Stabilization of the synthetic media for plant tissue and cell cultures

In standardMurashige-Skoog medium, particularly at pH higher than 5.0 and after heat sterilization, there is a tendency for turbidity or a sediment to appear, and for the acidity to increase by 0.2 to 0.5 degrees pH. The sediment is an amorphous precipitate of ferric phosphate and partly also of ferrous phosphate. In a stock iron solution prepared by chelation of ferrous sulphate with an equimolar quantity of the complexone Na₂EDTA. up to 10% free Fe^II ions could be detected. By titration of a concentrated complexon solution it was found that in the presence of an excess of Na₂EDTA (at the approximate molar ratio Fe^II: Na₂EDTA 1: 2) chelation of this free iron takes place to such an extent that its concentration falls to as little as 0.1%. Media with iron stabilized in this way are quite clear and maintain the adjusted pH for up to several weeks. The heat sterilization, too, does not lead to any precipitation or to a shift in pH within the broad range of adjusted values pH 4.8 – 6.0. We also attempted to increase the relatively low buffering capacity of Murashige-Skoog medium. The addition of sodium citrate (1.25 mmol 1^-1) and particularly of citrate-phosphate buffer (at a final concentration of 1.97 mmol citric acid and 6.07 mmol dibasic sodium phosphate per litre of medium) to the Murashige-Skoog medium considerably increased its buffering capacity, so that at the end of the subculture interval of tobacco cell suspensions the adjusted acidity changed only slightly (pH 5.40 ± 0.15). A thorough evaluation of the growth parameters of tobacco batch cultures (cell counts, vital staining, kinetics of DNA and protein synthesis) failed to reveal any negative effect either of additional chelation or of the buffering components.     

A rapid non-destructive fluorometric assay for gangliosides

An assay for gangliosides has been developed which is both rapid and non-destructive. The procedure is based on specific ganglioside serotonin binding and utilizes the inverse relationship between serotonin dialysis rates and ganglioside concentrations. The amount of serotonin in the diffusate after 30 minutes of dialysis is measured fluorometrically and converted, via a standard curve, to ganglioside content. Samples containing as little as 10 nanomoles of ganglioside have been assayed. A single assay is complete in 30 minutes and triplicate assays require less than an hour. Since no hydrolysis or other chemical reaction is involved, samples can be recovered intact by further dialysis and lyophilization.     

A fine-structural study of the freeze-preservation of plant tissue cultures

Summary Suspension culture cells of sycamore (Acer pseudoplatanus L.) and carrot (Daucus carota L.) were frozen to ultralow temperatures under rapid ( 100 °C s^–1) and slow, controlled (1 or 2 °C min^–1) rates, in the presence and absence of cryoprotective compounds. After storage at –196 °C, cells were recovered by thawing either slowly, in air at room temperature (ca. 20 °C min^–1) or rapidly, in a water bath at 40 °C (ca. 100 °C min^–1). The ultrastructure of the thawed cells was examined by thin-sectioning and compared with unfrozen controls and cells examined in the frozen state. Cells frozen rapidly, in the presence of cryoprotectants, or frozen slowly in their absence, suffered serious ultrastructural damage and a total loss of viability. Carrot cells frozen at a rate of 2 °C min^–1 in the presence of cryoprotectants and thawed at either rate, yielded up to 70% of viable cells. The recovered aggregates of carrot cells comprised some centrally located, seriously damaged cells and, at the periphery, groups of cells with a high electron opacity neighbouring well preserved cells, showing little ultrastructural modification compared with unfrozen controls. The highest rate of survival of sycamore cells (ca. 30%) was observed when they were frozen at a rate of 1 °C min^–1 and thawed rapidly. In all recoverd cells of sycamore some ultrastructural modifications were evident. These included: dilation of mitochondria, plastids, golgi and ER cisternae and the nuclear envelope, decrease in polysomes, increase in nuclear and cytoplasmic microfilaments and changes in nuclear and nucleolar granularity. The probable causes and timing of the ultrastructural changes and their effects on the potential for regrowth of the recovered cells are discussed.     

Alginate-based solid media for plant tissue culture

Summary A new method for solid medium plant tissue culture based on in situ gelation of alginate is proposed as an alternative to agar-based media. In situ gelation by the use of dispersed CaCO₃ and the slowly hydrolysing acid glucono--lactone (GDL) was the basis for the use of alginate as a gelling agent. Inexpensive alginate-based media can be made in a wide range of pH values. Biological tests of these gels, concerning sterile seed growth and microcalli plating of Brassica napus (cv. Topas) and biomass production of Panax ginseng callus, showed results equal to those achieved with agar-based gels.     

A method for estimating oxygen availability of stationary plant cell cultures in liquid media

A method for estimating the oxygen availability in plant cell cultures grown in stationary liquid media (e.g. many protoplast cultures) was developed. The method is based on short-term measurements of respiration rate versus oxygen concentration on a sample of cells, suspended in liquid media. From such data it is possible to estimate the oxygen concentration at the bottom of a stagnant liquid culture, by calculating the amount of oxygen reaching the cells by diffusion. As an example, rape (Brassica napus L. cv. Omega) hypocotyl protoplasts were grown with different oxygen concentrations at the site of the cells, obtained by varying the cell density, the height of the liquid layer and the oxygen content of the gas phase. The number of surviving calli was positively correlated with the estimated oxygen availability in the range between 60 and 350 M O₂, below 60 M all cells died. This indicates that oxygen availability can be a limiting factor in the range usually encountered in protoplast cultures, and that the method can be useful when designing optimal growth conditions for stationary cultures of plant cells.Abbreviations C₁ bulk oxygen concentration in agitated medium - C_o oxygen concentration in medium at the gas-liquid interface, in equilibrium with the gas - C_x oxygen concentration at cell level - D diffusion constant of oxygen in water - K_La oxygen transfer rate - l height of liquid above cells - n number of cells per ml - R_x respiration rate per cell     

Volatile emissions of plant tissue cultures

Summary The low-molecular-weight volatiles released by a variety of plant tissue cultures were examined by gas chromatography. Callus cultures invariably produced carbon dioxide, ethylene, acetaldehyde and ethanol. In cultures with developed shoots, ethanol was absent and acetaldehyde was detected only rarely.     

Foreign protein production in plant tissue cultures

Foreign proteins synthesised by plants are now in the marketplace, and clinical trials for plant-derived therapeutic proteins are underway. Economic analysis of plant production systems has helped identify the types of protein that would be most suitable for manufacture using tissue culture methods. The major advantages associated with in vitro plant systems include the ability to manipulate environmental conditions for better control over protein levels and quality, the rapidity of production compared with agriculture, and the use of simpler and cheaper downstream processing schemes for product recovery from the culture medium.     

Secondary product formation in plant tissue cultures

The formation of secondary products in plant tissue culturesis reviewed. The conditions for the enhanced production of secondaryproducts, which include alkaloids, terpenoids, steroids andphenolics, can be regulated in a number of ways. For example,manipulation of secondary product formation is possible by varyingthe nutrient composition of the growth medium, light, temperatureand pH, and by the use of elicitors, permeabilisation and two-stagesystems. Molecular engineering and the use of biomass and large-scaleculture are described along with future prospects for the commercialproduction of secondary products from cell suspension cultures.     

An improved MUG fluorescent assay for the determination of GUS activity within transgenic tissue of woody plants

Despite the success of the MUG fluorometric assay for quantitative analysis of beta-glucuronidase (GUS) activity within a vast array of transgenic plant species and tissues, attempts to apply this protocol for analysis of woody plants has been found to be problematic, primarily due to the interfering effects of phenolics and other secondary metabolites. Our analysis of transgenic spruce needles and poplar leaves illustrates that low tissue mass to extract volume, along with the inclusion of polyvinylpolypyrolidone and either beta-mercaptoethanol or metabisulphite, are essential for producing reliable results. The primary action of these additives was found to involve increased GUS extractability and the preservation of GUS activity during extract manipulation, but they were not completely effective in eliminating GUS enzymatic inhibitors. Normalization of GUS activity upon DNA concentration was also found to be an effective alternative to protein concentration, providing the ability to make cross-species and inter-tissue comparisons of gusA transgene activity.     

Microplate assay for detection of bacterial contamination in tissue culture media

In the present study, a rapid and simple colorimetric technique has been described to determine the presence of bacteria in tissue culture medium used in animal cell culture. The microplate assay is based on utilization of MTT [3(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide] by bacteria resulting in formation of formazan crystals which can be measured colorimetrically. Contaminated medium, a standard gram-negative and gram-positive bacteria produce formazan from MTT which is related to the bacterial load. The assay has utility in screening tissue culture reagents to detect the presence of bacteria.     

Bacterial growth in plant tissue culture media

C. LEIFERT AND W.M. WAITES. 1992. When Murashige and Skoog's liquid plant medium was inoculated with 10 different bacterial species in the absence of plants only Bacillus subtilis showed significant growth. The numbers of Lactobacillus plantarum, Pseudomonas maltophilia, Erwinia carotovora and Staphylococcus saprophyticus decreased rapidly and were not detected at 28 d.
Bacillus subtilis, Lact. plantarum, Ps. maltophilia, Erw. carotovora and Staph. saprophyticus grew and persisted in the same medium in the presence of Delphinium plants, while only Lact. plantarum and Erw. carotovora grew and persisted in the presence of Hemerocallis plants.
Hemerocallis plants lowered the pH of media from 5.6 to about 3.9 while Delphinium plants increased it to about 5.9 within 7 d after subculturing on fresh media. The pH drop in Hemerocallis media is thought to prevent the growth and persistence of bacteria such as B. subtilis, Staph. saprophyticus and Ps. maltophilia , which were found to be more sensitive to low pH than Lact. plantarum and Erw. carotovora. Bacterial growth in the medium altered the pH, reduced the plant growth and/or resulted in plant death.     

植物组织培养生产药物研究进展

植物是药物的天然宝库。本文综述了利用组织培养方法合成药物的优点和主要药物,探讨了植物组织生产药物的研究进展和前景。     

Lignosulfonates: Novel promoting additives for plant tissue cultures

Summary Lignosulfonates (LIGNs) are low-cost by-products from the paper industry and are already commercialized as fertilizers. Because earlier laboratory and glasshouse assays had shown a beneficial effect of LIGNs on rooting and general plant vigor, their incorporation in several plant tissue culture types was examined here. The present assays indicated that well-chosen concentrations of LIGNs, whether they were chelated with Ca or Fe, stimulated growth of a normal and an habituated sugarbeet callus, improved multiplication rate and vigor of a shoot-proliferating poplar cluster, and increased the rooting percentage of holly, ginseng, and poplar shoots. Complementing the exogenous rooting auxin with LIGNs enhanced the increases of endogenous levels of indoleacetic acid and its aspartate conjugate in the basal parts of poplar shoots at the rooting inductive phase. Although LIGNs exerted some effects in the absence of the growth regulators, they could not replace them. Their possible mode of action is discussed.     

Effect of silymarin on plant tissue cultures

The effect of silymarin complex on various types of expiants differing in their nutrition requirements was investigated. The growth of tumorous periwinkle (Catharanthus roseus [L.] G. Don) callus tissue was still identical with the control tissue at the silymarin concentration of 35 mg in 1000 ml of the nutrient medium. However, this silymarin concentration totally inhibited the growth of habituated periwinkle callus tissue; in the presence of 10 mg of silymarin, the growth of this tissue was similar to that of the corresponding tissue grown without silymarin. The growth of tobacco callus tissue (D-strain) requiring for its growth kinetin was reduced by 46.2% at the concentration 10 mg of silymarin in 1000 ml of nutrient medium, but its dry weight was increased by 21% in comparison with the control. Silymarin was most effective on the growth of callus derived from tobacco (Nicotiana glauca Grah:) stem pieces; callogenesis was observed in control tissue in 89.5% cases while in the presence of silymarin (10 mg) only in 48.6%. The primary callus growth was strongly inhibited, too (by 89.9%). The organogenesis onset was never observed on tobacco stem pieces cultured on a nutrient medium with kinetin and IAA in the presence of silymarin. When all types of expiants were transferred from the medium with silymarin on control medium, normal growth appeared very soon and the differences between the experimental and control expiants were smoothed out during two months. These results indicate that the observed changes might be due to the blocking of membrane system permeability leading to an insufficient supply of cells with nutrients and growth substances.     

Chemical sterilization of nutrient media for plant cell cultures using diethylpyrocarbonate

Summary Diethylpyrocarbonate (DPC), when added at about 1g/l to culture media is able to sterilize them. DPC kills all the contaminating microorganisms, and in contact with water it decomposes to ethanol and carbon dioxide in amounts not inhibiting the growth of plant cells. The plant cells cultivated on media treated by DPC did not show changes in their basic characteristics.