VK_3诱导悬浮培养的胡萝卜细胞凋亡

VK_3是一种醌类物质,在体内通过氧化还原产生超氧化自由基。我们的实验发现,VK_3对悬浮培养的胡萝卜细胞及原生质体有致死作用。这种作用是浓度依赖性的。100—800μmol/L的VK_3能引起10%—33%的细胞死亡。当VK_3浓度达到1mmol/L时,死亡率达到100%。DNA电泳分析发现,600和800μmol/L的VK_3处理过的细胞和原生质体产生大小为180bp整数倍的梯状条带。而在更高或更低浓度的处理中则没有观察到这种条带。用TUNEL方法检测,在400—800μmol/L浓度的处理中观察到细胞核DNA的断裂。而在更高或更低浓度的处理中则没有观察到这种断裂。由此我们推测,适当浓度的VK_3能诱导胡萝卜细胞及原生质体凋亡。     

寡聚糖诱导悬浮培养南方红豆衫细胞的凋亡

在真菌（Fusarium oxysporum f.vasinfectum(Atkinson)Snyder et Hansen）寡聚糖诱导悬浮培养南方红豆衫（Taxus chinensis(Pilger)Rehd.var.mairei(Lemee et Levl.）Cheng et L.K.Fu）细胞生产紫杉醇的体系中发现细胞出现凋亡，次生代谢增强，电镜观察到细胞核质和原生质出现凝集现象，液泡内出现大量的高电子致密林。核DNA器发达，但紫杉醇合成速率很低，加入寡聚糖后，细胞防御系统开启，细胞生长停止，次生代谢物酚类物质大量积累且次生壁加厚，多酚氧化酶活性迅速提高，苯丙烷类代谢途径的关键酶苯丙氨酸解氨酶的活性在1h后急速提高，目的产物紫杉醇在诱导后72h达到峰值，比对照组提高了6倍，且细胞凋亡的出现与紫杉醇合成的峰值具有时间上的一致性。     

DMSO对悬浮培养的东北红豆杉细胞凋亡和紫杉醇合成的影响

研究了不同浓度的DMSO对悬浮培养的东北红豆杉（Taxus cuspidata）细胞的增殖能力、细胞活性以及紫杉醇合成和释放等方面的影响,同时应用荧光指示剂双染法检测了细胞凋亡的发生情况。结果显示2％的DMSO处理能显著降低细胞活性,抑制细胞的增殖能力,使细胞核内DNA含量减少,培养中、后期在荧光显微镜下可见部分细胞核出现典型的凋亡形态,同时伴有紫杉醇产量的明显增加。对照组及1％以下浓度组未出现上述改变。结果表明一定浓度的DMSO能诱导细胞凋亡,促进细胞紫杉醇合成能力的提高。     

NAA对胡萝卜（Daucus carota）悬浮细胞茄红素合成的影响

通过对胡萝卜新透心红 DaucuscarotaCV.XinTouxinhong 细胞悬浮系单位鲜重细胞的茄红素含量、细胞总产量以及茄红素总产量的分析比较,研究了培养基中不同NAA浓度对胡萝卜细胞悬浮系茄红素代谢的影响.结果表明,NAA具有增强细胞茄红素代谢水平、提高单位鲜重细胞中茄红素的含量的作用,但对细胞的生长具有抑制作用.从茄红素单产和总产量综合评价,培养基中加入3.0mg·L-1NAA效果最佳.     

乙烯诱导胡萝卜原生质体凋亡

乙烯是一种参与多种重要生理学过程的植物激素。用乙烯利在密闭条件下处理胡萝卜（ＤａｕｃｕｓｃａｒｏｔａＬ．）原生质体（在ｐＨ＞４．１时释放乙烯）,发现随着乙烯利浓度增加,细胞死亡率逐渐增高。经乙烯利处理的胡萝卜原生质体出现核内染色质固缩,形成凋亡小体等典型的细胞凋亡的形态学特征。用中性法彗星电泳观测到彗星状的核ＤＮＡ片段的迁移。ＤＮＡ电泳分析观察到细胞凋亡时产生的典型的核小体间ＤＮＡ断裂所形成的梯状条带。结果表明,乙烯能诱导悬浮培养的胡萝卜原生质体凋亡     

外加紫杉醇对悬浮培养东北红豆杉细胞的诱导效应

研究表明外加紫杉醇能够诱导悬浮培养的东北红豆杉(Taxus cuspidata)细胞总DNA发生梯带化降解。利用mRNA差异显示技术比较了紫杉醇诱导凋亡与不诱导凋亡的东北红豆杉细胞基因表达的差异,得到了8个特异表达的cDNA克隆,经Northern杂交证实其中3个在不发生凋亡的细胞中表达,5个在凋亡的细胞中表达。对这8个cDNA克隆单向序列测定后,与GenBank/EMBL/DDBJ中同源序列进行了比较,结果表明：1个cDNA片段与拟南芥中ABA应答蛋白基因的保守区有86%的同源性;2个cDNA片段与番茄内切壳聚糖酶前体基因的保守区有50%的同源性;其他5个cDNA片段无明显的同源基因,可能是新基因。     

细胞色素c(Cyt c)诱导烟草悬浮细胞(BY-2)凋亡

用不同浓度细胞色素c(Cyt c)诱导继代时间不同的烟草悬浮细胞48 h后观察形态学特征的结果表明,继代培养10和13 d的细胞均在10 mmol·L-1Cyt c时出现最高的细胞凋亡率,而继代5 d的细胞在Cyt c浓度为12.5 mmol·L-1时细胞凋亡的诱导率仍表现上升趋势;DNA电泳检测结果显示凋亡处理的细胞中DNA呈现较明显的DNA梯度.     

胡萝卜培养细胞的盐适应蛋白

近年来植物离体培养细胞盐适应蛋白的发现以及盐影响下植物信使 RNA 体外转译产物的研究,为耐盐分子机理的研究和耐盐基因的鉴定和克隆开辟了道路。烟草细胞在适应1—2.5%NaCl 之后,细胞蛋白质合成发生变化,特别是26kD 的蛋白质大量积累。体内标记实验表明,细胞从不加 NaCl 的培养基转到加有1%     

除虫菊细胞的悬浮培养

除虫菊悬浮细胞从培养后第8天开始迅速生长,14 d达到最大生长量,其最适培养基为:MS 4mg·L-12,4-D 0.1mg·L-1NAA 0.3 mg·L-16-BA 30 g·L-1蔗糖,最适接种量为15 g·L-1,摇瓶装液的体积分数为40%,初始培养的最适pH值为5.8,收获时pH下降为4.6±1,最适培养温度为25℃.     

石竹细胞悬浮培养研究

石竹细胞继代周期为 7d时 ,悬浮细胞培养系生长最快 ,生长率最高 ,而且培养物中胚性细胞较多 ,并能保持较快的分裂和生长 ,能促进已形成的大细胞团的生长和分化。转代时接种物与新鲜培养基的体积比以1∶2较好 ,悬浮系细胞生长最快 ,生长率最高 ,以 1∶2和 1∶3的高倍稀释接种有利于胚性细胞的形成及产生小的胚性细胞团 ,对悬浮系添加椰乳和水解乳蛋白的混合物 ,可较大幅度地提高悬浮细胞系的生长速率 ,单独添加上述两种物质的效果均不如二者的综合效应好。在 6种不同激素组合中 ,配方 2 (2 ,4 D 1 .5mg/L +NAA0 .5mg/L +6 BA 0 .5mg/L)最好 ,生长率最高。配方 5 (2 ,4 D 1 .5mg/L +NAA 0 .5mg/L +6 BA 1 .0mg/L)其次 ;配方 1 (2 ,4 D 1 .0mg/L +NAA 0 .5mg/L +6 BA 0 .5mg/L)次之。     

Oligochitosan induces programmed cell death in tobacco suspension cells

Oligochitosan has been proved to trigger plant cell death. To gain some insights into the mechanisms of oligochitosan-induced cell death, the nature of oligochitosan-induced cell death and the role of calcium (Ca²⁺), nitric oxide (NO) and hydrogen peroxide (H₂O₂) were studied in tobacco suspension cells. Oligochitosan-induced cell death occurred in cytoplasmic shrinkage, phosphatidylserine externalization, chromatin condensation, TUNEL-positive nuclei, cytochrome c release and induction of programmed cell death (PCD)-related gene hsr203J, suggesting the activation of PCD pathway. Pretreatment cells with cyclosporin A, resulted in reducing oligochitosan-induced cytochrome c release and cell death, indicating oligochitosan-induced PCD was mediated by cytochrome c. In the early stage, cells undergoing PCD showed an immediate burst in free cytosolic Ca²⁺ ([Ca²⁺]_cyt) elevation, NO and H₂O₂ production. Further study showed that these three signals were involved in oligochitosan-induced PCD, while Ca²⁺ and NO played a negative role in this process by modulating cytochrome c release.     

A programmed cell death pathway activated in carrot cells cultured at low cell density

Programmed cell death (PCD) occurs in plants during development and defense, but the processes and mechanisms are not yet defined. Culture of carrot single cells at a cell density of <10⁴ cells ml⁻¹ activates a cell death process involving condensation and shrinkage of the cytoplasm and nucleus and fragmentation of the DNA. Modest abiotic stress treatments also cause cell condensation and shrinkage and the formation of DNA fragments, but the same abiotic stresses at high levels cause rapid necrosis with cell swelling and lysis. The common morphological features of cells dying at low cell density and following modest abiotic stress treatments suggest that these features reveal a PCD pathway in carrot. The addition of a cell-conditioned growth medium allows cells at low cell density to remain alive, demonstrating that cell-derived signal molecules suppress a pathway that is otherwise induced by default. Differences in the morphology of the dead cells suggest that proteolysis during PCD differs in detail in plants and animals; however, these findings show that plants, like animals, can control PCD by social signaling, and imply that the mechanism of PCD in plants and animals may be similar. Consistent with this, manipulation of signal pathway intermediates that regulate PCD in animals shows that Ca²⁺ and protein phosphorylation events are PCD pathway intermediates in carrot.     

Embryogenesis from microinjected single cells in a carrot cell suspension culture

A micrroinjection method was established for intact single cells with cell walls using a carrot suspension culture system in which selected single cells differentiate to embryos at high frequency. A solution of a fluorescent dye, Lucifer Yellow CH, was microinjected into those single cells, using an inverted microscope and a hydraulic micro-manipulator. In order to hold cells with cell walls and to overcome their turgor pressure, certain modification to conventional microinjection methods for protoplasts were necessary. The microinjected cells could divide and differentiate to embryos at a frequency of about 50%.     

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     

Induction of programmed cell death in human retinoblastoma Y79 cells by C2-ceramide

C₂-ceramide, a cell-permeable analogue of ceramide, induced significant, dose- and time-dependent death in human retinoblastoma Y79 cells. Dying cells strongly displayed the morphology of apoptosis as characterized by microscopic evidence of cell shrinkage, membrane blebbing, nuclear and chromatin condensation and degeneration of the nucleus into membrane-bound apoptotic bodies. Upon induction of apoptosis Y79 cells evidence early phosphatidylserine externalization, as shown by annexin V-FITC. Apoptosis was also assessed by monitoring changes in cell granularity by staining with the combined fluorescent dyes acridine orange and ethidium bromide. C₂-ceramide induced these morphological changes without a concomitant production of oligonucleosomal fragments responsible for the DNA ladder and without changes in p53 protein level. Apoptosis was accompanied by accumulation of a modified Bcl-2 protein with a slower-mobility form, and by proteolytic cleavage of PARP. The effect seemed to be specific for C₂-ceramide, as C₂-dihydroceramide, or other amphiphilic lipid analogues, or products of ceramide hydrolysis were ineffective. The effect also depended on mRNA and protein synthesis as it was markedly inhibited by actinomycin D and cycloheximide. Sphingomyelinase and interleukin-l, which are known to activate the sphingomyelin turnover leading to ceramide generation, also induced apoptosis mimicking the effects of ceramide. These findings propose ceramide as an activator of the suicidal program in Y79 cells.     

Synchronization of somatic embryogenesis in a carrot cell suspension culture

Synchronization of somatic embryogenesis was achieved in a carrot (Daucus carota L. cv. “Kurodagosun”) suspension culture by sieving the initial heterogeneous cell population, by density gradient centrifugation in Ficoll solutions, and by subsequent repeated centrifugations at a low speed (50g) for a short time (5 seconds), followed by transferring the cell clusters obtained, which were composed of 3 to 10 cells, to a medium containing zeatin (0.1 micromolar) but no auxin. The frequency of embryo formation reached more than 90%, and synchrony of the embryogenetic process was observed at least in the early stages of the process. The system established in the present work provides a useful system for biochemical research into the mechanisms of somatic embryogenesis.     

Potassium transport in suspension culture cells and protoplasts of carrot

The properties of potassium transport in carrot (Daucus carota L.) suspension culture cells and their isolated protoplasts were examined. Cells cultured in Murashige and Skoog (MS) medium (Plant Physiol 15: 473-497) were potassium saturated and, consequently, they exhibited little net potassium accumulation. Cells that transport and accumulate potassium were derived from the MS-grown cells by culturing them in a potassium-free modified medium. The transport properties of the modified medium cells included: (a) smooth nonsaturating kinetics with 80% of the maximum rates occurring at 0.1 millimolar KCl, (b) linear transport for at least 75 min, (c) alkaline pH optimum, (d) little accompanying anion uptake with increased malate concentrations balancing net increases in positive charge, and (3) little effect on transport by plasmolysis. Potassium transport activity appeared to be 50% lower in protoplasts isolated from the modified medium cells. Nevertheless, the protoplasts exhibited essentially the same kinetics, time course, pH response, and malate adjustment as the intact cells. We concluded from these results that the low potassium cells and their isolated protoplasts are ideally suited to investigating potassium transport at the cell level without the complications associated with multilayered and highly differentiated tissues.     

Detoxification of N-(phosphonoacetyl)-L-aspartate by carrot cells in suspension culture

Phototropic reversal of Phycomyces sporangiophores can be elicited by a change to darkness during steady-state phototropism. The reversal lasts 25–30 min under these conditions. Control experiments show that the reversal is not caused by gravitropism. Tropic reversal is also elicited by the removal of a barrier during an avoidance response, showing that the reversal occurs at the output of the sensory transduction chain.