Anticancer Activity of Rice Callus Suspension Cultures from Aromatic Varieties and Metabolites Regulated in Treated Cancer Cell Lines

Tissue culture techniques were used to produce large amounts of bioactive compounds with medicinal potential, overcoming space and time constraints for cancer prevention. Rice callus suspension cultures(RCSC) and seed extracts prepared from aromatic rice varieties were used to evaluate the cytotoxic impact on human colon and lung cancer cell lines, as well as a normal control cell line, using Taxol as a positive control. RCSC and seed extracts from two Indian aromatic rice varieties were applied at different concentrations to treat the cancer cell lines and normal lung fibroblasts over varying time intervals. Apoptosis was assessed in 1:5 dilutions of the A549 and HT-29 cell lines treated with RCSC for 72 h, using propidium iodide staining and flow cytometry. RCSC showed a more potent cytotoxic effect than seed extracts with minimal effect on the normal cell line, in contrast to Taxol. Confocal microscopy and flow cytometry further confirmed the apoptotic effect of RCSC. Gas chromatography-mass spectrometry-based metabolic profiling identified metabolites involved in cytotoxicity and highlighted altered pathways. RCSC is proposed as an alternative source for the development of novel anticancer drugs with reduced side effects.

Studies on Genetic Transformatiom of Embryogenic Suspension Cultures of Sweetpotato

Genetic transformation of embryogenic suspension cultures of sweetpotato cv. Lizixiang was conducted by using Agrobacterium tumefaciens strain A208SE harboring the binary vectors pROA93 with β-glucronidase(GUS)and neomycin phosphotransferase(NPTⅡ)genes. The results indicated that embryogenic suspension cultures precultured for 1 - 3 d were suitable for the transformation. The optimal cocultivation time was 4 - 5 d. The optimal concentration of kanamycin was 50-75 mg L-1 for suspension culture and 100 mg L-1 for embryogenic callus proliferation and plant regeneration. The optimal concentration of carbencillin was 100 mg L-1. Transgenic plants identified with GUS assays and PCR analyses were obtained.

Anticancer Activities of Plant Secondary Metabolites:Rice Callus Suspension Culture as a New Paradigm

Plant natural products including alkaloids,polyphenols,terpenoids and flavonoids have been reported to exert anticancer activity by targeting various metabolic pathways.The biological pathways regulated by plant products can serve as novel drug targets.Plant natural compounds or their derivatives used for cancer treatment and some novel plant-based compounds which are used in clinical trials were discussed.Callus suspension culture with secondary metabolites can provide a continuous source of plant pharmaceuticals without time and space limitations.Previous research has shown that rice callus suspension culture can kill>95%cancer cells with no significant effect on the growth of normal cells.The role of candidate genes and metabolites which are likely to be involved in the process and their potential to serve as anticancer and anti-inflammatory agents were discussed.Large scale production of plant callus suspension culture and its constituents can be achieved using elicitors which enhance specific secondary metabolites combined with bioprocess technology.

APOPTOSIS AND TAXOL PRODUCTION IN SUSPENSION CULTURES OF Taxus spp.CELLS

The hallmark of apoptosis, in suspension cultures of Taxus spp. cells induced by fungal extractive or by abiotic means, was studied by total DNA agarose gel electrophoresis and in situ end-labeling. The cleavage of nuclear DNA (nDNA) into oligonucleosomal fragments(DNA laddering) was a characteristic of apoptosis, which involved cell shrinkage, condensation of cytoplasm and tracheary elements differentiation. Terminal deoxynucleotidy transferase-mediated dUTP nick end in situ labeling (TUNEL) assay of Taxus spp. cells showed that fungal extractive or abiotic elicltors (Ce4+, Taxol, H2O2) induced TUNEL positive. Also, the increase of the apoptotic cell ratio was accompanied by the increase of secondary metabolites (especially Taxol). These results suggest that apoptosis may have some coincidence with biosynthesis of Taxol. The implication of apoptosis for the production of secondary metabolites in plant cell cultures is discussed.

Nitraria sibirica cell suspension culture:establishment,characterization and application

Nitraria sibirica Pall. is a shrub that grows in saline-alkali soil and has traditional medicinal value and potential commercial value. The objectives of this study include induction and multiplication of callus, establishment of a suspension cell line, and isolation of protoplasts from cell suspensions. Murashige and Skoog (MS) medium was used for callus induction from mature seeds of N. sibirica. Seed-derived calluses were further multiplied on MS medium augmented with 0.5 mg L-1 6-benzylaminopurine (6-BA) and 1.0 mg L-1 2,4-dichlorophenoxy (2,4-D) acetic acid. Suspension cultures of N. sibirica were initiated by transferring friable calli to the same liquid multiplication medium. Characterization of the suspension culture was assessed based on fresh mass, dry mass, cell viability and pH value of the culture. A typical growth curve was observed after inoculating 1.5 g of callus in 40 mL liquid medium, including a lag phase, an exponential growth phase, a stationary phase, and a negative acceleration phase. The effect of factors such as pre-plasmolysis, enzyme combination, enzymolysis time and mannitol concentration, on the isolation of cell-derived protoplasts were evaluated to determine the usefulness of suspension cultures. The maximum yield (9.79 x 10(6) cells/g) and highest viability (79.97%) of protoplast were reached when approximately 1 g of cell suspension (cultured for 6 days) was inoculated for 12 h in cell and protoplast washing solution made of 0.8 mol L-1 mannitol mixture solution, cellulose onozuka R-10 2% (w/v), hemicellulose 0.2%, macerozyme R-10 1%, and pectolyase Y-23 0.5%. Protoplast yield was significantly influenced by pre-plasmolysis and cellulose onozuka R-10 (P < 0.05).

Optimization of Transformation Efficiency of Suspension Cultured Vitis vinifera cv.Chardonnay Embryogenic Cells

Vitis vinifera cv.Chardonnay suspension cultures were established from proembryogenic mass and employed for optimizing Agrobacterium-mediated transformation system.One-factor-at-a-time experiment revealed that OD600 of Agrobacterium,time of inoculation,co-cultivation,and cell-drying before inoculation significantly affected the transformation efficiency which reached maximum 21.5% at the following conditions：0.8 of OD600,25 min of inoculation,2 d of co-cultivation,and 10 min of cell drying.Response surface methodology experiments based on a five-level,four-factor central-composite rotatable design were then used to optimize these selected factors.The optimized conditions for Chardonnay grape transformation were：0.8711 of OD600,28.9 min of inoculation,2.25 d of co-cultivation and 11.76 min of cell drying.After optimization,transformation efficiency was 26.2% and there were no interactions among different factors.

Optimization of Suspension Culture Conditions for Hairy Roots of Aconitum coreanum(Levl.) Raipaics

[ Objective] This study aimed to establish an optimized suspension culture for hairy roots of Aconitum coreanum （Levi.） Raipaics. [ Method ] Hairy root of Aconitum coreanum （ Levl. ） Raipaics A0489 was selected as experimental material, to screen the optimal medium for suspension culture. [ Result] Aconitum coreanum （Levl.） Raipaics hairy roots showed different growth conditions in various liquid medium; salt concentration, nitrogen content, sugar content, rotation speed and room temperature had great influences on the growth of Aconitum coreanum （Levi.） Raipaics hairy roots. [ Conclusion] The optimal suspension culture system for Aconitum coreanum （ Levi. ） Raipaics hairy roots was established ： B5 was used as basic medium, culture temperature was 20℃ under dark conditions, rotation speed was 140 r/m in the first 15 d and 110 r/m in the latter 15 d.

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.

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.

Embryoid Induction and Cell Suspension Culture of Premna microphylla Turca

In order to find suspension culture conditions suitable for Premna microphylla Turea, with the leaves of experimented P. microphylla as a material, em- bryogenic callus induction, subculture, dispersion of embryogenic callus and suspension culture were performed. The results showed that the calli induced from leaves were different. The medium most suitable for induction of loose embryogenic callus for suspension culture was MS ＋ 0.4 mg/L 2,4-D ＋ 0.4 mg/L NAA ＋ 3% sucrose ＋0.8% agar. A better cell line could be obtained with cellulase and pectinase for dispersion of P. microphyUa callus. The medium most suitable for sus- pension culture was MS ＋0.4 mg/L 2,4-D ＋0.8 mg/L KT ＋3% sucrose. In this medium, the fresh weight of the suspensian-cultured cells increased by 6.46 times per 7 d. Dark culture was more suitable for cell proliferation. The average contents of pectin and protein were, respectively, 6.57% and 0.12% in the sus- pended ceils of P. microphylla, which had been cultured for 3 weeks under the optimum culture conditions.

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.

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.

Cell Suspension Culture of Neem Tree

The establishment of suspension culture system for neem (Azadirachta indica A. Juss) cells and the suspension culture condition was studied. It shows that the neem cell suspension culture system was best in B5 liquid medium, 2.0~4.0mg/L NAA with direct spill method. Based on the integrated analysis of cell biomass, Azadirachtin content and productivity, the optimum culture conditions were B5 liquid medium, 2.0-4.0 mg/L NAA, 3% sucrose at 25 ℃. The optimum rotating speed of the shaker and broth content d...

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.

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.

Suspension state promotes extravasation of breast tumor cells by increasing integrin β1 expression

Mechanical microenvironment can strongly affect the metastatic efficiency of circulating tumor cells.However,the effect of suspension state on their extravasation and the mechanisms involved are still unclear.To explore the influence of suspension state on extravasation(including adhesion,spreading and transendothelial migration)of breast tumor cells and its relevant molecular mechanism,MDA-MB-231 cells were cultured on poly(2-hydroxyethyl methacrylate)coated 6-well plates to minic the suspension state.Suspension state promoted adhesion,spreading and transendothelial migration of MDA-MB-231 cells to EAhy926 endothelial cells(ECs)monolayer under both the static condition and 0.5 dyne/cm^(2) flow shear stress(FSS).The number of cells adhered to ECs monolayer reached 2.15(static condition,3 d)and 1.67(FSS,3 d)times,and the number of migration reached 10.60 times,respectively,as compared to that in adhesion state.Moreover,MDA-MB-231 cells knockdown of integrin β1 provoked poor adhesion and transendothelial migration,as compared with MDA-MB-231 cells.But it made no difference in cell spreading.Our results implied the increasing expression of integrin β1 induced by suspension culture promoted the adhesion and transendothelial migration of MDA-MB-231 cells,but had no significant influence on their spreading.

Effects of Cerium on Accumulation of Anthocyanins and Expression of Anthocyanin Biosynthetic Genes in Potato Cell Tissue Cultures

The effects of Ce （Ⅳ） on callus growth, anthocyanin content, and expression of anthocyanin biosynthetic genes in callus suspension cultures of Solanum tuberosum cv. Chieftain were studied by the measurement of fresh weight, spectrophotometric assays, and semiquantitative RT-PCR. The results indicate that 0.1 mmol·L^- 1 Ce （ Ⅳ ） can promote callus growth, increase the accumulation of anthocyanins, and enhance the expression of five anthocyanin biosynthetic genes （ CHS, F3H, F3＇5＇H, DFR, and 3 GT） most efficiently. At high concentrations of 1 mmol·L^- 1, Ce （Ⅳ） partially inhibits callus growth and at 2 mmol· L^-1 eventually lends to cell death. The results show that Ce（ Ⅳ ） can induce the expression of anthocyanin biosynthetic genes to produce and accumulate anthocyanins and increase the yield of anthocyanins.

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.

Somatic Embryogenesis and Plant Regeneration from Two Recalcitrant Genotypes of Gossypium hirsutum L.

An improved protocol has been developed for somatic embryogenesis and plant regeneration of recalcitrant cotton cultivars. High callus frequencies and embryogenic tissue were developed in MSB medium supplemented with gradient concentrations of KT and 2,4-D, their concentration decreasing from 0.1 to 0.01 mg·L^-1. Somatic embryos were successfully incubated in 1/2 macronutrient MSB suspension supplemented with 0.5 g· L^-1 glutamine and 0.5 g·L^-1 asparagine. Decrease in macronutrient concentration of MSB significantly alleviated browning and was beneficial to suspension cells. Transformation of somatic embryos into plants was induced in MSB medium supplemented with 3% sucrose, 0.5 g·L^-1 glutamine, 0.5 g·L^-1 asparagine, and 6.0 g·L^-1 agar. The effect of sucrose as carbohydrate was better than that of glucose for plant germination. Using this protocol, regenerated plantlets from the CCRI521 and Zhongzhi86-6 reached to as much as 19.6 and 18.5% somatic embryos, respectively.

Effect of La,Ce on Taxus Cuspidata Cell Growth,Biosynthesis and Release of Taxol

The effect of La, Ce was firstly tested on growth of the Taxus(T.) cuspidata cell suspensions, biosynthesis and release of taxol. The results indicate that the growth pattern of T.cuspidata cells is altered significantly by adding high concentration of rare earth in the medium. The lag and exponential phases of cell growth are shortened, the stationary phase disappears and the biomass fluctuates periodically during the decline phase. The rare earth compounds added in the exponential phase obviously increase the taxol biosyntheis and release yields of T.cuspidata cells, and the supplement of carbon source in the medium containing rare earth is also favorable to taxol biosynthesis.