Studies on Tissue Culture ef Medicinal Plants VI. The Cost Question in the Cell Culture of Anisodus acutangulus

Both increased dry wt. of the culture cells and contents of hyoscyamine and scopolamine in the culture cells were decreased slightly (decreased about 1.2% and 2.9% respectively) when non-ionic water was used instead of redistilled water in the cell suspension culture of Anisodus acutangulus. But the non-ionic water could reduce costs largely, e.g. power cost etc. (can be reduced about 1030 yuantonne medium). Since non-ionic water can now be mass-produced easily, it suits the needs of industrial production in the future. The increased dry wt. of culture cells was a litter high and contents of two alkaloids was decreased slightly (about 5.4%) in the cell suspension culture of A. acutangulus when store confectioner’s sugar was used instead of sucrose. But the applied quantities of store confectioner’s sugar and costs could be reduce 50% and 88% respectively. The suspension cells of A. acutangulus contain rich amylase (average amylase activity was 4.37 mg reducing sugar/g, fresh wt./hour), so it could use starck as their only carbon source for the growth.     

Cryopreservation of Calli of Arnebia euchroma

In this preliminary experiment two kinds of cryoprotectants were used: (1). 10% DM-SO + 0.5 mol/L sorbitol; (2). 7.5% DMSO +5% glycerin +5% sucrose. Calli samples were taken at day-10 and day-15 after subculture respectively. The specimens were cooled at a rate of l℃/min from 0℃, kept –40℃ for 2h, put into liquid nitrogen(–196℃) and then thawed in 40℃ water bath. Calli which bare been subcultured for 10 days showed normal growth by using cryoprotectant 2 and slow-freezing method mentioned above which indicated that they were effective methods for cryopreservation of Arnebia euchoma. Normal growth of the callus after cryopreservation could be restored, but the initial growth rate was somehow retarded. Using polycrylamide gel electrophoresis analysis no difference in esterase and peroxidase isozymes was found in both types of the treated calli. The present experiment indicates it is possible to use this slow-freezing method for germplasm cryopreservation of Arnebia euchroma.     

Submergence(Fermentation)Culture of Anisodus acutanguhts Cells and Identification of Hyoscyamine and Scopolamine in the Cultured Ceils

The growth rate of Anisodus acutangulus cells in the submergence culture was 1.5 g dry wt/l/day, a rate 3 times as that in the suspension culture and more than 10 times over that of the solid static culture: However, the contents of hyoscyamine (0.203 mg/g dry wt) and scopolamine (0.178 mg/g dry. wt) in submergence culture were only slightly higher than those in the two other cultures. But when the 12-day-old submergence culture was supplemented with phenylalanine (5m mol/l) and kinetin (0.1mg/l), it was observed that not only the cell growth rate was increased but also the cellular content of hyoscyamine was raised to a level of 0.217 mg/g dry wt., and that scopolamine to 0.412 mg/g dry wt. The contents of these two alkaloids represented 1.1 and 2.3 times respectively the value of the culture without phenylalanine and kinetin supplements. The optimum date for harvesting the A. acutangulus culture Was on the 14th day of the culture. The monomers of hyoscyamine and scopolamine isolated from the cultured cells were purified and recrystalized, and then identifited as the two compounds in question by the thin-layer chromatography, melting point determination, and ultraviolet, infra-red and nuclear magnetic resonance spectra. In this paper, we also summarize and discuss the results of A. acutangulus culture experiments performed in the past 8 years. Our finding seems to inclieate that following a pilot production trial, the tissue culture method could well be employed to produce hyoscyamine and scopolamine from A. acutangulus cells on an industrial scale.     

红豆杉细胞培养的研究

从红豆杉（Ｔａｘｕｓｃｈｉｎｅｎｓｉｓ（Ｐｉｌｇ）Ｒｅｈｄ）的嫩茎及针叶诱导的出愈伤组织,对愈伤组织培养及细胞悬浮培养进行了研究,利用ＨＰＬＣ方法测定它们合成紫杉醇的能力,发现了能够提高培养细胞生长速率及紫杉醇含量的一些因子,红豆杉愈伤组织及悬浮培养细胞的生长速率已分别达到０．２５ｇ／Ｌ．ｄ和０．２８ｇ／Ｌ．ｄ。而他们的紫杉醇含量分别是０．００２６％和０．０１２％。     

Development of cell line preservation method for research and industry producing useful metabolites by plant cell culture

The cell culture ofAngelica gigas Nakai producing decursin derivatives and immunostimulating polysaccharides was preserved in liquid nitrogen after pre-freezing in a deep freezer at −70°C for 480 min. The effects of the cryoprotectant and pretreatment before cooling were investigated to obtain the optimal procedure for cyropreservation. When compared to mannitol, sorbitol, or NaCl with a similar osmotic pressure, 0.7M sucrose was found to be the best osmoticum for the cryopreservation ofA. gigias cells. In the pre-culture medium, the cells in the exponential growth phase showed the best post-freezing survival after cryopre-servation. A mixture of sucrose, glycerol, and DMSO was found to be an effective cryoprotectant and a higher concentration of the cryoprotectant provided better cell viability. When compared with the vitrification, the optimum cryopreservation method proposed in this study would seem to be more effective for the long-term storage of suspension cells. The highest relative cell viability established with the optimal procedure was 89%.     

大麦胚性细胞悬浮系的建立及其生长特性

由春大麦品种“如车”种胚诱导的松脆型胚性愈伤组织经2个月的悬浮培养,成功建立分散性好、生长速度快的胚性细胞悬浮系。该系细胞直径为1-3mm,由富含淀粉粒的胚性薄壁细胞构成。经不同浓度2,4-D实验,发现2mg/L最适合该细胞系的生长。文中对成功建立大麦胚性细胞悬浮系的关键问题进行了讨论。     

Tumorsphere assay provides more accurate prediction of in vivo responses to chemotherapeutics

Although the sphere culture system has been widely used in stem cell biology, its application for drug screening is limited due to lack of standardized, rapid analytical tools. To optimize sphere cultures for in vitro screening of drugs, we evaluated the properties of primary tumor cells growing as tumorspheres and compared their chemosensitivity to those of cells growing in monolayer. Most cells in tumorsphere cultures were quiescent whereas cells in monolayer culture had a high mitotic index. Moreover, doxorubicin showed better cytotoxicity than paclitaxel in the sphere cultures, but their efficacy was reversed in the monolayer cultures. Importantly, the response of cytotoxic outcomes for suspension cultures matched the in vivo response better than monolayer cultures, providing support for the use of short term suspension cultures of primary cells as a model for drug testing.     

A METHOD FOR THE CRYOCONSERVATION OF DUNALIELLA SALINA (CHLOROPHYCEAE): EFFECT OF GLYCEROL AND COLD ADAPTATION1

The unicellular green alga Dunaliella salina Teod. was frozen according to the following procedure: 3 days cold adaptation at 4°C, addition of 3.5 M glycerol as a cryoprotectant, slow cooling to –40°C, immersion in liquid nitrogen, and rapid thawing. The survival rate was higher when cells were grown, before freezing, in the presence of 2 M NaCl instead of 1 M NaCl (78 and 48% survival, respectively). This difference is probably due to the intracellular amount of glycerol, which increases with external NaCl concentration and, therefore, may enhance cell protection. Although cells grown in 4 M NaCl accumulated a large amount of glycerol in response to osmotic stress, they did not withstand freezing. The use of cryoprotectant was absolutely necessary for the cells to recover from storage at –196°C. Glycerol was used because it is naturally produced by Dunaliella salina and therefore is not toxic. Provided it was added slowly to avoid osmotic shock, 3.5 M glycerol gave better results than 1M glycerol (48 and 18% survival, respectively). Cold adaptation in the dark increased postthaw viability. Cells grown in 1 M or 2 M NaCl had a survival rate of 48 and 78%, respectively, when cold-adapted, against 10 and 42% when not cold-adapted. This adaptation could be due to the synthesis, at low temperature, of specific proteins because two bands (28–29 kDa) appeared when electrophoretically separated proteins from cold-adapted cells and control cells were compared. Also, it could be due to the degradation of starch that occurs in the dark and leads to glycerol accumulation. Our procedure has never been used to cryopreserve microalgae and could enhance reported survival rates.     

Anticarcinogenic Alkaloids in the Cultured Cells of Cephalotaxus fortunei

Calli were induced from the leaves and stems of Cephalotaxus fortunei Hook. f. on MS medium supplemented with 0. 1 mg/L KT and 3 mg/L NAA, and from which the suspension culture cell line of this plant was established for the first time. Factors such as light, pH value of the medium, concentration of plant hormone, carbon resources and addition of substances to the medium, which affect the growth of suspension cells were investigated. The results showed that suspension cells grew appropriately at pH 5.8 with a low concentration of sucrose or glucose, and a low level of NAA. No difference effect on cell growth was seen between sucrose and glucose. Phenylalanine and protein hydrolysate were not suitable for cell growth in suspension cultures, and light inhibited cell growth. A sensitive and rapid high-performance liquid chromatographic method has been developed for detecting the alkaloids in cultured cells. The results revealed the following contents of cephalotaxine and its anticancer esters in cultured cells: harringtonine, isoharringtonine and homoharringtonine. The total alkaloid production in cell suspension cultures was doubled as that in solid cultures. The relative amounts of cephalotaxine, drupacine, harringtonine, homoharringtonine and isoharringtonine in suspension cells was 22%, 6%, 8%, 23% and 41% respectively. In addition, other alkaloid as deoxyharringtonine and some steroids, including ergdst-5-en-3-ol. stigmasta-5, 22-dien-3-ol, β-sitosterin and 2-naphthalenamine have also been detected in cell cultures using GC/MS combined technique.     

Osteogenic and adipogenic capacity of fibroblast-like progenitor cells derived from human fetal liver

The study of the differentiation potential of multipotent stromal progenitor cells (PC) in embryogenesis is a crucial issue for understanding their biology and role in the tissue regeneration of an adult organism. In this study, in monolayer culture, osteogenic and adipogenic potencies of fibroblast-like PCs derived from human fetal liver of 8–11 gestation weeks were investigated before and after exposure to cryoprotectant dimethyl sulphoxide (DMSO). It was shown that the primary suspension of human fetal liver cells includes immature stromal fibroblast-like PCs, which were able to induce osteogenic and adipogenic differentiation. The short-term exposure of recently isolated human fetal liver cells to cryoprotectant DMSO led to alterations in the properties of fibroblast-like PCs. Under subculture conditions, an increase in the number of fibroblast-like PCs capable of inducing osteogenic differentiation in vitro was discovered. It is necessary to take this established fact of DMSO influence on the differentiation capacity of fetal fibroblast-like PCs into consideration when developing cryopreservation methods for stem cells.     

Osteogenic and adipogenic capacity of fibroblast-like progenitor cells derived frow human fetal liver

The study of differentiation potential of multiponent stromal progenitor cells (PCs) in embryogenesis is a crucial issue for understanding their biology and role in tissue regeneration of an adult organism. In this study in monolayer culture there were investigated osteogenic and adipogenic capacities of fibroblast-like PCs derived from human fetal liver of 8-11 gestation weeks before and after exposure to cryoprotectant dimethyl sulphoxide (DMSO). It was shown that the primary suspension of human fetal liver cells included immature stromal fibroblast-like PCs which were able to be induced into osteogenic and adipogenic differentiation. A short-time exposure of freshly isolated human fetal liver cells to cryoprotectant DMSO led to altering properties of the fibroblast-like PCs. Under subculture conditions, it was found an increase in the number of fibroblast-like PCs which were able to be induced to osteogenic differentiation in vitro. The established fact of DMSO influence on the differentiation capacity of fetal fibroblast-like PCs is necessary to take into consideration while developing cryopreservation methods for stem cells.     

野葛细胞培养及其生长特性的研究

目的：以野葛不同器官来源的外植体为材料诱导愈伤组织,建立起悬浮细胞培养体系,研究愈伤组织及悬浮细胞的生长特性。方法：采用细胞生长测定、细胞培养物中有效成分含量测定及细胞的观察。结果：野葛细胞培养过程中异黄酮及葛根素含量随细胞生长逐渐积累,在细胞生长静止期含量最高,次生代谢物与细胞生长呈负相关。不同来源的愈伤组织以根愈伤组织中异黄酮及葛根素含量最高,其中总异黄酮含量为：47．60mg/g Dw、葛根素含量为：3．30mg／g Dw,其次叶、茎、子叶,悬浮细胞中含量最低。不同愈伤组织的细胞及悬浮细胞在生长过程中细胞形态有差异。     

Somatic embryogenesis and plantlet regeneration in the soybean Glycine max

A tissue culture procedure for the regeneration of somatic embryos and plantlets from somatic cells of the soybean Glycine max is described. Bean pods of soybean cv. TGM119 were immersed in liquid nitrogen for 20 minutes. Young embryos were excised from the immature seeds and cultured to form calli. Calli grown from the young embryos were incubated in liquid culture for two weeks. The liquid suspension culture was filtered to obtain single cells. The soybean cells were cultured for one month in a liquid medium in hanging drop cultures for development into proembryoids. The proembryoids were maintained on a solid growth medium for 40 days. The resultant callus tissue was transferred into MS media containing selected combinations and concentrations of 2,4-Dichlorophenoxyacetic acid, Naphthaleneacetic acid, Kinetin, Benzyladenine and Indoleacetic acid. In the presence of Benzyladenine (0.2 mg/l) and Indoleacetic acid (0.01 mg/l), globular and heart shaped somatic embryos were formed on the surface of the calli. Calli containing somatic embryos were transferred into liquid medium and incubated under low light conditions. After six months further incubation, more than 1,000 plantlets and a large number of somatic embryoids at various developmental stages were obtained per flask.Abbreviations KT kinetin - CM coconut milk - BA benzyladenine - NAA napthalene acetic acid - IAA indole acetic acid - 2,4-D 2,4 dichlorophenoxy acetic acid - MS Murashige and Skoog medium     

红藻糖苷对超低温冻存微藻的保护作用

为研究红藻糖苷对超低温冻存微藻细胞的保护作用,研究将3种不同的微藻置于含10% DMSO和不同浓度红藻糖苷的冻存液中,冻存并解冻后,以流式细胞仪检测细胞存活率,测定复养后藻株的生长曲线及相关生理参数。结果显示,由于冷冻损伤,冻存后各种藻细胞的生长速率、细胞密度及生理指标都显著性下降,而红藻糖苷协同DMSO能够显著增加细胞的存活率,尤其15%红藻糖苷能将紫球藻存活率提升20%（P0.05）;生长曲线得到明显改善;且对PSII最大光能转化效率也有显著性提高（P0.05）。总体结果来看,红藻糖苷对超低温冻存微藻,特别是紫球藻具有明显的保护作用,且效果强于蔗糖。     

Cryopreservation of the SB-M photosynthetic soybean [Glycine max (L.) Merr.] suspension culture

Summary The photosynthetic cell suspension culture of soybean [Glycine max (L.) Merr. cv. Corsoy] (SB-M) was successfully cryopreserved in liquid nitrogen using a preculture and controlled freezing to −40° C (two-step) freezing method. The effective method included a preculture treatment with gradually increasing levels of sorbitol added to the 3% sucrose already present in the medium. The cells were then placed in a cryoprotectant solution [10% DMSO (dimethylsulfoxide) and 9.1% sorbitol, or 10% DMSO and 8% sucrose], incubated for 30 min at 0° C, cooled at a rate of 1° C/min to −40° C, held at −40° C for 1 h, and then immersed directly into liquid nitrogen. The cells were thawed at 40° C and then immediately placed in liquid culture medium. The cell viabilities immediately after thawing were 75% or higher in all cases where cell growth resumed. The original growth rate and chlorophyll level of the cells was recovered within 40 to 47 d. If the sorbitol level was not high enough or the preculture period too short, growing cultures could not be recovered. Likewise, survival was not attained with cryoprotectant mixtures consisting of 15% DMSO, 15% glycerol, and 9.1% sucrose or 15% glycerol and 8% sucrose. The successful method was reproducible, thus allowing long-term storage of this and certain other unique photosynthetic suspension cultures in liquid nitrogen.     

Embryogenic cell suspensions of Triticum aestivum x Leymus angustus F1 hybrids: characterization and plant regeneration

Summary Embryogenic cell suspension cultures were established from Triticum aestivum X Leymus angustus F₁ hybrids, using compact nodular calli derived from inflorescence segments. Calli originating from leaf segments did not give rise to stable cell suspensions. Growth measurements of the cell suspensions revealed that they continued rapid growth up to 10 days after subculturing. Flow cytometric studies of the cell cycle over a 7 day culture period showed that the majority of cells were in G₁ phase while the rest were either in S or G₂. During the 7 days of culture, no significant differences in DNA distribution patterns were observed. The cells from suspension cultures produced somatic embryos when they were transferred to different solid media. The embryos germinated and gave rise to plantlets which were successfully rooted and transferred to soil.     

三分三悬浮培养细胞中苯丙氨酸、鸟氨酸和莨菪碱至东莨菪碱的生物转化

在三分三悬浮培养中分别补充前体苯丙氨酸、鸟氨酸和莨菪碱时,悬浮细胞的生长速率及东莨菪碱的转化率均以补充苯丙氨酸者为最高,分别比未加前体的对照增加50.2％和4.0％。 在上述处理培养两周后于培养液中分别加入70ppm的放线菌酮抑制蛋白质合成时,虽然细胞中东莨菪碱的转化按克干重计算均有明显增加,但是由于细胞生长受强烈抑制不利于东莨菪碱的总的转化,按每瓶总干重计算的转化率均比对照低。 在分别补充三种前体并培养两周后使营养缺乏时,细胞生长比对照稍有降低,但细胞中东莨菪碱的转化却显著增加。特别是补充莨菪碱并使营养缺乏时,大大利于莨菪碱转化为东莨菪碱,转化率高达27.4％。既降低了培养成本又提高了东莨菪碱产量,有利于未来的工业生产。     

Protoplast Culture and Plant Regeneration of Malus pumila

Calli were induced and suspension cell lines were set up from ovule of Malus pumila Mill. Protoplasts (5.40 × 106/g fr. wt) were isolated from suspension cell lines in enzyme mixture solution containing 2.0% Onozuka R-10, 0.5% pectinase, 0. 65 mol/L mannital, 0.01 mol/L CaC12, 0.7 mmol/L KH2 PO4, 0.3% dextran sulfate potassium salt, at pH 5.8 for 6 h at 26℃. The cell clumps were formed from protoplasts cultured in modified MS, K8p, D2 media. Calli were formed on MS solid medium containing 2.0 mg/L IAA, 2.0 mg/L NAA, 0.1 mg/L BA. Shoots were differentiated on differentiated medium after several changes of the medium. Eventually, shoots rooted and developed into whole plantlets on a rooting medium.     

油樟悬浮培养及次生代谢产物的诱导

以油樟（Cinnamomum longepaniculatum）叶片为外植体在附加6-BA和NAA不同激素浓度组合的MS培养基上筛选质地疏松、生长旺盛的愈伤组织,分别接种在MS、B5、WPM三种液体培养基中进行细胞悬浮培养,并检测诱导产生的次生代谢产物。结果表明：2mg·L^-16-BA＋0.5mg·L^-1NAA能诱导质地疏松、生长旺盛的愈伤组织,B5基本培养基中细胞长势最好;愈伤组织在B5＋2mg·L^-16-BA＋0.5mg·L^-1NAA中悬浮培养,继代2次后形成均一的单细胞;从油樟悬浮培养物中检测出50%以上的成分是苯甲醇。     

Optimization of growth and jaceosidin production in callus and cell suspension cultures of Saussurea medusa

Calli cultures derived from the leaves of Saussurea medusa were selected on the basis of colour into three callus, A, B and C, which suggested different levels of metabolite accumulation. An improved reversed phase high performance liquid chromatographic method provided selective determination of the jaceosidin content of these samples. The jaceosidin concentration in callus B was higher than that of the callus A and C. By using 12-day old culture and 9-day old inoculum, jaceosidin yield of 72.91 mg l^–1was obtained from cell line B in cell suspension cultures. The influence of some factors affecting jaceosidin formation, i.e. temperature, light, inoculum size, type of media, phytohormones, nitrogen and carbon source etc. were also examined. Light irradiation and combination of 3% (w/v) sucrose with 1% glucose brought about a marked increase of jaceosidin production. The effect of blue light on jaceosidin was markedly superior to other kinds of monochromatic light (red and far-red) or white light. Analysis of growth and jaceosidin content of callus cultures and cell suspension cultures demonstrated that the production of jaceosidin was growth-dependent in both cell solid culture and cell suspension culture.