Short-term radiorespirometry of cell suspensions

1. An apparatus and method are described with which the oxidation of labelled substrates to (14)CO(2) by cell suspensions may be examined. 2. The use of high-specific-radioactivity substrates at low concentration together with frequent quantitative collection of CO(2) permit a more detailed analysis of the appearance of various substrate carbon atoms as CO(2) than is possible by existing techniques. 3. Typical experiments with various cell types are reported, in which pathways of glucose oxidation are examined.     

Electropermeabilization of dense cell suspensions

This paper investigates the influence of cell density on cell membrane electropermeabilization. The experiments were performed on dense cell suspensions (up to 400 × 10⁶ cells/ml), which represent a simple model for studying electropermeabilization of tissues. Permeabilization was assayed with a fluorescence test using Propidium iodide to obtain the mean number of permeabilized cells (i.e. fluorescence positive) and the mean fluorescence per cell (amount of loaded dye). In our study, as the cell density increased from 10 × 10⁶ to 400 × 10⁶ cells/ml, the fraction of permeabilized cells decreased by approximately 50%. We attributed this to the changes in the local electric field, which led to a decrease in the amplitude of the induced transmembrane voltage. To obtain the same fraction of cell permeabilization in suspensions with 10 × 10⁶ and 400 × 10⁶ cells/ml, the latter suspension had to be permeabilized with higher pulse amplitude, which is in qualitative agreement with numerical computations. The electroloading of the cells also decreased with cell density. The decrease was considerably larger than expected from the differences in the permeabilized cell fractions alone. The additional decrease in fluorescence was mainly due to cell swelling after permeabilization, which reduced extracellular dye availability to the permeabilized membrane and hindered the dye diffusion into the cells. We also observed that resealing of cells appeared to be slower in dense suspensions, which can be attributed to cell swelling resulting from electropermeabilization.     

Absorption and efflux of glyphosate by cell suspensions

The absorption and efflux of [¹⁴C]-glyphosate (N-[phosphonomethyl]glycine) was studied in maize (Zea mays L. cv. Aussie) and soybean (Glycine max L. Merr. cv. Maple Arrow) cell suspensions. Glyphosate absorption was complex: at low external herbicide concentrations (3-250 M) there was evidence for a single active uptake system with an apparent Km of 31 M and Vmax of 11 nmol g^-1 fr. wt. 2 h^-1. The system was inhibited by carbonylcyanide m-chlorophenyl hydrazone (CCCP), orthovanadate, diethylstilbestrol (DES), phosphate, and phosphonoformic acid (PFA) suggesting the glyphosate carrier to be a phosphate transporter energized by the plant plasmalemma ATPase. At higher external glyphosate concentrations the operation of this carrier was masked as passive diffusion became the dominant absorption mechanism. Any non-specific binding of glyphosate to the cell surface during absorption was low (0.02-0.02 nmol g^-1 fr. wt). Efflux kinetics of [¹⁴C]-glyphosate suggests the herbicide to be located in the cells in three kinetically distinct compartments: after 24 h uptake of radiolabelled herbicide, 71% of absorbed glyphosate was found in the slow compartment (t1/2 162 h), 19% in the medium (t1/2 185 min) and 10% in the fast (t1/2 27 min). The implications of these results in relation to the delivery of glyphosate to its subcellular target site and subsequent phytotoxicity are discussed.Keywords: Zea mays, Glycine max, glyphosate (N-[phosphonomethyl]glycine), absorption, compartmentation.      

Plant regeneration from cell suspensions of spinach

Friable calluses induced from root segments of spinach (Spinacia oleracea L.) with a high amount of growth regulators (indole-3-acetic acid 48.52 μM and gibberellic acid 10 μM) were suspended in liquid medium. The cell fraction sized between 100 and 200 μm was used to establish suspension cultures. Adventitious shoots and roots were obtained from the suspensions (3.2 x 10⁵ cells per ml) by procedures comprising successive subcultures on two or three different media. In both of these procedures, the composition of the second culture medium (concentrations of plant growth regulators) had a key influence on the organogenesis of the suspensions. Regenerated shoots elongated and rooted on different solid media. Plantlets transplanted in soil grew and developed normally until flowering and produced seeds. This revised version was published online in June 2006 with corrections to the Cover Date.     

Modulation of auxin-binding proteins in cell suspensions

Summary Cultured cell lines from carrot (Daucus carota L.) with little or no embryogenic potential were examined for the auxin-binding capacity of their membranes. The lines belonged to different classes: (a) wild-type lines kept in culture for different periods (the longer the period, the lower being their embryogenic potential); (b) variants, isolated after mutagenesis, showing normal growth but a lack of embryogenic response; (c) auxin-resistant lines, isolated as colonies on solid media containing 45 M 2,4-d; (d) a previously described tumorous line (E9) isolated because of its resistance to hypomethylating drugs. All of these lines showed alterations in auxin-induced, auxin-binding capacity (modulation), i.e. in the non-embryogenic lines the addition of auxin increased the auxinbinding capacity to a very small degree, or removal of the hormone did not produce the proper decrease in that capacity, or both defects could be simultaneously present. Both types of defects were shown to be correctable: after treatments designed to increase the amplitude of modulation, embryogenic capacity was restored in a number of lines.     

Establishment of oil palm cell suspensions and plant regeneration

Primary globular callus from immature zygotic embryos and friable embryogenic tissue derived from mature zygotic embryos were used to establish suspension cultures. Callus cultures were established either on modified Y3 or MS medium containing 475–500 M 2,4-D or 250 M picloram and 0.3% (w/v) activated charcoal. Suspension cultures of both cell lines were established in modified Y3 medium containing 10 M 2,4-D. The establishment of cell suspensions from friable embryogenic tissue took only 2 months, in contrast with suspensions from primary globular callus which took 3–5 months to establish. Embryo differentiation was observed only in cell suspensions derived from the friable embryogenic tissue after plating aliquots on regeneration medium. Germinated embryos were recovered and plantlets were successfully established under greenhouse conditions.Abbreviations CET compact embryogenic tissue - FET friable embryogenic tissue - CIM callus induction medium - PGC primary globular callus - 2,3-D 2,4-dichlorphenoxyacetic acid Y3-Eeuwens' medium - MS Murashige & Skoog medium - PVP-40 polyvinylpyrrolidone - KM Kao & Michayluk vitamins - ABA abscisic acid     

Abnormal growth of habituated sugarbeet callus and cell suspensions

Summary Habituated sugarbeet callus and cell suspension cultures derived therefrom, compared to hormone-dependent normal cultures, exhibit a shorter linear growth phase, although they divide actively. Microscopic observations indicate deficiencies in cell expansion and absence of cell differentiation. Cell expansion apparently is interrupted by a cell “budding” process. Some of the cells seem to be empty due to ballooning out of the protoplasm and the bursting of the cell membrane by defective cell wall development. A low amount of cellulose was confirmed by microspectrophotometric estimation. Such cultures exhibit all the characteristics of vitrified tissue.     

Fluorimetric quantification of cell death in monolayer cultures and cell suspensions.

A fluorimetric assay using ethidium bromide (EB) was employed to quantify cell death in monolayer cell cultures (MA-104 cells) in situ and isolated cell suspensions (isolated colonic cells and Leishmania). Fluorescence of EB stained cells was measured with a photometer coupled to an inverted microscope for cell monolayers or in a spectrofluorometer for cell suspensions. Dead cells stained with trypan blue were fluorescent with EB in all preparations studied, but the latter gave an unequivocal signal. Staining with EB and fluorescein diacetate was mutually exclusive. The relationship between the number of EB fluorescent cells and the intensity of fluorescence measured in the microphotometer was linear for a large range of cell numbers (1-14000) from different types of preparations. Applicability of the method for measuring living and dead cells in two different time scales (minutes and hours) is shown using MA-104 cell monolayers infected with rotavirus and Leishmania suspensions treated with amphotericin B. The method is fast, simple, sensitive and reliable, enabling quantification of living and dead cells in monolayers and suspensions.