Auxin requirements of sycamore cells in suspension culture

Sycamore (Acer pseudoplatanus L.) cell suspension cultures (strain OS) require 2,4-dichlorophenoxyacetic acid (2,4-D) in their culture medium for normal growth. If the 2,4-D is omitted, rates of cell division are dramatically reduced and cell lysis may occur. Despite this `auxin requirement,' it has been shown by gas chromatography-mass spectrometry that the cells synthesize indol-3yl-acetic acid (IAA). Changes in free 2,4-D and IAA in the cells during a culture passage have been monitored.

There is a rapid uptake of 2,4-D by the cells during the lag phase leading to a maximum concentration per cell (125 nanograms per 10⁶ cells) on day 2 followed by a decline to 45 nanograms per 10⁶ cells by day 9 (middle of linear phase). The initial concentration of IAA (0.08 nanograms per 10⁶ cells) rises slowly to a peak of 1.4 nanograms per 10⁶ cells by day 9 then decreases rapidly to 0.2 nanograms per 10⁶ cells by day 15 (early declining phase) and 0.08 nanograms per 10⁶ cells by day 23 (early stationary phase).

     

Effects of Pluronic F-68 on plant cells in suspension culture

Summary The effects of the non-ionic surfactant, Pluronic F-68, on growth and structure ofSolanum dulcamara cells in suspension culture have been studied. Growth of cells, as measured by dry weight, was unaffected by low concentrations (0.01–1.0% w/v) of pluronic, while culture with higher concentrations (2.5–10.0%) resulted in cell death. It is suggested that low concentrations of pluronic may be valuable supplements in plant cell cultures to protect against mechanical damage and to manipulate membrane systems.     

Regulation of cytoplasmic and vacuolar volumes by plant cells in suspension culture

Quantitative microscopical measurements have been made of the proportion of cell volume occupied by cytoplasm in a cell suspension culture derived from cotyledons of bush bean (cv. Contender). On a 7-day culture cycle, the content of cytoplasm varies from 25% at the time of transfer to 45% at the start of the phase of rapid cell division. If the culture is continued beyond 7 days, the vacuole volume reaches 90% of cell volume by day 12.     

Slurry-producing agents provide buoyancy and allow growth of plant suspension cells in stationary culture

Summary Suspension cultures of carrot, soybean, tobacco, Black Mexican sweet corn, and photoautotrophic soybean have been successfully grown in stationary slurry culture. Cell colonies grew loosely and slowly in clumps which were easily dispersable, increasing in size only 4–5 fold by 48 days, which facilitated subculture.     

Effect of auxins on the formation of ubiquinone by tobacco plant cells in suspension culture

Ubiquinone (UQ) formation in BY-2 tobacco cells was especially promoted by a high concentration of 2,4-D. 2,4,5-T, MCP and NAA also promoted UQ formation in these cells. The UQ content in the cells cultured at high concentrations of 2,4-D was higher than that of controls throughout the culture period. The addition of 2,4-D at an early period in cell growth was very effective in promoting UQ formation, but addition at the stationary phase was ineffective. Cell growth was improved by adding phosphate to the medium but UQ content was decreased. UQ content decreased slowly during subculturing, whereas cell growth recovered gradually.     

Formation of a series of myo-inositol phosphates during growth of rice plant cells in suspension culture

A series of myo-inositol phosphates including myo-inositol mono-to hexa-phosphates was observed during growth of cultured riceplant cells. We also found that ³²Pi and myo-[2-³H] inositolwere incorporated into all these myo-inositol phosphates. myo-Inositolphosphorylating activity, which depended on ATP and Mg²⁺, wasdetected in the soluble fraction from the cells, and the reactionproduct was identified as myo-inositol-2-phosphate. (Received January 21, 1980; )     

The effects of amino acids and ammonium on the growth of plant cells in suspension culture

A suspension culture of soybean (Glycine max L.) was grown on a defined medium in which the nitrogen sources were nitrate (25 mM) and ammonium (2 mM). The cells did not grow on nitrate unless the medium was supplemented with ammonium or glutamine. The l- and d-isomers of 12 amino acids tested singly could not replace ammonium. Most amino acids (4 mM) inhibited growth when the cells were cultured on nitrate and ammonium. Cells from five other plants (Reseda luteoli L.; Triticum monococcum L.; flax, Linum usitatissimum L.; horseradish, Amoracia lapathifolia Gilib; Haplopappus gracilis L.) grew on the defined medium with nitrate (25 mM) as the sole nitrogen source. Higher cell yields were obtained when ammonium (2 mM) or glutamine also was present. Supplementing the defined medium with high concentrations of ammonium (20 mM) inhibited growth of soybean, Haplopappus, and wheat cells. Addition of citrate (5 mM) relieved the inhibitory effects of ammonium in soybean and wheat cells but not in the Haplopappus cells.     

Induced flocculation of animal cells in suspension culture

Recycle unit operations for suspension cell cultures may be improved by flocculation of the cells. A flocculant search was conducted, and it was found that where strongly cationic polymers were severly toxic to the cells, neutral and anionic polymers were nontoxic and ineffective at flocculating the cells. A weak and poorly soluble polycation, poly-L-histidine, was capable of flocculating cultures of CHO, HeLa, U-937, and CRL 1606 hybridoma cells with no toxicity to the majority of cells. In addition to the lowered acute toxicity, the polymer treatment left the cells in a growing state for recycle. Flocculation was found to be mediated by precipitates of the polymer. The low toxicity of poly-L-histidine is probably due to its low solubility and charge at physiological pH. Nonelectrostatic interactions may also play a role.     

Differentiation of Dictyostelium discoideum cells in suspension culture

Differentiation of Dictyostelium discoideum cells in suspension culture is reported, using a medium containing glucose, albumin, cyclic AMP, EDTA and streptomycin in a phosphate buffer. Production of UDPgalactose:polysaccharide transferase, an enzyme specifically present in prespore cells, and the formation of prespore-specific antigens in more than 60% of the cells, are demonstrated. Differentiation in this medium differs from that previously reported with other suspension systems in that (a) cells form only small, amorphous agglomerates, (b) there is an absolute requirement for cyclic AMP and (c) prior formation of loose cell mounds on a solid substratum is essential for subsequent differentiation in this medium. This last requirement indicates that the differentiation process, giving rise to the prespore-specific enzyme and antigen, can be resolved into two distinct stages, one requiring cell contact on a solid substratum and the other proceeding in small agglomerates incubated in the medium. This medium may be useful for elucidating the role of cyclic AMP and cell contact in slime mould development.     

Handling culture medium composition for optimizing plant cell suspension culture in shake flasks

Douglas-fir is a conifer species of major economic importance worldwide, including Western Europe and New Zealand. Herein we describe some characterization and significant refinement of somatic embryogenesis in Douglas-fir, with focus on maturation. The most typical structures observed in the embryonal masses were large polyembryogenic centres (up to 800–1500 µm) with a broad meristem, creating a compact cell “package” with suspensor cells. Singulated somatic embryos composed of both a embryonal head (300–400 µm) and long, tightly arranged suspensor were also frequent. Embryo development was enhanced following embryonal mass dispersion on filter paper discs at low density (50–100 mg fresh mass). Moreover, increasing gellan gum concentration in maturation medium (up to 10 g L^?1) improved both the quantity and quality of cotyledonary somatic embryos (SEs), which were subsequently able to germinate and develop into plantlets at high frequency. Embryogenic yield was highly variable among the seven embryogenic lines tested (27–1544 SE g^?1 fresh mass). Interestingly secondary somatic embryogenesis could be induced from cotyledonary SEs of both low- and highly-productive lines with some useful practical outcomes: secondary lines from low-performance lines (30–478 SE g^?1 fresh mass) displayed significantly higher embryogenic yield (148–1343 SE g^?1 fresh mass). In our best conditions, the total protein content in cotyledonary SEs increased significantly with maturation duration (up to 150 µg mg^?1 fresh mass after 7 weeks) but remained below that of mature zygotic embryos (300 µg mg^?1). The protein pattern was similar in both somatic and zygotic embryos, with major storage proteins identified as 7S-vicilin- and legumin-like proteins.     

The plant activation of m-phenylenediamine by Tradescantia clone 03 and clone 4430 cells in liquid suspension culture

In this study, we expanded the use of the genus Tradescantia to investigate the plant activation of promutagens and further refine the methodology of the plant cell/microbe coincubation assay. Liquid suspension cell cultures of Tradescantia clone 03 and Tradescantia clone 4430 were used to activate the promutagen m-phenylenediamine into a mutagenic compound which was detected by Salmonella typhimurium strain TA98 in the plant cell/microbe coincubation assay. Optimum treatment parameters were established for both plant cell lines. Optimum was defined as the lowest concentration or shortest time period that provided consistently positive results and high rates of revertants. Preliminary experiments with both cell lines defined 2.5 mumoles m-phenylenediamine per plate as the optimum concentration to be used in the determination of the optimal coincubation period and the optimal concentration of plant cells. These experiments also determined the optimal physiological stage at which both clones should be used in the coincubation assay. Differences were found in the optimal of coincubation (1h for clone 03, 2 h for clone 4430) and growth stage (mid-log for clone 03, mid- to late-log for clone 4430). Similar activation responses were seen for both clones when the concentration of plant cells (mg/ml) was varied. Under optimized conditions, clone 03 cells demonstrated an approximately 10% higher activation response than clone 4430.     

Growth and division of carrot cells in suspension culture

In a submerged culture of a strain of carrot cells, cellularmorphology and the mode of cell division were greatly affectedby growth factor(s) added to the medium. In the presence of2,4-D, cells showed two-dimensional growth and often formedtetrad-like structure after a set of two divisions. The sequenceof events was observed microscopically. Orientation of cellgrowth changed after the first division and the second cellplate formed at an oblique angle to the first. When IAA wasadded, instead of 2,4-D, cells showed one-dimensional growthand developed to a filamentous form. (Received June 1, 1970; )     

Carotenoid synthesis in a suspension culture of carrot cells

Biosynthesis of carotenoid in cultured carrot cells was studiedin relation to cell growth and acetate metabolism. Of the twostrains tested, one (GD-1) predominantly produces ß-caroteneand the other (GD-2) lycopene. In both strains, carotenoid wasproduced in parallel with cell growth. Incorporations of acetate-¹⁴Cinto carotenoids, organic acids and amino acids were acceleratedby increasing the concentration of 2,4-D in the medium. (Received November 17, 1970; )     

Pectic polysaccharides in carrot cells growing in suspension culture

Pectic polysaccharides in the cell wall of suspension-cultured carrot cells (Daucus carota L.) were fractionated into high- and low-molecular-weight components by molecular-sieve chromatography with a Sepharose 4B column. During the phase of cell-wall expansion, the relative content of low-molecular-weight polymers rapidly increased. Electrophoretic analyses of these fractions showed that the high-molecular-weight components were largely composed of neutral and weakly acidic polymers while the low-molecular-weight fraction contained, in addition to neutral polymers, strongly acidic polyuronides in which the content of neutral sugars was very small. The accumulation of a large amount of the strongly acidic polyuronides occurred in a late stage of cell-wall growth, concomitant with a marked decrease in the high-molecular-weight components.Abbreviation MW molecular weight     

Lipids,lipid turnover,and phospholipase D in plant suspension culture cells (Daucus carota)

The lipid pattern of Daucus carota L. suspension culture cells and other plant cell strains was analyzed. Phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and triacylglycerol were the main components. The characteristic plastidal and mitochondrial lipids could also be identified. All strains tested exhibited a phospholipase D activity. Several lipid precursors were found to be well utilized by the cells and to be special markers for certain lipids or parts of the lipid molecules. The half life times of the major lipids ranged at about half a generation time of the cells.