the putative soybean connexin in cultured soybean cells utilizing these probes demonstrated a peripherally localized punctate pattern of labeling at areas of contact between cells. Use of antibody to the soybean connexin as a probe on immunoblots of extracts from petals, fruits and leaves demonstrated that the soybean-type connexin is present in a large number of different plants.Abbreviations kDa kilodalton - IgG immunoglobulin G - NEPHGE non-equilibrium pH gradient electrophoresis - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Transplantation of isolated nuclei into plant protoplasts

The uptake of isolated nuclei from Vicia hajastana Grossh. cells into protoplasts of an auxotrophic cell line of Datura innoxia P. Mill. was induced under the influence of polyethylene glycol and Ca²⁺ at pH 6.8. The frequency of nuclear uptake varied from 0.8 to 2.3% and that of the recovery of prototrophic clones from 10^-5 to 6·10^-4. The prototrophic nuclear fusion products following nuclear uptake could be rescued by initial culture of the protoplasts in non-selective conditions and by the subsequent use of feeder cell layers to support the growth of surviving colonies on a selective medium. The presence of Vicia genomic DNA in some prototrophic clones was confirmed by dot-blot hybridization using Datura and Vicia DNA probes. In certain transformed clones, the recovery of prototrophy was accompanied by the restoration of morphogenetic potential. Welldeveloped shoots typical of wild-type Datura could be regenerated employing an appropriate regeneration medium.Abbreviations MS Murashige and Skoog (1962) - PEG polyethylene glycol     

Organelle loss in the endosymbiont ofGymnodinium acidotum (Dinophyceae)

Summary The freshwater dinoflagellateGymnodinium acidotum is known to harbor a cryptomonad endosymbiont whose chloroplasts give the organism its blue-green coloration. Every cell examined from a wild population possessed chloroplasts, mitochondria, and other organelles which are of endosymbiotic origin. Transmission electron microscopy and fluorescence microscopy revealed that only 33% of these cells possessed the nucleus of the endosymbiont. The lack of a cryptomonad nucleus in some cells did not appear to affect the cells' ability to photosynthesize or move in response to varying levels of illumination. This represents the first report of a host/endosymbiont relationship in which a significant number of individuals from a given population lack a major endosymbiont organelle.     

Expression of transferred genes during hairy root development in pea

Root border cell development and expression of reporter genes were evaluated in transgenic pea hairy roots. Successful induction of hairy roots in pea is conditioned by bacterial strain and plant genotype, as well as by developmental and environmental factors. Morphological changes sometimes occur when hairy roots are transferred from infected plants to tissue culture media, but such changes are confined to specific clones. Expression of reporter genes under the control of promoters from bean (Phaseolus vulgaris L.) stress genes encoding phenylalanine ammonia lyase and chalcone synthase were evaluated. Expression patterns vary between hairy roots taken directly from infected plants, and those grown in culture; most hairy roots taken from infected plants exhibit expression throughout all tissues, whereas expression in cultured hairy roots is most often localized to specific tissues. Patterns of expression that occur during different stages of hairy root development are very similar to those observed in transgenic plants expressing the same fusion genes. Border cell separation and release in hairy roots is normal, and expression of glucuronidase in border cells of some transgenic roots resulted in development of bright blue single cells. Cultured hairy roots should provide a very useful model for studying the effect of defined changes in root border cells on microbial associations with roots of this important legume.Abbreviations YEM yeast extract-mannitol - GUS glucuronidase - PAL phenylalanine ammonium lyase - CHS chalcone syntase     

Leaf anatomy and water loss of in vitro PEG-treated ‘Valiant’ grape

Polyethylene glycol was used to induce water stress of micropropagated Valiant grape. Reduced growth and slow rooting were observed in treated plantlets with 2, 4 and 6% polyethylene glycol as compared to control plantlets with no polyethylene glycol in the rooting medium. At high concentrations of 4 and 6%, leaves exhibited wilting and necrosis. At the 2% level, plantlets recovered and grew satisfactorily. Detached leaves of treated plantlets with 2% polyethylene glycol lost less water than controls when exposed to low humidity for 4 hours. Leaf anatomy of plantlets treated with 2% polyethylene glycol, control (in vitro plantlets) and greenhouse-grown plants were compared under light microscopy. Leaves from control plantlets contained larger mesophyll cells, lacked normal palisade layer formation, had greater intercellular pore spaces and fewer chloroplasts. Leaves from polyethylene glycol-treated plantlets, however, had smaller mesophyll cells, a more defined palisade layer, reduced intercellular pore space and the greatest number of chloroplasts. These results suggest that an osmoticum such as polyethylene glycol may be used to induce more normal leaf anatomy and reduced water loss in micropropagated Valiant grapes.Abbreviations BA 6-benzylaminopurine - FAA formalin-acetol-alcohol - MS Murashige & Skoog (1962) medium - MW molecular weight - NAA napthaleneacetic acid - PEG polyethylene glycol - TBA tertiary butyl alcohol