Protoplasmic Swelling as a Symptom of Freezing Injury in Onion Bulb Cells : Its Simulation in Extracellular KCl and Prevention by Calcium

Freezing injury, in onion bulb tissue, is known to cause enhanced K⁺ efflux accompanied by a small but significant loss of Ca²⁺ following incipient freezing injury and swelling of protoplasm during the postthaw secondary injury. The protoplasmic swelling of the cell is thought to be caused by the passive influx of extracellular K⁺ into the cell followed by water uptake. Using outer epidermal layer of unfrozen onion bulb scales (Allium cepa L. cv Big Red), we were able to stimulate the irreversible freezing injury symptoms, by bathing epidermal cells in 50 millimolar KCl. These symptoms were prevented by adding 20 millimolar CaCl₂ to the extracellular KCl solution. Our results provide evidence that loss of cellular Ca²⁺ plays an important role in the initiation and the progression of freezing injury.     

Changes of Onion Epidermal Cell Permeability Due to the Treatment with Alanap

The effect of Alanap, N-1-naphthylphthalamic acid (NPA), on the permeability of onion (Allium cepa) epidermal cells was studied by means of a plasmometric method. Urea, 1,3-dimethylurea, and water were used for the test of permeability. It was found that the treatment of onion epidermis with NPA at various concentration (5 × 10^–3 to 5 × 10^–6M) altered the deplasmolysis rates and the absolute permeability constants. However, NPA did not alter the osmotic value of the treated cells. It was suggested that NPA treatment altered the permeability of onion epidermal cells due to the effect of this herbicide on the plasma membrane.     

Micromanipulation of Cyanelles and a Cyanobacterium into Higher Plant Cells

A cyanobacterium (WPI-I) and cyanelles of Glaucocystis nostochinearum Itz, have been introduced into epidermal cells of Allium cepa L. cv. Downing Yellow Globe bulb scales by microinjection. Uptake of cyanelles into protoplasts of Allium cepa and Daucus carota L. cv. Imperator 58 has also been induced with polyethylene glycol. Protoplasts were cultured until new cell walls were synthesized.     

Beobachtungen zur Beteiligung des Tonoplasten an der Plasmaströmung

Summary Vital staining of inner epidermal cells (Allium cepa, onion bulbes) causes blue granular precipitations in cell sap which are moved by protoplasmatic streaming. Such and earlier observations proof that the tonoplast participates in all protoplasmatic streaming patterns which should be taken in consideration with regard to the unit-membrane concept of tonoplast.     

On the properties of fluorescing compounds in guard and epidermal cells of Allium cepa L.

Onion guard cells, in contrast to those of Vicia and Pisum, do not require an alkaline treatment in order to fluoresce. Fluorescing compounds of Allium cepa L. were characterized using in-vivo microspectrophotometry; furthermore, invitro chemical analysis for epidermal tissue, intact guard and epidermal cells, and isolated guard-cell protoplasts was performed. The emission intensity (_max 520 nm) decreased when intact onion guard cells were excited with 436 nm light, but increased (_max 470 nm) when excited at 365 nm. This photodecomposition at 436 nm is typical of flavins or flavoproteins whereas an increase in fluorescence intensity with excitation at 365 nm may be explained by the presence of other substances. The presence of flavins could not be unambiguously confirmed from these results. Indeed, the absorption spectra of the vacuolar area of guard cells did not show the peak at 445 nm which is characteristic for flavins. Furthermore, there was no decrease of absorption at the excitation wavelengths of 440 and 330 nm. Since spectral data indicate the presence at high amounts of flavonoids in guard and epidermal cells, this may reduce the sensitivity for the detection of flavins in guard cells. Using thin-layer chromatography and high-performance liquid chromatography together with hydrolytic procedures, flavonol glycosides with kaempferol and quercetin as aglycones substituted with sulphate and glucuronate were identified. Further studies on guard-cell metabolism should consider the presence of flavonoids in stomata of onion and other plants.Abbreviations GCP guard-cell protoplast - HPLC high-performance liquid chromatography - TLC thin-layer chromatography     

Evidence for nuclear-cytoplasmic incompatibility between Allium fistulosum and A. cepa

An F₂ population (Allium fistulosum x A. cepa) of 20plants, 10 BC₁,[(A. fistulosum x A. cepa) x A. cepa], and 50 BC₂ plants, [(A. fistulosum x A. cepa) x A. cepa] x A. cepa were studied cytogenetically and characterized for four isozyme alleles plus various morphological characteristics. All of the progenies were in A. fistulosum (the bunching onion) cytoplasm. In the F₂ population we observed non-random chromosomal and allelic segregation, suppression of bulb onion allelic expression, and abnormalities in mitosis and meiosis. Most BC₂ plants resembled A. cepa (the bulbing onion) morphologically, but anthers, filaments, pistils, and petals were abnormal. Only 3 plants, and these were most nearly like the F₁ hybrid morphologically, produced any seeds.The data and observations support the hypothesis of nuclear-cytoplasmic incompatibility interactions between the bunching and bulb onion species.Use of trade names does not imply endorsement of the products named nor criticism of similar ones not named. This research was supported by the New Mexico Agricultural Experiment Station.     

Investigation of viability of plant tissue in the environmental scanning electron microscopy

The advantages of environmental scanning electron microscopy (ESEM) make it a suitable technique for studying plant tissue in its native state. There have been few studies on the effects of ESEM environment and beam damage on the viability of plant tissue. A simple plant tissue, Allium cepa (onion) upper epidermal tissue was taken as the model for study. The change of moisture content of samples was studied at different relative humidities. Working with the electron beam on, viability tests were conducted for samples after exposure in the ESEM under different operating conditions to investigate the effect of electron beam dose on the viability of samples. The results suggested that without the electron beam, the ESEM chamber itself can prevent the loss of initial moisture if its relative humidity is maintained above 90%. With the electron beam on, the viability of Allium cepa (onion) cells depends both on the beam accelerating voltage and the electron dose/unit area hitting the sample. The dose can be controlled by several of the ESEM instrumental parameters. The detailed process of beam damage on cuticle-down and cuticle-up samples was investigated and compared. The results indicate that cuticular adhesion to the cell wall is relatively weak, but highly resistant to electron beam damage. Systematic study on the effect of ESEM operation parameters has been done. Results qualitatively support the intuitive expectations, but demonstrate quantitatively that Allium cepa epidermal cells are able to be kept in a hydrated and viable state under relevant operation condition inside ESEM, providing a basis for further in situ experiments on plant tissues.     

Bulb-type onion introgressants posessing Allium fistulosum L. genes recovered from interspecific hybrid backcrosses between A. cepa L. and A. fistulosum L.

Allium fistulosum possesses a number of traits which would be desirable in A. cepa. Thus far, no commercial A. cepa cultivars have been released which harbor Allium fistulosum traits. F₁BC₃ populations were generated for this study by backcrossing A. cepa to A. cepa×A. fistulosum hybrids. The F₁BC₃ plants were evaluated for plant morphology, floral characters, male- sterile cytoplasm, soluble solids and pungency, and isozymes. Overall growth habit and floral characters of the F₁BC₃ plants were much like A. cepa. We report here the recovery of recombinant, bulbing, and fertile A. cepa-type onions that exhibit A. fistulosum isozyme alleles and morphological markers. Recombination between A. cepa and A. fistulosum genomes was achieved using the introgression strategy of backcrossing A. fistulosum into A. cepa, thereby ameliorating the nuclear cytoplasmic barriers that occurred in previous less successful introgression attempts when plants were not in A. cepa cytoplasm. We believe this report to be the first demonstration of onion introgressants that are like A. cepa in appearance, are male- and female-fertile, and possess A. fistulosum genes. Received: 29 May 1999 / Accepted 22 June 1999     

Control of Ethylene Induced Permeability of Plant Cells by a Substituted Benzothiadiazole

The permeability of bulb tissues of onion (Allium cepa L.), amaryllis (Hippeastrum hybrids), tulip (Tulipa × hybrida) and daffodil (Narcissus tazetta L.) treated with Ethephon and 5-methyl-7-chloro-4-ethoxycarbonylmethoxy-2,1,3-benzothiadiazole (TH6241), to water and solutions of 1,3-dimethylurea was determined by the plasmometric methods. Ethylene treatments promoted, while those with TH6241 generally decreased the cell membrane permeability; TH6241 negated the effects of ethylene on cell permeability.     

Anti-mitotic and anti-genotoxic effects of Plantago lanceolata aqueous extract on Allium cepa root tip meristem cells

Plantago is the most important genus of Plantaginaceae family and is used in traditional medicine around the world for different purposes. Plantago coronopus L., Plantago major L., Plantago media L. and Plantago lanceolata L. are most commonly used species of Plantago in traditional medicine in Turkey. The main goal of this study was to investigate the eventual anti-mitotic and anti-genotoxic effects of P. lanceolata L. leaf aqueous extracts (15 g/L and 30 g/L) on Allium cepa L. root tip meristem cells which were treated with 0.7% hydrogen peroxide. For this purpose, two different experiments were performed under the same conditions. In the first experiment, Allium cepa onion bulbs were treated with 0.7% H₂O₂ for 1 h. After the H₂O₂ treatment, the onion bulbs were treated with two different concentrations (15 g/L and 30 g/L) of P. lanceolata extracts for 24 h. In the second experiment, A. cepa onion bulbs were treated with two different extract concentrations (15 g/L and 30 g/L) for 24 h and then with 0.7% H₂O₂ for 1 h. The test concentrations were determined according to doses which are recommended in alternative medicinal usage by people. As positive and negative control 0.7% H₂O₂ and tap water was used, respectively. As a result, it was determined that aqueous extracts reduced mitotic index and chromosome aberrations in treatment groups in comparison with controls. These results showed that P. lanceolata aqueous extracts have anti-mitotic and anti-genotoxic effects.     

Vacuolated plant cells as ideal osmometer: reversibility and limits of plasmolysis, and estimation of protoplasm volume in control and water-stress-tolerant cells

Abstract. The osmotic behaviour of vacuolated plant cells (adaxial epidermal cells of Allium cepa bulb scales, and epidermal as well as chloroplast containing subepidermal stem base cells of Pisum sativum) was studied over a wide range of CaCl₂ concentrations. The following results were obtained.

• a. Allium cepa and Pisum sativum plant cells behave as an ideal osmometer as far as plasmolytic contraction of the protoplast is concerned.
• b. The protoplasts of these cells could be plasmolysed to 15–45% of their original volume without the loss of membrane semi-permeability.
• c. Cells plasmolysed in 1.0 kmol m^?3 CaCl₂ could be completely deplasmolysed and upon deplasmolysis the cells resumed protoplasmic streaming.
• d. The above findings (a-c) indicate that during gradual plasmolysis and deplasmolysis membrane semi-permeability is maintained.
• e. At very high plasmolysing concentrations vacuoles covered with the tonoplast separated from the rest of the protoplasm in some cells whereas others showed systrophy. Extruded vacuoles were able to respond to osmotic shrinkage.
• f. The non-solvent space in Allium cells of about 3% also corresponded to the protoplasm volume calculated from the protoplast geometry (mean from results of direct measurement method and subtraction method).
• g. Subepidermal stem base cells of water-stress-tolerant Pisum plants had a 75% greater non-solvent space than the control cells indicating that a water-stress-tolerant cell may contain a larger amount of protoplasm and/or a vacuole with a higher content of colloidal material in the vacuole.
• h. Water-stress-tolerant cells showed greater tolerance to osmotic dehydration (volume reduction) than control cells.

     

Stimulation of seedling root growth by coal-derived sodium humate

Coal-derived sodium humate was found to stimulate primary root growth of seedlings of cantaloupe (Cucumis melo L.) at 1000 mg L^–1, lettuce (Lactuca sativa L.) and onion (Allium cepa L.) at 500 and 1000 mg L^–1, as well as hypocotyl growth of cantaloupe at 1000 mg L^–1. Growth enhancement was not due to release of nutrient elements by the product, and, in the case of lettuce and onion, was not due to increased availability or uptake of mineral elements. Growth stimulation of cantaloupe, however, was dependent on the provision of nutrient solution. Growth stimulation of onion roots under axenic conditions indicated an effect of the humate per se rather than a response mediated via microbial breakdown products.     

Gibberellin stabilizes microtubules in onion leaf sheath cells

Summary Colchicine and cremart (O-ethyl O-(3-methyl-6-nitrophenyl) N-sec-butylphosphorothioamidate) disrupt microtubules in leaf sheath cells of onion plants (Allium cepa L. cv. Senshu-Chuko) and cause cell swelling to make the basal parts of the plants bulbous. Gibberellin A₃(GA₃) protects microtubules from disruption by colchicine and cremart and suppresses the swelling caused by them. GA₃ also protects microtubules from disruption by low temperature.     

Sequence analysis of a chloroplast intergenic spacer for phylogenetic estimates in Allium section Cepa and a PCR-based polymorphism detecting mixtures of male-fertile and male-sterile cytoplasmic onion

Restriction-enzyme analysis of the chloroplast (cp) DNA yielded maternal phylogenies supporting a close phylogenetic relationship among normal (N) male-fertile and male-sterile (S) cytoplasmic bulb onion (Allium cepa), Allium altaicum, Allium fistulosum, Allium galanthum, Allium roylei, and Allium vavilovii. The S cytoplasm of onion is most likely an alien cytoplasm introduced in antiquity into onion populations. We previously showed that size differences in an intergenic spacer in the cp DNA distinguish N and S cytoplasms of onion. We cloned and sequenced this intergenic spacer from the N and S cytoplasms of onion, A. altaicum, A. fistulosum, A. galanthum, Allium pskemense, Allium oschaninii, A. roylei, and Allium ampeloprasm (outgroup) to identify the nature of previously described RFLPs and to develop a PCR-based marker revealing N-cytoplasmic contamination of S-cytoplasmic hybrid seed lots. Phylogenies based on restriction-enzyme analysis of the entire cp DNA were similar, but not identical, to those based on sequence divergence in this intergenic region. Received: 29 November 1999 / Accepted: 28 April 2000     

In Vivo Perturbation of Membrane-Associated Calcium by Freeze-Thaw Stress in Onion Bulb Cells : Simulation of This Perturbation in Extracellular KCl and Alleviation by Calcium

Incipient freeze-thaw stress in onion bulb scale tissue is known to cause enhanced efflux of K⁺, along with small but significant loss of cellular Ca²⁺. During the post-thaw period, irreversibly injured cells undergo a cytological aberration, namely, `protoplasmic swelling.' This cellular symptom is thought to be caused by replacement of Ca²⁺ from membrane by extracellular K⁺ and subsequent perturbation of K⁺ transport properties of plasma membrane. In the present study, onion (Allium cepa L. cv Sweet Sandwich) bulbs were slowly frozen to either −8.5°C or −11.5°C and thawed over ice. Inner epidermal peels from bulb scales were treated with fluorescein diacetate for assessing viability. In these cells, membrane-associated calcium was determined using chlorotetracycline fluorescence microscopy combined with image analysis. Increased freezing stress and tissue infiltration (visual water-soaking) were paralleled by increased ion leakage. Freezing injury (−11.5°C; irreversible) caused a specific and substantial loss of membrane-associated Ca²⁺ compared to control. Loss of membrane-associated Ca²⁺ caused by moderate stress (−8.5°C; reversible) was much less relative to −11.5°C treatment. Ion efflux and Ca²⁺-chlorotetracycline fluorescence showed a negative relationship. Extracellular KCl treatment simulated freeze-thaw stress by causing a similar loss of membrane-associated calcium. This loss was dramatically reduced by presence of extracellular CaCl₂. Our results suggest that the loss of membrane-associated Ca²⁺, in part, plays a role in initiation and progression of freezing injury.     

Onion microsatellites for germplasm analysis and their use in assessing intra- and interspecific relatedness within the subgenus Rhizirideum

We have identified a set of informative STMS markers in onion (Allium cepa L.) and report on their application for genotyping and for determining genetic relationships. The markers have been developed from a genomic library enriched for microsatellites. Integrity of the microsatellite polymorphism was confirmed by amplicon sequencing. The microsatellite genotypes of 83 onion accessions and landraces from living onion collections were compared. As few as four primer pairs were sufficient to assign unique microsatellite patterns to the 83 accessions. Some of the microsatellite markers can be used for interspecific taxonomic analyses among close relatives of Allium cepa. Generally, our data support and extend results obtained from recently performed analyses using ITS, RAPD and morphology. Received: 8 October 1999 / Accepted: 3 November 1999     

Cytogenetical evidences for hybrid structure and origin of diploid and triploid shallots (Allium cepa var.viviparum,Liliaceae) from Dalmatia (Croatia)

Two viviparous strains of common onion (Allium cepa) denoted asAllium cepa var.viviparum (syn.Allium ×proliferum), traditionally cultivated in the seaside regions of Croatia were found to be diploid (2n = 2x = 16) and triploid (2n = 3x = 24), respectively. The triploid cultivated onion Ljutika has not been previously reported in Europe. Using Feulgen and Giemsa C-banding methods both karyotypes were shown to be of hybrid constitution. Results obtained in this work indicate that Dalmatian viviparous onions arose from spontaneous hybridization ofA. cepa withA. fistulosum.     

Isolation and identification of soluble polysaccharides in epidermal tissue of Allium cepa

Because starch is absent from Allium-guard cells, another polysaccharide was sought that, in connection with stomatal opening, could be a source of organic anions. Analysis of isolated polysaccharides revealed xylose, arabinose, glucose, galactose, and galacturonic acid (3.4:1:1.6:0.7) to be components of the water-soluble mucilage of the epidermal strips of Allium cepa. However, the experiments gave no indication that the mucilage is the malate donator during the stomatal opening. After ¹⁴CO₂ fixation the following substances were labeled: organic acids, especially malate and citrate, amino acids and the polysaccharide mentioned above; radioactive 3-phosphoglyceric acid and sugar phosphates were not found. Therefore we conclude that the Calvin cycle does not operate in the guard cells of Allium cepa.Abbreviations ABA Abscisic acid     

Cell Wall Thickening of the Xylem Tracheary Elements in Different Zones of the Adventitious Root of Allium cepa L.

Cell wall thickness of the xylem tracheary elements was measuredin the proto- and metaxylem of the Allium cepa L. adventitiousroot. Measurements were taken in root fragments of known age(1, 3, 5, 7 and 9 d) located in either the basal or medio-apicalzone. Tracheary elements in the protoxylem matured within ashorter period of time than those in the metaxylem. Final cellwall thickness was greater in metaxylem than in protoxylem components.The cell wall thickening in the tracheary elements in both proto-and metaxylem was more rapid in the basal zone of the root thanin the medio-apical zone. Additionally, cell walls of the maturetracheary elements were thicker in the basal zone than in areasfurther from the bulb. Allium cepa, onion, root, cell wall, xylem maturation     

Influence of decenylsuccinic Acid on water permeability of plant cells

Decenylsuccinic acid altered permeability to water of epidermal cells of bulb scales of Allium cepa and of the leaf midrib of Rhoeo discolor. Water permeability, as determined by deplasmolysis time measurements, was related to the dose of undissociated decenylsuccinic acid (mm undissociated decenylsuccinic acid × minute). No relationship was found between permeability and total dose of decenylsuccinic acid, or dose of dissociated decenylsuccinic acid, suggesting that the undissociated molecule was the active factor in permeability changes and injury.