红花单细胞克隆的建立

KT,2,4-D 及 NAA 能提高红花(Cathamus tinctorius)细胞克隆平板培养的植板率。这三种激素对细胞生长的最佳搭配是2,4-D2.0mg/l,KT0.3mg/l,NAA 0.5mg/1。红花细胞悬浮继代培养代数不同,其植板率相差甚远,用悬浮培养第三代的细胞做材料最好,其植板率是第一代悬浮培养细胞做材料的8.5倍。红花细胞克隆的条件培养的植板率是普通平板培养的3.6倍。固-液双层培养的植板率是普通平板培养的4.7倍。对已建立的红花细胞克隆进行生长速率的比较表明,生长最漫的克隆的生长速率为3.08g/g/35天,生长最决的克隆的生长速率高达23.33g/g/35天。     

沙打旺悬浮培养细胞原生质体的体细胞胚胎发生再生植株(英文)

以继代培养5—24代的沙打旺(Astra-galus adsurgens Pall.)胚性悬浮系为材料,从生长4d的细胞培养物中可游离出大量有活力的原生质体。细胞预质壁分离或低温预处理对原生质体得率和活力没有影响,但可提高原生质体培养的植板率。预处理的原生质体培养在NH_4NO_3浓度降至2.5mmol/L,并附加0.5mg/L NAA、1.0mg/L 2,4-D、0.7mg/L BA和0.4mol/L葡萄糖的KM8P培养基中,经持续分裂形成细胞克隆,植板率高达16%—20%。细胞克隆在附加1.0mg/L 2,4-D和0.5mg/L BA的MS培养基中增殖后,转至含0.1mg/L NAA和1.0mg/L BA的MS分化培养基中可形成体细胞胚,平均体细胞胚发生频率约为40%,每个克隆产生20—40个体细胞胚。在无激素的1/2MS培养基中,体细胞胚发育成形态正常和染色体数稳定的小植株。     

沙打旺胚性原生质体培养优化及高频再生植株

外植体类型和光照条件决定沙打旺胚性愈伤组织的形成。用生长10d的胚性愈伤组织可分离到1.2×10⁶个/g(原生质体/细胞),活力超过80%。当原生质体以1.0×10⁵/mL的植板密度培养在含0.6%琼脂糖附加1.5mg/L 2,4-D、0.5mg/L BA和0.5mol/L葡萄糖的培养基(无机盐降为1/4)中,植板率为16.8%。条件培养基显著促进原生质体的生长发育。长大的细胞克隆经2周4℃低温处理后转到含0.1mg/L NAA和1.0mg/L BA分化培养基上,体细胞胚胎发生频率高达70%,每克细胞产生的体细胞胚数在200个以上。成熟的体细胞胚转到无激素的1/2MS培养基中即分化成苗,再生植株为正常的二倍体。     

人参培养细胞的单细胞克隆

培养基组成成分能影响人参培养细胞单细胞克隆的植板率。Ms培养基中2,4－D和KT需要一个适合的浓度比才能有效地促进细胞克隆的形成,其最佳浓度组合是2,4－D1．5mg／L和KT O．5mg／L。适合人参细胞克隆形成的NH₄N0₃浓度是400mg／L,CaCl₂.2H₂O浓度是750mg／L。向培养基中补加适量的琥珀酸、精氨酸和维生素等都能明显提高植板率。通过优化培养基的组成成分,细胞克隆的植板率可增加到2.34倍。悬浮培养两周左右的细胞平板培养最有利克隆形成,当细胞植板密度低于4×10³个细胞／ml时,几乎没有克隆形成。     

银杏致密细胞团颗粒悬浮培养生产银杏内酯研究

以MS培养基上培养的银杏高产细胞系MH－3为材料,SH培养基为出发培养基,通过优化营养元素和激素配比,建立银杏致密细胞团悬浮培养体系,在培养过程中添加前体异戊二烯和香叶醇有助于提高银杏内酯产量。结果表明：颗粒粒径大小影响银杏内酯的产生,选用0．8SH培养基,添加浓度为3mg/L的2,4－D和2mg/L的6－BA,致密颗粒的平均粒径为3．36mm,细胞中银杏内酯的含量为557μg/g.dw.在培养过程中的10d添加100mg/L的香叶醇,细胞中银杏内酯的含量达到676μg/g.dw。     

藏红花细胞悬浮培养体系的建立及优化

基于诱导的藏红花细胞系,通过摇瓶法,优化了其液体培养基、接种量和种龄等培养条件,以建立藏红花细胞悬浮培养体系。结果表明,将生长在固体培养基上的藏红花愈伤组织接种在MS液体培养基(添加了2mg/L2,4-D,1mg/L6-BA和300mg/LCH)中,于(22±0.3)℃,120r/min的摇床上,暗培养30d,便可获得藏红花的悬浮细胞系。经优化其培养基、接种量和种龄,将种龄为20d的细胞系,按照5%接种量接种在液体B5培养基(添加了2mg/LNAA,1mg/L6-BA和300mg/LCH)中,于(22±0.3)℃,120r/min的摇床上,培养36d,细胞生物量(13.4g/L)和藏红花素产量(0.91g/L)均达到最高。本研究建立的藏红花细胞悬浮培养体系为其生物反应器放大培养奠定了基础。     

表达重组抗CD52单克隆抗体CHO细胞灌流培养基的筛选

目的筛选重组抗CD52单克隆抗体CHO细胞株培养和连续灌流表达用培养基,以提高抗体表达量。方法通过调整原有批培养用培养基中谷氨酰胺和植物水解蛋白,获得5种培养基配比。使用模拟灌注方式进行细胞培养,分析细胞密度、活细胞比率和目标蛋白表达,筛选连续灌流细胞培养和表达用培养基。最后在7 L反应器中采用灌注培养方式对筛选获得的培养基进行验证。结果使用50 mL细胞培养管进行模拟灌注培养时,活细胞比率较高,达到90%以上;CHO细胞在添加谷氨酰胺至4.0 mmol/L和植物水解蛋白至5.0 g/L的批培养用培养基中生长速度最快;在基础培养基中抗体表达量比优化前高15%。20 d培养周期内,优化的培养基在7 L反应器中可以维持CHO细胞密度在(2 727±253)万个/mL,活细胞比率在95%以上。结论通过模拟灌注培养,筛选获得了一种在7 L反应器灌流培养中适宜于重组抗CD52单克隆抗体CHO细胞表达的培养基。     

人NK细胞克隆化培养条件的初步探讨

为探讨NK细胞克隆化培养的条件,我们首先将人外周血单个核细胞经rhIL-2诱导,使NK细胞得以扩增后,去除T细胞,得到相对纯化的NK细胞.经有限稀释,在饲养细胞及rhIL-2、PHA及LCM等培养条件下,获得NK细胞的单个克隆并进行鉴定.结果表明,每96孔板可获4-16个CD3-CD56+NK克隆,每个克隆的细胞数最多可达2.35×104,存活约3-5周.以丝裂霉素C(25μg/m1)作为饲养细胞抑制剂,以rhIL-2 200u/ml、PHA 10μg/ml及10%LCM培养,可获得较多的克隆和细胞数.     

高产莪术细胞悬浮系建立及产物前体对挥发油合成的调控研究

研究了高产莪术细胞悬浮系培养的条件及前体物质添加对挥发油合成的调控。结果表明：淡黄色颗粒状愈伤组织是建立高产细胞悬浮系的最佳供试愈伤组织;最佳培养基成分是MS培养基添加葡萄糖与蔗糖各15—30g／L(1：1),氮源为NH4^ 和NO3^-,比例为1：3,总量为80mmol／L;激素组合为6-BA3．0—5．0mg／L、2,4-D1．0mg／L;光下培养10—15天再转入优化条件下的暗培养,可形成稳定的高产细胞悬浮系;其细胞周期中的最大细胞生长量及挥发油含量分别是248g／L和2．28％;前体物质泛酸钙、乙酸铵、乙酸钾的添加均可有效提高培养细胞合成挥发油的百分含量,其中乙酸铵最有效,在指数生长中期添加0．5mmol／L乙酸铵,挥发油的最高含量可达3．11％,产量为8．27g/L,分别是添加前的1.25倍及1.2倍。     

建立金线莲原球茎悬浮体系生产次生代谢产物

研究植物激素浓度和培养周期对金线莲原球茎悬浮培养生长及其代谢产物积累的影响,以增加金线莲悬浮培养的生长量,提高次生代谢产物的生产。结果表明,MS培养基添加S-3307 1.0mg/L,6-BA0.5mg/L和3%的蔗糖适合总生物量的生长(214.45g/L,FW和18.23g/L DW)。而MS培养基添加S-3307 1.0mg/L,6-BA 3.0mg/L和5%的蔗糖,总黄酮,总酚和多糖的干重(5.43mg/g,2.87mg/g和243.23mg/g)达到最大化。研究原球茎悬浮培养过程,发现经过7个星期培养就能获得最大的生物质总量(225.98 g/L的FW和18.53 g/L的DW)、总黄酮干重(5.09mg/g)和总酚干重(2.04mg/g),而多糖生产达到其峰值(229.36mg/g干重)是在培养后5个星期。     

低密度和条件培养对红豆杉细胞生长的影响

红豆杉种胚来源的细胞,在改良Ｂ５液体培养基中继代培养的临界接种密度为鲜重４０ｇ／Ｌ．低密度培养下,１０－１６ｄ的条件培养液（ＣＭ）与新鲜培养液按５７：４３的比例混合时,能显著缩短细胞生长的延迟期,提高生长率,１００Ｌ生物反应器中,按４５．５％体积分数添加条件培养液,在鲜重２７ｇ／Ｌ低接种密度下培养５周,生物量增长９倍,达干重１４．３ｇ／Ｌ．对内源植物激素、精胺、维生素和氨基酸等的比较分析表明,吲哚     

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％。     

聚乙二醇对东北红豆杉培养细胞的

在改良的B     

Lobeline production by hairy root culture of Lobelia inflata L.

Hairy roots were obtained following inoculation of the stems of Lobelia inflata L. with Agrobacterium rhizogenes strain ATCC 15834. These hairy roots contained agropine and mannopine. In addition, lobeline was detected by HPLC and confirmed by mass spectrometry. Various media were tested for the growth of hairy roots as well as for the content of lobeline in hairy roots. The growth rate of hairy roots cultured in Nitsch and Nitsch's medium was approximately one third of those cultured in other media. The lobeline content of hairy roots (18–54 g/g dry weight) cultured in these media was the same order of magnitude compared with that of roots of L. inflata (24 g/g dry weight) cultivated in pots. The hairy roots cultured in Nitsch and Nitsch's medium were morphologically different from those cultured in other media.Abbreviations MS medium Murashige and Skoog's medium - 1/2 MS medium one-half strength of the standard Murashige and Skoog's medium - B5 medium Gamborg's B5 medium - NN medium Nitsch and Nitsch's medium - FW fresh weight - DW dry weight     

尖顶羊肚菌液体培养基质与条件的研究

通过对尖顶羊肚菌液体培养基质与条件的研究,明确其菌丝生长的最适pH值、最适温度、适宜光照条件、适宜葡萄糖和蛋白胨浓度、适宜培养基,以便应用于尖顶羊肚菌液体菌种的生产和工业发酵。结果表明:菌丝的最适生长温度为2 5℃;最适生长pH值为6 ;葡萄糖和蛋白胨最适浓度分别为2 0 0g/L和10g/L ;菌丝在黑暗环境下生长良好,光照对菌丝生长具有抑制作用;用胡萝卜酵母膏培养基振荡培养形成的菌丝球多,菌丝生长量大;菌丝球在不同培养基中生长,可引起培养液pH值的上升或者下降;菌丝球可利用培养基内的氨基酸,使氨基酸降解,在胡萝卜酵母膏培养基中振荡培养8d的菌液总氨基酸含量较原液减少了36 71% ,亮氨酸、异亮氨酸和甲硫氨酸含量的下降幅度最大     

Ethanolic fermentation of hydrolysates from ammonia fiber expansion (AFEX) treated corn stover and distillers grain without detoxification and external nutrient supplementation

External nutrient supplementation and detoxification of hydrolysate significantly increase the production cost of cellulosic ethanol. In this study, we investigated the feasibility of fermenting cellulosic hydrolysates without washing, detoxification or external nutrient supplementation using ethanologens Escherichia coli KO11 and the adapted strain ML01 at low initial cell density (16 mg dry weight/L). The cellulosic hydrolysates were derived from enzymatically digested ammonia fiber expansion (AFEX)-treated corn stover and dry distiller's grain and solubles (DDGS) at high solids loading (18% by weight). The adaptation was achieved through selective evolution of KO11 on hydrolysate from AFEX-treated corn stover. All cellulosic hydrolysates tested (36-52 g/L glucose) were fermentable. Regardless of strains, metabolic ethanol yields were near the theoretical limit (0.51 g ethanol/g consumed sugar). Volumetric ethanol productivity of 1.2 g/h/L was achieved in fermentation on DDGS hydrolysate and DDGS improved the fermentability of hydrolysate from corn stover. However, enzymatic hydrolysis and xylose utilization during fermentation were the bottlenecks for ethanol production from corn stover at these experimental conditions. In conclusion, fermentation under the baseline conditions was feasible. Utilization of nutrient-rich feedstocks such as DDGS in fermentation can replace expensive media supplementation.     

影响油菜子叶外植体不定芽高频率再生的因素

采用７个甘蓝型和２个白菜型油菜品种研究了影响子叶外植体芽再生的一些因素和不同基因型的子叶外植体的离子体培养反应和芽再生能力。结果表明,苗龄４ｄ的幼苗叶子含２,４－Ｄ０．５－１．２ｍｇ／Ｌ和６－ＢＡ０．２ｍｇ／Ｌ的培养基培养２或６ｄ后再转到分化培养基上培养,芽再生率３４％－４６％。     

Selection and proliferation of rapid growing cell lines from embryo derived cell cultures of yew tree (Taxus cuspidata Sieb. et Zucc)

Calli were induced from 300,000 embryos isolated from immature to mature stage of seeds collected on late September from 14 elite trees. When the embryos were cultured onto plastic Petri-dish containing 20 mL of modified B5 basal medium supplemented with 3% (w/v) sucrose, 500 mg/L casein hydrolysate, 250 mg/L myo-inositol, 0.5% (w/v) polyvinyl polypyrrolidon (PVPP), 2×MS vitamins, 0.5 mg/L gibberellic acid, and 10 mg/L 2,4-D after 2 weeks of culture, yellowish-white calli were immediately formed on the surfaces of embryos, and subcultured for 4 weeks in same culture medium. Because most of calli maintained for more than 3 months were revealed differences in their colors, surface texture, and growth rate, visual selection was made for first round screening. When the size of visually selected calli larger than 19 mm in their diameter were inoculated, persistent proliferation was observed. Among the plating methods tested for the selection of rapid growing cell lines at single cell and/or small cell aggregate level, 2-layer spread plating revealed as the best for single cell cloning. To enhance cell growth and maintain high rate of viability for long-term culture of yew cells in bioreactor, final cell volume less than 50% in SCV seemed to be the best. Time course study revealed that 30% of inoculum density was suitable for fed batch culture. Among the tested conditional media, the rate of 1∶2 (old medium: fresh medium) was recorded at the best for cell growth.     

An novel immobilization method of Saccharomyces cerevisiae to sorghum bagasse for ethanol production

Natural sorghum bagasse without any treatment was used to immobilize Saccharomyces cerevisiae at 0.6+/-0.2g dry cell weight (DCW)/g dry sorghum bagasse weight (DSW) through solid-state or semi-solid state incubation. The scanning electron microscopy (SEM) of the carriers revealed the friendship between yeast cells and sorghum bagasse are adsorption and embedding. The ethanol productivity of the immobilized cells was 2.24 times higher than the free cells. In repeated batch fermentation with an initial sugar concentration of 200g/L, nearly 100% total sugar was consumed after 16 h. The ethanol yield and productivity were 4.9 g/g consumed sugar on average and 5.72 g/(Lh), respectively. The immobilized cell reactor was operated over a period of 20 days without breakage of the carriers, while the free cell concentration in the effluent remained less than 5 g/L thoughout the fermentation. The maximum ethanol productivity of 16.68 g/(Lh) appeared at the dilution rate of 0.3h(-1).     

In vitro-selection of hydroxyproline-resistant cell lines of wheat (Triticum aestivum): accumulation of proline, decrease in osmotic potential, and increase in frost tolerance

Five hundred hydroxyproline-resistant cell lines were selected from cell cultures of wheat ( Triticum aestivum L. cv. Koga II) after plating on 10 to 30 m M hydroxyproline (Hyp) containing solid Gamborg B 5 medium. All selected cell lines from 30 m M Hyp-medium contained increased (up to 17-fold) levels of free proline. Seventy-four cell lines were transferred to Hyp-free medium and subcultivated 25 times, for 12 months altogether, after which 80% still had increased proline levels. Fourteen cell lines with increased proline levels were further investigated in liquid media with regard to their frost tolerance, which was measured by means of electrolyte leakage. Ten of them showed increased fros tolerance, with LT 50 values as low as 2.7°C below that of the wild type (-4.7°C). Besides increased proline levels and increased percentage dry weight, the Hyp-resistant cell lines had lower osmotic potentials. Osmotic potentials correlated better than levels of free proline with the increase in frost tolerance.