Characteristics of Cultured Tobacco Cell Strains Producing High Levels of Ubiquinone-10 Selected by a Cell Cloning Technique

Characteristic differences were examined between tobacco cell strains producing high levels of ubiquinone (UQ) and the original tobacco cell line (Nicotiana tabacum L. cv BY-2). The growth rate of strains producing high levels of UQ was about half of that of the original cells. The maximum yield in cell dry weight was about two-thirds of that of original cells. The time-course of UQ formation by selected strains and the respiratory rates were similar to those in the original cells. The UQ contents were much higher than those in original cells, not only per g-dry weight but also per cell. Most UQ in the selected strains were also localized in mitochondria, as well as in the original cells. On protein basis, the yield of purified mitochondria from strains producing high levels of UQ was 4.3 times as much as that from original cells. UQ formation per mg of mitochondrial protein and the molar ratios of UQ to the other electron transport components in selected cells were similar to those in original cells. The ratio of the mitochondrial protein yield in strains producing high levels of UQ to the yield in original cells correlated closely with the ratio of UQ content per g-dry weight in UQ-producing strains to UQ content in original cells.     

Effects of Nutritional Factors on the Formation of Ubiquinone by Tobacco Plant Cells in Suspension Culture

The ubiquinone (UQ) content of BY–2 cells on surface culture was examined to compare with that in suspension culture. The UQ content on surface culture was a little lower than that in suspension culture, but the pattern of the time-course of the UQ formation on surface culture was similar.

The changes of UQ content in BY–2 cells during autolysis were also examined. UQ in the cells subjected to autolysis was not rapidly metabolized nor excreted into the medium.

In order to obtain basic information for UQ formation by BY–2 cells in suspension culture, the cultural conditions, especially nutritional ones were investigated. Addition of 2,4-D was remarkably effective on UQ formation and a higher UQ content was observed with a higher 2,4-D concentration. Sucrose and glucose concentrations in the original medium were also influential factors. The UQ content tends to increase with the decrease of the sugar content. Precursors of UQ, amino acids, vitamins and organic acids were not effective on the UQ formation.     

Cell Growth and Flavonoids Production in Suspension Culture of Saussurea medusa

Cell suspension cultures were established from Saussurea medusa Maxim. callus cultures. The effects of different rotation speeds of the gyratory shaker, different inoeulum sizes and different pH values of the medium on cell growth and flavonoid formation were studied. The result showed that the optimum rotation speed, inoeulum size and initial pH value of the medium were 90–120 r/min,50– 80 g FW/L and 5.5–6.0 respectively for cell growth and flavonoids formation in the suspension cultures. Sucrose was better than glucose and fructose for the suspension cultures. The optimum concentration of sucrose for cell growth and flavonoid production was 40 g/L, and the concentration of flavonoids could be as high as 1 423.25 mg/L. High performance liquid chromatographic analysis of cell suspension culture extracts showed that the concentrations of jaceosidin and hispidulin in the flavonoids were 22.11% and 0. 15% respectively.     

Setting up a Single Cell Clone Lines of Cathamus tinctorius

Different concentrations of 2,4-D, KT and NAA were able to influence the plating efficiency (PE) of single cells of Cathamus tinctorius. The best combination of these three hormones for the growth of single cells was 2.0, 0.3 and 0.5 mg/l, respectively. The PE was obviously different as cells came from different generations of suspension subculture and the third generation of suspension culture cells, had the best PE which 8.5 times as high as that of the first generation of suspension culture cells. Single cell growth in condition medium or in solid-liquid dual layer culture was better than in normal plate culture. The PE of single cell clones in condition culture was 3.6 times as high as in normal plate culture. The PE of single cell clones in solid-liquid dual layer culture was 4.7 times as high as in normal plate culture. Many clones from single cells were set up. Different growth rates were observed in different single-cell clones. The lowest growth rate in these clones was 3.08 g/g/35 days, the highest growth rate in these clones was 23.33 g/g/35 days.     

樟叶越桔细胞悬浮培养条件的优化

为了提高樟叶越桔(Vacciniumdunalianum)悬浮培养细胞的生物量,以樟叶越桔叶片愈伤组织为试材,通过单因素试验探究不同蔗糖浓度、培养基pH值、培养基体积、初始接种量和摇床转速对悬浮培养细胞生长的影响,并根据响应面法Box-Behnken试验设计原理进行组合试验以优化培养条件。结果显示,以改良WPM培养基为基础培养基,樟叶越桔细胞悬浮培养的最优条件为40 g·L^–1蔗糖、培养基pH5.2、培养基体积45 mL、初始接种量2.64 g和摇床转速为149 r·min^–1,其细胞生物量干重为0.184 4 g,与理论预测值0.184 5 g较为接近,且细胞的生长曲线呈S型。研究结果为樟叶越桔悬浮培养细胞次生代谢产物的生产调控奠定了技术基础。     

Cell culture establishment and regulation of two phenylethanoid glycosides accumulation in cell suspension culture of desert plant <Emphasis Type="Italic">Cistanche tubulosa</Emphasis>

Cistanche tubulosa is one of the most valuable desert medicinal plants, whose cell culture investigations have been rarely reported before. Phenylethanoid glycosides (PhGs) are its major components with a wide range of pharmacological activities. In this article, callus culture and cell suspension of C. tubulosa were established. Fleshy stems were found to be the most suitable explants for callus induction, and the optimal medium for induction was B5 solid medium supplemented with 0.8 g/L casein hydrolysate, 20 g/L sucrose, 2 mg/L naphthaleneacetic acid (NAA), and 1 mg/L 6-benzyladenine (6-BA). Based on qualitative and quantitative determination of two PhGs (echinacoside and acteoside) contents, the effects of carbon source concentration, precursor feeding, and elicitor treatments on cell growth and two PhGs accumulation in cell suspension cultures were investigated. Thirty g/L was the optimal initial sucrose concentration to obtain the high yield of biomass (9.29 g dry weight, DW) per liter cell suspension culture, echinacoside (12.14%, based on DW cells) and acteoside (2.17%). Precursor feeding also had a positive effect on PhGs accumulation. Feeding of precursor tyrosine (1 g/L) to the cell cultures increased the levels of echinacoside to 18.83% and acteoside to 2.92%, which were approximate 1.5 times of the corresponding levels in the control group. Methyl jasmonate (MJ) was the ideal elicitor for PhGs accumulations in C. tubulosa, particularly for eliciting acteoside production. The maximum echinacoside and acteoside contents reached 21.18 and 5.24% after 12 h of treatment with 200 µM MJ, respectively, which were approximate twofold higher than those in wild plant.     

Ubiquinone-10 production using Agrobacterium tumefaciens dps gene in Escherichia coli by coexpression system

Ubiquinone (Coenzyme Q; abbreviation, UQ) acts as a mobile component of the respiratory chain by playing an essential role in the electron transport system, and has been widely used in pharmaceuticals. The biosynthesis of UQ involves 10 sequential reactions brought about by various enzymes. In this study we have cloned, expressed the decaprenyl diphosphate synthase, designated dps gene, from Agrobacterium tumefaciens, and succeeded in detecting UQ-10 in addition to innate UQ-8 in Escherichia coli. Furthermore, the production of UQ-10 was higher than UQ-8. To establish an efficient expression system for UQ-10 production, we used genes, including ubiC, ubiA, and ubiG involved in UQ biosynthesis in E. coli, to construct a better co-expression system. The expression coupled by dps and ubiCA was effective for increasing UQ-10 production by five times than that by expressing single dps gene in the shake flask culture. To study for a large-scale production of UQ-10 in E. coli, fed-batch fermentations were implemented to achieve a high cell density culture. A cell concentration of 85.40 g/L and 94.58 g/L dry cell weight (DCW), and UQ-10 content of 50.29 mg/L and 45.86 mg/L was obtained after 32.5 h and 27.5 h of cultivation, subsequent to isopropyl-β-d-thiogalactopy ranoside and lactose induction, respectively. In addition, plasmid stability was maintained at high level throughout the fermentation.     

Rheological characteristics of cell suspension and cell culture of Perilla frutescens

Physical properties such as viscosity, fluid dynamic behavior of cell suspension, and size distribution of cell aggregates of a plant, Perilla frustescens, cultured in a liquid medium were studied. As a result of investigations using cells harvester after 12 days of cultivation in a flask, it was found that the apparent viscosity of the cell suspension did not change with any variation of cell concentration below 5 g dry cell/L but markedly increased when the cell concentration increased over 12.8 g dry cell/L. The cell suspension exhibited the characteristics of a Bingham plastic fluid with a small yield stres. The size of cell aggregates in the range 74 to 500 mum did not influence the rheological characteristics of the cell suspension. The rheological characteristics of cultivation mixtures of P. frutescens cultivated in a flask and in a bioreactor were also investigated. The results showed that the flow characteristics of the cell culture could be described by a Bingham plastic model. At the later stage of cultivation, the apparent viscosity increased steadily, even though the biomass concentration (by dry weight) decreased, due to the increase of individual cell size. (c) 1992 John Wiley & Sons, Inc.     

杜仲细胞悬浮培养产黄酮及其动力学研究

本文应用正交设计对杜仲细胞悬浮培养的基本培养基和植物生长物质浓度进行了筛选,并对影响杜仲细胞悬浮培养和总黄酮含量的不同因素进行了考察。结果表明,B5培养基+0.5mg/L NAA+0.6mg/L 6-BA、蔗糖30g/L、初始pH 5.0-5.5、接种量20g(FW)/L以及摇床转速110r/min为杜仲细胞悬浮培养的适宜条件。通过对杜仲悬浮细胞生长和代谢动力学的分析表明：杜仲细胞悬浮培养生长符合Logistic生长模型,最大比生长速率( m)为0.417d-1;细胞基于蔗糖的真正比生长得率(YG)与维持系数(m)分别为0.619g/g和0.0206g/(g·d-1);黄酮合成属部分生长耦联型,可用Luedeking-Piret模型进行描述。研究结果为杜仲细胞大规模悬浮培养生产天然活性成分奠定了基础。     

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.     

Studies on Cell Suspension Culture and Fermentation Culture in Arnebia euchroma

This paper reports some characteristics of cell suspension and fermentation culture in Arnebia euchroma (Royle) Johnst. The yield of suspension culture reached 22.0g dry wt/L per month when inoculum quantity was 2.50 g dry wt/L. Time-course study showed that cell growith lagged in 0–3 days and enhanced greatly in 3–12 days, and almost ceased after 12 days of culture, pH value changed during the culture period and peaked on the 12th day after inoculation. When cells were cultured in liquid production medium, the contents of shikonin derivatives increased quickly and reached to the maximum about the 25th day. The cell yield of 9.47 and 9.34 g dry wt/L per month was obtained in fermentation culture. Timecourse of cell growth in fermentation culture was similar to that in suspension culture. The total content of shikonin derivatives in fermentation culture was 14.26% dry weight from 10 L bioreactor. The yield of shikonin derivatives was 1.93 g/L.     

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。     

Establishment of Orthosiphon stamineus Cell Suspension Culture for Cell Growth

Callus of Orthosiphon stamineus could be induced successfully from petiole, leaf and stem tissues but not roots when cultured on MS medium containing different concentration of NAA (0–4.0 mg l^–1) and 2,4-D (0–2.0 mg l^–1). Highest fresh weight callus production was obtained from leaf explants and those with best friability were obtained on MS medium plus 1.0 mg l^–1 2,4-D plus 1.0 mg l^–1 NAA. Cell suspension cultures were established from these cultures. The appropriate cell inoculum size for the best cell growth was 0.75 g of cells in 20 ml culture medium. Cell suspension culture using MS medium supplemented with 1.0 mg l^–1 2,4-D promoted the best cell growth with maximum biomass of 8.609 g fresh weight and 0.309 g dry weight 24 days after inoculation. Cells that grew in MS medium supplemented with 1.0 mg l^–1 2,4-D reached the stationary growth phase in 15 days as compared to the cells that grew in MS medium supplemented with 1.0 mg l^–1 2,4-D + 1.0 mg l^–1 NAA reached the stationary phase in 24 days. MS medium supplemented with 1.0 mg l^–1 2,4-D was considered as the maintenance medium for maintaining the optimum cell growth of O. stamineus in the cell suspension cultures with 2-week interval subculture.     

搅拌式生物反应器悬浮培养水母雪莲细胞的研究

应用 2L通气搅拌式生物反应器一步批式培养水母雪莲细胞。采用倾斜式搅拌桨代替透平桨 ,研究了搅拌转速、通气量和接种量对细胞生长和黄酮合成的影响 ,发现在 75r min、70 0～1000L min和 4.0～ 5.0gDCW L接种量下细胞生长和黄酮合成比较好。经过 12d培养细胞干重达 13.8gDCW L ,黄酮产量 416mg L ,黄酮含量占细胞干重的 30%。水母雪莲细胞生长及黄酮合成的进程表明 ,黄酮积累与细胞生长呈正相关。对细胞聚集体分布的研究发现 ,流变压力使细胞聚集体分裂 ,使反应器中细胞生长受到影响 ,黄酮产量较摇瓶中降低     

Rosmarinic acid production in perfused Anchusa officinalis culture: Effect of inoculum size

Summary High cell density and rosmarinic acid (RA) productivity have been achieved by applying periodic culture perfusion to the Anchusa officinalis cell suspension. In this study, the effect of inoculum size on cell growth and RA productivity in the perfused Anchusa culture was investigated. Experimental results showed that RA productivity increased with the inoculum size, up to 4 g dry weight/L. Further increases in the inoculum size did not yield a higher RA productivity regardless of culture perfusion. Moreover, the maximum cell concentration was not affected by the inoculum sizes, from 1 to 11 g dry weight/L. Cell crowding, indicated by high culture packed cell volumes, is believed to be the predominant cause of low productivity in perfused cultures with high inoculum sizes.     

濒危植物珙桐愈伤组织的诱导及悬浮细胞培养初探

以珙桐（Davidia involucrate）无菌苗的下胚轴为外植体,以LSD分析和正交设计与分析为主要研究方法。诱导和筛选愈伤组织,进行悬浮培养,初步建立了珙桐悬浮培养体系,并对珙桐悬浮培养中生物量和pH值的变化进行了初步研究。结果表明：KT和2,4-D的组合诱导出的愈伤组织更适合进行细胞悬浮培养,最佳愈伤组织诱导培养基为WPM＋KT 1.0 mg/L＋2,4-D 0.5 mg/L＋Vc 1.0 mg/L;悬浮细胞培养中,接种量和Ca2＋浓度是影响生物量的主要因素,最佳接种量为25 g/L,最佳Ca2＋浓度为标准WPM培养基中Ca2＋浓度的2倍;黑暗条件下,珙桐悬浮培养生物量变化曲线呈＂S＂型,在21 d时可获得最大生物量;pH值的变化与生物量的变化有一定的相关性。     

培养基和培养条件对栀子悬浮细胞合成多糖的影响

对栀子悬浮细胞合成多糖的调控因子研究表明 :B5为最适培养基 ;5～10d继代周期的细胞可以保持良好的生长状态和多糖的合成能力 ;80g L的鲜细胞的接种量有利于栀子细胞的生长和多糖的合成 ;使用单一碳源时 ,葡萄糖比蔗糖对细胞生长更有益 ,但葡萄糖成本高 ,因而混合碳源 45g L(葡萄糖 :蔗糖 =1∶1)是最佳配方 ;氮源种类对细胞生长和多糖合成没有明显的影响 ,但氮源浓度是主要因素 ,40～50mmol L是最佳浓度 ,同时运用悬浮细胞生产栀子多糖可以通过在不同时间收获的细胞来避免提取时黄色素的干扰 ,具有很好的实际意义。     

藏红花细胞悬浮培养动力学研究

基于已经建立的藏红花细胞悬浮培养体系,研究了其悬浮培养动力学.结果表明,悬浮培养时细胞的生长周期约为20 d,在20 d时生物量达到最大,为12.3 g DW/L,但是藏红花素的合成周期大约为28 d,在28 d时藏红花素的含量和产量达到最大,分别为95.8 mg/g和0.92 g/L.藏红花素的积累与细胞的生长之间的关系为半生长偶联型,为其生物反应器放大培养提供了参数等理论依据.     

Effects of Inorganic Nitrogen Sources and Physical Factors on the Formation of Ubiquinone by Tobacco Plant Cells in Suspension Culture

In order to obtain basic information on ubiquinone (UQ) formation by BY-2 cells in suspension culture, effects of inorganic phosphate and nitrogen sources and such physical factors as initial pH, light irradiation, shaking condition and temperature were investigated. The concentration of phosphate and nitrogen sources had no marked effect, but the ratio of ammonium nitrogen to nitrate nitrogen was significantly effective on UQ formation. The UQ content in BY-2 cells tends to increase at higher ratios of ammonium nitrogen. The increase in the UQ content was recognized at higher concentrations of 2, 4-d, especially with lower concentrations of sucrose. Physical factors had no marked effect on UQ formation except temperature. The UQ content was considerably raised at higher temperatures.     

葡萄糖的添加对昆虫细胞Sf21悬浮生长的影响

添加葡萄糖对昆虫细胞Sf21(Spodoptera frugiperda)悬浮生长的影响,发现补加糖量在lg／L时·能明显提高细胞生长的速度和最高密度,细胞最高密度由2．5×10⁴／ml增加到4.9×10⁶／ml; 朴加糖量在2g／L时,则有显著的抑制作用,即使增加接种密度．细胞生长的最高密度也只有2．1×10⁶／ml。当采取流加葡萄糖方法来培养细胞时,则其生长的最大密度可提高到5．2×10⁶／ml。