Atomic force microscopy and laser confocal scanning microscopy analysis of callose fibers developed from protoplasts of embryogenic cells of a conifer

Efficiency of novel fiber formation was much improved in protoplast culture of embryogenic cells (ECs) of a conifer, Larix leptolepis (Sieb. et Zucc.) Gord., by pre-culturing ECs in a medium containing a high concentration of glutamine (13.7 mM). The fibrillar substructures of large and elongated fibers of protoplasts isolated from Larix ECs were investigated by laser confocal scanning microscopy (LCSM) after Aniline Blue staining and atomic force microscopy (AFM) using a micromanipulator without any pre-treatment. Fibers were composed of bundles of fibrils and subfibrils, whose diameters were defined as 0.7 and 0.17 μm, respectively, by image analysis after LCSM and AFM. These fibers were proven to be composed of callose by using specific degrading enzymes for β-1,4-glucan and β-1,3-glucan.     

Studies of cotyledon protoplast cultures from B napus,B. campestris and B. oleracea. II: Callus formation and plant regeneration

Cotyledons from twelve cultivars of Brassica; B. napus (Westar, Eureka, Global, Pivot and Narc 82); B. campestris: (Arlo, Sonja, Bunyip and Wonk Bok) and B. oleracea (Phenomenal Early, Sugar Loaf and Earliball) were used for protoplast isolation and culture in a comparative study of cell colony and callus formation, and plant regeneration. The formation of cell colonies and callus from protoplast cultures were significantly influenced by the light conditions of seed germination. All twelve cultivars showed callus formation from protoplast cultures derived from cotyledons of seedlings grown in dark for 3 days followed by 1 day dim light (dark/dim light-grown). Callus was obtained in all five liquid media used: modified K8P(1), modified K8P(2), modified MS, modified B and modified NN. In contrast, only six cultivars exhibited callus formation from the protoplasts isolated from cotyledons of seedlings germinated under light conditions for 7 days (light-grown) and in only three media: modified K8P(1), modified MS, modified B.Callus, derived from protoplast cultures isolated from dark/dim light-grown cotyledons and grown on K3 or MS series solid media for about 1 month, could develop shoots when further transferred onto MS series regeneration media. All five cultivars of B. napus, three of the four cultivars of B. campestris (Arlo, Sonja and Bunyip) and one of the three cultivars of B. oleracea (Sugar Loaf) exhibited shoot regeneration from protoplast cultures within 2–3 months after protoplast isolation. The frequency of shoot regeneration ranged among 1–22.5%. A high degree of reproducibility was observed in cultivars Westar, Eureka, Global, Arlo, Bunyip and Sugar Loaf. In contrast, among the six cultivars that formed callus in protoplast culture derived from light-grown cotyledons, only three cultivars from B. napus (Westar, Eureka, Global) exhibited shoot regeneration 5.5 months after protoplast isolation. Regenerated shoots from cultivars Westar, Eureka and Bunyip and Sugar Loaf, which derived from protoplasts of dark/dim light germinated seedling and were induced to root on rooting media, survived in soil and grew to produce silique and set seeds.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - BA benzylaminopurine - EDTA ethylenediaminetetraacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - KT kinetin - GA₃ gibberellic acid - MS Murashige and Skoog medium - NAA -naphthaleneacetic acid - PAR photosynthetically active radiation     

A novel cottong ovule culture: Induction, growth, and characterization of submerged cotton fibers (Gossypium hirsutum L.)

Summary The growth of submerged cotton (Gossypium hirsutum L.) fibers from cultured ovules has been investigated. The results indicate that exogenous plant hormone levels regulate the induction of submerged fiber growth. The age of ovules at induction is also important. Cell diameter, wall thickness, and cell length of submerged fibers were measured and compared with air-grown fibers and fibers grown in vivo (produced by cotton plants grown in the greenhouse). Various cellwall thickening patterns were observed among submerged fibers, while only one predominant cell-wall deposition pattern was produced in air-grown fibers and in fibers produced in vivo. The diameter of submerged fibers was about the same as that of air-grown fibers but about 22% less than that of fibers grown, in vivo. It appears that the secondary cell wall thickenings are initiated earlier in submerged fibers. The cell-wall thickness of submerged fibers, at 41 d post anthesis (DPA), was 51% greater than that of fibers grown in vivo, whereas the cell-wall thickness of air-grown fibers was 42% less than that of fibers produced in vivo. The cell length of submerged fibers was approximately half that of fibers grown in vivo. and the air-grown fiber length was about two-thirds of fibers grown in vivo. The age of ovules at induction affects the outcome of the air-grown fiber-cell length, but does not appear to affect the length of submerged fiber cells. To produce submerged fiber growth, we found that the optimal age of ovules at induction was 0 DPA, and the optimal medium (with a GA₃ of 0.5 μM and an IAA range of 5-20 μM) depends on the time of ovule induction (−2 to+2DPA). We conclude that conditions leading to submerged cotton fiber growth have great potential for (a) direct monitoring of growth and making precise, detailed measurements during fiber growth and development; (b) producing cellulose and fibers in vitro more efficiently than earlier ovule-culture methods; and (c) using these unique cultures to obtain a better understanding of signal transduction and gene expression leading to growth, development, and programmed cell death in the life history of the cotton fiber.     

DYNAMICS OF ARBUSCULE DEVELOPMENT AND DEGENERATION IN A ZEA MAYS MYCORRHIZA

A quantitative light and electron microscope study of developing and degenerating mycorrhizal arbuscules of Glomus fasciculatum in Zea mays was carried out in order to estimate three parameters during the colonization cycle. These were: 1) V_v(f,c), the fraction of the host cell volume occupied by a volume of fungus; 2) V_v(cy,c), the fraction of the host cell volume occupied by host cytoplasm; 3) S_v(pr,c), the surface-area-to-volume ratio of the host protoplast to the whole host cell. Uninfected cortical cells had an S_v(pr,c) of 0.13 μm²/μm³. As the fungus penetrates the cell wall, the protoplast invaginates, causing a decrease in protoplast volume and an increase in protoplast S_v. The S_v(pr,c) of a cell containing a mature arbuscule is 1.275 μm²/μm³. Because of the shrinkage of the protoplast, the S_v of the protoplast to its own volume rather than the original cell volume is 2.55 μm²/μm³, or almost a 20-fold increase. Total cell size is unaffected. When the arbuscule is mature, the fungus occupies 42% of the cell, with 24% as 1-μm-diam branches, and 18% as trunk. Arbuscular branch formation progresses at a linear rate and is the most important factor in causing the increased host S_v. The correlation coefficient for V_v(br,c) the volume fraction for arbuscular branches, vs. Sv(pr,c) is r = 0.932 (P < 0.001). Degeneration of the arbuscule is marked by a rapid decrease in branches, host S_v, and host cytoplasm. The trunk develops and degenerates at a slower rate than the branches.     

Cadmium biosorption by a cadmium resistant strain of Bacillus thuringiensis: regulation and optimization of cell surface affinity for metal cations

A marine bacterial strain putatively identified asBacillus thuringiensis strain DM55, showed multiple heavy metal resistance and biosorption phenotypes. Electron microscopic studies revealed that DM55 cells are encased in anionic cell wall polymers that can immobilize discrete aggregates of cations. Factors affecting cell surface affinity for metal cations, monitored by means of Cd²⁺ binding capability, are investigated. The mechanisms of cadmium resistance and Cd²⁺ biosorption by the bacterium appeared to be inducible and coincident. Medium components affecting metal removal under cadmium-stressed growth conditions were explored based on the application of two sequential multi-factorial statistical designs. Concentrations of potassium phosphates and peptone were the most significant variables. Optimized culture conditions allowed DM55 cells grown in the presence of 0.25 mM CdCl₂ to remove about 79% of the metal ions within 24 h with a specific biosorption capacity of 21.57 mg g^–1 of biomass. Both fresh and dry cells of DM55 prepared under cadmium-free optimal nutrient condition were also able to biosorb Cd²⁺. In addition to the concentration of phosphate in the medium, KinA, a major phosphate provider in the phosphorelay of Bacillus cells, was also demonstrated to regulate the magnitude of cell surface affinity for cadmium ions.     

Somatic embryogenesis and plant regeneration from isolated protoplasts of Lavatera thuringiaca

Embryogenic cell suspensions of Lavatera thuringiaca L. were established from leaf petiole and shoot regeneration was achieved when cells were plated on medium without growth regulators. We tested three methods for protoplast culture, isolated from a one-year old embryogenic cell suspension, to determine the best conditions for L. thuringiaca protoplast culture and shoot regeneration. The highest protoplast plating efficiency was obtained with the agaroseembedded method, reaching 30%, while the nursing culture method gave 5% when the protoplasts were plated over Whatman paper No. 2. However, the same nursing culture failed to produce protoplast-derived microcalluses when the protoplasts were plated on a nitrocellulose filter. The liquid thin layer method gave the lowest plating efficiency with only 0.5%. Shoot regeneration from protoplast-derived microcalluses was achieved in two steps; first, globular embryo development was favored in medium low in auxin (2,4-d and BA at 0.01 and 0.05 mg 1^-1, respectively), second, the globular embryos further differentiate into shoots in medium without growth regulators or in medium containing GA₃ (0.5 to 1.0 mg 1^-1).Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - BA benzyladenine - GA₃ gibberellic acid - NAA -naphthaleneacetic acid - IBA indole-3-butyric acid     

Isolation and culture of anucleate protoplasts from cotton fiber; assessment of viability and analysis of regenerated wall polymers

Procedures were developed for the isolation and culture of an anucleate protoplast system from cotton fibers actively undergoing secondary wall synthesis. Because the fibers at this stage are elongated single cells (30 m × 1–2 cm), most of the cellular vesicles released in the process of isolation are anucleate. After purification, the protoplast population was nuclei-free. When transferred to culture medium, the anucleate protoplasts (cytoplasts) synthesized starch, hydrolyzed fluorescene diacetate for up to 9 days and formed cell wall material for at least 7 days. The composition of the regenerated cell walls was dependent upon the substrate supplied in the medium: -1,3-linked glucans were predominantly synthesized when 1 mM UDP[¹⁴C]glucose was supplied; -1,4-linked glucans were predominantly synthesized when 1 mM [¹⁴C]-glucose was supplied. Thus the composition of the regenerated cell walls formed by the anucleate protoplasts was similar to the secondary cell wall synthesized by intact cotton fibers under the same culture conditions.     

Mitochondrial DMA variation in somatic embryogenic cultures ofLarix

outhern hybridization analysis using wheat mitochondrial gene-specific probes indicates that changes in mitochondrial genomic organization and the relative representation of certain genomic regions occur during in vitro somatic embryogenic cell culture ofLarix species. We observed differences in the mitochondrial (mt)DNA hybridization patterns between somatic embryogenic cell cultures and trees grown from seed forLarix leptolepis,L. decidua, and the reciprocal hybrids of these twoLarix species. This is the first study to describe the correlation of molecular changes in a gymnosperm mitochondrial genome with in vitro somatic embryogenic cell culture. Quantitative differences in mtDNA hybridization signals were also observed among a 4-year-old somatic embryogenic cell culture ofLarix ×eurolepis trees regenerated from this culture, and the seed source tree from which the somatic embryogenic cell cultures were initiated.     

Comparative studies on the mycorrhization of Larix decidua and Picea abies by Suillus grevillei

Summary Mycorrhization of Picea abies has been achieved, for the first time, with six strains of Suillus grevillei by a new culture method, using activated charcoal paper and liquid medium as a substrate. Mycorrhization of P. abies and Larix decidua was compared, and the process was found to be significantly different in the two tree species. S. grevillei is not incompatible with P. abies, but it forms mycorrhizae more readily with L. decidua. Hyphal growth was clearly stimulated on the surface of roots of Larix but retarded on Picea. A well organized Hartig net was formed with both tree species, but wall protuberances were frequently observed on the outer cell walls of Picea cortex cells when the Hartig net was not fully developed. No conspicuous cell wall reactions occurred in Larix roots. Cell wall protuberances may be comparable to those in transfer cells and are interpreted as an alternative to Hartig net development. Anatomical differences between roots of Larix and Picea, and physiologically active substances such as recognition factors on the root surfaces, are discussed with respect to their responsibility for the different reactions of S. grevillei.     

The isolation and culture of protoplasts from an embryogenic cell suspension culture of Picea glauca (moench) voss

Conditions were standardized for the isolation and culture of protoplasts from an embryogenic cell suspension culture of Picea glauca. A combination of 0.5% Cellulase R-10, 0.25% Macerozyme, 0.25% Driselase, 0.25% Rhozyme HP-150 with 0.5M mannitol and 5 mM CaCl₂.2H₂O produced an average of 4.5 × 10⁶ protoplasts per gram fresh weight of cells. Of the several protoplast culture media tested, von Arnold and Eriksson and Kao and Michayluk (KM8P) media best supported mitotic divisions of protoplasts. A density of 10⁵ protoplasts per ml and the addition of 5 mM glutamine to the culture medium was necessary to induce sustained divisions and microcallus formation. Microcalli grew into subculturable callus using a nurse culture technique.Abbreviations BAP benzylaminopurine - 2,4-D 2,4-dichlorophenoxy-acetic acid - FDA fluorescein diacetate NRCC No. 27937     

The relation between recalcitrancy of a mangrove plant, Kandelia obovata, and high endogenous level of abscisic acid

Kandelia obovata Sheue, Liu & Yong sp. nov. is one of the cold tolerant mangrove plants. Some callus formation was obtained from the leaves of K. obovata in liquid medium containing 10 μM of 2,4-dichlorophenoxyacetic acid. However, recalcitrancy was found when subculturing them. Endogenous levels of gibberellins (GAs) and abscisic acid (ABA) in leaf protoplasts of K. obovata were determined using micro-scale extraction and purification steps, including thin layer chromatography and quantification by micro-bioassay or enzyme linked immunosorbent assays. Very high amounts of ABA and low activities of GAs were found in leaf protoplasts of K. obovata. Low concentrations of gibberellic acid and uniconazole-P were effective of enhancing cell enlargement in protoplast cultures. Exogenous application of ABA was inhibitory to protoplast culture. All cytokinins tested were inhibitory to both leaf and protoplast cultures. The high endogenous level of ABA is most likely the underlying cause of recalcitrancy of mangrove cultures.     

Hemoglobin regulates the migration of glioma cells along poly(ε‐caprolactone)‐aligned nanofibers

Aligned fibers have been shown to facilitate cell migration in the direction of fiber alignment while oxygen (O₂)‐carrying solutions improve the metabolism of cells in hypoxic culture. Therefore, U251 aggregate migration on poly(ε‐caprolactone) (PCL)‐aligned fibers was studied in cell culture media supplemented with the O₂ storage and transport protein hemoglobin (Hb) obtained from bovine, earthworm and human sources at concentrations ranging from 0 to 5 g/L within a cell culture incubator exposed to O₂ tensions ranging from 1 to 19% O₂. Individual cell migration was quantified using a wound healing assay. In addition, U251 cell aggregates were developed and aggregate dispersion/cell migration quantified on PCL‐aligned fibers. The results of this work show that the presence of bovine or earthworm Hb improved individual cell viability at 1% O₂, while human Hb adversely affected cell viability at increasing Hb concentrations and decreasing O₂ levels. The control data suggests that decreasing the O₂ tension in the incubator from 5 to 1% O₂ decreased aggregate dispersion on the PCL‐aligned fibers. However, the addition of bovine Hb at 5% O₂ significantly improved aggregate dispersion. At 19% O₂, Hb did not impact aggregate dispersion. Also at 1% O₂, aggregate dispersion appeared to increase in the presence of earthworm Hb, but only at the latter time points. Taken together, these results show that Hb‐based O₂ carriers can be utilized to improve O₂ availability and the migration of glioma spheroids on nanofibers. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1214–1220, 2014     

The effect of oxygen tension on rat hepatocytes in short-term culture

Summary Cell viability, cytochrome P-450 content, cell respiration, and lipid peroxidation were all investigated as a function of oxygen tension in adult rat hepatocytes in short-term culture (less than 9 h). The various oxygen tensions used in this study were obtained by equilibrating culture medium with air, air + nitrogen, or air + oxygen. Cell viability, as assessed by trypan blue exclusion, was significantly greater at all time points tested when hepatocytes were cultured in Ham's F12 medium containing 132 μM O₂, as compared to medium equilibrated with air (220 μM O₂) or air + oxygen (298 μM O₂). Cells cultured in 220 μM O₂ (air) also exhibited a gradual loss of cytochrome P-450, so that by 9 h of incubation less than 60% of the active material remained. This loss of P-450 was minimized when cells were cultured in 163 μM O₂ and abolished when cells were cultured in 132 μM O₂. The 132 μM O₂ exposure conditions also maintained cell respiration at the 1 h incubation values, whereas there was a continuous loss in cell respiration over time when the cells were cultured in either 220 μM O₂ (air) or 298 μM O₂ (air:O₂). These cytotoxicity findings may be related to oxidative cell damage inasmuch as it was additionally demonstrated that lipid peroxidation (as measured by malondieldehyde equivalents) was consistantly lower in hepatocytes cultured in air:N₂ as compared to air or air:O₂. These results suggest that hepatocyte culture in low oxygen tension improves not only cell viability but also maintains other functional characteristics of the cell. This work was supported by a Biomedical Research Support Grant S-S07-RR 05448 awarded to the University of Minnesota School of Public Health by the Biomedical Research Grant Program, Division of Research and Resources, National Institutes of Health, Bethesda, MD.     

Excretion of peroxidase from horseradish hairy root in combination with ion supplementation

Summary The effect of ion-supplemented medium on peroxidase excretion from horseradish (Armoracia rusticana) hairy roots was studied. Supplementation of mannitol instead of ions revealed that the excretion was stimulated not by osmotic pressure in the medium but by ionic properties. Extracellular peroxidase activity per dry cell was proportionally correlated with the ionic strength of the cations. CaCl₂ or MgCl₂ was found to be the most effective agent for excretion among other combinations. CaCl₂ supplementation at the beginning of the culture caused higher peroxidase production in the medium without a significant loss of final cell mass compared with CaCl₂ addition during the culture. Repeated batch culture with 50 mM CaCl₂ supplementation allowed a continuous retention of cell viability over 149 days and produced a great amount of extracellular peroxidase, 12-fold higher than that achieved in a 40-day-old batch culture with 50 mM CaCl₂ supplementation. Correspondence to: T. Kobayashi     

Formation,regeneration, and fusion of protoplasts in aCellulomonas strain

The effect of different conditions on protoplast formation was studied in the streptomycin-resistant strainCellulomonas sp.M32Bo. The greatest efficiency (75% protoplasts) was achieved by use of 0.5M sodium succinate as osmotic stabilizer, supplemented with 20 mM MgCl₂, 200 µg/ml of lysozyme, and 0.01M EDTA at pH 7.4. Cells harvested at the midexponential growth phase were more suitable for protoplast formation than those of the stationary phase. Electron microscopy observations showed the presence of both protoplasts and spheroplasts in the treated samples, some of them still showing a rod shape. Two regeneration media were developed that showed similar regeneration frequencies (52%). StrainM32Bo was fused with a tetracycline-resistant strain (Cellulomonas sp. Sz). Segregation analysis of fusant colonies suggested the existence of a temporary diploid stage in which both parental genotypes were expressed.     

Electrical properties and transmitter output associated with growth and transformation in shrimp muscle fibers

Summary Comparisons were made of the passive electrical properties of closer muscle fibers in the dimorphic claws of snapping shrimp,Alpheus armillatus. During claw transformation the small fibers of pincer claws grow to become much larger snapper claw fibers. As muscle fibers grow, the relationship of fiber input resistance (R ₀) to fiber diameter (d) is predicted by the proportionality,R ₀d ^–3/2. Muscle fiber membrane resistance,R _m, is independent of fiber diameter, but membrane capacitance,C _m, grows with diameter. This results in a 40 to 50 fold reduction in fiber input impedance as fiber diameter enlarges during transformation. Reductions of muscle fiber impedance are partially compensated by 2–5 fold increases in quantal content at excitatory synapses on snapper muscle fibers. However, changes in quantal content during transformation apparently are independent of fiber diameter per se. Excitatory junction potentials in both pincer and snapper muscle fibers have equal amplitude. Because fiber input impedance decreases precipitously during transformation, and in view of the relatively small compensatory changes in quantal content at excitatory synapses, additional pre- or post-synaptic modifications must supplement increased quantal content to maintain synaptic efficacy in transformed muscle fibers.Abbreviations ejp excitatory junctional potential - epp endplate potential - mepp miniature endplate potential     

Anchorage-dependent cell expansion in fiber-shaped microcarrier aggregates

This article describes a three-dimensional culture system for the expansion of anchorage-dependent cells using fiber-shaped microcarrier (MC; Cytodex3) aggregates, termed “MC fibers.” The fiber encapsulates the cells, the MC aggregates, and collagen and is covered with a poly-l -lysine membrane. The thin structure of the fiber enables sufficient supply of O₂ and nutrients to the cell. Using the MC fiber, we demonstrated the efficient expansion of C2C12 cells with high viability through serial passaging. Therefore, our culture system is useful for various applications where large-scale cell expansion is required, such as in pharmaceutical technologies, regenerative medicine, and cultured meat production. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2755, 2019.     

The Role of MIP in Lens Fiber Cell Membrane Transport

MIP has been hypothesized to be a gap junction protein, a membrane ion channel, a membrane water channel and a facilitator of glycerol transport and metabolism. These possible roles have been indirectly suggested by the localization of MIP in lens gap junctional plaques and the properties of MIP when reconstituted into artificial membranes or exogenously expressed in oocytes. We have examined lens fiber cells to see if these functions are present and whether they are affected by a mutation of MIP found in Cat ^Fr mouse lens. Of these five hypothesized functions, only one, the role of water channel, appears to be true of fiber cells in situ. Based on the rate of volume change of vesicles placed in a hypertonic solution, fiber cell membrane lipids have a low water permeability (p _H2O ) on the order of 1 μm/sec whereas normal fiber cell membrane p _H2O was 17 μm/sec frog, 32 μm/sec rabbit and 43 μm/sec mouse. Cat ^Fr mouse lens fiber cell p _H2O was reduced by 13 μm/sec for heterozygous and 30 μm/sec for homozygous mutants when compared to wild type. Lastly, when expressed in oocytes, the p _H2O conferred by MIP is not sensitive to Hg²⁺ whereas that of CHIP28 (AQP1) is blocked by Hg²⁺. The fiber cell membrane p _H2O was also not sensitive to Hg²⁺ whereas lens epithelial cell p _H2O (136 μm/sec in rabbit) was blocked by Hg²⁺. With regard to the other hypothesized roles, fiber cell membrane or lipid vesicles had a glycerol permeability on the order of 1 nm/sec, an order of magnitude less than that conferred by MIP when expressed in oocytes. Impedance studies were employed to determine gap junctional coupling and fiber cell membrane conductance in wild-type and heterozygous Cat ^Fr mouse lenses. There was no detectable difference in either coupling or conductance between the wild-type and the mutant lenses. Received: 17 February 1999/Revised: 16 April 1999     

Factors Influencing Protoplast Viability of Suspension-Cultured Rice Cells during Isolation Process

Callus cells of rice (Oryza sativa L.) that were actively dividing in suspension culture had lost the ability to divide during the isolation process of protoplasts. Factors influencing the protoplast viability were examined using highly purified preparations of cellulase C₁, xylanase, and pectin lyase, which were essential enzymes for the isolation of protoplasts from the rice cells. The treatment of the cells with xylanase and pectin lyase, both of which are macerating enzymes, caused cellular damage. Xylanase treatment was more detrimental to the cells. Osmotic stress, cell wall fragments solubilized by xylanase, and disassembly of cortical microtubules were not the primary factors which damaged the rice cells and protoplasts. The addition of AgNO₃, an inhibitor of ethylene action, to the protoplast isolation medium increased the number of colonies formed from the cultured protoplasts, although the yield of protoplasts was reduced by the addition. Superoxide radical (O₂-) was generated from the cells treated with xylanase or pectin lyase. The addition of superoxide dismutase and catalase to the protoplast isolation medium resulted in a marked improvement in protoplast viability especially when the non-additive control protoplasts formed colonies with a low frequency. The addition of glutathione peroxidase and phospholipase A₂, which have been known to reduce and detoxify lipid hydroperoxides in membranes, to the protoplast culture medium significantly increased the frequency of colony formation. These results suggested that some of the damage to rice protoplasts may be caused by oxygen toxicity.