Detection of Sugar-Regulated Gene Expression and Signaling in Suspension-Cultured Rice Cells

To better understand the mechanism of sugar signaling in rice cell, the suspension-cultured rice cells were transferred from sucrose-containing (+S) to sucrose-free (-S) of MS culture medium, we found that ribosomal RNAs (rRNAs) were degraded progressively. This suggests that carbon, nitrogen, and phosphate were recycled in this process and the reduction in cellular rRNAs might lead to decreased translation to save energy in response to sugar starvation. Differential screening revealed that two groups of genes, sugar-starvation-repressed (SSR) and sugar-starvation-activated (SSA) genes, were regulated by sugar in an opposing manner. Northern-blot analysis showed that two major hybridization signals of 0.8 and 1.9 kb were induced strongly under sugar starvation. The two populations of genes corresponded with homologs of α-amylases (1.9 kb) and the glycine-rich proteins (GRPs) gene family (0.8 kb), and all were SSA genes. Expression of GRP genes was strongly induced in sugar-starved cells, which suggests that GRPs may help to protect cells against nutritional stress. Treatment of +S and -S cells with the protein kinase (PK) inhibitor staurosporine (St) and the serine/theronine phosphoprotein phosphatases 1 (PP1) and 2A (PP2A) inhibitor okadaic acid (OA) revealed that PP1 and PP2A (PPs) might be involved in increasing SSR gene expression in +S cells, and that activation of the majority of the SSA genes in -S cells might be due to PKs activity. These results suggested that PKs and PPs might be involved in the sugar regulation of SSR and SSA gene expression. An in-gel PK activity assay demonstrated that the activity of two classes of PKs (50 and 66 kDa) may be induced rapidly after transfer of +S cells to -S medium. Following transfer of -S cells to +S medium, a novel class of 38 kDa PK was induced rapidly and showed high activity. The 38 kDa PK might play a role in sugar sensing, and the 50 and 66 kDa PKs might play roles in signal sensing under sugar starvation in rice cells. These results provide valuable information on three classes of protein kinases that might play key roles in sugar sensing and signaling in rice.

Ce^(4+)-Induced Apoptosis of Taxus cuspidata Cells in Suspension Culture

The standard detection hallmarks of apoptosis of Taxus cuspidata cells in suspension culture with Ce 4+ were studied. The condensation and margination of chromatin were observed under the electron microscopy. DNA fragmentation ranged 'DNA ladder' on agarose gel electrophoresis. TdT mediated dUTP nick end labeling (TUNEL) analysis of the cells reveals that the nuclear DNA strand breaks can be identified by labeling free 3′ OH termini. These results suggest that Ce 4+ can induce apoptosis of Taxus cuspidata cells and also indicate that there is a certain relationship between apoptosis and secondary metabolite product Taxol.

Influences of Plant Growth Regulators， Basal Media and Carbohydrate Levels on Cell Suspension Culture of Panax ginseng

A cell suspension culture of Panax ginseng which may be continuously subcultured has been established. Embryogenic callus derived from clutured young leaves was used to initiate the culture.Plant growth regulators, basal medium formula and carbohydrate levels were examined to determine their various effects on suspension culture cell growth and development. The best selection of plant growth regulator, hasal medium and carbohydrate level is 2 mg / L 2,4-D: 0.5 mg / L KT,MS and 3% sucrose respectively.

Biomass accumulation of Panax vietnamensis in cell suspension cultures varies with addition of plant growth regulators and organic additives

Objective: To evaluate the impact of plant growth regulators including kinetin(KN),benzyl adenine and naphthalene acetic acid, yeast extract and casein hydrolyzate on biomass accumulation of Vietnamese ginseng Panax vietnamensis(P. vietnamensis) in cell suspension culture.Methods: Cell suspension cultures were established from friable calluses derived from leaves and petioles of 3-year-old in-vitro P. vietnamensis plants. The cell suspension cultures were grown in Murashige and Skoog basal media supplemented with various concentrations of KN, benzyl adenine, naphthalene acetic acid, and yeast extract and casein hydrolyzate.Results: All tested factors generated an increase in the cell biomass of P. vietnamensis in suspension culture, but the impact of each varies depended on the factor type, concentration, and incubation period. Addition of 2.0 mg/L KN resulted in the largest biomass increase after 24 d,(57.0 ± 0.9) and(3.1 ± 0.1) mg/m L fresh and dry weight, respectively,whereas addition of benzyl adenine or naphthalene acetic acid produced optimum levels of Panax cell biomass at 1.0 and 1.5 mg/L, respectively. Addition of the elicitor yeast extract led to a 1.4–2.4 fold increase in biomass of P. vietnamensis, while addition of casein hydrolyzate enhanced biomass accumulation 1.8–2.6 fold.Conclusions: The addition of each factor causes significant changes in biomass accumulation of P. vietnamensis. The largest biomass accumulation is from cultures grown in MS media containing 2.0 mg/L KN for 24 d. The outcome of the present study provides new insights into the optimal suspension culture conditions for studies on the in vitro cell biomass production of P. vietnamensis.

Ultrastructural and Extracellular Protein Changes in Cell Suspension Cultures of Populus euphratica Associated with Low Temperature-induced Cold Acclimation

Populus euphratica Olive is the only tree species that can grow in the saline land and also survive cold winters in northwest China, and it plays a very important role in stabilizing the vulnerable ecosystem there. A cell suspension culture was initiated from callus derived from plantlets of Populus euphratica. Cold acclimation was induced (LT50 of 17.5 ℃) in cell suspension at 45 ℃ in the dark for 30 days and the freezing tolerance increased from LT50 of 12.5 ℃ in nonacclimated cells to LT50 of 17.5 ℃ in cold-acclimated cells. Microvacuolation, cytoplasmic augmentation and accumulation of starch granules were observed in cells that were cold-acclimated by exposure to low temperatures. Several qualitative and quantitative changes in proteins were noted during cold acclimation. Antibodies to carrot extracellular (apoplastic) 36 kD antifreeze protein did not cross react on immunoelectroblots with extracellular proteins in cell suspension culture medium of Populus euphratica, indicating no common epitopes in the carrot 36 kD antifreeze protein and P. euphratica extracellular proteins. The relationship of these changes to cold acclimation in Populus euphratica cell cultures was discussed.

Alcohol Dehydrogenase Activity in Cultured Cells from Different Tobacco (Nicotiana tabacum L.) Varieties

The activity of alcohol dehydrogenase (ADH) in cultured cells of various tobacco was determined. It was found that significant differences existed in cells of different varieties cultured under normal conditions and as well after treated with exogenous ethanol. The ADH activity had positive relation with the ability of the cells to catabolize exogenous ethanol, indicating that the main function of the ADH in tobacco cells was in the direction of converting ethanol to acetaldehyde.

Study on Cell Suspension Culture of Floribunda Rose

Friable callus was induced when immature seeds of floribunda rose were inoculated on MS medium supplemented with 2,4-D 3.0 mg.L^-1. When transfered onto subculture media, friable callus developed into embryogenic callus, which was used to establish cell suspension lines. Cell suspensions had to be subcultured at a interval of 4-5 days at the first several culture cycles. The best subculturing cycle for the stable cell suspensions was 8-10 days. The best inoculum quantity was 1 mL PCV（Packed Cell Volume） per 40 mL culture fluid.

A <i>β</i>-Glucosidase Activity Potentially Involved in Cell Division and Wall Development of <i>Phyllostachys</i>Bamboo Suspension Cells

We propose a novel Madake (Phyllostachys bambusoides) bamboo suspension culture model for investigation of key enzyme(s) activity involved in growth/differentiation. Sedimented Cell Volume (SCV) and fresh weight (FW) of the suspension cultured cells reached 34% (v/v) and 8.7 g in 10 μM 2,4-dichlorophenoxyacetic acid (2,4-D)-containing medium while only 7% (v/v) SCV and 1.9 g FW of the cells in 10 μM gibberellic acid (GA3)-containing medium in 14 days. Proportion of mitotically active cells (S to G2/M phases) at a log phase was identified as 29.5% in the former cells with tiny cytoplasmic features while 5.4% in the latter cells with elongation, wall thickening, and lignification by using flow cytometry and laser scanning microscopic analysis. The total β-glucosidase (BGL) activity under the 2,4-D condition increased from 4.8 U in day 2 to 26.2 U in day 14 (ca. 5.5-fold) while a slight reduction, from 4.4 U in day 2 to 2.1 U in day 14 (ca. 0.5-fold), occurred when cell division was suppressed under the GA3 condition. Ratio of the BGL activity of the soluble fractions to the membrane-associated fractions varied depending of the culture condition. The ratio was stable (2 to 8) during the culture period under the 2,4-D condition. Interestingly, the activity of the soluble enzyme fractions increased up to ca. 65% under the GA3 condition in inverse proportion to the membrane-associated enzymes. All together, it was strongly suggested that the detected specificity/variability of BGL activity is potentially involved in cell division and lignification in Madake bamboo cells.

Coscinium fenestratum: Callus and Suspension Cell Culture of the Endangered Medicinal Plant Using Vermicompost Extract and Coelomic Fluid as Plant Tissue Culture Media

In vitro tissue culture of hard woody, endangered, medicinal plant Coscinium fenestratum is most challenging to plant tissue culturists. In the present study, petiole and leaf explants of Coscinium fenestratum were induced to form callus when cultured on vermicompost extract media along with coelomic fluid. Suspension medium was developed using vermicompost extract and coelomic fluid in 3:1 ratio. Phytochemical analysis of the alkaloid berberine was confirmed from callus, suspension cell culture and suspension medium by Thin Layer Chromatography and High Performance Liquid Chromatography. Vermicompost and its extracts with coelomic fluid have shown maximum (100 per cent) response of callus induction. Callus mass enlarged with increasing concentration of coelomic fluid and callus growth was assessed from the biomass. Incubation of culture tubes in dark supported callus development significantly. The Rf value of 0.36 confirmed the presence of berberine by Thin Layer Chromatography. Qualitative analysis confirmed the presence of alkaloid berberine with the retention time of 2.8 minutes similar to that of standard reference sample from Sigma chemicals, USA. The suspension medium turned deep yellow because of the release of the alkaloid. Vermicompost and its extracts along with coelomic fluid have shown the economical approach for micropropagation of economically and medicinally important plants.

Direct Differentiation of Plants from Small Cell Clusters of Indica Rice in Suspension Culture

The major factors affecting plant regeneration in suspension culture were investigated.The resultsshow that,in order for cell clusters to differentiate directly in liquid medium,it is essential toestablish an embryogenic callus line with high potential for plant regeneration.Embryogenic calliwere suspended in AA basic medium supplemented with 2,4-D 1 mg/L,6-benzylaminopurine 0.5mg/L,CH 300 mg/L,sucrose 3％ and mannitol 3％ and subcultured 7 days for each passage.Aftermore than 6 months of culturing,a fine suspension culture of small cell clusters(SCC)wasestablished.The SCC,80-270μin diameter,were then transferred to a liquid medium(MSsupplemented with NAA 0.01 mg/L and 4-pyridylurae(4-PU)0.5 mg/L)and allowed to grow instationary culture.Finally direct differentiation from SCC was observed.Several factors in suspension-subculture exerted strong after-effects on differentiation ofSCC.Basicmedia,kinds and concentration combinations of auxin and cytokinin in the subculture media,duration of shaking at each subculture passage,and amount of packed cell volume transferred to newmedium at each subculture passage and so on,all these affected the frequency of differentiation ofSCC.Higher concentrations of NAA and kinetin in the differentiation medium inhibited the directdifferentiation of SCC.Low concentrations(0.01-0.1 mg/L)of NAA with 4-PU in thedifferentiation medium were helpful for the direct differentiation of SCC.The differentiated clusterspossessed typical embryogenic structure from which normal plantlets could develop after transferringto agar medium.

Studies on Single Cell Culture in vitro in Wheat——The variation of grain protein content and its fractions from regenerated plants

On the basis of previous studies dealing with the variation of major agronomic and yield characteristics of regenerated plants derived from single cell culture in vitro of common wheat (Triticum aestivum L.Cultivar NE 7742), the grain protein content and its fractions from regenerated plants with stable agronomic characteristics were studied from 1992 to 1995. The results showed that the variation of grain protein content and its fractions in somaclones from single cell culture in vitro were very significant and the range was very wide (11531770%). Several types of variation were found in the studies, especially the type with higher protein content than that of cultivar NE 7742 (non-culture parent). Among them, -2069% of lines the grain protein content was significantly higher than that of NE 7742 and combined with high yielding potential. The tendency of variation of the four protein fractions showed that the variation of albumin was not obvious and maintained the same level as NE774 increased in some somaclones and decreased in others. However, the percentages both globulin and glutenin tended to increase. The variation of total amount of structural protein and the ratio between globulin and glutenin tended to increase. The variation of total amount of structural protein and the ratio between globulin and albumm was mainly influenced by globulin under the condition of culture in vitro. The variation of total amount of storage protein and the ratio between gliadin and glutenin was mainly affected by glutenin. The results mentioned above demonstrated that the induction and screening of somaclonal variation could be an effective way in wheat improvement in combining high protein content with high yield.

Production of asiaticoside from centella(Centella asiatica L.Urban)cells in bioreactor

Objective:To investigate the effects of some culture conditions on production of asialicoside from centella(Centella asiatica L.Urban)cells cultured in 5-L bioreactor.Methods:The centell cell suspension culture was conducted in 5-L bioreactor to investigate the growth and asiaticoside accumulation under various conditions.Asiaticoside content was determined by HPLC analysis.Results:The results showed that the cell growth and asiaticoside accumulation peaked after 24d of culture at an agitation speed of 150 r/min and aeration rate of 2.5 L/min.The cell biomass reached a maximum value of 302.45 g fresh weight(31.43 g dry weight)and growth index of 3.03with inoculum size of 100 g.However,asiaticoside content was the highest(60.08 mg/g dry weight)when culture was initiated with an inoculum size of 50 g.Conclusions:The present study found the suitable conditions for growth of centella cells and their asiaticoside production in bioreactor.

An Efficient Rice Mutagenesis System Based on Suspension-Cultured Cells

Plant mutants are important bio-resources for crop breeding and gene functional studies. Conventional methods for generating mutant libraries by mutagenesis of seeds with physical or chemical agents are of low efficiency. Here, we developed a highly-efficient ethyl methanesulfonate （EMS） mutagenesis system based on suspension-cultured cells, with rice （Oryza sativa L.） as an example. We show that treatment of suspension-cultured tiny cell clusters with 0.4% EMS for 18-22 h followed by differentiation and regeneration produced as high as 29.4% independent mutant lines with visible phenotypic variations, including a number of important agronomic traits such as grain size, panicle size, grain or panicle shape, tiller number and angle, heading date, male sterility, and disease sensitivity. No mosaic mutant was observed in the mutant lines tested. In this mutant library, we obtained a mutant with an abnormally elongated uppermost internode. Sequencing and functional analysis revealed that this is a new allelic mutant of eui （elongated uppermost internode） caused by two point mutations in the first exon of the EUI gene, representing a successful example of this mutagenesis system.

Biotransformation of 14-Deacetoxy-13-oxo sinenxan A by Ginkgo Cell Cultures

Deacetoxy-13-oxo sinenxan A (1) was converted to 9a-hydroxy-13-oxo-2a, 5a, 10b-triacetoxy-4(20),11-taxadiene (2) and 10b-hydroxy-13-oxo-2a,5a,9a-triacetoxy- 4(20), 11- taxadiene (3) by Ginkgo cell suspension cultures in 45% and 15% yields, respectively.

Actomyosin is Involved in the Organization of the Microtubule Preprophase Band in Arabidopsis Suspension Cultured Cells

The microtubule preprophase bands （PPBs） participate in the sequence of events to position cell plates in most plants. However, the mechanism of PPB formation remains to be clarified. In the present study, the organization of PPBs in Arabidopsis suspension cultured cells was investigated by confocal laser scanning microscopy combined with pharmacological treatments of reagents specific for the cytoskeleton elements. Double staining of F-actin and microtubules （MTs） showed that actin filaments were arranged randomly and no colocalization with cortical MTs was observed in the interphase cells. However, cortical actin filaments showed colocalization with MTs during the formation of PPBs. A broad actin band formed with the broad MT band in the initiation of PPB and narrowed down together with the MT band to form the PPB. Nevertheless, broad MT bands were formed but failed to narrow down in cells treated with the F-actin disruptor latrunculin A. In contrast, in the presence of the F-actin stabilizer phalloidin, PPB formation did not exhibit any abnormality. Therefore, the integrity, but not the dynamics, of the actin cytoskeleton is necessary for the formation of normal PPBs. Treatment with 2, 3-butanedine monoxime, a myosin inhibitor, also resulted in the formation of broad MT bands, indicating that actomyosin may be involved in the rearrangement of MTs to form the PPBs. Double staining of MTs and myosin revealed that myosin concentrated on the PPB region during PPB formation. It is suggested that the actin cytoskeleton at the PPB site may serve as a rack to transport cortical MTs by using myosin when the broad MT band narrows down to form the PPB.

Acceleration of Cell Growth by Xyloglucan Oligosaccharides in Suspension-Cultured Tobacco Cells

The incorporation of xyloglucan oligosaccharide （XXXG） into the walls of suspension-cultured tobacco cells accelerated cell expansion followed by cell division, changed cell shape from cylindrical to spherical, decreased cell size, and caused cell aggregation. Fluorescent XXXG added to the culture medium was found to be incorporated into the surface of the entire wall, where strong incorporation occurred not only on the surface, but also in the interface walls between cells during cell division. Cell expansion was always greater in the transverse direction than in the longitudinal direction and then, immediately, expansion led to cell division in the presence of XXXG; this process might result in the high level of cell aggregation seen in cultured tobacco cells. We concluded that the integration of this oligosaccharide into the walls could accelerate not only cell expansion, but also cell division in cultured cells.

Multiplication of the Recombinant Strain Re-7 of Avian Influenza Virus Subtype H5 in MDCK Cells

This study was conducted to explore the multiplication pattern of the recombinant strain Re-7 of avian influenza virus subtype H5 in Madin Darby Canine Kidney （MDCK） cells and to determine the optimal multiplicity of infection （MOI） and the optimal time for virus harvest. The recombinant strain Re-7 was inoculated at different MOIs into MDCK cells grown in serum-free medium in 100 L bioreactors for replication. Then, the hemagglutination（HA） titer, 50% tissue culture infectious dose （TCID50） and 50% embryo infectious dose （EID50） of culture medium were measured once every 12 h from 24 h after virus inoculation to determine the optimal MOI. After that, virus was inoculated at the optimal MOI determined above into MDCK cells for large-scale virus replication to determine the optimal time for virus harvest. The results showed that the optimal MOI was 10 2, and the optimal time for virus harvest was 60 h after inoculation. Under these conditions, the HA titer, TCIDso per 1 mL and EIDso per 0.1 mL were increased to 1：102 4, 10^7.33 and 10^6.83, respectively. This study provides relatively stable parameters for large-scale production of the recombinant strain Re-7 of avian influenza virus subtype H5.

Regenerative Properties of Fetal n. Raphe Cells in Modeling Injuries of Serotonergic Neurite-Glial Fibers in Vitro

It is considered that renewal of growth in serotoninergic caudal fibers their sprouting to injury site and formation ofoutgrowth net is factor for locomotion recovery. The aim of our work was to study the influence of rat fetal neural cells enriched withserotonin-producing cells, on morphogenesis, gene expression of serotogenesis （Pet1, Nk2.2, Lmxlb, Tphl, Tph2, Sert） andserotonin level in organotypic n. raphe cultures in injury of neurite-glial fibers in vitro. Injury was modeled by the way of crossingneurite-glial fibers after their formation under long-term culture. The cell accumulation was observed in the injury zone when fetal n.raphe cells, previously enriched with serotonin-producing cells were added into organotypic culture of n. raphe with crossing fibersunder the culture conditions. As a result, crossing site was filled with these cells after 14 days of culture. The activation of expressionin cascade of regulatory genes-regulators of serotogenesis Pet1, Lmxlb, Tphl, Tph2, Sert and increased serotonin content in theculture material were also observed. The activation of gene expression of serotogenesis Pet1 and Sert, and increased serotonincontent by 1.7 times were revealed when conditioned medium from culture of fetal rat cells was used. An eviction of single cells wasobserved in control samples of organotypic n. raphe culture after injury. Crossing site was remained without signs of cell filling inthese samples at day 14 after culture. The activation of regulatory gene expression of serotogenesis Pet1, Nkx2.2, Lmxlb, Tphl,Tph2, Sert and restoration of serotonin content were also absent in the culture material.

小麦小孢子培养影响因素研究

为优化小麦小孢子培养技术,建立实用化的小麦小孢子培养技术体系,以冬小麦品种新麦35和西农059及春小麦品种Bobwhite为供试材料,研究了预处理方式和不同分离液对小麦小孢子活力及形成胚性小孢子的影响,并对不同培养密度下小孢子形成愈伤组织的诱导率进行分析,比较了两种分化培养基对胚状体和愈伤组织分化率的影响。结果表明,与高温加甘露醇预处理相比,对麦穗进行低温预处理12 d的胚性小孢子数量多,小孢子活力最高;采用NPB-99培养基作为小孢子分离液不影响梯度离心,得到的小孢子活力较高,活力小孢子比率达31.96%,远高于单一甘露醇分离液和NPB-99无机盐分离液的3.62%和4.59%;小孢子培养密度为0.5×10^(4)个·mL^(-1)时诱导产生的愈伤组织较多,诱导率为60.22%;在GEM培养基上愈伤组织分化率相对较高,分化率为6.80%。因此,低温预处理12 d的麦穗以NPB-99培养基作为小孢子分离液操作简单、效果好,小孢子培养密度对胚状体/愈伤组织诱导率具有显著性影响,基因型对愈伤组织诱导率和分化率具有极显著影响,培养基对分化率有显著影响。

293T细胞生产慢病毒工艺优化

随着细胞治疗的快速发展,大规模慢病毒的生产成为工艺环节中的瓶颈,因此,优化293T高滴度和高纯度的CAR慢病毒载体生产工艺显得至关重要。本研究旨在优化包装慢病毒的293T贴壁细胞,节省时间,节约成本,提高慢病毒包装的能力。同时对优化慢病毒载体中出现悬浮细胞结团生长的现象进行探索,检测其影响结团的因素。分别采用快速、慢速驯化方式将293T贴壁细胞驯化为悬浮培养,并比较其细胞形态、细胞密度、细胞活率、慢病毒包装能力和冻存复苏后稳定一致性,筛选出最优的悬浮驯化条件。通过调节Ca^(2+)浓度和EDTA添加量来研究比较细胞结团生长状况。结果证明,使用无血清培养基OPM-293 CD05溶剂(medium)可以将293T贴壁细胞快速驯化为293T悬浮细胞,并能制备出慢病毒滴度且优于贴壁细胞的包装滴度(^(*)P<0.05)。Ca^(2+)浓度会影响细胞结团大小,添加EDTA能有效分离分散非必要的细胞抱团生长。研究结果显示,传统293T贴壁细胞可以使用无血清培养基OPM-293 CD05溶剂快速驯化成悬浮细胞;在一定范围内,Ca^(2+)浓度越高细胞所结团块及粒径越大,EDTA添加量越高细胞所结团块及粒径变小。这为优化慢病毒载体包装工艺和悬浮培养条件,同时为体外规模化细胞培养放大和生产奠定了理论基础,具有一定的实用价值。