高产人参寡糖素培养细胞克隆系的筛选

人参（ＰａｎａｘｇｉｎｓｅｎｇＣ．Ａ．Ｍｅｙ．）培养细胞经细胞平板克隆,获得近３００个克隆系。克隆系在细胞生长速率和寡糖素含量及产率上均存在显著差异,且寡糖素产率和细胞生长之间有明显的相关性。经１１代连续继代培养观察及过氧化物酶同工酶谱特征分析,筛选到一株寡糖素产率高且稳定的克隆系ＰＧ－１８０,其平均生长速率是０．４９５ｇ于重／Ｌ·天,为原始株系的１．３９倍,平均寡糖素含量为１４．６９％干重,是亲本的１．６５倍,平均寡糖素产率是２．１８３ｇ／Ｌ,为原始株系的２．３２倍。比较克隆系ＰＧ－１８０和原始株系细胞悬浮培养时间进程发现,由人参培养细胞生产寡糖素的最佳细胞收获期为３周左右。     

高产人参寡糖素培养细胞克隆系的诱变筛选

紫外辐射能显著地降低人参培养细胞单细胞克隆的植板率。当紫外辐射悬浮细胞３０ｓ后,细胞克隆的植林率是对照组的２１．４３％。细胞克隆平板培养６０ｄ,挑取克隆连续转移培养３次,共获得克隆系１２２株。对所有克隆系进行变异分析并经１０代连续继代培养,从中筛选到一株稳定的高产寡糖素克隆系ＰＧＵＡ－０８,而且它的过氧化物酶同工酶谱特征也保持稳定。克隆系ＰＧＵＡ－０８的生长速率为０．５３７ｇＤＷＬ－１ｄ－１,是亲本的１．４６倍,寡糖素含量为１７．１６％ＤＷ,是亲本的１．８１倍,寡糖素产率为２．７６４ｇ／Ｌ,是亲本的２．６２倍。     

细胞培养生产人参寡糖素降低成本的途径

在人参（Ｐａｎａｘｇｉｎｓｅｎｇ）细胞悬浮培养中,以无离子水代替重蒸馏水,细胞生长速率和寡糖素产率分别降低２．３％和２．９％。用白糖代替蔗糖,细胞生长速率和寡糖素产生率分别降低１．７４％和１．２３％。综合上述两方面结果,以无离子水和白糖分别替代原培养基中的重蒸馏水和蔗糖组成替代培养基,用替代培养基培养人参培养细胞,其生长速率可达０．５０９ｇＤＷ／Ｌ．ｄ．寡糖素产率可达１．４４３ｇ／Ｌ,和原培养基相     

粉叶小檗愈伤组织单细胞克隆

以粉叶小檗愈伤组织为材料 ,用B5液体培养基进行悬浮培养建立悬浮细胞系。经 3～ 4次继代培养即可得到悬浮的单细胞。悬浮细胞通过细胞平板克隆 (一般B5培养基平板克隆 ,优化培养基平板克隆和条件培养基平板克隆 ) ,经 5代连续继代培养观察和薄板层析 -分光光度法分析 ,发现用优化培养基进行平板克隆植板率最高 ,且克隆最易成功 ,并且还筛选到一株小檗碱产率高且稳定的克隆CV 5 7,其平均生长速率为 14 .4 12mg .Fw/L .d ,为原始株系的 1.91倍 ,平均小檗碱含量为 2 .17%干重 ,是原始株系的 2 .2 6倍。     

三七.人参和西洋参细胞悬浮培养的比较研究

用薄层层析对三七、人参和西洋参愈伤组织进行的初步鉴定表明,三种愈伤组织都含有皂甙和主要皂甙成分Rb_1、Rg_1,三七愈伤组织还含有一种抗癌皂甙Rh_1。对愈伤组织的生长,三七低于人参高于西洋参;对愈伤组织中总皂甙含量,三七均高于人参和西洋参。三种植物细胞悬浮培养结果类似于他们的愈伤组织培养,但生长又进一步提高。三七细胞悬浮培养中皂甙产生的时间进程几乎与生长平行,合适的收获期为培养30天。寡糖素不仅增强三七培养细胞的皂甙形成而且促进细胞生长,较合适的浓度为1.25 ppm。通过以上研究,使三七悬浮培养细胞的生长(干重增加178毫克)为最初培养愈伤组织的4倍以上,总皂甙产率高达20.6毫克,为最初培养愈伤组织的8.5倍。     

人参寡糖素对三七悬浮培养细胞生长的效应

从人参培养细胞的细胞壁中分离纯化到不同分子量的单体人参寡糖素。试验结果表明命名为人参寡糖素Ⅶ和人参寡糖素Ⅷ的两种寡糖素对三七悬浮培养细胞的生长具有明显的促进作用,其增长率分别为１９．３４％和１０．５８％,人参寡糖素Ⅶ的适宜浓度为５—１０ｍｇ／ｌ。在高浓度下（大于２５ｍｇ／ｌ）稍抑制培养细胞生长。在细胞培养２２天（指数生长期）后．加入１０ｍｇ／ｌ的人参寡糖素Ⅶ．然后再培养２天。其生长速率即提高,加入人参寡糖素Ⅷ后．缩短了三七细胞悬浮培养生长的延缓期．提前进入对数生长期和指数生长期,并在对数生长期和指数生长期作用最明显,因而最终收获时培养细胞的产率增加。     

Studies on Fermentation Culture of Cells of Panax notoginseng

The saponin content of Panax notoginseng cell cultures-was 11.14% of dry weight, the saponin yield was 1513.3mg· L-1, and yield of cell cultures was 13.58g dry wt · L-1 per month in fermentation culture, which were all better than those obtained from in suspension culture. Increasing inoculum quantity could obviously increase growth rate, saponin content and yield of cell cultures. An aeration rate of 0.8vvm was optimal for fermentation culture of the cells. The pH value of the culture broth went down from 5.80 to 3.92 gradually and never returned in fermentation culture of P. notoginseng cells.     

Rosmarinic acid synthesis in transformed callus culture of Coleus blumei benth

Agrobacteria mediated Coleus blumei tumour tissues were cultured in vitro on MS medium. Sixteen diversified transformed callus cultures were maintained for several years in the absence of plant growth regulators and antibiotics without affecting the growth rate. Rosmarinic acid was detected spectrophotometrically in all tissue lines but in different quantities. The highest rosmarinic acid accumulation detected was 11% of dry tissue mass. The relation between culture growth and rosmarinic acid production was investigated in three callus lines. The lines showed different rosmarinic acid accumulation in relation to their growth rate; it was either parallel or inversely related to the tissue growth. The effects of certain medium constituents on the callus growth and rosmarinic acid accumulation were examined in four tumour cell lines. Addition of 4% or 5% sucrose stimulated rosmarinic acid synthesis and decreased callus growth. Nitrogen reduction to one half or one quarter of initial concentration did not affect rosmarinic acid synthesis and decreased callus growth in three lines, while it increased rosmarinic acid accumulation and callus growth in one line. Addition of 0.1 mg/l Phe stimulated rosmarinic acid production in two lines but had little effect on the rosmarinic acid level in others. Rosmarinic acid production was significantly improved on modified macronutrients, where the Ac2 line produced 16.5 mg of rosmarinic acid per tube (0.2 g of dry wt) after being in culture for 35 days.     

寡糖素对红花及三七培养细胞的生理作用

三种寡糖素,即来自人参(Panax ginseng)培养细胞的人参寡糖素、红花(Carthamus tinctorius)培养细胞的红花寡糖素、黑节草(Dendrobium candidum)植物的黑节草寡糖素对红花及三七 (Panax notoginseng)的培养细胞的生长及代谢产物的含量均有显著的促进作用。寡糖素可耐高温高压(121℃、1.2bs／cm2)灭菌15分钟而不失活,其对植物培养细胞的影响与利用过滤方法灭菌的效果相似。红花寡糖素对红花悬浮培养细胞作用的适宜浓度是5-10mg/L,而在愈伤组织中为15mg/L,在三七培养细胞进入生长旺盛(培养至22天)时加入黑节草寡糖素,再培养2天后其生长即提高。黑节草寡糖素均能缩短红花及三七培养细胞生长的延缓期,提前进入对数生长期及指数生长期。并且使红花培养细胞中a-生育酚在细胞生长最活跃的指数生长期大量积累,最终增加了培养细胞及代谢产物的产率。     

Establishment of Salvia officinalis L. hairy root cultures for the production of rosmarinic acid

Shoots of Salvia officinalis, a medicinally important plant, were infected with Agrobacterium rhizogenes strains ATCC 15834 and A4 which led to the induction of hairy roots in 57% and 37% of the explants, respectively. Seven lines of hairy roots were established in WP liquid medium under light and dark conditions. The transformed nature of the root lines was confirmed by polymerase chain reaction using rolB and rolC specific primers. Transformed root cultures of Salvia officinalis showed variations in biomass and rosmarinic acid production depending on the bacterial strain used for transformation and the root line analyzed. Both parameters (growth and rosmarinic acid content) of ATCC 15834-induced lines were significantly higher than the A4-induced lines. The maximum accumulation of rosmarinic acid (about 45 mg g(-1) of dry weight) was achieved by hairy root line 1 (HR-1) at the end of the culture period (45-50 days). The level was significantly higher than that found in untransformed root culture (19 mg g(-10 of dry wt).     

High berberine-producing cultures of coptis japonica cells

The highest berberine content of unselected Coptis cells cultured under the best conditions for berberine production (darkness, high aeration, 3% sucrose and White's basal medium) was about 5% on a dry wt basis. Fluoromicroscopy showed that cultured Coptis cells had heterogeneous characters; therefore, selection was used to establish a high berberine-producing culture of Coptis cells. When small cell aggregates were cloned, high berberine-producing cell lines were produced. Repeated cloning, however, was needed to obtain stable cell lines that produced large amounts of berberine. The highest berberine production in a selected cell line was 13.2% on a dry wt basis (1.39 g/l. culture). The average production was 8.2% (0.90 g/l. culture).     

人参悬浮细胞系的建立及其生长特性的研究

从人参幼叶的培养中,筛选出了质地松疏、生长迅速、易于分散、可以长期进行继代培养的淡黄色半透明状愈伤组织系。将这种愈伤组织接种在液体培养基中进行振荡培养．建立起分散程度好的人参悬浮细胞系。在此基础上,测定了人参细胞悬浮培养物的生长曲线。实验表明,水解酪蛋白（ＬＨ）对人参悬浮细胞的生长有利。滋养培养可以使人参悬浮细胞的愈伤组织形成率提高,并在低密度下达到较高的植板率。这为有效地筛选出适合于工业化生产的高产人参细胞株提供了方便。     

Production of tilapia insulin-like growth factor-2 in high cell density cultures of recombinant Escherichia coli

An improved expression plasmid pET-insulin-like growth factor-2 (IGF2) was constructed and transferred into Escherichia coli BL21(DE3) for the expression of tilapia insulin-like growth factor-2. The recombinant insulin-like growth factor-2 was produced as inclusion bodies, and the recombinant insulin-like growth factor-2 content was as high as 10.3% of the total protein content. For production of recombinant insulin-like growth factor-2 in E. coli, pH-stat fed-batch cultures were used to achieve a high cell density culture. A cell concentration 183gl(-1) dry cell weight (DCW) was obtained after 30h cultivation and plasmid stability was maintained at high levels. Expression of insulin-like growth factor-2 was induced at three different cell concentrations, 50, 78.5, and 114.5gl(-1) dry cell weight. When cells were induced at a cell concentration of 114.5gl(-1) dry cell weight, the amount of insulin-like growth factor-2 produced was 9.69gl(-1) (11.3% of the total protein). Using a simple purification process including inclusion body isolation, denaturation, refolding and Ni-NTA affinity chromatography, 19.51mg of insulin-like growth factor-2 was obtained from a 22.5ml of culture, and the recovery yield was 20.5%. The biological activity of the purified IGF-2 was demonstrated as promoting the growth of four different cell lines by the colorimetric bioassay and the best growth stimulation ratio was obtained for the Balb/3T3 clone 31A cell line.     

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.     

Characteristics of transformedPanax ginseng C.A. Meyer hairy roots: Growth and nutrient profile

Ginseng (Panax ginseng C.A. Meyer) hairy root cultures, which are established via the infection of ginseng root discs withRhizobium rhizogenes, have been used to construct profiles of both biomass growth and nutrient consumption in flask cultures. In a 250 mL shake flask culture, the maximum biomass was observed on the 59th day of the culture period, at 216.8 g (fresh wt) per liter or 11.4 g (dry wt) per liter. The hairy roots were determined to have a growth rate of 0.355 g-DW/g cells/day during the exponential growth phase and a maximum specific growth rate on day 7. Total ginseng saponin and phenolic compound contents were noted to have increased within the latter portion of the culture period. Linear correlations between increases in biomass weight and nutrient uptake were used to imply the conductivity yield 2.60 g-DW/(L·mS) and carbon yield 0.45 g-DW/(g sugar) in the 250 mL flask cultures. The biomass yield when two different nitrogen sources were used (ammonia and nitrate) was shown to remain approximately constant, at 0.47 g-DW/(l·mM NH₄) and 0.33 g-DW/(L·mM NO₃); it remained at these levels for 16 days with the ammonia, and for 24 days with the nitrate. The biomass yield when a phosphate source was used was also shown to remain approximately constant for 9 days, at 3.17 g-DW/(L·mM PO₄), with an R² of 0.99.     

The role of oxygen limitation in the formation of poly-β-hydroxybutyrate during batch and continuous culture of Azotobacter beijerinckii

Azotobacter beijerinckii was grown in ammonia-free glucose-mineral salts media in batch culture and in chemostat cultures limited by the supply of glucose, oxygen or molecular nitrogen. In batch culture poly-beta-hydroxybutyrate was formed towards the end of exponential growth and accumulated to about 74% of the cell dry weight. In chemostat cultures little poly-beta-hydroxybutyrate accumulated in organisms that were nitrogen-limited, but when oxygen limited a much increased yield of cells per mol of glucose was observed, and the organisms contained up to 50% of their dry weight of poly-beta-hydroxybutyrate. In carbon-limited cultures (D, the dilution rate,=0.035-0.240h(-1)), the growth yield ranged from 13.1 to 19.8g/mol of glucose and the poly-beta-hydroxybutyrate content did not exceed 3.0% of the dry weight. In oxygen-limited cultures (D=0.049-0.252h(-1)) the growth yield ranged from 48.4 to 70.1g/mol of glucose and the poly-beta-hydroxybutyrate content was between 19.6 and 44.6% of dry weight. In nitrogen-limited cultures (D=0.053-0.255h(-1)) the growth yield ranged from 7.45 to 19.9g/mol of glucose and the poly-beta-hydroxybutyrate content was less than 1.5% of dry weight. The sudden imposition of oxygen limitation on a nitrogen-limited chemostat culture produced a rapid increase in poly-beta-hydroxybutyrate content and cell yield. Determinations on chemostat cultures revealed that during oxygen-limited steady states (D=0.1h(-1)) the oxygen uptake decreased to 100mul h(-1) per mg dry wt. compared with 675 for a glucose-limited culture (D=0.1h(-1)). Nitrogen-limited cultures had CO(2) production values in situ ranging from 660 to 1055mul h(-1) per mg dry wt. at growth rates of 0.053-0.234h(-1) and carbon-limited cultures exhibited a variation of CO(2) production between 185 and 1328mul h(-1) per mg dry wt. at growth rates between 0.035 and 0.240h(-1). These findings are discussed in relation to poly-beta-hydroxybutyrate formation, growth efficiency and growth yield during growth on glucose. We suggest that poly-beta-hydroxybutyrate is produced in response to oxygen limitation and represents not only a store of carbon and energy but also an electron sink into which excess of reducing power can be channelled.     

Characterization of porcine stable kidney cell line adapted to hyperthermic temperature

The optimum temperature for the growth of porcine stable (PS) kidney cell line is 37 degrees C. We have adapted the cell line to grow at 40 degrees C. The original cell line grown at 37 degrees C has been denoted as PS-37, and the adapted new strain has been denoted as PS-40. Both the cell lines were screened for mycoplasma by Hoechst staining and tritiated uridine-uracil uptake and were found to be negative. Comparative characterization of PS-40 and its progenitor PS-37 cell line was done by using various parameters. The antigenic studies indicated that the new cell strain was not cross-contaminated with any other cell lines. It was observed that PS-40 cells were more fibroblastic with clean cytoplasm and appeared healthy. The growth of PS-40 cells was faster than the original cell line. The karyological study showed heteroploid chromosome number in PS-40 cells. The modal chromosome number of PS-40 cells was 58, whereas that of PS-37 cell line was 38. The lactic dehydrogenase isoenzyme pattern showed a cathodal shift of bands. The PS-40 cell strain could be cryopreserved and revived. The viability of PS-37 as well as PS-40 cell lines is in the range of 90-95%, and the growth characteristics of thawed cells showed six- to eightfold multiplications within 5 d. The virus susceptibility study revealed that the cytopathic effect was more profound and observed 1 d earlier in PS-40 cell line. Increased yields of Japanese encephalitis, Sindbis, and Semliki forest viruses were obtained by 1.8, 1.75, and 1.5 log plaque-forming units/ml, respectively. The yield of West Nile virus was, however, comparable to that in PS-37 cell line. Both the cell lines were refractory to Dengue viruses.     

Effects of oligosaccharins on callus growth and saponin content of Panax notoginseng

This work provides some evidences for the saponinproduction of Panax notoginseng callus by using biologi-cally active,wall-related oligosaccharins.In anappropriate concentration,three kinds of oligosaccharinsstimulated saponin formation or callus growth.Theconcentration of DO,GO and CO for saponin productionof Panax notoginseng callus culture were 15ppm,15ppmaud 20ppm respectively by comparing saponin yield.Itwas very obvious for DO to increase saponin contentwhen the concentration was 10ppm,and for GO tostimulate callus growth when the concentration was20ppm.It would be a good way to produce saponin byusing oligosaccharins in large scale culture in thefuture.     

条斑紫菜抗高温和快速生长细胞株系HB的建立及栽培

本文主要应用单细胞培养技术培育紫菜新品系,以建立紫菜细胞工程快速育苗系统。通过酶解技术分离出紫菜叶状体细胞,并进行多克隆,从中获得4个细胞纯系植株(HA,HB,HC,HD)。对该4个细胞株系进行了纯系培养,并对其细胞苗和丝状体的生长速度和抗高温(19℃,21℃,23℃,25℃)性进行了测定。结果显示,船株系具有较高的抗高温性和最快生长速度。在1998年到2000年间,在江苏省启东县海丰海区对HB株系进行了海上栽培实验,结果显示,实验组的产量高于当地对照组产量,证明了条斑紫菜船株系不仅具有较高的抗高温性,而且也有较快生长速度。本实验说明应用细胞工程进行条斑紫菜育种是一条很好的快速育种途径。     

Continued growth of Escherichia coli after stopping medium addition to a K+-limited chemostat culture

The steady-state bacterial dry wt of Escherichia coli, growing under K+-limited conditions in the chemostat, was inversely dependent on the growth rate. This phenomenon was more carefully investigated in medium-flow stop experiments. Growth did not stop immediately but continued for a time, initially at the same rate as before. The dry wt increased to a value corresponding to a steady-state growth rate near zero, independent of the initial specific growth rate. This was observed in both the wild-type strain and a mutant that lacked the high-affinity K+ uptake system. The wild-type strain maintained a low extracellular K+ concentration both in the chemostat under steady-state conditions and after stopping the medium flow. The mutant, on the other hand, maintained a much higher extracellular K+ concentration in the steady state, which decreased to much lower values after stopping the medium flow. From the increase in bacterial dry wt and the low external K+ concentration after stopping the medium flow it is concluded that the intracellular K+ is redistributed among the cells, including new cells. The growth yield on K+ was highest in the stationary growth phase of a batch culture and all steady-state cultures converged ultimately to this yield value after the medium flow had been stopped. It is proposed that the growth rate of E. coli under K+-limited conditions is determined by the intracellular K+ concentration.