条件培养液对红豆杉细胞Paclitaxel生产的促进作用

在两步法红豆杉（Taxus chinensis)细胞悬浮培养体系的生产阶段,加入从生长阶段悬浮培养物中制得的条件培养液（conditioned Medium,CM)既能促进细胞的生长,又能提高紫杉醇（paclitaxel)的产率,解决了生产培养时,细胞生长受抑制的问题,特别是,取自生长12天的细胞悬浮培养物的CM按体积分数为25％添加到新鲜生产培养基中时,可使细胞紫杉醇最高产量达28．5mg/L,细胞干重达32．3g/L,分别是对照的2．4倍和2．2倍,对CM中的蔗糖,果糖,NO3－和PO4－3等的含量的进行了分析。     

Stimulation of Cultured Iridophores by Amphibian Ventral Conditioned Medium

That the ventral integument of adult frogs (Rana pipiens) contains factor(s) that stimulate iridophore expression (adhesion, morphologic appearance, proliferation) was demonstrated on iridophores derived from tadpoles of R. pipiens and Pachymedusa dacnicolor, and maintained in primary culture in a growth medium based upon Leibovitz's L-15. Experimental growth medium (VCM) conditioned by a one-hour exposure to pieces of ventral skin of adult R. pipiens induced iridophores to assume a broad and stellate appearance, to form confluent sheets, and to proliferate over a nine-day period. Iridophores in control medium assumed long thin profiles, detached easily, and exhibited no signs of proliferation. Unknown cells containing reflecting platelets and unusual other organelles appeared uniquely in chromatophore cultures of P. dacnicolor in VCM. The intense stimulation of iridophore expression in VCM is consistent with the known inhibitory effect of this medium on melanization and with its purported role in the determination of dorsal/ventral pigment patterns of amphibians. The results are discussed in terms of a prevailing theory about pigment cell origins and development.     

Growth Behaviour of Suspension Cultures of Rice and Transient Expression of Electroporated Gene in Intact Cells

Suspension cultures of Indics (Basmati 370 and Improved Sabarmati) and Japonica (Taipei 309) rice were established from scutellum-derived callus obtained by culture of the mature seeds. The suspension cultures contain groups of small and cytoplasmically rich cells with a Qubling time of 3.6–5.1 days, as measured by Increase In packed cell volume. The suspension cells of Indica rice var Improved Sabarmati were employed to optimize electroporation-mediated delivery of the plasmid pBl 221 having β-glucuronidase gene under the control of eukaryotic expression signals. GUS activity was determined both fluorometrically and hisotchemlcally. Maximum expression was obtained when cells were electroporated at 600 V cm^-1 with capacitance selected at 400 μF.     

Oxidative Stimulation of Glutathione Synthesis in Arabidopsis thaliana Suspension Cultures

A system based on Arabidopsis thaliana suspension cultures was established for the analysis of glutathione (GSH) synthesis in the presence of hydrogen peroxide. Mild oxidative stress was induced by use of the catalase inhibitor, aminotriazole, and its development was monitored by measurement of the oxidative inactivation of aconitase. Addition of 2 mM aminotriazole resulted in a 25% decrease in activity of aconitase over 4 h. During the subsequent 10 h, no further decrease in aconitase activity was measured despite a sustained inhibition of catalase. In combination with our failure to detect significant increases in the level of lipid peroxidation, another marker indicative of oxidative injury, these data suggest that although hydrogen peroxide initially leaked into the cytosol, its accumulation was limited by a cytosolic catalase-independent mechanism. A 4-fold increase in the level of GSH, which was almost exclusively in the reduced form, was observed under the same treatment. To determine to what extent this increase in reduced GSH played a role in limiting the accumulation of hydrogen peroxide in the cytosol, we inhibited GSH synthesis with buthionine sulfoximine (BSO), a specific inhibitor of [gamma]-glutamylcysteine synthetase. No significant oxidative injury was detected as a result of treatment with 50 [mu]M BSO alone, and furthermore, this treatment had no effect on cell viability, However, addition of 2 mM aminotriazole to cells preincubated with 50 [mu]M BSO for 15 h led to a rapid loss of aconitase activity (75% in 4 h), and significant accumulation of products of lipid peroxidation. Within 72 h, cell viability was lost completely. After removal of BSO from the growth medium, GSH levels recovered to normal over a period of 20 h. Addition of 2 mM aminotriazole to cells at different time points during this recovery period demonstrated a strong correlation between the level of reduced GSH and the degree of protection against oxidative injury. These data strongly suggest that the induction of GSH synthesis by an oxidative stimulus plays a crucial role in determining the susceptibility of cells to oxidative stress.     

Photoautotrophic Growth and Photosynthesis in Cell Suspension Cultures of Chenopodium rubrum

A method is described for growing cell suspension cultures of Chenopodium rubrum photoautotrophically for prolonged periods of time. By using a two-tier culture vessel the growth medium with the cells was separated from the CO₂ reservoir. Definite CO₂ concentrations were established by a K₂CO₃/KHCO₃ buffer. Photoautotrophic growth in C. rubrum cell suspension cultures was correlated with the CO₂ level. At 0.5% CO₂ the cell cultures contained 68 μg chlorophyll/g fresh weight and showed an increase in fresh weight of about 80% in 18 days. At 1% CO₂ an increase in fresh weight of 165% in 18 days was observed. The chlorophyll content rose up to 84 μg/g fresh weight. The photoautotrophic growth was also greatly influenced by the 2,4-D content of the medium. Cell growth was enhanced by lowering the auxin concentration. Best growth was attained (210% increase in fresh weight) at 10^?8M 2,4-D. The photosynthetic activity of the cells was measured by the light dependent ¹⁴CO₂ incorporation. At 0.5% CO₂ the cell suspensions assimilated about 100 μmol CO₂/mg chlorophyll × h. In the presence of 1% CO₂ the light driven assimilation was raised up to 185 μmol CO₂/mg chlorophyll × h. In both cases, the dark incorporation of CO₂ was merely 1.8% of the values obtained in light.     

Growth and Plating of Cell Suspension Cultures of Datura innoxia

Suspension cultures of Datura innoxia Mill, were successfully grown on a modified Murashige and Skoog medium with 2,4–D, NAA or BAP as growth substances, provided the micronutrient levels were reduced to 1/10. Normal amounts of micronutrients were toxic. Attempts to identify the toxic elements did not succeed. Cultures grew exponentially on a shaker at 27°C in the light. Their doubling times varied from 1.1 days on 2,4–D (10^–6M) or NAA (10^?5M)+ 1 g/1 casein hydrolysate to 2.7 days on BAP (3 × 10^?7M) and 5.1 days on supraoptimal levels of 2,4-D (10^?5M). Cultures grew on NH₄⁺-N alone (from ammonium malate) or on NO₃^?-N alone. Dry weight yield was proportional to the amount of nitrate-N added (47 mg/mg N). Filtered suspension cultures containing single cells (plating cultures) could be grown in agar in petri dishes when NAA or 2,4-D were used as growth substances. Cells grew at densities above 500 units/ml in the agar. Most colonies grew from cell aggregates but division in single cells was observed. The highest plating efficiency was about 50% on 10^?6 M 2,4-D + 1 g/1 casein hydrolysate.     

Stimulation of Sanguinarine Production by Combined Fungal Elicitation and Hormonal Deprivation in Cell Suspension Cultures of Papaver bracteatum

Fungal elicitor preparations from either homogenized mycelia of Dendryphion penicillatum (Cda.) Fr., a specific pathogen of Papaver species, or conidia of Verticillium dahliae Kleb., a general pathogen, were added to 14-day-old suspension cultures of Papaver bracteatum. Plant tissue cultures were grown either in the presence or absence of 0.1 milligram of 2,4-dichlorophenoxyacetic acid per liter and 0.5 milligram of 6-benzylam-inopurine per liter. Dendryphion extracts elicited an accumulation of the benzophenanthridine alkaloid, sanguinarine, which was not greatly influenced by hormone deprivation. Millimolar concentrations of dopamine were detected under all conditions. Thebaine was found when cells were cultured in hormone-free media, but it was not elicitor dose dependent. Verticillium-elicited cultures accumulated sanguinarine in an elicitor-dose-dependent manner only under conditions of hormonal deprivation, resulting in an elevation of sanguinarine levels 5- to 500-fold greater than controls (2-10% dry weight). Most of the sanguinarine accumulated in the medium (23 milligrams per liter), with 85% of the alkaloid associated with a 100g sedimenting fraction that, upon light microscopic inspection, proved to be devoid of cells. In bioassays, sanguinarine showed significant biological activity at concentrations as low as 5 to 10 micrograms per milliliter against three general plant pathogens, Verticillium dahliae, Botrytis cinerea Pers. ex Fr., and Rhizoctonia solani Kuehn. Dendryphion was less affected by sanguinarine addition and displayed an ability to metabolize the alkaloid as evidenced by its loss from the media, subsequent accumulation in the mycelia, and ultimate disappearance over a 48-hour period. By comparison, dopamine and thebaine were less toxic to the general plant pathogens.     

Growth of Suspension Cultures of Sugarcane Cells in Chemically Defined Media

A chemically defined medium is desirable for nutritional studies and is frequently necessary for biochemical investigations. Several defined media are available for use with tissue and cell cultures from dicotyledonous plants. A fully defined medium has now been developed for cell suspension cultures from sugarcane. Prior to this, the only medium successfully used for cell cultures of monocotyledonous plants was a modification of Straus' synthetic medium (used to grow cell suspensions of corn). Cell suspension cultures from sugarcane stalk parenchyma, originally established in complex media containing coconut milk or yeast extract, can be grown in this synthetic medium, which consists of inorganic salts, vitamins, sucrose, 2.4-dicliloropheuoxyacetic acid, and a mixture of 13 amino acids. The most important of the amino acids are arginine aspartic acid, and glutamic acid. This simplified medium wilt aid in the investigation of the unusual and important role of arginine in sugar-cane growth and metabolism.     

The Potassium Requirement for Growth and Embryogenesis in Wild Carrot Suspension Cultures

A requirement for potassium for growth and forembryogenesis in suspension cultures of wild carrot (Daucus carota L.) was demonstrated. The concentration of K⁺ required for maximal growth (1 mM) was less than that required for maximal embryogenesis (20 mM). Neither Na⁺ nor NH₄⁺ could replace K⁺. Ammonium ion enhanced embryogenesis when K⁺ was present at suboptimal levels greater than 1 mM. Nitrogen sources strongly influenced growth and embryogenesis, but the effects of nitrogen were separable from those of K⁺. Subline differences were noted. Subline CSC-29 produced nearly half the maximum embryo number in 1 mM K⁺ while CSC-31 produced no embryos at that K⁺ concentration. Growth of CSC-29 was slightly repressed by Na⁺, but no more than by similarconcentrations of K⁺. Growth of CSC-31 in 1 mM K⁺ was strongly repressed by Na⁺. Embryogenesis in CSC-29 was unaffected by Na⁺. In CSC-31, Na⁺ repressed embryogenesis at lower concentrations of K⁺.     

Increased Production of IAA by Rhizoctonia solani is Induced by Culture Filtrate from Rice Suspension Cultures

To investigate the role of the plant hormones produced by fungi,we tried to construct a system to examine the interaction betweenRhizoctonia solani Kühn MAFF305219 and rice cells in suspensionculture (Oc). R. solani was previously found to produce IAA,with the main biosynthetic pathway via the indole-3-pyruvatepathway. The amount of IAA in the medium produced by R. solaniwas increased by cocultivation with rice cells (Oc) and by culturefiltrate (CF) of Oc. Further analysis revealed that the factor(s)that induced the enhanced accumulation of IAA was sensitiveto heat, to freezing and thawing and lyophilization, and themolecular weight was estimated to more than 10,000. These resultssuggest that the active agent(s) in the medium was (a) proteinor a proteinous substance. Among suspension cultures of variousplants, Oc and another line of rice cells (Ok) had the abilityto induce the accumulation of IAA in the fungal medium 4 h afterinoculation but other cultures of plant cells were ineffective.The promotive effect of rice CF on the accumulation of IAA wasalso observed with some strains of R. solani that belong toa different anastmosis group from MAFF305219. Thus, the accumulationof IAA was not related to the host specificity. (Received July 28, 1997; Accepted October 27, 1997)     

Neurite-Promoting Factor in Conditioned Medium from RN22 Schwannoma Cultures: Bioassay, Fractionation, and Properties

Abstract: On polyornithine (PORN) substrata dissociated 8-day chick embryo ciliary ganglionic neurons will survive if the culture medium is supplemented with Ciliary Neuronotrophic Factor. However, neuritic growth will not occur unless the substratum is derivatized with a PORN-bindable Neurite Promoting Factor (PNPF). In this preliminary study we report that soluble PNPF can be (1) assayed by a convenient in vitro system; (2) obtained in relatively large amounts from serum-free media conditioned over RN22 Schwannoma cultures; (3) concentrated by using Amicon XM100 ultrafiltration; and (4) separated from nearly all of the non-active protein by using ion-exchange chromatography. The partially purified PNPF can be concentrated using XM100 and is heat- and protease-sensitive. In the course of these fractionation studies we observed in some cases a concentration-dependent interference with the expression of PNPF activity in the bioassay; we propose graphical methods to permit the simultaneous determination of PNPF and the extent of such interference. Different treatments that affected the interference property did not always affect PNPF activity in a reciprocal manner, leaving open the possibility that the interference with PNPF activity results from reversible alteration of the PNPF molecule, or that there exists a separate interfering agent.     

Modulation of Extracellular Matrix Production by Conditioned Medium

This report describes some general features of chick embryochondrocyte cultures and some methods for measuring matrix production.It is reported that as cultures grow, the average chondrocyteacquires an increased ability to synthesize matrix components.In part this increased ability is caused by conditioning ofthe culture medium, since conditioned medium from chondrocytecultures can rapidly stimulate mucopolysaccharide and collagensynthesis, but not growth. The cells condition the medium byreleasing a factor that has the following characteristics: non-dialyzable,heat and trypsin-sensitive, sensitive to treatment with mercaptoethanol,p-chloromercuribenzoate, and periodate, and a molecular weightgreater than 30,000 but less than 150,000. The factor is a specializedproduct of chondrocytes, since it is not made by unexpressedchondrocytes nor by differentiated pigmented retina cells. Conditionedmedium acts rapidly (2 hr) to produce a significant stimulationof the incorporation of sulfate into hyaluronidase-sensitivematerial. This action is not sensitive to treatment with actinomycinD, and it is suggested that conditioned medium might act onthe cell surface. The action of conditioned medium factor representsan example of positive feedback of one specialized product madeby a particular cell type on the synthesis of other specializedproducts made by the same cell type.     

Catechol Oxidase in Suspension Cultures of Apple Fruit--The Effects of Growth Regulators

Growth and catechol oxidase activity were followed in suspensioncultures of cells derived from apple fruit. In cultures whichrequired the addition of hormones, enzyme activity was affectedby 2, 4-D (2, 4-dichlorophenoxyacetic acid) and kinetin viatheir effect on growth. Exogenous hormones did not affect catecholoxidase activity of habituated cultures. Ethylene and PCIB (-4-chlorophenoxyisobutyricacid) rapidly depressed enzyme activity in all cell fractions,but only at concentrations which repressed growth. Ethioninecaused an immediate decrease in enzyme activity which precededthe repression of growth. Ethionine did not inhibit enzyme activityin vitro. Possible mechanisms of the action of ethionine arediscussed and the formation of a specific inhibitor is hypothesized.     

Heat-stable Chemically Defined Medium for Growth of Animal Cells in Suspension

Procedures are described for preparation of a chemically defined medium, capable of being autoclaved, for the cultivation of animal cells in suspension. Yields of 34.1 x 10(5), 33.8 x 10(5), and 47.1 x 10(5) cells per ml were recorded for cat kidney, HeLa, and mouse fibroblast (L) cells after 6, 10, and 10 days of incubation, respectively.     

磷对霍山石斛类原球茎悬浮培养细胞生长和多糖合成的影响

在确定了最适接种量和外植体细胞生理时间的基础上,研究了在不同起始磷浓度下,霍山石斛类原球茎生长、碳、氮消耗和多糖积累的动力学特性。以生长30d的类原球茎为材料,在接种量为100g/L时,类原球茎生长的最佳起始磷浓度为2.5mmol/L,培养36d时,类原球茎鲜重达496.5g/L。动力学分析表明,磷是霍山石斛类原球茎生长的限制性因素,胞内磷的积累水平与细胞生长具有相关性,2.5mmol/L的磷酸盐有利于碳、氮等营养物质的吸收;而多糖积累的最佳起始磷浓度为0.312mmol/L,培养36d时,其产量为2.22g/L。     

发根农杆菌A4菌株转化苜蓿悬浮培养物

将苜蓿无菌苗下胚轴切割后,在附加２ｍｇ／Ｌ２,４Ｄ的ＭＳ培养基上诱导愈伤组织。愈伤组织在附加０５ｍｇ／Ｌ２,４Ｄ的ＳＨ培养基中悬浮培养。悬浮培养物在用于转化之前,用０４５ｍｏｌ／Ｌ甘露醇处理１ｈ,然后用０１６ｍｏｌ／ＬＣａＣｌ２·２Ｈ２Ｏ洗涤两次。预处理后的悬浮培养物用ＳＨ培养基悬浮（１０ｍｌ／ｇ悬浮培养物）,再加０２ｍｌ农杆菌悬浮液,于２５±２℃共培养２ｄ。共培养的悬浮培养物洗涤后在附加０５ｍｇ／Ｌ羧苄青霉素的无激素培养基上选择培养。悬浮培养天数、悬浮培养基激素组成和选择培养基种类明显影响转化频率。纸电泳分析表明７０％的转化体可以合成农杆碱和甘露碱。染色体观察显示转化组织细胞存在严重的数目和结构变异