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Sample records for cotton suspension culture

  1. Cell suspension culture-mediated incorporation of the rice bel gene into transgenic cotton.

    Directory of Open Access Journals (Sweden)

    Liping Ke

    Full Text Available Cotton plants engineered for resistance to the herbicides, glyphosate or glufosinate have made a considerable impact on the production of the crop worldwide. In this work, embryogenic cell cultures derived from Gossypium hirsutum L. cv Coker 312 hypocotyl callus were transformed via Agrobacterium tumefaciens with the rice cytochrome P450 gene, CYP81A6 (bel. In rice, bel has been shown to confer resistance to both bentazon and sulfanylurea herbicides. Transformed cells were selected on a liquid medium supplemented alternately or simultaneously with kanamycin (50mg/L and bentazon (4.2 µmol. A total of 17 transgenic cotton lines were recovered, based on the initial resistance to bentazon and on PCR detection of the bel transgene. Bel integration into the cotton genome was confirmed by Southern blot and expression of the transgene was verified by RT-PCR. In greenhouse and experimental plot trials, herbicide (bentazon tolerance of up to 1250 mg/L was demonstrated in the transgenic plants. Transgenic lines with a single copy of the bel gene showed normal Mendelian inheritance of the characteristic. Importantly, resistance to bentazon was shown to be stably incorporated in the T1, T2 and T3 generations of self-fertilised descendents and in plants outcrossed to another upland cotton cultivar. Engineering resistance to bentazon in cotton through the heterologous expression of bel opens the possibility of incorporating this trait into elite cultivars, a strategy that would give growers a more flexible alternative to weed management in cotton crops.

  2. Site of clomazone action in tolerant-soybean and susceptible-cotton photomixotrophic cell suspension cultures.

    Science.gov (United States)

    Norman, M A; Liebl, R A; Widholm, J M

    1990-10-01

    Studies were conducted to determine the herbicidal site of clomazone action in tolerant-soybean (Glycine max [L.] Merr. cv Corsoy) (SB-M) and susceptible-cotton (Gossypium hirsutum [L.] cv Stoneville 825) (COT-M) photomixotrophic cell suspension cultures. Although a 10 micromolar clomazone treatment did not significantly reduce the terpene or mixed terpenoid content (microgram per gram fresh weight) of the SB-M cell line, there was over a 70% reduction in the chlorophyll (Chl), carotenoid (CAR), and plastoquinone (PQ) content of the COT-M cell line. The tocopherol (TOC) content was reduced only 35.6%. Reductions in the levels of Chl, CAR, TOC, and PQ indicate that the site of clomazone action in COT-M cells is prior to geranylgeranyl pyrophosphate (GGPP). The clomazone treatment did not significantly reduce the flow of [(14)C]mevalonate ([(14)C]MEV) (nanocuries per gram fresh weight) into CAR and the three mixed terpenoid compounds of SB-M cells. Conversely, [(14)C]MEV incorporation into CAR and the terpene moieties of Chl, PQ, and TOC in COT-M cells was reduced at least 73%, indicating that the site of clomazone action must be after MEV. Sequestration of clomazone away from the chloroplast cannot account for soybean tolerance to clomazone since chloroplasts isolated from both cell lines incubated with [(14)C]clomazone contained a similar amount of radioactivity (disintegrations per minute per microgram of Chl). The possible site(s) of clomazone inhibition include mevalonate kinase, phosphomevalonate kinase, pyrophosphomevalonate decarboxylase, isopentenyl pyrophosphate isomerase, and/or a prenyl transferase.

  3. Site of Clomazone Action in Tolerant-Soybean and Susceptible-Cotton Photomixotrophic Cell Suspension Cultures 1

    Science.gov (United States)

    Norman, Michael A.; Liebl, Rex A.; Widholm, Jack M.

    1990-01-01

    Studies were conducted to determine the herbicidal site of clomazone action in tolerant-soybean (Glycine max [L.] Merr. cv Corsoy) (SB-M) and susceptible-cotton (Gossypium hirsutum [L.] cv Stoneville 825) (COT-M) photomixotrophic cell suspension cultures. Although a 10 micromolar clomazone treatment did not significantly reduce the terpene or mixed terpenoid content (microgram per gram fresh weight) of the SB-M cell line, there was over a 70% reduction in the chlorophyll (Chl), carotenoid (CAR), and plastoquinone (PQ) content of the COT-M cell line. The tocopherol (TOC) content was reduced only 35.6%. Reductions in the levels of Chl, CAR, TOC, and PQ indicate that the site of clomazone action in COT-M cells is prior to geranylgeranyl pyrophosphate (GGPP). The clomazone treatment did not significantly reduce the flow of [14C]mevalonate ([14C]MEV) (nanocuries per gram fresh weight) into CAR and the three mixed terpenoid compounds of SB-M cells. Conversely, [14C]MEV incorporation into CAR and the terpene moieties of Chl, PQ, and TOC in COT-M cells was reduced at least 73%, indicating that the site of clomazone action must be after MEV. Sequestration of clomazone away from the chloroplast cannot account for soybean tolerance to clomazone since chloroplasts isolated from both cell lines incubated with [14C]clomazone contained a similar amount of radioactivity (disintegrations per minute per microgram of Chl). The possible site(s) of clomazone inhibition include mevalonate kinase, phosphomevalonate kinase, pyrophosphomevalonate decarboxylase, isopentenyl pyrophosphate isomerase, and/or a prenyl transferase. PMID:16667768

  4. Uptake and metabolism of clomazone in tolerant-soybean and susceptible-cotton photomixotrophic cell suspension cultures

    Energy Technology Data Exchange (ETDEWEB)

    Norman, M.A.; Liebl, R.A.; Widholm, J.M. (Univ. of Illinois, Urbana (USA))

    1990-03-01

    Studies were conducted to determine the uptake and metabolism of the pigment synthesis inhibiting herbicide clomazone in tolerant-soybean (Glycine max (L.) Merr. cv Corsoy) and susceptible-cotton (Gossypium hirsutum (L.) cv Stoneville 825) photomixotrophic cell suspensions. Soybean and cotton on a whole plant level are tolerant and susceptible to clomazone, respectively. Preliminary studies indicated that I{sub 50} values for growth, chlorophyll (Chl), {beta}-carotene, and lutein were, respectively, >22, 14, 19, and 23 times greater for the soybean cell line (SB-M) 8 days after treatment (DAT) compared to the cotton cell line (COT-M) 16 DAT. Differences in ({sup 14}C)clomazone uptake cannot account for selectivity since there were significantly greater levels of domazone absorbed by the SB-M cells compared to the COT-M cells for each treatment. The percentage of absorbed clomazone converted to more polar metabolite(s) was significantly greater by the SB-M cells relative to COT-M cells at 6 and 24 hours after treatment, however, only small differences existed between the cell lines by 48 hours after treatment. Nearly identical levels of parental clomazone was recovered from both cell lines for all treatments. A pooled metabolite fraction isolated from SB-M cells had no effect on the leaf pigment content of susceptible velvetleaf or soybean seedlings. Conversely, a pooled metabolite fraction from COT-M cells reduced the leaf Chl content of velvetleaf. Soybean tolerance to clomazone appears to be due to differential metabolism (bioactivation) and/or differences at the site of action.

  5. Uptake and metabolism of clomazone in tolerant-soybean and susceptible-cotton photomixotrophic cell suspension cultures.

    Science.gov (United States)

    Norman, M A; Liebl, R A; Widholm, J M

    1990-03-01

    Studies were conducted to determine the uptake and metabolism of the pigment synthesis inhibiting herbicide clomazone in tolerant-soybean (Glycine max [L.] Merr. cv Corsoy) and susceptible-cotton (Gossypium hirsutum [L.] cv Stoneville 825) photomixotrophic cell suspensions. Soybean and cotton on a whole plant level are tolerant and susceptible to clomazone, respectively. Preliminary studies indicated that I(50) values for growth, chlorophyll (Chl), beta-carotene, and lutein were, respectively, >22, 14, 19, and 23 times greater for the soybean cell line (SB-M) 8 days after treatment (DAT) compared to the cotton cell line (COT-M) 16 DAT. Differences in [(14)C]clomazone uptake cannot account for selectivity since there were significantly greater levels of clomazone absorbed by the SB-M cells compared to the COT-M cells for each treatment. The percentage of absorbed clomazone converted to more polar metabolite(s) was significantly greater by the SB-M cells relative to COT-M cells at 6 and 24 hours after treatment, however, only small differences existed between the cell lines by 48 hours after treatment. Nearly identical levels of parental clomazone was recovered from both cell lines for all treatments. A pooled metabolite fraction isolated from SB-M cells had no effect on the leaf pigment content of susceptible velvetleaf (Abutilon theophrasti Medic.) or soybean seedlings. Conversely, a pooled metabolite fraction from COT-M cells reduced the leaf Chl content of velvetleaf. Soybean tolerance to clomazone appears to be due to differential metabolism (bioactivation) and/or differences at the site of action.

  6. Uptake and Metabolism of Clomazone in Tolerant-Soybean and Susceptible-Cotton Photomixotrophic Cell Suspension Cultures 1

    Science.gov (United States)

    Norman, Michael A.; Liebl, Rex A.; Widholm, Jack M.

    1990-01-01

    Studies were conducted to determine the uptake and metabolism of the pigment synthesis inhibiting herbicide clomazone in tolerant-soybean (Glycine max [L.] Merr. cv Corsoy) and susceptible-cotton (Gossypium hirsutum [L.] cv Stoneville 825) photomixotrophic cell suspensions. Soybean and cotton on a whole plant level are tolerant and susceptible to clomazone, respectively. Preliminary studies indicated that I50 values for growth, chlorophyll (Chl), β-carotene, and lutein were, respectively, >22, 14, 19, and 23 times greater for the soybean cell line (SB-M) 8 days after treatment (DAT) compared to the cotton cell line (COT-M) 16 DAT. Differences in [14C]clomazone uptake cannot account for selectivity since there were significantly greater levels of clomazone absorbed by the SB-M cells compared to the COT-M cells for each treatment. The percentage of absorbed clomazone converted to more polar metabolite(s) was significantly greater by the SB-M cells relative to COT-M cells at 6 and 24 hours after treatment, however, only small differences existed between the cell lines by 48 hours after treatment. Nearly identical levels of parental clomazone was recovered from both cell lines for all treatments. A pooled metabolite fraction isolated from SB-M cells had no effect on the leaf pigment content of susceptible velvetleaf (Abutilon theophrasti Medic.) or soybean seedlings. Conversely, a pooled metabolite fraction from COT-M cells reduced the leaf Chl content of velvetleaf. Soybean tolerance to clomazone appears to be due to differential metabolism (bioactivation) and/or differences at the site of action. PMID:16667349

  7. Site of clomazone action in tolerant-soybean and susceptible-cotton photomixotrophic cell suspension cultures. [Glycine max (L. ); Gossypium hirsutum

    Energy Technology Data Exchange (ETDEWEB)

    Norman, M.A.; Liebl, R.A.; Widholm, J.M. (Univ. of Illinois, Urbana (USA))

    1990-10-01

    Studies were conducted to determine the herbicidal site of clomazone action in tolerant-soybean (Glycine max (L.) Merr. cv Corsoy) (SB-M) and susceptible-cotton (Gossypium hirsutum (L.) cv Stoneville 825) (COT-M) photomixotrophic cell suspension cultures. Although a 10 micromolar clomazone treatment did not significantly reduce the terpene or mixed terpenoid content (microgram per gram fresh weight) of the SB-M cell line, there was over a 70% reduction in the chlorophyll (Chl), carotenoid (CAR), and plastoquinone (PQ) content of the COT-M cell line. The tocopherol (TOC) content was reduced only 35.6%. Reductions in the levels of Chl, CAR, TOC, and PQ indicate that the site of clomazone action in COT-M cells is prior to geranylgeranyl pyrophosphate (GGPP). The clomazone treatment did not significantly reduce the flow of ({sup 14}C)mevalonate (({sup 14}C)MEV) (nanocuries per gram fresh weight) into CAR and the three mixed terpenoid compounds of SB-M cells. Conversely, ({sup 14}C)MEV incorporation into CAR and the terpene moieties of Chl, PQ, and TOC in COT-M cells was reduced at least 73%, indicating that the site of clomazone action must be after MEV. Sequestration of clomazone away from the chloroplast cannot account for soybean tolerance to clomazone since chloroplasts isolated from both cell lines incubated with ({sup 14}C)clomazone contained a similar amount of radioactivity (disintegrations per minute per microgram of Chl). The possible site(s) of clomazone inhibition include mevalonate kinase, phosphomevalonate kinase, pyrophosphomevalonate decarboxylase, isopentenyl pyrophosphate isomerase, and/or a prenyl transferase.

  8. Carbon source dependent somatic embryogenesis and plant regeneration in cotton, Gossypium hirsutum L. cv. SVPR2 through suspension cultures.

    Science.gov (United States)

    Ganesan, M; Jayabalan, N

    2005-10-01

    Highly reproducible and simple protocol for cotton somatic embryogenesis is described here by using different concentrations of maltose, glucose, sucrose and fructose. Maltose (30 g/l) is the best carbon source for embryogenic callus induction and glucose (30 g/l) was suitable for induction, maturation of embryoids and plant regeneration. Creamy white embryogenic calli of hypocotyl explants were formed on medium containing MS basal salts, myo-inositol (100 mg/l), thiamine HCI (0.3 mg/l), picloram (0.3 mg/l), Kin (0.1 mg/l) and maltose (30 g/l). During embryo induction and maturation, accelerated growth was observed in liquid medium containing NH3NO4 (1 g/l), picloram (2.0 mg/l), 2 ip (0.2 mg/l), Kin (0.1 mg/l) and glucose (30 g/l). Before embryoid induction, large clumps of embryogenic tissue were formed. These tissues only produced viable embryoids. Completely matured somatic embryos were germinated successfully on the medium fortified with MS salts, myo-inositol (50 mg/l), thiamine HCl (0.2 mg/l), GA3 (0.2 mg/l), BA (1.0 mg/l) and glucose (30 g/l). Compared with earlier reports, 65% of somatic embryo germination was observed. The abnormal embryo formation was highly reduced by using glucose (30 g/l) compared to other carbon sources. The regenerated plantlets were fertile but smaller in height than the seed derived control plants.

  9. Somatic embryogenesis in suspension cultures of Gossypium klotzschianum anderss.

    Science.gov (United States)

    Price, H J; Smith, R H

    1979-01-01

    Somatic embryoids differentiated in suspension cultures of G. klotzschianum after 3-4 weeks of culture in a liquid medium containing glutamine (optimally, 10-15 mM). Embryogenesis occurred after a preculture of callus on a medium containing 10 mg/l of the cytokinin, 2iP. The embryoids had meristematic regions, a well formed epidermis, and formed roots and vestigial leaves. Asparagine was much less effective than glutamine in promoting embryoid differentiation. The presence of 2,4-D in the medium resulted in increased vigor of the suspension cultures and subsequently in the formation of many embryoids, but does not seem to be necessary for somatic embryogenesis in cotton.

  10. Cell Suspension Culture of Neem Tree

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    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...

  11. Somatic embryogenesis in Lolium multiflorum suspension culture

    Directory of Open Access Journals (Sweden)

    Margarita Pavlova

    2014-01-01

    Full Text Available The embryogenic cell suspension was obtained from immature embryos of Lolium multiflorum through a callus culture. Somatic embryogenesis was induced by addition of 2,4-D, dicamba and picloram in 0,5 mg/l concentrations in MS liquid nutrient medium. It was shown that somatic embryos arised from single cells. In globular embryoids, the meristematic cells are characterized by the presence of phytoferritin inclusions in the leucoplasts.

  12. Studies on suspension culture of virginia mallow

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    Anna Kasprzyk

    2014-04-01

    Full Text Available Virginia mallow (Sida hermaphrodita (L. Rusby belongs to the Malvaceae family. It is a very important industrial and energetic crop (Kasprzyk et al. 2013. In our studies, we used plant cell suspension cultures due to the fact that it is a useful tool to investigate biochemical, molecular and physiological aspects of many cellular functions (Dong et al. 2010. Virginia mallow seeds, obtained from Prof. Borkowska (University of Life Sciences in Lublin, Poland, were used in this investigation to obtain plants which were grown in sterile conditions in the Department of Plant Anatomy and Cytology, Maria Curie-Skłodowska University in Lublin, Poland. The seeds were surface sterilized and washed three times in sterile, distilled water. After 3 weeks of in vitro culture, young seedlings were used as a source of explants (to callus induction. Two types of explants were used to form callus culture: leaf and petiole. Callus tissues were then aseptically transferred to an Erlenmeyer flask with liquid medium and placed on an orbital shaker moving at 120 rpm. The observations of this suspense culture were conducted under light and confocal LSM microscopes. The authors observed that depending on the type of explants and composition of medium, callus tissue has varied in color and character of growth.

  13. Regeneration of soybean via embryogenic suspension culture

    Directory of Open Access Journals (Sweden)

    Droste Annette

    2001-01-01

    Full Text Available In an attempt to establish an alternative plant regeneration system for soybean [Glycine max (L. Merrill] cultivars used in Brazilian breeding programs, ten genotypes were tested for their embryogenic potential. Cotyledons were removed as explants from immature seeds harvested from field-grown plants. After 45 days on induction medium, the number of responding cotyledons and the number of somatic embryos per immature cotyledon were evaluated. The percentage of explants that produced somatic embryos varied from 1 to 70% among cultivars. The average number of somatic embryos produced per cotyledon pair ranged from 0.01 to 10.3 with a mean of 3.4. Suspension cultures were initiated with three Agrobacterium tumefaciens susceptible cultivars. Suspensions were successfully developed from Bragg and IAS5 cultivars. The packed cell volume, in one-month growth, increased 8.1 fold for Bragg and 3.5 fold for IAS5 and the fresh weight increased 6.6 and 2.8 fold, respectively. The cultivars differed for the analysed parameters. All tissue from each cultivar was transferred to the maturation medium and subsequently to the germination medium. The germination frequency was 45.7 and 54.9% for Bragg and IAS5, respectively. Plants were gradually exposed to ambient humidity over one week and then planted in soil. All plants yielded seeds in the greenhouse.

  14. Study on Cell Suspension Culture of Floribunda Rose

    Institute of Scientific and Technical Information of China (English)

    ZHANG Chun'ai; WANG Jingang; FAN Jinping; GONG Shufang; CHE Daidi

    2008-01-01

    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,fi-iable 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.

  15. Large-scale production of monoclonal antibodies in suspension culture.

    Science.gov (United States)

    Backer, M P; Metzger, L S; Slaber, P L; Nevitt, K L; Boder, G B

    1988-10-01

    Monoclonal antibodies are being manufactured for clinical trials in suspension culture at the 1300-L scale. Suspension culture offers some advantages relative to high-density mammalian cell culture methods; in particular, the ability to closely monitor the behavior of cells in a homogeneous environment. Computer control and on-line mass spectrography of exit gases provide instantaneous information about the culture metabolic activity. Air sparging and agitation by marine impeller provide aeration sufficient to maintain a constant dissolved oxygen tension at cell concentrations up to 5.0 x 10(6) cells/mL without causing apparent cell damage.

  16. Studies on Genetic Transformatiom of Embryogenic Suspension Cultures of Sweetpotato

    Institute of Scientific and Technical Information of China (English)

    ZHAI Hong; LIU Qing-chang

    2003-01-01

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

  17. Visnagin: biosynthesis and isolation from Ammi visnagi suspension cultures.

    Science.gov (United States)

    Kaul, B; Staba, E J

    1965-12-24

    During an examination of Ammi visnaga Lam. suspension cultures for the biosynthesis of furanochromones and related medicinal compounds, visnagin was isolated in crystalline form and identified. Thus, certain medicinally important secondary plant metabolites may be produced in appreciable amounts by plant tissue cultures.

  18. SUSPENSION CULTURE AND PLANT REGENERATION OF TYPHA LATIFOLIA

    Science.gov (United States)

    This study is the first reported attempt to generate a growth curve from Typha latifolia L. (broadleaf cattail) callus cells in suspension culture. Several media and hormone combinations were tested for their capacity to induce callus cell formation from T. latifolia leaf section...

  19. SUSPENSION CULTURE AND PLANT REGENERATION OF TYPHA LATIFOLIA

    Science.gov (United States)

    This study is the first reported attempt to generate a growth curve from Typha latifolia L. (broadleaf cattail) callus cells in suspension culture. Several media and hormone combinations were tested for their capacity to induce callus cell formation from T. latifolia leaf section...

  20. Production of recombinant proteins in suspension-cultured plant cells.

    Science.gov (United States)

    Plasson, Carole; Michel, Rémy; Lienard, David; Saint-Jore-Dupas, Claude; Sourrouille, Christophe; de March, Ghislaine Grenier; Gomord, Véronique

    2009-01-01

    Plants have emerged in the past decade as a suitable alternative to the current production systems for recombinant pharmaceutical proteins and, today their potential for low-cost production of high quality, much safer and biologically active mammalian proteins is largely documented. Among various plant expression systems being explored, genetically modified suspension-cultured plant cells offer a promising system for production of biopharmaceuticals. Indeed, when compared to other plant-based production platforms that have been explored, suspension-cultured plant cells have the advantage of being totally devoid of problems associated with the vagaries of weather, pest, soil and gene flow in the environment. Because of short growth cycles, the timescale needed for the production of recombinant proteins in plant cell culture can be counted in days or weeks after transformation compared to months needed for the production in transgenic plants. Moreover, recovery and purification of recombinant proteins from plant biomass is an expensive and technically challenging business that may amount to 80-94% of the final product cost. One additional advantage of plant cell culture is that the recombinant protein fused with a signal sequence can be expressed and secreted into the culture medium, and therefore recovered and purified in the absence of large quantities of contaminating proteins. Consequently, the downstream processing of proteins extracted from plant cell culture medium is less expensive, which may/does balance the higher costs of fermentation. When needed for clinical use, recombinant proteins are easily produced in suspension-cultured plant cells under certified, controllable and sterile conditions that offer improved safety and provide advantages for good manufacturing practices and regulatory compliance. In this chapter, we present basic protocols for rapid generation of transgenic suspension-cultured cells of Nicotiana tabacum, Oriza sativa and Arabidopis

  1. Growth and Plating of Cell Suspension Cultures of Datura Innoxia

    DEFF Research Database (Denmark)

    Engvild, Kjeld Christensen

    1974-01-01

    Suspension cultures of Datura innoxia Mill, were successfully grown on a modified Murashige and Skoog medium with 2,4–D, NAA or BAP as growth substances, provided the micronutrient levels were reduced to 1/10. Normal amounts of micronutrients were toxic. Attempts to identify the toxic elements did...... malate) or on NO3−-N alone. Dry weight yield was proportional to the amount of nitrate-N added (47 mg/mg N). Filtered suspension cultures containing single cells (plating cultures) could be grown in agar in petri dishes when NAA or 2,4-D were used as growth substances. Cells grew at densities above 500...... units/ml in the agar. Most colonies grew from cell aggregates but division in single cells was observed. The highest plating efficiency was about 50% on 10−6 M 2,4-D + 1 g/1 casein hydrolysate....

  2. Embryo forming cells in carrot suspension cultures.

    OpenAIRE

    Toonen, M.A.J.

    1997-01-01

    Somatic cells of many plant species can be cultured in vitro and induced to form embryos that are able to develop into mature plants. This process, termed somatic embryogenesis, was originally described in carrot (Daucus carota L.). Somatic embryos develop through the same characteristic morphological stages, i.e. the globular-, heartand torpedo-stage respectively, as their zygotic counterparts. Due to the different cellular origin of somatic embryos, it is less clear to what extent the earli...

  3. Callus and cell suspension cultures of carnation

    DEFF Research Database (Denmark)

    Engvild, Kjeld Christensen

    1972-01-01

    Callus cultures of carnation, Dianthus caryophyllus L. ev. G. J. Sim, were grown on a synthetic medium of half strength Murashige and Skoog salts, 3 % sucrose, 100 mg/l of myo-inositol, 0.5 mg/l each of thiamin, HCl, pyridoxin, HCl and nicotinic acid and 10 g/l agar. Optimal concentrations of gro......, but all attempts to induce formation of shoots or em-bryoids gave negative results....

  4. Production of plant virus inhibitor by Phytolacca americana suspension culture.

    Science.gov (United States)

    Misawa, M; Hayashi, M; Tanaka, H

    1975-09-01

    The inhibitory activity of tobacco mosaic virus (TMV) infection was assayed with the extracts of various callus tissues derived from the intact plants. Phytolacca americana callus was selected as a producer of the virus inhibitor and its cultural conditions in suspension were examined for cell growth and the inhibitor production. A modified liquid medium containing twofold concentrations of all components in that of Murashige and Skoog plus2,4-D (1.0 mg/liter) and sucrose (6%), but without any vitamins and glycine was chosen for production of higher levels of the inhibitor. TMV infections in tobacco, bean, and tomato plants were markedly inhibited by the introduction of the disrupted whole broth of suspension cultured P. americana.

  5. Putting the spotlight back on plant suspension cultures

    Directory of Open Access Journals (Sweden)

    Rita B. Santos

    2016-03-01

    Full Text Available Plant cell suspension cultures have several advantages that make them suitable for the production of recombinant proteins. They can be cultivated under aseptic conditions using classical fermentation technology, they are easy to scale-up for manufacturing, and the regulatory requirements are similar to those established for well-characterized production systems based on microbial and mammalian cells. It is therefore no surprise that taliglucerase alfa (Elelyso® – the first licensed recombinant pharmaceutical protein derived from plants – is produced in plant cell suspension cultures. But despite this breakthrough, plant cells are still largely neglected compared to transgenic plants and the more recent plant-based transient expression systems. Here, we revisit plant cell suspension cultures and highlight recent developments in the field that show how the rise of plants cells parallels that of Chinese hamster ovary cells, currently the most widespread and successful manufacturing platform for biologics. These developments include medium optimization, process engineering, statistical experimental designs, scale-up/scale-down models and process analytical technologies. Significant yield increases for diverse target proteins will encourage a gold rush to adopt plant cells as a platform technology, and the first indications of this breakthrough are already on the horizon.

  6. Characterization of aggregate size in Taxus suspension cell culture.

    Science.gov (United States)

    Kolewe, Martin E; Henson, Michael A; Roberts, Susan C

    2010-05-01

    Plant cells grow as aggregates in suspension culture, but little is known about the dynamics of aggregation, and no routine methodology exists to measure aggregate size. In this study, we evaluate several different methods to characterize aggregate size in Taxus suspension cultures, in which aggregate diameters range from 50 to 2,000 microm, including filtration and image analysis, and develop a novel method using a specially equipped Coulter counter system. We demonstrate the suitability of this technology to measure plant cell culture aggregates, and show that it can be reliably used to measure total biomass accumulation compared to standard methods such as dry weight. Furthermore, we demonstrate that all three methods can be used to measure an aggregate size distribution, but that the Coulter counter is more reliable and much faster, and also provides far better resolution. While absolute measurements of aggregate size differ based on the three evaluation techniques, we show that linear correlations are sufficient to account for these differences (R(2) > 0.99). We then demonstrate the utility of the novel Coulter counter methodology by monitoring the dynamics of a batch process and find that the mean aggregate size increases by 55% during the exponential growth phase, but decreases during stationary phase. The results indicate that the Coulter counter method can be routinely used for advanced process characterization, particularly to study the relationship between aggregate size and secondary metabolite production, as well as a source of reliable experimental data for modeling aggregation dynamics in plant cell culture.

  7. Isolation and culture of Celosia cristata L cell suspension protoplasts

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    Retno Mastuti

    2003-06-01

    Full Text Available Developmental competence of Celosia cristata L. cell suspension-derived protoplasts was investigated. The protoplasts were isolatedfrom 3- to 9-d old cultures in enzyme solution containing 2% (w/v Cellulase YC and 0.5% (w/v Macerozyme R-10 which was dissolvedin washing solution (0.4 M mannitol and 10 mM CaCl2 at pH 5.6 for 3 hours. The highest number of viable protoplasts was releasedfrom 5-d old culture of a homogenous cell suspension. Subsequently, three kinds of protoplast culture media were simultaneously examinedwith four kinds of concentration of gelling agent. Culturing the protoplasts on KM8p medium solidified with 1.2% agarose significantlyenhanced plating efficiency as well as microcolony formation. Afterwards, the microcalli actively proliferated into friable watery calluswhen they were subcultured on MS medium supplemented with 0.3 mg/l 2,4-D and 1.0 mg/l kinetin. Although the plant regenerationfrom the protoplasts-derived calli has not yet been obtained, the reproducible developmental step from protoplasts to callus in thisstudy may facilitate the establishment of somatic hybridization using C. cristata as one parent.

  8. Somatic Embryogenesis from Cell Suspension Cultures of Aspen Clone

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Suspension cultures initiated from callus derived from petiole explants of aspen hybrid (Populus tremuloides × P.tremula) produced somatic embryos. Callus was induced on a MS medium supplemented with 5 mg·L-1 2,4-D and 0.05 mg·L-1 zeatin under light conditions. Embryogenic calli were obtained when a subsequent subculture of calli was suspended in the same basal medium with 10 mg·L-1 2,4-D. The highest number of globular embryos were induced from embryogenic calli by cell suspension culture in a MS liquid medium supplemented with 10 mg·L-1 2,4-D. Genotype and 2,4-D concentration were vital to the induction of embryogenic calli producing competent cells. Embryogenic calli for each genotype were heterogeneous. Green calli with gel-like consistency could yield more competent cells than light yellow embryogenic calli. However, some globular embryos broke into slices and some developed abnormally after one month of culture under the same or other hormonal conditions.

  9. A Versatile Bioreactor for Dynamic Suspension Cell Culture. Application to the Culture of Cancer Cell Spheroids.

    Science.gov (United States)

    Massai, Diana; Isu, Giuseppe; Madeddu, Denise; Cerino, Giulia; Falco, Angela; Frati, Caterina; Gallo, Diego; Deriu, Marco A; Falvo D'Urso Labate, Giuseppe; Quaini, Federico; Audenino, Alberto; Morbiducci, Umberto

    2016-01-01

    A versatile bioreactor suitable for dynamic suspension cell culture under tunable shear stress conditions has been developed and preliminarily tested culturing cancer cell spheroids. By adopting simple technological solutions and avoiding rotating components, the bioreactor exploits the laminar hydrodynamics establishing within the culture chamber enabling dynamic cell suspension in an environment favourable to mass transport, under a wide range of tunable shear stress conditions. The design phase of the device has been supported by multiphysics modelling and has provided a comprehensive analysis of the operating principles of the bioreactor. Moreover, an explanatory example is herein presented with multiphysics simulations used to set the proper bioreactor operating conditions for preliminary in vitro biological tests on a human lung carcinoma cell line. The biological results demonstrate that the ultralow shear dynamic suspension provided by the device is beneficial for culturing cancer cell spheroids. In comparison to the static suspension control, dynamic cell suspension preserves morphological features, promotes intercellular connection, increases spheroid size (2.4-fold increase) and number of cycling cells (1.58-fold increase), and reduces double strand DNA damage (1.5-fold reduction). It is envisioned that the versatility of this bioreactor could allow investigation and expansion of different cell types in the future.

  10. Protoplast culture and protoplast symmetric fusion in cotton%棉花原生质体培养和原生质体对称融合研究

    Institute of Scientific and Technical Information of China (English)

    孙玉强

    2011-01-01

    In light of the critical need to increase genetic diversity in the gene pool, cotton improvement programs are increasingly turning to the application of molecular approaches to breeding and germplasm utilizations. The abundant species of wild cotton (Gossypium spp. ) are an important renewable resources and have been the valuable genetic germplasms for cotton genentic improvement,and which has been significant in the reality and theory, potential in the application. It is very difficult to widecross between cul-tivars and wild species for the distant relationship,or no fertility of Fi hybrids,or very low fertility. Application of biotechnology is an effective way for developing new germplasm in cotton,and we aim to develop new sources of cotton germplasm via somatic cell culture, protoplast culture and protoplast fusion. Our studies involved somatic embryogenesis and plant regeneration in wild cotton species,protoplast culture in Gossypium hirsutum L. And wild species, protoplast fusion between cultivars and wild species. The main results of this research were as follows:1. Calli were induced from 9 wild cotton species. Among them,the normally regenerated plants were obtained from G. Davidsonii ,G. Klotzschianum, G. Raimondii and G. Stocksii via somatic embryogenesis, regenerated plants with abnormal morphology from G. Aridum. Only non-embryogenic calli were obtained from G. Anomalum,G. Ajricanum,G. Thurberi and G. Bickii. We studied the methods and factors for embryogenic callus induction and conservation,improving somatic embryos maturation and germination, plant regeneration in detailed, and then a new and elementary protocol has been developed for somatic cell culture,mainly somatic embryogenesis and plant regeneration in wild cotton species. The combination of 2,4-D/KT was very useful for callus induction in all tested wild species. Different combinations of PGR,sugar sources,suspension culture and environmental stress etc improved the formation of embryogenic

  11. Induction of Apoptosis in Protoplasts and Suspension Cultures of Plant Cells

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Many studies have showed that apoptosis exists in plants. Our study shows that (1) menadione(VK3) induces apoptosis in suspension cultures of carrot cells; (2) heat shock induces apoptosis in suspension cultures of tobacco cells; and (3) ethrel induces apoptosis in carrot protoplasts. Some important indications of apoptosis were observed, including DNA laddering, TUNEL-positive reaction, condensation and degradation of nuclei.

  12. Auxin requirements of sycamore cells in suspension culture.

    Science.gov (United States)

    Moloney, M M; Hall, J F; Robinson, G M; Elliott, M C

    1983-04-01

    Sycamore (Acer pseudoplatanus L.) cell suspension cultures (strain OS) require 2,4-dichlorophenoxyacetic acid (2,4-D) in their culture medium for normal growth. If the 2,4-D is omitted, rates of cell division are dramatically reduced and cell lysis may occur. Despite this ;auxin requirement,' it has been shown by gas chromatography-mass spectrometry that the cells synthesize indol-3yl-acetic acid (IAA). Changes in free 2,4-D and IAA in the cells during a culture passage have been monitored.There is a rapid uptake of 2,4-D by the cells during the lag phase leading to a maximum concentration per cell (125 nanograms per 10(6) cells) on day 2 followed by a decline to 45 nanograms per 10(6) cells by day 9 (middle of linear phase). The initial concentration of IAA (0.08 nanograms per 10(6) cells) rises slowly to a peak of 1.4 nanograms per 10(6) cells by day 9 then decreases rapidly to 0.2 nanograms per 10(6) cells by day 15 (early declining phase) and 0.08 nanograms per 10(6) cells by day 23 (early stationary phase).

  13. Enhanced catharanthine and vindoline production in suspension cultures of Catharanthus roseus by ultraviolet-B light

    OpenAIRE

    Ramani, Shilpa; Jayabaskaran, Chelliah

    2008-01-01

    Suspension cultures of Catharanthus roseus were used to evaluate ultraviolet-B (UV-B) treatment as an abiotic elicitor of secondary metabolites. A dispersed cell suspension culture from C. roseus leaves in late exponential phase and stationary phase were irradiated with UV-B for 5 min. The stationary phase cultures were more responsive to UV-B irradiation than late exponential phase cultures. Catharanthine and vindoline increased 3-fold and 12-fold, respectively, on treatment with a 5-min UV-...

  14. Establishment of callus, cell suspension and shoot cultures of Leonurus cardiaca L. and diterpene analysis.

    Science.gov (United States)

    Knöss, W

    1995-10-01

    Callus cultures, cell suspension cultures and shoot cultures of Leonurus cardiaca L. (Motherwort) were established and growth conditions optimized. Shoot cultures showed constant growth whether in the dark or under continuous light, accumulating varying amounts of the furanic labdane diterpenes leosibiricin, preleosibirin, leosibirin and isoballotenol acetate, which are also present in the soil-grown plants. Only traces of leosibiricin were detected in callus cultures, while cell suspension cultures did not produce any furanic diterpenes. A small amount of furanic labdane diterpenes was found in the medium of shoot cultures. Callus and shoot culture induction of several other Lamiaceae species is also described.

  15. Isolation and culture of suspension protoplasts of vetiver

    Directory of Open Access Journals (Sweden)

    Somporn Prasertsongskun

    2005-05-01

    Full Text Available In this research, protoplasts were isolated from cell suspension derived from inflorescence of vetiver (Vetiveria zizanioides Nash Surat Thani germplasm. The optimum condition for protoplast isolation was established by using 2% cellulase Onozuka R10, 2% macerozyme R10, 0.5% pectinase in 0.4 M mannitol and 7 mM CaCl2.2H2O at pH 5.8 and incubated for 10 hours in the dark on the rotary shaker at 50 rpm. Maximum protoplast yields were 8.4 × 104 protoplasts/ml PCV. Division of protoplasts was observed only in liquid medium. The first cell division was observed after 3 days of culture initiation, and the average division was 5.0% in the N6 medium supplemented with 1.0 mg/l 2,4-D (2,4-dichlorophenoxyacetic acid and 0.5 mg/l BA (Benzyladenine. An optimal density for culture division was 1 × 105 protoplasts/ml.

  16. Nitration of plant apoplastic proteins from cell suspension cultures.

    Science.gov (United States)

    Szuba, Agnieszka; Kasprowicz-Maluśki, Anna; Wojtaszek, Przemysław

    2015-04-29

    Nitric oxide causes numerous protein modifications including nitration of tyrosine residues. This modification, though one of the greatest biological importance, is poorly recognized in plants and is usually associated with stress conditions. In this study we analyzed nitrotyrosines from suspension cultures of Arabidopsis thaliana and Nicotiana tabacum, treated with NO modulators and exposed to osmotic stress, as well as of BY2 cells long-term adapted to osmotic stress conditions. Using confocal microscopy, we showed that the cell wall area is one of the compartments most enriched in nitrotyrosines within a plant cell. Subsequently, we analyzed nitration of ionically-bound cell-wall proteins and identified selected proteins with MALDI-TOF spectrometry. Proteomic analysis indicated that there was no significant increase in the amount of nitrated proteins under the influence of NO modulators, among them 3-morpholinosydnonimine (SIN-1), considered a donor of nitrating agent, peroxynitrite. Moreover, osmotic stress conditions did not increase the level of nitration in cell wall proteins isolated from suspension cells, and in cultures long-term adapted to stress conditions; that level was even reduced in comparison with control samples. Among identified nitrotyrosine-containing proteins dominated the ones associated with carbon circulation as well as the numerous proteins responding to stress conditions, mainly peroxidases. High concentrations of nitric oxide found in the cell wall and the ability to produce large amounts of ROS make the apoplast a site highly enriched in nitrotyrosines, as presented in this paper. Analysis of ionically bound fraction of the cell wall proteins indicating generally unchanged amounts of nitrotyrosines under influence of NO modulators and osmotic stress, is noticeably different from literature data concerning, however, the total plant proteins analysis. This observation is supplemented by further nitroproteome analysis, for cells long

  17. Use of attenuated total reflection Fourier transform infrared (ATR FT-IR) spectroscopy in direct, non-destructive, and rapid assessment of developmental cotton fibers grown in planta and in culture

    Science.gov (United States)

    Cotton fibers are routinely harvested from cotton plants (in planta), and their end-use qualities depend on their development stages. Cotton fibers are also cultured at controlled laboratory environments, so that cotton researchers can investigate many aspects of experimental protocols in cotton bre...

  18. Effects of pigment glands andgossypol on somatic cell cul-ture of upland cotton (Gos-sypium hirsutum L.)

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The effects of pigment glands and gossypol on the somatic cell culture of upland cotton were studied, using the materials as follows: three pairs of glanded and glandless upland cotton near isogenic lines, TM-1, and Coker 312. The results showed that the pigment glands and gossypol contents in the explants had great inhibiting effect on the induction and growth of callus in somatic cell culture of upland cotton, and the induction rate of callus and the single callus weight of glandless cotton were much higher than those of their glanded near isogenic lines. It was easier to obtain regeneration plants from glandless cotton than from their glanded near isogenic lines. There was a significant inverse correlation between the gossypol contents in the explants and callus induction rate, with the correlation coefficient of ?0.84. The vitro gossypol in the medium had some inhibiting effect on the induction and growth of callus, especially for the glandless cotton. However, a certain concentration of vitro gossypol in the medium (0.1 mg/L) was an aid to the steadiness growth of callus in glandless cotton somatic cell culture, with a high rate of embryogenic cells which was in favor of plant regeneration, and it was also relatively easy to obtain regeneration plants when they were transferred into differentiation medium with 0.1 mg/L of vitro gossypol, even for some cultivars which are difficult in somatic cell culture. In addition, the gossypol content and its variation in the seedlings and callus during culture of Coker 312 were discussed, as well as the relationship between gossypol variation in the explants and its somatic cell culture. The probability of vitro gossypol used in cotton somatic cell culture for the improvement of somatic cell culture was suggested.

  19. Growth arrest of vascular smooth muscle cells in suspension culture using low-acyl gellan gum.

    Science.gov (United States)

    Natori, Tomomi; Fujiyoshi, Masachika; Uchida, Masashi; Abe, Natsuki; Kanaki, Tatsuro; Fukumoto, Yasunori; Ishii, Itsuko

    2017-03-01

    The proliferation of vascular smooth muscle cells (SMCs) causes restenosis in biomaterial vascular grafts. The purposes of this study were to establish a suspension culture system for SMCs by using a novel substrate, low-acyl gellan gum (GG) and to maintain SMCs in a state of growth inhibition. When SMCs were cultured in suspension with GG, their proliferation was inhibited. Their viability was 70% at day 2, which was maintained at more than 50% until day 5. In contrast, the viability of cells cultured in suspension without GG was 5.6% at day 2. By cell cycle analysis, the ratio of SMCs in the S phase when cultured in suspension with GG was lower than when cultured on plastic plates. In SMCs cultured in suspension with GG, the ratio of phosphorylated retinoblastoma (Rb) protein to Rb protein was decreased and p27(Kip1) expression was unchanged in comparison with SMCs cultured on plastic plates. In addition, SMCs could be induced to proliferate again by changing the culture condition from suspension with GG to plastic plates. These results suggest that our established culturing method for SMCs is useful to maintain SMCs in a state of growth inhibition with high viability.

  20. Beryllium toxicity testing in the suspension culture of mouse fibroblasts.

    Science.gov (United States)

    Rössner, P; Bencko, V

    1980-01-01

    Suspension culture of mouse fibroblast cell line L-A 115 was used to test beryllium toxicity in the presence of magnesium ions. Beryllium added to the MEM cultivation medium was bound in a complex with sulphosalicylic acid BeSSA complex, because the use of beryllium chloride turned out to yield ineffective beryllium phosphate that formed macroscopically detectable insoluble opacities. The BeSSA complex was used in the concentration range: 10(-3)--10(-9)M, magnesium was used in 3 concentrations: 10(-1)M, 5 x 10(-2)M and 10(-2)M. Growth curve analysis revealed pronounced beryllium toxicity at the concentration of 10(-3)M, magnesium-produced toxic changes were observed only at the concentration of 10(-1)M. No competition between the beryllium and magnesium ions was recorded. It is assumed that the possible beryllium-magnesium competition was significantly modified by the use of BeSSA complex-bound beryllium.

  1. Cryopreservation of transformed and wild-type Arabidopsis and tobacco cell suspension cultures.

    Science.gov (United States)

    Menges, Margit; Murray, James A H

    2004-02-01

    We have recently described Arabidopsis cell suspension cultures that can be effectively synchronised. Here, we describe procedures that allow clonal-transformed cell suspension lines to be produced using Agrobacterium-mediated transformation, and an optimised and straightforward procedure for the cryopreservation and recovery of both parental and transformed lines. Frozen cultures show 90% viability and rapid re-growth after recovery. We show that the cryopreservation procedure is equally applicable to the frequently used tobacco bright yellow (BY)2 cell suspension culture, and that cell cycle synchronisation capacity of parental lines is maintained after both transformation and recovery from cryopreservation. The techniques require no specialised equipment, and are suitable for routine laboratory use, greatly facilitating the handling and maintenance of cell cultures and providing security against both contamination and cumulative somaclonal variation. Finally, the ability to store easily large numbers of transformed lines opens the possibility of using Arabidopsis cell suspension cultures for high-throughput analysis.

  2. A phytochemical study of lignans in whole plants and cell suspension cultures of Anthriscus sylvestris

    NARCIS (Netherlands)

    Koulman, A; Kubbinga, M.E.; Batterman, S; Woerdenbag, H.J.; Pras, N.; Woolley, J.G.; Quax, Wim

    2003-01-01

    In the roots of Anthriscus sylvestris 12 different lignans were detected. Arctigenin, dimethylmatairesinol, dimethylthujaplicatin, podophyllotoxin, 7-hydroxyyatein and 7-hydroxyanhydropodorhizol have not been previously reported to be present in A. sylvestris. In the cell suspension cultures, which

  3. Measuring NO Production by Plant Tissues and Suspension Cultured Cells

    Institute of Scientific and Technical Information of China (English)

    Jan Vitecek; Vilem Reinohl; Russell L.Jones

    2008-01-01

    We describe an inexpensive and reliable detector for measuring NO emitted in the gas phase from plants.The method relies on the use of a strong oxidizer to convert NO to NO2 and subsequent capture of NO2 by a Griess reagent trap.The set-up approaches the sensitivity for NO comparable to that of instruments based on chemiluminescence and photoacoustic detectors.We demonstrate the utility of our set-up by measuring NO produced by a variety of well established plant sources.NO produced by nitrate reductase (NR) in tobacco leaves and barley aleurone was readily detected,as was the production of NO from nitrite by the incubation medium of barley aleurone.Arabidopsis mutants that overproduce NO or lack NO-synthase (AtNOS1) also displayed the expected NO synthesis phenotype when assayed by our set-up.We could also measure NO production from elicitor-treated suspension cultured cells using this set-up.Further,we have focused on the detection of NO by a widely used fluorescent probe 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM).Our work points to the pitfalls that must be avoided when using DAF-FM to detect the production of NO by plant tissues.In addition to the dramatic effects that pH can have on fluorescence from DAF-FM,the widely used NO scavengers 2-phenyl-4,4,5,5-tetramethylimidazoline-l-oxyl-3-oxide (PTIO) and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) can produce anomalous and unexpected results.Perhaps the most serious drawback of DAF-FM is its ability to bind to dead cells and remain NO-sensitive.

  4. Growth and accumulation of flavan-3-ol in Camellia sinensis through callus culture and suspension culture method

    Directory of Open Access Journals (Sweden)

    Sutini Sutini

    2017-02-01

    Full Text Available This study was aimed to assess flavan-3-ol biomass in C. sinensis through callus cultures and suspension cultures derived from leaf explants. Callus initiation of both cultures were using Murashige and Skoog medium were enriched with plant growth regulators Naphtha-lene Acetic Acid 3.0 mg/L and kinetin 2.0 mg/L. The procedures in this study were: (1 callus initiation by cutting the leaves of C. sinen-sis shoots then planted on Murashige and Skoog medium that were enriched with plant growth regulators, (2 sub callus culture on fresh medium that enriched with the same growth regulators, (3 suspension culture initiation of liquid callus, (4 growth examination of callus and suspension cultures in week 12, (5 examination of qualitative-quantitative content of flavan-3-olin suspension cultures at week 4. The results show that suspension cultures contain biomass flavan-3-ol that increase in the same manner of the increase of callus age and weight

  5. In-ovulo embryo culture and seedling development of cotton (Gossypium hirsutum L.).

    Science.gov (United States)

    Stewart, J M; Hsu, C L

    1977-01-01

    A convenient and reliable method for culturing cotton embryos is needed to obtain interspecific hybrids of this genus. C.A. Beasley and I.P. Ting (Amer. J. Bot. 60, 130, 1973) developed a phytohormone-supplemented medium (BTP) upon which the growth of ovules was similar that of in situ ovules. This medium was examined for in-ovulo embryo culture. Although good ovule growth occurred on BTP no embryos developed to maturity. However, when the medium was supplemented with NH 4 (+) , more than 50% of the ovules produced mature embryos, and many of these germinated precociously after 8-10 weeks of culture. After germination seedlings were established on a separate medium designed to give balanced root and shoot growth. Subsequently young plants could be transferred to pots for greenhouse culture.

  6. Chromatographic study of marmesin and visnagin occurrence in Ammi visnaga Lam. suspension tissue cultures

    Directory of Open Access Journals (Sweden)

    Jadwiga H. Supniewska

    2015-05-01

    Full Text Available Chromatographic examination of tissue from suspension cultures of A. visnaga proved their ability to biosynthesis of furanochromone-visnagin and furanocoumarin-marmesin. The occurrence of these two compounds depends on the composition of medium which also influences culture growth and embryogenesis, after subculture for at least l year

  7. Production of secondary metabolites trimethyl xanthina by Camellia sinensis L suspension culture

    Science.gov (United States)

    Sutini, Sodiq, Mochamad; Muslihatin, Wirdhatul; Indra, Mochamad Rasjad

    2017-06-01

    Bioactive trimethyl xanthina can be obtained from the plant Camellia sinensis L. To obtain bioactive plant of which there are several hurdles for instance to wait up to five years to be harvested, also it needs land at a certain height from the sea level. Therefore, the production of secondary metabolites trimethyl xanthina need to be developed with suspense culture techniques. The purpose of this study obtained the production of bioactive trimethyl xanthina way culturally suspense in large scale with a relatively short time, potentially as anti-oxidants. Research methods include: (1) initiation of callus from pieces of leaves, shoots the youngest of the plant Camellia sinensis L in the media MS with the optimization of the addition of growth regulators, (2) the subculture of callus on media and plant growth regulator that is equal to the stage of initiation, (3) initiation of suspension culture using explants of callus Camellia sinensis L, (4) Analysis of secondary metabolites trimethyl xanthina growth in suspension culture, (5) the isolation and identification of trimethyl xanthina qualitatively and quantitatively using thin layer chromatography/high performance chromatography column. The results of the study suspension cultures containing bioactive trimethyl xanthina candidates that can be used as an antioxidant.

  8. Development of a scalable suspension culture for cardiac differentiation from human pluripotent stem cells

    Directory of Open Access Journals (Sweden)

    Vincent C. Chen

    2015-09-01

    Full Text Available To meet the need of a large quantity of hPSC-derived cardiomyocytes (CM for pre-clinical and clinical studies, a robust and scalable differentiation system for CM production is essential. With a human pluripotent stem cells (hPSC aggregate suspension culture system we established previously, we developed a matrix-free, scalable, and GMP-compliant process for directing hPSC differentiation to CM in suspension culture by modulating Wnt pathways with small molecules. By optimizing critical process parameters including: cell aggregate size, small molecule concentrations, induction timing, and agitation rate, we were able to consistently differentiate hPSCs to >90% CM purity with an average yield of 1.5 to 2 × 109 CM/L at scales up to 1 L spinner flasks. CM generated from the suspension culture displayed typical genetic, morphological, and electrophysiological cardiac cell characteristics. This suspension culture system allows seamless transition from hPSC expansion to CM differentiation in a continuous suspension culture. It not only provides a cost and labor effective scalable process for large scale CM production, but also provides a bioreactor prototype for automation of cell manufacturing, which will accelerate the advance of hPSC research towards therapeutic applications.

  9. Development of a scalable suspension culture for cardiac differentiation from human pluripotent stem cells.

    Science.gov (United States)

    Chen, Vincent C; Ye, Jingjing; Shukla, Praveen; Hua, Giau; Chen, Danlin; Lin, Ziguang; Liu, Jian-chang; Chai, Jing; Gold, Joseph; Wu, Joseph; Hsu, David; Couture, Larry A

    2015-09-01

    To meet the need of a large quantity of hPSC-derived cardiomyocytes (CM) for pre-clinical and clinical studies, a robust and scalable differentiation system for CM production is essential. With a human pluripotent stem cells (hPSC) aggregate suspension culture system we established previously, we developed a matrix-free, scalable, and GMP-compliant process for directing hPSC differentiation to CM in suspension culture by modulating Wnt pathways with small molecules. By optimizing critical process parameters including: cell aggregate size, small molecule concentrations, induction timing, and agitation rate, we were able to consistently differentiate hPSCs to >90% CM purity with an average yield of 1.5 to 2×10(9) CM/L at scales up to 1L spinner flasks. CM generated from the suspension culture displayed typical genetic, morphological, and electrophysiological cardiac cell characteristics. This suspension culture system allows seamless transition from hPSC expansion to CM differentiation in a continuous suspension culture. It not only provides a cost and labor effective scalable process for large scale CM production, but also provides a bioreactor prototype for automation of cell manufacturing, which will accelerate the advance of hPSC research towards therapeutic applications.

  10. Suspension culture of pluripotent stem cells: effect of shear on stem cell fate.

    Science.gov (United States)

    Keller, Kevin C; Rodrigues, Beatriz; zur Nieden, Nicole I

    2014-01-01

    Despite significant promise, the routine usage of suspension cell culture to manufacture stem cell-derived differentiated cells has progressed slowly. Suspension culture is an innovative way of either expanding or differentiating cells and sometimes both are combined into a single bioprocess. Its advantages over static 2D culturing include a homogeneous and controllable culture environment and producing a large quantity of cells in a fraction of time. This feature makes suspension cell culture ideal for use in stem cell research and eventually ideal in the large-scale production of differentiated cells for regenerative medicine. Because of their tremendous differentiation capacities and unlimited growth properties, pluripotent stem cells (PSCs) in particular are considered potential sources for future cell-replacement therapies. Currently, expansion of PSCs is accomplished in 2D, which only permits a limited amount of cell growth per culture flask before cells need to be passaged. However, before stem cells can be applied clinically, several aspects of their expansion, such as directed growth, but also differentiation, need to be better controlled. This review will summarize recent advantages in suspension culture of PSCs, while at the same time highlighting current challenges.

  11. Production of beta-thujaplicin in Cupressus lusitanica suspension cultures fed with organic acids and monoterpenes.

    Science.gov (United States)

    Zhao, J; Fujita, K; Sakai, K

    2001-05-01

    Effects of some organic acids and monoterpenes on production of beta-thujaplicin were studied in Cupressus lusitanica suspension cultures. The fungal elicitor-induced biosynthesis of beta-thujaplicin was promoted by the feedings of malate, pyruvate, fumarate, succinate, and acetate. These results suggest some relationships between acetate/pyruvate metabolism and beta-thujaplicin biosynthesis, or between tricarboxylic acid cycle and beta-thujaplicin biosynthesis. Feedings of C. lusitanica suspension cultures with some monoterpenes inhibited elicitor-triggered beta-thujaplicin biosynthesis, but 2-carene and terpinyl acetate feedings significantly improved the beta-thujaplicin production of C. lusitanica suspension cultures. These results indicate a possible involvement of terpinyl acetate and 2-carene in beta-thujaplicin biosynthesis, as well as potential uses of these monoterpenes in large-scale beta-thujaplicin production.

  12. Putrescine facilitated enhancement of capsaicin production in cell suspension cultures of Capsicum frutescens.

    Science.gov (United States)

    Sudha, Govindaswamy; Ravishankar, Gokare A

    2003-04-01

    Putrescine treatment (0.1 mmol/L) influenced enhancement of growth and capsaicin production in the cell suspension cultures of C. frutescens. The administration of polyamine inhibitor DFMA (alpha-DL-difluoromethylarginine) resulted in a reduction of the growth, capsaicin content and the endogenous titres of polyamines (PAs). The capsaicin synthase activity was also higher in the putrescine (Put) treated cultures. Ethylene levels were lower in the cultures treated with putrescine. This study suggested that Put facilitates growth and capsaicin production.

  13. Determination of cardiac glycosides and total phenols in different generations of Securigera securidaca suspension culture

    Directory of Open Access Journals (Sweden)

    Z. Tofighi

    2016-04-01

    Full Text Available Background and objectives: The seeds of Securigera securidaca (L. Deg. & Dorf. (Fabaceae are used as anti-diabetic remedy in Iranian folk medicine. The aim of the present study was to establish the callus and suspension culture of S. securidaca seeds for the first time and to determine the major secondary metabolites including cardiac glycosides and total phenols. Methods: The culture of S. securidaca from seeds was initiated in hormone-supplemented MS medium containing 1 and 0.1 ppm 2, 4-D solution for solid and suspension cultures, respectively, sucrose and vitamins (B1, B2, B6, Folic acid, Biotin, Nicotinamide and Ca pantothenate at 25 °C and 12 h photoperiods. The cardiac glycosides were determined based on the calibration curve of securidaside which was isolated from the seeds extract of S. securidaca. Total phenolic compounds of different generations of suspension culture were determined using Folin Ciocalteu reagent. Results: Callus culture of S. securidaca was grown light cream to pale yellow in color and soft in texture while the cells of suspension culture grew cream to yellow with isolated cells and small aggregates. The production of cardiac glycosides in the 7th generation were more than the seeds extract (p

  14. Production of dammarenediol-II triterpene in a cell suspension culture of transgenic tobacco.

    Science.gov (United States)

    Han, Jung-Yeon; Wang, Hong-Yan; Choi, Yong-Eui

    2014-02-01

    Dammarenediol-II is biologically active tetracyclic triterpenoid, which is basic compound of ginsenoside saponin. Here, we established the dammarenediol-II production via a cell suspension culture of transgenic tobacco overexpressing PgDDS. Dammarenediol-II synthase catalyzes the cyclization of 2,3-oxidosqualene to dammarenediol-II, which is the basic triterpene skeleton in dammarene-type saponin (ginsenosides) in Panax ginseng. Dammarenediol-II is a useful candidate both for pharmacologically active triterpenes and as a defense compound in plants. Dammarenediol-II is present in the roots of P. ginseng in trace amounts because it is an intermediate product in triterpene biosynthesis. In this work, we established the production of dammarenediol-II via cell suspension culture of transgenic tobacco. The dammarenediol-II synthase gene (PgDDS) isolated from P. ginseng was introduced into the Nicotiana tobacum genome under the control of 35S promoter by Agrobacterium-mediated transformation. Accumulation of dammarenediol-II in transgenic tobacco plants occurred in an organ-specific manner (roots > stems > leaves > flower buds), and transgenic line 14 (T14) exhibited a high amount (157.8 μg g⁻¹ DW) of dammarenediol-II in the roots. Dammarenediol-II production in transgenic tobacco plants resulted in reduced phytosterol (β-sitosterol, campesterol, and stigmasterol) contents. A cell suspension culture was established as a shake flask culture of a callus derived from root segments of transgenic (T14) plants. The amount of dammarenediol-II production in the cell suspension reached 573 μg g⁻¹ dry weight after 3 weeks of culture, which is equivalent to a culture volume of 5.2 mg dammarenediol-II per liter. Conclusively, the production of dammarenediol-II in a cell suspension culture of transgenic tobacco can be applied to the large-scale production of this compound and utilized as a source of pharmacologically active medicinal materials.

  15. Culture medium refinement by dialysis for the expansion of human induced pluripotent stem cells in suspension culture.

    Science.gov (United States)

    Nath, Suman Chandra; Nagamori, Eiji; Horie, Masanobu; Kino-Oka, Masahiro

    2017-01-01

    Human induced pluripotent stem cells (hiPSCs) secrete essential autocrine factors that are removed along with toxic metabolites when the growth medium is exchanged daily. In this study, after determining the minimum inhibitory level of lactic acid for hiPSCs, a medium refining system was constructed by which toxic metabolites were removed from used culture medium and autocrine factors as well as other growth factors were recycled. Specifically, about 87 % of the basic fibroblast growth factor and 80 % of transforming growth factor beta 1 were retained in the refined medium after dialysis. The refined medium efficiently potentiated the proliferation of hiPS cells in adherent culture. When the refining system was used to refresh medium in suspension culture, a final cell density of (1.1 ± 0.1) × 10(6) cells mL(-1) was obtained, with 99.5 ± 0.2 % OCT 3/4 and 78.3 ± 1.1 % TRA-1-60 expression, on day 4 of culture. These levels of expression were similar to those observed in the conventional suspension culture. With this method, culture medium refinement by dialysis was established to remove toxic metabolites, recycle autocrine factors as well as other growth factors, and reduce the use of macromolecules for the expansion of hiPSCs in suspension culture.

  16. Establishment of sorghum cell suspension culture system for ...

    African Journals Online (AJOL)

    STORAGESEVER

    2008-03-18

    Mar 18, 2008 ... Total soluble proteins (TSP) and culture filtrate (CF) proteins were extracted from the cell culture ... Even though. Arabidopsis provides for an excellent model system for .... stained, destained and imaged using a Molecular Imager PharosFX ... system, are dynamic and heterogeneous, being com- posed of a ...

  17. Isolation of plasmodesmata from Arabidopsis suspension culture cells.

    Science.gov (United States)

    Grison, Magali S; Fernandez-Calvino, Lourdes; Mongrand, Sébastien; Bayer, Emmanuelle M F

    2015-01-01

    Due to their position firmly anchored within the plant cell wall, plasmodesmata (PD) are notoriously difficult to isolate from plant tissue. Yet, getting access to isolated PD represents the most straightforward strategy for the identification of their molecular components. Proteomic and lipidomic analyses of such PD fractions have provided and will continue to provide critical information on the functional and structural elements that define these membranous nano-pores. Here, we describe a two-step simple purification procedure that allows isolation of pure PD-derived membranes from Arabidopsis suspension cells. The first step of this procedure consists in isolating cell wall fragments containing intact PD while free of contamination from other cellular compartments. The second step relies on an enzymatic degradation of the wall matrix and the subsequent release of "free" PD. Isolated PD membranes provide a suitable starting material for the analysis of PD-associated proteins and lipids.

  18. DIGLUCOSYLATION OF SALICYL ALCOHOL BY CELL SUSPENSION CULTURES OF SOLANUM LACINIATUM

    Institute of Scientific and Technical Information of China (English)

    ACHMAD SYAHRANI; FRANSISCA HARTUTI; GUNAWAN INDRAYANTO; ALISTAIR L.WILKINS

    2001-01-01

    A new biotransformation product, salicyl alcohol-7-O-β-D-(β-l,6-D-glucopyranosyl)-gluco pyranoside was isolated from cell suspension cultures of Solanum laciniatum, following administration of salicyl alcohol, and its structure was elucidated using a combination of one and two-dimensional 1H and 13C-NMR data, and positive and negative ion ESMS data.

  19. Accumulation of podophyllotoxin and related lignans in cell suspension cultures of Linum album

    NARCIS (Netherlands)

    Smollny, T.; Wichers, H.; Kalenberg, S.; Shahsavari, A.; Petersen, M.; Alfermann, A.W.

    1998-01-01

    Cell suspension cultures of Linum album were established, which were able to synthesize and accumulate lignans. Podophyllotoxin and 5-methoxypodophyllotoxin were the main products and were present as glycosides, together with small amounts of deoxypodophyllotoxin, 5′-demethoxy-5-methoxypodophyllotox

  20. Comparative metabolite profiling of the insecticide thiamethoxam in plant and cell suspension culture of tomato.

    Science.gov (United States)

    Karmakar, Rajib; Bhattacharya, Ramcharan; Kulshrestha, Gita

    2009-07-22

    The metabolism of thiamethoxam [(EZ)-3-(2-chloro-1,3-thiazol-5-yl-methyl)-5-methyl-1,3,5-oxadiazinan-4-ylidene (nitro) amine] was investigated in whole plant, callus, and heterotrophic cell suspension culture of aseptically and field grown tomato (Lycopersicon esculentum Mill.) plants. The structure of the metabolites was elucidated by chromatographic (HPLC) and spectroscopic (IR, NMR, and MS) methods. Thiamethoxam metabolism proceeded by the formation of a urea derivative, a nitroso product, and nitro guanidine. Both urea and nitro guanidine metabolites further degraded in plants, and a mechanism has been proposed. In the plant, organ-specific differences in thiamethoxam metabolism were observed. Only one metabolite was formed in whole plant against four in callus and eight metabolites in cell suspension culture under aseptic conditions. Out of six metabolites of thiamethoxam in tomato fruits in field conditions, five were similar to those formed in the cell suspension culture. In the cell suspension culture, thiamethoxam degraded to maximum metabolites within 72 h, whereas in plants, such extensive conversion could only be observed after 10 days.

  1. Abiotic elicitation of gymnemic acid in the suspension cultures of Gymnema sylvestre.

    Science.gov (United States)

    Ch, Bhuvaneswari; Rao, Kiranmayee; Gandi, Suryakala; Giri, Archana

    2012-02-01

    Elicitation is one of the few strategies that find commercial application in the enhancement of secondary metabolite production from plants as well as cell culture systems. Due to their immense medicinal value, production of saponins in suspension cultures has been attempted by many researchers. Gymnema sylvestre is a rich source of gymnemic acids (saponins) that find application in the treatment of diabetes. The present study is an attempt to evaluate the effect of various metal salts (cadmium chloride, mercuric chloride, silver nitrate, cupric chloride, cobaltous chloride and calcium chloride) in eliciting the response from G. sylvestre suspension cultures. The maximum gymnemic acid production in the suspensions was achieved on day 12 of culture, though the maximum biomass was obtained on day 16. Among the different salts, CdCl(2) gave maximum response (59.97 mg/gDCW) at 2 mM concentration after a 24 h time period, while, AgNO(3) gave the least response (18.35 mg/gDCW) on incubation of 48 h at 1 mM concentration, in terms of gymnemic acid accumulation. The accumulation of gymnemic acid was found to be dependent on treatment time and concentration of the elicitor. The enhanced gymnemic acid production shown by the suspensions in response to the metal salts indicates their role in evoking the plant defense mechanisms. These elicitation studies help in providing a platform for improved commercial supply of bioactive gymnemic acids.

  2. Surfactant-induced non-lethal release of anthraquinones from suspension culture of Morinda citrifolia.

    NARCIS (Netherlands)

    Bassetti, L.; Hagendoorn, M.J.M.; Tramper, J.

    1995-01-01

    A new approach based on the use of the surfactant Pluronic F-68 to obtain non-lethal release of plant cell intracellular products was investigated. Suspension cultures of Morinda citrifolia (Rubiaceae), producing anthraquinones as secondary metabolites, were selected as model system. By supplementin

  3. Feruloyl oligosaccharides from cell walls of suspension-cultured spinach cells and sugar beet pulp.

    Science.gov (United States)

    Ishii, T

    1994-06-01

    Cell walls of suspension-cultured spinach cells and sugar beet pulp were separately hydrolyzed with Driselase. A feruloyl arabinobiose was isolated from both spinach cells and sugar beet. Four feruloyl oligosaccharides were obtained from sugar beet. The four oligosaccharides were characterized by NMR spectroscopy, methylation analysis and FAB-MS.

  4. Surfactant-induced non-lethal release of anthraquinones from suspension culture of Morinda citrifolia.

    NARCIS (Netherlands)

    Bassetti, L.; Hagendoorn, M.J.M.; Tramper, J.

    1995-01-01

    A new approach based on the use of the surfactant Pluronic F-68 to obtain non-lethal release of plant cell intracellular products was investigated. Suspension cultures of Morinda citrifolia (Rubiaceae), producing anthraquinones as secondary metabolites, were selected as model system. By

  5. Culture of isolated single cells from Taxus suspensions for the propagation of superior cell populations.

    Science.gov (United States)

    Naill, Michael C; Roberts, Susan C

    2005-11-01

    Single cells isolated from aggregated Taxus cuspidata cultures via enzymatic digestion were grown in suspension culture. High seeding density (4 x 10(5 )cells/ml) and the addition of cell-free conditioned medium were essential for growth. Doubling the concentration of the nutrients [ascorbic acid (150 g/l), glutamine (6.25 mM: ), and citric acid (150 g/l)] had no effect on single cell growth or viability. A specific growth rate of 0.11 days(-1) was achieved, which is similar to the observed growth rate of aggregated Taxus suspensions. The biocide, Plant Preservative Mixture, added at 0.2% (v/v) to all single cell cultures to prevent microbial contamination, had no significant effect on growth or viability. Following cell sorting, single cell cultures can be used to establish new cell lines for biotechnology applications or provide cells for further study.

  6. Optimization of Lycopene Extraction from Tomato Cell Suspension Culture by Response Surface Methodology

    OpenAIRE

    Lu, Chi-Hua; Engelmann, Nancy J.; Lila, Mary Ann; Erdman, John W

    2008-01-01

    Radioisotope-labeled lycopene is an important tool for biomedical research but currently is not commercially available. A tomato cell suspension culture system for the production of radioisotope-labeled lycopene was previously developed in our laboratory. In the current study, the goal was to optimize the lycopene extraction efficiency from tomato cell cultures for preparatory high-performance liquid chromatography (HPLC) separation. We employed response surface methodology (RSM), which combi...

  7. Improving Cotton Embryo Culture by Simulating In Ovulo Nutrient and Hormone Levels

    Energy Technology Data Exchange (ETDEWEB)

    Rodney Fuller; Vincent Liddiard; J. Hess; John Carman

    2011-06-01

    Plant ovules provide zygotes with a physicochemical environment that supports embryo differentiation, growth, and maturation. The exact nature of this embryogenesis-enabling environment is not well characterized, as evidenced by failed attempts to induce normal embryony from zygotes or proembryos (precotyledonary) on defined media. To identify factors required for cotton (Gossypium hirsutum L.) zygotic embryony in vitro, we previously performed chemical and dissolved oxygen tension analyses of cotton ovule fluids and tissues at multiple stages of embryony in situ. Based on these analyses, we report herein the development of procedures that normalize embryo differentiation, growth, maturation, and germination in vitro, starting with proembryos. Our medium differed from Murashige and Skoog (MS) medium as follows (percentage of MS): N (30%, mostly from ten amino acids), P (815%), K (237%), Mg (85%), Ca (267%), S (506%), Fe (88%), and myoinositol (883%). Levels of other MS nutrients and vitamins, except sucrose, were kept at MS levels. Additionally, we included 100 mg L-1 casein hydrolysate plus the following (mmol L-1): d-glucose (1.8), fructose (4.7), sucrose (62.0), arabinose (7.1), melibiose (3.5), malic acid (11.6), and citric acid (3.8). Mannitol was added to achieve a medium osmotic potential of -1.10 MPa, and an atmospheric O2 tension of 3.3 mol m-3 at the surface of embryos was maintained during culture. When cultured on medium containing 8.0 µmol L-1 indole-3-acetic acid, 80-90% of proembryos (as small as 100 cells) of cultivars HS-26 and B-27 increased four- to eightfold in surface area during the first 18 d in culture and germinated thereafter to produce viable plants. Increases in surface area of proembryos cultured on a modified MS medium previously used for somatic embryogenesis were from 0.2- to 0.6-fold. The described embryo culture medium should be useful for studying nutritional and molecular aspects of early embryony and possibly for plant zygote

  8. Structure and organ specificity of an anionic peroxidase from Arabidopsis thaliana cell suspension culture

    DEFF Research Database (Denmark)

    Ostergaard, L; Abelskov, A K; Mattsson, O

    1996-01-01

    The predominant peroxidase (pI 3.5) (E.C. 1.11.1.7) of an Arabidopsis thaliana cell suspension culture was purified and partially sequenced. Oligonucleotides were designed and a specific probe was obtained. A cDNA clone was isolated from an Arabidopsis cell suspension cDNA library and completely...... sequenced. The cDNA clone comprised 1194 bp and encodes a 30 residue signal peptide and a 305 residue mature protein (Mr 31,966). The sequence of the mature protein is 95% identical to the well-characterized horseradish peroxidase HRP A2 and has therefore been designated ATP A2. Three introns at positions...

  9. Impact of stirred suspension bioreactor culture on the differentiation of murine embryonic stem cells into cardiomyocytes

    Science.gov (United States)

    2011-01-01

    Background Embryonic stem cells (ESCs) can proliferate endlessly and are able to differentiate into all cell lineages that make up the adult organism. Under particular in vitro culture conditions, ESCs can be expanded and induced to differentiate into cardiomyocytes in stirred suspension bioreactors (SSBs). However, in using these systems we must be cognizant of the mechanical forces acting upon the cells. The effect of mechanical forces and shear stress on ESC pluripotency and differentiation has yet to be clarified. The purpose of this study was to investigate the impact of the suspension culture environment on ESC pluripotency during cardiomyocyte differentiation. Results Murine D3-MHC-neor ESCs formed embyroid bodies (EBs) and differentiated into cardiomyocytes over 25 days in static culture and suspension bioreactors. G418 (Geneticin) was used in both systems from day 10 to enrich for cardiomyocytes by eliminating non-resistant, undifferentiated cells. Treatment of EBs with 1 mM ascorbic acid and 0.5% dimethyl sulfoxide from day 3 markedly increased the number of beating EBs, which displayed spontaneous and cadenced contractile beating on day 11 in the bioreactor. Our results showed that the bioreactor differentiated cells displayed the characteristics of fully functional cardiomyocytes. Remarkably, however, our results demonstrated that the bioreactor differentiated ESCs retained their ability to express pluripotency markers, to form ESC-like colonies, and to generate teratomas upon transplantation, whereas the cells differentiated in adherent culture lost these characteristics. Conclusions This study demonstrates that although cardiomyocyte differentiation can be achieved in stirred suspension bioreactors, the addition of medium enhancers is not adequate to force complete differentiation as fluid shear forces appear to maintain a subpopulation of cells in a transient pluripotent state. The development of successful ESC differentiation protocols within

  10. Impact of stirred suspension bioreactor culture on the differentiation of murine embryonic stem cells into cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Shafa Mehdi

    2011-12-01

    Full Text Available Abstract Background Embryonic stem cells (ESCs can proliferate endlessly and are able to differentiate into all cell lineages that make up the adult organism. Under particular in vitro culture conditions, ESCs can be expanded and induced to differentiate into cardiomyocytes in stirred suspension bioreactors (SSBs. However, in using these systems we must be cognizant of the mechanical forces acting upon the cells. The effect of mechanical forces and shear stress on ESC pluripotency and differentiation has yet to be clarified. The purpose of this study was to investigate the impact of the suspension culture environment on ESC pluripotency during cardiomyocyte differentiation. Results Murine D3-MHC-neor ESCs formed embyroid bodies (EBs and differentiated into cardiomyocytes over 25 days in static culture and suspension bioreactors. G418 (Geneticin was used in both systems from day 10 to enrich for cardiomyocytes by eliminating non-resistant, undifferentiated cells. Treatment of EBs with 1 mM ascorbic acid and 0.5% dimethyl sulfoxide from day 3 markedly increased the number of beating EBs, which displayed spontaneous and cadenced contractile beating on day 11 in the bioreactor. Our results showed that the bioreactor differentiated cells displayed the characteristics of fully functional cardiomyocytes. Remarkably, however, our results demonstrated that the bioreactor differentiated ESCs retained their ability to express pluripotency markers, to form ESC-like colonies, and to generate teratomas upon transplantation, whereas the cells differentiated in adherent culture lost these characteristics. Conclusions This study demonstrates that although cardiomyocyte differentiation can be achieved in stirred suspension bioreactors, the addition of medium enhancers is not adequate to force complete differentiation as fluid shear forces appear to maintain a subpopulation of cells in a transient pluripotent state. The development of successful ESC

  11. Effects of Selected Physicochemical Parameters on Zerumbone Production of Zingiber zerumbet Smith Cell Suspension Culture

    Directory of Open Access Journals (Sweden)

    Mahanom Jalil

    2015-01-01

    Full Text Available Zingiber zerumbet Smith is an important herb that contains bioactive phytomedicinal compound, zerumbone. To enhance cell growth and production of this useful compound, we investigated the growth conditions of cell suspension culture. Embryogenic callus generated from shoot bud was used to initiate cell suspension culture. The highest specific growth rate of cells was recorded when it was cultured in liquid Murashige and Skoog basal medium containing 3% sucrose with pH 5.7 and incubated under continuous shaking condition of 70 rpm for 16 h light and 8 h dark cycle at 24°C. Our results also revealed that the type of carbohydrate substrate, light regime, agitation speed, and incubation temperature could affect the production of zerumbone. Although the zerumbone produced in this study was not abundant compared to rhizome of Z. zerumbet, the possibility of producing zerumbone during early stage could serve as a model for subsequent improvement.

  12. Rheological characteristics of cell suspension and cell culture of Perilla frutescens.

    Science.gov (United States)

    Zhong, J J; Seki, T; Kinoshita, S; Yoshida, T

    1992-12-01

    Physical properties such as viscosity, fluid dynamic behavior of cell suspension, and size distribution of cell aggregates of a plant, Perilla frustescens, cultured in a liquid medium were studied. As a result of investigations using cells harvester after 12 days of cultivation in a flask, it was found that the apparent viscosity of the cell suspension did not change with any variation of cell concentration below 5 g dry cell/L but markedly increased when the cell concentration increased over 12.8 g dry cell/L. The cell suspension exhibited the characteristics of a Bingham plastic fluid with a small yield stress. The size of cell aggregates in the range 74 to 500 mum did not influence the rheological characteristics of the cell suspension. The rheological characteristics of cultivation mixtures of P. frutescens cultivated in a flask and in a bioreactor were also investigated. The results showed that the flow characteristics of the cell culture could be described by a Bingham plastic model. At the later stage of cultivation, the apparent viscosity increased steadily, even though the biomass concentration (by dry weight) decreased, due to the increase of individual cell size.

  13. Out-of-School Suspensions of Black Youths: Culture, Ability, Disability, Gender, and Perspective.

    Science.gov (United States)

    Haight, Wendy; Kayama, Misa; Gibson, Priscilla Ann

    2016-07-01

    Racial disproportionality in out-of-school suspensions is a persistent social justice issue in public schools. This article examines out-of-school suspensions of four black youths from the perspectives of the youths, their caregivers, and educators. The case involving David, a 14-year-old African American with a learning disability, illustrates the challenges of students experiencing the intersection of disability and race. The case involving George, a 14-year-old Liberian immigrant, illustrates how parents and teachers may form alliances around shared goals and values despite profound cultural differences in understanding of youths' misbehavior. The case involving Nina, a 12-year-old African American, illustrates how educators' failure to consider the context of her misbehaviors as responses to sexual harassment, along with their subsequent harsh punishment and failure to protect her, led to her disengagement from school. The case involving Craig, a 16-year-old African American, provides a glimpse into how the use of criminal justice language to refer to youths' misbehaviors can support the development of a criminalized self- and social identity. These cases illustrate the diversity of black students--including ability, disability, culture, and gender--and how events surrounding suspensions are interpreted by students, caregivers, and educators. Understanding such diversity will undergird implementation of effective alternatives to suspensions.

  14. [Massive multiplication of coffee (Coffee arabica L. cv. Catimor) through embryogenic cell suspension culture].

    Science.gov (United States)

    Flermoso-Gallardo, L; Menóndez-Yuffá, A

    2000-01-01

    Cell suspensions offer several advantages as a system for massive propagation because of the high rates of multiplication, the higher homogeneity in the culture conditions and the possibility of automatization. In this study, different experimental conditions were analyzed to establish embryogenic cell suspension cultures of coffee. The best conditions to establish the embryogenic cell suspension cultures of coffee were as follows: coffee leaf sections were cultivated during 12 weeks (Stage I) in a solid medium with the Murashige and Skoog salts, 2 mg/l kinetin and 0.5 mg/l 2,4-dichlorophenoxiacetic acid (medium 1). Under these conditions the explants formed a callus tissue that was transferred to a liquid medium containing 5 mg/l of 6-benzylamlno-purine (medium 2). After 12 days in a shaking liquid medium (Stage II), the cultures were sieved and were maintained In the same media, which was renewed every eight days (Stage III). This method yielded 1884 embryos in 50 ml; placing the embryos under conditions for germination yielded plantlets of normal appearance.

  15. Evaluation of limonoid production in suspension cell culture of Citrus sinensis

    Directory of Open Access Journals (Sweden)

    Elisângela Fumagali Gerolino

    2015-10-01

    Full Text Available ABSTRACTThe use of cell and plant tissue culture techniques to produce economically important active metabolites has been growing. Among these substances are total limonoid aglycones, which are produced by "pera" orange (Citrus sinensis (L. Osbeck, Rutaceae and have received considerable attention because of their anticancer actions. The main objective of the present study was to analyze and compare the levels of limonoid aglycones in seeds, callus cultures (originating from seeds, callus cultures (originating from hypocotyls, cell suspensions from hypocotyls cells, and cell suspensions from cotyledons. The cell cultures or C. sinensis were obtained by inoculating two strains of callus in MS medium supplemented with 2.0 µM 2,4-dichlorophenoxyacetic acid, 7.0 µM benzyl aminopurine, and 3% (w/v sucrose in the dark. The highest concentrations of limonoid aglycone that were obtained were observed in cotyledon cell lines (240 mg/100 g dry weight that were produced on day 21 of culture and hypocotyl cell lines on day 7 (210 mg/100 g dry weight. Explants of different origins under the same culture conditions had different limonoid aglycone content. The present results may suggest strategies for enhancing the productivity of biologically important limonoid aglycones and investigating the complex pathways of these secondary metabolites in plant tissue cultures.

  16. Cotton fabric coated with nano TiO{sub 2}-acrylate copolymer for photocatalytic self-cleaning by in-situ suspension polymerization

    Energy Technology Data Exchange (ETDEWEB)

    Jiang Xue, E-mail: jiangx@jiangnan.edu.cn [Key Laboratory of Eco-textiles of Ministry of Education, College of textiles and Clothing, Jiangnan University, Wuxi, Jiangsu 214122 (China); State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou, Guangdong, 510640 (China); Tian Xiuzhi; Gu Jian; Huang Dan [Key Laboratory of Eco-textiles of Ministry of Education, College of textiles and Clothing, Jiangnan University, Wuxi, Jiangsu 214122 (China); Yang Yiqi [Key Laboratory of Eco-textiles of Ministry of Education, College of textiles and Clothing, Jiangnan University, Wuxi, Jiangsu 214122 (China); Department of Textiles, Clothing and Design, 234 HECO Bldg, University of Nebraska-Lincoln, Lincoln, NE 68583-0802 (United States)

    2011-08-01

    Two kinds of nano TiO{sub 2}-polyacrylate hybrid dispersions, TBM-w and TBM-e were synthesized by in-situ suspension polymerization and solution polymerization respectively, in order to fix the nano TiO{sub 2} on fabrics. The photocatalytic self-cleaning fabrics have received much attention in recent years for its water-saving and environment-protection advantages. However, the fixation of the photocatalyst on fabrics is still a key problem that inhibits industrialization of these eco-friendly fabrics. The cotton fabric was treated by the two hybrid dispersions. The photocatalytic self-cleaning property was characterized. Infrared spectroscopy, burning loss test and thermogravimetry showed that some copolymer chains entangled with the nano TiO{sub 2}. Transmission electron microscope illustrated that there was a polymeric layer on the surface of nano TiO{sub 2}. The average diameter of TBM-w was smaller than that of TBM-e based on size analysis. The photocatalytic decoloration of the grape syrup indicated that the fabric with TiO{sub 2}-polymer hybrid had excellent self-cleaning property.

  17. Phytophthora elicitor PB90 induced apoptosis in suspension cultures of tobacco

    Institute of Scientific and Technical Information of China (English)

    JI Rui; ZHANG Zhengguang; WANG Yuanchao; ZHENG Xiaobo

    2005-01-01

    The protein elicitor PB90 secreted by Phytophthora boehmeriae is an efficient elicitor inducing the hypersensitive response and systemic acquired resistance in tobacco plants. Here, we observed cell death in suspension-cultured cells of Nicotiana tabacum BY-2 with PB90 treatment using Trypan blue staining method. And this cell death could be suppressed by cycloheximide, an inhibitor of proteins synthesis, which implies that PB90-induced cell death was an active cell death process requiring new protein synthesis. DAPI staining revealed that PB90 induce rapid chromatin condensation, margination, apoptotic bodies' formation and DNA laddering, further TUNEL assay also observed the specific breakage of 3′-OH ends. All of the above common morphological characteristics indicated that PB90 induced apoptosis in suspension cultures of tobacco, suggesting that hypersensitive response induced by PB90 is an apoptotic process.

  18. A versatile platform for three-dimensional dynamic suspension culture applications

    OpenAIRE

    Isu, Giuseppe

    2016-01-01

    In the last decades, the rapid upgrading in cell biological knowledge has bumped the interest in using cell-based therapeutic approaches as well as cell-based model systems for the treatment of diseases. Given the rapid translation towards cell-based clinical treatments and the consequent increasing demand of cell sources, three-dimensional (3D) suspension cultures have demonstrated to be an advantageous alternative to monolayer techniques for large scale expansion of cells and for the genera...

  19. C-27 AND C-3 GLUCOSYLATION OF DIOSGENIN BY CELL SUSPENSION CULTURES OF COSTUS SPECIOSUS

    Institute of Scientific and Technical Information of China (English)

    GUNAWAN INDRAYANTO; SITI ZUMAROH; ACHMAD SYAHRANI; ALISTAIR L. WILKINS

    2001-01-01

    3-O-[β-D-glucopyranosyl-(l″→ 2′)-β-D-glucopyranosyl], 27-O-β-D-glucopyranosyl-(25R)-spir ost-5-ene-3β,27-diol was isolated from cell suspension cultures of Costus speciosus, following incubation with diosgenin, and its structure was elucidated using a combination of one- and two-dimensional 1H and 13C NMR spectral data, and positive and negative ion ESMS spectral data.

  20. Establishment of cell suspension cultures of two Costa Rican Jatropha species (Euphorbiaceae)

    OpenAIRE

    Solís Ramos, Laura Yesenia; Miranda Carballo, Laura; Valdez Melara, Marta

    2013-01-01

    J. curcas has been studied in different countries and some interesting agronomic, pharmacological and industrial properties have been reported. More recently, it has been considered an important alternative source for biofuel production. The objective of this study was to establish a long-term method for the maintenance of calli and cell suspension cultures of the local species J. curcas and J. gossypifolia, in order to allow future studies for novel compounds with pharmaceutical or industria...

  1. Flavonoids and darkness lower PCD in senescing Vitis vinifera suspension cell cultures.

    Science.gov (United States)

    Bertolini, Alberto; Petrussa, Elisa; Patui, Sonia; Zancani, Marco; Peresson, Carlo; Casolo, Valentino; Vianello, Angelo; Braidot, Enrico

    2016-10-26

    Senescence is a key developmental process occurring during the life cycle of plants that can be induced also by environmental conditions, such as starvation and/or darkness. During senescence, strict control of genes regulates ordered degradation and dismantling events, the most remarkable of which are genetically programmed cell death (PCD) and, in most cases, an upregulation of flavonoid biosynthesis in the presence of light. Flavonoids are secondary metabolites that play multiple essential roles in development, reproduction and defence of plants, partly due to their well-known antioxidant properties, which could affect also the same cell death machinery. To understand further the effect of endogenously-produced flavonoids and their interplay with different environment (light or dark) conditions, two portions (red and green) of a senescing grapevine callus were used to obtain suspension cell cultures. Red Suspension cell Cultures (RSC) and Green Suspension cell Cultures (GSC) were finally grown under either dark or light conditions for 6 days. Darkness enhanced cell death (mainly necrosis) in suspension cell culture, when compared to those grown under light condition. Furthermore, RSC with high flavonoid content showed a higher viability compared to GSC and were more protected toward PCD, in accordance to their high content in flavonoids, which might quench ROS, thus limiting the relative signalling cascade. Conversely, PCD was mainly occurring in GSC and further increased by light, as it was shown by cytochrome c release and TUNEL assays. Endogenous flavonoids were shown to be good candidates for exploiting an efficient protection against oxidative stress and PCD induction. Light seemed to be an important environmental factor able to induce PCD, especially in GSC, which lacking of flavonoids were not capable of preventing oxidative damage and signalling leading to senescence.

  2. Isolation of fatty acids and aromatics from cell suspension cultures of Lavandula angustifolia.

    Science.gov (United States)

    Topçu, Gülaçti; Herrmann, Gabriele; Kolak, Ufuk; Gören, C; Porzel, Andrea; Kutchan, Toni M

    2007-02-01

    Cell suspension cultures of Lavandula angustifolia Mill. ssp. angustifolia (syn.: L. officinalis Chaix.) afforded a fatty acid composition, cis and trans p-coumaric acids (=p-hydroxy cinnamic acids), and beta-sitosterol. The fatty acid composition was analyzed by GC-MS, and the structures of the isolated three compounds were determined by 1H- and 13C-NMR, and MS spectroscopic techniques.

  3. The isolation and culture of protoplasts from an embryogenic cell suspension culture of Picea glauca (moench) voss.

    Science.gov (United States)

    Bekkaoui, F; Saxena, P K; Attree, S M; Fowke, L C; Dunstan, D I

    1987-12-01

    Conditions were standardized for the isolation and culture of protoplasts from an embryogenic cell suspension culture of Picea glauca. A combination of 0.5% Cellulase R-10, 0.25% Macerozyme, 0.25% Driselase, 0.25% Rhozyme HP-150 with 0.5M mannitol and 5 mM CaCl2.2H2O produced an average of 4.5 × 10(6) protoplasts per gram fresh weight of cells. Of the several protoplast culture media tested, von Arnold and Eriksson and Kao and Michayluk (KM8P) media best supported mitotic divisions of protoplasts. A density of 10(5) protoplasts per ml and the addition of 5 mM glutamine to the culture medium was necessary to induce sustained divisions and microcallus formation. Microcalli grew into subculturable callus using a nurse culture technique.

  4. Induction of phytic acid synthesis by abscisic acid in suspension-cultured cells of rice.

    Science.gov (United States)

    Matsuno, Koya; Fujimura, Tatsuhito

    2014-03-01

    A pathway of phytic acid (PA) synthesis in plants has been revealed via investigations of low phytic acid mutants. However, the regulation of this pathway is not well understood because it is difficult to control the environments of cells in the seeds, where PA is mainly synthesized. We modified a rice suspension culture system in order to study the regulation of PA synthesis. Rice cells cultured with abscisic acid (ABA) accumulate PA at higher levels than cells cultured without ABA, and PA accumulation levels increase with ABA concentration. On the other hand, higher concentrations of sucrose or inorganic phosphorus do not affect PA accumulation. Mutations in the genes RINO1, OsMIK, OsIPK1 and OsLPA1 have each been reported to confer low phytic acid phenotypes in seeds. Each of these genes is upregulated in cells cultured with ABA. OsITPK4 and OsITPK6 are upregulated in cells cultured with ABA and in developing seeds. These results suggest that the regulation of PA synthesis is similar between developing seeds and cells in this suspension culture system. This system will be a powerful tool for elucidating the regulation of PA synthesis.

  5. Establishment of forskolin yielding transformed cell suspension cultures of Coleus forskohlii as controlled by different factors.

    Science.gov (United States)

    Mukherjee, S; Ghosh, B; Jha, S

    2000-01-07

    Suspension cultures derived from gall calli which were obtained following infection with Agrobacterium tumefaciens (C58) were established in Coleus forskohlii. Cell line selection following single cell cloning or cell aggregate cloning was carried out to select cell lines capable of fast growth and for producing high level of forskolin. A fast growing cell line (GSO-5/7) thus selected was found to accumulate 0.021% forskolin in 42 days. The effect of cultural conditions on cell growth was studied to identify factors influencing biomass yield. Cell growth in suspension was found to be influenced significantly by carbon source, initial cell density and light or dark condition. Optimal cell growth (20 fold increase in biomass in a 42 day period) was obtained when the cells were grown in dark condition in B5O media containing 3% sucrose as sole carbon source with an initial cell density of 1.5 x 10(5) cells per ml. Forskolin accumulation was maximum (0.021%) in the stationary phase of cell growth. These suspension cultures showed continuous and stable production of forskolin.

  6. A novel xylogenic suspension culture model for exploring lignification in Phyllostachys bamboo

    Directory of Open Access Journals (Sweden)

    Ogita Shinjiro

    2012-09-01

    Full Text Available Abstract Background Some prominent cultured plant cell lines, such as the BY-2 cell line of tobacco (Nicotiana tabacum cv. ‘Bright Yellow 2’ and the T87 cell line of Arabidopsis (Arabidopsis thaliana L. Heynh., ecotype Columbia are used as model plant cells. These suspension cell culture systems are highly applicable for investigating various aspects of plant cell biology. However, no such prominent cultured cell lines exist in bamboo species. Results We standardized a novel xylogenic suspension culture model in order to unveil the process of lignification in living bamboo cells. Initial signs of lignin deposition were able to be observed by a positive phloroglucinol-HCl reaction at day 3 to 5 under lignification conditions (LG, i.e., modified half-strength Murashige and Skoog medium (m1/2MS containing 10 μM 6-benzyladenine (BA and 3% sucrose. Two types of xylogenic differentiation, both fiber-like elements (FLEs with cell wall thickening and tracheary elements (TEs with formation of perforations in the cell wall, were observed under these conditions. The suspension cells rapidly formed secondary cell wall components that were highly lignified, making up approximately 25% of the cells on a dry weight basis within 2 weeks. Detailed features involved in cell growth, differentiation and death during lignification were characterized by laser scanning microscopic imaging. Changes in transcript levels of xylogenesis-related genes were assessed by RT-PCR, which showed that the transcription of key genes like PAL1, C4H, CCoAOMT, and CCR was induced at day 4 under LG conditions. Furthermore, interunit linkage of lignins was compared between mature bamboo culms and xylogenic suspension cells by heteronuclear single quantum coherence (HSQC NMR spectroscopy. The presence of the most common interunit linkages, including β-aryl ether (β-O-4, phenylcoumaran (β-5 and resinol (β-β structures was identified in the bamboo cultured cell lignin (BCCL

  7. Regulation of Cytoplasmic and Vacuolar Volumes by Plant Cells in Suspension Culture

    DEFF Research Database (Denmark)

    Owens, Trevor; Poole, Ronald J

    1979-01-01

    Quantitative microscopical measurements have been made of the proportion of cell volume occupied by cytoplasm in a cell suspension culture derived from cotyledons of bush bean (cv. Contender). On a 7-day culture cycle, the content of cytoplasm varies from 25% at the time of transfer to 45......% at the start of the phase of rapid cell division. If the culture is continued beyond 7 days, the vacuole volume reaches 90% of cell volume by day 12.Attempts to measure relative cytoplasmic volumes by compartmental analysis of nonelectrolyte efflux were unsuccessful. The proportion of cell volume occupied...... by cytoplasm is roughly correlated with protein content, but shows no correlation with cell size or with intracellular concentrations of K or Na. The most striking observation is that the growth of cytoplasmic volume for the culture as a whole appears to be constant throughout the culture cycle, despite...

  8. Survival of Suspension-cultured Sycamore Cells Cooled to the Temperature of Liquid Nitrogen.

    Science.gov (United States)

    Sugawara, Y; Sakai, A

    1974-11-01

    Suspension-cultured cells of sycamore (Acer pseudoplatanus L.) which were immersed in liquid nitrogen after prefreezing to the temperatures from -30 to -50 C in the presence of dimethylsulfoxide and glucose as cryoprotective additive could proliferate vigorously when rewarmed rapidly in water at 40 C. For maintaining high viability of the cells after immersion in liquid nitrogen, it seems to be essential to use the cells at the later lag phase or the early cell division phase. This study provides a possibility for long term preservation in liquid nitrogen of plant-cultured lines.

  9. Establishment and optimization of cell growth in suspension culture of Papaver bracteatum: a biotechnology approach for thebaine production

    OpenAIRE

    FARJAMINEZHAD, Reza; Nasser ZARE; ASGHARI-ZAKARIA, Rasool; Farjaminezhad, Manoochehr

    2013-01-01

    Iranian poppy (Papaver bracteatum) is an important medicinal plant that is the main source of the opium alkaloids codeine, morphine, and thebaine. To establish an efficient protocol for cell suspension culture and growth, the effects of different plant growth regulators (2,4-D, NAA, BAP, and kinetin) on callus induction and cell suspension culture of Iranian poppy were evaluated. The maximum percentage of callus induction (86.67%) and fresh weight of callus were obtained in MS medium suppleme...

  10. Molecular farming of pharmaceutical proteins using plant suspension cell and tissue cultures.

    Science.gov (United States)

    Schillberg, Stefan; Raven, Nicole; Fischer, Rainer; Twyman, Richard M; Schiermeyer, Andreas

    2013-01-01

    Plants have been used for more than 20 years to produce recombinant proteins but only recently has the focus shifted away from proof-of-principle studies (i.e. is my protein expressed and is it functional?) to a serious consideration of the requirements for sustainable productivity and the regulatory approval of pharmaceutical products (i.e. is my protein safe, is it efficacious, and does the product and process comply with regulatory guidelines?). In this context, plant tissue and cell suspension cultures are ideal production platforms whose potential has been demonstrated using diverse pharmaceutical proteins. Typically, cell/tissue cultures are grown in containment under defined conditions, allowing process controls to regulate growth and product formation, thus ensuring regulatory compliance. Recombinant proteins can also be secreted to the culture medium, facilitating recovery and subsequent purification because cells contain most of the contaminating proteins and can be removed from the culture broth. Downstream processing costs are therefore lower compared to whole plant systems, balancing the higher costs of the fermentation equipment. In this article, we compare different approaches for the production of valuable proteins in plant cell suspension and tissue cultures, describing the advantages and disadvantages as well as challenges that must be overcome to make this platform commercially viable. We also present novel strategies for system and process optimization, helping to increase yields and scalability.

  11. A simple and efficient method for the long-term preservation of plant cell suspension cultures

    Directory of Open Access Journals (Sweden)

    Boisson Anne-Marie

    2012-01-01

    Full Text Available Abstract Background The repeated weekly subculture of plant cell suspension is labour intensive and increases the risk of variation from parental cells lines. Most of the procedures to preserve cultures are based on controlled freezing/thawing and storage in liquid nitrogen. However, cells viability after unfreezing is uncertain. The long-term storage and regeneration of plant cell cultures remains a priority. Results Sycamore (Acer pseudoplatanus and Arabidopsis cell were preserved over six months as suspensions cultures in a phosphate-free nutrient medium at 5°C. The cell recovery monitored via gas exchange measurements and metabolic profiling using in vitro and in vivo 13C- and 31P-NMR took a couple of hours, and cell growth restarted without appreciable delay. No measurable cell death was observed. Conclusion We provide a simple method to preserve physiologically homogenous plant cell cultures without subculture over several months. The protocol based on the blockage of cell growth and low culture temperature is robust for heterotrophic and semi-autotrophic cells and should be adjustable to cell lines other than those utilised in this study. It requires no specialized equipment and is suitable for routine laboratory use.

  12. Jasmonic and salicylic acids enhanced phytochemical production and biological activities in cell suspension cultures of spine gourd (Momordica dioica Roxb).

    Science.gov (United States)

    Chung, Ill-Min; Rekha, Kaliyaperumal; Rajakumar, Govindasamy; Thiruvengadam, Muthu

    2017-03-01

    In vitro cell suspension culture was established for the production of commercially valuable phytochemicals in Momordica dioica. The influence of elicitors in jasmonic acid (JA) and salicylic acid (SA) increased their effect on phytochemical production and biomass accumulation in M. dioica. The results indicate that compared with non-elicited cultures, JA- and SA-elicited cell suspension cultures had significantly enhanced phenolic, flavonoid, and carotenoid production, as well as antioxidant, antimicrobial, and antiproliferative activities. Furthermore, elicited cultures produced 22 phenolic compounds, such as flavonols, hydroxycinnamic acids, and hydroxybenzoic acids. Greater biomass production, phytochemical accumulation, and biological activity occurred in JA- than in SA-elicited cell cultures. This study is the first to successfully establish M. dioica cell suspension cultures for the production of phenolic compounds and carotenoids, as well as for biomass accumulation.

  13. Isolation of protoplasts and regeneration of callus from suspension cultures of cultivated beets.

    Science.gov (United States)

    Bhat, S R; Ford-Lloyd, B V; Callow, J A

    1985-12-01

    Conditions necessary for the isolation and culture of protoplasts from suspension cultures of sugar, fodder and garden beets were investigated. Good yields of protoplasts were obtained by treating cells with a mixture of cellulase, Macerozyme and Driselase enzymes. Nutritional requirements of beet protoplasts were found to be quite simple: protoplasts could be cultured in MS, B5 or PGo based media with 0.4 M glucose with the optimum result being produced on KM8p medium. Plating efficiency (P.E) was genotype-dependent with the sugar beet giving better P.E. than the fodder or garden beets used, and higher values being achieved with the use of desalted Driselase for isolation followed by culture on KMBp medium.

  14. Evaluation of the oxidative burst in suspension cell culture of Phaseolus vulgaris.

    Science.gov (United States)

    Janisch, Kerstin; Schempp, Harald

    2004-01-01

    Plants respond to the attack of pathogens with the oxidative burst, a production of reactive oxygen species (ROS). In this work a cell culture suspension of Phaseolus vulgaris was used to investigate the oxidative burst triggered by a conidia suspension of different races of Colletotrichum lindemuthianum. As a defence response of the cells a two-phase peak was observed with all used races of Colletotrichum lindemuthianum, varying only in the produced amounts of hydrogen peroxide. Findings with additives such as superoxide dismutase (SOD), diphenyleneiodonium (DPI) and catalase gave rise to the conclusion that more superoxide radicals were produced than be detectable with Amplex Red as hydrogen peroxide. It is assumed that the conversion of the superoxide radical is spontaneous and not driven via a cell-derived superoxide dismutase. The addition of low-molecular cell wall components (ergosterol, glucosamine, galactosamine) showed clearly that compounds like this act as elicitors and thus are involved in triggering the burst. Furthermore, an evaluation of the metabolizing capacities of hydrogen peroxide of the suspension culture cells revealed the enormous capacity of the cells to detoxify this ROS.

  15. Quantitative proteome changes in Arabidopsis thaliana suspension-cultured cells in response to plant natriuretic peptides

    KAUST Repository

    Turek, Ilona

    2015-06-30

    Proteome changes in the Arabidopsis thaliana suspension cells in response to the A. thaliana plant natriuretic peptide (PNP), AtPNP-A (At2g18660) were assessed using quantitative proteomics employing tandem mass tag (TMT) labeling and tandem mass spectrometry (LC–MS/MS). In this study, we characterized temporal responses of suspension-cultured cells to 1 nM and 10 pM AtPNP-A at 0, 10 and 30 min post-treatment. Both concentrations we found to yield a distinct differential proteome signature. The data shown in this article are associated with the article “Plant natriuretic peptides induce a specific set of proteins diagnostic for an adaptive response to abiotic stress” by Turek et al. (Front. Plant Sci. 5 (2014) 661) and have been deposited to the ProteomeXchange with identifier PXD001386.

  16. Somatic embryogenesis and plant regeneration from cell suspension and tissue cultures of mature himalayan poplar (Populus ciliata).

    Science.gov (United States)

    Cheema, G S

    1989-02-01

    Somatic embryogenesis and plantlet formation were obtained from callus and cell suspension cultures of 40-year- old Himalayan Poplar (Populus ciliata Wall ex Royle). Callus and cell suspensions were obtained by transfer of inoculum of semiorganized leaf cultures, which were maintained on Murashige and Skoog (MS) medium supplemented with benzylaminopurine (BAP), to MS with 2,4-dichlorophenoxyacetic acid (2,4-D). Reduction of 2,4-D concentration during subsequent subculture of cell suspensions resulted in the formation of embryoids. These embryoids developed further only after being transferred to agar-based MS medium supplemented with BAP and naphthalene acetic acid. Loss of embryogenic potential was observed in cell suspensions after 6 subcultures. However, callus cultures retained the embryogenic potential even after repeated subcultures for more than a year. Plantlets could be successfully hardened and grown in natural outdoor conditions.

  17. [Research on ursolic acid production of Eriobotrya japonica cell suspension culture in WAVE bioreactor].

    Science.gov (United States)

    Li, Hui-hua; Yao, De-heng; Xu, Jian; Wang, Wei; Chang, Qiang; Su, Ming-hua

    2015-05-01

    Through scale-up cultivation of Eriobotrya japonica suspension cells using WAVE bioreactor, the cell growth and ursolic acid (UA) accumulation were studied. The comparison test was carried out in the flask and the reactor with cell dry weight (DW) and UA content as evaluation indexes. The culture medium, DW and UA content were compared in 1 L and 5 L working volumes of bioreactor. The orthogonal test with main actors of inoculation amount, speed and angle of rotation was developed to find the optimal combination, in 1 L working volume of bioreactor. DW of the cell growth and the UA content in bioreactor were higher than those of the shaker by 105.5% and 27.65% respectively. In bioreactor, the dynamic changes of elements in the fluid culture, the dry weight of the cell growth and the UA content in 1 L and 5 L working volumes were similar. Inoculation of 80 g, rotational speed of 26 r · min(-1), and angle of 6 ° was the optimal combination, and the cell biomass of 19.01 g · L(-1) and the UA content of 27.750 mg · g(-1) were achieved after 100 h cultivation in 1 L working volume of bioreactor. WAVE Bioreactor is more suitable than flasks for the E. japonica cell suspension culture, and culture parameters can be achieved from 1 L to 5 L amplification.

  18. Preparation of single cells from aggregated Taxus suspension cultures for population analysis.

    Science.gov (United States)

    Naill, Michael C; Roberts, Susan C

    2004-06-30

    A method for the isolation of single plant cells from Taxus suspension cultures has been developed for the analysis of single cells via rapid throughput techniques such as flow cytometry. Several cell wall specific enzymes, such as pectinase, pectolyase Y-23, macerozyme, Driselase(R), and cellulase were tested for efficacy in producing single cell suspensions. The method was optimized for single cell yield, viability, time, and representivity of aggregated cell cultures. The best combination for single cell isolation was found to be 0.5% (w/v) pectolyase Y-23 and 0.04% (w/v) cellulase. High viability (>95%) and high yields of single cell aggregates (>90%) were obtained following 4 hours of digestion for four separate Taxus cell lines. In addition, methyl jasmonate elicitation (200 microM) was found to have no effect on three of the four tested Taxus lines. Isolated single cells were statistically similar to untreated cell cultures for peroxidase activity (model cell wall protein) and paclitaxel content (secondary metabolite produced in Taxus cell cultures). In comparison, protoplasts showed marked changes in both peroxidase activity and paclitaxel content as compared to untreated cultures. The use of flow cytometry was demonstrated with isolated cells that were found to have > 99% viability upon staining with fluorescein diacetate. The development of a method for the isolation of single plant cells will allow the study of population dynamics and culture variability on a single cell level for the development of population models of plant cell cultures and secondary metabolism. Copyright 2004 Wiley Periodicals, Inc.

  19. Maintenance of undifferentiated mouse embryonic stem cells in suspension by the serum- and feeder-free defined culture condition

    Science.gov (United States)

    Tsuji, Yukiiko; Yoshimura, Naoko; Aoki, Hitomi; Sharov, Alexei A.; Ko, Minoru S.H.; Motohashi, Tsutomu; Kunisada, Takahiro

    2008-01-01

    The proven pluripotency of ES cells is expected to allow their therapeutic use for regenerative medicine. We present here a novel suspension culture method that facilitates the proliferation of pluripotent ES cells without feeder cells. The culture medium contains polyvinyl alcohol (PVA), free of either animal-derived or synthetic serum, and contains very low amounts of peptidic or proteinaceous materials, which are favorable for therapeutic use. ES cells showed sustained proliferation in the suspension culture, and their undifferentiated state and pluripotency were experimentally verified. DNA microarray analyses showed a close relationship between the elevated expression of genes related to cell adhesions. We suggest that this suspension culture condition provides a better alternative to the conventional attached cell culture condition, especially for possible therapeutic use, by limiting the exposure of ES cells to feeder cells and animal products. PMID:18624284

  20. Comparison of mesencephalic free-floating tissue culture grafts and cell suspension grafts in the 6-hydroxydopamine-lesioned rat

    DEFF Research Database (Denmark)

    Meyer, Morten; Widmer, H R; Wagner, B

    1998-01-01

    improvements in terms of significant reductions in amphetamine-induced rotations were observed in rats grafted with FFRT cultures (127%) and rats grafted with cell suspensions (122%), while control animals showed no normalization of rotational behavior. At 84 days after transplantation, there were similar...... days in culture or directly as dissociated cell suspensions, and compared with regard to neuronal survival and ability to normalize rotational behavior in adult rats with unilateral 6-hydroxydopamine (6-OHDA) lesions. Other lesioned rats received injections of cell-free medium and served as controls...... numbers of TH-immunoreactive (TH-ir) neurons in grafts of cultured tissue (775 +/- 98, mean +/- SEM) and grafts of fresh, dissociated cell suspension (806 +/- 105, mean +/- SEM). Cell counts in fresh explants, 7-day-old cultures, and grafted cultures revealed a 68.2% loss of TH-ir cells 7 days after...

  1. Effect of subculture and elicitation on instability of taxol production in Taxus sp. suspension cultures.

    Science.gov (United States)

    Kim, Beum Jun; Gibson, Donna M; Shuler, Michael L

    2004-01-01

    The production of secondary metabolites through plant cell suspension cultures is challenging because the level and pattern of production is often unstable and unpredictable. To investigate the factors affecting instability of secondary metabolite production, high Taxol (paclitaxel)-producing Taxus cultures induced by methyl jasmonate elicitation and their low Taxol-producing counterparts were compared with respect to growth and Taxol production kinetics. With Taxus subcultures we observe alternating states of high and low productivity. Parental cultures and their subcultures from five different cell lines were used to test whether a high-producing culture grows more slowly or dies more rapidly than a low-producing one. These cell lines were of three types: (1) Taxol-producing with and without methyl jasmonate, (2) Taxol-producing only upon elicitation, and (3) nonproducing. High-producing cultures show growth inhibition upon subculture, whereas nonproducing elicited cultures show little growth inhibition. Thus, growth inhibition is primarily due to Taxol or taxane accumulation and not a direct result of methyl jasmonate treatment. Through media exchange between high- and low-producing cultures, it appears that culture components generated by cells alter culture properties. To assess variability as a function of culture lineage, two groups of replicate cultures were generated either with a mixing of the parental flasks or segregation of parental flasks at each subculture. Although parental culture mixing did not reduce flask-to-flask variation, the production level of Taxol in subcultures resulting from mixing inocula was sustained at a higher level relative to segregated subcultures. The results are consistent with the possibility of cell signaling within the population that can induce Taxol production.

  2. Towards high-yield production of pharmaceutical proteins with plant cell suspension cultures.

    Science.gov (United States)

    Xu, Jianfeng; Ge, Xumeng; Dolan, Maureen C

    2011-01-01

    "Molecular farming" in plants with significant advantages in cost and safety is touted as a promising platform for the production of complex pharmaceutical proteins. While whole-plant produced biopharmaceuticals account for a significant portion of the preclinical and clinical pipeline, plant cell suspension culture, which integrates the merits of whole-plant systems with those of microbial fermentation, is emerging as a more compliant alternative "factory". However, low protein productivity remains a major obstacle that limits extensive commercialization of plant cell bioproduction platform. This review highlights the advantages and recent progress in plant cell culture technology and outlines viable strategies at both the biological and process engineering levels for advancing the economic feasibility of plant cell-based protein production. Approaches to overcome and solve the associated challenges of this culture system that include non-mammalian glycosylation and genetic instability will also be discussed.

  3. Defense gene expression in elicitor-treated cell suspension cultures of french bean cv. Imuna.

    Science.gov (United States)

    Ellis, J S; Jennings, A C; Edwards, L A; Mavandad, M; Lamb, C J; Dixon, R A

    1989-12-01

    Cell suspension cultures of bean (Phaseolus vulgaris) cv. Imuna accumulated isoflavonoid phytoalexins on exposure to elicitor from the phytopathogenic fungus Colletotrichum lindemuthianum (CL). This was preceeded by rapid increases in the activities of phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS). However, the patterns of expression of PAL and CHS genes differed from those observed in cultures of a previously studied bean cultivar. The relative levels of transcripts from individual members of the CHS multigene family differed significantly at 1.5 h compared to 22.5 h after elicitation. More strikingly, three PAL genes were expressed in cultivar Imuna in response to fungal elicitor, whereas two are expressed in elicitor-treated cell cultures of cultivar Canadian Wonder.

  4. Suspension cell culture in microgravity and development of a space bioreactor

    Science.gov (United States)

    Morrison, Dennis R.

    1987-01-01

    NASA has methodically developed unique suspension type cell and recovery apparatus culture systems for bioprocess technology experiments and production of biological products in microgravity. The first space bioreactor has been designed for microprocessor control, no gaseous headspace, circulation and resupply of culture medium, and slow mixing in very low shear regimes. Various ground based bioreactors are being used to test reactor vessel design, on-line sensors, effects of shear, nutrient supply, and waste removal from continuous culture of human cells attached to microcarriers. The small (500 ml) bioreactor is being constructed for flight experiments in the Shuttle middeck to verify systems operation under microgravity conditions and to measure the efficiencies of mass transport, gas transfer, oxygen consumption, and control of low shear stress on cells.

  5. Histology of embryoid development in oil palm (Elaeis guineensis Jacq. cell suspension culture

    Directory of Open Access Journals (Sweden)

    Songrat Tinnongjig

    2001-11-01

    Full Text Available Embryos of oil palm (Elaeis guineensis Jacq. variety tenera were cultured on Eeuwens or Y3 (1976; 1978 medium supplemented with 2 mg/l 2,4-D. Calluses were initiated from these embryos. The eight-weekold calluses derived from embryos were transferred to modified Y3 liquid medium devoid of 2,4-D and supplemented with NAA, BA and coconut water to establish cell suspension culture. After a period of culture,these cells were then subcultured to the same medium without plant growth regulators to induce embryoid formation. The calluses and embryoids were harvested at various times, fixed, sectioned, stained and examined microscopically. Histological study revealed that embryoid occurred from meristematic cells with dense cytoplasm along the callus clumps.

  6. Suspension Culture Alters Insulin Secretion in Induced Human Umbilical Cord Matrix-Derived Mesenchymal Cells

    Directory of Open Access Journals (Sweden)

    Fatemeh Seyedi

    2016-04-01

    Full Text Available Objective: Worldwide, diabetes mellitus (DM is an ever-increasing metabolic disorder. A promising approach to the treatment of DM is the implantation of insulin producing cells (IPC that have been derived from various stem cells. Culture conditions play a pivotal role in the quality and quantity of the differentiated cells. In this experimental study, we have applied various culture conditions to differentiate human umbilical cord matrix-derived mesenchymal cells (hUCMs into IPCs and measured insulin production. Materials and Methods: In this experimental study, we exposed hUCMs cells to pancreatic medium and differentiated them into IPCs in monolayer and suspension cultures. Pancreatic medium consisted of serum-free Dulbecco’s modified eagle’s medium Nutrient mixture F12 (DMEM/F12 medium with 17.5 mM glucose supplemented by 10 mM nicotinamide, 10 nM exendin-4, 10 nM pentagastrin, 100 pM hepatocyte growth factor, and B-27 serum-free supplement. After differentiation, insulin content was analyzed by gene expression, immunocytochemistry (IHC and the chemiluminesence immunoassay (CLIA. Results: Reverse transcription-polymerase chain reaction (RT-PCR showed efficient expressions of NKX2.2, PDX1 and INSULIN genes in both groups. IHC analysis showed higher expression of insulin protein in the hanging drop group, and CLIA revealed a significant higher insulin production in hanging drops compared with the monolayer group following the glucose challenge test. Conclusion: We showed by this novel, simple technique that the suspension culture played an important role in differentiation of hUCMs into IPC. This culture was more efficient than the conventional culture method commonly used in IPC differentiation and cultivation.

  7. Characterization of transmembrane auxin transport in Arabidopsis suspension-cultured cells.

    Science.gov (United States)

    Seifertová, Daniela; Skůpa, Petr; Rychtář, Jan; Laňková, Martina; Pařezová, Markéta; Dobrev, Petre I; Hoyerová, Klára; Petrášek, Jan; Zažímalová, Eva

    2014-03-15

    Polar auxin transport is a crucial process for control and coordination of plant development. Studies of auxin transport through plant tissues and organs showed that auxin is transported by a combination of phloem flow and the active, carrier-mediated cell-to-cell transport. Since plant organs and even tissues are too complex for determination of the kinetics of carrier-mediated auxin uptake and efflux on the cellular level, simplified models of cell suspension cultures are often used, and several tobacco cell lines have been established for auxin transport assays. However, there are very few data available on the specificity and kinetics of auxin transport across the plasma membrane for Arabidopsis thaliana suspension-cultured cells. In this report, the characteristics of carrier-mediated uptake (influx) and efflux for the native auxin indole-3-acetic acid and synthetic auxins, naphthalene-1-acetic and 2,4-dichlorophenoxyacetic acids (NAA and 2,4-D, respectively) in A. thaliana ecotype Landsberg erecta suspension-cultured cells (LE line) are provided. By auxin competition assays and inhibitor treatments, we show that, similarly to tobacco cells, uptake carriers have high affinity towards 2,4-D and that NAA is a good tool for studies of auxin efflux in LE cells. In contrast to tobacco cells, metabolic profiling showed that only a small proportion of NAA is metabolized in LE cells. These results show that the LE cell line is a useful experimental system for measurements of kinetics of auxin carriers on the cellular level that is complementary to tobacco cells.

  8. Effect of Magnetic Nanoparticles on Tobacco BY-2 Cell Suspension Culture

    Directory of Open Access Journals (Sweden)

    Rene Kizek

    2012-12-01

    Full Text Available Nanomaterials are structures whose exceptionality is based on their large surface, which is closely connected with reactivity and modification possibilities. Due to these properties nanomaterials are used in textile industry (antibacterial textiles with silver nanoparticles, electronics (high-resolution imaging, logical circuits on the molecular level and medicine. Medicine represents one of the most important fields of application of nanomaterials. They are investigated in connection with targeted therapy (infectious diseases, malignant diseases or imaging (contrast agents. Nanomaterials including nanoparticles have a great application potential in the targeted transport of pharmaceuticals. However, there are some negative properties of nanoparticles, which must be carefully solved, as hydrophobic properties leading to instability in aqueous environment, and especially their possible toxicity. Data about toxicity of nanomaterials are still scarce. Due to this fact, in this work we focused on studying of the effect of magnetic nanoparticles (NPs and modified magnetic nanoparticles (MNPs on tobacco BY-2 plant cell suspension culture. We aimed at examining the effect of NPs and MNPs on growth, proteosynthesis — total protein content, thiols — reduced (GSH and oxidized (GSSG glutathione, phytochelatins PC2-5, glutathione S-transferase (GST activity and antioxidant activity of BY-2 cells. Whereas the effect of NPs and MNPs on growth of cell suspension culture was only moderate, significant changes were detected in all other biochemical parameters. Significant changes in protein content, phytochelatins levels and GST activity were observed in BY-2 cells treated with MNPs nanoparticles treatment. Changes were also clearly evident in the case of application of NPs. Our results demonstrate the ability of MNPs to negatively affect metabolism and induce biosynthesis of protective compounds in a plant cell model represented by BY-2 cell suspension

  9. Effect of magnetic nanoparticles on tobacco BY-2 cell suspension culture.

    Science.gov (United States)

    Krystofova, Olga; Sochor, Jiri; Zitka, Ondrej; Babula, Petr; Kudrle, Vit; Adam, Vojtech; Kizek, Rene

    2012-12-20

    Nanomaterials are structures whose exceptionality is based on their large surface, which is closely connected with reactivity and modification possibilities. Due to these properties nanomaterials are used in textile industry (antibacterial textiles with silver nanoparticles), electronics (high-resolution imaging, logical circuits on the molecular level) and medicine. Medicine represents one of the most important fields of application of nanomaterials. They are investigated in connection with targeted therapy (infectious diseases, malignant diseases) or imaging (contrast agents). Nanomaterials including nanoparticles have a great application potential in the targeted transport of pharmaceuticals. However, there are some negative properties of nanoparticles, which must be carefully solved, as hydrophobic properties leading to instability in aqueous environment, and especially their possible toxicity. Data about toxicity of nanomaterials are still scarce. Due to this fact, in this work we focused on studying of the effect of magnetic nanoparticles (NPs) and modified magnetic nanoparticles (MNPs) on tobacco BY-2 plant cell suspension culture. We aimed at examining the effect of NPs and MNPs on growth, proteosynthesis - total protein content, thiols - reduced (GSH) and oxidized (GSSG) glutathione, phytochelatins PC2-5, glutathione S-transferase (GST) activity and antioxidant activity of BY-2 cells. Whereas the effect of NPs and MNPs on growth of cell suspension culture was only moderate, significant changes were detected in all other biochemical parameters. Significant changes in protein content, phytochelatins levels and GST activity were observed in BY-2 cells treated with MNPs nanoparticles treatment. Changes were also clearly evident in the case of application of NPs. Our results demonstrate the ability of MNPs to negatively affect metabolism and induce biosynthesis of protective compounds in a plant cell model represented by BY-2 cell suspension culture. The

  10. Evaluation of Simulated Microgravity Environments Induced by Diamagnetic Levitation of Plant Cell Suspension Cultures

    Science.gov (United States)

    Kamal, Khaled Y.; Herranz, Raúl; van Loon, Jack J. W. A.; Christianen, Peter C. M.; Medina, F. Javier

    2016-06-01

    Ground-Based Facilities (GBF) are essetial tools to understand the physical and biological effects of the absence of gravity and they are necessary to prepare and complement space experiments. It has been shown previously that a real microgravity environment induces the dissociation of cell proliferation from cell growth in seedling root meristems, which are limited populations of proliferating cells. Plant cell cultures are large and homogeneous populations of proliferating cells, so that they are a convenient model to study the effects of altered gravity on cellular mechanisms regulating cell proliferation and associated cell growth. Cell suspension cultures of the Arabidopsis thaliana cell line MM2d were exposed to four altered gravity and magnetic field environments in a magnetic levitation facility for 3 hours, including two simulated microgravity and Mars-like gravity levels obtained with different magnetic field intensities. Samples were processed either by quick freezing, to be used in flow cytometry for cell cycle studies, or by chemical fixation for microscopy techniques to measure parameters of the nucleolus. Although the trend of the results was the same as those obtained in real microgravity on meristems (increased cell proliferation and decreased cell growth), we provide a technical discussion in the context of validation of proper conditions to achieve true cell levitation inside a levitating droplet. We conclude that the use of magnetic levitation as a simulated microgravity GBF for cell suspension cultures is not recommended.

  11. Biochemical precursor effects on the fatty acid production in cell suspension cultures of Theobroma cacao L.

    Science.gov (United States)

    Parra, O; Gallego, A M; Urrea, A; Rojas, L F; Correa, C; Atehortúa, L

    2017-02-01

    Cocoa butter (CB) is composed of 96% palmitic, stearic, oleic, linoleic and linolenic fatty acids that are responsible for the hardness, texture and fusion properties of chocolate. Through in vitro plant cell culture it is possible to modify CB lipid profiles and to study the fatty acid biosynthesis pathway on a subcellular level, evaluating fundamental aspects to enhance in vitro fatty acid production in a specific and controlled way. In this research, culture media was supplemented with acetate, biotin, pyruvate, bicarbonate and glycerol at three different concentrations and the effects on the biomass production (g/L), cell viability, and fatty acids profile and production was evaluated in in vitro cell suspensions culture. It was found that biotin stimulated fatty acid synthesis without altering cell viability and cell growth. It was also evident a change in the lipid profile of cell suspensions, increasing middle and long chain fatty acids proportion, which are unusual to those reported in seeds; thus implying that it is possible to modify lipid profiles according to the treatment used. According to the results of sucrose gradients and enzyme assays performed, it is proposed that cacao cells probably use the pentose phosphate pathway, mitochondria being the key organelle in the carbon flux for the synthesis of reductant power and fatty acid precursors. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  12. Saccharomyces cerevisiae gene expression changes during rotating wall vessel suspension culture

    Science.gov (United States)

    Johanson, Kelly; Allen, Patricia L.; Lewis, Fawn; Cubano, Luis A.; Hyman, Linda E.; Hammond, Timothy G.

    2002-01-01

    This study utilizes Saccharomyces cerevisiae to study genetic responses to suspension culture. The suspension culture system used in this study is the high-aspect-ratio vessel, one type of the rotating wall vessel, that provides a high rate of gas exchange necessary for rapidly dividing cells. Cells were grown in the high-aspect-ratio vessel, and DNA microarray and metabolic analyses were used to determine the resulting changes in yeast gene expression. A significant number of genes were found to be up- or downregulated by at least twofold as a result of rotational growth. By using Gibbs promoter alignment, clusters of genes were examined for promoter elements mediating these genetic changes. Candidate binding motifs similar to the Rap1p binding site and the stress-responsive element were identified in the promoter regions of differentially regulated genes. This study shows that, as in higher order organisms, S. cerevisiae changes gene expression in response to rotational culture and also provides clues for investigations into the signaling pathways involved in gravitational response.

  13. Transformation of soybean protoplasts from permanent suspension cultures by cocultivation with cells of Agrobacterium tumefaciens.

    Science.gov (United States)

    Baldes, R; Moos, M; Geider, K

    1987-03-01

    Cell wall regenerating protoplasts from soybean cells kept in suspension culture were cocultivated with bacteria which were derived from the nopaline strain C58 of Agrobacterium tumefaciens. When the bacteria carried an oncogenic Ti-plasmid, about 5% of the surviving protoplasts were able to form calli on hormone-free agar in contrast to controls, where bacteria without Ti-plasmid were applied, and where no calli were formed. After isolation of DNA from hormone-independently growing cells further evidence for transformation was obtained by hybridization to Ti-plasmid specific RNA and by rescue of a segment with a bacterial resistance gene which had been inserted before into the T-DNA. Transfer of T-DNA harboring a neomycin-resistance gene activated by the nos-promoter resulted in calli growing on kanamycin. Verification of segments located at the left and the right part of the T-DNA indicated the presence of its entire length in transformed soybean cells. Expression of T-DNA genes was measured by the assay of nopaline-synthase. Cells cultured on agar had a much higher level of nopaline-synthase than fast growing cells in suspension culture. Transferring them to agar or treatment with azacytidine strongly increased synthesis of nopaline-synthase indicating a reversible repression presumably via a methylation mechanism.

  14. Serum-free spheroid suspension culture maintains high proliferation and differentiation potentials of mesenchymal stem cells

    Science.gov (United States)

    Alimperti, Stella; Wen, Yuan; Lei, Pedro; Tian, Jun; Campbell, Andrew; Andreadis, Stelios T.

    2016-01-01

    There have been many clinical trials recently using ex vivo-expanded human mesenchymal stem cells (MSCs) to treat several indications such as graft-versus-host disease, acute myocardial infarction, Crohn’s disease, and multiple sclerosis. However, the conventional 2-dimensional (2D) culture of MSCs is laborious and limited in scale potential. The large dosage requirement for many of the indications further exacerbates this manufacturing challenge. In contrast, spheroid MSC culture does not require a cell attachment surface and is amenable to large-scale suspension cell culture techniques, such as stirred-tank bioreactors. In this present study, we developed and optimized serum free media for culturing MSC spheroids. We used Design of Experiment (DoE)-based strategies to systematically evaluate media mixtures and a panel of different components. The optimization yielded two prototype media that could allow MSCs to form aggregates and proliferate in both static cultures and dynamic cultures. The expanded MSCs expressed the expected surface markers for mesenchymal cells (CD73, CD90 and CD105). In addition, the expanded cells demonstrated multipotency and differentiated to the osteocyte, chondrocyte and adipocyte lineages, which showed similar or enhanced differentiation levels compared with serum-containing adherent cultures. PMID:24616445

  15. Induced accumulation of oleanolic acid and ursolic acid in cell suspension cultures of Uncaria tomentosa.

    Science.gov (United States)

    Feria-Romero, Iris; Lazo, Elizabeth; Ponce-Noyola, Teresa; Cerda-García-Rojas, Carlos M; Ramos-Valdivia, Ana C

    2005-06-01

    Increasing sucrose from 20 to 50 g l(-1) in Uncaria tomentosa cell suspension cultures enhanced ursolic acid and oleanolic acid production from 129 +/- 61 to 553 +/- 193 microg g(-1) cell dry wt. The maximal concentration of both triterpenes (1680 +/- 39 microg g(-1) cell dry wt) was 8 days after elicitation by jasmonic acid, while yeast extract or citrus pectin treatments produced 1189 +/- 20 or 1120 +/- 26 microg g(-1) cell dry wt, respectively. The ratio of ursolic acid:oleanolic acid was constant at 70:30.

  16. Ce4+-Induced Apoptosis of Taxus cuspidata Cells in Suspension Culture

    Institute of Scientific and Technical Information of China (English)

    葛志强; 元英进; 王艳东; 马振毅; 胡宗定

    2002-01-01

    The standard detection hallmarks of apoptosis of Taxus cuspidata cells in suspension culture with Ce4+ 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 Ce4+ can induce apoptosis of Taxus cuspidata cells and also indicate that there is a certain relationship between apoptosis and secondary metabolite product-Taxol.

  17. Proteomic characterization of golgi membranes enriched from Arabidopsis suspension cell cultures

    DEFF Research Database (Denmark)

    Hansen, Sara Fasmer; Ebert, Berit; Rautengarten, Carsten

    2016-01-01

    The plant Golgi apparatus has a central role in the secretory pathway and is the principal site within the cell for the assembly and processing of macromolecules. The stacked membrane structure of the Golgi apparatus along with its interactions with the cytoskeleton and endoplasmic reticulum has...... from an Arabidopsis cell suspension culture that can be used to investigate the proteome of this organelle. We also provide a useful workflow for the examination of proteomic data as the result of multiple analyses. Finally, we highlight a simple technique to validate the subcellular localization...

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

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    l.lntroductionSuspension cultures of Taxal chinensis var.mairei frequently accumulate Taxol (paclitaxel),which is clinically effective amineoplanic agent.TSXol is known to act by enhancing thePOlymeriZation of tubulin in the initiation andextension of microtubules, ac has been shown toinduce apoptosis in human and animal cellslll.Apoptosis, also known as programmed cell death, isthe active process of cell death which occurs duringdevelopment and in resPOnse tO enviboental cues ofa multicellular organism. In...

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

    Institute of Scientific and Technical Information of China (English)

    TangWei; WuJiongyuan; 等

    1995-01-01

    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,basal medium and carbohydrate level is 2mg/L 2,4-D:0.5mg/L KT,MS and 3% sucrose respectively.

  20. Growth characteristics and nutrient depletion of Miscanthus x ogiformis Honda 'Giganteus' suspension cultures

    DEFF Research Database (Denmark)

    Holme, Inger Bæksted

    1998-01-01

    The growth characteristics and nutrient depletion in suspension cultures of Miscanthus ogiformis Honda ‘Giganteus' grown in media containing either Murashige and Skoog or N6 basal nutrient salts were studied during a culture period of 15 days. Proline was added to both media in concentrations from...

  1. Evaluation of Antioxidant and Antibacterial Potentials of Nigella sativa L. Suspension Cultures under Elicitation

    Directory of Open Access Journals (Sweden)

    Hera Chaudhry

    2015-01-01

    Full Text Available Nigella sativa L. (family Ranunculaceae is an annual herb of immense medicinal properties because of its major active components (i.e., thymoquinone (TQ, thymohydroquinone (THQ, and thymol (THY. Plant tissue culture techniques like elicitation, Agrobacterium mediated transformation, hairy root culture, and so on, are applied for substantial metabolite production. This study enumerates the antibacterial and antioxidant potentials of N. sativa epicotyl suspension cultures under biotic and abiotic elicitation along with concentration optimization of the elicitors for enhanced TQ and THY production. Cultures under different concentrations of pectin and manganese chloride (MnCl2 elicitation (i.e., 5 mg/L, 10 mg/L, and 15 mg/L showed that the control, MnCl2 10 mg/L, and pectin 15 mg/L suspension extracts greatly inhibited the growth of E. coli, S. typhimurium, and S. aureus (MIC against E. coli, i.e., 2.35±0.8, 2.4±0.2, and 2.46±0.5, resp.. Elicitation decreased SOD enzyme activity whereas CAT enzyme activity increased remarkably under MnCl2 elicitation. MnCl2 10 mg/L and pectin 15 mg/L elicitation enhanced the DPPH radical inhibition ability, but ferric scavenging activity was comparable to the control. TQ and THY were quantified by LC-MS/MS in the cultures with high bioactive properties revealing maximum content under MnCl2 10 mg/L elicitation. Therefore, MnCl2 elicitation can be undertaken on large scale for sustainable metabolite production.

  2. Determination of triterpenic acids and screening for valuable secondary metabolites in Salvia sp. suspension cultures.

    Science.gov (United States)

    Kümmritz, Sibylle; Haas, Christiane; Pavlov, Atanas I; Geib, Doris; Ulber, Roland; Bley, Thomas; Steingroewer, Juliane

    2014-01-01

    Plant in vitro cultures are a prospective alternative for biochemicals production, for example the triterpenes oleanolic and ursolic acid present in plants and cell cultures of Salvia sp. Our objective was to develop a suitable analysis protocol for evaluation of triterpenic acid yield in plant raw material and in vitro cultures supporting selection processes. Moreover, valuable bioactive compounds had to be revealed. Thus, different strategies enhancing the separation for a sensitive and effective HPLC-UV method were investigated and the developed method was validated for linearity, precision, accuracy, limits of detection and quantification. A baseline separation of these isomers enabled detection limits of below 0.4 microg/mL and quantification limits of about 1.2 microg/mL. Over the tested concentration range a good linearity was observed (R2 > 0.9999). The variations in the method were below 6% for intra- and inter-day assays of concentration. Recoveries were between 85-98% for both compounds using ethanol as extraction solvent. Additionally, metabolite profiling of cell suspension culture extracts by GC-MS has shown the production variability of different plant metabolites and especially the presence of plant phenols and sterols. These studies provide a method suitable for screening plant and cell culture productivity of triterpenic acids and highlighted interesting co-products of plant cell cultures.

  3. Impact of UV-B radiation on some biochemical changes and growth parameters in Echinacea purpurea callus and suspension culture

    Directory of Open Access Journals (Sweden)

    Hossam H. Manaf

    2016-12-01

    Full Text Available The effects of UV-B light force, exposure time and incubation period on producing caffeic acid derivatives and growth parameters in Echinacea purpurea callus and suspension culture were assessed. UV-B led to an increment of all growth parameters and antioxidant activity in callus and cell suspension and caffeic acid derivatives in cell suspension by increasing incubation period. The reverse was true for G-POD activity in cell suspension and PAL activity in both types of cultures. Incubation period 2 weeks was more effective in caffeic acid, total phenols and G-POD activity in callus cells and incubation period one week only for total phenols in cell suspension. The two exposure times 2 and 4 h increased antioxidant activity in the two types of cultures. Exposure time 2 h led to increase caffeic acid and total phenols in callus cells. The maximum increase in caffeic acid, total phenols and PAL activity in cell suspension was achieved by 4 h exposure time. Likewise, using 2 UV-B lamps for 2 h was the most effective in creating more biochemical components than the other treatments.

  4. Isolation of transcription factor complexes from Arabidopsis cell suspension cultures by tandem affinity purification.

    Science.gov (United States)

    Van Leene, Jelle; Eeckhout, Dominique; Persiau, Geert; Van De Slijke, Eveline; Geerinck, Jan; Van Isterdael, Gert; Witters, Erwin; De Jaeger, Geert

    2011-01-01

    Defining protein complexes is critical to virtually all aspects of cell biology because most cellular processes are regulated by stable or more dynamic protein interactions. Elucidation of the protein-protein interaction network around transcription factors is essential to fully understand their function and regulation. In the last decade, new technologies have emerged to study protein-protein interactions under near-physiological conditions. We have developed a high-throughput tandem affinity purification (TAP)/mass spectrometry (MS) platform for cell suspension cultures to analyze protein complexes in Arabidopsis thaliana. This streamlined platform follows an integrated approach comprising generic Gateway-based vectors with high cloning flexibility, the fast generation of transgenic suspension cultures, TAP adapted for plant cells, and tandem matrix-assisted laser desorption ionization MS for the identification of purified proteins. Recently, we evaluated the GS tag, originally developed to study mammalian protein complexes, that combines two IgG-binding domains of protein G with a streptavidin-binding peptide, separated by two tobacco etch virus cleavage sites. We found that this GS tag outperforms the traditional TAP tag in plant cells, regarding both specificity and complex yield. Here, we provide detailed protocols of the GS-based TAP platform that allowed us to characterize transcription factor complexes involved in signaling in response to the plant phytohormone jasmonate.

  5. Production of Gymnemic Acid from Cell Suspension Cultures of Gymnema sylvestre.

    Science.gov (United States)

    Nagella, Praveen; Dandin, Vijayalaxmi S; Murthy, Hosakatte Niranjana

    2016-01-01

    Gymnema sylvestre R. Br. is a popular herbal medicine. It has been used in ayurvedic system of medicine for thousands of years. It is popularly called as "Gur-mar" for its distinctive property of temporarily destroying the taste of sweetness and is used in the treatment of diabetes. The leaves of gymnema possess antidiabetic, antimicrobial, anti-hypercholesterolemic, anti-sweetener, anti-inflammatory, and hepatoprotective properties and have traditional uses in the treatment of asthma, eye complaints, and snake bite. The leaves contain triterpene saponins such as gymnemic acid which is an active ingredient of Gymnema. Since the cultivation of G. sylvestre is a very slow process and the content of gymnemic acid depends on the environmental factors, cell suspension culture is sought as an alternative means for the production of Gymnema biomass and to enhance the gymnemic acid content. In this chapter, the methods employed for the induction of callus and subsequent establishment of cell suspension cultures for the production of biomass and analysis of gymnemic acid using high performance liquid chromatography are described.

  6. Five 2-(2-Phenylethylchromones from Sodium Chloride-Elicited Aquilaria sinensis Cell Suspension Cultures

    Directory of Open Access Journals (Sweden)

    Zhongxiu Zhang

    2016-04-01

    Full Text Available Five 2-(2-phenylethylchromones including a new one, (5S,6R,7S,8R-5,8-dichloro-6,7-dihydroxy-2-phenylethyl-5,6,7,8-tetrahydro-4H-chromen-4-one (1, and four known ones (2–5, were isolated from 150 mM NaCl-elicited Aquilaria sinensis cell suspension cultures. In addition, three feruloyl amides (6–8, six nucleosides (9–14, (+-syringaresinol (15, indole-3-carboxaldehyde (16, and two glycosides (17–18 were also obtained. The structures were unambiguously identified by analysis of their UV, IR, NMR, and HRESIMS data. The absolute configuration of the new 2-(2-phenylethylchromone (1 was established by a dimolybdenum tetraacetate-induced circular dichroism experiment. Compared to un-elicited cell lines, the appearance of 2-(2-phenylethylchromones in NaCl-treated cells occurred on the 3rd and 5th days of their treatment. 2-(2-Phenylethylchromones, feruloyl amides, nucleosides, and lignins have been reported to be closely related to plant defense; therefore, the identification of these compounds from NaCl-elicited A. sinensis cell suspension cultures would be useful for further exploring the mechanism of agarwood formation.

  7. Enhanced camptothecin production by ethanol addition in the suspension culture of the endophyte, Fusarium solani.

    Science.gov (United States)

    Venugopalan, Aarthi; Srivastava, Smita

    2015-01-01

    Ethanolic extract of a non-camptothecin producing plant, Catharanthus roseus when added in the suspension culture of the endophyte Fusarium solani known to produce camptothecin, resulted in enhanced production of camptothecin by 10.6-fold in comparison to that in control (2.8 μg/L). Interestingly, addition of pure ethanol (up to 5% v/v) in the suspension culture of F. solani resulted in maximum enhancement in camptothecin production (up to 15.5-fold) from that obtained in control. In the presence of ethanol, a reduced glucose uptake (by ∼ 40%) and simultaneous ethanol consumption (up to 9.43 g/L) was observed during the cultivation period (14 days). Also, the total NAD level and the protein content in the biomass increased by 3.7- and 1.9-fold, respectively, in comparison to that in control. The study indicates a dual role of ethanol, presumably as an elicitor and also as a carbon/energy source, leading to enhanced biomass and camptothecin production. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. An Efficient Rice Mutagenesis System Based on Suspension-Cultured Cells

    Institute of Scientific and Technical Information of China (English)

    Yuan-Ling Chen; Hui-Lin Liang; Xing-Liang Ma; Su-Lin Lou; Yong-Yao Xie; Zhen-Lan Liu; Le-Tian Chen; Yao-Guang Liu

    2013-01-01

    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.

  9. Methods for suspension culture, protoplast extraction, and transformation of high-biomass yielding perennial grass Arundo donax.

    Science.gov (United States)

    Pigna, Gaia; Dhillon, Taniya; Dlugosz, Elizabeth M; Yuan, Joshua S; Gorman, Connor; Morandini, Piero; Lenaghan, Scott C; Stewart, C Neal

    2016-12-01

    Arundo donax L. is a promising biofuel feedstock in the Mediterranean region. Despite considerable interest in its genetic improvement, Arundo tissue culture and transformation remains arduous. The authors developed methodologies for cell- and tissue culture and genetic engineering in Arundo. A media screen was conducted, and a suspension culture was established using callus induced from stem axillary bud explants. DBAP medium, containing 9 µM 2,4-D and 4.4 µM BAP, was found to be the most effective medium among those tested for inducing cell suspension cultures, which resulted in a five-fold increase in tissue mass over 14 days. In contrast, CIM medium containing 13 µM 2,4-D, resulted in just a 1.4-fold increase in mass over the same period. Optimized suspension cultures were superior to previously-described solidified medium-based callus culture methods for tissue mass increase. Suspension cultures proved to be very effective for subsequent protoplast isolation. Protoplast electroporation resulted in a 3.3 ± 1.5% transformation efficiency. A dual fluorescent reporter gene vector enabled the direct comparison of the CAMV 35S promoter with the switchgrass ubi2 promoter in single cells of Arundo. The switchgrass ubi2 promoter resulted in noticeably higher reporter gene expression compared with that conferred by the 35S promoter in Arundo.

  10. Improved beta-thujaplicin production in Cupressus lusitanica suspension cultures by fungal elicitor and methyl jasmonate.

    Science.gov (United States)

    Zhao, J; Fujita, K; Yamada, J; Sakai, K

    2001-04-01

    Production of a novel antimicrobial tropolone, beta-thujaplicin, in Cupressus lusitanica suspension cultures was studied by using a variety of chemicals and fungal elicitors. Sodium alginate, chitin, and methyl jasmonate resulted in 2-, 2.5-, and 3-fold higher beta-thujaplicin production, respectively, than in the control. Significantly improved beta-thujaplicin production (187 mg l(-1)) was obtained using a high cell density (180-200 g l(-1)) and fungal elicitor treatment [10 mg (g fresh cells)(-1)] in a production medium with a high ferrous ion concentration (0.3 mM). This improved volumetric productivity was 3- to 4-fold higher than obtained under standard conditions. A synergistic effect of fungal elicitor and ferrous ion on beta-thujaplicin production was also suggested by our study. Plant cell culture technology is a promising alternative for producing a large variety of secondary metabolites that are widely used as food additives, pharmaceuticals, and dairy products (Verpoorte et al. 1999). Thus, beta-thujaplicin production by plant cell cultures was developed with the goal of commercial application (Berlin and Witte 1988; Itose and Sakai 1997; Ono et al. 1998). However, the production of beta-thujaplicin by plant cell cultures is still not competitive for use in industrial applications. In this study, we assessed the effects of methyl jasmonate, alginate, chitin, and fungal elicitor on beta-thujaplicin production; we obtained a significantly elevated beta-thujaplicin production by using an improved culture strategy.

  11. [Impact of subculture cycles and inoculum sizes on suspension cultures of Vitis vinifera].

    Science.gov (United States)

    Qu, Jun-Ge; Zhang, Wei; Hu, Quan-Li; Jin, Mei-Fang

    2006-11-01

    The commercial application of plant cell cultures is often hindered by the instability of secondary metabolite biosynthesis, where the metabolite yield fluctuates and decline dramatically over subcultures. This study proposed that such instability is due to the fluctuations of culture variables. To validate this hypothesis, the effects of the fluctuations of two culture variables (subculture cycle and inoculum size) on the biomass, anthocyanin biosynthesig, intracellular carbon, nitrogen and phosphate during continuous 10 subculture cycles were investigated. The subculture cycle was fluctuated for 12h in a 7 day cycle (6.5, 7 and 7.5 d), and the inoculum size was fluctuated by 20% on basis of 2.00 g (1.60, 2.00 and 2.40 g). It was found that all the measured culture parameters fluctuated over the 10 subculture cycles. The fluctuation in terms of inoculum sizes had a greater effect on the stability of anthocyanin biosynthesis in suspension cultures of V. vinifera. Among all the subculture conditions investigated, 7d-subculture cycle and 1.60 g-inoculum size was the best one to hold the relatively stable anthocyanin production. The anthocyanin yield presented a negative correlation with intracellular sucrose content or intracellular total phosphate content.

  12. Dose responses for Colletotrichum lindemuthianum elicitor-mediated enzyme induction in French bean cell suspension cultures.

    Science.gov (United States)

    Dixon, R A; Dey, P M; Murphy, D L; Whitehead, I M

    1981-03-01

    The induction of L-phenylalanine ammonialyase (PAL, EC 4.3.1.5) and flavanone synthase in French bean cell suspension cultures in response to heat-released elicitor from cell walls of the phytopathogenic fungus Colletotrichum lindemuthianum is highly dependent upon elicitor concentration. The elicitor dose-response curve for PAL induction shows two maxima at around 17.5 and 50 μg elicitor carbohydrate per ml culture, whereas the flavanone synthase response shows one maximum at around 100 μg ml(-1). The PAL response is independent of the elicitor concentration present during the lag phase of enzyme induction; if the initial elicitor concentration is increased after 2 h by addition of extra elicitor, or decreased by dilution of the cultures, the dose response curves obtained reflect the concentration of elicitor present at the time of harvest. PAL induction is not prevented by addition of methyl sugar derivatives to the cultures; α-methyl-D-glucoside, itself a weak elicitor of PAL activity, elicits a multiphasic PAL response when increasing concentrations are added in the presence of Colletotrichum elicitor. Eight fractions with different monosaccharide compositions, obtained from the crude elicitor by gel-filtration, each elicit different dose-responses for PAL induction; the response to unfractionated elicitor is not the sum of the response to the isolated fractions. There is no correlation between the ability of the fractions to induce PAL in the cultures and their ability to act as elicitors of isoflavonoid phytoalexin accumulation in bean hypocotyls.

  13. The Effect of Calcium on Early Fiber Elongation in Cotton Ovule Culture

    Science.gov (United States)

    Cotton fibers are single-cell trichomes that initiate on the ovule epidermis. Fiber initials accumulate calcium and membranes, including ER. Multiple calcium sensors, and small GTPase proteins that may act in calcium signaling pathways and/or primary cell wall biosynthesis were present in fiber init...

  14. More for less: Improving the biomass yield of a pear cell suspension culture by design of experiments.

    Science.gov (United States)

    Rasche, Stefan; Herwartz, Denise; Schuster, Flora; Jablonka, Natalia; Weber, Andrea; Fischer, Rainer; Schillberg, Stefan

    2016-03-18

    Plant cell suspension cultures are widely used for the production of recombinant proteins and secondary metabolites. One of the most important steps during process development is the optimization of yields by testing different cultivation parameters, including the components of the growth medium. However, we have shown that the biomass yield of a cell suspension culture derived from the pear cultivar Pyrus communis cv. Champagner Bratbirne can be significantly improved solely by varying the temperature, inoculum density, illumination, and incubation time. In contrast to medium optimization, these simple physical factors are easily controlled and varied, thereby reducing the effort required. Using an experimental design approach, we improved the biomass yield from 146 g fresh weight (FW)/L to 407 g FW/L in only 5 weeks, simultaneously reducing the costs of goods sold per kg biomass from € 125 to € 45. Our simple approach therefore offers a rapid, efficient and economical process for the optimization of plant cell suspension cultures.

  15. Induction of linalool as a pharmaceutical and medicinal metabolite via cell suspension culture of cumin (Cuminum cyminum L.).

    Science.gov (United States)

    Kazemi, N; Kahrizi, D; Mansouri, M; Karim, H; Vaziri, S; Zargooshi, J; Khanahmadi, M; Shokrinia, M; Mohammadi, N

    2016-05-30

    Cumin is an important medicinal plant in Iran. Plant cell suspension culture is a method for the production of medicinal and secondary metabolites. The linalool is a plant secondary metabolite that has been recognized as a neuroprotective agent. The purpose of this study was to evaluate the effects of salicylic acid elicitor on induction of linalool in cell suspension culture of cumin. For this purpose, the cumin seeds were prepared, to obtain sterile seedling, were disinfected with sodium hypochlorite and alcohol, and were cultured on MS basal medium. This research was conducted in two separate experiments including callus induction and suspension cultures. Leaf explants were prepared from sterile seedlings and used to produce callus on MS medium supplemented with 1 mg/l NAA and 0.5 mg/l BAP. In order to establish suspension culture, the appropriate calli were transferred to liquid medium. Then cell cultures were treated with elicitors. The effects of elicitor on the production of linalool secondary metabolite and cell viability were assessed by GC-Mass and tetrazolium test respectively. For this purpose, the salicylic acid (at concentrations of 0, 1, 2, 4 and 8 mg/l) was used. The experimental design was a completely randomized design with five treatments and three replications. The results of cell culture and GC-Mass analysis showed that salicylic acid had significant effects on the linalool production (suspension culture experiments was lower than control. Increasing the elicitor concentrations lead to reduction in cell survival. In conclusion it is possible to produce linalool as a secondary metabolite and pharmaceutical agent in cell culture of cumin. It is necessary to determine the best combination of medium and elicitor.

  16. Suspension cell culture as a tool for the characterization of class III peroxidases in sugarcane.

    Science.gov (United States)

    Cesarino, Igor; Araújo, Pedro; Paes Leme, Adriana Franco; Creste, Silvana; Mazzafera, Paulo

    2013-01-01

    Secreted class III peroxidases (EC 1.11.1.7) are implicated in a broad range of physiological processes throughout the plant life cycle. However, the unambiguous determination of the precise biological role of an individual class III peroxidase isoenzyme is still a difficult task due to genetic redundancy and broad substrate specificity in vitro. In addition, many difficulties are encountered during extraction and analysis of cell wall proteins. Since class III peroxidases are also secreted into the apoplast, the use of suspension cell cultures can facilitate isolation and functional characterization of individual isoforms. Here, we report on the characterization of class III peroxidases secreted in the spent medium of sugarcane suspension cell cultures. After treatment with specific inducers of cell wall lignification, peroxidases were isolated and activities assayed with guaiacol, syringaldazine and coniferyl alcohol. Enzymatic activity was not significantly different after treatments, regardless of the substrate, with the exception of methyl-jasmonate treatment, which led to a decreased guaiacol peroxidase activity. Remarkably, peroxidases isolated from the medium were capable of oxidizing syringaldazine, an analog to sinapyl alcohol, suggesting that sugarcane cultures can produce peroxidases putatively correlated to lignification. A proteomic approach using activity staining of 2-DE gels revealed a complex isoperoxidase profile, composed predominantly of cationic isoforms. Individual spots were excised and analyzed by LC-ESI-Q-TOF and homology-based search against the Sugarcane EST Database resulted in the identification of several proteins. Spatio-temporal expression pattern of selected genes was determined for validation of identified class III peroxidases that were preferentially expressed during sugarcane stem development.

  17. An embryogenic suspension cell culture system for Agrobacterium-mediated transformation of citrus.

    Science.gov (United States)

    Dutt, M; Grosser, J W

    2010-11-01

    A method for the genetic transformation of several citrus cultivars is described, including cultivars observed to be recalcitrant to conventional epicotyl-mediated transformation. Embryogenic cell suspension cultures, established from unfertilized ovules were used as target tissues for Agrobacterium-mediated transformation. Several modifications were made to the culture environment to investigate factors required for efficient transfer of the T-DNA and the subsequent regeneration of transgenic citrus plants. It was determined that co-cultivation of citrus cells and Agrobacterium in EME medium supplemented with maltose (EME-M) and 100 μM acetosyringone for 5 days at 25°C was optimum for transformation of each of the citrus cultivars. Efficient selection was obtained and escapes were prevented when the antibiotic hygromycin B was used as a selection antibiotic following transformation with an Agrobacterium strain containing hptII in the T-DNA region. Transgenic embryo regeneration and development was enhanced in medium that contained a liquid overlay consisting of a 1:2 mixture of 0.6 M BH3 and 0.15 M EME-M media. PCR and Southern blot analyses confirmed the presence of the T-DNA and the stable integration into the genome of regenerated plants, while RT-PCR demonstrated variable amounts of RNA being transcribed in different transgenic lines. This protocol can create an avenue for insertion of useful traits into any polyembryonic citrus cultivar that can be established as embryogenic cell suspension cultures, including popular specialty mandarins and seedless cultivars.

  18. Assessment of cultivation factors that affect biomass and geraniol production in transgenic tobacco cell suspension cultures.

    Directory of Open Access Journals (Sweden)

    Nikolay Vasilev

    Full Text Available A large-scale statistical experimental design was used to determine essential cultivation parameters that affect biomass accumulation and geraniol production in transgenic tobacco (Nicotiana tabacum cv. Samsun NN cell suspension cultures. The carbohydrate source played a major role in determining the geraniol yield and factors such as filling volume, inoculum size and light were less important. Sucrose, filling volume and inoculum size had a positive effect on geraniol yield by boosting growth of plant cell cultures whereas illumination of the cultures stimulated the geraniol biosynthesis. We also found that the carbohydrates sucrose and mannitol showed polarizing effects on biomass and geraniol accumulation. Factors such as shaking frequency, the presence of conditioned medium and solubilizers had minor influence on both plant cell growth and geraniol content. When cells were cultivated under the screened conditions for all the investigated factors, the cultures produced ∼ 5.2 mg/l geraniol after 12 days of cultivation in shaking flasks which is comparable to the yield obtained in microbial expression systems. Our data suggest that industrial experimental designs based on orthogonal arrays are suitable for the selection of initial cultivation parameters prior to the essential medium optimization steps. Such designs are particularly beneficial in the early optimization steps when many factors must be screened, increasing the statistical power of the experiments without increasing the demand on time and resources.

  19. Elicitor-mediated induction of chalcone isomerase in Phaseolus vulgaris cell suspension cultures.

    Science.gov (United States)

    Dixon, R A; Gerrish, C; Lamb, C J; Robbins, M P

    1983-12-01

    Approximately fourfold increases in the extractable activity of the enzyme chalcone isomerase (CHI, EC 5.5.1.6) were observed within 24 h of treatment of cell suspension cultures of Phaseolus vulgaris with a crude elicitor preparation heatreleased from the cell walls of the bean pathogen Colletotrichum lindemuthianum. The induction of CHI activity was highly dependent upon elicitor concentration, with maximum induction occurring in two discrete concentration ranges. A basal half-life for CHI>32 h in control cultures was determined by labelling with (2)H from (2)H2O followed by analysis of the equilibrium distribution of enzyme activity in CsCl density gradients. Comparative density labelling indicated that at both the lower and higher effective elicitor concentrations, the induced appearance of CHI activity was the result of an apparent initial activation of pre-existing enzyme followed by an increase in the rate of de-novo synthesis of the enzyme as compared with non-elicited controls. The increased appearance of the enzyme over the first 8 h in elicitor-treated cultures was inhibited by cycloheximide, cordycepin and actinomycin D. The results are discussed in relation to the mechanisms of co-ordinate enzyme induction operating in French-bean cell cultures exposed to fungal elicitors.

  20. Sterol and sesquiterpenoid biosynthesis during a growth cycle of tobacco cell suspension cultures.

    Science.gov (United States)

    Chappell, J; Von Lanken, C; Vögeli, U; Bhatt, P

    1989-05-01

    The accumulation and biosynthesis of sterols and fungal elicitor-inducible sesquiterpenoids by tobacco (Nicotiana tabacum) cell suspension cultures were examined as a function of a 10 day culture cycle. Sterols accumulated concomitantly with fresh weight gain. The rate of sterol biosynthesis, measured as the incorporation rate of [(14)C]acetate and [(3)H]mevalonate, was maximal when the cultures entered into their rapid phase of growth. Changes in squalene synthetase enzyme activity correlated more closely with thein vivo synthesis rate and accumulation of sterols than 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) enzyme activity. Cell cultures entering into the rapid phase of growth also responded maximally to fungal elicitor as measured by the production of capsidiol, an extracellular sesquiterpenoid. However, the rate of sesquiterpenoid biosynthesis, measured as the incorporation rate of [(14)C]acetate and [(3)H]mevalonate, could not be correlated with elicitor-inducible HMGR or sesquiterpene cyclase enzyme activities, nor elicitor-suppressible squalene synthetase enzyme activity.

  1. Erythrocytic malaria growth or invasion inhibition assays with emphasis on suspension culture GIA.

    Science.gov (United States)

    Haynes, J David; Moch, J Kathleen; Smoot, Douglas S

    2002-01-01

    Erythrocytic cycle malaria parasite growth or invasion inhibition assays (GIA) compare the effects of various test and control substances on malaria parasite growth in erythrocytes or invasion into erythrocytes in vitro. Although inhibitions by antimalarial drugs in vitro correlate well with drug protective levels required in vivo, as yet there are too few data to know how well inhibitions by antibodies in vitro correlate with the types and degrees of immune protection in vivo. Antibody-mediated GIA is frequently complicated by parasite strain-specific inhibitions, as well as nonspecific inhibitory factors generated in sera collected or stored under nonoptimal conditions. In this chapter, we describe methods for collecting and processing sera, for using different strains of parasite, and a simplified method for staining parasite DNA with Hoechst dye 33342 before quantitating parasites using ultraviolet (UV)-excited flow cytometry. We also describe a new type of GIA using suspension cultures in a 48-well plate. Critical to this method is enclosing the plate in a gassed, heat-sealed plastic bag, which, being low mass, can easily be rested at a 13.5 degrees angle on a rotor platform (114 rpm with 1-in. displacement) to produce gentle pulsatile waves of media in each well. The suspension GIA, which, relative to the static GIA, increased inhibition by one antibody and decreased inhibition by another (Table 1), may better simulate in vivo blood flow and may thus better predict in vivo efficacy.

  2. Isolation and purification of total RNA from Streptococcus mutans in suspension cultures and biofilms

    Directory of Open Access Journals (Sweden)

    Jaime Aparecido Cury

    2008-09-01

    Full Text Available The presence of extracellular polysaccharides matrix makes extraction and purification of RNA from Streptococcus mutans within biofilms challenging. In this study, several approaches to purify RNA extracted from S. mutans in suspension cultures and biofilms were examined. The combination of sonication (3 pulses of 30 s at 7 W, suspension in NAES buffer (50 mM sodium acetate buffer, 10 mM EDTA and 1% SDS; pH 5.0 and homogenization-mechanical cells disruption in NAES- acid phenol:chloroform, yielded 9.04 mg (or 0.52 mg of crude preparation of RNA per 100 mg of total cell (or biofilm dry-weight. The crude RNA preparations were subjected to various DNAse I treatments. The combination of DNAse I in silica-gel based column followed by recombinant DNase I in solution provided the best genomic DNA removal, resulting in 4.35 mg (or 0.06 mg of purified RNA per 100 mg of total cell (or biofilm dry-weight. The cDNAs generated from the purified RNA sample were efficiently amplified using gtfB S. mutans-specific primers. The results showed a method that yields high-quality RNA from both planktonic cells and biofilms of S. mutans in sufficient quantity and quality for real-time RT-PCR analyses.

  3. Suspension-cultured plant cells as a tool to analyze the extracellular proteome.

    Science.gov (United States)

    Sabater-Jara, Ana B; Almagro, Lorena; Belchí-Navarro, Sarai; Martínez-Esteso, María J; Youssef, Sabry M; Casado-Vela, Juan; Vera-Urbina, Juan C; Sellés-Marchart, Susana; Bru-Martínez, Roque; Pedreño, María A

    2014-01-01

    Suspension-cultured cells (SCC) are generally considered the most suitable cell systems to carry out scientific studies, including the extracellular proteome (secretome). SCC are initiated by transferring friable callus fragments into flasks containing liquid culture medium for cell biomass growth, and they are maintained in an orbital shaker to supply the sufficient oxygen that allows cell growth. SCC increase rapidly during the exponential phase and after 10-20 days (depending on the cell culture nature), the growth rate starts to decrease due to limitation of nutrients, and to maintain for decades these kinds of cell cultures is needed to transfer a portion of these SCC into a fresh culture medium. Despite the central role played by extracellular proteins in most processes that control growth and development, the secretome has been less well characterized than other subcellular compartments, meaning that our understanding of the cell wall physiology is still very limited. Useful proteomic tools have emerged in recent years to unravel metabolic network that occurs in cell walls. With the recent progress made in mass spectrometry technology, it has become feasible to identify proteins from a given organ, tissue, cells, or even a subcellular compartment. Compared with other methods used to isolate cell wall proteins, the spent medium of SCC provides a convenient, continuous, and reliable and unique source of extracellular proteins. Therefore, this biological system could be used as a large-scale cell culture from which these proteins can be secreted, easily separated from cells without cell disruption, and so, without any cytosolic contamination, easily recovered from the extracellular medium. This nondestructive cell wall proteome approach discloses a set of proteins that are specifically expressed in the remodelling of the cell wall architecture and stress defense.

  4. Cell Size Clues for the Allee Effect in Vegetative Amoeba Suspension Culture

    Science.gov (United States)

    Franck, Carl; Rappazzo, Brendan; Wang, Xiaoning; Segota, Igor

    That cells proliferate at higher rates with increasing density helps us appreciate and understand the development of multicellular behavior through the study of dilute cell systems. However, arduous cell counting with a microscope reveals that in the model eukaryote, Dictyostelium discoideum this transition is difficult to ascertain and thereby further explore despite our earlier progress (Phys. Rev. E 77, 041905, (2008)). Here we report preliminary evidence that the slow proliferation phase is well characterized by reduced cell size compared to the wide distribution of cell sizes in the familiar exponential proliferation phase of moderate densities. This observation is enabled by a new system for characterizing cells in stirred suspension cultures. Our technique relies on quickly acquiring magnitude distributions of detected flashes of laser light scattered in situ by cell targets.

  5. β-Secretase (BACE1)-inhibiting C-methylrotenoids from Abronia nana suspension cultures.

    Science.gov (United States)

    Park, Se-Hoon; Yang, Eun-Ju; Kim, Sang-In; Song, Kyung-Sik

    2014-07-01

    Suspension cultures of Abronia nana were established to produce C-methylisoflavones. A new C-methylrotenoid, named abronione A (2), was isolated along with three known rotenoids, boeravinone D (1), boeravinone A methyl ether (3), and mirabijalone D (4). The IC50 values of compounds 1, 2, and 4 on β-secretase (BACE1) were 4.77, 62.21, and 4.24 μM, respectively, whereas 3 was inactive. At concentrations up to 1.0 mM, the compounds did not inhibit other proteases such as trypsin, chymotrypsin, and elastase, indicating that they were specific inhibitors of β-secretase. Compounds 1 and 4 were non-competitive inhibitors based on the Dixon plot and with Ki values of 5.01 and 4.28 μM, respectively. At 50 μM, compound 4 inhibited Aβ1-42 production by 43.7% in APPSW-N2a cells.

  6. The isolation of plasma membrane from protoplasts of soybean suspension cultures.

    Science.gov (United States)

    Galbraith, D W; Northcote, D H

    1977-04-01

    A procedure for the isolation of plasma membranes from protoplasts of suspension-cultured soybean is described. Protoplasts were prepared by enzymic digestion of the cell wall and the plasma membrane was labelled with radioactive diazotized sulphanilic acid. The membrane systems from broken protoplasts were separated by continuous isopycnic sucrose gradient centrifugation. Radioactivity was localized in a band possessing a buoyant density of 1-14 g ml-1. The activities of NADPH- and NADH-cytochrome c reductase, fumarase, Mg2+-ATPase, IDPase and acid phosphodiesterase in the various regions of the density gradient were determined. A plasma membrane fraction was selected which was relatively uncontaminated with membranes derived from endoplasmic reticulum, tonoplasts and mitochondria. The results indicated that Mg2+-ATPase and possibly acid phosphodiesterase were associated with the plasma membrane.

  7. Effects of oligosaccharides from endophytic Fusarium oxysporum Dzf17 on activities of defense-related enzymes in Dioscorea zingiberensis suspension cell and seedling cultures

    Directory of Open Access Journals (Sweden)

    Peiqin Li

    2014-07-01

    Conclusions: Both EOS and WOS significantly increased the activities of PAL, PPO and POD in the suspension cell and seedling cultures of D. zingiberensis. The results suggested that the oligosaccharides from the endophytic fungus F. oxysporum Dzf17 may be related to the activation and enhancement of the defensive mechanisms of D. zingiberensis suspension cell and seedling cultures.

  8. Biosynthesis of sterols and triterpenes in cell suspension cultures of Uncaria tomentosa.

    Science.gov (United States)

    Flores-Sánchez, Isvett J; Ortega-López, Jaime; del Carmen Montes-Horcasitas, María; Ramos-Valdivia, Ana C

    2002-12-01

    Pectin administered to Uncaria tomentosa cell suspension cultures, was found to increase the production of triterpene acids (ursolic and oleanolic acid), however, neither growth nor sterol accumulation were affected. Cell cultures showed that pectin treatment caused a rapid threefold increase in the activities of enzymes involved in the biosynthesis of C(5) and C(30 )isoprenoid, such as isopentenyl diphosphate isomerase and squalene synthase. The activity of a farnesyl diphosphatase, which could divert the flux of farnesyl diphosphate to farnesol, was two times lower in elicited than in control cells. Elicited cells also transformed more rapidly a higher percentage of [5-(3)H]mevalonic acid into triterpene acids. Interestingly, addition of terbinafine, an inhibitor of squalene epoxidase, to elicited cell cultures inhibited sterol accumulation while triterpene production was not inhibited. These results suggest that in U. tomentosa cells, both the previously mentioned enzymes and those involved in squalene 2,3-oxide formation play an important regulatory role in the biosynthesis of sterols and triterpenes.

  9. [Effect of calcium on medium alkalinization induced by salicylic acid in Salvia miltiorrhiza suspension cultures].

    Science.gov (United States)

    Liu, Liancheng; Wang, Cong; Dong, Juan'e; Su, Hui; Zhuo, Zequn; Xue, Yaxin

    2013-07-01

    We studied medium alkalinization in Salvia miltiorrhiza suspension cultures treated with salicylic acid and the effect of Ca2+ in this process through application of calcium channel antagonists (Verapamil, LaCl3, LiCl, 2-APB) and ionophore A23187. The results show that salicylic acid could induce significant medium alkalinization in S. miltiorrhiza culture. Verapamil and LaCl3 or LiCl and 2-APB, two different groups of calcium channel antagonist, significantly inhibited the medium alkalinization induced by salicylic acid. However, the suppression effect of verapamil or LaCl3 on medium alkalinization induced by salicylic acid was higher than that of LiCl or 2-APB. When two types of calcium channel inhibitor (LaCl3 and 2-APB) were used together, the medium alkalinization induced by salicylic acid was completely suppressed and even reduced the pH in medium. On the other hand, A23187 could promote the medium alkalinization. Based on the results above, we speculated that salicylic acid could induce significant medium alkalinization in S. miltiorrhiza culture, depending on the calcium from both extracell and intracell. Moreover, calcium from extracell plays a more dominant role in this process. Reveal of relationship in this research between Ca2+ and medium alkalinization can provide theory evidence for mechanism of the plant secondary metabolism.

  10. Dynamics of indole-3-acetic acid oxidase activity in suspension culture of sunflower crown-gall

    Directory of Open Access Journals (Sweden)

    Zofia Chirek

    2014-02-01

    Full Text Available IAA oxidase activity was determined in several growth phases of a suspension culture of sunflower crown-gall. During the short phase of intensive growth (zero passage - PO a negative correlation was noted between enzymatic activity and the rate of growth. IAA oxidase activity increased to a certain level is not a factor limiting cell division. For protraction of the phase of intensive growth (first passage - P1, however, a decrease in the activity of this enzyme seems indispensable. IAA oxidase activity in the tested culture is under the control of inhibitors present in the cells and medium. High enzyme inhibition was observed in PO cells during the phase, of intensive growth and in P1 at the beginning and in the middle part of this phase. These results suggest' that the -auxin level determined in earlier studies in sunflower crown-gall culture is controlled by the IAA oxidase set. During the long phase of intensive growth (P1 this control is of negative feedback type.

  11. Controlling Expansion and Cardiomyogenic Differentiation of Human Pluripotent Stem Cells in Scalable Suspension Culture

    Directory of Open Access Journals (Sweden)

    Henning Kempf

    2014-12-01

    Full Text Available To harness the potential of human pluripotent stem cells (hPSCs, an abundant supply of their progenies is required. Here, hPSC expansion as matrix-independent aggregates in suspension culture was combined with cardiomyogenic differentiation using chemical Wnt pathway modulators. A multiwell screen was scaled up to stirred Erlenmeyer flasks and subsequently to tank bioreactors, applying controlled feeding strategies (batch and cyclic perfusion. Cardiomyogenesis was sensitive to the GSK3 inhibitor CHIR99021 concentration, whereas the aggregate size was no prevailing factor across culture platforms. However, in bioreactors, the pattern of aggregate formation in the expansion phase dominated subsequent differentiation. Global profiling revealed a culture-dependent expression of BMP agonists/antagonists, suggesting their decisive role in cell-fate determination. Furthermore, metallothionein was discovered as a potentially stress-related marker in hPSCs. In 100 ml bioreactors, the production of 40 million predominantly ventricular-like cardiomyocytes (up to 85% purity was enabled that were directly applicable to bioartificial cardiac tissue formation.

  12. Optimization of lycopene extraction from tomato cell suspension culture by response surface methodology.

    Science.gov (United States)

    Lu, Chi-Hua; Engelmann, Nancy J; Lila, Mary Ann; Erdman, John W

    2008-09-10

    Radioisotope-labeled lycopene is an important tool for biomedical research but currently is not commercially available. A tomato cell suspension culture system for the production of radioisotope-labeled lycopene was previously developed in our laboratory. In the current study, the goal was to optimize the lycopene extraction efficiency from tomato cell cultures for preparatory high-performance liquid chromatography (HPLC) separation. We employed response surface methodology (RSM), which combines fractional factorial design and a second-degree polynomial model. Tomato cells were homogenized with ethanol, saponified by KOH, and extracted with hexane, and the lycopene content was analyzed by HPLC-PDA. We varied five factors at five levels: ethanol volume (1.33-4 mL/g); homogenization period (0-40 s/g); saturated KOH solution volume (0-0.67 mL/g); hexane volume (1.67-3 mL/g); and vortex period (5-25 s/g). Ridge analysis by SAS suggested that the optimal extraction procedure to extract 1 g of tomato cells was at 1.56 mL of ethanol, 28 s homogenization, 0.29 mL of KOH, 2.49 mL of hexane, and 17.5 s vortex. These optimal conditions predicted by RSM were confirmed to enhance lycopene yield from standardized tomato cell cultures by more than 3-fold.

  13. Development of a low capital investment reactor system: application for plant cell suspension culture

    Science.gov (United States)

    Hsiao; Bacani; Carvalho; Curtis

    1999-01-01

    Growth of plant cell cultures is demonstrated in an uncontrolled, simple, and inexpensive plastic-lined vessel. Sustained specific growth rates of 0.22 day-1 for Hyoscyamus muticus cell suspension cultures are achieved in a low-cost gas-sparged bioreactor configuration (6.5 L working volume, wv) which is comparable to an "optimized" 5 L wv mechanically agitated fermentor. In an effort to reduce bioreactor costs, the need for an autoclavable vessel was eliminated. Sterilization is achieved by separate autoclaving of the plastic liner and by gas-phase sterilization using ethylene oxide. The initial run sterilized with ethylene oxide displayed a long lag, apparently due to residual sterilant gas. Because ethylene oxide could eliminate costs associated with autoclave rated vessels, a quantitative basis for aeration time was developed by experimental measurements and modeling of diffusion in the polymer liner. Operational techniques to eliminate toxicity are implemented to grow 0.2 kg dry weight of plant cells in 13 days in a 40 L (28.5 L wv) air-lift bioreactor without autoclave sterilization. The biomass yields for all reactors were statistically indistinguishable from shake flask culture.

  14. Uncertainties Mounting, Cotton Price Becomes Volatile

    Institute of Scientific and Technical Information of China (English)

    Huang Junfei

    2010-01-01

    @@ In the domestic market, the unre-mitting foul weather has delayed cotton picking by two weeks with downgraded quality; in the inter-national market, factors such as sus-pension of cotton export in India and disaster-affecting cotton yield in Paki-stan have led to such a market anticipa-tion that cotton stock across the world is to show another decline trend in the upcoming year. The unanimous market anticipation has resulted in a surge in cotton price during the Mid-autumn Festival: the transaction price for un-loading cotton inventories has increased by nearly RMB 3,000/ton, the price for purchasing new cotton has gone beyond RMB 25,000/ton and the cost for the imported cotton with owned quota (effect shipment after the next Spring Festival)has exceeded RMB 21,000/ton.

  15. Differentiation and selection of hepatocyte precursors in suspension spheroid culture of transgenic murine embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Elke Gabriel

    Full Text Available Embryonic stem cell-derived hepatocyte precursor cells represent a promising model for clinical transplantations to diseased livers, as well as for establishment of in vitro systems for drug metabolism and toxicology investigations. This study aimed to establish an in vitro culture system for scalable generation of hepatic progenitor cells. We used stable transgenic clones of murine embryonic stem cells possessing a reporter/selection vector, in which the enhanced green fluorescent protein- and puromycin N-acetyltransferase-coding genes are driven by a common alpha-fetoprotein gene promoter. This allowed for "live" monitoring and puromycin selection of the desired differentiating cell type possessing the activated alpha-fetoprotein gene. A rotary culture system was established, sequentially yielding initially partially selected hepatocyte lineage-committed cells, and finally, a highly purified cell population maintained as a dynamic suspension spheroid culture, which progressively developed the hepatic gene expression phenotype. The latter was confirmed by quantitative RT-PCR analysis, which showed a progressive up-regulation of hepatic genes during spheroid culture, indicating development of a mixed hepatocyte precursor-/fetal hepatocyte-like cell population. Adherent spheroids gave rise to advanced differentiated hepatocyte-like cells expressing hepatic proteins such as albumin, alpha-1-antitrypsin, cytokeratin 18, E-cadherin, and liver-specific organic anion transporter 1, as demonstrated by fluorescent immunostaining. A fraction of adherent cells was capable of glycogen storage and of reversible up-take of indocyanine green, demonstrating their hepatocyte-like functionality. Moreover, after transplantation of spheroids into the mouse liver, the spheroid-derived cells integrated into recipient. These results demonstrate that large-scale hepatocyte precursor-/hepatocyte-like cultures can be established for use in clinical trials, as well as in

  16. Feruloyl Oligosaccharides from Cell Walls of Suspension-Cultured Spinach Cells and Sugar Beet Pulp : STRUCTURE AND FUNCTION OF CELLS

    OpenAIRE

    Tadashi, Ishii; Forestry and Forest Products Research Institute

    1994-01-01

    Cell walls of suspension-cultured spinach cells and sugar beet pulp were separately hydrolyzed with Driselase. A feruloyl arabinobiose was isolated from both spinach cells and sugar beet. Four feruloyl oligosaccharides were obtained from sugar beet. The four oligosaccharides were characterized by NMR spectroscopy, methylation analysis and FAB-MS.

  17. Maintenance of undifferentiated mouse embryonic stem cells in suspension by the serum- and feeder-free defined culture condition

    OpenAIRE

    Tsuji, Yukiiko; Yoshimura, Naoko; Aoki, Hitomi; Sharov, Alexei A; Minoru S.H. Ko; Motohashi, Tsutomu; KUNISADA, Takahiro

    2008-01-01

    The proven pluripotency of ES cells is expected to allow their therapeutic use for regenerative medicine. We present here a novel suspension culture method that facilitates the proliferation of pluripotent ES cells without feeder cells. The culture medium contains polyvinyl alcohol (PVA), free of either animal-derived or synthetic serum, and contains very low amounts of peptidic or proteinaceous materials, which are favorable for therapeutic use. ES cells showed sustained proliferation in the...

  18. Monoterpenoid oxindole alkaloid production by Uncaria tomentosa (Willd) D.C. cell suspension cultures in a stirred tank bioreactor.

    Science.gov (United States)

    Trejo-Tapia, Gabriela; Cerda-García-Rojas, Carlos M; Rodríguez-Monroy, Mario; Ramos-Valdivia, Ana C

    2005-01-01

    Cell growth, monoterpenoid oxindole alkaloid (MOA) production, and morphological properties of Uncaria tomentosa cell suspension cultures in a 2-L stirred tank bioreactor were investigated. U. tomentosa (cell line green Uth-3) was able to grow in a stirred tank at an impeller tip speed of 95 cm/s (agitation speed of 400 rpm), showing a maximum biomass yield of 11.9 +/- 0.6 g DW/L and a specific growth rate of 0.102 d(-1). U. tomentosa cells growing in a stirred tank achieved maximum volumetric and specific MOA concentration (467.7 +/- 40.0 microg/L, 44.6 +/- 5.2 microg/g DW) at 16 days of culture. MOA chemical profile of cell suspension cultures growing in a stirred tank resembled that of the plant. Depending on culture time, from the total MOA produced, 37-100% was found in the medium in the bioreactor culture. MOA concentration achieved in a stirred tank was up to 10-fold higher than that obtained in Erlenmeyer flasks (agitated at 110 rpm). In a stirred tank, average area of the single cells of U. tomentosa increased up to 4-fold, and elliptical form factor increased from 1.40 to 2.55, indicating enlargement of U. tomentosa single cells. This work presents the first report of U. tomentosa green cell suspension cultures that grow and produce MOA in a stirred tank bioreactor.

  19. Effects of mercury (II) species on cell suspension cultures of catharanthus roseus

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, L. (Hangzhou Univ. (China)); Cullen, W.R. (Univ. of British Columbia, Vancouver, British Columbia (Canada))

    1994-11-01

    Mercury has received considerable attention because of its high toxicity. Widespread contamination with mercury poses severe environmental problems despite our extensive knowledge of its toxicity in living systems. It is generally accepted that the toxicity of mercury is related to its oxidation states and species, the organic forms being more toxic than the inorganic forms. In the aquatic environment, the toxicity of mercury depends on the aqueous speciation of the mercuric ion (Hg[sup 2+]). Because of the complex coordination chemistry of mercury in aqueous systems, the nature of the Hg[sup 2+] species present in aquatic environments is influenced greatly by water chemistry (e. g, pH, inorganic ion composition, and dissolved organics). Consequently, the influence of environmental factors on the aqueous speciation of mercury has been the focus of much attention. However, there is very little information available regarding the effects of the species and speciation on Hg (II) toxicity in plant-tissue cultures. Catharanthus roseus (C. roseus), commonly called the Madagascar Periwinkle, is a member of the alkaloid rich family Apocynaceae. The present investigation was concerned with the toxicity of mercury on the growth of C. roseus cell suspension cultures as influenced by mercury (II) species and speciation. The specific objectives of the study were to (a) study the effects of mercury species on the growth of C. roseus cultures from the point of view of environmental biology and toxicology; (b) evaluate the effects of selenate, selenite and selected ligands such as chloride, 1-cysteine in the media on the acute toxicity of mercuric oxide; (c) determine the impact of the initial pH of the culture media on the toxicities of mercuric compounds; (d) discuss the dependence of the toxicity on the chemical species and speciation of Hg (II). 11 refs., 7 figs., 2 tabs.

  20. Establishment of suspension cell culture of Gymnema sylvestre R.Br.- A threatened anti-diabetic plant

    Directory of Open Access Journals (Sweden)

    Karthic Raju

    2012-04-01

    Full Text Available A cell suspension culture was established from leaf explants of wild Gymnema sylvestre plants collected from Muniyankudisai,Tamilnadu, India. Murashige and Skoog medium supplemented with 9.0 μM l-1 of 2, 4- Dichlorophenoxy acetic acid and 2.1 μM l-1Benzyl adenine produced yellow friable callus with green patches.The cells were subcultured conscientiously twelve times to getconsistent growth of the cells in suspension. From the 10thsubculture onwards callus cells acclimatized to grow in suspensionwith aggregation and reached 168.6 g l-1 fw and 5.16 g l-1 dw of cellbiomass.

  1. High yield derivation of enriched glutamatergic neurons from suspension-cultured mouse ESCs for neurotoxicology research

    Directory of Open Access Journals (Sweden)

    Hubbard Kyle S

    2012-10-01

    Full Text Available Abstract Background Recently, there has been a strong emphasis on identifying an in vitro model for neurotoxicity research that combines the biological relevance of primary neurons with the scalability, reproducibility and genetic tractability of continuous cell lines. Derived neurons should be homotypic, exhibit neuron-specific gene expression and morphology, form functioning synapses and consistently respond to neurotoxins in a fashion indistinguishable from primary neurons. However, efficient methods to produce neuronal populations that are suitable alternatives to primary neurons have not been available. Methods With the objective of developing a more facile, robust and efficient method to generate enriched glutamatergic neuronal cultures, we evaluated the neurogenic capacity of three mouse embryonic stem cell (ESC lines (R1, C57BL/6 and D3 adapted to feeder-independent suspension culture. Neurogenesis and neuronal maturation were characterized as a function of time in culture using immunological, genomic, morphological and functional metrics. The functional responses of ESNs to neurotropic toxins with distinctly different targets and mechanisms of toxicity, such as glutamate, α-latrotoxin (LTX, and botulinum neurotoxin (BoNT, were also evaluated. Results Suspension-adapted ESCs expressed markers of pluripotency through at least 30 passages, and differentiation produced 97×106 neural progenitor cells (NPCs per 10-cm dish. Greater than 99% of embryonic stem cell-derived neurons (ESNs expressed neuron-specific markers by 96 h after plating and rapidly developed complex axodendritic arbors and appropriate compartmentalization of neurotypic proteins. Expression profiling demonstrated the presence of transcripts necessary for neuronal function and confirmed that ESN populations were predominantly glutamatergic. Furthermore, ESNs were functionally receptive to all toxins with sensitivities and responses consistent with primary neurons

  2. Anaerobic bacteria grow within Candida albicans biofilms and induce biofilm formation in suspension cultures.

    Science.gov (United States)

    Fox, Emily P; Cowley, Elise S; Nobile, Clarissa J; Hartooni, Nairi; Newman, Dianne K; Johnson, Alexander D

    2014-10-20

    The human microbiome contains diverse microorganisms, which share and compete for the same environmental niches. A major microbial growth form in the human body is the biofilm state, where tightly packed bacterial, archaeal, and fungal cells must cooperate and/or compete for resources in order to survive. We examined mixed biofilms composed of the major fungal species of the gut microbiome, Candida albicans, and each of five prevalent bacterial gastrointestinal inhabitants: Bacteroides fragilis, Clostridium perfringens, Escherichia coli, Klebsiella pneumoniae, and Enterococcus faecalis. We observed that biofilms formed by C. albicans provide a hypoxic microenvironment that supports the growth of two anaerobic bacteria, even when cultured in ambient oxic conditions that are normally toxic to the bacteria. We also found that coculture with bacteria in biofilms induces massive gene expression changes in C. albicans, including upregulation of WOR1, which encodes a transcription regulator that controls a phenotypic switch in C. albicans, from the "white" cell type to the "opaque" cell type. Finally, we observed that in suspension cultures, C. perfringens induces aggregation of C. albicans into "mini-biofilms," which allow C. perfringens cells to survive in a normally toxic environment. This work indicates that bacteria and C. albicans interactions modulate the local chemistry of their environment in multiple ways to create niches favorable to their growth and survival. Copyright © 2014 Elsevier Ltd. All rights reserved.

  3. Manipulation of culture strategies to enhance capsaicin biosynthesis in suspension and immobilized cell cultures of Capsicum chinense Jacq. cv. Naga King Chili.

    Science.gov (United States)

    Kehie, Mechuselie; Kumaria, Suman; Tandon, Pramod

    2014-06-01

    Manipulation of culture strategies was adopted to study the influence of nutrient stress, pH stress and precursor feeding on the biosynthesis of capsaicin in suspension and immobilized cell cultures of C. chinense. Cells cultured in the absence of one of the four nutrients (ammonium and potassium nitrate for nitrate and potassium stress, potassium dihydrogen orthophosphate for phosphorus stress, and sucrose for sugar stress) influenced the accumulation of capsaicin. Among the stress factors studied, nitrate stress showed maximal capsaicin production on day 20 (505.9 ± 2.8 μg g(-1) f.wt) in immobilized cell, whereas in suspension cultures the maximum accumulation (345.5 ± 2.9 μg g(-1) f.wt) was obtained on day 10. Different pH affected capsaicin accumulation; enhanced accumulation of capsaicin (261.6 ± 3.4 μg g(-1) f.wt) was observed in suspension cultures at pH 6 on day 15, whereas in case of immobilized cultures the highest capsaicin content (433.3 ± 3.3 μg g(-1) f.wt) was obtained at pH 5 on day 10. Addition of capsaicin precursors and intermediates significantly enhanced the biosynthesis of capsaicin, incorporation of vanillin at 100 μM in both suspension and immobilized cell cultures resulted in maximum capsaicin content with 499.1 ± 5.5 μg g(-1) f.wt on day 20 and 1,315.3 ± 10 μg g(-1) f.wt on day 10, respectively. Among the different culture strategies adopted to enhance capsaicin biosynthesis in cell cultures of C. chinense, cells fed with vanillin resulted in the maximum capsaicin accumulation. The rate of capsaicin production was significantly higher in immobilized cells as compared to freely suspended cells.

  4. Design of serum-free medium for suspension culture of CHO cells on the basis of general commercial media.

    Science.gov (United States)

    Miki, Hideo; Takagi, Mutsumi

    2015-08-01

    The design of serum-free media for suspension culture of genetically engineered Chinese hamster ovary (CHO) cells using general commercial media as a basis was investigated. Subcultivation using a commercial serum-free medium containing insulin-like growth factor (IGF)-1 with or without FCS necessitated additives other than IGF-1 to compensate for the lack of FCS and improve cell growth. Suspension culture with media containing several combinations of growth factors suggested the effectiveness of addition of both IGF-1 and the lipid signaling molecule lysophosphatidic acid (LPA) for promoting cell growth. Subcultivation of CHO cells in suspension culture using the commercial serum-free medium EX-CELL™302, which contained an IGF-1 analog, supplemented with LPA resulted in gradually increasing specific growth rate comparable to the serum-containing medium and in almost the same high antibody production regardless of the number of generations. The culture with EX-CELL™302 supplemented with LPA in a jar fermentor with pH control at 6.9 showed an apparently higher cell growth rate than the cultures without pH control and with pH control at 6.8. The cell growth in the medium supplemented with aurintricarboxylic acid (ATA), which was much cheaper than IGF-1, in combination with LPA was synergistically promoted similarly to that in the medium supplemented with IGF-1 and LPA. In conclusion, the serum-free medium designed on the basis of general commercial media could support the growth of CHO cells and antibody production comparable to serum-containing medium in suspension culture. Moreover, the possibility of cost reduction by the substitution of IGF-1 with ATA was also shown.

  5. Influence of auxins and sucrose in monoterpenoid oxindole alkaloid production by Uncaria tomentosa cell suspension cultures.

    Science.gov (United States)

    Luna-Palencia, Gabriela R; Cerda-García-Rojas, Carlos M; Rodríguez-Monroy, Mario; Ramos-Valdivia, Ana C

    2005-01-01

    Growth and alkaloid production in Uncaria tomentosa cell suspension cultures were studied in Murashige and Skoog medium supplemented with 10 microM 2,4-dichlorophenoxyacetic acid, 10 microM kinetin, and 58 mM sucrose for maintenance and with 10 microM indole-3-acetic acid, 10 microM kinetin, and 58 mM sucrose for production. A U. tomentosa pale Uth-3 cell line, cultured in the production medium, showed a reduced lag phase and a specific growth rate (mu) of 0.27 day(-1), while cells growing in the maintenance medium showed mu = 0.20 day(-1). U. tomentosa cells growing in the production medium produced monoterpenoid oxindole alkaloids (MOA) in amounts of 10.2 +/- 1.6 microg g(-1) dry weight (DW). The chemical profile of MOA produced by in vitro cell cultures was similar to that found in the plant. After 10 subcultures, maximum MOA production decreased to 2.0 +/- 0.7 microg g(-1) DW, while tryptamine alkaloids (TA) were produced with a maximum of 6.2 +/- 0.4 microg g(-1) DW. The increase of initial sucrose concentration up to 145 mM in the production medium enhanced the cell biomass by 3.2-fold (from 10.2 +/- 0.1 to 32.8 +/- 1.1 g DW L(-1)), reduced mu from 0.27 to 0.23 day(-1), and provoked a substantial accumulation of TA (23.1 +/- 4.7 microg g(-1) DW). A high sucrose concentration stimulated MOA production in the maintenance medium (2.7 +/- 0.5 microg g(-1) DW), even in the presence of 2,4-dichlorophenoxyacetic acid.

  6. Oxygen transport and stem cell aggregation in stirred-suspension bioreactor cultures.

    Directory of Open Access Journals (Sweden)

    Jincheng Wu

    Full Text Available Stirred-suspension bioreactors are a promising modality for large-scale culture of 3D aggregates of pluripotent stem cells and their progeny. Yet, cells within these clusters experience limitations in the transfer of factors and particularly O2 which is characterized by low solubility in aqueous media. Cultured stem cells under different O2 levels may exhibit significantly different proliferation, viability and differentiation potential. Here, a transient diffusion-reaction model was built encompassing the size distribution and ultrastructural characteristics of embryonic stem cell (ESC aggregates. The model was coupled to experimental data from bioreactor and static cultures for extracting the effective diffusivity and kinetics of consumption of O2 within mouse (mESC and human ESC (hESC clusters. Under agitation, mESC aggregates exhibited a higher maximum consumption rate than hESC aggregates. Moreover, the reaction-diffusion model was integrated with a population balance equation (PBE for the temporal distribution of ESC clusters changing due to aggregation and cell proliferation. Hypoxia was found to be negligible for ESCs with a smaller radius than 100 µm but became appreciable for aggregates larger than 300 µm. The integrated model not only captured the O2 profile both in the bioreactor bulk and inside ESC aggregates but also led to the calculation of the duration that fractions of cells experience a certain range of O2 concentrations. The approach described in this study can be employed for gaining a deeper understanding of the effects of O2 on the physiology of stem cells organized in 3D structures. Such frameworks can be extended to encompass the spatial and temporal availability of nutrients and differentiation factors and facilitate the design and control of relevant bioprocesses for the production of stem cell therapeutics.

  7. Simple suspension culture system of human iPS cells maintaining their pluripotency for cardiac cell sheet engineering.

    Science.gov (United States)

    Haraguchi, Yuji; Matsuura, Katsuhisa; Shimizu, Tatsuya; Yamato, Masayuki; Okano, Teruo

    2015-12-01

    In this study, a simple three-dimensional (3D) suspension culture method for the expansion and cardiac differentiation of human induced pluripotent stem cells (hiPSCs) is reported. The culture methods were easily adapted from two-dimensional (2D) to 3D culture without any additional manipulations. When hiPSCs were directly applied to 3D culture from 2D in a single-cell suspension, only a few aggregated cells were observed. However, after 3 days, culture of the small hiPSC aggregates in a spinner flask at the optimal agitation rate created aggregates which were capable of cell passages from the single-cell suspension. Cell numbers increased to approximately 10-fold after 12 days of culture. The undifferentiated state of expanded hiPSCs was confirmed by flow cytometry, immunocytochemistry and quantitative RT-PCR, and the hiPSCs differentiated into three germ layers. When the hiPSCs were subsequently cultured in a flask using cardiac differentiation medium, expression of cardiac cell-specific genes and beating cardiomyocytes were observed. Furthermore, the culture of hiPSCs on Matrigel-coated dishes with serum-free medium containing activin A, BMP4 and FGF-2 enabled it to generate robust spontaneous beating cardiomyocytes and these cells expressed several cardiac cell-related genes, including HCN4, MLC-2a and MLC-2v. This suggests that the expanded hiPSCs might maintain the potential to differentiate into several types of cardiomyocytes, including pacemakers. Moreover, when cardiac cell sheets were fabricated using differentiated cardiomyocytes, they beat spontaneously and synchronously, indicating electrically communicative tissue. This simple culture system might enable the generation of sufficient amounts of beating cardiomyocytes for use in cardiac regenerative medicine and tissue engineering.

  8. LC/MS profiling of flavonoid glycoconjugates isolated from hairy roots, suspension root cell cultures and seedling roots of Medicago truncatula

    OpenAIRE

    Staszków, Anna; Swarcewicz, Barbara; Banasiak, Joanna; Muth, Dorota; Jasiński, Michał; Stobiecki, Maciej

    2011-01-01

    Hairy roots and suspension cell cultures are commonly used in deciphering different problems related to the biochemistry and physiology of plant secondary metabolites. Here, we address about the issue of possible differences in the profiles of flavonoid compounds and their glycoconjugates derived from various plant materials grown in a standard culture media. We compared profiles of flavonoids isolated from seedling roots, hairy roots, and suspension root cell cultures of a model legume plant...

  9. Biomass Yield and Steviol Glycoside Production in Callus and Suspension Culture of Stevia rebaudiana Treated with Proline and Polyethylene Glycol.

    Science.gov (United States)

    Gupta, Pratibha; Sharma, Satyawati; Saxena, Sanjay

    2015-06-01

    Enhanced production of steviol glycosides (SGs) was observed in callus and suspension culture of Stevia rebaudiana treated with proline and polyethylene glycol (PEG). To study their effect, yellow-green and compact calli obtained from in vitro raised Stevia leaves were sub-cultured on MS medium supplemented with 2.0 mg l(-1) NAA and different concentrations of proline (2.5-10 mM) and PEG (2.5-10 %) for 2 weeks, and incubated at 24 ± 1 °C and 22.4 μmol m(-2) s(-1) light intensity provided by white fluorescent tubes for 16 h. Callus and suspension culture biomass (i.e. both fresh and dry weight content) was increased with 5 mM proline and 5 % PEG, while at further higher concentrations, they got reduced. Further, quantification of SGs content in callus (collected at 15th day) and suspension culture (collected at 10th and 15th day) treated with and without elicitors was analysed by HPLC. It was observed that chemical stress enhanced the production of SGs significantly. In callus, the content of SGs increased from 0.27 (control) to 1.09 and 1.83 % with 7.5 mM proline and 5 % PEG, respectively, which was about 4.0 and 7.0 times higher than control. However, in the case of suspension culture, the same concentrations of proline and polyethylene glycol enhanced the SG content from 1.36 (control) to 5.03 and 6.38 %, respectively, on 10th day which were 3.7 times and 4.7 times higher than control.

  10. UV-B-induced signaling events leading to enhanced-production of catharanthine in Catharanthus roseus cell suspension cultures

    Directory of Open Access Journals (Sweden)

    Chelliah Jayabaskaran

    2007-11-01

    Full Text Available Abstract Background Elicitations are considered to be an important strategy towards improved in vitro production of secondary metabolites. In cell cultures, biotic and abiotic elicitors have effectively stimulated the production of plant secondary metabolites. However, molecular basis of elicitor-signaling cascades leading to increased production of secondary metabolites of plant cell is largely unknown. Exposure of Catharanthus roseus cell suspension culture to low dose of UV-B irradiation was found to increase the amount of catharanthine and transcription of genes encoding tryptophan decarboxylase (Tdc and strictosidine synthase (Str. In the present study, the signaling pathway mediating UV-B-induced catharanthine accumulation in C. roseus suspension cultures were investigated. Results Here, we investigate whether cell surface receptors, medium alkalinization, Ca2+ influx, H2O2, CDPK and MAPK play required roles in UV-B signaling leading to enhanced production of catharanthine in C. roseus cell suspension cultures. C. roseus cells were pretreated with various agonists and inhibitors of known signaling components and their effects on the accumulation of Tdc and Str transcripts as well as amount of catharanthine production were investigated by various molecular biology techniques. It has been found that the catharanthine accumulation and transcription of Tdc and Str were inhibited by 3–4 fold upon pretreatment of various inhibitors like suramin, N-acetyl cysteine, inhibitors of calcium fluxes, staurosporine etc. Conclusion Our results demonstrate that cell surface receptor(s, Ca2+ influx, medium alkalinization, CDPK, H2O2 and MAPK play significant roles in UV-B signaling leading to stimulation of Tdc and Str genes and the accumulation of catharanthine in C. roseus cell suspension cultures. Based on these findings, a model for signal transduction cascade has been proposed.

  11. Birds, seals and the suspension culture of mussels in Bantry Bay, a non-seaduck area in Southwest Ireland

    Science.gov (United States)

    Roycroft, D.; Kelly, T. C.; Lewis, L. J.

    2004-12-01

    Concerns about the environmental impacts of mariculture have grown in recent years in response to the rapid expansion of the industry. The blue mussel ( Mytilus edulis) is the main product of shellfish mariculture in the Northeast Atlantic and Baltic Sea, with approximately one third of the harvest cultured using suspended longlines within sheltered marine areas. The main aim of this study was to examine the interactions, and assess the impacts (if any) of mussel suspension culture on the seabird and seal community, employing a simultaneous study of culture and control sites. The study spanned a 20-month period (from November 2001 to August 2003) and encompassed six sites in Bantry Bay (Southwest Ireland). There was no significant difference in species richness between mussel and control sites. Similarly, species diversity did not significantly differ between the mussel and control sites although control sites were generally more diverse than mussel sites, the latter particularly dominated by large numbers of Laridae. Significantly higher numbers of Phalacrocoracidae, Laridae and Alcidae were recorded in mussel sites than in control sites. However, no significant difference was found between Gaviidae or common seal ( Phoca vitulina) numbers in mussel and control sites. Seasonal patterns of abundance were similar in mussel and control sites, with peak numbers of most species groups occurring in spring. Mussel suspension culture does not appear to have an adverse effect on the abundance of seabirds or common seals in this area. The safe perching platforms provided by suspension culture floats, combined with a number of other factors, contribute to an increased abundance of a number of seabird species, particularly Laridae. The possible interactions between vertebrate predators and mussel suspension aquaculture are discussed and possible explanations for the increased seabird abundance observed in these areas are offered.

  12. Ectopic lignification in primary cellulose-deficient cell walls of maize cell suspension cultures

    Institute of Scientific and Technical Information of China (English)

    Hugo Melida; Antonio Encina; Asier Largo-Gosens; Esther Novo-Uzal; Rogelio Santiago; Federico Pomar; Pedro Garca; Penelope Garca-Angulo; Jose Luis Acebes; Jesus Alvarez

    2015-01-01

    Maize (Zea mays L.) suspension-cultured cells with up to 70% less cellulose were obtained by stepwise habituation to dichlobenil (DCB), a cellulose biosynthesis inhibitor. Cellulose deficiency was accompanied by marked changes in cell wall matrix polysaccharides and phenolics as revealed by Fourier transform infrared (FTIR) spectroscopy. Cell wall compositional analysis indicated that the cellulose-deficient cell walls showed an enhancement of highly branched and cross-linked arabinoxylans, as well as an increased content in ferulic acid, diferulates and p-coumaric acid, and the presence of a polymer that stained positive for phloroglucinol. In accordance with this, cellulose-deficient cell walls showed a fivefold increase in Klason-type lignin. Thioacidolysis/GC-MS analysis of cellulose-deficient cell walls indicated the presence of a lignin-like polymer with a Syringyl/Guaiacyl ratio of 1.45, which differed from the sensu stricto stress-related lignin that arose in response to short-term DCB-treatments. Gene expression analysis of these cells indicated an overexpression of genes specific for the biosynthesis of monolignol units of lignin. A study of stress signaling pathways revealed an overexpression of some of the jasmonate signaling pathway genes, which might trigger ectopic lignification in response to cell wall integrity disruptions. In summary, the structural plasticity of primary cell walls is proven, since a lignification process is possible in response to cellulose impoverishment.

  13. Treatment strategies for high resveratrol induction in Vitis vinifera L. cell suspension culture

    Directory of Open Access Journals (Sweden)

    Thu V. Vuong

    2014-06-01

    Full Text Available Bioprocesses capable of producing large scales of resveratrol at nutraceutical grade are in demand. This study herein investigated treatment strategies to induce the production of resveratrol in Vitis vinifera L. cell suspension cultures. Among seven investigated elicitors, jasmonic acid (JA, salicylic acid, β-glucan (GLU, and chitosan enhanced the production of intracellular resveratrol manyfold. The combined treatment of JA and GLU increased extracellular resveratrol production by up to tenfold. The application of Amberlite XAD-7 resin for in situ removal and artificial storage of secreted resveratrol further increased resveratrol production by up to four orders of magnitude. The level of resveratrol produced in response to the combined treatment with 200 g/L XAD-7, 10 μM JA and 1 mg/mL GLU was approximately 2400 mg/L, allowing the production of resveratrol at an industrial scale. The high yield of resveratrol is due to the involvement of a number of mechanisms working in concert.

  14. A lipochito-oligosaccharide, Nod factor, induces transient calcium influx in soybean suspension-cultured cells.

    Science.gov (United States)

    Yokoyama, T; Kobayashi, N; Kouchi, H; Minamisawa, K; Kaku, H; Tsuchiya, K

    2000-04-01

    Lipochito-oligosaccharides (Nod factors) produced by Rhizobium or Bradyrhizobium are the key signal molecules for eliciting nodulation in their corresponding host legumes. To elucidate the signal transduction events mediated by Nod factors, we investigated the effects of Nod factors on the cytosolic [Ca2+] of protoplasts prepared from roots and suspension-cultured cells of soybean (Glycine max and G. soja) using a fluorescent Ca2+ indicator, Fura-PE3. NodBj-V (C18:1, MeFuc), which is a major component of Nod factors produced by Bradyrhizobium japonicum, induces transient elevation of cytosolic [Ca2+] in the cells of soybean within a few minutes. This effect is specific to soybean cells and was not observed in the tobacco BY-2 cells. Furthermore, NodBj-V without MeFuc did not induce any cytosolic [Ca2+] elevation in soybean cells. Exclusion of Ca2+ from the medium, as well as pre-treatment of the cells with an external Ca2+ chelator or with a plasma membrane voltage-dependent Ca2+ channel inhibitor, suppressed the Nod factor-dependent cytosolic [Ca2+] elevation. These results indicate that transient Ca2+ influx from extracellular fluid is one of the earliest responses of soybean cells to NodBj-V (C18:1, MeFuc) in a host-specific manner.

  15. The reduction of starch accumulation in transgenic sugarcane cell suspension culture lines.

    Science.gov (United States)

    Ferreira, Stephanus J; Kossmann, Jens; Lloyd, James R; Groenewald, Jan-Hendrik

    2008-11-01

    Starch only occurs in small amounts in sugarcane, but is, nevertheless an unwanted product because it reduces the amount of sucrose that can be crystallized from molasses. In an attempt to reduce the starch content of sugarcane, the activities of ADP-glucose pyrophosphorylase (AGPase) and beta-amylase were manipulated using transgenic approaches. Transformation vectors to reduce AGPase activity and to increase plastidial beta-amylase activity were constructed and used for the transformation of sugarcane calli. The results of the manipulations were analyzed in suspension cultures. AGPase activity was reduced down to between 14 and 54% of the wild-type control. This led to a reduction in starch concentration down to 38% of the levels of the wild-type control. beta-Amylase activity was increased in the transgenic lines by 1.5-2 times that of the wild-type control. This increase in activity led to a reduction in starch amounts by 90% compared to wild-type control cells. In both experiments, the changes in starch concentrations could be correlated with the change in enzyme activity. There were no significant effects on sucrose concentrations in either experiment, indicating that these approaches might be useful to engineer regenerated sugarcane for optimized sucrose production.

  16. Programmed cell death features in apple suspension cells under low oxygen culture

    Institute of Scientific and Technical Information of China (English)

    徐昌杰; 陈昆松; FERGUSONIanB

    2004-01-01

    Suspension-cultured apple fruit cells (Malus pumila Mill. cv. Braeburn) were exposed to a low oxygen atmosphere to test whether programmed cell death (PCD) has a role in cell dysfunction and death under hypoxic conditions. Protoplasts were prepared at various times after low oxygen conditions were established, and viability tested by triple staining with fluorescein diacetate (FDA), propidium iodide (PI) and Hoechst33342 (HO342). DNA breakdown and phosphatidylserine exposure on the plasma membrane were observed using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), and annexin V binding. About 30% of protoplasts from cells after 48 h under low oxygen showed an increased accumulation of HO342, indicating increased membrane permeability. Positive TUNEL and annexin V results were also only obtained with protoplasts from cells under low oxygen. The results suggest that apple celi death under low oxygen is at least partially PCD mediated, and may explain tissue breakdown under controlled atmosphere (low oxygen) conditions in apple fruit.

  17. Programmed cell death features in apple suspension cells under low oxygen culture.

    Science.gov (United States)

    Xu, Chang-jie; Chen, Kun-song; Ferguson, Ian B

    2004-02-01

    Suspension-cultured apple fruit cells (Malus pumila Mill. cv. Braeburn) were exposed to a low oxygen atmosphere to test whether programmed cell death (PCD) has a role in cell dysfunction and death under hypoxic conditions. Protoplasts were prepared at various times after low oxygen conditions were established, and viability tested by triple staining with fluorescein diacetate (FDA), propidium iodide (PI) and Hoechst33342 (HO342). DNA breakdown and phosphatidylserine exposure on the plasma membrane were observed using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), and annexin V binding. About 30% of protoplasts from cells after 48 h under low oxygen showed an increased accumulation of HO342, indicating increased membrane permeability. Positive TUNEL and annexin V results were also only obtained with protoplasts from cells under low oxygen. The results suggest that apple cell death under low oxygen is at least partially PCD mediated, and may explain tissue breakdown under controlled atmosphere (low oxygen) conditions in apple fruit.

  18. Programmed cell death features in apple suspension cells under low oxygen culture

    Institute of Scientific and Technical Information of China (English)

    XU Chang-jie(徐昌杰); CHEN Kun-song(陈昆松); FERGUSON Ian B.

    2004-01-01

    Suspension-cultured apple fruit cells (Malus pumila Mill. cv. Braeburn) were exposed to a low oxygen atmosphere to test whether programmed cell death (PCD) has a role in cell dysfunction and death under hypoxic conditions. Protoplasts were prepared at various times after low oxygen conditions were established, and viability tested by triple staining with fluorescein diacetate (FDA), propidium iodide (PI) and Hoechst33342 (HO342). DNA breakdown and phosphatidylserine exposure on the plasma membrane were observed using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), and annexin V binding. About 30% of protoplasts from cells after 48 h under low oxygen showed an increased accumulation of HO342, indicating increased membrane permeability. Positive TUNEL and annexin V results were also only obtained with protoplasts from cells under low oxygen. The results suggest that apple cell death under low oxygen is at least partially PCD mediated, and may explain tissue breakdown under controlled atmosphere (low oxygen) conditions in apple fruit.

  19. Solubilization, partial purification, and immunodetection of squalene synthetase from tobacco cell suspension cultures.

    Science.gov (United States)

    Hanley, K; Chappell, J

    1992-01-01

    Squalene synthetase, an integral membrane protein and the first committed enzyme for sterol biosynthesis, was solubilized and partially purified from tobacco (Nicotiana tabacum) cell suspension cultures. Tobacco microsomes were prepared and the enzyme was solubilized from the lipid bilayer using a two-step procedure. Microsomes were initially treated with concentrations of octyl-beta-d-thioglucopyranoside and glycodeoxycholate below their critical micelle concentration, 4.5 and 1.1 millimolar, respectively, to remove loosely associated proteins. Complete solubilization of the squalene synthetase enzyme activity was achieved after a second treatment at detergent concentrations above or at their critical micelle concentration, 18 and 2.2 millimolar, respectively. The detergent-solubilized enzyme was further purified by a combination of ultrafiltration, gel permeation, and Fast Protein Liquid Chromatography anion exchange. A 60-fold purification and 20% recovery of the enzyme activity was achieved. The partially purified squalene synthetase protein was used to generate polyclonal antibodies from mice that efficiently inhibited synthetase activity in an in vitro assay. The apparent molecular mass of the squalene synthetase protein as determined by immunoblot analysis of the partially purified squalene synthetase protein separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 47 kilodaltons. The partially purified squalene synthetase activity was optimal at pH 6.0, exhibited a K(m) for farnesyl diphosphate of 9.5 micromolar, and preferred NADPH as a reductant rather than NADH.

  20. Loss of competence for glyoxysome formation during somatic embryogenesis in anise (Pimpinella anisum L.) suspension cultures.

    Science.gov (United States)

    Kudielka, R A; Theimer, R R

    1983-10-01

    Somatic embryogenesis in anise (Pimpinella anisum L.) suspension cultures induced by transfer to hormone-free growth medium may be synchronized by previous selection of cell aggregates with diameters between 100-240 μm. Around 80-90% of the embryoids are globular after 2-3 d, heart-shaped after 5-7 d and torpedo-shaped after 9 d. In embryogenic medium without source of carbon or with 20 mmol/l acetate differentiation and growth cease. But like in dedifferentiated cell aggregates the key enzyme activities for glyoxysomes such as isocitrate lyase and malate synthase are induced in globular (3 d old) and heart-shaped (5 d old) embryoids, but not in embryoids at day 7 or later. Similarly, in explants from anise hypocotyl glyoxysomes cannot be derepressed by such treatment. It is concluded that during differentiation of heart-shaped embryoids to torpedo forms the competence of the cells for the yet unknown inducing principle for glyoxysomes is lost.

  1. Agrobacterium-mediated transformation of Vitis Cv. Monastrell suspension-cultured cells: Determination of critical parameters.

    Science.gov (United States)

    Chu, Mingyu; Quiñonero, Carmen; Akdemir, Hülya; Alburquerque, Nuria; Pedreño, María Ángeles; Burgos, Lorenzo

    2016-05-01

    Although some works have explored the transformation of differentiated, embryogenic suspension-cultured cells (SCC) to produce transgenic grapevine plants, to our knowledge this is one of the first reports on the efficient transformation of dedifferentiated Vitis vinifera cv Monastrell SCC. This protocol has been developed using the sonication-assisted Agrobacterium-mediated transformation (SAAT) method. A construct harboring the selectable nptII and the eyfp/IV2 marker genes was used in the study and transformation efficiencies reached over 50 independent transformed SCC per gram of infected cells. Best results were obtained when cells were infected at the exponential phase. A high density plating (500 mg/dish) gave significantly better results. As selective agent, kanamycin was inefficient for the selection of Monastrell transformed SCC since wild type cells were almost insensitive to this antibiotic whereas application of paromomycin resulted in very effective selection. Selected eyfp-expressing microcalli were grown until enough tissue was available to scale up a new transgenic SCC. These transgenic SCC lines were evaluated molecularly and phenotypically demonstrating the presence and integration of both transgenes, the absence of Agrobacterium contamination and the ability of the transformed SCC to grow in highly selective liquid medium. The methodology described here opens the possibility of improving the production of valuable metabolites. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:725-734, 2016.

  2. Rapamycin treatment inhibits CHO cell death in a serum-free suspension culture by autophagy induction.

    Science.gov (United States)

    Lee, Jae Seong; Lee, Gyun Min

    2012-12-01

    Rapamycin, a specific mTOR inhibitor, has been used as a chemical activator in autophagy research both in vitro and in vivo. Recently, autophagy has received attention as an anti-cell death engineering target in addition to apoptosis in the Chinese hamster ovary (CHO) cell engineering field. Here, the effect of rapamycin and the subsequent autophagy induction is investigated on two CHO cell lines, DG44 host and an antibody-producing recombinant CHO (rCHO), in a serum-free suspension culture. In both cell lines, the rapamycin treatment delayed the viability drop and apoptosis induction. In particular, the improved cell viability of the antibody-producing rCHO cell line resulting from the rapamycin treatment led to a 21% increase in the maximum antibody concentration. From observations that a rapamycin derivative, everolimus, demonstrated similar positive effects in both cell lines, but not FK-506, which forms the same complex as rapamycin, but does not inhibit mTOR, it was demonstrated that the positive effects of rapamycin appear to be mTOR-dependent. In addition, the cultivation with rapamycin and/or an autophagy inhibitor, bafilomycin A1, indicated that the autophagy induction is related to the positive effects of rapamycin. The genetic perturbation of the autophagy pathway through the regulation of the expression level of Beclin-1, an important autophagy regulator, resulted in a delayed autophagy induction and apoptosis inhibition in response to the rapamycin treatment in the DG44 host cell line. Taken together, the results obtained in this study imply a positive role for autophagy and predict the usefulness of pro-autophagy engineering in CHO cell cultures.

  3. Effects of aluminum on growth, polyamine metabolism, and inorganic ions in suspension cultures of red spruce (Picea rubens)

    Science.gov (United States)

    Rakesh Minocha; Walter C. Shortle; Daniel J. Jr. Coughin; Subhash C. Minocha

    1996-01-01

    The influence of age of red spruce (Picea rubens Sarg.) cell suspensions on aluminum (Al) effects was studied by adding AICI3 (0.2, 0.5, and 1.0 mM) to the media on each day of a 7-day culture period and analyzing for changes in total cell mass, polyamines, arginine decarboxylase activity, and inorganic ions after 24 h of...

  4. Proper selection of 1 g controls in simulated microgravity research as illustrated with clinorotated plant cell suspension cultures

    Science.gov (United States)

    Kamal, Khaled Y.; Hemmersbach, Ruth; Medina, F. Javier; Herranz, Raúl

    2015-04-01

    Understanding the physical and biological effects of the absence of gravity is necessary to conduct operations on space environments. It has been previously shown that the microgravity environment induces the dissociation of cell proliferation from cell growth in young seedling root meristems, but this source material is limited to few cells in each row of meristematic layers. Plant cell cultures, composed by a large and homogeneous population of proliferating cells, are an ideal model to study the effects of altered gravity on cellular mechanisms regulating cell proliferation and associated cell growth. Cell suspension cultures of Arabidopsis thaliana cell line (MM2d) were exposed to 2D-clinorotation in a pipette clinostat for 3.5 or 14 h, respectively, and were then processed either by quick freezing, to be used in flow cytometry, or by chemical fixation, for microscopy techniques. After long-term clinorotation, the proportion of cells in G1 phase was increased and the nucleolus area, as revealed by immunofluorescence staining with anti-nucleolin, was decreased. Despite the compatibility of these results with those obtained in real microgravity on seedling meristems, we provide a technical discussion in the context of clinorotation and proper 1 g controls with respect to suspension cultures. Standard 1 g procedure of sustaining the cell suspension is achieved by continuously shaking. Thus, we compare the mechanical forces acting on cells in clinorotated samples, in a control static sample and in the standard 1 g conditions of suspension cultures in order to define the conditions of a complete and reliable experiment in simulated microgravity with corresponding 1 g controls.

  5. Nucleotide sequences of chloroplast 5S ribosomal RNA from cell suspension cultures of the liverworts Marchantia polymorpha and Jungermannia subulata.

    OpenAIRE

    Yamano, Y; Ohyama, K; Komano, T

    1984-01-01

    The nucleotide sequences of chloroplast 5S rRNAs from cell suspension cultures of the liverworts Marchantia polymorpha and Jungermannia subulata were determined. Their nucleotide sequences, 119 nucleotides long, were highly homologous to each other (96% identity) and had high homology with those from chloroplast 5S rRNAs of two higher plants, tobacco (92% identity) and spinach (92-91% identity), but less homology (87-85% identity) with that from a lower plant, the fern Dryopteris acuminata.

  6. In vitro induction of α-pinene, pulegone, menthol, menthone and limonene in cell suspension culture of pennyroyal (Mentha pulegium).

    Science.gov (United States)

    Darvishi, E; Kahrizi, D; Bahraminejad, S; Mansouri, M

    2016-03-20

    Medicinal plants are known as important sources of secondary metabolites. Because of the economic value of pennyroyal [Mentha pulegium L. (Lamiaceae)] in food industries, propagation of this valuable plant has special importance. Plant cell suspension culture can increase some produced components. The aim of this research was performing cell culture for induction of some secondary metabolites of M. pulegium and compares it with native one. The MS medium was used for suspension culture. To investigate quantitative materials, 4 levels of yeast extract elicitor (20, 40, 60 and 80 mg/L) and salicylic acid in 4 levels (2, 4, 6 and 8 mg/L) were used. Obtained extracts were analyzed by GC-MS. Statistical analysis showed that the amount of limonene, menthone, menthol and α-pinene were more than mentioned compounds in natural plant as control. The maximum amount of this metabolites were obtained as limonene (in 60 mg/l yeast extract), menthone (in 40 mg/l yeast extract and 2 mg/l salicylic acid), menthol (in 6 mg/l salicylic acid) and α-pinene (in 4 mg/l salicylic acid) in the M. pulegium cell culture. The Pulegone was fond more in natural plants than cell culture mass. The most important secondary metabolites were increased by cell culture containing of salicylic acid and yeast extract elicitors in M. pulegume.

  7. Possible Involvement of NADPH Oxidase in Lanthanide Cation-Induced Superoxide Anion Generation in BY-2 Tobacco Cell Suspension Culture

    Institute of Scientific and Technical Information of China (English)

    Yang Shengchang

    2006-01-01

    A rapid and concentration-dependent generation of superoxide anion (·O-2), measured with a superoxide-specific Cypridina luciferin-derived chemiluminescent reagent, was observed when two lanthanide salts (LaCl3 and GdCl3) were added to tobacco (Nicotiana tabacum) cell suspension culture.Addition of superoxide dismutase (480 U·ml-1) and Tiron (5 μmol·L-1) to cell culture suspension decreases the level of lanthanide cation-induced ·O-2 generation, suggesting that ·O-2 generation is extra-cellular.Pretreatment of the cell culture suspension with diphenyleneiodonium (10 and 50 μmol·L-1), quinacrine (1 and 5 mmol·L-1) and imidazol (10 mmol·L-1), inhibitors of NADPH oxidase, notably inhibits the generation of superoxide induced by lanthanide cation, implying the possible involvement of activation of NADPH oxidase.In addition, addition of SHAM (1 and 5 mmol·L-1), azide (0.2 and 1 mmol·L-1), inhibitor of peroxidase, has no influence on ·O-2 generation.

  8. Immune suppression of human lymphoid tissues and cells in rotating suspension culture and onboard the International Space Station.

    Science.gov (United States)

    Fitzgerald, Wendy; Chen, Silvia; Walz, Carl; Zimmerberg, Joshua; Margolis, Leonid; Grivel, Jean-Charles

    2009-12-01

    The immune responses of human lymphoid tissue explants or cells isolated from this tissue were studied quantitatively under normal gravity and microgravity. Microgravity was either modeled by solid body suspension in a rotating, oxygenated culture vessel or was actually achieved on the International Space Station (ISS). Our experiments demonstrate that tissues or cells challenged by recall antigen or by polyclonal activator in modeled microgravity lose all their ability to produce antibodies and cytokines and to increase their metabolic activity. In contrast, if the cells were challenged before being exposed to modeled microgravity suspension culture, they maintained their responses. Similarly, in microgravity in the ISS, lymphoid cells did not respond to antigenic or polyclonal challenge, whereas cells challenged prior to the space flight maintained their antibody and cytokine responses in space. Thus, immune activation of cells of lymphoid tissue is severely blunted both in modeled and true microgravity. This suggests that suspension culture via solid body rotation is sufficient to induce the changes in cellular physiology seen in true microgravity. This phenomenon may reflect immune dysfunction observed in astronauts during space flights. If so, the ex vivo system described above can be used to understand cellular and molecular mechanisms of this dysfunction.

  9. Inter-microcarrier transfer and phenotypic stability of stem cell-derived Schwann cells in stirred suspension bioreactor culture.

    Science.gov (United States)

    Shakhbazau, Antos; Mirfeizi, Leila; Walsh, Tylor; Wobma, Holly M; Kumar, Ranjan; Singh, Bhagat; Kallos, Michael S; Midha, Rajiv

    2016-02-01

    Emerging bioreactor technologies offer an effective way for scaled-up production of large numbers of cells for cell therapy applications. One of the clinical paradigms where cell therapy can be an asset is restorative neurosciences. Nerve repair can benefit from the injections of stem cells and/or Schwann cells, acting as a source for axon myelination, myelin debris clearance, and trophic support. We have adapted microcarrier-based suspension bioreactor culture for Schwann cells (SCs) differentiated from a new stem cell source - skin-derived precursors (SKPs). SKP-derived SCs attach and grow on different types of microcarriers in both static and stirred culture, with Cytodex 3 and CultiSpher-S found most effective. Inter-microcarrier migration of SKP-SCs represents a key mechanism for rapid expansion and colonization in stirred suspension culture. We have shown that microcarrier-expanded SKP-SCs cells express Schwann cell markers p75-NTR, GFAP and S100 and retain their key ability to myelinate axons both in vitro and in vivo. Scaled-up microcarrier-based production of SKP-SCs in suspension bioreactors appears feasible for timely generation of sufficient cell numbers for nerve repair strategies.

  10. Embryogenic callus formation, growth and regeneration in callus and suspension cultures of Miscanthus x ogiformis Honda 'Giganteus' as affected by proline

    DEFF Research Database (Denmark)

    Holme, Inger Bæksted; Krogstrup, Peter; Hansen, Jürgen

    1997-01-01

    The effects of proline additions to culture systems of Miscanthus x ogiformis Honda Giganteus' were investigated. Proline was added in concentrations of 0, 12.5, 25, 50, 100 or 300 mM to the callus induction and suspension culture media containing either Murashige and Skoog or N6 basal salts and 22...... with Murashige and Skoog salts increased embryogenic callus formation on shoot apices and leaf explants while proline had no effect on embryogenic callus formation in medium with N6 salts. Increased growth with increasing proline concentration was obtained in suspension aggregates grown in medium with N6 salts......, whereas proline only increased growth of suspension aggregates grown in medium with Murashige and Skoog salts at concentrations of 12.5 or 25 mM. A stimulating effect of proline on plant regeneration was observed in short-term cultures of callus as well as in long-term cultures of suspension aggregates...

  11. Enhanced biosynthesis of withanolides by elicitation and precursor feeding in cell suspension culture of Withania somnifera (L.) Dunal in shake-flask culture and bioreactor.

    Science.gov (United States)

    Sivanandhan, Ganeshan; Selvaraj, Natesan; Ganapathi, Andy; Manickavasagam, Markandan

    2014-01-01

    The present study investigated the biosynthesis of major and minor withanolides of Withania somnifera in cell suspension culture using shake-flask culture and bioreactor by exploiting elicitation and precursor feeding strategies. Elicitors like cadmium chloride, aluminium chloride and chitosan, precursors such as cholesterol, mevalonic acid and squalene were examined. Maximum total withanolides detected [withanolide A (7606.75 mg), withanolide B (4826.05 mg), withaferin A (3732.81 mg), withanone (6538.65 mg), 12 deoxy withanstramonolide (3176.63 mg), withanoside IV (2623.21 mg) and withanoside V (2861.18 mg)] were achieved in the combined treatment of chitosan (100 mg/l) and squalene (6 mM) along with 1 mg/l picloram, 0.5 mg/l KN, 200 mg/l L-glutamine and 5% sucrose in culture at 4 h and 48 h exposure times respectively on 28th day of culture in bioreactor. We obtained higher concentrations of total withanolides in shake-flask culture (2.13-fold) as well as bioreactor (1.66-fold) when compared to control treatments. This optimized protocol can be utilized for commercial level production of withanolides from suspension culture using industrial bioreactors in a short culture period.

  12. Improved Production of Paclitaxel from Suspension Culture of Taxus chinensis var.mairei by in situ Extraction with Organic Solvents

    Institute of Scientific and Technical Information of China (English)

    未作君; 元英进; 吴兆亮; 吴金川

    2003-01-01

    The production of paclitaxel from suspension culture of Taxus chinensis var,mairei was improved by in situ extraction with organic solvents to avoid feedback repression and product degradation.Oleic acid and dibutyl phthalate were proved to be suitable solvents .The optimal volumetric percentage of organic solvents in the culture medium was found to be around 8%,and the favorable time for their introduction was at the exponential phase of cell growth,Paclitaxel production with the in situ extraction was ca 3-fold of that without extraction.

  13. Cotton Fever: Does the Patient Know Best?

    Science.gov (United States)

    Xie, Yingda; Pope, Bailey A; Hunter, Alan J

    2016-04-01

    Fever and leukocytosis have many possible etiologies in injection drug users. We present a case of a 22-year-old woman with fever and leukocytosis that were presumed secondary to cotton fever, a rarely recognized complication of injection drug use, after an extensive workup. Cotton fever is a benign, self-limited febrile syndrome characterized by fevers, leukocytosis, myalgias, nausea and vomiting, occurring in injection drug users who filter their drug suspensions through cotton balls. While this syndrome is commonly recognized amongst the injection drug user population, there is a paucity of data in the medical literature. We review the case presentation and available literature related to cotton fever.

  14. Antioxidant metabolism of coffee cell suspension cultures in response to cadmium.

    Science.gov (United States)

    Gomes-Junior, Rui A; Moldes, Carlos A; Delite, Fabricio S; Pompeu, Georgia B; Gratão, Priscila L; Mazzafera, Paulo; Lea, Peter J; Azevedo, Ricardo A

    2006-11-01

    The antioxidant responses of coffee (Coffea arabica L.) cell suspension cultures to cadmium (Cd) were investigated. Cd accumulated very rapidly in the cells and this accumulation was directly correlated with an increase in applied CdCl(2) concentration in the external medium. At 0.05mM CdCl(2), growth was stimulated, but at 0.5mM CdCl(2), the growth rate was reduced. An alteration in activated oxygen metabolism was detected by visual analysis as well as by an increase in lipid peroxidation at the higher CdCl(2) concentration. Catalase (CAT; EC 1.11.1.6), glutathione reductase (GR; EC 1.6.4.2) and superoxide dismutase (SOD; EC 1.15.1.1) activity increased, particularly at the higher concentration of CdCl(2). Ascorbate peroxidase (APX; EC 1.11.1.11) activity was increased at the lower CdCl(2) concentration used, but could not be detected in cells growing in the higher CdCl(2) concentration after 24h of growth, whilst guaiacol peroxidase (GOPX; EC 1.11.1.7) did not show a clear response to Cd treatment. An analysis by non-denaturing PAGE followed by staining for enzyme activity, revealed one CAT isoenzyme, nine SOD isoenzymes and four GR isoenzymes. The SOD isoenzymes were differently affected by CdCl(2) treatment and one GR isoenzyme was shown to specifically respond to CdCl(2). The results suggest that the higher concentrations of CdCl(2) may lead to oxidative stress. The main response appears to be via the induction of SOD and CAT activities for the removal of reactive oxygen species (ROS), and by the induction of GR to ensure the availability of reduced glutathione for the synthesis of Cd-binding peptides, which may also be related to the inhibition of APX activity probably due to glutathione and ascorbate depletion.

  15. Programmed cell death activated by Rose Bengal in Arabidopsis thaliana cell suspension cultures requires functional chloroplasts.

    Science.gov (United States)

    Gutiérrez, Jorge; González-Pérez, Sergio; García-García, Francisco; Daly, Cara T; Lorenzo, Oscar; Revuelta, José L; McCabe, Paul F; Arellano, Juan B

    2014-07-01

    Light-grown Arabidopsis thaliana cell suspension culture (ACSC) were subjected to mild photooxidative damage with Rose Bengal (RB) with the aim of gaining a better understanding of singlet oxygen-mediated defence responses in plants. Additionally, ACSC were treated with H2O2 at concentrations that induced comparable levels of protein oxidation damage. Under low to medium light conditions, both RB and H2O2 treatments activated transcriptional defence responses and inhibited photosynthetic activity, but they differed in that programmed cell death (PCD) was only observed in cells treated with RB. When dark-grown ACSC were subjected to RB in the light, PCD was suppressed, indicating that the singlet oxygen-mediated signalling pathway in ACSC requires functional chloroplasts. Analysis of up-regulated transcripts in light-grown ACSC, treated with RB in the light, showed that both singlet oxygen-responsive transcripts and transcripts with a key role in hormone-activated PCD (i.e. ethylene and jasmonic acid) were present. A co-regulation analysis proved that ACSC treated with RB exhibited higher correlation with the conditional fluorescence (flu) mutant than with other singlet oxygen-producing mutants or wild-type plants subjected to high light. However, there was no evidence for the up-regulation of EDS1, suggesting that activation of PCD was not associated with the EXECUTER- and EDS1-dependent signalling pathway described in the flu mutant. Indigo Carmine and Methylene Violet, two photosensitizers unable to enter chloroplasts, did not activate transcriptional defence responses in ACSC; however, whether this was due to their location or to their inherently low singlet oxygen quantum efficiencies was not determined.

  16. Characterization of the secretome of suspension cultures of Medicago species reveals proteins important for defense and development.

    Science.gov (United States)

    Kusumawati, Lucia; Imin, Nijat; Djordjevic, Michael A

    2008-10-01

    Molecular events occurring in the plant apoplast contribute to important developmental and defense responses. To define the secretome of Medicago, we used suspension cultures to isolate and identify secreted proteins as a first step to determining their functions. Proteins in the extracellular medium of the suspension cultures were examined using SDS-PAGE, tandem mass spectrometry (MALDI-TOF/TOF) and bioinformatics tools. There were 39 proteins identified in the cultures derived from M. sativa, M. truncatula 2HA (an embryogenic line), and M. truncatula sickle (an ethylene-insensitive mutant). N-Terminal secretion signals were detected in 34 proteins and five other proteins were predicted to be secreted via a nonclassical (ER-independent) route. All samples possessed defense related proteins including pathogenesis related (PR) proteins. The glycoprotein, SIEP1L, was found only in M. sativa. Three secreted proteinases were identified in M. truncatula, including a serine carboxypeptidase detected only in 2HA. Some proteins were unique to a cell culture line. Quantitative real time RT-PCR was used to determine mRNA expression of selected genes corresponding to proteins found only in 2HA or sickle or in both. The results correlate well with the proteomic data. For instance, a GDSL-lipase gene known to be regulated by ethylene was found only in 2HA but not in the ethylene insensitive mutant. Similarly, the PR1a protein, expressed from a well recognized ethylene-regulated gene, was found in 2HA but not sickle. These experiments indicate that the suspension culture systems established here are useful to avoid contamination from cytoplasmic proteins and to identify secreted proteins in Medicago, and should have application in other plant systems.

  17. Enhanced stem cell-derived cardiomyocyte differentiation in suspension culture by delivery of nitric oxide using S-nitrosocysteine.

    Science.gov (United States)

    Hodge, Alexander J; Zhong, Juming; Lipke, Elizabeth A

    2016-04-01

    The development of cell-based treatments for heart disease relies on the creation of functionally mature stem cell-derived cardiomyocytes employing in vitro culture suspension systems, a process which remains a formidable and expensive endeavor. The use of nitric oxide as a signaling molecule during differentiation has demonstrated the potential for creating increased numbers of spontaneously contracting embryoid bodies in culture; however, the effects of nitric oxide signaling on the function and maturation of stem cell-derived cardiomyocytes is not well understood. In this study, the effects of nitric oxide on mouse embryonic stem cell-derived cardiomyocyte contractile activity, protein, and gene expression, and calcium handling were quantified. Embryoid bodies (EBs) formed using the hanging drop method, were treated with the soluble nitric oxide donor S-nitrosocysteine (CysNO) over a period of 18 days in suspension culture and spontaneous contractile activity was assessed. On day 8, selected EBs were dissociated to form monolayers for electrophysiological characterization using calcium transient mapping. Nitric oxide treatment led to increased numbers of stem cell-derived cardiomyocytes (SC-CMs) relative to non-treated EBs after 8 days in suspension culture. Increased incidence of spontaneous contraction and frequency of contraction were observed from days 8-14 in EBs receiving nitric oxide treatment in comparison to control. Expression of cardiac markers and functional proteins was visualized using immunocytochemistry and gene expression was assessed using qPCR. Cardiac-specific proteins were present in both CysNO-treated and control SC-CMs; however, CysNO treatment during differentiation significantly increased βMHC gene expression in SC-CMs relative to control SC-CMs. Furthermore, increased calcium transient velocity and decreased calcium transient duration was observed for CysNO-treated SC-CMs in comparison to control SC-CMs. Soluble nitric oxide donors

  18. ""Thames Valley cotton pickers"": Race and youth in London blues culture

    OpenAIRE

    2005-01-01

    This study addresses the reception of African American blues music and the ensuing production of English blues in London from 1955 to 1966. It concentrates on London adolescents' unexpected fascination with a musical style that they virtually had no contact with prior to 1955, while analyzing how this immersion in African American culture shaped their cultural identity. Analysis of the influence of African American blues music in London during this period highlights the BBC's weakened influen...

  19. Rheological properties of mammalian cell culture suspensions: Hybridoma and HeLa cell lines.

    Science.gov (United States)

    Shi, Y; Ryu, D D; Ballica, R

    1993-03-25

    Data on viscous (eta') and elastic (eta'') components of the complex viscosity versus oscillatory angular frequency (0.01 to 4.0 rad/s) with increasing strains were obtained for hybridoma cell (62'D3) and HeLa cell (S3) suspensions in PBS at 0.9 (mL/mL) cell volume fraction using a Weissenberg rheogoniometer equipped with two parallel plate geometry at ambient temperature. Both cell suspensions exhibited shear thinning behavior. From the measured viscoelastic properties, the yield stress was calculated. Hybridoma cell suspension (15 microm as the mean diameter of cells) showed the yield stress at 550 dyne/cm(2) that was 1.8 times higher than the value of HeLa cell suspension (22 microm mean diameter) as measured at the oscillatory angular frequency, 4.0 rad/s. The apparent viscosities of HeLa cell suspension at four concentrations and varying steady shear rate were also determined using the Brookfield rotational viscometer. The yield stress to steady shear test was about 130 dyne/cm(2) for HeLa cell suspension at 0.9 (mL/mL) cell volume fraction. The apparent viscosity was in the range about 1 approximately 1000 Poise depending on the cell concentration and shear rate applied. A modified semiempirical Mooney equation, eta = eta(0) exp[K gamma(.)(-beta)phi(c)(1 - K'' sigmaphi(c) /D)] was derived based on the cell concentration, the cell morphology, and the steady shear rate. The beta, shear rate index, was estimated as 0.159 in the range of shear rate, 0.16 to 22.1 s(-1), for the cell volume fractions from 0.6 to 0.9 (mL/mL). In this study, the methods of determining the shear sensitivity and the viscous and the elastic components of mammalian cell suspensions are described under the steady shear field.

  20. Glycyrrhiza glabra (Linn.) and Lavandula officinalis (L.) cell suspension cultures-based biotransformation of β-artemether.

    Science.gov (United States)

    Patel, Suman; Gaur, Rashmi; Upadhyaya, Mohita; Mathur, Archana; Mathur, Ajay K; Bhakuni, Rajendra S

    2011-07-01

    The biotransformation of β-artemether (1) by cell suspension cultures of Glycyrrhiza glabra and Lavandula officinalis is reported here for the first time. The major biotransformed product appeared as a grayish-blue color spot on thin-layer chromatography (TLC) with transparent crystal-like texture. Based on its infrared (IR) and (1)H nuclear magnetic resonance (NMR) spectra, the product was characterized as a tetrahydrofuran (THF)-acetate derivative (2). The highest conversion efficiencies of 57 and 60% were obtained when 8-9-day-old cell suspensions of G. glabra and L. officinalis were respectively fed with 4-7 mg of compound 1 in 40 ml of medium per culture and the cells were harvested after 2-5 days of incubation. The addition of compound 1 at the beginning of the culture cycle caused severe growth depression in a dose-dependent manner, resulting in poor bioconversion efficiency of ~25% at 2-5 mg/culture dose only.

  1. A genotype of modified vaccinia Ankara (MVA) that facilitates replication in suspension cultures in chemically defined medium.

    Science.gov (United States)

    Jordan, Ingo; Horn, Deborah; John, Katrin; Sandig, Volker

    2013-01-21

    While vectored vaccines, based on hyperattenuated viruses, may lead to new treatment options against infectious diseases and certain cancers, they are also complex products and sometimes difficult to provide in sufficient amount and purity. To facilitate vaccine programs utilizing host-restricted poxviruses, we established avian suspension cell lines (CR and CR.pIX) and developed a robust, chemically defined, culturing process for production of this class of vectors. For one prominent member, modified vaccinia Ankara (MVA), we now describe a new strain that appears to replicate to greater yields of infectious units, especially in the cell-free supernatant of cultures in chemically defined media. The new strain was obtained by repeated passaging in CR suspension cultures and, consistent with reports on the exceptional genetic stability of MVA, sequencing of 135 kb of the viral genomic DNA revealed that only three structural proteins (A3L, A9L and A34R) each carry a single amino acid exchange (H639Y, K75E and D86Y, respectively). Host restriction in a plaque-purified isolate of the new genotype appears to be maintained in cell culture. Processing towards an injectable vaccine preparation may be simplified with this strain as a complete lysate, containing the main burden of host cell contaminants, may not be required anymore to obtain adequate yields.

  2. Elicitation of gymnemic acid production in cell suspension cultures of Gymnema sylvestre R.Br. through endophytic fungi.

    Science.gov (United States)

    Netala, Vasudeva Reddy; Kotakadi, Venkata Subbaiah; Gaddam, Susmila Aparna; Ghosh, Sukhendu Bikash; Tartte, Vijaya

    2016-12-01

    The enhancement of plant secondary metabolite production in cell suspension cultures through biotic or abiotic elicitation has become a potential biotechnological approach for commercialization or large-scale production of bioactive compounds. Gymnema sylvestre R.Br. is an important medicinal plant, rich in a group of oleanane triterpenoid saponins called gymnemic acid, well known for its anti-diabetic activity. Two endophytic fungal strains were isolated from the leaves of G. sylvestre and identified as Polyancora globosa and Xylaria sp. based on the PCR amplification and internal transcribed spacer (ITS 1-5.8S-ITS 2) sequencing of 18S rRNA gene. The process of elicitation of cell suspension cultures of G. sylvestre with dried powder of fungal mycelia (DPFM) and extracellular culture filtrate (ECF) of endophytic fungi consistently enhanced the accumulation of gymnemic acid and the DPFM was proved to be an effective elicitor when compared to the ECF. The DPFM elicited the gymnemic acid content in the range of 2.57-10.45-fold, while the ECF elicited the gymnemic acid content in the range of 2.39-7.8-fold. P. globosa, a novel and a rare endophytic fungal strain, has shown a great influence on the production of gymnemic acid. Cell suspension cultures elicited with DPFM of P. globosa produced higher amount of gymnemic acid content (124.23 mg/g dried cell weight) compared to the cultures elicited with DPFM of Xylaria sp. (102.24 mg/g DCW). But the cultures treated with consortium of DPFM of both fungi showed great influence on the production of gymnemic acid (139.98 mg/g DCW) than the cultures treated with DPFM alone. Similarly, cultures treated with consortium of ECF of both fungi produced more gymnemic acid content (94.86 mg/g DCW) compared with cultures treated with ECF of Xylaria sp. (77.93 mg/g DCW) and ECF of P. globosa (78.65 mg/g DCW) alone.

  3. Optimization of the basal medium for improving production and secretion of taxanes from suspension cell culture of Taxus baccata L

    Directory of Open Access Journals (Sweden)

    Kajani Abolghasem

    2012-10-01

    Full Text Available Abstract Background and purpose of the study Taxol is one of the most effective anticancer drugs that isolated from Taxus sp. due to the slow growth of Taxus trees and low concentration of Taxol in the tissues, the biotechnological approaches especially plant cell culture have been considered to produce Taxol in commercial scale. Methods We investigated the effects of basal medium type used in culture media on production of Taxol and other taxane compounds from cell suspension culture of T. baccata L. Briefly, five commonly basal media including Gamborg, Murashige and Skoog, Woody Plant, Schenk and Hildebrandt, and Driver and Kuniyuki medium were used for preparing separate suspension culture media. The intra- and extra-cellular yields of taxanes were analyzed by using HPLC after 21 days period of culturing. Results The yields of taxanes were significantly different for the cultures prepared by different basal media. Moreover, the effects of basal medium on the yield of products differed for varius taxane compounds. Maximum yields of Baccatin III (10.03 mgl-1 and 10-deacetyl baccatin III (4.2 mgl-1 were achieved from the DKW basal media, but the yield of Taxol was maximum (16.58 mgl-1 in the WPM basal media. Furthermore, the secretion of taxanes from the cells into medium was also considerably affected by the type of basal medium. The maximum extra-cellular yield of Taxol (7.81 mgl-1, Baccatin III (5.0 mgl-1, and 10-deacetyl baccatin III (1.45 mgl-1 were also obtained by using DKW basal medium that were significantly higher than those obtained from other culture media.

  4. Optimization of the basal medium for improving production and secretion of taxanes from suspension cell culture of Taxus baccata L

    Directory of Open Access Journals (Sweden)

    Abolghasem Abbasi Kajani

    2012-10-01

    Full Text Available Background and purpose of the study Taxol is one of the most effective anticancer drugs that isolated from Taxus sp. due to the slow growth of Taxus trees and low concentration of Taxol in the tissues, the biotechnological approaches especially plant cell culture have been considered to produce Taxol in commercial scale.MethodsWe investigated the effects of basal medium type used in culture media on production of Taxol and other taxane compounds from cell suspension culture of T. baccata L. Briefly, five commonly basal media including Gamborg, Murashige and Skoog, Woody Plant, Schenk and Hildebrandt, and Driver and Kuniyuki medium were used for preparing separate suspension culture media. The intra- and extra-cellular yields of taxanes were analyzed by using HPLC after 21 days period of culturing.ResultsThe yields of taxanes were significantly different for the cultures prepared by different basal media. Moreover, the effects of basal medium on the yield of products differed for varius taxane compounds. Maximum yields of Baccatin III (10.03 mgl-1 and 10-deacetyl baccatin III (4.2 mgl-1 were achieved from the DKW basal media, but the yield of Taxol was maximum (16.58 mgl-1 in the WPM basal media. Furthermore, the secretion of taxanes from the cells into medium was also considerably affected by the type of basal medium. The maximum extra-cellular yield of Taxol (7.81 mgl-1, Baccatin III (5.0 mgl-1, and 10-deacetyl baccatin III (1.45 mgl-1 were also obtained by using DKW basal medium that were significantly higher than those obtained from other culture media.

  5. Induction and analysis of the alkaloid mitragynine content of a Mitragyna speciosa suspension culture system upon elicitation and precursor feeding.

    Science.gov (United States)

    Mohamad Zuldin, Nor Nahazima; Said, Ikram Md; Mohd Noor, Normah; Zainal, Zamri; Jin Kiat, Chew; Ismail, Ismanizan

    2013-01-01

    This study aimed to determine the effects of different concentrations and combinations of the phytohormones 2,4-dichlorophenoxy acetic acid (2,4-D), kinetin, 6-benzylaminopurine (BAP), and 1-naphthaleneacetic acid (NAA) on callus induction and to demonstrate the role of elicitors and exogenous precursors on the production of mitragynine in a Mitragyna speciosa suspension culture. The best callus induction was achieved from petiole explants cultured on WPM that was supplemented with 4 mg L⁻¹ 2,4-D (70.83%). Calli were transferred to liquid media and agitated on rotary shakers to establish Mitragyna speciosa cell suspension cultures. The optimum settled cell volume was achieved in the presence of WPM that contained 3 mg L⁻¹ 2,4-D and 3% sucrose (9.47 ± 0.4667 mL). The treatment of cultures with different concentrations of yeast extract and salicylic acid for different inoculation periods revealed that the highest mitragynine content as determined by HPLC was achieved from the culture treated with 250 mg L⁻¹ yeast extract (9.275 ± 0.082 mg L⁻¹) that was harvested on day 6 of culturing; salicylic acid showed low mitragynine content in all concentrations used. Tryptophan and loganin were used as exogenous precursors; the highest level of mitragynine production was achieved in cultures treated with 3  μM tryptophan and harvested at 6 days (13.226 ± 1.98 mg L⁻¹).

  6. Induction and Analysis of the Alkaloid Mitragynine Content of a Mitragyna speciosa Suspension Culture System upon Elicitation and Precursor Feeding

    Directory of Open Access Journals (Sweden)

    Nor Nahazima Mohamad Zuldin

    2013-01-01

    Full Text Available This study aimed to determine the effects of different concentrations and combinations of the phytohormones 2,4-dichlorophenoxy acetic acid (2,4-D, kinetin, 6-benzylaminopurine (BAP, and 1-naphthaleneacetic acid (NAA on callus induction and to demonstrate the role of elicitors and exogenous precursors on the production of mitragynine in a Mitragyna speciosa suspension culture. The best callus induction was achieved from petiole explants cultured on WPM that was supplemented with 4 mg L−1 2, 4-D (70.83%. Calli were transferred to liquid media and agitated on rotary shakers to establish Mitragyna speciosa cell suspension cultures. The optimum settled cell volume was achieved in the presence of WPM that contained 3 mg L−1 2,4-D and 3% sucrose (9.47±0.4667 mL. The treatment of cultures with different concentrations of yeast extract and salicylic acid for different inoculation periods revealed that the highest mitragynine content as determined by HPLC was achieved from the culture treated with 250 mg L−1 yeast extract (9.275±0.082 mg L−1 that was harvested on day 6 of culturing; salicylic acid showed low mitragynine content in all concentrations used. Tryptophan and loganin were used as exogenous precursors; the highest level of mitragynine production was achieved in cultures treated with 3 μM tryptophan and harvested at 6 days (13.226±1.98 mg L−1.

  7. Propagation of Brazilian Zika virus strains in static and suspension cultures using Vero and BHK cells.

    Science.gov (United States)

    Nikolay, Alexander; Castilho, Leda R; Reichl, Udo; Genzel, Yvonne

    2017-03-23

    The recent spread of Zika virus (ZIKV) in the Americas and the Pacific has reached alarming levels in more than 60 countries. However, relatively little is known about the disease on a virological and epidemiological level and its consequences for humans. Accordingly, a large demand for in vitro derived Brazilian ZIKV material to support in vitro and in vivo studies has arisen. However, a prompt supply of ZIKV and ZIKV antigens cannot be guaranteed as the production of this virus typically using Vero or C6/36 cell lines remains challenging. Here we present a production platform based on BHK-21 suspension (BHK-21SUS) cells to propagate Brazilian ZIKV at larger quantities in perfusion bioreactors. Scouting experiments performed in tissue culture flasks using adherent BHK-21 and Vero cells have demonstrated similar permissivity and virus yields for four different Brazilian ZIKV isolates. The cell-specific yield of infectious virus particles varied between respective virus strains (1-48PFU/cell), and the ZIKV isolate from the Brazilian state Pernambuco (ZIKV(PE)) showed to be a best performing isolate for both cell lines. However, infection studies of BHK-21SUS cells with ZIKV(PE) in shake flasks resulted in poor virus replication, with a maximum titer of 8.9×10(3)PFU/mL. Additional RT-qPCR measurements of intracellular and extracellular viral RNA levels revealed high viral copy numbers within the cell, but poor virus release. Subsequent cultivation in a perfusion bioreactor using an alternating tangential flow filtration system (ATF) under controlled process conditions enabled cell concentrations of about 1.2×10(7)cells/mL, and virus titers of 3.9×10(7)PFU/mL. However, while the total number of infectious virus particles was increased, the cell-specific yield (3.3PFU/cell) remained lower than determined in adherent cell lines. Nevertheless, the established perfusion process allows to provide large amounts of ZIKV material for research and is a first step towards

  8. Effects of medium nutrition on cell growth and isocamptothecin A and B production by suspension cell culture of Camptotheca acuminata

    Institute of Scientific and Technical Information of China (English)

    Zhang Dongyan; Yu Fang; Bai Fengwu; An Lijia

    2006-01-01

    The effects of initial sucrose concentration, nitrate to ammonium ratio, total N concentration and phosphate concentration in medium on cell growth and isocamptothecin A and B synthesis by suspension cell culture of Camptotheca acuminata were investigated in 250 mL shake flasks. 30 g L-1 sucrose concentration was beneficial to secondary metabolites synthesis. The cell growth and metabolites synthesis were also affected by the ratio of NO-3/NH+4, and nitrate was favourable for cell growth. The maximum dry weight was achieved when nitrate was used as the sole N source. The effect of total initial N on the cell cultures was also investigated with NO-3/NH+4 ratio of 1∶2. The final dry cell weight was similar throughout culture period and 50 mM initial N was favourable for secondary metabolite synthesis. 50 mM initial phosphate concentration facilitated both cell growth and secondary metabolites synthesis.

  9. A promising approach on biomass accumulation and withanolides production in cell suspension culture of Withania somnifera (L.) Dunal.

    Science.gov (United States)

    Sivanandhan, Ganeshan; Kapil Dev, Gnanajothi; Jeyaraj, Murugaraj; Rajesh, Manoharan; Muthuselvam, Manickam; Selvaraj, Natesan; Manickavasagam, Markandan; Ganapathi, Andy

    2013-08-01

    Withanolide is one of the most extensively exploited steroidal lactones, which are biosynthesized in Withania somnifera. Its production from cell suspension culture was analyzed to defeat limitations coupled with its regular supply from the plant organs. In order to optimize the different factors for sustainable production of withanolides and biomass accumulations, different concentrations of auxins or cytokinins and their combinations, carbon sources, agitation speed, organic additives and seaweed extracts was studied in cell suspension culture. Maximum biomass accumulation (16.72 g fresh weight [FW] and 4.18 g dry weight [DW]) and withanolides production (withanolide A 7.21 mg/g DW, withanolide B 4.23 mg/g DW, withaferin A 3.88 mg/g DW and withanone 6.72 mg/g DW) were achieved in the treatment of Gracilaria edulis extract at 40 % level. Organic additive L-glutamine at 200 mg/l in combination with picloram (1 mg/l) and KN (0.5 mg/l) promoted growth characteristics (11.87 g FW and 2.96 g DW) and withanolides synthesis (withanolide A 5.04 mg/g DW, withanolide B 2.59 mg/g DW, withaferin A 2.36 mg/g DW and withanone 4.32 mg/g DW). Sucrose at 5 % level revolved out to be a superior carbon source yielded highest withanolides production (withanolide A 2.88 mg/g DW, withanolide B 1.48 mg/g DW, withaferin A 1.35 mg/g DW and withanone 2.47 mg/g DW), whereas biomass (7.28 g FW and 1.82 g DW) was gratefully increased at 2 % level of sucrose in cell suspension culture. This optimized protocol can be utilized for large scale cultivation of W. somnifera cells in industrial bioreactors for mass synthesis of major withanolides.

  10. Partially acetylated chitosan oligo- and polymers induce an oxidative burst in suspension cultured cells of the gymnosperm Araucaria angustifolia.

    Science.gov (United States)

    dos Santos, André Luis Wendt; El Gueddari, Nour Eddine; Trombotto, Stéphane; Moerschbacher, Bruno Maria

    2008-12-01

    Suspension-cultured cells were used to analyze the activation of defense responses in the conifer A. angustifolia , using as an elicitor purified chitosan polymers of different degrees of acetylation (DA 1-69%), chitin oligomers of different degrees of polymerization (DP 3-6), and chitosan oligomer of different DA (0-91%). Suspension cultured cells elicited with chitosan polymers reacted with a rapid and transient generation of H2O2, with chitosans of high DA (60 and 69%) being the most active ones. Chitosan oligomers of high DA (78 and 91%) induced substantial levels of H2O2, but fully acetylated chitin oligomers did not. When cultivated for 24-72 h in the presence of 1-10 microg mL(-1) chitosan (DA 69%), cell cultures did not show alterations in the levels of enzymes related to defense responses, suggesting that, in A. angustifolia , the induction of an oxidative burst is not directly coupled to the induction of other defense reactions.

  11. Pretreatment of Parsley (Petroselinum crispum L.) Suspension Cultures with Methyl Jasmonate Enhances Elicitation of Activated Oxygen Species.

    Science.gov (United States)

    Kauss, H.; Jeblick, W.; Ziegler, J.; Krabler, W.

    1994-05-01

    Suspension-cultured cells of parsley (Petroselinum crispum L.) were used to demonstrate an influence of jasmonic acid methyl ester (JAME) on the elicitation of activated oxygen species. Preincubation of the cell cultures for 1 d with JAME greatly enhanced the subsequent induction by an elicitor preparation from cell walls of Phytophtora megasperma f. sp. glycinea (Pmg elicitor) and by the polycation chitosan. Shorter preincubation times with JAME were less efficient, and the effect was saturated at about 5 [mu]M JAME. Treatment of the crude Pmg elicitor with trypsin abolished induction of activated oxygen species, an effect similar to that seen with elicitation of coumarin secretion. These results suggest that JAME conditioned the parsley suspension cells in a time-dependent manner to become more responsive to elicitation, reminiscent of developmental effects caused by JAME in whole plants. It is interesting that pretreatment of the parsley cultures with 2,6-dichloroisonicotinic and 5-chlorosalicylic acid only slightly enhanced the elicitation of activated oxygen species, whereas these substances greatly enhanced the elicitation of coumarin secretion. Therefore, these presumed inducers of systemic acquired resistance exhibit a specificity different from JAME.

  12. The Structure of Plant Cell Walls: II. The Hemicellulose of the Walls of Suspension-cultured Sycamore Cells.

    Science.gov (United States)

    Bauer, W D; Talmadge, K W; Keegstra, K; Albersheim, P

    1973-01-01

    The molecular structure, chemical properties, and biological function of the xyloglucan polysaccharide isolated from cell walls of suspension-cultured sycamore (Acer pseudoplatanus) cells are described. The sycamore wall xyloglucan is compared to the extracellular xyloglucan secreted by suspension-cultured sycamore cells into their culture medium and is also compared to the seed "amyloid" xyloglucans.Xyloglucan-or fragments of xyloglucan-and acidic fragments of the pectic polysaccharides are released from endopolygalacturonase-pretreated sycamore walls by treatment of these walls with 8 m urea, endoglucanase, or 0.5 n NaOH. Some of the xyloglucan thus released is found to cochromatograph with the acidic pectic fragments on diethylaminoethyl Sephadex. The chemical or enzymic treatments required for the release of xyloglucan from the walls and the cochromatography of xyloglucan with the acidic pectic fragments indicate that xyloglucan is covalently linked to the pectic polysaccharides and is noncovalently bound to the cellulose fibrils of the sycamore cell wall.The molecular structure of sycamore xyloglucan was characterized by methylation analysis of the oligosaccharides obtained by endoglucanase treatment of the polymer. The structure of the polymer is based on a repeating heptasaccharide unit which consists of 4 residues of beta-1-4-linked glucose and 3 residues of terminal xylose. A single xylose residue is glycosidically linked to carbon 6 of 3 of the glucosyl residues.

  13. Incorporation and Degradation of 14C and 3H-labeled Thymidine by Sugarcane Cells in Suspension Culture 12

    Science.gov (United States)

    Lesley, Stanley M.; Maretzki, Andrew; Nickell, Louis G.

    1980-01-01

    Sugarcane cells growing in suspension culture degrade exogenous thymidine, releasing thymine. Thymine is not utilized for DNA synthesis. Thymine is rapidly catabolized to β-aminoisobutyric acid which is found within the cell. Thymidine in the medium is used for DNA synthesis. The label of [2-14C]thymidine is lost as 14CO2, but the label of [3H]methylthymidine is found in the cell as [3H]β-aminoisobutyric acid, some of which is used for the synthesis of other cell components. The degradation of thymidine can be partially inhibited by addition of certain substituted pyrimidines. PMID:16661365

  14. Dynamic Effects of Cerium on Syntheses of Soluble Protein and Taxol in Suspension Culture of Taxus Chinensis Var. Mairei Cells

    Institute of Scientific and Technical Information of China (English)

    李景川; 马忠海; 元英进; 孙安慈; 胡昌序

    2001-01-01

    The dynamic effects of Ce4+ on the syntheses of soluble protein and taxol in suspension cultures of Taxus chinensis var. mairei cells were studied. The phenomena of “partition” and “bifurcation” were observed in studying the dynamic effect of Ce4+ on soluble protein synthesis and cell activity. That is, Ce4+ of low concentration improves the soluble protein synthetic strength and cell activity, while Ce4+of high concentration is harmful to protein synthesis and cell activity. In addition, Ce4+ of appropriate concentration enhances taxol synthesis.

  15. Correlation of cotyledonary node shoot proliferation and somatic embryoid development in suspension cultures of soybean (Glycine max L. Merr.).

    Science.gov (United States)

    Kerns, H R; Barwale, U B; Meyer, M M; Widholm, J M

    1986-04-01

    Suspension cultures of soybean were initiated from hypocotyl or cotyledon callus tissue of several soybean genotypes. When these were grown on L2 medium with 0.4 mg/liter 2,4-D several genotypes produced numerous embryoids while others produced only a few such structures. Due to internal anatomy, no embryoid developed into a complete plant. A genotype's propensity to form normal appearing embryoids was correlated with the ability to proliferate shoots at the cotyledonary node on a medium with benzylaminopurine as determined in previous testing.

  16. Development of desiccation tolerance and vitrification by preculture treatment in suspension-cultured cells of the liverwort Marchantia polymorpha.

    Science.gov (United States)

    Hatanaka, Rie; Sugawara, Yasutake

    2010-03-01

    Some cultured plant cells are able to acquire tolerance to various stresses when they are cultured under suitably controlled conditions. Induction of a high level of desiccation tolerance in suspension-cultured cells of the liverwort Marchantia polymorpha was examined for studying the mechanisms of desiccation tolerance and vitrification at the cellular level. Desiccation tolerance level of cells was very low and the survival rate was less than 10% after exposure to drying below 0.1 g H(2)O g(-1) dry weight (DW). Preculture treatment in 0.5 M sucrose medium was the most effective method for inducing a high level of desiccation tolerance in cells and the survival rate was 87% even after being desiccated to below 0.1 g H(2)O g(-1) DW. Preculture treatment caused alteration of cell structures and accumulation of a large amount of sucrose and newly synthesized proteins in cells. Abundant sucrose and preculture-induced proteins were necessary for full development of desiccation tolerance in the cells. When water content decreased to below 0.1 g H(2)O g(-1) DW, desiccation-tolerant cells that had been precultured were vitrified above 0 degrees C and maintained stable viability. We have succeeded in the induction of desiccation tolerance that allows formation of intracellular glass with cell viability at ambient temperatures by controlling culture conditions, and our results suggest that suspension-cultured cells of M. polymorpha are useful for studying cellular mechanisms for the development of desiccation tolerance and the stabilization of vitrified cells.

  17. Establishment of Suspension Cell Culture from Agrobacterium-transformed Hairy Root Cells of Psammosilene tunicoides, an Endangered and Rare Medicinal Plant of China

    Directory of Open Access Journals (Sweden)

    Zhang Zong-Shen

    2015-08-01

    Full Text Available Psammosilene tunicoides is an important medicinal plant endemic in China. Its annual yield is severely limited due to slow growth, poor seed germination and excessive collection. To satisfy the growing market demands, it’s necessary to seek alternatives to field cultivation and wild resources of this endangered plant. Using Agrobacterium -transformed hairy roots as initial explants, here, we reported the development of a suspension cell culture system for P. tunicoides. Results showed the Agrobacterium -transformed hairy roots-derived suspension cells are fast in growth and strong in capacity for accumulation of bioactive metabolites. We established that 1/2MS was a suitable medium for culturing the hairy root-derived suspension cells and the optimal combination of phytohormones is 1.5 mg/L 2, 4-D+0.5 mg/L 6-BA+0.25 mg/L NAA+0.1 mg/L KT. Under this condition, the maximal biomass was achieved at the 20th day of culture with an average growth rate of 0.72 g/L/d; and the intracellular saponine content reached 0.92%, comparable to that of mother hairy roots. Compared with the normal P. tunicoides suspension cells, the hairy roots-derived suspension cells exhibited features of fast growth, short culture period and high concentration of saponines, suggesting that the large scale culture of hairy root-derived cells could be a feasible alternative to the wild resources of P. tunicoides.

  18. Phytoalexin Induction in French Bean : Intercellular Transmission of Elicitation in Cell Suspension Cultures and Hypocotyl Sections of Phaseolus vulgaris.

    Science.gov (United States)

    Dixon, R A; Dey, P M; Lawton, M A; Lamb, C J

    1983-02-01

    Treatment of hypocotyl sections or cell suspension cultures of dwarf French bean (Phaseolus vulgaris L.) with an abiotic elicitor (denatured ribonuclease A) resulted in increased extractable activity of the enzyme l-phenylalanine ammonia-lyase. This induction could be transmitted from treated cells through a dialysis membrane to cells which were not in direct contact with the elicitor. In hypocotyl sections, induction of isoflavonoid phytoalexin accumulation was also transmitted across a dialysis membrane, although levels of insoluble, lignin-like phenolic material remained unchanged in elicitor-treated and control sections. In bean cell suspension cultures, the induction of phenylalanine ammonia-lyase in cells separated from ribonuclease-treated cells by a dialysis membrane was also accompanied by increases in the activities of chalcone synthase and chalcone isomerase, two enzymes previously implicated in the phytoalexin defense response. Such intercellular transmission of elicitation did not occur in experiments with cells treated with a biotic elicitor preparation heat-released from the cell walls of the bean pathogen Colletotrichum lindemuthianum. The results confirm and extend previous suggestions that a low molecular weight, diffusible factor of host plant origin is involved (in French bean) in the intercellular transmission of the elicitation response to abiotic elicitors.

  19. Differential patterns of arabinosylation by membranes of suspension-cultured cells of Phaseolus vulgaris (French bean) after subculture or elicitation.

    Science.gov (United States)

    Bolwell, G P

    1984-09-01

    Suspension-cultured cells of Phaseolus vulgaris (French bean) incorporated [1-3H] arabinose in vivo into high-Mr polymers that could be separated into glycoprotein and polysaccharide. Microsomal membranes from suspension-cultured cells of beans incorporated arabinose from UDP-beta-L-arabinose in vitro into both polysaccharide and glycoprotein. The enzyme involved in arabinan synthesis, arabinan synthase, appeared to be immunologically distinct from the protein:arabinosyltransferase system. Both these activities are inducible, but behave differently with either plant-growth-regulator or fungal-elicitor treatments. After subculture of cells entering the stationary growth phase the arabinan synthase activity reaches much higher values than does that of the protein transferase system during the initial period of cell division and growth, whereas after elicitation at the same growth stage, all the increased incorporation of arabinose occurs into glycoprotein of Mr higher than 200 000 and to a greater extent into a specific glycoprotein of Mr 42 500. Preliminary characterization of these glycoproteins prepared under non-reducing conditions and after acid and alkaline hydrolysis suggests that the high-Mr glycoprotein material is similar to arabinogalactan protein, whereas the lower-Mr material may be a hydroxyproline-rich protein existing as a dimer and that specifically increases during the hypersensitive response of the cells to the fungal elicitor from Colletotrichum lindemuthianum.

  20. A β-secretase (BACE1)-inhibiting C-methylrotenoid induced by yeast elicitation in Abronia nana suspension cultures.

    Science.gov (United States)

    Kim, Sang-In; Yang, Eun-Ju; Park, Se-Hoon; Kim, Chang-Kil; Song, Kyung-Sik

    2014-04-01

    Suspension cultures of Abronia nana were established to produce C-methylisoflavones. Treatment of the A. nana cultures with yeast elicitor induced boeravinone E (1), with maximum induction at 24 h after elicitor treatment. Of the biotic and abiotic elicitors tested, yeast extract gave the strongest induction of 1. The IC50 value of 1 against β-secretase (β-amyloid cleaving enzyme-1) was 5.57 μM. Other proteases such as trypsin, chymotrypsin, and elastase were not inhibited by concentrations up to 1.0 mM, indicating that inhibition of β-secretase was specific. 1 was noncompetitive in Dixon plot, and Ki value was 3.79 μM.

  1. Maize black Mexican sweet suspension cultured cells are a convenient tool for studying aquaporin activity and regulation.

    Science.gov (United States)

    Cavez, Damien; Hachez, Charles; Chaumont, François

    2009-09-01

    Aquaporins (AQPs) are channel proteins that facilitate and regulate the permeation of water across biological membranes. Black Mexican sweet suspension cultured cells are a convenient model for studying the regulation of maize AQP expression and activity. Among other advantages, a single cell system allows the contribution of plasma membrane AQPs (PIPs, plasma membrane intrinsic proteins) to the membrane water permeability coefficient (P(f)) to be determined using biophysical measurement methods, such as the cell pressure probe or protoplast swelling assay. We generated a transgenic cell culture line expressing a tagged version of ZmPIP2;6 and used this material to demonstrate that the ZmPIP2;6 and ZmPIP2;1 isoforms physically interact. This kind of interaction could be an additional mechanism for regulating membrane water permeability by acting on the activity and/or trafficking of PIP hetero-oligomers.

  2. Regulation of glutamate dehydrogenase activity in relation to carbon limitation and protein catabolism in carrot cell suspension cultures.

    Science.gov (United States)

    Robinson, S A; Stewart, G R; Phillips, R

    1992-03-01

    Glutamate dehydrogenase (GDH) specific activity and function have been studied in cell suspension cultures of carrot (Daucus carota L. cv Chantenay) in response to carbon and nitrogen supply in the culture medium. The specific activity of GDH was derepressed in sucrose-starved cells concomitant with protein catabolism, ammonium excretion, and the accumulation of metabolically active amino acids. The addition of sucrose led to a rapid decrease in GDH specific activity, an uptake of ammonium from the medium, and a decrease in amino acid levels. The extent of GDH derepression was correlated positively with cellular glutamate concentration. These findings strengthen the view that the function of GDH is the catabolism of glutamate, which under conditions of carbon stress provides carbon skeletons for tricarboxylic acid cycle activity.

  3. Expression of hemagglutinin protein from the avian influenza virus H5N1 in a baculovirus/insect cell system significantly enhanced by suspension culture

    Directory of Open Access Journals (Sweden)

    Spencer Lynn

    2006-02-01

    Full Text Available Abstract Background Prevention of a possible avian influenza pandemic necessitates the development of rapid diagnostic tests and the eventual production of a vaccine. Results For vaccine production, hemagglutinin (HA1 from avian influenza H5N1 was expressed from a recombinant baculovirus. Recombinant HA1 was expressed in monolayer or suspension culture insect cells by infection with the recombinant baculovirus. The yield of rHA1 from the suspension culture was 68 mg/l, compared to 6 mg/l from the monolayer culture. Immunization of guinea pigs with 50 μg of rHA1 yielded hemagglutinin inhibition and virus neutralization titers of 1:160 after two times vaccination with rHA1 protein. Conclusion Thus, the production of rHA1 using an insect suspension cell system provides a promising basis for economical production of a H5 antigen.

  4. Opposite extremes in ethylene/nitric oxide ratio induce cell death in suspension culture and root apices of tomato exposed to salt stress.

    Science.gov (United States)

    Poór, P; Borbély, P; Kovács, Judit; Papp, Anita; Szepesi, Ágnes; Takács, Z; Tari, Irma

    2014-12-01

    The plant hormone ethylene or the gaseous signalling molecule nitric oxide (NO) may enhance salt stress tolerance by maintaining ion homeostasis, first of all K+/Na+ ratio of tissues. Ethylene and NO accumulation increased in the root apices and suspension culture cells of tomato at sublethal salt stress caused by 100 mM NaCl, however, the induction phase of programmed cell death (PCD) was different at lethal salt concentration. The production of ethylene by root apices and the accumulation of NO in the cells of suspension culture did not increase during the initiation of PCD after 250 mM NaCl treatment. Moreover, cells in suspension culture accumulated higher amount of reactive oxygen species which, along with NO deficiency contributed to cell death induction. The absence of ethylene in the apical root segments and the absence of NO accumulation in the cell suspension resulted in similar ion disequilibrium, namely K+/Na+ ratio of 1.41 ± 0.1 and 1.68 ± 0.3 in intact plant tissues and suspension culture cells, respectively that was not tolerated by tomato.

  5. Elicitation of Jerusalem artichoke (Helianthus tuberosus L.) cell suspension culture for enhancement of inulin production and altered degree of polymerisation.

    Science.gov (United States)

    Ma, Chunquan; Zhou, Dong; Wang, Haitao; Han, Dongming; Wang, Yang; Yan, Xiufeng

    2017-01-01

    Plant cell suspension cultures have emerged as a potential source of secondary metabolites for food additives and pharmaceuticals. In this study inulin accumulation and its degree of polymerisation (DP) in the treated cells in the same medium were investigated after treatment with six types of elicitors. An in vitro cell suspension culture of Jerusalem artichoke (Helianthus tuberosus L.) was optimised by adding an extra nitrogen source. According to the growth kinetics, a maximum biomass of 5.48 g L(-1) was obtained from the optimal cell suspension medium consisted of Murashige and Skoog basic medium (MS) + 1.0 mg L(-1) α-naphthalene acetic acid (NAA) + 1.0 mg L(-1) 6-benzylaminopurine (6-BA) + 0.5 mg L(-1) proline + 1.0 mg L(-1) glutamine. Methyl jasmonate (MeJA, 250 µmol L(-1) ) treatment for 15 days led to the highest levels of inulin (2955.27 ± 9.81 mg L(-1) compared to control of 1217.46 ± 0.26 mg L(-1) ). The elicited effect of five elicitors to the suspension cells of Jerusalem artichoke is as follows: AgNO3 (Ag, 10 µmol L(-1) ), salicylic acid (SA, 75 µmol L(-1) ), chitosan (KJT, 40 mg L(-1) ), Trichoderma viride (Tv, 90 mg L(-1) ), yeast extract (YE, 0.25 mg L(-1) ), and the corresponding content of inulin is increased by 2.05-, 1.93-, 1.76-, 1.44- and 1.18-fold compared to control, respectively. The obvious effect on the percentage of lower DP in inulin was observed in cells treated with 40 mg L(-1) KJT, 0.25 mg L(-1) YE and 10 µmol L(-1) Ag. Among the six types of elicitors, the descending order of inulin content is MeJA > Ag > SA > KJT > Tv > YE. For the purpose inulin with lower DP and its application to prebiotic food, three elicitors, including KJT, YE and Ag, can be used for the elicitation. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  6. Inducible packaging cells for large-scale production of lentiviral vectors in serum-free suspension culture.

    Science.gov (United States)

    Broussau, Sophie; Jabbour, Nadine; Lachapelle, Guillaume; Durocher, Yves; Tom, Rosanne; Transfiguracion, Julia; Gilbert, Rénald; Massie, Bernard

    2008-03-01

    We have developed new packaging cell lines (293SF-PacLV) that can produce lentiviral vectors (LVs) in serum-free suspension cultures. A cell line derived from 293SF cells, expressing the repressor (CymR) of the cumate switch and the reverse transactivator (rtTA2(S)-M2) of the tetracycline (Tet) switch, was established first. We next generated clones stably expressing the Gag/Pol and Rev genes of human immunodeficiency virus-1, and the glycoprotein of vesicular stomatitis virus (VSV-G). Expression of Rev and VSV-G was tightly regulated by the cumate and Tet switches. Our best packaging cells produced up to 2.6 x 10(7) transducing units (TU)/ml after transfection with the transfer vector. Up to 3.4 x 10(7) TU/ml were obtained using stable producers generated by transducing the packaging cells with conditional-SIN-LV. The 293SF-PacLV was stable, as shown by the fact that some producers maintained high-level LV production for 18 weeks without selective pressure. The utility of the 293SF-PacLV for scaling up production in serum-free medium was demonstrated in suspension cultures and in a 3.5-L bioreactor. In shake flasks, the best packaging cells produced between 3.0 and 8.0 x 10(6) TU/ml/day for 3 days, and the best producer cells, between 1.0 and 3.4 x 10(7) TU/ml/day for 5 days. In the bioreactor, 2.8 liters containing 2.0 x 10(6) TU/ml was obtained after 3 days of batch culture following the transfection of packaging cells. In summary, the 293SF-PacLV possesses all the attributes necessary to become a valuable tool for scaling up LV production for preclinical and clinical applications.

  7. Characterization of an immunomodulatory Der p 2-FIP-fve fusion protein produced in transformed rice suspension cell culture.

    Science.gov (United States)

    Su, Chin-Fen; Kuo, I-Chun; Chen, Peng-Wen; Huang, Chiung-Hui; Seow, See Voon; Chua, Kaw Yan; Yu, Su-May

    2012-02-01

    Der p 2, a major allergen of Dermatophagoides pteronyssinus mites, is one of the most clinically relevant allergens to allergic patients worldwide. FIP-fve protein (Fve) from the golden needle mushroom (Flammulina velutipes) is an immunomodulatory protein with potential Th1-skewed adjuvant properties. Here, we produced and immunologically evaluated a Der p 2-Fve fusion protein as a potential immunotherapeutic for allergic diseases. Using an inducible expression system in cultured rice suspension cells, the recombinant Der p 2-Fve fusion protein (designated as OsDp2Fve) was expressed in rice cells under the control of an α-amylase gene (αAmy8) promoter and secreted under sucrose starvation. OsDp2Fve was partially purified from the cultured medium. The conformation of Der p 2 in OsDp2Fve remains intact as reflected by its unaltered allergenicity, as assessed by human IgE ELISA and histamine release assays, compared to non-fusion Der p 2 protein. Furthermore, the Fve protein expressed in OsDp2Fve retains its in vitro lymphoproliferative activity but loses its hemagglutination and lymphoagglutination effects compared to the native protein. Notably, in vivo evaluation showed that mice administered with OsDp2Fve possessed an enhanced production of Der p 2-specific IgG antibodies without potentiating the production of Der p 2-specific IgE and Th2 effector cytokines in comparison with mice co-administered with native Fve and Der p 2 proteins. These results suggest that the recombinant Der p 2-Fve fusion protein produced in rice suspension cell cultures has a great potential for allergy immunotherapy.

  8. Three-dimensional Expansion: In Suspension Culture of SD Rat's Osteoblasts in a Rotating Wall Vessel Bioreactor

    Institute of Scientific and Technical Information of China (English)

    KE-DONG SONG; TIAN-QING LIU; XIANG-QIN LI; ZHAN-FENG CUI; XIANG-YU SUN; XUE-HU MA

    2007-01-01

    Objective To study large-scale expansion of SD (Sprague-Dawley) rat's osteoblasts in suspension culture in a rotating wall vessel bioreactor (RWVB). Methods The bioreactor rotation speeds were adjusted in the range of 0 to 20 rpm,which could provide low shear on the microcarriers around 1 dyn/cm2. The cells were isolated via sequential digestions of neonatal (less than 3 days old) SD rat calvaria. After the primary culture and several passages, the cells were seeded onto the microcarriers and cultivated in T-flask, spinner flask and RWVB respectively. During the culture period, the cells were counted and observed under the inverted microscope for morphology every 12 h. After 7 days, the cells were evaluated with scanning electron microscope (SEM) for histological examination of the aggregates. Also, the hematoxylin-eosin (HE) staining and alkaline phosphatase (ALP) staining were performed. Moreover, von-Kossa staining and Alizarin Red S staining were carried out for mineralized nodule formation. Results The results showed that in RWVB, the cells could be expanded by more than ten times and they presented better morphology and vitality and stronger ability to form bones. Conclusions The developed RWVB can provide the culture environment with a relatively low shear force and necessary three-dimensional (3D)interactions among cells and is suitable for osteopath expansion in vitro.

  9. Identification and quantitative determination of pinoresinol in Taxus ×media Rehder needles, cell suspension and shoot cultures

    Directory of Open Access Journals (Sweden)

    Paulina Mistrzak

    2015-01-01

    Full Text Available The aim of our study was to investigate the presence and quantitative contents of lignans in the tissues of Taxus ×media. The presence of the lignans: pinoresinol, matairesinol and secoisolariciresinol was assessed in needles, shoots cultures and suspension culture. Pinoresinol was the only lignan found in the tissue of T. ×media. The total pinoresinol content in the needles and in the shoots was 1.24 mg/g dry weight (dw and 0.69 mg/g dw, respectively. Most of the pinoresinol identified was appeared glycosidically bound. In needles, the amount of glycosidically bound pinoresinol (0.81 mg/g dw was about twice as high as that of free pinoresinol (0.43 mg/g dw. The content of free and glycosidically bound pinoresinol showed the level of 0.18 mg/g dw and 0.51 mg/g dw, respectively in the in vitro shoot cultures. In the cell culture, no pinoresinol was found.

  10. Changes in auxin level in the course of growth of a sunflower crown-gall suspension culture

    Directory of Open Access Journals (Sweden)

    Zofia Chirek

    2014-01-01

    Full Text Available The auxin level in the cell mass and culture medium was determined by means of the Avena straight caleoptile test in various periods of the suspension culture cycle of the sunflower crown-gall tumour. The investigations were performed in the course of the zero passage (PO and first one (Pl, differing in their time of duration of maximum growth and its intensity. In both passages the intra- and extra-cellular auxin levels reach values of the same order. At the beginning of the maximal growth phase the activity corresponding to IAA in the cells prevails over that of the other auxin-like compounds. This disproportion diminishes with further development of the culture, and with the beginning of the stationary phase the cellular IAA level is lower than that of the remaining auxin-like compounds. The short phase of maximal growth (PO occurs with an auxin level decreasing in the cell mass and increasing in the medium, and towards the end of the cycle these levels become equal. During the long phase of maximal growth (Pl the total amount of auxins in the cells increases and is 2-3 times higher than in the medium, whereas IAA in the cells remains at a constant level. These results suggest that the participation of IAA in the intracellular pool of auxin-like substances is decisive for the mitotic activity of the cells and maintenance of growth in the culture.

  11. Feedback regulation of beta-thujaplicin production and formation of its methyl ether in a suspension culture of Cupressus lusitanica.

    Science.gov (United States)

    Yamada, Junko; Fujita, Koki; Sakai, Kokki

    2002-07-01

    Suspension cell cultures of Cupressus lusitanica produce beta-thujaplicin, a tropolone found mostly in Cupressaceae heartwood. The factors controlling beta-thujaplicin accumulation in this cell culture system were investigated. Initial cell density of the cultures did not affect beta-thujaplicin levels, though initial addition of beta-thujaplicin suppressed its de novo production. When beta-thujaplicin accumulation reached a certain level (ca. 40 mg/l) in the medium, the cultures seemed to cease beta-thujaplicin production. However, beta-thujaplicin productivity was restored when the beta-thujaplicin-containing medium was exchanged for fresh medium; the formation of 2-methoxy-6-(methylethyl)cyclohepta-2,4,6-trien-1-one, an isomer of methylated beta-thujaplicin, in medium was also observed. These results suggest that beta-thujaplicin synthesis was regulated by product feedback mechanism in this cell line, and that excess accumulation of beta-thujaplicin is relieved by conversion of beta-thujaplicin to its methyl ether.

  12. [Establishment of embryogenic cell suspension culture and plant regeneration of edible banana Musa acuminata cv. Mas (AA)].

    Science.gov (United States)

    Wei, Yue-Rong; Huang, Xue-Lin; Li, Jia; Huang, Xia; Li, Zhe; Li, Xiao-Ju

    2005-01-01

    Conventional breeding for dual resistance of disease and pest of Musa cultivars remains a difficult endeavor, as the plant is polyploidic and high in sterility. Biotechnological techniques, eg., genetic engineering, in vitro mutation breeding, or protoplast fusion, may overcome the difficulties and improve the germplasm. Establishment of a stable embryogenic cell suspension (ECS) is a prerequisite for any of the biotechnological breeding methods. In this study an embryogenic cell suspension was established from immature male flower of Musa acuminata cv. Mas (AA), a popular commercial variety of banana in the South-East Asian region. After culture for 5-6 months on callus induction media, which consisted of MS salts, different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), 4.1 micromol/L biotin, 5.7 micromol/L indoleacetic acid (IAA), 5.4 micromol/L naphthaleneacetic acid (NAA), other vitamins, 87 mmol/L sucrose, and solidified with 7 g/L agarose, meristematic globules and yellow, friable embryogenic cultures were induced from the explants of 1-15th row young floral hands of immature male flowers. Of the four treatments of 2,4-D, 9 micromol/L was the most effective on the callus induction, it transformed 40.96% and 7.45% of the cultivated male floral hands into callus and embryogenic callus respectively. The explants to produce highest frequency of the embryogenic calli were floral hands of 6 to 12th rows, which generated 5.79% of the embryogenic calli. Suspension cultures were initiated from these embryogenic calli in liquid medium supplemented with 4.5 micromol/L 2, 4-D. After sieving selection of the cultures using a stainless steel metallic strainer with pore sizes of 154 microm at 15 day intervals for 3 months, homogeneous and yellow embryogenic cell suspensions, composed of single cells and small cell aggregates, were established. Based upon the growth quantity and growth rate of ECS, it was determined that the appropriate inoculum was 2.0 mL PCV

  13. Effect of phenylalanine on Taxol production and antioxidant activity of extracts of suspension-cultured hazel (Corylus avellana L.) cells.

    Science.gov (United States)

    Bemani, Ebrahim; Ghanati, Faezeh; Rezaei, Ayatollah; Jamshidi, Mitra

    2013-07-01

    Taxol is produced by a few microorganisms and plants such as yew (Taxus sp.). Recent researches have shown that hazel (Corylus avellana L.) is also able to produce Taxol. In the present study, effects of different concentrations of phenylalanine (Phe) on the production of Taxol, antioxidant activity, and cytotoxic effects of extracts of suspension-cultured hazel cells were investigated. The cells were treated with different concentrations of Phe on day 7 of subculture and were harvested on day 14. The results showed that the amounts of Taxol and antioxidant activity were increased by increasing the phenylalanine supply. Interestingly, the cytotoxic effects of hazel cell extract were even stronger than that of pure Taxol (standard), suggesting hazel cell extract as a novel and suitable probe for treating human cancer. Application of phenylalanine to hazel cells exaggerates their effects.

  14. Distinct isoforms of ADPglucose pyrophosphatase and ADPglucose pyrophosphorylase occur in the suspension-cultured cells of sycamore (Acer pseudoplatanus L.).

    Science.gov (United States)

    Baroja-Fernández, E; Zandueta-Criado, A; Rodríguez-López, M; Akazawa, T; Pozueta-Romero, J

    2000-09-01

    The intracellular localizations of ADPglucose pyrophosphatase (AGPPase) and ADPglucose pyrophosphorylase (AGPase) have been studied using protoplasts prepared from suspension-cultured cells of sycamore (Acer pseudoplatanus L.). Subcellular fractionation studies revealed that all the AGPPase present in the protoplasts is associated with amyloplasts, whereas more than 60% of AGPase is in the extraplastidial compartment. Immunoblots of amyloplast- and extraplastid-enriched extracts further confirmed that AGPase is located mainly outside the amyloplast. Experiments carried out to identify possible different isoforms of AGPPase in the amyloplast revealed the presence of soluble and starch granule-bound isoforms. We thus propose that ADPglucose levels linked to starch biosynthesis in sycamore cells are controlled by enzymatic reactions catalyzing the synthesis and breakdown of ADPglucose, which take place both inside and outside the amyloplast.

  15. Structure of Plant Cell Walls : XVIII. An Analysis of the Extracellular Polysaccharides of Suspension-Cultured Sycamore Cells.

    Science.gov (United States)

    Stevenson, T T; McNeil, M; Darvill, A G; Albersheim, P

    1986-04-01

    The water-soluble polysaccharides (SEPS) secreted into the medium by suspension-cultured sycamore cells were examined to determine whether the polysaccharides were the same as those present in the walls of sycamore cells. The SEPS were made more amenable to fractionation by treatment with a highly purified alpha-1,4-endopolygalacturonase (EPG). The EPG-treated SEPS were fractionated by anion-exchange and gelpermeation chromatography. The following polysaccharides were found: xyloglucan, arabinoxylan, at least two arabinogalactans, a rhamnogalacturonan-II-like polysaccharide, and a polygalacturonic acid-rich polysaccharide. The oligogalacturonide fragments expected from EPG-digested homogalacturonan were also identified. Evidence was obtained for the presence of a rhamnogalacturonan-I-like polysaccharide. All of the above polysaccharides have been isolated from or are believed to be present in sycamore cell walls. Furthermore, all of the noncellulosic polysaccharides known to be present in sycamore cell-walls appear to be present in the SEPS.

  16. Im"plant"ing of Mammalian Glycosyltransferase Gene into Plant Suspension-Cultured Cells Using Agrobacterium-Mediated Transformation.

    Science.gov (United States)

    Kajiura, Hiroyuki; Fujiyama, Kazuhito

    2015-01-01

    Enzymatic activity assay of exogenous glycosyltransferase (GT) and glycosylhydrolase (GH) expressed in plants is an important analysis for determination of the expression of the gene of interest. However, generations and establishment of in planta transgenic lines are time-consuming. Furthermore, the expression levels and the activities of the exogenous GTs and GHs are quite low and weak, the radiolabeled donor substrate had to be used to analyze the enzymatic activity. Here, we describe a protocol for the generation of transgenic plants using suspension-cultured cells and a high sensitive assay for GT, especially β1,4-galactosyltransferase, using microsomal fraction from plant cells and fluorescent-labeled sugar chains as an acceptor substrate. This method enables less-time-consuming preparation of stable transgenic plants, non-radiolabeled, high-throughput detail analysis which includes mass spectrometric analysis and exo-glycosidase digestions.

  17. Statistical optimization of influenza H1N1 production from batch cultures of suspension Vero cells (sVero).

    Science.gov (United States)

    Paillet, Cristian; Forno, Guillermina; Soldano, Nicolas; Kratje, Ricardo; Etcheverrigaray, Marina

    2011-09-22

    Efficient vaccine production requires the growth of large quantities of virus produced with high yield from a safe host system. Human influenza vaccines are produced in embryonated chicken eggs. However, over the last decade many efforts have allowed the establishment of cell culture-derived vaccines. We generated a Vero cell line adapted to grow in suspension (sVero) in a serum-free medium and evaluated it for its potential as host cell for influenza vaccine production. Initially we studied the capacity of sVero cells to grow in the presence of incremental concentrations of trypsin. In comparison with adherent Vero cells (aVero), we found that sVero cells maintain their growth kinetics even with a three-fold increase in trypsin concentration. The influence of the conditions of infection on the yield of H1N1 produced in serum-free suspension cultures of sVero cells was investigated by a 2(2) full factorial experiment with center point. Each experiment tested the influence of the multiplicity of infection (m.o.i.) and trypsin concentration, on production yields at two levels, in four possible combinations of levels and conditions, plus a further combination in which each condition was set in the middle of its extreme levels. On the basis of software analysis, a combination of m.o.i. of 0.0066TCID(50%)/cell and trypsin concentration of 5μg/1.0×10(6) cells with a desirability of 0.737 was selected as the optimized condition for H1N1 production in sVero cells. Our results show the importance of proper selection of infection conditions for H1N1 production on sVero cells in serum-free medium. Copyright © 2011 Elsevier Ltd. All rights reserved.

  18. Improved Biodegradation of 1,2,4-Trichlorobenzene by Adapted Microorganisms in Agricultural Soil and in Soil Suspension Cultures

    Institute of Scientific and Technical Information of China (English)

    SONG Yang; WANG Fang; F. O. KENGARA; BIAN Yong-Rong; YANG Xing-Lun; LIU Cui-Ying; JIANG Xin

    2011-01-01

    Inoculating soil with an adapted microbial community is a more effective bioaugrnentation approach than inoculation with pure strains in bioremediation.However,information on the potential of different inocula from sites with varying contamination levels and pollution histories in soil remediation is lacking.The objective of the study was to investigate the potential of adapted microorganisms in soil inocula,with different contamination levels and pollution histories,to degrade 1,2,4-trichlorobenzene (1,2,4-TCB).Three different soils from chlorobenzene-contaminated sites were inoculated into agricultural soils and soil suspension cultures spiked with 1,2,4-TCB.The results showed that 36.52% of the initially applied 1,2,4-TCB was present in the non-inoculated soil,whereas about 19.00% of 1,2,4-TCB was present in the agricultural soils inoculated with contaminated soils after 28 days of incubation.The soils inoculated with adapted microbial biomass (in the soil inocula) showed higher respiration and lower 1,2,4-TCB volatilization than the non-inoculated soils,suggesting the existence of 1,2,4-TCB adapted degraders in the contaminated soils used for inoculation.It was further confirmed in the contaminated soil suspension cultures that the concentration of inorganic chloride ions increased continuously over the entire experimental period.Higher contamination of the inocula led not only to higher degradation potential but also to higher residue formation.However,even inocula of low-level contamination were effective in enhancing the degradation of 1,2,4-TCB.Therefore,applying adapted microorganisms in the form of soil inocula,especially with lower contamination levels,could be an effective and environment-friendly strategy for soil remediation.

  19. In vitro production of azadirachtin from cell suspension cultures of Azadirachta indica

    Indian Academy of Sciences (India)

    S Sujanya; B Poornasri Devi; Isha Sai

    2008-03-01

    The present study aimed to elucidate the effect of nutritional alteration on biomass content and azadirachtin production in cell suspensions of the elite neem variety crida-8. Variations in total nitrogen availability in the medium in terms of different ratios of nitrate:ammonium showed that the ratio 4:1 revealed a profound effect, leading to a 1.5-fold increase in the total extracellular azadirachtin production (5.59 mg/l) over the standard MS medium. Reduction in sucrose (15 mg/l) in the medium exhibited a reduction in biomass and absence of azadirachtin, whereas total phosphate reduction raised intracellular azadirachtin production (6.98 mg/l). An altered medium with a nitrate:ammonium ratio of 4:1 coupled with complete elimination of phosphate enhanced biomass by 36% (59.36 g/l).

  20. [Enhanced production of taxuyunnanine c in cell suspension cultures of Taxus chinensis by methyl jasmonate elicitation and in situ absorption].

    Science.gov (United States)

    Gao, Mingbo; Zhang, Wei; Yu, Xingju

    2010-02-01

    A bioprocess intensification strategy that combines both elicitation and in situ absorption was developed to improve the production of taxuyunnanine c (Tc) in cell suspension cultures of Taxus chinensis. When 100 micromol/L methyl jasmonate was added as an elicitor on Day 7, the Tc content and yield increased 3.6 and 3.3 times respectively, however the cell growth was reduced by 10%-30%. Significant improvement in Tc yield was observed when an absorbent XAD-7 was added on different time of the culture period. The optimum Tc yield was achieved when 100 g/L XAD-7 was added simultaneously with 100 micromol/L methyl jasmonate on Day 7. The maximum Tc yield of 477.4 mg/L was obtained on Day 21 of the culture, being 6.3-fold of the control and 1.9-fold of the 100 micromol/L methyl jasmonate treatment alone. In the combined treatment, 94% of the Tc produced was secreted outside of the cells and absorbed on XAD-7 absorbents. The results demonstrated that the process strategy combining elicitation and in situ absorption was effective to intensify the Tc biosynthesis via elicitation with the removal of product feedback inhibition via absorption, presenting a great potential in commercial applications.

  1. Hydrodynamic stress induces monoterpenoid oxindole alkaloid accumulation by Uncaria tomentosa (Willd) D. C. cell suspension cultures via oxidative burst.

    Science.gov (United States)

    Trejo-Tapia, Gabriela; Sepúlveda-Jiménez, Gabriela; Trejo-Espino, José Luis; Cerda-García-Rojas, Carlos M; de la Torre, Mayra; Rodríguez-Monroy, Mario; Ramos-Valdivia, Ana C

    2007-09-01

    Uncaria tomentosa cell suspension cultures were grown in a 2-L stirred tank bioreactor operating at a shear rate gamma(.)(avg)=86 s(-1). The cultures showed an early monophasic oxidative burst measured as H2O2 production (2.15 micromol H2O2 g(-1) dw). This response was followed by a transient production of monoterpenoid oxindole alkaloids (178 +/- 40 microg L(-1) at 24 h). At the stationary phase (144 h), the increase of the shear rate gamma(.)(avg) up to 150 s(-1) and/or oxygen tension up to 85% generated H2O2, restoring oxindole alkaloid production. U. tomentosa cells cultured in Erlenmeyer flasks also exhibited the monophasic oxidative burst but the H2O2 production was 16-fold lower and the alkaloids were not detected. These cells exposed to H2O2 generated in situ produced oxindole alkaloids reaching a maximum of 234 +/- 40 microg L(-1). A positive correlation was observed between the oxindole alkaloid production and the endogenous H2O2 level. On the other hand, addition of 1 microM diphenyleneiodonium (NAD(P)H oxidase inhibitor) or 10 microM sodium azide (peroxidases inhibitor) reduced both H2O2 production and oxindole alkaloids build up, suggesting that these enzymes might play a role in the oxidative burst induced by the hydrodynamic stress.

  2. Role of Changes in Cell Fatty Acids Composition in the Increasing of Frost Resistance of Winter Wheat Suspension Culture

    Directory of Open Access Journals (Sweden)

    I.V. Lyubushkina

    2013-11-01

    Full Text Available Influences of low temperatures (4 and 8 ° С on the frost tolerance and fatty acid compositions of cells in a winter wheat suspension culture have been studied. It has been found that treatment of the culture with 4 °C (7 days did not protect cells from subsequent freezing temperature action (-8 °С, 6 h and was not accompanied significant changes in the fatty acid composition. On the contrary, the treatment of the culture with the temperature 8 °C (7 days prevented the death caused by freezing temperature and the content of saturated fatty acids decreased: pentadecanoic acid (by 35,0%, palmitic acid (by 19,9% and stearic acid (by 65,4%, and the content of α-linolenic acid increased by 94%. That was the cause of the double bond index (DBI increase by 16%. The role of fatty acids composition changes in the process of increasing frost tolerance in plants are discussed.

  3. Widespread occurrence of a covalent linkage between xyloglucan and acidic polysaccharides in suspension-cultured angiosperm cells.

    Science.gov (United States)

    Popper, Zoë A; Fry, Stephen C

    2005-07-01

    Covalent linkages between xyloglucan and rhamnogalacturonan-I (RG-I) have been reported in the primary cell walls of cultured Rosa cells and may contribute to wall architecture. This study investigated whether this chemical feature is general to angiosperms or whether Rosa is unusual. * Xyloglucan was alkali-extracted from the walls of l-[1-3H]arabinose-fed suspension-cultured cells of Arabidopsis, sycamore, rose, tomato, spinach, maize and barley. The polysaccharide was precipitated with 50 % ethanol and subjected to anion-exchange chromatography in 8 m urea. Eluted fractions were Driselase-digested, yielding [3H]isoprimeverose (diagnostic of [3H]xyloglucan). The Arabidopsis cells were also fed [6-14C]glucuronic acid, and radiolabelled pectins were extracted with ammonium oxalate. * [3H]Xyloglucan was detected in acidic (galacturonate-containing) as well as non-anionic polysaccharide fractions. The proportion of the [3H]isoprimeverose units that were in anionic fractions was: Arabidopsis, 45 %; sycamore, 60 %; rose, 44 %; tomato, 75 %; spinach, 70 %; maize, 50 %; barley, 70 %. In Arabidopsis cultures fed d-[6-(14)C]glucuronate, 20 % of the (galacturonate-14C)-labelled pectins were found to hydrogen-bond to cellulose, a characteristic normally restricted to hemicelluloses such as xyloglucan. * Alkali-stable, anionic complexes of xyloglucan (reported in the case of Rosa to be xyloglucan-RG-I covalent complexes) are widespread in the cell walls of angiosperms, including gramineous monocots.

  4. Investigation and characterization of the duct cell-enriching process during serum-free suspension and monolayer culture using the human exocrine pancreas fraction.

    Science.gov (United States)

    Klein, Tino; Heremans, Yves; Heimberg, Harry; Pipeleers, Daniel; Madsen, Ole D; Serup, Palle; Heller, R Scott

    2009-01-01

    We aimed to characterize a serum-free culture system resulting in highly enriched duct cells from human exocrine pancreas. In addition, we tested the effect of vascular endothelial growth factor (VEGF) on endothelial cell proliferation and endocrine differentiation of the duct cells. The exocrine pellet fraction was cultivated in suspension followed by monolayer culture. Time course analysis of multiple acinar and duct cell markers was performed using reverse transcription-polymerase chain reaction and immunocytochemistry. The effects of VEGF and placental growth factor on the quantities of endothelial, duct, and endocrine cells and fibroblasts were investigated using computerized imaging analysis. Suspension culture of the exocrine material efficiently enriched the cultures for duct cells. Frequent acinar cell death as well as cell selective adherence of acinar cells to the culture dish was the underlying cause of the enrichment. Confocal microscopy demonstrated the virtual absence of cells coexpressing duct cell- and acinar cell-specific markers. The endothelial immunoreactivity of the suspension culture system could be increased 2-fold by VEGF treatment, yet no effect was observed on endocrine cell numbers. We have characterized a serum-free in vitro culture system to enrich human duct cells and further show that the contribution of acinoductal transdifferentiation to the enrichment of duct cells is negligible.

  5. Novel O-D-Galacturonoyl Esters in the Pectic Polysaccharides of Suspension-Cultured Plant Cells

    National Research Council Canada - National Science Library

    John A. Brown; Stephen C. Fry

    1993-01-01

    Driselase digestion of uronate-6- C-labeled primary walls of cultured spinach (Spinacia oleracea L.) cells yielded about 18 novel uronate-containing compounds, most of which could be hydrolyzed by cold dilute alkali to yield oligo...

  6. Optimization of callus and cell suspension cultures of Barringtonia racemosa (Lecythidaceae family) for lycopene production

    OpenAIRE

    Behbahani, Mandana; Shanehsazzadeh, Mehrnaz; Hessami,Mohamad Javad

    2011-01-01

    Lycopene is present in a range of fresh fruits and vegetables, especially in the leaves of Barringtonia racemosa. The traditional lycopene extraction from the plant is being employed instead of an easy propagation technique like cell culture process from the leaf explants. We intend to assess how lycopene could be extracted via tissue culture under light (illuminance: 8,200 lux under white fluorescent lamps, photoperiod 16 h per day at 25ºC) and dark. Leaf explants of Barringtonia racemosa we...

  7. INSECTICIDAL POTENTIALITY OF FLAVONOIDS FROM CELL SUSPENSION CULTURE OF MARCHANTIA LINEARIS LEHM. & LINDENB AGAINST SPODOPTERA LITURA F.

    Directory of Open Access Journals (Sweden)

    Remya Krishnan

    2015-05-01

    Full Text Available Bryophytes were diverse, primitive non vascular am phibious taxa distributed worldwide and form the second largest category of plants. Bryophytes synthesize an array of phytochemicals to combat against the unhospitable environmental conditions including predation, UV radiation, high temperature and pest and pathogens. The present investigation was undertaken to elucidate flavonoids from in vitro cell cultures of the liverwort Marchantia linearis Lehm. & Lindenb. its fractionation and analysis of insecticidal potentialities. Initially, callus culture was initiated from spores in MS/5 media containing gr owth regulators BAP and NAA at the concentration of 2 mg/L and 0.5 mg/L. Agitation of the friable callus at lowe r rpm bring about lower leve l of cell dispersion, on the contrary at higher rpm might have risk of cell collision that is why rpm was kept at moderate speed i.e., 110 rpm. Continuous sub culturing process substantially improves cell growth and biomass. In the second phase, the flavonoids were isolated from cell suspension cultures of M. linearis and were fractionated by TLC and HPLC PAD chromatogram, which revealed the presence of quer cetin, luteolin, apigenin , rutin and kaempferol. In vivo insecticidal analysis revealed significant antifeedan t, larvicidal and pupicidal activities at all the concentrations against 5 th instar larvae of Spodoptera litura . The extract also exhibited feeding deterrent activity with M. linearis. Similarly, the nutritional parameters were also affected i.e., reduced ECI (Efficiency of conversion of ingested food and ECD (Efficiency of conversion of digested food and increased AD (Approximate digestibility and metabolic cost for the larvae, when compared with the control. The consumption of the basal diet with the incorporation of flavonoids by S. litura larvae was not significantly different compared to the co nsumption of the control diet by the larvae. Faecal production reduced proportionally with

  8. Biochemical and morphological changes during the growth kinetics of Araucaria angustifolia suspension cultures

    Directory of Open Access Journals (Sweden)

    André Luis Wendt dos Santos

    2010-06-01

    Full Text Available Embryogenic cultures of Araucaria angustifolia were established in a BM liquid medium supplemented with 2 µM 2,4-dichlorophenoxyacetic acid, 1 µM 6-benzylaminopurine and 1 µM kinetin (BM2 and in a BM medium free of growth regulators (BM0. During 42 days in culture, the cell growth pattern of both cultures was similar. The pH of the culture medium of both BM0 and BM2 underwent progressive reduction during culture time. For both the embryogenic cultures a preferential uptake of glucose in the late stages of cell growth kinetics was observed. The extracellular protein content was similar for both the embryogenic cultures. Acetocarmine and Evan's blue double stain showed major differences for early somatic embryo organisation, in which only the embryogenic culture grown in a liquid culture medium free of plant growth regulators showed the presence of bipolar somatic pro-embryos.Culturas embriogênicas de Araucaria angustifolia foram estabelecidas em meio de cultura líquido BM suplementado com 2 µM Ácido 2,4 Diclorofenoxiacético, 1 µM 6-Benzilaminopurina e 1 µM Cinetina (BM2 e em meio BM isento de reguladores de crescimento (BM0. Durante 42 dias de cultivo, o padrão de crescimento celular em ambas as culturas foi similar. O pH do meio de cultura BM0 e BM2 sofreu uma progressiva redução durante o período de cultivo. Em ambas as culturas embriogênicas foram observadas um consumo preferencial de glicose no período final da curva de crescimento celular. O nível de proteínas extracelulares foi similar para ambas as culturas embriogênicas. A dupla coloração com carmin acético e azul de Evans revelou diferenças na organização das linhagens celulares embriogênicas, sendo que a presença de proembriões somáticos bipolares foi apenas evidenciada nas culturas embriogênicas mantidas em meio de cultura líquido sem reguladores de crescimento.

  9. 五加科药用植物悬浮培养技术研究进展%Advances in Study of Medicinal Plant Suspension Culture in Araliaceae

    Institute of Scientific and Technical Information of China (English)

    宋娟; 雷秀娟; 尹红新; 王英平

    2014-01-01

    The suspension culture of tissue or cells of medicinal plants in vitro is a very important means to realize the sustainable utilization of the medicinal plants in Araliaceae .Suspension cultivation is affected by the plants'own characters and the conditions of culture ,to identify the elements that influence the efficiency of culture clearly is essential .This study mainly introduced the characteristics of medicinal plant suspen-sion culture of Araliaceae and reactor culture ,analyzed the factors which affecte the cultivation and various culture conditions that may influence the medicinal composition .We also proposed the research direction of medicinal plant suspension culture of Araliaceae .%利用悬浮培养技术对组织或细胞进行离体培养,是实现五加科药用植物可持续利用的重要途径。本文主要介绍了五加科药用植物悬浮培养的特点和反应器培养进展,分析了影响悬浮培养的条件以及各种培养条件对药效成分的影响,认为植物自身特点和培养条件对悬浮培养的成效影响较大,因此,明确影响不同植物培养效率的内、外因素十分必要,并提出了未来五加科药用植物悬浮培养研究的方向。

  10. Selection and optimization of transfection enhancer additives for increased virus-like particle production in HEK293 suspension cell cultures.

    Science.gov (United States)

    Cervera, Laura; Fuenmayor, Javier; González-Domínguez, Irene; Gutiérrez-Granados, Sonia; Segura, Maria Mercedes; Gòdia, Francesc

    2015-12-01

    The manufacturing of biopharmaceuticals in mammalian cells typically relies on the use of stable producer cell lines. However, in recent years, transient gene expression has emerged as a suitable technology for rapid production of biopharmaceuticals. Transient gene expression is particularly well suited for early developmental phases, where several potential therapeutic targets need to be produced and tested in vivo. As a relatively new bioprocessing modality, a number of opportunities exist for improving cell culture productivity upon transient transfection. For instance, several compounds have shown positive effects on transient gene expression. These transfection enhancers either facilitate entry of PEI/DNA transfection complexes into the cell or nucleus or increase levels of gene expression. In this work, the potential of combining transfection enhancers to increase Gag-based virus-like particle production levels upon transfection of suspension-growing HEK 293 cells is evaluated. Using Plackett-Burman design of experiments, it is first tested the effect of eight transfection enhancers: trichostatin A, valproic acid, sodium butyrate, dimethyl sulfoxide (DMSO), lithium acetate, caffeine, hydroxyurea, and nocodazole. An optimal combination of compounds exhibiting the highest effect on gene expression levels was subsequently identified using a surface response experimental design. The optimal consisted on the addition of 20 mM lithium acetate, 3.36 mM valproic acid, and 5.04 mM caffeine which increased VLP production levels 3.8-fold, while maintaining cell culture viability at 94%.

  11. Enhanced extracellular production of trans-resveratrol in Vitis vinifera suspension cultured cells by using cyclodextrins and coronatine.

    Science.gov (United States)

    Almagro, Lorena; Belchí-Navarro, Sarai; Martínez-Márquez, Ascensión; Bru, Roque; Pedreño, María A

    2015-12-01

    In the present work the effect of cyclodextrin and coronatine on both trans-resveratrol production and the expression of stilbene biosynthetic genes in Vitis vinifera L. cv Monastrell suspension cultured cells were evaluated. The results showed the maximum level of trans-resveratrol produced by cells and secreted to the culture medium with 50 mM cyclodextrins and 1 μM coronatine. Since the levels of trans-resveratrol produced in the combined treatment were higher than the sum of the individual treatments, a synergistic effect between both elicitors was assumed. In addition, all the analysed genes were induced by cyclodextrins and/or coronatine. The expression of the phenylalanine ammonia lyase and stilbene synthase genes was greatly enhanced by coronatine although an increase in the amount of trans-resveratrol in the spent medium was not detected. Therefore, despite the fact that trans-resveratrol production is related with the expression of genes involved in the biosynthetic process, other factors may be involved, such as post-transcriptional and post-traductional regulation. The expression maximal levels of cinnamate 4-hydroxylase and 4-coumarate-CoA ligase genes were found with cyclodextrins alone or in combination with coronatine suggesting that the activity of these enzymes could be not only important for the formation of intermediates of trans-R biosynthesis but also for those intermediates involved in the biosynthesis of lignins and/or flavonoids. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  12. Elicitor-induced biosynthesis of psoralens in Ammi majus L. suspension cultures. Microsomal conversion of demethylsuberosin into (+)marmesin and psoralen.

    Science.gov (United States)

    Hamerski, D; Matern, U

    1988-01-15

    Suspension cultures of Ammi majus L. cells produce various linear furanocoumarins in response to treatment with elicitor preparations from either Alternaria carthami Chowdhury or Phytophthora megasperma f.sp. glycinea. Microsomes which were isolated from these cells 14 h after addition of the elicitor efficiently catalyzed the conversion of demethyl [3-14C]suberosin into labelled (+)marmesin in the presence of NADPH and oxygen. In contrast to the chemical cyclization of demethylsuberosin by m-chloroperoxybenzoic acid, the reaction catalyzed by the marmesin synthase proceeded rapidly and no intermediate demethylsuberosin epoxide could be recovered. Significant blue-light-reversible inhibition by carbon monoxide and inhibition by various chemicals known to inhibit reactions dependent on cytochrome P450 suggested that the marmesin synthase is a cytochrome-P450-dependent monooxygenase. Upon prolonged incubation, a subsequent major labelled product originated from (+)marmesin, which was identified as psoralen. The psoralen synthase was also characterized as a cytochrome-P450-dependent monooxygenase. Both the marmesin synthase and the psoralen synthase, as well as enzymes catalyzing the formation of demethylsuberosin and O-prenylumbelliferone from umbelliferone and dimethylallyl diphosphate, were associated with the endoplasmic reticulum in Ammi majus cells and their activities were concomitantly induced by elicitor treatment of the cells. We propose that in vivo these enzymes are active in the lumen of the endoplasmic reticulum from where the furanocoumarin phytoalexins are excreted into the cell culture fluid.

  13. Increased sesquiterpenoid biosynthesis and an apparent decrease in sterol biosynthesis in elicitor-treated tobacco cell suspension cultures

    Energy Technology Data Exchange (ETDEWEB)

    Voegeli, U.; Bhatt, P.N.; Chappell, J.

    1987-04-01

    Addition of fungel elicitor prepared from Phytophthora parasitica to tobacco cell suspension cultures leads to an increased production of the phytoalexin capsidiol. Capsidiol is a sesquiterpenoid which is most likely synthesized from farnesylpyrophosphat (FPP) by a bicyclic cyclase reaction. Because FPP is also a substrate for squalene synthetase and therefore a precursor of sterol biosynthesis, the question arises whether or not the accumulation of capsidiol in elicitor-treated cells occurs at the expense of sterol biosynthesis. (/sup 14/C)-acetate was given to elicitor-treated and control (no treatment) cell cultures and incorporation into sterols and capsidiol determined. No labeled capsidiol was detected in control cells. In elicitor-treated cells about 12-15% of the radioactivity taken up by the cells was incorporated into capsidiol. In contrast, control cells incorporated 4 times more radioactivity into sterols than elicitor-treated cells. Similar results were obtained using (/sup 3/H)-mevalonate as a precursor of capsidiol and sterol biosynthesis. Likely explanations for the apparently decline in sterol biosynthesis in elicitor-treated cells include: (1) inhibition of squalene synthetase; (2) induction of capsidiol synthesizing enzymes; and (3) metabolic channeling of FPP into capsidiol versus sterols. These possibilities will be discussed further together with other results.

  14. Biotransformation of perfumery terpenoids, (−-ambrox® by a fungal culture Macrophomina phaseolina and a plant cell suspension culture of Peganum harmala

    Directory of Open Access Journals (Sweden)

    Musharraf Syed Ghulam

    2012-08-01

    Full Text Available Abstract Background Biotransformation offers chemo enzymatic system to modify the compounds into their novel analogues which are difficult to synthesize by chemical methods. This paper describes the biotransformational studies of ambrox, one of the most important components of natural Ambergris (wale sperm with fungal and plant cell culture. Results Biotransformation of (−-ambrox (1 with a fungal cell culture of Macrophomina phaseolina and a plant cell suspension cultures of Peganum harmala yielded oxygenated products, 3β-hydroxyambrox (2, 6β-hydroxyambrox (3, 1α-hydroxy-3oxoambrox (4, 1α,3β-dihydroxyambrox (5, 13,14,15,16-tetranorlabdane-3-oxo-8,12-diol (6, 3-oxoambrox (7, 2α-hydroxyambrox (8, 3β-hydroxysclareolide (9, and 2α,3β-dihydroxyambrox (10. Metabolite 4 was found to be new compound. These metabolites were structurally characterized on the basis of spectroscopic studies. Conclusion Nine oxygenated metabolites of (−-ambrox (1 were obtained from Macrophomina phaseolina and Peganum harmala. Enzymatic system of screened organisms introduced hydroxyl and keto functionalities at various positions of compound 1 in a stereo- and regio-controlled manner.

  15. Biotransformation of perfumery terpenoids, (-)-ambrox® by a fungal culture Macrophomina phaseolina and a plant cell suspension culture of Peganum harmala.

    Science.gov (United States)

    Musharraf, Syed Ghulam; Naz, Sheeba; Najeeb, Asma; Khan, Saifullah; Choudhary, M Iqbal

    2012-08-05

    Biotransformation offers chemo enzymatic system to modify the compounds into their novel analogues which are difficult to synthesize by chemical methods. This paper describes the biotransformational studies of ambrox, one of the most important components of natural Ambergris (wale sperm) with fungal and plant cell culture. Biotransformation of (-)-ambrox (1) with a fungal cell culture of Macrophomina phaseolina and a plant cell suspension cultures of Peganum harmala yielded oxygenated products, 3β-hydroxyambrox (2), 6β-hydroxyambrox (3), 1α-hydroxy-3oxoambrox (4), 1α,3β-dihydroxyambrox (5), 13,14,15,16-tetranorlabdane-3-oxo-8,12-diol (6), 3-oxoambrox (7), 2α-hydroxyambrox (8), 3β-hydroxysclareolide (9), and 2α,3β-dihydroxyambrox (10). Metabolite 4 was found to be new compound. These metabolites were structurally characterized on the basis of spectroscopic studies. Nine oxygenated metabolites of (-)-ambrox (1) were obtained from Macrophomina phaseolina and Peganum harmala. Enzymatic system of screened organisms introduced hydroxyl and keto functionalities at various positions of compound 1 in a stereo- and regio-controlled manner.

  16. Biotransformation of perfumery terpenoids, (−)-ambrox® by a fungal culture Macrophomina phaseolina and a plant cell suspension culture of Peganum harmala

    Science.gov (United States)

    2012-01-01

    Background Biotransformation offers chemo enzymatic system to modify the compounds into their novel analogues which are difficult to synthesize by chemical methods. This paper describes the biotransformational studies of ambrox, one of the most important components of natural Ambergris (wale sperm) with fungal and plant cell culture. Results Biotransformation of (−)-ambrox (1) with a fungal cell culture of Macrophomina phaseolina and a plant cell suspension cultures of Peganum harmala yielded oxygenated products, 3β-hydroxyambrox (2), 6β-hydroxyambrox (3), 1α-hydroxy-3oxoambrox (4), 1α,3β-dihydroxyambrox (5), 13,14,15,16-tetranorlabdane-3-oxo-8,12-diol (6), 3-oxoambrox (7), 2α-hydroxyambrox (8), 3β-hydroxysclareolide (9), and 2α,3β-dihydroxyambrox (10). Metabolite 4 was found to be new compound. These metabolites were structurally characterized on the basis of spectroscopic studies. Conclusion Nine oxygenated metabolites of (−)-ambrox (1) were obtained from Macrophomina phaseolina and Peganum harmala. Enzymatic system of screened organisms introduced hydroxyl and keto functionalities at various positions of compound 1 in a stereo- and regio-controlled manner. PMID:22863186

  17. Oxidative stress in plant cell culture: a role in production of beta-thujaplicin by Cupresssus lusitanica suspension culture.

    Science.gov (United States)

    Zhao, Jian; Fujita, Koki; Sakai, Kokki

    2005-06-05

    Oxidative stress is a common physiological stress that often challenges plants. Reactive oxygen species (ROS) are major factors in oxidative stress that significantly affect plant cell growth and secondary metabolism. Here we used beta-thujaplicin production by Cupressus lusitanica cell culture as an example to demonstrate the common occurrence of oxidative stress in cultivated plant cells and its effect on multiple aspects of cell culture process. C. lusitanica cells cultivated under Fe(2+) stress generate a significant level of ROS, and oxidative stress also occurs at late stages of C. lusitanica cell cultures under normal conditions. ROS production inhibited cell growth, induced lipid peroxidation and cell death, and enhanced ethylene and beta-thujaplicin production. It is demonstrated that Fe(2+) stress enhances ROS production via the Fenton reaction and promotes beta-thujaplicin production via ROS-induced lipid peroxidation that may activate cyclic oxylipin and ethylene pathways. Results further indicate that H(2)O(2) is a positive signal for beta-thujaplicin production, whereas superoxide anion radical (O(2) (- )) negatively affects beta-thujaplicin induction and strongly induces cell death. The study suggests that evaluating the oxidative stress and plant responses in a cell culture process is very necessary and important for understanding biochemical processes and for gaining the maximal productivity of target secondary metabolites.

  18. Establishment of Cell Suspension Culture and Plant Regeneration in Abrus precatorius L., a Rare Medicinal Plant

    Directory of Open Access Journals (Sweden)

    Mohammad Serajur RAHMAN

    2012-02-01

    Full Text Available A new protocol has been developed for cell culture and in vitro regeneration of Abrus precatorius that holds enormous potentiality for preparation of medicines. In vitro grown calli were cultured in Murashige and Skoog (MS liquid media in agitated condition fortified with 0.5 mg/l 6-Benzylaminopurine. Growth curve of cells revealed that the cells continued to grow until 12 days of culture and got the highest peak from day 6-8. Isolated cell was found to produce highest 8.2% calli when suspended on MS medium supplemented with 0.5 mg/l 6-Benzylaminopurine and 0.1 mg/l 1-Naphthaleneacetic acid. Callus derived from single cell produced highest number of embryo (25-28% cultured on MS medium fortified with 2.0 mg/l 6-Benzylaminopurine and 0.2 mg/l 1-Naphthaleneacetic acid. The bipolar embryos were selected and optimum shoot formation was recorded on MS medium supplemented with 2.0 mg/l 6-Benzylaminopurine and 0.1 mg/l 1-Naphthaleneacetic acid. The optimum root induction was noticed in MS medium supplemented with 1.0 mg/l 3-Indolebutyric acid. Rooted plantlets were successfully transferred to potting soil and acclimatized to outdoor conditions.

  19. Stable lentiviral transformation of CHO cells for the expression of the hemagglutinin H5 of avian influenza virus in suspension culture

    Directory of Open Access Journals (Sweden)

    Alaín González Pose

    2014-09-01

    Full Text Available Avian influenza virus H5N1 has caused extensive damage worldwide among poultry and humans. Effective expression systems are needed for the production of viral proteins required for monitoring this devastating disease. The present study deals with the establishment of a stable expression system for the hemagglutinin H5 (HAH5 of avian influenza virus using CHO cells in suspension culture transduced with a recombinant lentiviral vector. The synthetic gene coding the HAH5 protein was inserted in a lentiviral vector with the aim of performing a stable transduction of CHO cells. After the selection of recombinant clones, the one with the highest expression level was adapted to suspension culture and the HAH5 protein was purified by immunoaffinity chromatography from the culture supernatant. There were no significant differences when this protein, purified or direct from the culture supernatant of CHO or SiHa cells, was utilized in an immunologic assay using positive and negative sera as reference. It was also demonstrated that the HAH5 protein in its purified form is able to bind anti-HAH5 antibodies generated with proper and non-proper folded proteins. The results demonstrate that the CHO cell line stably transduced with a lentiviral vector coding the sequence of the HAH5 protein and cultured in suspension can be a suitable expression system to obtain this protein for diagnostic purpose in a consistent and reliable manner.

  20. The mycorrhizal fungus Amanita muscaria induces chitinase activity in roots and in suspension-cultured cells of its host Picea abies.

    Science.gov (United States)

    Sauter, M; Hager, A

    1989-08-01

    A cell-wall fraction of the mycorrhizal fungus Amanita muscaria increased the chitinase activity in suspension-cultured cells of spruce (Picea abies (L.) Karst.) which is a frequent host of Amanita muscaria in nature. Chitinase activity was also increased in roots of spruce trees upon incubation with the fungal elicitor. Non-induced levels of chitinase activity in spruce were higher in suspension cells than in roots whereas the elicitorinduced increase of chitinase activity was higher in roots. Treatment of cells with hormones (auxins and cytokinin) resulted in a severalfold depression of enzyme activity. However, the chitinase activity of hormone-treated as well as hormone-free cells showed an elicitor-induced increase. Suspension cells of spruce secreted a large amount of enzyme into the medium. It is postulated that chitinases released from the host cells in an ectomycorrhizal system partly degrade the fungal cell walls, thus possibly facilitating the exchange of metabolites between the symbionts.

  1. Effect of salts (NaCl and Na2CO3) on callus and suspension culture of Stevia rebaudiana for Steviol glycoside production.

    Science.gov (United States)

    Gupta, Pratibha; Sharma, Satyawati; Saxena, Sanjay

    2014-03-01

    Steviol glycosides are natural non-caloric sweeteners which are extracted from the leaves of Stevia rebaudiana plant. Present study deals the effect of salts (NaCl and Na2CO3) on callus and suspension culture of Stevia plant for steviol glycoside (SGs) production. Yellow-green and compact calli obtained from in vitro raised Stevia leaves sub-cultured on MS medium supplemented with 2.0 mg l(-1) NAA and different concentrations of NaCl (0.05-0.20%) and Na2CO3 (0.0125-0.10%) for 2 weeks, and incubated at 24 ± 1 °C and 22.4 μmol m(-2) s(-1) light intensity provided by white fluorescent tubes for 16 h. Callus and suspension biomass cultured on salts showed less growth as well as browning of medium when compared with control. Quantification of SGs content in callus culture (collected on 15th day) and suspension cultures (collected at 10th and 15th days) treated with and without salts were analyzed by HPLC. It was found that abiotic stress induced by the salts increased the concentration of SGs significantly. In callus, the quantity of SGs got increased from 0.27 (control) to 1.43 and 1.57% with 0.10% NaCl, and 0.025% Na2CO3, respectively. However, in case of suspension culture, the same concentrations of NaCl and Na2CO3 enhanced the SGs content from 1.36 (control) to 2.61 and 5.14%, respectively, on the 10th day.

  2. Identification and expression analysis of methyl jasmonate responsive ESTs in paclitaxel producing Taxus cuspidata suspension culture cells

    Directory of Open Access Journals (Sweden)

    Lenka Sangram K

    2012-04-01

    Full Text Available Abstract Background Taxol® (paclitaxel promotes microtubule assembly and stabilization and therefore is a potent chemotherapeutic agent against wide range of cancers. Methyl jasmonate (MJ elicited Taxus cell cultures provide a sustainable option to meet the growing market demand for paclitaxel. Despite its increasing pharmaceutical importance, the molecular genetics of paclitaxel biosynthesis is not fully elucidated. This study focuses on identification of MJ responsive transcripts in cultured Taxus cells using PCR-based suppression subtractive hybridization (SSH to identify genes involved in global pathway control. Results Six separate SSH cDNA libraries of paclitaxel-accumulating Taxus cuspidata P991 cell lines were constructed at three different post-elicitation time points (6h, 18h and 5 day to identify genes that are either induced or suppressed in response to MJ. Sequencing of 576 differentially screened clones from the SSH libraries resulted in 331 unigenes. Functional annotation and Gene Ontology (GO analysis of up-regulated EST libraries showed enrichment of several known paclitaxel biosynthetic genes and novel transcripts that may be involved in MJ-signaling, taxane transport, or taxane degradation. Macroarray analysis of these identified genes unravelled global regulatory expression of these transcripts. Semi-quantitative RT-PCR analysis of a set of 12 candidate genes further confirmed the MJ-induced gene expression in a high paclitaxel accumulating Taxus cuspidata P93AF cell line. Conclusions This study elucidates the global temporal expression kinetics of MJ responsive genes in Taxus suspension cell culture. Functional characterization of the novel genes identified in this study will further enhance the understanding of paclitaxel biosynthesis, taxane transport and degradation.

  3. Comparison of use of Vero cell line and suspension culture of murine macrophage to attenuation of virulence of Neospora caninum.

    Science.gov (United States)

    Khordadmehr, Monireh; Namavari, Mehdi; Khodakaram-Tafti, Azizollah; Mansourian, Maryam; Rahimian, Abdollah; Daneshbod, Yahya

    2013-10-01

    In this study the tachyzoite yields of Neospora caninum were compared in two cell lines: Vero (African Green Monkey Kidney) and suspension culture of murine macrophage (J774) cell lines. Then, N. caninum were continuously passaged in these cell lines for 3 months and the effect of host cells on virulence of tachyzoites was assessed by broiler chicken embryonated eggs. Inoculation was performed in the chorioallantoic (CA) liquid of the embryonated eggs with different dilutions (0.5 × 10(4), 1.0 × 10(4), 1.5 × 10(4)) of tachtzoites isolated from these cell cultures. The mortality pattern and pathological changes of the dead embryos and hatched chickens were noted. Tissue samples of brain, liver and heart were examined by histopathological and detection of DNA of parasite by polymerase chain reaction (PCR). Also, consecutive sections of the tissues examined histologically were used for immunohistochemical (IHC) examination. Embryos inoculated with tachyzoites derived from Vero cell line (group V) showed a higher mortality rate (100%) than the embryos that received tachyzoites derived from J774 cell line (group J) (10% mortality rate). The results of this study indicated that the culture of N. caninum in J774 cell led to a marked increase in the number of tachyzoite yields and rapid attenuation in comparison to Vero, so the results were confirmed by IHC and PCR. This study is the first report of the significant effect of host cell on the attenuation of virulence of N. caninum tachyzoites. These findings could potentially provide a practical approach in the mass production of N. caninum tachyzoites, and also in producing live attenuated vaccine.

  4. Manipulating anthocyanin composition in Vitis vinifera suspension cultures by elicitation with jasmonic acid and light irradiation.

    Science.gov (United States)

    Curtin, Chris; Zhang, Wei; Franco, Chris

    2003-07-01

    Jasmonic acid altered the accumulation of major anthocyanins in Vitis vinifera cell culture. Peonidin 3-glucoside content at day three was increased from 0.3 to 1.7 mg g(-1) dry cell wt while other major anthocyanins were increased by smaller increments. By day 14, the content of methylated and acylated anthocyanins (peonidin 3-p-coumaroylglucoside and malvidin 3-p-coumaroylglucoside) was 6.3 mg g(-1) DCW, in response to treatment with jasmonic acid, and comprising approximately 45% (w/w) of total anthocyanins. In comparison, the untreated control culture contained 1.2 mg g(-1) DCW which made up approximately 32% (w/w) of total anthocyanins. Light further enhanced anthocyanin accumulation induced by jasmonic acid elicitation. The content of peonidin 3-glucoside at day 3 was 6.6 mg g(-1) DCW, 22-fold higher than control cultures while the content in response to light irradiation alone was 0.6 mg g(-1) DCW. When a highly pigmented cell line was elicited with jasmonic acid total anthocyanins increased from 9.2 to 20.7 mg g(-1) DCW, but there was no change in the anthocyanin composition.

  5. Factorial experimental design for the culture of human embryonic stem cells as aggregates in stirred suspension bioreactors reveals the potential for interaction effects between bioprocess parameters.

    Science.gov (United States)

    Hunt, Megan M; Meng, Guoliang; Rancourt, Derrick E; Gates, Ian D; Kallos, Michael S

    2014-01-01

    Traditional optimization of culture parameters for the large-scale culture of human embryonic stem cells (ESCs) as aggregates is carried out in a stepwise manner whereby the effect of varying each culture parameter is investigated individually. However, as evidenced by the wide range of published protocols and culture performance indicators (growth rates, pluripotency marker expression, etc.), there is a lack of systematic investigation into the true effect of varying culture parameters especially with respect to potential interactions between culture variables. Here we describe the design and execution of a two-parameter, three-level (3(2)) factorial experiment resulting in nine conditions that were run in duplicate 125-mL stirred suspension bioreactors. The two parameters investigated here were inoculation density and agitation rate, which are easily controlled, but currently, poorly characterized. Cell readouts analyzed included fold expansion, maximum density, and exponential growth rate. Our results reveal that the choice of best case culture parameters was dependent on which cell property was chosen as the primary output variable. Subsequent statistical analyses via two-way analysis of variance indicated significant interaction effects between inoculation density and agitation rate specifically in the case of exponential growth rates. Results indicate that stepwise optimization has the potential to miss out on the true optimal case. In addition, choosing an optimum condition for a culture output of interest from the factorial design yielded similar results when repeated with the same cell line indicating reproducibility. We finally validated that human ESCs remain pluripotent in suspension culture as aggregates under our optimal conditions and maintain their differentiation capabilities as well as a stable karyotype and strong expression levels of specific human ESC markers over several passages in suspension bioreactors.

  6. Blister roof grafting, cultured melanocytes transplantation and non-cultured epidermal cell suspension transplantation in treating stable vitiligo: A mutual self-control study.

    Science.gov (United States)

    Bao, Huaye; Hong, Weisong; Fu, Lifang; Wei, Xiaodong; Qian, Guopei; Xu, Aie

    2015-01-01

    To compare the efficacy of blister roof grafting (BG), cultured melanocytes transplantation (CMT) and non-cultured epidermal cell suspension transplantation (NCES) in the treatment of stable vitiligo. In each person of 83 vitiligo patients one vitiligo macule was selected and divided in three areas for separate treatment with BG, CMT and NCES in the same session. The results were evaluated 12-month post-surgery for the extent of repigmentation and color match. A satisfactory result (>50% repigmentation) was achieved in 92%, 82% and 81% of the 83 patients with the BG, CMT and NCES methods, respectively. Significant differences between the BG and CMT groups (p = 0.038), and between BG and NCES groups (p = 0.017) were observed, but not between the CMT and NCES groups (p = 0.986). The extent of repigmentation on the head neck and trunk was superior to that of the extremities by all the three methods. A difference in the time of onset of repigmentation was observed, with repigmentation first appearing after 10 days, 20-30 days and >30 days in the BG, CMT and NCES groups, respectively. All the three methods are safe and effective to treat vitiligo. Future studies with larger groups are warranted to confirm our results.

  7. LC/MS profiling of flavonoid glycoconjugates isolated from hairy roots, suspension root cell cultures and seedling roots of Medicago truncatula.

    Science.gov (United States)

    Staszków, Anna; Swarcewicz, Barbara; Banasiak, Joanna; Muth, Dorota; Jasiński, Michał; Stobiecki, Maciej

    2011-12-01

    Hairy roots and suspension cell cultures are commonly used in deciphering different problems related to the biochemistry and physiology of plant secondary metabolites. Here, we address about the issue of possible differences in the profiles of flavonoid compounds and their glycoconjugates derived from various plant materials grown in a standard culture media. We compared profiles of flavonoids isolated from seedling roots, hairy roots, and suspension root cell cultures of a model legume plant, Medicago truncatula. The analyses were conducted with plant isolates as well as the media. The LC/MS profiles of target natural products obtained from M. truncatula seedling roots, hairy roots, and suspension root cell cultures differed substantially. The most abundant compounds in seedlings roots were mono- and diglucuronides of isoflavones and/or flavones. This type of glycosylation was not observed in hairy roots or suspension root cell cultures. The only recognized glycoconjugates in the latter samples were glucose derivatives of isoflavones. Application of a high-resolution mass spectrometer helped evaluate the elemental composition of protonated molecules, such as [M + H](+). Comparison of collision-induced dissociation MS/MS spectra registered with a quadrupole time-of-flight analyzer for tissue extracts and standards allowed us to estimate the aglycone structure on the basis of the pseudo-MS(3) experiment. Structures of these natural products were described according to the registered mass spectra and literature data. The analyses conducted represent an overview of flavonoids and their conjugates in different types of plant material representing the model legume, M. truncatula. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11306-011-0287-2) contains supplementary material, which is available to authorized users.

  8. Naturally Colored Cotton

    Institute of Scientific and Technical Information of China (English)

    履之

    1994-01-01

    Instead of using dye to color cotton, an Arizona cotton breeder is letting nature do the work. Through crossbreeding, Sally Fox of Natural Cotton Colours in Wickenberg is creating plants that yield fiber in an array

  9. Methyl Jasmonate and Salicylic Acid Induced Oxidative Stress and Accumulation of Phenolics in Panax ginseng Bioreactor Root Suspension Cultures

    Directory of Open Access Journals (Sweden)

    Kee-Yoeup Paek

    2007-03-01

    Full Text Available To investigate the enzyme variations responsible for the synthesis of phenolics, 40 day-old adventitious roots of Panax ginseng were treated with 200 μM methyl jasmonate (MJ or salicylic acid (SA in a 5 L bioreactor suspension culture (working volume 4 L. Both treatments caused an increase in the carbonyl and hydrogen peroxide (H2O2 contents, although the levels were lower in SA treated roots. Total phenolic, flavonoid, ascorbic acid, non-protein thiol (NPSH and cysteine contents and 1,1-diphenyl-2-picrylhydrazyl (DPPH radical reducing activity were increased by MJ and SA. Fresh weight (FW and dry weight (DW decreased significantly after 9 days of exposure to SA and MJ. The highest total phenolics (62%, DPPH activity (40%, flavonoids (88%, ascorbic acid (55%, NPSH (33%, and cysteine (62% contents compared to control were obtained after 9 days in SA treated roots. The activities of glucose 6-phosphate dehydrogenase, phenylalanine ammonia lyase, substrate specific peroxidases (caffeic acid peroxidase, quercetin peroxidase and ferulic acid peroxidase were higher in MJ treated roots than the SA treated ones. Increased shikimate dehydrogenase, chlorogenic acid peroxidase and β-glucosidase activities and proline content were observed in SA treated roots than in MJ ones. Cinnamyl alcohol dehydrogenase activity remained unaffected by both MJ and SA. These results strongly indicate that MJ and SA induce the accumulation of phenolic compounds in ginseng root by altering the phenolic synthesis enzymes.

  10. Enhancement of anthraquinone production in Morinda citrifolia cell suspension cultures after stimulation of the proline cycle with two proline analogs.

    Science.gov (United States)

    Quevedo, Carla V; Perassolo, María; Giulietti, Ana M; Rodríguez Talou, Julián

    2012-03-01

    Synthesis of anthraquinones (AQs) involves the shikimate and 2-C-methyl-D-erythritol 4-phosphate pathways. The proline cycle is linked to the pentose phosphate pathway (PPP) to generate NADPH needed in the first steps of this pathway. The effect of two proline analogs, azetidine-2-carboxylic acid (A2C) and thiazolidine-4-carboxylic acid (T4C), were evaluated in Morinda citrifolia suspension cultures. Both analogs gave higher proline accumulation after 6 and 10 days (68 and 179% after 6 days with A2C at 25 and 50 μM, respectively, and 111% with T4C added at 100 μM). Induction of the proline cycle increased the AQ content after 6 days (~40% for 50 μM A2C and 100 μM T4C). Whereas A2C (50 μM) increased only AQ production, T4C also enhanced total phenolics. However, no induction of the PPP was observed with any of the treatments. This pathway therefore does not limit the supply of carbon skeletons to secondary metabolic pathways.

  11. Nitric Oxide Functions as a Signal in Ultraviolet-B-Induced Baicalin Accumulation in Scutellaria baicalensis Suspension Cultures

    Directory of Open Access Journals (Sweden)

    Jin-Jie Zhang

    2014-03-01

    Full Text Available Stress induced by ultraviolet-B (UV-B irradiation stimulates the accumulation of various secondary metabolites in plants. Nitric oxide (NO serves as an important secondary messenger in UV-B stress-induced signal transduction pathways. NO can be synthesized in plants by either enzymatic catalysis or an inorganic nitrogen pathway. The effects of UV-B irradiation on the production of baicalin and the associated molecular pathways in plant cells are poorly understood. In this study, nitric oxide synthase (NOS activity, NO release and the generation of baicalin were investigated in cell suspension cultures of Scutellaria baicalensis exposed to UV-B irradiation. UV-B irradiation significantly increased NOS activity, NO release and baicalin biosynthesis in S. baicalensis cells. Additionally, exogenous NO supplied by the NO donor, sodium nitroprusside (SNP, led to a similar increase in the baicalin content as the UV-B treatment. The NOS inhibitor, Nω-nitro-l-arginine (LNNA, and NO scavenger, 2-(4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO partially inhibited UV-B-induced NO release and baicalin accumulation. These results suggest that NO is generated by NOS or NOS-like enzymes and plays an important role in baicalin biosynthesis as part of the defense response of S. baicalensis cells to UV-B irradiation.

  12. A two-stage process with temperature-shift for enhanced anthocyanin production in strawberry cell suspension cultures

    Institute of Scientific and Technical Information of China (English)

    张卫; Shintaro; Furusaki; Chris; Franco

    1999-01-01

    A two-stage process with temperature-shift has been developed to enhance the anthocyanin yield in suspension cultures of strawberry cells. The effect of the temperature-shift interval and the shift-time point was investigated for the optimization of this strategy. In this process, strawberry cells were grown at 30℃ (the optimum temperature for cell growth) for a certain period as the first stage, with the temperature shifted to a lower temperature for the second stage. In response to the temperature shift-down, anthoeyanin synthesis was stimulated and a higher content could be achieved than that at both boundary temperatures but cell growth was suppressed. When the lower boundary temperature was deereased, cell growth was lowered and a delayed, sustained maximum anthocyanin content was achieved. Anthocyanin synthesis was strongly influeneed by the shift-time point but cell growth was not. Consequently, the maximum anthocyanin content of 2.7 mg(?)g-fresh cell-1 was obtained on day 9 by a temperature-

  13. Induction of two prenyltransferases for the accumulation of coumarin phytoalexins in elicitor-treated Ammi majus cell suspension cultures.

    Science.gov (United States)

    Hamerski, D; Schmitt, D; Matern, U

    1990-01-01

    Two dimethylallyl diphosphate:umbelliferone dimethylallyltransferase (prenyltransferase) activities, catalysing the 6-prenylation and the 7-O-prenylation, respectively, of umbelliferone in the course of phytoalexin synthesis, increased in Ammi majus cell suspension cultures in response to elicitor treatment. Both enzyme activities were dependent on Mg2+ or Mn2+ with significant preference for Mg2+ in the 6-prenylation reaction. Whereas dark-grown cells did not contain these activities, both prenyltransferase activities were induced rapidly by the addition of elicitor reaching a first maximum after 10-14 hr and a second maximum beyond 30 hr. Other coumarin specific, elicitor-induced enzyme activities of A. majus cells, in contrast, showed only one maximum of activity within the 50 hr experimental period, while the pattern of induction of phenylalanine ammonia-lyase activity resembled that of the prenyltransferases with maxima at ca 8 hr and 20-30 hr. Preliminary data suggest that the apparent biphasic induction of these enzyme activities is due to post-translational enzyme modifications.

  14. Highly protein-resistant coatings and suspension cell culture thereon from amphiphilic block copolymers prepared by RAFT polymerization.

    Science.gov (United States)

    Haraguchi, Kazutoshi; Kubota, Kazuomi; Takada, Tetsuo; Mahara, Saori

    2014-06-09

    Novel amphiphilic block copolymers composed of hydrophobic (poly(2-methoxyethyl acrylate): M) and hydrophilic (poly(N,N-dimethylacrylamide): D) segments were synthesized by living radical polymerization: a reversible addition-fragmentation chain-transfer polymerization. Two types of amphiphilic block copolymers, triblock (MDM) and 4-arm block ((MD)4) copolymers with specific compositions (D/M = (750-1500)/250), were prepared by a versatile one-pot synthesis. These copolymers show good adhesion to various types of substrates (e.g., polystyrene, polycarbonate, polypropylene, Ti, and glass), and the surface coating showed high protein repellency and a low contact angle for water, regardless of the substrate. The two opposing characteristics of high protein repellency and good substrate adhesion were achieved by the combined effects of the molecular architecture of the block copolymers, the high molecular weight, and the characteristics of each segment, that is, low protein adsorption capability of both segments and low glass transition temperature of the hydrophobic segment. Further, a polystyrene dish coated with the MDM block copolymer could be sterilized by γ-ray irradiation and used as a good substrate for a suspension cell culture that exhibits low cell adhesion and good cell growth.

  15. Enhanced accumulation of phytosterols and phenolic compounds in cyclodextrin-elicited cell suspension culture of Daucus carota.

    Science.gov (United States)

    Miras-Moreno, Begoña; Almagro, Lorena; Pedreño, M A; Sabater-Jara, Ana Belén

    2016-09-01

    In this work, suspension-cultured cells of Daucus carota were used to evaluate the effect of β-cyclodextrins on the production of isoprenoid and phenolic compounds. The results showed that the phytosterols and phenolic compounds were accumulated in the extracellular medium (15100μgL(-1) and 477.46μgL(-1), respectively) in the presence of cyclodextrins. Unlike the phytosterol and phenolic compound content, β-carotene (1138.03μgL(-1)), lutein (25949.54μgL(-1)) and α-tocopherol (8063.82μgL(-1)) chlorophyll a (1625.13μgL(-1)) and b (9.958 (9958.33μgL(-1)) were mainly accumulated inside the cells. Therefore, cyclodextrins were able to induce the cytosolic mevalonate pathway, increasing the biosynthesis of phytosterols and phenolic compounds, and accumulate them outside the cells. However, in the absence of these cyclic oligosaccharidic elicitors, carrot cells mainly accumulated carotenoids through the methylerythritol 4-phosphate pathway. Therefore, the use of cyclodextrins would allow the extracellular accumulation of both phytosterols and phenolic compounds by diverting the carbon flux towards the cytosolic mevalonate/phenylpropanoid pathway.

  16. Golgi-specific localization of transglycosylases engaged in glycoprotein biosynthesis in suspension-cultured cells of sycamore (Acer pseudoplatanus L.).

    Science.gov (United States)

    Ali, M S; Mitsui, T; Akazawa, T

    1986-12-01

    Golgi complex and endoplasmic reticulum (ER) were isolated from suspension-cultured cells of sycamore (Acer pseudoplatanus L.) by stepwise sucrose density gradient centrifugation using protoplasts as starting material. The purity of the two organelle fractions isolated was assessed by measuring marker enzyme activities. Localization of glycolipid and glycoprotein glycosyltransferase activities in the isolated Golgi and ER fractions was examined; three glycosyltransferases, i.e., galactosyltransferase, fucosyltransferase, and xylosyltransferase, proved to be almost exclusively confined to the Golgi, whereas the ER fractions contained glycolipid glycosyltransferase. The Golgi complex was further subfractionated on a discontinuous sucrose density gradient into two components, migrating at densities of 1.118 and 1.127 g/cm3. The two fractions differed in their compositional polypeptide bands discernible from Na-dodecylsulfate gel electrophoresis. Galactosyltransferase distributed nearly equally between the two protein peaks and xylosyltransferase activities using the endogenous acceptor also appeared to be localized in the two subcompartments. By contrast, fucosyltransferase, engaged in the terminal stage of glycosylation, banded in the lower density fractions. Golgi-specific alpha-mannosidase, which is presumably engaged in the sugar trimming of Asn-N-linked glycoprotein carbohydrate core, was enriched fourfold in specific activity in the fractions of the higher density. The overall experimental results indicate that the cotranslational glycosylation of Asn-N-linked glycoproteins, e.g., polyphenol oxidase (laccase), takes place in the ER, while subsequent post-translational processing of the oligosaccharide moiety proceeds successively in the two physically separable compartments of the Golgi complex.

  17. Immunogold localization of xyloglucan and rhamnogalacturonan I in the cell walls of suspension-cultured sycamore cells.

    Science.gov (United States)

    Moore, P J; Darvill, A G; Albersheim, P; Staehelin, L A

    1986-11-01

    PLANT CELL WALLS SERVE SEVERAL FUNCTIONS: they impart rigidity to the plant, provide a physical and chemical barrier between the cell and its environment, and regulate the size and shape of each cell. Chemical studies have provided information on the biochemical composition of the plant cell walls as well as detailed knowledge of individual cell wall molecules. In contrast, very little is known about the distribution of specific cell wall components around individual cells and throughout tissues. To address this problem, we have produced polyclonal antibodies against two cell wall matrix components; rhamnogalacturonan I (RG-I), a pectic polysaccharide, and xyloglucan (XG), a hemicellulose. By using the antibiodies as specific markers we have been able to localize these polymers on thin sections of suspension-cultured sycamore cells (Acer pseudoplatanus). Our results reveal that each molecule has a unique distribution. XG is localized throughout the entire wall and middle lamella. RG-I is restricted to the middle lamella and is especially evident in the junctions between cells. These observations indicate that plant cell walls may have more distinct chemical (and functional?) domains than previously envisaged.

  18. 接种菌悬液培养方式对纺织品抗菌试验的影响%Effect of Inoculation Suspension Culture Method on Textile Antibacterial Test

    Institute of Scientific and Technical Information of China (English)

    张红利; 刘会; 蒋芳

    2011-01-01

    探讨纺织品抗茵性能检测中接种菌悬液的培养方式对抗菌试验的影响.分析了接种茵悬液浓度与试验有效性的关系,分别采用振荡培养和静止培养方式制取金黄色葡萄球茵和大肠杆菌悬液,通过对比所制得接种茵悬液的浓度,认为在接种量相同的情况下,采用静置培养所得的金黄色葡萄球菌的接种菌悬液浓度符合标准要求,而采用振荡培养所得的大肠杆菌的接种菌悬液符合标准要求.%Effect of inoculation suspension culture method in textile antibacterial test on antibacterial test was discussed. Relationship between inoculation suspension concentration and test effectiveness was analyzed, staphylococcus aureus suspension and bacillus coli suspension were cultured by oscillation culture and static culture respectively.Through contrasting the suspensions concentration,it is considered that in condition of same inoculum amount, staphylococcus aureus suspension concentration got in static culture could fit standard demand while bacillus coli suspension got by oscillation culture could fit standard demand.

  19. Effects of Medium Constituents on Growth and Canthinone Accumulation in Cell Suspension Cultures of Eurycoma longifolia Jack

    Directory of Open Access Journals (Sweden)

    LUTHFI AZIZ MAHMUD SIREGAR

    2009-06-01

    Full Text Available The effect of various macronutrients, micronutrients and sucrose on growth and canthinone alkaloid production in cell suspension cultures of Pasak Bumi (Eurycoma longifolia Jack was investigated. The optimum macronutrients and micronutrients content for the high alkaloid production of E. longifolia Jack was different to that found in the Murashige and Skoog (MS medium. The highest amount of alkaloids, 9-hydroxycanthin-6-one and 9-methoxycanthin-6-one, could be obtained from E. longifolia Jack cells cultured in modified MS liquid medium that containing macronutrients: 21.50 mM NH4NO3, 14.25 mM KNO3, 7.50 mM CaCl2•2H2O, 2.50 mM MgSO4•7H2O, 1.45 mM KH2PO4, while content of micronutrients was 0.233 mM FeNa-EDTA, 0.215 mM MnSO4•4H2O and without CuSO4•5H2O. Increased sucrose concentration to 4.00% (w/v in modified MS liquid medium could increase total of two-alkaloid. The modification of macronutrients and micronutrients concentration based the optimum production of biomass was obtained MSBs medium that producing high biomass but also increasing the production of 9-hydroxycanthin-6-one. The modification of macronutrients or macronutrients and micronutrients based the optimum total of two-alkaloid was obtained MSC and MSD medium that producing low fresh weight but producing the high 9-hydroxycanthin-6-one.

  20. Novel O-D-galacturonoyl esters in the pectic polysaccharides of suspension-cultured plant cells.

    Science.gov (United States)

    Brown, J A; Fry, S C

    1993-11-01

    Driselase digestion of uronate-6-14C-labeled primary walls of cultured spinach (Spinacia oleracea L.) cells yielded about 18 novel uronate-containing compounds, most of which could be hydrolyzed by cold dilute alkali to yield oligo-[14C]galacturonides. One typical Driselase digestion product (compound 17) yielded alpha-(1-->4)-D-[14C]galacturonotriose(GalA3) upon very mild treatment with alkali (50% yield of GalA3 in 7.2 min at pH 11 and 25 degrees C). One of the three galacturonate residues in compound 17 was reducible to a galactose residue with sodium borohydride, indicating that that GalA residue was esterified, via its--COOH group, to a putative alcohol. Compound 17 had a higher mobility than GalA3 on paper chromatography, indicating that the putative alcohol was relatively nonpolar. The putative alcohol could not have been methanol because Driselase readily hydrolyzed mono-, di-, and trimethyl esters of GalA3 to yield free galacturonic acid. Another Driselase digestion product (compound 12) was a derivative of GalA3 that apparently possessed two nonpolar esterified substituents: one about as labile as in compound 17, and the other approximately 10 times more stable. Compounds 12 and 17 could not labeled by in vivo feeding of [U-14C]cinnamate, suggesting that they were not phenolic conjugates. Similar but chromatographically distinguishable uronate-14C-labeled esters were obtained by Driselase digestion of walls of cultured carrot (Daucus carota L.), Paul's Scarlet rose (Rosa sp.), and tall fescue (Festuca arundinacea Schreber) cells. In spinach, the novel compounds constituted about 5% of the total galacturonate residues of the cell wall. The observations suggest that pectic polysaccharides are linked, via O-D-galacturonoyl ester bonds, to relatively hydrophobic constituents of the primary cell wall. Their possible role in wall architecture is discussed.

  1. Detection of Changes in the Medicago sativa Retinoblastoma-Related Protein (MsRBR1) Phosphorylation During Cell Cycle Progression in Synchronized Cell Suspension Culture.

    Science.gov (United States)

    Ayaydin, Ferhan; Kotogány, Edit; Ábrahám, Edit; Horváth, Gábor V

    2017-01-01

    Deepening our knowledge on the regulation of the plant cell division cycle depends on techniques that allow for the enrichment of cell populations in defined cell cycle phases. Synchronization of cell division can be achieved using different plant tissues; however, well-established cell suspension cultures provide large amount of biological sample for further analyses. Here, we describe the methodology of the establishment, propagation, and analysis of a Medicago sativa suspension culture that can be used for efficient synchronization of the cell division. A novel 5-ethynyl-2'-deoxyuridine (EdU)-based method is used for the estimation of cell fraction that enters DNA synthesis phase of the cell cycle and we also demonstrate the changes in the phosphorylation level of Medicago sativa retinoblastoma-related protein (MsRBR1) during cell cycle progression.

  2. Fast Filtration of Bacterial or Mammalian Suspension Cell Cultures for Optimal Metabolomics Results.

    Science.gov (United States)

    Bordag, Natalie; Janakiraman, Vijay; Nachtigall, Jonny; González Maldonado, Sandra; Bethan, Bianca; Laine, Jean-Philippe; Fux, Elie

    2016-01-01

    The metabolome offers real time detection of the adaptive, multi-parametric response of the organisms to environmental changes, pathophysiological stimuli or genetic modifications and thus rationalizes the optimization of cell cultures in bioprocessing. In bioprocessing the measurement of physiological intracellular metabolite levels is imperative for successful applications. However, a sampling method applicable to all cell types with little to no validation effort which simultaneously offers high recovery rates, high metabolite coverage and sufficient removal of extracellular contaminations is still missing. Here, quenching, centrifugation and fast filtration were compared and fast filtration in combination with a stabilizing washing solution was identified as the most promising sampling method. Different influencing factors such as filter type, vacuum pressure, washing solutions were comprehensively tested. The improved fast filtration method (MxP® FastQuench) followed by routine lipid/polar extraction delivers a broad metabolite coverage and recovery reflecting well physiological intracellular metabolite levels for different cell types, such as bacteria (Escherichia coli) as well as mammalian cells chinese hamster ovary (CHO) and mouse myeloma cells (NS0).The proposed MxP® FastQuench allows sampling, i.e. separation of cells from medium with washing and quenching, in less than 30 seconds and is robustly designed to be applicable to all cell types. The washing solution contains the carbon source respectively the 13C-labeled carbon source to avoid nutritional stress during sampling. This method is also compatible with automation which would further reduce sampling times and the variability of metabolite profiling data.

  3. Zinc tolerance and accumulation in stable cell suspension cultures and in vitro regenerated plants of the emerging model plant Arabidopsis halleri (Brassicaceae).

    Science.gov (United States)

    Vera-Estrella, Rosario; Miranda-Vergara, Maria Cristina; Barkla, Bronwyn J

    2009-03-01

    Arabidopsis halleri is increasingly employed as a model plant for studying heavy metal hyperaccumulation. With the aim of providing valuable tools for studies on cellular physiology and molecular biology of metal tolerance and transport, this study reports the development of successful and highly efficient methods for the in vitro regeneration of A. halleri plants and production of stable cell suspension lines. Plants were regenerated from leaf explants of A. halleri via a three-step procedure: callus induction, somatic embryogenesis and shoot development. Efficiency of callus proliferation and regeneration depended on the initial callus induction media and was optimal in the presence of 1 mg L(-1) 2,4-dichlorophenoxyacetic acid, and 0.05 mg L(-1) benzylaminopurine. Subsequent shoot and root regeneration from callus initiated under these conditions reached levels of 100% efficiency. High friability of the callus supported the development of cell suspension cultures with minimal cellular aggregates. Characterization of regenerated plants and cell cultures determined that they maintained not only the zinc tolerance and requirement of the whole plant but also the ability to accumulate zinc; with plants accumulating up to 50.0 micromoles zinc g(-1) FW, and cell suspension cultures 30.9 micromoles zinc g(-1) DW. Together this work will provide the experimental basis for furthering our knowledge of A. halleri as a model heavy metal hyperaccumulating plant.

  4. Expression of Amyloplast and Chloroplast DNA in Suspension-Cultured Cells of Sycamore (Acer pseudoplatanus L.).

    Science.gov (United States)

    Ngernprasirtsiri, J; Macherel, D; Kobayashi, H; Akazawa, T

    1988-01-01

    Green mutant cells of sycamore (Acer pseudoplatanus L.), which had been selected by mutagenic treatment of the white wild type, grow photoheterotrophically in auxin-depleted culture medium. In contrast to the wild-type cells, mutant cells exhibit photosynthetic O(2)-evolution activity during their growth coincident with increases of (a) chlorophyll, (b) protein, and (c) ribulose-1,5-bisphosphate (RuBP) carboxylase activity. Functionally competent chloroplasts were isolated from the green cells. Mechanism(s) governing gene expression of amyloplast DNA in the heterotrophically grown white cells were compared with those of the chloroplast DNA isolated from the mutant cells. We have demonstrated in both amyloplast and chloroplast DNAs the presence of sequences homologous to the maize chloroplast genes for photosynthesis, including the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO)(rbcL), the 32 kDa Q(B) protein (PG32) (psbA), the apoprotein of P700 (psaA) and subunits of CF(1) (atpA, atpB, and atpE). However, employing either enzyme assays or immunological techniques, RuBisCO and CF(1) cannot be detected in the white wild type cells. Northern blot hybridization of the RNA from the white cells showed high levels of transcripts for the 16S rRNA gene and low level of transcripts for psbA; based on comparison with results obtained using the green mutant cells, we propose that the amyloplast genome is mostly inactive except for the 16S rRNA gene and psbA which is presumably regulated at the transcriptional level.

  5. Fast Filtration of Bacterial or Mammalian Suspension Cell Cultures for Optimal Metabolomics Results.

    Directory of Open Access Journals (Sweden)

    Natalie Bordag

    Full Text Available The metabolome offers real time detection of the adaptive, multi-parametric response of the organisms to environmental changes, pathophysiological stimuli or genetic modifications and thus rationalizes the optimization of cell cultures in bioprocessing. In bioprocessing the measurement of physiological intracellular metabolite levels is imperative for successful applications. However, a sampling method applicable to all cell types with little to no validation effort which simultaneously offers high recovery rates, high metabolite coverage and sufficient removal of extracellular contaminations is still missing. Here, quenching, centrifugation and fast filtration were compared and fast filtration in combination with a stabilizing washing solution was identified as the most promising sampling method. Different influencing factors such as filter type, vacuum pressure, washing solutions were comprehensively tested. The improved fast filtration method (MxP® FastQuench followed by routine lipid/polar extraction delivers a broad metabolite coverage and recovery reflecting well physiological intracellular metabolite levels for different cell types, such as bacteria (Escherichia coli as well as mammalian cells chinese hamster ovary (CHO and mouse myeloma cells (NS0.The proposed MxP® FastQuench allows sampling, i.e. separation of cells from medium with washing and quenching, in less than 30 seconds and is robustly designed to be applicable to all cell types. The washing solution contains the carbon source respectively the 13C-labeled carbon source to avoid nutritional stress during sampling. This method is also compatible with automation which would further reduce sampling times and the variability of metabolite profiling data.

  6. Kinetics in Suspension Culture of Acer ginnala%茶条槭悬浮培养的动力学

    Institute of Scientific and Technical Information of China (English)

    董杰; 詹亚光; 任健

    2012-01-01

    In this paper we investigated kinetics parameters during the cell culture of Acer ginnala , such as cell growth , consumption of the carbon, nitrogen and phosphorus, and change in the pH and electrical conductivity in the medium, as well as changes in cell fresh weight and dry weight at the various stages in the culture procedure. 1 ) The cell suspension culture cycle lasted about 15 days by which the maximum biomass in dry weight and the gallic acid content reached to 11.3 g·L-1 and 0.49% , and the maximum specific rates of cell growth and gallic acid synthesis were 0.541 d and 0. 682 d-1 , respectively. The specific gallic acid production rate was relatively high when the cell growth rate was 0. 3 -0. 4 d-1. Gallic acid accumulation was partially-growth-associated. 2) The electrical conductivity of the culture medium gradually descended during culture procedure, reached to the lowest point on the 21* day, and then slightly increased. 3) After 15 days of the cell culture, sucrose and phosphate of the medium almost all were consumed. Ammonium was rapidly adsorbed at the early stage and was consumed on the 12th day. Compared with ammonium, the absorption of nitrate was slow, it was not absorbed fastly until the 6th day and the absorption rate reached to the lowest point on the 15th day.%对茶条槭细胞培养动力学进行研究,在培养周期内不同的培养阶段测定茶条槭细胞生长和培养基中碳源、氮源、磷源的消耗,电导率的变化,以及细胞的鲜质量与干质量的变化,从而了解细胞生长、营养消耗与次生代谢产物积累的基本规律,为建立结构化动力学模型奠定基础.研究结果表明:1)茶条槭细胞悬浮培养周期约为15天,经过15天的悬浮培养,最大生物量和没食子酸含量分别达到了11.3 g·L-1和0.49%.细胞的最大比生长速率和没食子酸的最大比生成速率分别为0.541 d-1和0.682 d-1.没食子酸的比生成速率当细胞比生长速率在0.3~0.4d-1

  7. Factors influencing cucumber (Cucumis sativus L. somatic embryogenesis. I. The crucial role of pH and nitrogen in suspension culture

    Directory of Open Access Journals (Sweden)

    Tadeusz Wróblewski

    2014-01-01

    Full Text Available A method of obtaining and the characteristics of an embryogenic stabilised cucumber (Cucumis sativus L. suspension culture which has many similarities to the carrot model are presented. The Specific Type I cells and proembryogenic mass were present in such a suspension. The maintenance of the proembryogenic stage took place in medium containing 2,4-D as the sole growth regulator, subsequent stages of embryogenesis occurred in hormone-free medium. Embryonic structures were also observed in medium with auxin in the late stages of growth, probably due to the depletion of 2,4-D in the medium during subculture. The choice of the proper inorganic nitrogen sources and the maintenance of correct proportions between them had a significant effect on the formation of these structures. We have shown that the pH of the medium with an embryogenic culture became stabilized regardless of the initial pH value and depended on the medium composition. The inoculum used for the initiation of subsequent subcultures of the stable suspension culture was 1 part tissue to 300 parts medium and was small in comparison to the systems described for the cucumber so far. From 1 ml of basic suspension 7 embryos were obtained on medium without growth regulators 10 days after inoculation, and this amount increased to 21 after 3 weeks. From 3.2% of the somatic embryos it was posible to regenerate plants. The high yield and synchronisation of the process and the development of embryos without passing through callus tissue create the possibility of using this system for molecular investigations and in the technology of somatic seed production.

  8. The impact of CdSe/ZnS Quantum Dots in cells of Medicago sativa in suspension culture

    Directory of Open Access Journals (Sweden)

    Maycock Christopher

    2010-10-01

    Full Text Available Abstract Background Nanotechnology has the potential to provide agriculture with new tools that may be used in the rapid detection and molecular treatment of diseases and enhancement of plant ability to absorb nutrients, among others. Data on nanoparticle toxicity in plants is largely heterogeneous with a diversity of physicochemical parameters reported, which difficult generalizations. Here a cell biology approach was used to evaluate the impact of Quantum Dots (QDs nanocrystals on plant cells, including their effect on cell growth, cell viability, oxidative stress and ROS accumulation, besides their cytomobility. Results A plant cell suspension culture of Medicago sativa was settled for the assessment of the impact of the addition of mercaptopropanoic acid coated CdSe/ZnS QDs. Cell growth was significantly reduced when 100 mM of mercaptopropanoic acid -QDs was added during the exponential growth phase, with less than 50% of the cells viable 72 hours after mercaptopropanoic acid -QDs addition. They were up taken by Medicago sativa cells and accumulated in the cytoplasm and nucleus as revealed by optical thin confocal imaging. As part of the cellular response to internalization, Medicago sativa cells were found to increase the production of Reactive Oxygen Species (ROS in a dose and time dependent manner. Using the fluorescent dye H2DCFDA it was observable that mercaptopropanoic acid-QDs concentrations between 5-180 nM led to a progressive and linear increase of ROS accumulation. Conclusions Our results showed that the extent of mercaptopropanoic acid coated CdSe/ZnS QDs cytotoxicity in plant cells is dependent upon a number of factors including QDs properties, dose and the environmental conditions of administration and that, for Medicago sativa cells, a safe range of 1-5 nM should not be exceeded for biological applications.

  9. Jasmonic Acid Effect on the Fatty Acid and Terpenoid Indole Alkaloid Accumulation in Cell Suspension Cultures of Catharanthus roseus

    Directory of Open Access Journals (Sweden)

    Guitele Dalia Goldhaber-Pasillas

    2014-07-01

    Full Text Available The stress response after jasmonic acid (JA treatment was studied in cell suspension cultures of Catharanthus roseus. The effect of JA on the primary and secondary metabolism was based on changes in profiles of fatty acids (FA and terpenoid indole alkaloids (TIA. According to multivariate data analyses (MVDA, three major time events were observed and characterized according to the variations of specific FA and TIA: after 0–30 min of induction FA such as C18:1, C20:0, C22:0 and C24:0 were highly induced by JA; 90–360 min after treatment was characterized by variations of C14:0 and C15:0; and 1440 min after induction JA had the largest effect on both group of metabolites were C18:1, C18:2, C18:3, C16:0, C20:0, C22:0, C24:0, catharanthine, tabersonine-like 1, serpentine, tabersonine and ajmalicine-like had the most significant variations. These results unambiguously demonstrate the profound effect of JA particularly on the accumulation of its own precursor, C18:3 and the accumulation of TIA, which can be considered as late stress response events to JA since they occurred only after 1440 min. These observations show that the early events in the JA response do not involve the de novo biosynthesis of neither its own precursor nor TIA, but is due to an already present biochemical system.

  10. Laminin-adherent versus suspension-non-adherent cell culture conditions for the isolation of cancer stem cells in the DAOY medulloblastoma cell line.

    Science.gov (United States)

    de la Rosa, Javier; Sáenz Antoñanzas, Ander; Shahi, Mehdi H; Meléndez, Bárbara; Rey, Juan A; Castresana, Javier S

    2016-09-01

    Medulloblastoma (MB) is a highly malignant tumor of childhood. MB seems to be initiated and maintained by a small group of cells, known as cancer stem cells (CSCs). The CSC hypothesis suggests that a subset of tumor cells is able to proliferate, sustain the tumor, and develop chemoresistance, all of which make of CSC an interesting target for new anticancer therapies. The MB cell line DAOY was cultured in suspension by a medullosphere traditional culturing method and in adherent conditions by laminin-pre-coated flasks and serum-free medium enriched with specific growth factors. An increase in the stem features was shown when cells were successively cultured in hypoxia conditions. By contrast, a reduction in these properties was appreciated when cells were exposed to differentiation conditions. In addition, the CD133+ and CD133- subpopulations were isolated from cells grown in laminin-pre-coated flasks, and in vitro experiments showed that the CD133+ fraction represented the stem population and it could have CSC with a higher probability than the CD133- fraction. We can conclude that the laminin culture method in adherent conditions and the medullosphere traditional culturing method in suspension are similarly good for obtaining stem-like cells in the DAOY cell line.

  11. Plant tissue culture independent Agrobacterium tumefaciens mediated In-planta transformation strategy for upland cotton (Gossypium hirsutum

    Directory of Open Access Journals (Sweden)

    Bipinchandra B. Kalbande

    2016-06-01

    Full Text Available A new method of transgenic development called “In-planta” transformation method, where Agrobacterium is used to infect the plantlets but the steps of in vitro regeneration of plants is totally avoided. In this study, we have reported a simple In-planta method for efficient transformation of diploid cotton Gossypium hirsutum cv LRK-516 Anjali using Agrobacterium tumefaciens EHA-105 harbouring recombinant binary vector plasmid pBinAR with Arabidopsis At-NPR1 gene. Four day old plantlets were used for transformation. A vertical cut was made at the junction of cotyledonary leaves, moderately bisecting the shoot tip and exposing meristem cells at apical meristem. This site was infected with Agrobacterium inoculum. The transgenic events obtained were tested positive for the presence of At-NPR1 gene with promoter nptII gene. They are also tested negative for vector backbone integration and Agrobacterium contamination in T0 events. With this method a transformation frequency of 6.89% was reported for the cv LRK-516.

  12. Elicitor modulation of the turnover of L-phenylalanine ammonia-lyase in French bean cell suspension cultures.

    Science.gov (United States)

    Lawton, M A; Dixon, R A; Lamb, C J

    1980-12-01

    (1) The mechanisms underlying the transient increase in phenylalanine ammonia-lyase activity during phaseollin accumulation in cell suspension cultures of Dwarf French bean (Phaseolus volgaris) have been investigated using density labelling with 3H from 2H2O coupled with residual analysis of the equilibrium distribution of enzyme activity in high-resolution KBr density gradients. (2) The resolution achieved in this system is sufficient to allow quantitative analysis of the relative proportions of light, unlabelled, pre-existing enzyme and heavy, labelled, newly synthesised enzyme. (3) Elicitor released by heat treatment of cell walls of Colletotrichum lindemuthianum, the causal agent of anthracnose disease of French bean, caused a marked but transient increase in phenylalanine ammonia-lyase activity concomitant with the onset of phaseollin accumulation in the bean cultures. The induction of enzyme activity was highly dependent on elicitor concentration, with maximum induction occurring in two discrete concentration ranges; at an intermediate elicitor concentration, or at supra-optimal elicitor concentrations, no enzyme induction was observed. (4) At low concentrations of elicitor the induction of enzyme was entirely a result of elicitor stimulation of the rate of de novo enzyme production. In contrast, at higher elicitor concentrations the increase in enzyme activity was accompanied by a marked apparent stabilization of the enzyme in vivo, and the rapid but transient increase in enzyme activity was achieved by a programme of reciprocal changes in the rate constant for de novo enzyme production and the rate constant for removal of enzyme activity. Such reciprocal control of the rates of enzyme production and removal may be crucial in determining the magnitude and duration of the phytoalexin defense response. (5) Information on the specific activity of 2H label in the amino acid pools was obtained from analysis of the equilibrium distribution of residual, labelled

  13. Single cell dual adherent-suspension co-culture micro-environment for studying tumor-stromal interactions with functionally selected cancer stem-like cells.

    Science.gov (United States)

    Chen, Yu-Chih; Zhang, Zhixiong; Fouladdel, Shamileh; Deol, Yadwinder; Ingram, Patrick N; McDermott, Sean P; Azizi, Ebrahim; Wicha, Max S; Yoon, Euisik

    2016-08-07

    Considerable evidence suggests that cancer stem-like cells (CSCs) are critical in tumor pathogenesis, but their rarity and transience has led to much controversy about their exact nature. Although CSCs can be functionally identified using dish-based tumorsphere assays, it is difficult to handle and monitor single cells in dish-based approaches; single cell-based microfluidic approaches offer better control and reliable single cell derived sphere formation. However, like normal stem cells, CSCs are heavily regulated by their microenvironment, requiring tumor-stromal interactions for tumorigenic and proliferative behaviors. To enable single cell derived tumorsphere formation within a stromal microenvironment, we present a dual adherent/suspension co-culture device, which combines a suspension environment for single-cell tumorsphere assays and an adherent environment for co-culturing stromal cells in close proximity by selectively patterning polyHEMA in indented microwells. By minimizing dead volume and improving cell capture efficiency, the presented platform allows for the use of small numbers of cells (cells). As a proof of concept, we co-cultured single T47D (breast cancer) cells and primary cancer associated fibroblasts (CAF) on-chip for 14 days to monitor sphere formation and growth. Compared to mono-culture, co-cultured T47D have higher tumorigenic potential (sphere formation rate) and proliferation rates (larger sphere size). Furthermore, 96-multiplexed single-cell transcriptome analyses were performed to compare the gene expression of co-cultured and mono-cultured T47D cells. Phenotypic changes observed in co-culture correlated with expression changes in genes associated with proliferation, apoptotic suppression, tumorigenicity and even epithelial-to-mesechymal transition. Combining the presented platform with single cell transcriptome analysis, we successfully identified functional CSCs and investigated the phenotypic and transcriptome effects induced by

  14. Serum replacement with albumin-associated lipids prevents excess aggregation and enhances growth of induced pluripotent stem cells in suspension culture.

    Science.gov (United States)

    Horiguchi, Ikki; Sakai, Yasuyuki

    2016-07-08

    Suspension culture systems are currently under investigation for the mass production of pluripotent stem (PS) cells for tissue engineering; however, the control of cell aggregation in suspension culture remains challenging. Existing methods to control aggregation such as microwell culture are difficult to scale up. To address this issue, in this study a novel method that incorporates the addition of KnockOut Serum Replacement (KSR) to the PS cell culture medium was described. The method regulated cellular aggregation and significantly improved cell growth (a 2- to 10-fold increase) without any influence on pluripotency. In addition, albumin-associated lipids as the major working ingredient of KSR responsible for this inhibition of aggregation were identified. This is one of the simplest methods described to date to control aggregation and requires only chemically synthesizable reagents. Thus, this method has the potential to simplify the mass production process of PS cells and thus lower their cost. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1009-1016, 2016.

  15. Induction of trans-resveratrol and extracellular pathogenesis-related proteins in elicited suspension cultured cells of Vitis vinifera cv Monastrell.

    Science.gov (United States)

    Belchí-Navarro, Sarai; Almagro, Lorena; Sabater-Jara, Ana Belén; Fernández-Pérez, Francisco; Bru, Roque; Pedreño, Maria Angeles

    2013-02-15

    Suspension-cultured cells of Vitis vinifera cv Monastrell were used to investigate the effects of methyljasmonate, ethylene and salicylic acid separately or in combination with cyclodextrins on both trans-resveratrol production and the induction of defense responses. The results showed that the addition of methyljasmonate or ethylene to suspension-cultured cells jointly treated with cyclodextrins and salicylic acid provoked a decrease of trans-resveratrol levels suggesting that salicylic acid has a negative and antagonistic effect with methyljasmonate or ethylene on trans-resveratrol production. Likewise, the exogenous application of these compounds induced the accumulation of pathogenesis-related proteins. Analysis of the extracellular proteome showed the presence of amino acid sequences homologous to an specific β-1,3-glucanase, class III peroxidases and a β-1,4-mannanase, which suggests that these signal molecules could play a role in mediating defense-related gene product expression in V. vinifera cv Monastrell. Apart from these inducible proteins, other proteins were found in both the control and elicited cell cultures of V. vinifera. These included class IV chitinase, polygalacturonase inhibitor protein and reticuline oxidase-like protein, suggesting that their expression is constitutive being involved in the modification of the cell wall architecture during cell culture growth and in the prevention of pathogen attack. Copyright © 2012 Elsevier GmbH. All rights reserved.

  16. Rapid Identification of Shiga Toxin-Producing Escherichia coli O Serogroups from Fresh Produce and Raw Milk Enrichment Cultures by Luminex Bead-Based Suspension Array.

    Science.gov (United States)

    Kase, Julie A; Maounounen-Laasri, Anna; Lin, Andrew

    2016-09-01

    The U.S. Food and Drug Administration's Bacteriological Analytical Manual (BAM) Chapter 4a describes a Luminex microbead-based suspension array used to screen colonies for 11 clinically relevant Shiga toxin-producing Escherichia coli (STEC) serogroups: O26, O45, O91, O103, O104, O111, O113, O121, O128, O145, and O157. We evaluated the usefulness of this method to identify STEC-positive enrichment samples before agar plating. Twelve E. coli strains were added to three types of fresh produce (bagged baby spinach, alfalfa sprouts, and cilantro) at levels near the detection limit of the test. A subset of these strains (six O serogroups) was similarly evaluated in raw milk. For comparison, portions of each of the 168 enrichment cultures were analyzed for serogroup by a real-time PCR assay and a Bio-Plex 200 assay with the bead-based suspensions. No false-positive results were obtained. Of the 112 samples with a reported cycle threshold (CT) value, 101 undiluted, diluted, or extracted enrichment cultures also produced ratios above 5.0 in the Bio-Plex assay. When PCR CT values approached or were greater than 35, Bio-Plex detection became less reliable. Using undiluted or extracted enrichment cultures resulted in a significantly larger number of positive results. With the same enrichment material prepared for real-time PCR analysis as described in the BAM Chapter 4a, the STEC microbead-based suspension array can accurately screen food enrichment cultures.

  17. Effect of sucrose and methyl jasmonate on biomass and anthocyanin production in cell suspension culture of Melastoma malabathricum (Melastomaceae

    Directory of Open Access Journals (Sweden)

    Koay Suan See

    2011-06-01

    Full Text Available Melastoma malabathricum, belongs to the Melastomaceae family, is an important medicinal plant widely distributed from Madagascar to Australia, that is used in traditional remedies for the treatment of variousailments. Besides its medicinal properties, it has been identified as a potential source of anthocyanin production.The present study was carried out to investigate the effect of sucrose and methyl jasmonate and feeding time oncell biomass yield and anthocyanin production in cell suspension culture of M. malabathricum. Addition of differentconcentrations of sucrose into the cell culture of M. malabathricum influenced cell biomass and pigment accumulation. The addition of methyl jasmonate was found to have no effect on cell biomass but the presence of higher amount (12.5-50mg/L had caused a reduction in anthocyanin production and accumulation. MS medium supplemented with 30g/L sucrose and 3.5 mg/L of MeJA added on cero day and 3rd day produced high fresh cell mass at the end of nine days of culture but did not support the production of anthocyanins. However, cells cultured in the medium supplemented with 45g/L sucrose without MeJA showed the highest pigment content (0.69±0.22Cv/g-FCM. The cells cultured in MS medium supplemented with 30 g/L sucrose with 3.5mg/L MeJA added on the 3rd and 6th day of culture, showed the lowest pigment content (0.37-0.40Cv/g-FCM. This study indicated that MeJA was not necessary but sucrose was needed for the enhancement of cell growth and anthocyanin production in M. malabathricum cell cultures. Rev. Biol. Trop. 59 (2: 597-606. Epub 2011 June 01.elastoma malabathricum pertenece a la familia de las melastomáceas, es una planta medicinal importante ampliamente distribuida desde Madagascar hasta Australia, que se utiliza en remedios tradicionales para el tratamiento de diversas dolencias. Además de sus propiedades medicinales, se ha identificado como una fuente potencial de producción de antocianinas. En esta

  18. Somatic embryogenesis in wild relatives of cotton (Gossypium Spp.)

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Wild cotton species can contribute a valuable gene pool for agronomically desirable cultivated tetraploid cultivars. In order to exploit diploid cotton a regeneration system is required to achieve transformation based goals. The present studies aimed at optimizing the conditions for regeneration of local varieties as well as wild species of cotton. Different callus induction media were tested with varying concentrations of hormones in which sucrose was used as nutritional source. Different explants (hypocotyls, cotyledon, root) were used to check the regeneration of both local cotton plants and wild relatives using T & G medium,BAP medium, CIM medium, EMMS medium, and cell suspension medium. Different stages of embryogenicity such as early torpedo stage, late torpedo stage, heart stage, globular stage and cotyledonary stage were observed in wild relatives of cotton. The results of this study pave the way for establishing future transformation methods.

  19. Establishment of the in vitro CottonRoot Tip Culture System%棉花离体根尖培养体系的建立

    Institute of Scientific and Technical Information of China (English)

    祝水金; 季道藩

    2000-01-01

    从培养基成分着手,研究了影响棉花离体根尖培养的各种因素,以建立棉花离体根尖培养体系。研究结果表明,棉花根尖培养的培养基为:MS大量元素+1/2 MS微量元素+100mg·L。肌醇+1.0mg·L。烟酸+10.0mg·L。盐酸硫胺素+L 0mg·L。盐酸吡哆辛+20mg·L。蔗糖+0.125mg·L。吲哚丁酸,用0.8%的琼脂为支撑物,调节pH值为6.4。培养温度为(28±2)℃,每天光照14 h,光照强度为2000 Lx。用此方法进行棉花根尖培养,离体根系可在培养基中生长达2个月而不影响其生活力。该棉花根尖培养体系适用于陆地棉和海岛棉,但基因型之间培养效果存在一定差异。%Conditions of cotton root tip culture invitro were studied in this paper. The recom-mended medium for cotton root tip culture invitro was macronutrients and iron salt of MS ba-sic medium, 1/2 micronutruents of MS basicmedium, vitamins and organic materials of B6basic medium containing 100mg · L-1 of inositol,1.0 mg· L1 of nicotonic acid, 10.0 mg· L-1 ofvitamin B1, 1.0 mg· L4 of vitamin B6, 20 mg ·L-1 of saccharose as the carbon resource and 0. 125 mg · L-t of IBA, using 0. 8% of agar asthe solid material and adjusting pH to 6.4. Thecotton root tips were cultured at a photoperiodof 14 hours daily with a light intensity of 2000Lx and temperature of (28±2)C . The excitedcotton roots could grow very well in the mediumfor more than two months with this culturemethod. This culture system was suitable forthe materials of both G. hirsutum and G. bar-badense, though some differences were presentamong the genotypes.

  20. 陆地棉抗虫遗传工程%Genetic Engineering of Cotton (Gossypium hirsutum L. ) for Insect-resistance

    Institute of Scientific and Technical Information of China (English)

    Shengwei ZHU; Jingsan SUN; Yinchuan TIAN

    2002-01-01

    @@ In order to improve insect-resistance of cotton and cultivate new cotton varieties ,tissue culture and plant regeneration of cotton (Gossypium hirsutum L. ) were studied with Xinluzao 4,Xi 550,Jizi 492,Hengwu 89-30,Han 93-2 and Jizi 123 . A system of cotton tissue culture for rapid plant regeneration was developed.

  1. An inverse relationship between allelopathic activity and salt tolerance in suspension cultures of three mangrove species, Sonneratia alba, S. caseolaris and S. ovata: development of a bioassay method for allelopathy, the protoplast co-culture method.

    Science.gov (United States)

    Hasegawa, Ai; Oyanagi, Tomoya; Minagawa, Reiko; Fujii, Yoshiharu; Sasamoto, Hamako

    2014-11-01

    A bioassay method for allelopathy, the 'protoplast co-culture method' was developed to study the relationship between salt tolerance and allelopathy of three mangrove species, Sonneratia alba, S. caseolaris, and S. ovata. Plants of S. alba grow in the seaward-side high salinity region and plants of the latter two species grow in upstream-side regions of a mangrove forest, respectively. Effects of five sea salts (NaCl, KCl, MgCl2, MgSO4 and CaCl2) on the growth of the suspension cells of the latter two species were first investigated by a small-scale method using 24-well culture plates. S. ovata cells showed higher tolerance than S. caseolaris cells to NaCl and other salts, but were not as halophilic as S. alba cells. Protoplasts isolated from suspension cells were co-cultured with lettuce protoplasts in Murashige and Skoog's (MS) basal medium containing 1 μM 2,4-dichlorophenoxyacetic acid, 0.1 μM benzyladenine, 3% sucrose and 0.6-0.8 M osmoticum. S. caseolaris protoplasts had a higher inhibitory effect on lettuce protoplast cell divisions than S. alba protoplasts at any lettuce protoplast density, and the effect of S. ovata was intermediate between the two. These results were similar to those obtained from a different in vitro bioassay method for allelopathy, the 'sandwich method' with dried leaves. The inverse relationship between allelopathic activity and salt tolerance in suspension cells of Sonneratia mangroves is discussed.

  2. Production of Limonoids with Insect Antifeedant Activity in a Two-Stage Bioreactor Process with Cell Suspension Culture of Azadirachta indica.

    Science.gov (United States)

    Vásquez-Rivera, Andrés; Chicaiza-Finley, Diego; Hoyos, Rodrigo A; Orozco-Sánchez, Fernando

    2015-09-01

    Neem tree (Azadirachta indica) cell suspension culture is an alternative for the production of limonoids for insect control that overcomes limitations related to the supply of neem seeds. To establish conditions for cell growth and azadiracthin-related limonoid production, the effect of different sucrose concentrations, nitrate and phosphate in Murashige and Skoog (MS) medium, and the addition of one precursor and three elicitors was evaluated in shake flasks. The process was scaled up to a 3-l stirred tank bioreactor in one- and two-stage batch cultivation. In shake flasks, more than fivefold increase in the production of limonoids with the modified MS medium was observed (increase from 0.77 to 4.52 mg limonoids/g dry cell weight, DCW), while an increase of more than fourfold was achieved by adding the elicitors chitosan, salicylic acid, and jasmonic acid together (increase from 1.03 to 4.32 mg limonoids/g DCW). In the bioreactor, the volumetric production of limonoids was increased more than threefold with a two-stage culture in day 18 (13.82 mg limonoids/l in control single-stage process and 41.44 mg/l in two-stage process). The cultivation and operating mode of the bioreactor reported in this study may be adapted and used in optimization and process plant development for production of insect antifeedant limonoids with A. indica cell suspension cultures.

  3. Diadenosine triphosphate is a novel factor which in combination with cyclodextrins synergistically enhances the biosynthesis of trans-resveratrol in Vitis vinifera cv. Monastrell suspension cultured cells.

    Science.gov (United States)

    Pietrowska-Borek, Małgorzata; Czekała, Łukasz; Belchí-Navarro, Sarai; Pedreño, María Angeles; Guranowski, Andrzej

    2014-11-01

    Dinucleoside polyphosphates are considered as signal molecules that may evoke response of plant cells to stress. Other compounds whose biological effects have been recognized are cyclodextrins. They are cyclic oligosaccharides that chemically resemble the alkyl-derived pectic oligosaccharides naturally released from the cell walls during fungal attack, and they act as true elicitors, since, when added to plant cell culture, they induce the expression of genes involved in some secondary metabolism pathways. Previously, we demonstrated that some dinucleoside polyphosphates triggered the biosynthesis of enzymes involved in the phenylpropanoid pathway in Arabidopsis thaliana. In Vitis vinifera suspension cultured cells, cyclodextrins were shown to enhance the accumulation of trans-resveratrol, one of the basic units of the stilbenes derived from the phenylpropanoid pathway. Here, we show that diadenosine triphosphate, applied alone or in combination with cyclodextrins to the grapevine suspension-cultured cells, increased the transcript level of genes encoding key phenylpropanoid-pathway enzymes as well as the trans-resveratrol production inside cells and its secretion into the extracellular medium. In the latter case, these two compounds acted synergistically. However, the accumulation of trans-resveratrol and its glucoside trans-piceid inside cells were stimulated much better by diadenosine triphosphate than by cyclodextrins. Copyright © 2014. Published by Elsevier Masson SAS.

  4. Host-Pathogen Interactions : XXIV. Fragments Isolated from Suspension-Cultured Sycamore Cell Walls Inhibit the Ability of the Cells to Incorporate [C]Leucine into Proteins.

    Science.gov (United States)

    Yamazaki, N; Fry, S C; Darvill, A G; Albersheim, P

    1983-07-01

    A bioassay to measure the incorporation of [(14)C]leucine into acid-precipitable polymers of suspension-cultured sycamore (Acer pseudoplatanus L.) cells is described. Using this assay, cell wall fragments solubilized from sycamore cell walls by partial acid hydrolysis are shown to contain components that inhibit the incorporation of [(14)C]leucine into the acid-precipitable polymers. This inhibition was not attributable to a suppression of [(14)C]leucine uptake. The effectiveness of the wall fragments in inhibiting [(14)C]leucine incorporation was substantially relieved by plasmolysis of the cells. Fragments released from starch and citrus pectin are shown not to possess such inhibitory activities.

  5. Producing biodiesel from cotton seed oil using Rhizopus oryzae ATTC #34612 whole cell biocatalysts: Culture media and cultivation period optimization

    Science.gov (United States)

    The effect of culture medium composition and cultivation time on biodiesel production by Rhizopus oryzae ATCC #34612 whole cell catalysts, immobilized on novel rigid polyethylene biomass supports, was investigated. Supplementation of the medium with carbon sources led to higher lipase activity and i...

  6. Cotton fertilization using PGPR Bacillus amyloliquefaciens FZB42 and compost: Impact on insect density and cotton yield in North Benin, West Africa

    Directory of Open Access Journals (Sweden)

    Thiery B. Charles Alavo

    2015-12-01

    Full Text Available This work has compared the effects of the biofertilizer Bacillus amyloliquefaciens FZB42 with that of compost for cotton production. The population dynamics of pests and predators have been studied in order to check whether the use of both fertilization materials can contribute to pest management in cotton. Three treatments were considered: (i dressing of seeds in rhizobacteria suspension, (ii introduction of rhizobacterial suspension directly in the pocket, same time with the seeds, and (iii fertilization with compost. The study was carried out in northwest Benin (West Africa. Results showed that cotton aphids, Aphis gossypii, pink bollworm, Pectinophora gossypiella, leaf roller, Sylepta derogata, and cotton bugs, Dysdercus sp. are the major insect pests encountered in the experimental plots. Cotton bollworm, Helicoverpa armigera, was present but under the economic threshold. The coccinellid predators, Cheilomenes spp., occurred in the experimental plots and almost suppressed aphid proliferation. Other natural enemies such as chrysopids and ant species also occurred and probably contributed to maintain the cotton bollworm under the economic threshold. The treatment with seeds dressed with the rhizobacteria suspension yielded 39% more cotton compared to the compost fertilization. The use of both fertilization materials without application of chemicals can contribute to pest management in cotton.

  7. Smart textiles: Tough cotton

    Science.gov (United States)

    Avila, Alba G.; Hinestroza, Juan P.

    2008-08-01

    Cotton is an important raw material for producing soft textiles and clothing. Recent discoveries in functionalizing cotton fibres with nanotubes may offer a new line of tough, wearable, smart and interactive garments.

  8. Improved Agrobacterium-mediated transformation protocol for VgDGAT1a gene based on shoot tip culture of cotton%农杆菌介导的VgDGAT1a基因棉花茎尖转化体系优化研究

    Institute of Scientific and Technical Information of China (English)

    王安可; 何秋伶; 潘晶晶; 孙英超; 祝水金; 陈进红

    2013-01-01

    In past two decades , abundant researches have been published on transformation , regeneration , and genetic enhancement of cotton , especially Gossypium hirsutum . Generally , genes conferring agronomic advantages have been introduced into plants via A grobacterium‐mediation or particle bombardment , and then the transgenic plants are regenerated through somatic embryogenesis from callus . However , the embryogenic callus‐based regeneration is difficult and time‐consuming in cotton . Therefore , it is necessary to establish an effective system for the A grobacterium‐mediated genetic transformation of shoot tip in cotton . Triacylglycerols ( TAG) are a heterogeneous group of molecules with a glycerol backbone and three fatty acids attached by ester bones . Diacylglycerol acyltransferase ( DGAT ; EC3 .2 .1 .20) catalyzes the last step of TAG biosynthesis and it is the only key enzyme evolved in . DGA T1 and DGA T2 , as two types of DGA T in eukaryotes , belong to different gene families . And previous studies have reported that the expression of DGA T1 increased seed oil content and mass . In order to get new cotton germplasm with high oil content , we used the shoot tips of Zhongmiansuo 49 as explants and introduced an improved vector carrying a selection marker H ptⅡ gene and a target V gDGA T1a gene into cotton via A grobacterium‐mediated transformation . This improved A grobacterium‐mediated transformation and regeneration system were established by optimizing different parameters such as pre‐culture period of seeds , concentration of A grobacterium in solution , immersing time , co‐cultivation period and components of MSB [ Murashige and Skoog ( MS) medium + vitamins of Gamborg s (B5) medium] . Cotton seeds ( Gossypium hirsutum L . cv . Zhongmiansuo 49 ) were decorticated manually and surface sterilized in 0 .1% HgCl for 8 min , followed by rinsing with sterile distilled water for five times . The cotyledons were removed from 3‐day‐old to

  9. Targeted Gene Deletion Using DNA-Free RNA-Guided Cas9 Nuclease Accelerates Adaptation of CHO Cells to Suspension Culture.

    Science.gov (United States)

    Lee, Namil; Shin, JongOh; Park, Jin Hyoung; Lee, Gyun Min; Cho, Suhyung; Cho, Byung-Kwan

    2016-11-18

    Chinese hamster ovary (CHO) cells are the preferred host for the production of a wide array of biopharmaceuticals. Thus, efficient and rational CHO cell line engineering methods have been in high demand to improve quality and productivity. Here, we provide a novel genome engineering platform for increasing desirable phenotypes of CHO cells based upon the integrative protocol of high-throughput RNA sequencing and DNA-free RNA-guided Cas9 (CRISPR associated protein9) nuclease-based genome editing. For commercial production of therapeutic proteins, CHO cells have been adapted for suspension culture in serum-free media, which is highly beneficial with respect to productivity and economics. To engineer CHO cells for rapid adaptation to a suspension culture, we exploited strand-specific RNA-seq to identify genes differentially expressed according to their adaptation trajectory in serum-free media. More than 180 million sequencing reads were generated and mapped to the currently available 109,152 scaffolds of the CHO-K1 genome. We identified significantly downregulated genes according to the adaptation trajectory and then verified their effects using the genome editing method. Growth-based screening and targeted amplicon sequencing revealed that the functional deletions of Igfbp4 and AqpI gene accelerate suspension adaptation of CHO-K1 cells. The availability of this strand-specific transcriptome sequencing and DNA-free RNA-guided Cas9 nuclease mediated genome editing facilitates the rational design of the CHO cell genome for efficient production of high quality therapeutic proteins.

  10. Hydrogen sulfide donor sodium hydrosulfide-induced heat tolerance in tobacco (Nicotiana tabacum L) suspension cultured cells and involvement of Ca(2+) and calmodulin.

    Science.gov (United States)

    Li, Zhong-Guang; Gong, Ming; Xie, Hong; Yang, Lan; Li, Jing

    2012-04-01

    Hydrogen sulfide (H(2)S) is considered as a new emerging cell signal in higher plants. Hydrogen sulfide donor, sodium hydrosulfide, pretreatment significantly increased survival percentage of tobacco suspension cultured cells under heat stress and regrowth ability after heat stress, and alleviated decrease in vitality of cells, increase in electrolyte leakage and accumulation of malondialdehyde (MDA). In addition, sodium hydrosulfide-induced heat tolerance was markedly strengthened by application of exogenous Ca(2+) and its ionophore A23187, respectively, while this heat tolerance was weakened by addition of Ca(2+) chelator ethylene glycol-bis(b-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), plasma membrane channel blocker La(3+), as well as calmodulin (CaM) antagonists chlorpromazine (CPZ) and trifluoperazine (TFP), respectively, but intracellular channel blocker ruthenium red (RR) did not. These results suggested that sodium hydrosulfide pretreatment could improve heat tolerance in tobacco suspension cultured cells and the acquisition of this heat tolerance requires the entry of extracellular Ca(2+) into cells across the plasma membrane and the mediation of intracellular CaM.

  11. Structure of Plant Cell Walls : XXVI. The Walls of Suspension-Cultured Sycamore Cells Contain a Family of Rhamnogalacturonan-I-Like Pectic Polysaccharides.

    Science.gov (United States)

    Ishii, T; Thomas, J; Darvill, A; Albersheim, P

    1989-02-01

    Considerable information has been obtained about the primary structures of suspension-cultured sycamore (Acer pseudoplatanus) cell-wall pectic polysaccharides, i.e. rhamnogalacturonan I, rhamnogalacturonan II, and homogalacturonan. However, these polysaccharides, which are solubilized from the walls by endo-alpha-1,4-polygalacturonase, account for only about half of the pectic polysaccharides known to be present in sycamore cell walls. We now report that, after exhaustive treatment with endo-alpha-1,4-polygalacturonase, additional pectic polysaccharides were extracted from sycamore cell walls by treatment with Na(2)CO(3) at 1 and 22 degrees C. These previously uncharacterized polysaccharides accounted for approximately 4% of the cell wall. Based on the glycosyl and glycosyl-linkage compositions and the nature of the products obtained by treating the quantitatively predominant NaCO(3)-extracted polysaccharides with lithium metal dissolved in ethylenediamine, the polysaccharides were found to strongly resemble rhamnogalacturonan I. However, unlike rhamnogalacturonan I that characteristically had equal amounts of 2- and 2,4-linked rhamnosyl residues in its backbone, the polysaccharides extracted in Na(2)CO(3) at 1 degrees C had markedly disparate ratios of 2- to 2,4-linked rhamnosyl residues. We concluded that polysaccharides similar to rhamnogalacturonan I but with different degrees of branching are present in the walls of suspension-cultured sycamore cells.

  12. Cotton Trip in China

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    From September 6th to 12th,a National Cotton CouncilCotton Council International 2010 China leadership team,led by Charles Parker,Vice Chairman of NCC,visited China to see its cotton industrial development and continue building a good relationship with U.S.raw cotton’s largest consumer.

  13. World Collection of Cotton

    Institute of Scientific and Technical Information of China (English)

    KHAKIMJON Saydaliyev; ALISHER Amanturdiev; MALOXAT Halikova

    2008-01-01

    @@ Achievements of selection and other theoretical researches on cotton not only in our country,but also world-wide depend on the presence of genetic resources.Uzbek Scientific Research Institute of Selection and Seed Growing of Cotton is a leading center of science on breeding and production of cotton across Central Asia.

  14. Cotton Pricing Discussion

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    @@ Cotton prices have received a lot of attention recently.Cotton Incorporated especically designed this Special Edition of Supply Chain Insights to frame the discussion concerning prices throughout the cotton supply chain in terms of the cyclical events that contributed to recent volatility and how a return to long-term averages over time can be expected.

  15. Dictionary of Cotton

    Science.gov (United States)

    The Dictionary of Cotton has over 2,000 terms and definitions that were compiled by 33 researchers. It reflects the ongoing commitment of the International Cotton Advisory Committee, through its Technical Information Section, to the spread of knowledge about cotton to all those who have an interest ...

  16. Instability of anthocyanin composition under different subculture conditions during long-term suspension cultures of Vitis vinifera L. var. Gamay Fréaux.

    Science.gov (United States)

    Qu, Junge; Zhang, Wei; Yu, Xingju

    2011-11-01

    The instability of secondary metabolite production is a ubiquitous problem in plant cell culture. In order to understand the instability in plant cell culture, we investigated anthocyanin accumulation in suspension cultures of Vitis vinifera, as a model system, in our laboratory. Not only the anthocyanin contents but also its composition exhibited instability along with the long-term subculture. New methods were developed to indicate the instability of plant cell culture. Both the definition of instability coefficient (delta) and the application of factor scores were the first time in this field. To examine the effects of culture conditions on instability of anthocyanin biosynthesis, different subculture cycles and inoculum sizes had been investigated. Subculture cycle and inoculum size were both environmental cues driving the instability. Compared with subculture cycle, inoculum size was more effective in working on the instability of anthocyanin accumulation. Among all the conditions investigated in our study, (6.5 d, 2.00 g), (7 d, 2.00 g), (7.5 d, 2.00 g), (7 d, 1.60 g) and (7 d, 2.40 g), the condition of 7 d-subculture cycle together with 1.60 g-inoculum size was the best one to keep the stable production of anthocyanins.

  17. Effects of pulsed electric field on secondary metabolism of Vitis vinifera L. cv. Gamay Fréaux suspension culture and exudates.

    Science.gov (United States)

    Cai, Zhenzhen; Riedel, Heidi; Thaw Saw, Nay Min Min; Kütük, Onur; Mewis, Inga; Jäger, Henry; Knorr, Dietrich; Smetanska, Iryna

    2011-06-01

    Plant cell cultures provide a large potential for the production of secondary metabolites. Through the application of different physical and chemical cell stress factors, we investigated the production of the secondary metabolites in plant cell cultures. The effects of pulsed electric field (PEF) and ethephon on growth and secondary metabolism, particularly anthocyanins and phenolic acids synthesis, were investigated by using suspension culture of Vitis vinifera L. cv. Gamay Fréaux as a model system. Anthocyanins were measured by spectrophotometer and extracellular phenolic acids were determined by high-performance liquid chromatography. The compounds were identified by liquid chromatography-mass spectrometry and nuclear magnetic resonance. After the treatments with PEF and ethephon, the concentrations of anthocyanins and phenolic acids in cell culture were higher than in the control, without loss of biomass. The combination of PEF treatment and ethephon improved secondary metabolites formation. Production levels of extracellular phenolic acids, 3-O-glucosyl-resveratrol were increased by PEF and ethephon treatments. The results show that PEF induced a defense response of plant cells and may have altered the cell/membrane's dielectric properties. PEF, an external stimulus or stress, is proposed as a promising new abiotic elicitor for stimulating secondary metabolites biosynthesis in plant cell cultures.

  18. Enhancement of recombinant human EPO production and glycosylation in serum-free suspension culture of CHO cells through expression and supplementation of 30Kc19.

    Science.gov (United States)

    Park, Ju Hyun; Wang, Zesong; Jeong, Hee-Jin; Park, Hee Ho; Kim, Byung-Gee; Tan, Wen-Song; Choi, Shin Sik; Park, Tai Hyun

    2012-11-01

    We previously reported that the expression of Bombyx mori 30Kc19 gene in CHO cells significantly improved both the production and sialylation of recombinant human EPO (rHuEPO) in adhesion culture mode. In this study, the effects of 30Kc19 expression and supplementation of 30Kc19 recombinant protein on the productivity and glycosylation pattern of rHuEPO were investigated in the serum-free suspension culture mode. Especially, glycosylation pattern was examined in detail using a quantitative MALDI-TOF MS method. The expression of 30Kc19 increased the EPO production by 2.5-folds and the host cells produced rHuEPO with more complex glycan structures and a larger content of sialic acid and fucose. The glycan structures of rHuEPO in the 30Kc19-expressing cell consisted of bi-, tri-, tetra-, and penta-antennary branching (35, 18, 33, and 14 %, respectively), while the control cells produced predominantly bi-antennary branching (70 %). About 53 % of the glycans from rHuEPO in the 30Kc19-expressing cell was terminally sialylated, while no obvious sialylated glycan was found in the control cells. The percentage of fucosylated glycans from the 30Kc19-expressing cell culture was 77 %, whereas only 61 % of the glycans from the control cell were fucosylated glycans. We also examined whether these effects were observed when the recombinant 30Kc19 protein produced from Escherichia coli was supplemented into the culture medium for CHO cells. In the control cell line without the 30Kc19 gene, EPO production increased by 41.6 % after the addition of 0.2 mg/mL of the recombinant 30Kc19 protein to the culture medium. By the Western blot analysis after two-dimensional electrophoresis (2-DE) of isoforms of EPO, we confirmed that 30Kc19 enhanced the sialylation of EPO glycans. These results demonstrated that both 30Kc19 gene expression and the recombinant 30Kc19 protein addition enhanced rHuEPO productivity and glycosylation in suspension culture. In conclusion, the utilization of

  19. Studies on the Characteris of the Induction of Cotton Fiber Derived from Cotton Ovule Callus Cells%棉胚珠愈伤组织诱导成纤维实验系统的研究

    Institute of Scientific and Technical Information of China (English)

    汤清秀; 赵旌旌; 王隆华

    2000-01-01

    Cotton nbers were induced from cotton ovule callus cells by suspension culture,floatculture or filter paper bridge culture.h was found that float culture or filter paper bridge culture was bener than suspension culture.APM and actinomycin D inhibit the fibe elongation.Cellobiose promotes its development. Polarity affects its culture.%用棉花胚珠切块诱导愈伤组织,经悬浮振荡培养、漂浮培养、滤纸桥法等方法诱导成纤维细胞。发现漂浮培养和改进的滤纸桥法对纤维的诱导效果比悬浮振荡培养的效果好。微管解聚剂APM和核酸抑制剂抑制纤维的生长,纤维二糖有一定的促进作用。极性对纤维的生长有影响。

  20. Genetic transformation of cry1EC gene into cotton (Gossypium ...

    African Journals Online (AJOL)

    welcome

    2013-04-10

    Apr 10, 2013 ... sp is not susceptible to Bt cotton containing Cry1Ac toxin so, there is ... encodes an insecticidal protein Cry1EC, (ii) 35S promoter from cauliflower ... the embryogenic calli were treated with the bacterial suspension by shaking ...

  1. Comparative study of withanolide production and the related transcriptional responses of biosynthetic genes in fungi elicited cell suspension culture of Withania somnifera in shake flask and bioreactor.

    Science.gov (United States)

    Ahlawat, Seema; Saxena, Parul; Ali, Athar; Khan, Shazia; Abdin, Malik Z

    2017-05-01

    Ashwagandha (Withania somnifera) is one of the most reputed medicinal plants in the traditional medicinal system. In this study, cell suspension culture of W. somnifera was elicited with cell homogenates of fungi (A. alternata, F. solani, V. dahliae and P. indica) in shake flask and the major withanolides like withanolide A, withaferin A and withanone were analysed. Simultaneously expression levels of key pathway genes from withanolides biosynthetic pathways were also checked via quantitative PCR in shake flask as well as in bioreactor. The results show that highest gene expression of 10.8, 5.8, 4.9, and 3.3 folds were observed with HMGR among all the expressed genes in cell suspension cultures with cell homogenates of 3% P. indica, 5% V. dahliae, 3% A. alternata and 3% F. solani, respectively, in comparison to the control in shake flask. Optimized concentration of cell homogenate of P. indica (3% v/v) was added to the growing culture in 5.0-l bioreactor under optimized up-scaling conditions and harvested after 22 days. The genes of MVA, MEP and withanolides biosynthetic pathways like HMGR, SS, SE, CAS, FPPS, DXR and DXS were up-regulated by 12.5, 4.9, 2.18, 4.65, 2.34, 1.89 and 1.4 folds, respectively in bioreactor. The enhancement of biomass (1.13 fold) and withanolides [withanolide A (1.7), withaferin A (1.5), and withanone (1.5) folds] in bioreactor in comparison to shake flask was also found to be in line with the up-regulation of genes of withanolide biosynthetic pathways. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  2. Structure of Plant Cell Walls: XI. GLUCURONOARABINOXYLAN, A SECOND HEMICELLULOSE IN THE PRIMARY CELL WALLS OF SUSPENSION-CULTURED SYCAMORE CELLS.

    Science.gov (United States)

    Darvill, J E; McNeil, M; Darvill, A G; Albersheim, P

    1980-12-01

    The isolation, purification, and partial characterization of a glucuronoarabinoxylan, a previously unobserved component of the primary cell walls of dicotyledonous plants, are described. The glucuronoarabinoxylan constitutes approximately 5% of the primary walls of suspension-cultured sycamore cells. This glucuronoarabinoxylan possesses many of the structural characteristics of analogous polysaccharides that have been isolated from the primary and secondary cell walls of monocots as well as from the secondary cell walls of dicots. The glucuronoarabinoxylan of primary dicot cell walls has a linear beta-1,4-linked d-xylopyranosyl backbone with both neutral and acidic sidechains attached at intervals along its length. The acidic sidechains are terminated with glucuronosyl or 4-O-methyl glucuronosyl residues, whereas the neutral sidechains are composed of arabinosyl and/or xylosyl residues.

  3. Efficient and high-throughput vector construction and Agrobacterium-mediated transformation of Arabidopsis thaliana suspension-cultured cells for functional genomics.

    Science.gov (United States)

    Ogawa, Yoichi; Dansako, Tomoko; Yano, Kentaro; Sakurai, Nozomu; Suzuki, Hideyuki; Aoki, Koh; Noji, Masaaki; Saito, Kazuki; Shibata, Daisuke

    2008-02-01

    We established a large-scale, high-throughput protocol to construct Arabidopsis thaliana suspension-cultured cell lines, each of which carries a single transgene, using Agrobacterium-mediated transformation. We took advantage of RIKEN Arabidopsis full-length (RAFL) cDNA clones and the Gateway cloning system for high-throughput preparation of binary vectors carrying individual full-length cDNA sequences. Throughout all cloning steps, multiple-well plates were used to treat 96 samples simultaneously in a high-throughput manner. The optimal conditions for Agrobacterium-mediated transformation of 96 independent binary vector constructs were established to obtain transgenic cell lines efficiently. We evaluated the protocol by generating transgenic Arabidopsis T87 cell lines carrying individual 96 metabolism-related RAFL cDNA fragments, and showed that the protocol was useful for high-throughput and large-scale production of gain-of-function lines for functional genomics.

  4. Establishing Embryogenic Callus Suspension Culture System in Black Locust%刺槐胚性细胞悬浮体系的建立1)

    Institute of Scientific and Technical Information of China (English)

    冯玥; 习洋; 陈串; 喻娃亚雄; 王少明; 徐惠敏; 孙宇涵; 李云

    2014-01-01

    以刺槐( Robinia pseudoacacia L.)未成熟合子胚为外植体材料,通过对外植体取材时期、基本培养基营养物质质量浓度、细胞起始密度、2,4-二氯苯氧乙酸(2,4-D)质量浓度等影响悬浮培养的因素的研究,建立了良好的刺槐胚性细胞悬浮系。研究结果表明:授粉后55 d的未成熟合子胚是为刺槐细胞悬浮培养提供胚性愈伤组织的最佳起始材料;添加有2.5 mg· L-1 NAA和0.5 mg· L-16-BA的1/2MS培养基为刺槐细胞悬浮培养的最适培养基;建立悬浮系的最适pH值范围在5.0~5.7;起始质量为2.0 g的悬浮系统其生长量增速较快,且细胞保持持续增长;当起始质量为3.0 g时,培养16 d后细胞进入缓慢增长期,且生长量有回落的趋势,起始质量为1.0 g时,细胞增殖速度非常缓慢,且容易出现褐化死亡的现象。%We employed embryogenic cell suspension ( ECS) induced from immature seeds of black locust ( Robinia pseudoaca-cia L.) as materials to study the effecting factors as explants period , nutrient concentration of basic medium , initial cell density and 2,4-D concentration of suspension culture medium to establish a successful embryogenic callus suspension cul -ture system for black locust.The seeds collected at eight weeks (about 55 days) post-anthesis obtained the highest ability to initiate embryogenic culture for embryogenic callus cell suspension establishment .The best callus initiation medium was 1/2MS+2.5 mg· L-1 NAA+0.5 mg· L-1 6-BA and the suitable pH ranged in 5.0-5.7.The cells maintained a sustained growth as initial density of suspension system was 2.0 g, the cells proliferation was slowed down after 16 days when the ini-tial density was 3.0 g, and the growth rate was with down trend .The cells were brown and died in slow proliferation rate with the initial density of 1.0 g.

  5. Metabolic analysis of elicited cell suspension cultures of Cannabis sativa L. by (1)H-NMR spectroscopy.

    Science.gov (United States)

    Pec, Jaroslav; Flores-Sanchez, Isvett Josefina; Choi, Young Hae; Verpoorte, Robert

    2010-07-01

    Cannabis sativa L. plants produce a diverse array of secondary metabolites. Cannabis cell cultures were treated with jasmonic acid (JA) and pectin as elicitors to evaluate their effect on metabolism from two cell lines using NMR spectroscopy and multivariate data analysis. According to principal component analysis (PCA) and partial least square-discriminant analysis (PLS-DA), the chloroform extract of the pectin-treated cultures were more different than control and JA-treated cultures; but in the methanol/water extract the metabolome of the JA-treated cells showed clear differences with control and pectin-treated cultures. Tyrosol, an antioxidant metabolite, was detected in cannabis cell cultures. The tyrosol content increased after eliciting with JA.

  6. Effects of Polysaccharide Elicitors from Endophytic Fusarium oxysporium Dzf17 on Growth and Diosgenin Production in Cell Suspension Culture of Dioscorea zingiberensis

    Directory of Open Access Journals (Sweden)

    Ligang Zhou

    2011-10-01

    Full Text Available Three polysaccharides, namely exopolysaccharide (EPS, water-extracted mycelial polysaccharide (WPS and sodium hydroxide-extracted mycelial polysaccharide (SPS, were prepared from the endophytic fungus Fusarium oxysporium Dzf17 isolated from the rhizomes of Dioscorea zingiberensis. The effects of the time of addition and polysaccharide concentration on the growth and diosgenin accumulation in cell suspension culture of D. zingiberensis were studied. Among them, WPS was found to be the most effective polysaccharide. When WPS was added to the medium at 20 mg/L on the 25th day of culture, the cell dry weight was increased 1.34-fold, diosgenin content 2.85-fold, and diosgenin yield 3.83-fold in comparison to those of control. EPS and SPS showed moderate and relatively weak enhancement effects on cell growth and diosgenin accumulation, respectively. The dynamics of cell growth and diosgenin accumulation when WPS was added to the medium at 20 mg/L on the 25th day of culture were investigated, and results showed that dry weight of cells reached a maximum value on day 30 but the maximum diosgenin content was achieved on day 31.

  7. Effects of polysaccharide elicitors from endophytic Fusarium oxysporium Dzf17 on growth and diosgenin production in cell suspension culture of Dioscorea zingiberensis.

    Science.gov (United States)

    Li, Peiqin; Mou, Yan; Shan, Tijiang; Xu, Jianmei; Li, Yan; Lu, Shiqiong; Zhou, Ligang

    2011-10-26

    Three polysaccharides, namely exopolysaccharide (EPS), water-extracted mycelial polysaccharide (WPS) and sodium hydroxide-extracted mycelial polysaccharide (SPS), were prepared from the endophytic fungus Fusarium oxysporium Dzf17 isolated from the rhizomes of Dioscorea zingiberensis. The effects of the time of addition and polysaccharide concentration on the growth and diosgenin accumulation in cell suspension culture of D. zingiberensis were studied. Among them, WPS was found to be the most effective polysaccharide. When WPS was added to the medium at 20 mg/L on the 25th day of culture, the cell dry weight was increased 1.34-fold, diosgenin content 2.85-fold, and diosgenin yield 3.83-fold in comparison to those of control. EPS and SPS showed moderate and relatively weak enhancement effects on cell growth and diosgenin accumulation, respectively. The dynamics of cell growth and diosgenin accumulation when WPS was added to the medium at 20 mg/L on the 25th day of culture were investigated, and results showed that dry weight of cells reached a maximum value on day 30 but the maximum diosgenin content was achieved on day 31.

  8. The age-dependent epigenetic and physiological changes in an Arabidopsis T87 cell suspension culture during long-term cultivation

    Energy Technology Data Exchange (ETDEWEB)

    Kwiatkowska, Aleksandra, E-mail: A.Kwiatkows@gmail.com [Department of Botany, University of Rzeszow, Kolbuszowa (Poland); Zebrowski, Jacek [Department of Plant Physiology, University of Rzeszow, Kolbuszowa (Poland); Oklejewicz, Bernadetta [Department of Genetics, University of Rzeszow, Kolbuszowa (Poland); Czarnik, Justyna [Department of Botany, University of Rzeszow, Kolbuszowa (Poland); Halibart-Puzio, Joanna [Department of Plant Physiology, University of Rzeszow, Kolbuszowa (Poland); Wnuk, Maciej [Department of Genetics, University of Rzeszow, Kolbuszowa (Poland)

    2014-05-02

    Highlights: • A decrease in proliferation rate during long-term cultivation of Arabidopsis cells. • Age-dependent increase in senescence-associated gene expression in Arabidopsis cells. • Age-related increase in DNA methylation, H3K9me2, and H3K27me3 in Arabidopsis cells. • High potential of photosynthetic efficiency of long-term cultured Arabidopsis cells. - Abstract: Plant cell suspension cultures represent good model systems applicable for both basic research and biotechnological purposes. Nevertheless, it is widely known that a prolonged in vitro cultivation of plant cells is associated with genetic and epigenetic instabilities, which may limit the usefulness of plant lines. In this study, the age-dependent epigenetic and physiological changes in an asynchronous Arabidopsis T87 cell culture were examined. A prolonged cultivation period was found to be correlated with a decrease in the proliferation rate and a simultaneous increase in the expression of senescence-associated genes, indicating that the aging process started at the late growth phase of the culture. In addition, increases in the heterochromatin-specific epigenetic markers, i.e., global DNA methylation, H3K9 dimethylation, and H3K27 trimethylation, were observed, suggesting the onset of chromatin condensation, a hallmark of the early stages of plant senescence. Although the number of live cells decreased with an increase in the age of the culture, the remaining viable cells retained a high potential to efficiently perform photosynthesis and did not exhibit any symptoms of photosystem II damage.

  9. American Cotton Development Strategy

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    The 30th International Cotton Conference took place March 24 - 27 in the historic city of Bremen,Germany this year.Worldwide high-ranking experts from cotton production, trade,spinning,weaving and some other fields of textile industries gathered together in the Bremen Town Hall.Allen A.Terhaar,Executive Director of Cotton Council International(CCI), Washington,presented a speech on the future development strategy of American cotton industry,and the development schedule in Chinese market.In the following part,let’s share his opinions and foresighted views.

  10. American Cotton Development Strategy

    Institute of Scientific and Technical Information of China (English)

    Wang Ting

    2010-01-01

    @@ When we celebrated 2009 as the International Year of Natural Fiber, the global cotton industry joined hands in bringing recognition to cotton and all natural fibers. As we move into 2010 and beyond we must continue to engage the global consumer with messages that highlight the natural, renewable and biodegradable benefits of our product However, we must also go beyond what nature has provided and work toward true sustainability throughout the cotton supply chain. If some major brands and suppliers cannot achieve "sustainability" with cotton, they will do so with other fibers.

  11. Weed hosts of cotton mealybug, Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae).

    Science.gov (United States)

    Vennila, S; Prasad, Y G; Prabhakar, M; Agarwal, Meenu; Sreedevi, G; Bambawale, O M

    2013-03-01

    The exotic cotton mealybug, Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae) invaded India during 2006, and caused widespread infestation across all nine cotton growing states. P. solenopsis also infested weeds that aided its faster spread and increased severity across cotton fields. Two year survey carried out to document host plants of P. solenopsis between 2008 and 2010 revealed 27, 83, 59 and 108 weeds belonging to 8, 18, 10 and 32 families serving as alternate hosts at North, Central, South and All India cotton growing zones, respectively. Plant species of four families viz., Asteraceae, Amaranthaceae, Malvaceae and Lamiaceae constituted almost 50% of the weed hosts. While 39 weed species supported P. solenopsis multiplication during the cotton season, 37 were hosts during off season. Higher number of weeds as off season hosts (17) outnumbering cotton season (13) at Central over other zones indicated the strong carryover of the pest aided by weeds between two cotton seasons. Six, two and seven weed hosts had the extreme severity of Grade 4 during cotton, off and cotton + off seasons, respectively. Higher number of weed hosts of P. solenopsis were located at roadside: South (12) > Central (8) > North (3) zones. Commonality of weed hosts was higher between C+S zones, while no weed host was common between N+S zones. Paper furnishes the wide range of weed hosts of P. solenopsis, discusses their significance, and formulated general and specific cultural management strategies for nationwide implementation to prevent its outbreaks.

  12. Cloning, expression, and characterization of (+)-delta-cadinene synthase: a catalyst for cotton phytoalexin biosynthesis.

    Science.gov (United States)

    Chen, X Y; Chen, Y; Heinstein, P; Davisson, V J

    1995-12-20

    In cotton, sesquiterpene phytoalexins are elicited in response to bacterial or fungal infection. A Gossypium arboreum cell suspension culture which produces the sesquiterpene phytoalexin gossypol showed a time-dependent 10-fold increase in a 1.9-kb mRNA in response to a challenge by a preparation from Verticillium dahliae. The mRNA prepared from these elicited cultures was used to isolated two cDNA clones that contain open frames coding for proteins of 554 amino acids with M(r) 64,096 and 64,118. The encoded protein shows a significant degree of sequence identity with the other known plant terpene cyclases. Western blot analyses with a cross-reactive monoclonal antibody from a related sesquiterpene synthase in Nicotiana tabacum showed a time-dependent increase of a 65-kDa protein which reached a maximal level 24 h post elicitor treatment. The encoded protein from the pXC1 cDNA was produced in Escherichia coli and purified by affinity column chromatography. The enzymatic properties of this protein were identified by a radiochemical assay for cyclization of farnesyldiphosphate and a product structure was assigned by GC-MS, chiral phase GC, and NMR analyses as (+)-delta-cadinene. The fungal-elicited production of a (+)-delta-cadinene synthase is consistent with a role for this enzyme as the first committed step in the pathways leading to the related phytoalexins gossypol and lacinilene C in cotton.

  13. Generation of HIV-1 Gag VLPs by transient transfection of HEK 293 suspension cell cultures using an optimized animal-derived component free medium.

    Science.gov (United States)

    Cervera, Laura; Gutiérrez-Granados, Sonia; Martínez, Marta; Blanco, Julià; Gòdia, Francesc; Segura, María Mercedes

    2013-07-20

    Virus-like particles (VLPs) offer great promise as candidates for new vaccine strategies. Large-scale approaches for the manufacturing of HIV-1 Gag VLPs have mainly focused on the use of the baculovirus expression system. In this work, the development and optimization of an HIV-1 Gag VLP production protocol by transient gene expression in mammalian cell suspension cultures is reported. To facilitate process optimization, a Gag-GFP fusion construct enabling the generation of fluorescent VLPs was used. The great majority of Gag-GFP present in cell culture supernatants was shown to be correctly assembled into virus-like particles of the expected size and morphology consistent with immature HIV-1 particles. Medium optimization was performed using design of experiments (DoE). Culture medium supplementation with non-animal derived components including recombinant proteins and lipids of synthetic or non-animal-derived origin resulted in improved HEK 293 cell growth and VLP production. The maximum cell density attained using the optimized Freestyle culture medium was 5.4×10(6)cells/mL in batch mode, almost double of that observed using the unsupplemented medium (2.9×10(6)cells/mL). Best production performance was attained when cells were transfected at mid-log phase (2-3×10(6)cells/mL) with medium exchange at the time of transfection using standard amounts of plasmid DNA and polyethylenimine. By using an optimized production protocol, VLP titers were increased 2.4-fold obtaining 2.8μg of Gag-GFP/mL or 2.7×10(9)VLPs/mL according to ELISA and nanoparticle tracking quantification analyses, respectively. Copyright © 2013 Elsevier B.V. All rights reserved.

  14. Characterization of a cartilage-like engineered biomass using a self-aggregating suspension culture model: molecular composition using FT-IRIS.

    Science.gov (United States)

    Kim, Minwook; Kraft, Jeffrey J; Volk, Andrew C; Pugarelli, Joan; Pleshko, Nancy; Dodge, George R

    2011-12-01

    Maintenance of chondrocyte phenotype and robust expression and organization of macromolecular components with suitable cartilaginous properties is an ultimate goal in cartilage tissue engineering. We used a self-aggregating suspension culture (SASC) method to produce an engineered cartilage, "cartilage tissue analog" (CTA). With an objective of understanding the stability of phenotype of the CTA over long periods, we cultured chondrocytes up to 4 years and analyzed the matrix. Both early (eCTAs) (6 months) and aged (aCTAs) (4 years) showed type II collagen throughout with higher concentrations near the edge. Using Fourier transform-infrared imaging spectroscopy (FT-IRIS), proteoglycan/collagen ratio of eCTA was 2.8 times greater than native cartilage at 1 week, but the ratio was balanced to native level (p = 0.017) by 36 weeks. Surprisingly, aCTAs maintained the hyaline characteristics, but there was evidence of calcification within the tissue with a distinct range of intensities. Mineral/matrix ratio of those aCTA with "intensive" calcification was significantly higher (p = 0.017) than the "partial," but when compared to native bone the ratio of "intensive" aCTAs was 2.4 times lower. In this study we utilized the imaging approach of FT-IRIS and have shown that a biomaterial formed is compositionally closely related to natural cartilage for long periods in culture. We show that this culture platform can maintain a CTA for extended periods of time (4 years) and under those conditions signs of mineralization can be found. This method of cartilage tissue engineering is a promising method to generate cartilaginous biomaterial and may have potential to be utilized in both cartilage and boney repairs. Copyright © 2011 Orthopaedic Research Society.

  15. Biolistic transformation of cotton zygotic embryo meristem

    Science.gov (United States)

    Biolistic transformation of cotton meristems, isolated from mature seed is detailed in this book chapter. This method is simple and avoids the necessity to use genotype-dependent regenerable cell cultures. However, identification of germ line transformation using this method is laborious and time-c...

  16. Production of high-titer human influenza A virus with adherent and suspension MDCK cells cultured in a single-use hollow fiber bioreactor.

    Science.gov (United States)

    Tapia, Felipe; Vogel, Thomas; Genzel, Yvonne; Behrendt, Ilona; Hirschel, Mark; Gangemi, J David; Reichl, Udo

    2014-02-12

    Hollow fiber bioreactors (HFBRs) have been widely described as capable of supporting the production of highly concentrated monoclonal antibodies and recombinant proteins. Only recently HFBRs have been proposed as new single-use platforms for production of high-titer influenza A virus. These bioreactors contain multiple hollow fiber capillary tubes that separate the bioreactor in an intra- and an extra-capillary space. Cells are usually cultured in the extra-capillary space and can grow to a very high cell concentration. This work describes the evaluation of the single-use hollow fiber bioreactor PRIMER HF (Biovest International Inc., USA) for production of influenza A virus. The process was setup, characterized and optimized by running a total of 15 cultivations. The HFBRs were seeded with either adherent or suspension MDCK cells, and infected with influenza virus A/PR/8/34 (H1N1), and the pandemic strain A/Mexico/4108/2009 (H1N1). High HA titers and TCID₅₀ of up to 3.87 log₁₀(HA units/100 μL) and 1.8 × 10(10)virions/mL, respectively, were obtained for A/PR/8/34 influenza strain. Influenza virus was collected by performing multiple harvests of the extra-capillary space during a virus production time of up to 12 days. Cell-specific virus yields between 2,000 and 8,000 virions/cell were estimated for adherent MDCK cells, and between 11,000 and 19,000 virions/cell for suspension MDCK.SUS2 cells. These results do not only coincide with the cell-specific virus yields obtained with cultivations in stirred tank bioreactors and other high cell density systems, but also demonstrate that HFBRs are promising and competitive single-use platforms that can be considered for commercial production of influenza virus.

  17. Cotton-based nonwovens

    Science.gov (United States)

    This article is an abbreviated description of a new cotton-based nonwovens research program at the Southern Regional Research Center, which is one of the four regional research centers of the Agricultural Research Service, U.S. Department of Agriculture. Since cotton is a significant cash crop inte...

  18. An Optimized Method for Suspension Culture of CHO Cells to Produce Recombinant Human Erythropoietin (EPO)%悬浮培养CHO细胞生产重组人促红细胞生成素条件的优化

    Institute of Scientific and Technical Information of China (English)

    杨栋; 牛红军; 陆刚; 史嘉林; 孙浩明; 李晖

    2012-01-01

    Objective: To screen and domesticate the adherent cultured CHO cells to obtain high expression of cell suspension culture for production of recombinant human erythropoietin erythropoietin (rHuEPO). Methods: Using 96-well and 24-well plates culture method to screen and domesticate the highly expressing CHO cell strain. Acclimate the high expression cell strain and make it suitable for suspension culture. It's inoculated into the bioreactor in serum-free culture after amplified by the shake flask, and monitoring of glucose content, measuring rHuEPO expression of daily. Results: The suspension culture of CHO cell production of rHuEPO has short production period, higher expression than adherent culture. On the other hand, it is easy to operate and scale-up, but not easy to pollute. Furthermore, we established of the CHO cell strain for suspension culture,which provided a technical basis for industrial production of CHO cells the rHuEPO. Conclusion: After process optimization, the use of serum-free suspension culture production of erythropoietin average expression has high, short production period, low cost of production.than adherent culture.%目的:通过对贴壁培养CHO细胞筛选驯化,得到高表达的细胞后进行悬浮培养生产重组人促红细胞生成素(rHuEPO).方法:利用96孔板和24孔板对CHO细胞进行筛选,得到高表达细胞株后进行驯化,使其适合悬浮培养,经过摇瓶扩增后接种到生物反应器中无血清培养,每天监测葡萄糖含量,测rHuEPO表达量.结果:悬浮培养CHO细胞生产rHuEPO,生产周期短,表达量比贴壁培养高出很多,操作方便,减少污染,易于放大,并建立了适合悬浮培养的CHO细胞株,为工业化悬浮培养CHO细胞生产rHuEPO提供了技术基础.结论:经过工艺优化后利用无血清悬浮培养生产促红细胞生成素平均表达量较贴壁培养高,生产周期短,有利于降低生产成本.

  19. Cotton Demand Dropping in China

    Institute of Scientific and Technical Information of China (English)

    2012-01-01

    The ICAC claimed, global cotton market outlook is bleak in the 2012/2013 annual. Global cotton production is estimated at 25.9 million tons and cotton usage is estimated at 23.4 million tons. Cotton supply will exceed demand; the excess volume will reach 2.4 million tons.

  20. Dictionary of cotton: Picking & ginning

    Science.gov (United States)

    Cotton is an essential commodity for textiles and has long been an important item of trade in the world’s economy. Cotton is currently grown in over 100 countries by an estimated 100 producers. The basic unit of the cotton trade is the cotton bale which consists of approximately 500 pounds of raw c...

  1. About Viscosity of Cotton Fiber

    Institute of Scientific and Technical Information of China (English)

    SAGDULLAEV Ahror

    2008-01-01

    @@ The biological variety is mainly connected with presence of the field ecosites,which determine the mechanism of interaction (the symbiosis,pathogenesis,and etc.) that differ typically of such niches of live organism.The biological,forming on sowing of the cultural plants,including cotton plant are the example for this.Their formation is conditioned presence of the separations of aphids,consisting of different sugar,squirrel,ferment,pigment and other component natural substrata.Simultaneously with creation of in natural,it begins shaping the system with determined by balance insect and successes of microorganism.

  2. Effect of light wavelength on cell growth, content of phenolic compounds and antioxidant activity in cell suspension cultures of Thevetia peruviana.

    Science.gov (United States)

    Arias, J P; Zapata, K; Rojano, B; Arias, M

    2016-10-01

    Thevetia peruviana (T. peruviana) has been considered as a potentially important plant for industrial and pharmacological application. Among the number of compounds which are produced by T. peruviana, antioxidants and polyphenols are of particular interest due to their benefits on human health. Cell suspension cultures of T. peruviana were established under different conditions: 1) constant illumination (24h/day) at different light wavelengths (red, green, blue, yellow and white), 2) darkness and 3) control (12h/12h: day light/dark) to investigate their biomass, substrate uptake, polyphenols production and oxidizing activity. The results showed biomass concentrations between 17.1g dry weight (DW)/l (green light) and 18.2g DW/l (control) after 13days. The cultures that grew under green light conditions consumed completely all substrates after 10days, while other cultures required at least 13days or more. The total phenolic content was between 7.21 and 9.46mg gallic acid (GA)/g DW for all light conditions. In addition the ferric reducing antioxidant power and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid antioxidant activity ranged from 5.41-6.58mg ascorbic acid (AA)/g DW and 82.93-110.39μmol Trolox/g DW, respectively. Interestingly, the samples which grew under the darkness presented a higher phenolic content and antioxidant capacity when compared to the light conditions. All together, these results demonstrate the extraordinary effect of different lighting conditions on polyphenols production and antioxidant compounds by T. peruviana. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Divergence of secondary metabolism in cell suspension cultures and differentiated plants of Piper cernuum and P. crassinervium

    Energy Technology Data Exchange (ETDEWEB)

    Danelutte, Ana P.; Costantin, Mara B.; Kato, Massuo J. [Sao Paulo Univ., SP (Brazil). Inst. de Quimica]. E-mail: majokato@iq.usp.br; Delgado, Guillermo E. [Universidad Nacional Pedro Ruiz Gallo, Lambayeque (Peru); Braz-Filho, Raimundo [Universidade do Norte Fluminense, Campos dos Goytacazes, RJ (Brazil)

    2005-11-15

    The secondary metabolism in the leaves of P. cernuum produces cinnamic and dihydrocinnamic acid derivatives and the lignan cubebin. In case of P. crassinervium flavonoids and prenylated hydroquinones were characterized as major compounds. The cell cultures showed the production of the phenylethylamines dopamine and tyramine in P. cernuum, while in case of P. crassinervium four alkamides were isolated as major compounds, including the new 2,3,4- trimethoxy-N-methyl-aristolactam and 3-hydroxy-2-methoxy-N-methyl-aristolactam. (author)

  4. Reprogramming of enteroendocrine K cells to pancreatic β-cells through the combined expression of Nkx6.1 and Neurogenin3, and reaggregation in suspension culture

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Esder; Ryu, Gyeong Ryul; Moon, Sung-Dae; Ko, Seung-Hyun; Ahn, Yu-Bae; Song, Ki-Ho, E-mail: kihos@catholic.ac.kr

    2014-01-17

    Highlights: •K cells were selected from STC-1 cells, a heterogeneous enteroendocrine cell line. •K cells did not express Nkx6.1 and Neurogenin3. •Combined expression of Nkx6.1 and Neurogenin3 reprogrammed K cells to β-cells. •Reprogramming of K cells to β-cells was not complete. -- Abstract: Recent studies have demonstrated that adult cells such as pancreatic exocrine cells can be converted to pancreatic β-cells in a process called cell reprogramming. Enteroendocrine cells and β-cells share similar pathways of differentiation during embryonic development. Notably, enteroendocrine K cells express many of the key proteins found in β-cells. Thus, K cells could be reprogrammed to β-cells under certain conditions. However, there is no clear evidence on whether these cells convert to β-cells. K cells were selected from STC-1 cells, an enteroendocrine cell line expressing multiple hormones. K cells were found to express many genes of transcription factors crucial for islet development and differentiation except for Nkx6.1 and Neurogenin3. A K cell clone stably expressing Nkx6.1 (Nkx6.1{sup +}-K cells) was established. Induction of Neurogenin3 expression in Nkx6.1{sup +}-K cells, by either treatment with a γ-secretase inhibitor or infection with a recombinant adenovirus expressing Neurogenin3, led to a significant increase in Insulin1 mRNA expression. After infection with the adenovirus expressing Neurogenin3 and reaggregation in suspension culture, about 50% of Nkx6.1{sup +}-K cells expressed insulin as determined by immunostaining. The intracellular insulin content was increased markedly. Electron microscopy revealed the presence of insulin granules. However, glucose-stimulated insulin secretion was defective, and there was no glucose lowering effect after transplantation of these cells in diabetic mice. In conclusion, we demonstrated that K cells could be reprogrammed partially to β-cells through the combined expression of Nkx6.1 and Neurogenin3, and

  5. Functional compartmentation of the Golgi apparatus of plant cells : immunocytochemical analysis of high-pressure frozen- and freeze-substituted sycamore maple suspension culture cells.

    Science.gov (United States)

    Zhang, G F; Staehelin, L A

    1992-07-01

    The Golgi apparatus of plant cells is engaged in both the processing of glycoproteins and the synthesis of complex polysaccharides. To investigate the compartmentalization of these functions within individual Golgi stacks, we have analyzed the ultrastructure and the immunolabeling patterns of high-pressure frozen and freeze-substituted suspension-cultured sycamore maple (Acer pseudoplatanus L.) cells. As a result of the improved structural preservation, three morphological types of Golgi cisternae, designated cis, medial, and trans, as well as the trans Golgi network, could be identified. The number of cis cisternae per Golgi stack was found to be fairly constant at approximately 1, whereas the number of medial and trans cisternae per stack was variable and accounted for the varying number of cisternae (3-10) among the many Golgi stacks examined. By using a battery of seven antibodies whose specific sugar epitopes on secreted polysaccharides and glycoproteins are known, we have been able to determine in which types of cisternae specific sugars are added to N-linked glycans, and to xyloglucan and polygalacturonic acid/rhamnogalacturonan-I, two complex polysaccharides. The findings are as follows. The beta-1,4-linked d-glucosyl backbone of xyloglucan is synthesized in trans cisternae, and the terminal fucosyl residues on the trisaccharide side chains of xyloglucan are partly added in the trans cisternae, and partly in the trans Golgi network. In contrast, the polygalacturonic/rhamnogalacturonan-I backbone is assembled in cis and medial cisternae, methylesterification of the carboxyl groups of the galacturonic acid residues in the polygalacturonic acid domains occurs mostly in medial cisternae, and arabinose-containing side chains of the polygalacturonic acid domains are added to the nascent polygalacturonic acid/rhamnogalacturonan-I molecules in the trans cisternae. Double labeling experiments demonstrate that xyloglucan and polygalacturonic acid

  6. Cotton School Tells Us More--The Fourth COTTON USA Cotton School Convened in Qingdao

    Institute of Scientific and Technical Information of China (English)

    By Wang Ting

    2012-01-01

    Since the year of 2006, Cotton Council International has already convened the Cotton School for three times in China. This year, in 2012, CCI held the Cotton School in the city of Qingdao for the fourth time, generously shared with international buyers, especially the Chinese domestic purchases, the knowledge of qualified U.S. cotton.

  7. Favorable conditions of cotton straw composting using as soilless culture substrate%棉秆作为无土栽培基质的适宜发酵条件

    Institute of Scientific and Technical Information of China (English)

    张晔; 余宏军; 杨学勇; 蒋卫杰

    2013-01-01

      The aim of this study was to find the optimal conditions for cotton straw composting as a Soilless Culture Substrate. Cotton straw size, carbon-nitrogen ratio, and nitrogen source were investigated to determine their effects on the process of cotton straw composting by using an orthogonal design method. Each factor was set at three levels:C/N ratios were 25:1, 30:1, 35:1, cotton straw sizes were 1, 2, and 3 cm, and nitrogen sources were chicken manure、urea, and a mixture of chicken manure and urea. Cotton straw applied in this trial was bought from the famers in a Beijing suburb and was broken into 1-3cm particles by machine. The C/N ratio of cotton straw was 38:1. The dry chicken manure and urea as the nitrogen resource were used to adjust the C/N ratio. The cotton straw weight of each treatment was 5 kg, and the water content of each treatment was adjusted to 60%-70%. Plastic weaving bags of 70-liter capacity were used as composting containers and were placed in three layers with a randomized design. Each treatment had one bag and three replications. The bags were turned over every 10 days during the maturation phase in order to improve the O2 level inside the bags. The trial lasted 30days.   The parameters included composting temperature, C/N ratio, bulk density, pH, EC, accumulated temperature, water holding capacity, and air filled porosity. A temperature meter recorded the temperature in each bag every day. Bulk density and porosity were determined following the Byrne method and conventional method, respectively. The pH and electrical conductivity (EC) were determined by IQ150 Portable pH/mV/thermometer measurement.   The results from the study indicated that during the composting period, C/N ratio of 25:1, 1 cm straw particle size, the mixture of chicken manure and urea as an added nitrogen source were the optimal conditions to sustain high temperature (>50℃) in the composting pile of cotton straw, and the days keeping high temperature in

  8. Cotton and its interaction with cotton morphology

    Science.gov (United States)

    The morphological plasticity of the cotton plant enables it to be produced in a wide variety of agro-ecological regions (Oosterhuis and Jernstedt 1999). This plasticity essentially translates to the lengthening, shortening, or interruption of its effective flowering period in response to season leng...

  9. CottonDB Enhancement

    Institute of Scientific and Technical Information of China (English)

    YU Jing; KOHEL Russell; HINZE Lori; FRELICHOWSKI James; XU Zhan-you; YU John Z; PERCY Richard

    2008-01-01

    @@ CottonDB (www.cottondb,org) was initiated in 1995.It is a database that contains genomic,genetic,and taxonomic information for cotton (Gossypium spp.).It serves both as an archival database and as a dynamic database,which incorporates new data and user resources.CottonDB is maintained at the Southern Plains Agricultural Research Center in College Station,TX.The project includes a website and database creating a repository of information for over 450,000 gene,EST,and conting sequences; genetic and physical map data; nearly 10,000 DNA primers; and 9,000 germplasm accessions.

  10. Purification and characterization of three chitinases and one beta-1,3-glucanase accumulating in the medium of cell suspension cultures of barley (Hordeum vulgare L.)

    DEFF Research Database (Denmark)

    Kragh, K.M.; Jacobsen, S.; Dalgaard Mikkelsen, J.

    1991-01-01

    Three basic chitinases and one basic beta-1,3-glucanase were secreted into the medium when embryogenic cell suspensions of barley (Hordeum vulgare L.) cv. 'Igri' were cultured as undifferentiated aggregates in the presence of 2,4-D. The enzymes were purified by affinity and ion exchange...... chromatography. Two of the chitinases were identified as the previously described endochitinases T and C from barley grain. The third and novel chitinase, designated K, was the major basic chitinase in the medium constituting 4% of the soluble protein. Chitinase K was found to be a 33-kDa endochitinase with p...... chitinases from barley aleurone and barley, bean and potato leaves. The purified beta-1,3-glucanase with a molecular weight (MW) of 32 kDa and pI greater-than-or-equal-to 9.8 constituted 1% of the soluble protein in the medium. Based on similar MW, pI and amino acid composition as well as identical N...

  11. NMR spectroscopic search module for Spektraris, an online resource for plant natural product identification--Taxane diterpenoids from Taxus × media cell suspension cultures as a case study.

    Science.gov (United States)

    Fischedick, Justin T; Johnson, Sean R; Ketchum, Raymond E B; Croteau, Rodney B; Lange, B Markus

    2015-05-01

    Development and testing of Spektraris-NMR, an online spectral resource, is reported for the NMR-based structural identification of plant natural products (PNPs). Spektraris-NMR allows users to search with multiple spectra at once and returns a table with a list of hits arranged according to the goodness of fit between query data and database entries. For each hit, a link to a tabulated alignment of (1)H NMR and (13)C NMR spectroscopic peaks (query versus database entry) is provided. Furthermore, full spectroscopic records and experimental meta information about each database entry can be accessed online. To test the utility of Spektraris-NMR for PNP identification, the database was populated with NMR data (total of 466 spectra) for ∼ 250 taxanes, which are structurally complex diterpenoids (including the anticancer drug taxol) commonly found in the genus Taxus. NMR data generated with metabolites purified from Taxus cell suspension cultures were then used to search Spektraris-NMR, and enabled the identification of eight taxanes with high confidence. A ninth isolated metabolite could be assigned, based on spectral searches, to a taxane skeletal class, but no high confidence hit was produced. Using various spectroscopic methods, this metabolite was characterized as 2-deacetylbaccatin IV, a novel taxane. These results indicate that Spektraris-NMR is a valuable resource for rapid and reliable identification of known metabolites and has the potential to contribute to de-replication efforts in novel PNP discovery.

  12. Assessment of cytotoxic and genotoxic activity of alcohol extract of Polyscias filicifolia shoot, leaf, cell biomass of suspension culture and saponin fraction.

    Science.gov (United States)

    Marczewska, Jadwiga; Karwicka, Ewa; Drozd, Janina; Anuszewskal, Elzbieta; Sliwińska, Anita; Nosov, Aleksander; Olszowska, Olga

    2011-01-01

    Some medicinal plants are the object of biotechnologists' special interest owing to their content of secondary metabolites, which have a strong pharmacological effect. Polyscias filicifolia is a plant known for long in traditional medicine of the Southeast Asia. Literature data suggest that it acts on the endocrine system, has adaptogenic and antiulcerative activity, shows bactericidal and insecticidal properties, restores the activity of the protein synthesis system in the conditions of long- and short-term anoxia, as well as reduces the effect of many mutagens in vitro. The purpose of the studies was to assess the cytotoxic and genotoxic effect of ethanol extracts from Polyscias filicifolia dry shoots and leaves obtained in vitro, as well as cell biomass from suspension culture. Saponin fraction from dried shoots was also tested. Initially, the cytotoxic effect was evaluated using the murine connective tissue cell line C3H/AN - L929. The genotoxic properties of the extracts were assessed using standard screening tests: the Ames test and the micronucleus test. Based on the obtained results it can be concluded that none of the extracts increases the number of revertants, both in tests with and without metabolic activation. The lack of in vitro genotoxic and mutagenic activity of tested shoot, dried leaf, cell biomass extracts, as well as the saponin fraction from dried shoots allows us to hope that Polyscias filicifolia could be used as a possible pharmaceutical raw material showing therapeutic properties.

  13. Prevention of copper-induced calcium influx and cell death by prion-derived peptide in suspension-cultured tobacco cells.

    Science.gov (United States)

    Kagenishi, Tomoko; Yokawa, Ken; Kuse, Masaki; Isobe, Minoru; Bouteau, François; Kawano, Tomonori

    2009-01-01

    Impact of copper on the oxidative and calcium signal transductions leading to cell death in plant cells and the effects of the copper-binding peptide derived from the human prion protein (PrP) as a novel plant-protecting agent were assessed using a cell suspension culture of transgenic tobacco (Nicotiana tabacum L., cell line BY-2) expressing the aequorin gene. Copper induces a series of biological and chemical reactions in plant cells including the oxidative burst reflecting the production of reactive oxygen species (ROS), such as hydroxyl radicals, and stimulation of calcium channel opening, allowing a transient increase in cytosolic calcium concentrations. The former was proven by the action of specific ROS scavengers blocking the calcium responses and the latter was proven by an increase in aequorin luminescence and its inhibition by specific channel blockers. Following these early events completed within 10 min, the development of copper-induced cell death was observed during additional 1 h in a dose-dependent manner. Addition of a synthetic peptide (KTNMKHMA) corresponding to the neurotoxic sequence in human PrP, prior to the addition of copper, effectively blocked both calcium influx and cell death induced by copper. Lastly, a possible mechanism of peptide action and future applications of this peptide in the protection of plant roots from metal toxicity or in favour of phytoremediation processes are discussed.

  14. Excretion of polyamines in alfalfa and tobacco suspension-cultured cells and its possible role in maintenance of intracellular polyamine contents.

    Science.gov (United States)

    Cvikrová, Milena; Gemperlová, Lenka; Eder, Josef; Zazímalová, Eva

    2008-07-01

    Changes in polyamines (PAs) in cells and cultivation media of alfalfa (Medicago sativa L.) and tobacco bright yellow 2 (BY-2) (Nicotiana tabacum L.) cell suspension cultures were studied over their growth cycles. The total content of PAs (both free and conjugated forms) was nearly 10 times higher in alfalfa, with high level of free putrescine (Put) (in exponential growth phase it represented about 65-73% of the intracellular Put pool). In contrast, the high content of soluble Put conjugates was found in tobacco cells (in exponential phase about 70% of the intracellular Put). Marked differences occurred in the amount of PAs excreted into the cultivation medium: alfalfa cells excreted at the first day after inoculation 2117.0, 230.5, 29.0 and 88.0 nmol g(-1) of cell fresh weight (FW) of Put, spermidine (Spd), spermine (Spm) and cadaverine (Cad), respectively, while at the same time tobacco cells excreted only small amount of Put and Spd (12.7 and 2.4 nmol g(-1) FW, respectively). On day 1 the amounts of Put, Spd, Spm and Cad excreted by alfalfa cells represented 21, 38, 12 and 15% of the total pool (intra- plus extra-cellular contents) of Put, Spd, Spm and Cad, respectively. In the course of lag-phase and the beginning of exponential phase the relative contents of extracellular PAs continually decreased (with the exception of Cad). On day 10, the extracellular Put, Spd, Spm and Cad still represented 11.3, 10.9, 2.1 and 27% of their total pools. The extracellular PAs in tobacco cells represented from day 3 only 0.1% from their total pools. The possible role of PA excretion into the cultivation medium in maintenance of intracellular PA contents in the cells of the two cell culture systems, differing markedly in growth rate and PA metabolism is discussed.

  15. Oxidative stress and senescence-like status of pear calli co-cultured on suspensions of incompatible quince microcalli.

    Science.gov (United States)

    Nocito, Fabio F; Espen, Luca; Fedeli, Chiara; Lancilli, Clarissa; Musacchi, Stefano; Serra, Sara; Sansavini, Silviero; Cocucci, Maurizio; Sacchi, Gian Attilio

    2010-04-01

    This work presents a simple in vitro system to study physiological, biochemical and molecular changes occurring in a pear callus (Pyrus communis L., cv. Beurré Bosc) grown in close proximity to spatially separated undifferentiated homologous (pear) or heterologous (quince; Cydonia oblonga Mill., East Malling clone C) cells in its neighboring environment. After a 7-day co-culture period, the presence of heterologous cells produced negative effects on the pear callus, whose relative weight increase and adenylate energy charge decreased by 30 and 24%, respectively. Such behavior was associated with a higher O(2) consumption rate (+125%) which did not seem to be coupled to adenosine triphosphate synthesis. Analyses of alternative oxidase and enzymatic activities involved in reactive oxygen species (ROS) detoxification strongly suggested that the higher O(2) consumption rate, measured in the pear callus grown in the heterologous combination, may probably be ascribed to extra-respiratory activities. These, in turn, might contribute to generate metabolic scenarios where ROS-induced oxidative stresses may have the upper hand. The increase in the levels of 2-thiobarbituric acid reactive metabolites, considered as diagnostic indicators of ROS-induced lipid peroxidation, seemed to confirm this hypothesis. Moreover, reverse transcription polymerase chain reaction analysis revealed that the expression levels of a few senescence-associated genes were higher in the pear callus grown in the heterologous combination than in the homologous one. Taken as a whole, physiological and molecular data strongly suggest that undifferentiated cells belonging to a pear graft-incompatible quince clone may induce an early senescence-like status in a closely co-cultured pear callus.

  16. Increasing the cotton yield and improving the ecology in cotton fields by utilizing the properties of natural resources in Xinjiang, China

    Science.gov (United States)

    Tian, Changyan; Lu, Zhaozhi; Song, Yudong; Zhang, Henian

    2003-07-01

    The area of aeolian sand soil in Xinjiang is 3.7189×107 hm2 and occupies 25% of the total land area. Traditionally, it is considered that aeolian sand soil has low yield of crops due to its poor retention power of soil moisture and soil fertility. However, the stems of cotton growing on aeolian sand soil are small and their fictile shape is easy to be controlled. Thus, a culture mode of "increasing stems and bolls, double-layer and double-stem" of cotton is developed by scientific irrigation and fertilizer spread as well as artificial control of fictile shape based on the growth laws of cotton and the properties of aeolian sand soil, and a lint yield of over 3,750 kg/hm2 has been reaped in successive 3 years. Currently, the cotton culture in Xinjiang is rapidly developed, the proportion of cotton-culture areas occupies 40~60%, the cultivating areas of other crops are reduced, the ecosystems are simplified, and the natural enemies in cotton fields are reduced. Alfalfa belts of 8~10 m in width are planted in the zones affected by shelter forests, the occurrence of Therioaphis maculata (Buckton) in alfalfa belts is 10~15 days earlier than that of cotton aphids (Aphis gossypii Glover), and in the alfalfa belts the quantity of herioaphis maculata (Buckton), the natural enemies, is 13.65 times of that in cotton fields when the cotton aphids occur. To resect the alfalfa this moment makes the natural enemies in the alfalfa belts enter the cotton fields and eat cotton aphids, which has good effects for preventing and controlling cotton aphids.

  17. STRUCTURAL INVESTIGATIONS OF VARIOUS COTTON FIBERS AND COTTON CELLULOSES

    Directory of Open Access Journals (Sweden)

    Michael Ioelovich

    2008-02-01

    Full Text Available Macro- and crystalline structure, as well as chemical composition of fibers related to various types and sorts of Israeli cottons, both white and naturally colored, were investigated. The differences in structural parameters and chemical compositions of the cotton fibers were evaluated. Samples of cotton of the “Pima”-type had long, thin and strong fibers with highly ordered supermolecular structure. Fibers of middle-long and hybrid cottons had some lower-ordered structural organization in comparison to long-length cotton, while fibers of naturally colored cotton were characterized with disordered supermolecular and crystalline structure. Dependence of tensile strength on orientation of nano-fibrils towards the fiber axis was found. Conditions of cellulose isolation from the different cotton fibers were studied. Structural characteristics of isolated cotton celluloses and obtained MCC are discussed.

  18. Analysis of the Components in Callus and Cell Suspension Cultures of Ligusticum chuanxiong Hort. By Gas Chromatography-Mass Spectrometry%川芎愈伤组织及悬浮培养物化学成分气相色谱-质谱联用分析

    Institute of Scientific and Technical Information of China (English)

    詹玉莲; 马小军; 戴均贵; 刘德华

    2006-01-01

    Objective To analyze the components in callus and cell suspension cultures of Ligusticum chuanxiong Hort. by gas chromatography-mass spectrometry (GC-MS). Methods Initiate the callus and cell suspension cultures of Ligusticum chuanxiong Hort.. Collect the callus and three sequent generations cell suspension cultures, and the components in callus and cell suspension cultures were analyzed by gas chromatography-mass spectrometry. Results The callus and cell suspension cultures of Ligusticum chuanxiong Hort. contained similar constituenents, but the components in callus and cell suspension cultures were quite different from those of the plant, and the main bioactive constituentes of the plant were not found in callus and cell suspension cultures. Conclusion The characteristics of callus and cell suspension cultures were different from those of the plant.%目的用气相色谱-质谱联用法分析川芎愈伤组织及悬浮培养物中的化学成分.方法建立川芎愈伤组织及悬浮培养体系,收集愈伤组织及连续三代的悬浮培养物,采用气相色谱-质谱联用法进行川芎愈伤组织及悬浮培养物化学成分的分析.结果川芎愈伤组织及悬浮培养物含有相似的成分,但与原植物中的成分区别很大,原植物中的主要活性成分在愈伤组织及悬浮培养物中也未发现.结论愈伤组织及悬浮培养物的特性与原植物有很大的区别.

  19. O-glycans and O-glycosylation sites of recombinant human GM-CSF derived from suspension-cultured rice cells, and their structural role.

    Science.gov (United States)

    Kim, Jihye; Park, Heajin; Park, Byung Tae; Hwang, Hye Seong; Kim, Jae Il; Kim, Dae Kyong; Kim, Ha Hyung

    2016-10-14

    Recombinant human GM-CSF (rhGM-CSF) from yeast has been clinically applied to immunosuppressed patients. The production of suspension-cultured rice-cell-derived rhGM-CSF (rrhGM-CSF), which has a longer blood clearance time and the same bioactivity as yeast-derived rhGM-CSF, and the analysis of its N-glycans have been reported recently. However, there are no previous reports of the O-glycosylation of rhGM-CSF from plant cells, and so this study investigated O-glycans, O-glycosylation sites, and their structural role in rrhGM-CSF. Monosaccharide analysis revealed the presence of O-glycans comprising arabinose and galactose. Eight O-glycans comprising four arabinose residues with zero to seven galactose residues along with their relative quantities were analyzed. Analysis of pronase-digested glycopeptides indicated that the O-glycans are partially attached to Ser 5, Ser 7, Ser 9, or Thr 10 residues, and glycan heterogeneity was confirmed at each site. Pro-to-hydroxyproline conversions occurred at Pro 2, Pro 6, and Pro 8 residues. The preparation of deglycosylated rrhGM-CSFs revealed that deglycosylation greatly affects their α-helix structures. These findings indicate that O-glycans of rrhGM-CSF are essential for maintaining its structural stability and result in an extended in vivo half-life, but without affecting its biological function. This is the first report on the O-glycosylation of rhGM-CSF derived from plant cells.

  20. The Structure of Plant Cell Walls: I. The Macromolecular Components of the Walls of Suspension-cultured Sycamore Cells with a Detailed Analysis of the Pectic Polysaccharides.

    Science.gov (United States)

    Talmadge, K W; Keegstra, K; Bauer, W D; Albersheim, P

    1973-01-01

    This is the first in a series of papers dealing with the structure of cell walls isolated from suspension-cultured sycamore cells (Acer pseudoplatanus). These studies have been made possible by the availability of purified hydrolytic enzymes and by recent improvements in the techniques of methylation analysis. These techniques have permitted us to identify and quantitate the macromolecular components of sycamore cell walls. These walls are composed of 10% arabinan, 2% 3,6-linked arabinogalactan, 23% cellulose, 9% oligo-arabinosides (attached to hydroxyproline), 8% 4-linked galactan, 10% hydroxyproline-rich protein, 16% rhamnogalacturonan, and 21% xyloglucan.The structures of the pectic polymers (the neutral arabinan, the neutral galactan, and the acidic rhamnogalacturonan) were obtained, in part, by methylation analysis of fragments of these polymers which were released from the sycamore walls by the action of a highly purified endopolygalacturonase. The data suggest a branched arabinan and a linear 4-linked galactan occurring as side chains on the rhamnogalacturonan. Small amounts or pieces of a xyloglucan, the wall hemicellulose, appear to be covalently linked to some of the galactan chains. Thus, the galactan appears to serve as a bridge between the xyloglucan and rhamnogalacturonan components of the wall.The rhamnogalacturonan consists of an alpha-(1 --> 4)-linked galacturonan chain which is interspersed with 2-linked rhamnosyl residues. The rhamnosyl residues are not randomly distributed in the chain but probably occur in units of rhamnosyl- (1 --> 4)-galacturonosyl- (1 --> 2)-rhamnosyl. This sequence appears to alternate with a homogalacturonan sequence containing approximately 8 residues of 4-linked galacturonic acid. About half of the rhamnosyl residues are branched, having a substituent attached to carbon 4. This is likely to be the site of attachment of the 4-linked galactan.The hydroxyprolyl oligo-arabinosides of the hydroxyproline-rich glycoprotein

  1. Association of H-Translocating ATPase in the Golgi Membrane System from Suspension-Cultured Cells of Sycamore (Acer pseudoplatanus L.).

    Science.gov (United States)

    Ali, M S; Akazawa, T

    1986-05-01

    The Golgi complex and the disrupted vesicular membranes were prepared from suspension-cultured cells of sycamore (Acer pseudoplatanus L.) using protoplasts as the starting material and employing linear sucrose density gradient centrifugation followed by osmolysis (Ali et al. [1985] Plant Cell Physiol 26: 1119-1133). The isolated Golgi fraction was found to be enriched with marker enzyme activities and depleted of the activity of a typical mitochondrial marker enzyme, cytochrome c oxidase. Golgi complex, and vesicular membranes derived thereof were found to contain the specific ATPase (specific activity of about 0.5 to 0.7 micromoles per minute per milligram protein). Inhibitor studies suggested that the ATPase of Golgi was different from plasma membrane, tonoplast and mitochondrial ATPases as it was not inhibited by sodium vanadate, potassium nitrate, oligomycin and sodium azide. The sensitivity to N-ethylmaleimide further distinguished the Golgi ATPase from F(0) to F(1) ATPase of mitochondria. The internal acidification was measured by monitoring the difference in absorbance at 550 nanometers minus 600 nanometers using neutral red as a probe. The maximum rate detected with Golgi and disrupted membrane system was 0.49 and 0.61 optical density unit per minute per milligram protein, at pH 7.5, respectively, indicating that the proton pump activity was tightly associated with the Golgi membranes. In both cases, the acidification was inhibited 70 to 90% by various ionophores, indicating that the proton pump was electrogenic in nature. Both the Golgi ATPase activity and ATP-dependent acidification were profoundly inhibited by N,N'-dicyclohexylcarbodiimide, which also indicate that the two activities are catalyzed by the same enzyme.

  2. Rectal culture (image)

    Science.gov (United States)

    A rectal culture test is performed by inserting a cotton swab in the rectum. The swab is rotated gently, and withdrawn. A smear of the swab is placed in culture media to encourage the growth of microorganisms. The ...

  3. Cotton (Gossypium hirsutum L.).

    Science.gov (United States)

    Rathore, Keerti S; Campbell, LeAnne M; Sherwood, Shanna; Nunes, Eugenia

    2015-01-01

    Cotton continues to be a crop of great economic importance in many developing and some developed countries. Cotton plants expressing the Bt gene to deter some of the major pests have been enthusiastically and widely accepted by the farmers in three of the major producing countries, i.e., China, India, and the USA. Considering the constraints related to its production and the wide variety of products derived from the cotton plant, it offers several target traits that can be improved through genetic engineering. Thus, there is a great need to accelerate the application of biotechnological tools for cotton improvement. This requires a simple, yet robust gene delivery/transformant recovery system. Recently, a protocol, involving large-scale, mechanical isolation of embryonic axes from germinating cottonseeds followed by direct transformation of the meristematic cells has been developed by an industrial laboratory. However, complexity of the mechanical device and the patent restrictions are likely to keep this method out of reach of most academic laboratories. In this chapter, we describe the method developed in our laboratory that has undergone further refinements and involves Agrobacterium-mediated transformation of cotton cells, selection of stable transgenic callus lines, and recovery of plants via somatic embryogenesis.

  4. Cotton, biotechnology, and economic development

    OpenAIRE

    Baffes, John

    2011-01-01

    During the past decade, cotton prices remained considerably below other agricultural prices (although they recovered toward the end of 2010). Yet, between 2000-04 and 2005-09 world cotton production increased 13 percent. This paper conjectures that biotechnology-induced productivity improvements increased supplies by China and India, which, in addition to keeping cotton prices low, aided t...

  5. Distribuição nos compartimentos ambientais dos herbicidas utiilizados nas culturas de algodão, café e citros Distribution of environmental compartments of herbicides used in the cotton, coffee and citrus cultures

    Directory of Open Access Journals (Sweden)

    L.P.M. Plese

    2009-03-01

    Full Text Available O objetivo deste trabalho foi avaliar o destino ambiental dos herbicidas acetochlor, 2,4-D, diuron, clomazone, thidiazuron, paraquat, simazine, fluazifop-p-butil, clethodim, oxyfluorfen, flumioxazin, carfentrazone-ethyl, ametrina, trifluralin e MSMA em áreas de cultivo de algodão, café e citros, utilizando o modelo de fugacidade nível I. Na metodologia foram utilizadas basicamente as características físico-químicas dos herbicidas, os compartimentos ambientais e as equações de fugacidade. A avaliação preliminar do risco de contaminação pelo uso de herbicidas nas culturas de algodão, café e citros pode ser feita de forma expedita a partir das propriedades físico-químicas desses produtos, aplicando o modelo de fugacidade nível I. Para a maioria dos herbicidas avaliados, o compartimento água foi o mais vulnerável. O estudo de avaliação da predição em que se empregou o nível de fugacidade I demonstrou ser uma ferramenta importante no destino ambiental dos herbicidas estudados para as culturas de algodão, café e citros.The aim of this paper was to evaluate the environmental fate of herbicides (acetochlor, 2,4-D, diuron, clomazone, thidiazuron, paraquat, simazine, fluazifo-p-butil, clethodim, oxufluorfen, flumioxazin, carfentrazone-ethyl, ametrina, trifluralin and MSMA in cotton, coffee and citrus cultivation areas, applying the level I fugacity model. The methodology basically used the chemical and physical characteristics of the pesticides, environmental compartments and the fugacity equations. The preliminary evaluation of risk of contamination due to the use of these pesticides on the cultures studied was carried out swiftly, based on the chemical and physical properties of these products as the level I fugacity model was applied. For most of the herbicides evaluated, the water compartment was the most vulnerable. The prediction evaluation study using fugacity level I was found to be a relevant tool for the environmental

  6. Bacterial blight of cotton

    Directory of Open Access Journals (Sweden)

    Aïda JALLOUL

    2015-04-01

    Full Text Available Bacterial blight of cotton (Gossypium ssp., caused by Xanthomonas citri pathovar malvacearum, is a severe disease occurring in all cotton-growing areas. The interactions between host plants and the bacteria are based on the gene-for-gene concept, representing a complex resistance gene/avr gene system. In light of the recent data, this review focuses on the understanding of these interactions with emphasis on (1 the genetic basis for plant resistance and bacterial virulence, (2 physiological mechanisms involved in the hypersensitive response to the pathogen, including hormonal signaling, the oxylipin pathway, synthesis of antimicrobial molecules and alteration of host cell structures, and (3 control of the disease.

  7. Comparative properties of cellulose nano-crystals from native and mercerized cotton fibers

    Science.gov (United States)

    Stable aqueous suspensions of cellulose nano-crystals (CNCs) were fabricated from both native and mercerized cotton fibers by sulfuric acid hydrolysis, followed by high-pressure homogenization. Fourier Transform Infrared Spectrometry and Wide-angle X-Ray Diffraction data showed that the fibers had b...

  8. Systematic secretome analyses of rice leaf and seed callus suspension-cultured cells: workflow development and establishment of high-density two-dimensional gel reference maps.

    Science.gov (United States)

    Jung, Young-Ho; Jeong, Seung-Hee; Kim, So Hee; Singh, Raksha; Lee, Jae-Eun; Cho, Yoon-Seong; Agrawal, Ganesh Kumar; Rakwal, Randeep; Jwa, Nam-Soo

    2008-12-01

    Secreted proteins control a multitude of biological and physiological processes in multicellular organisms such as plants. Identification of secreted proteins in reference plants like Arabidopsis and rice under normal growth conditions and adverse environmental conditions will help better understand the secretory pathways. Here, we have performed a systematic in planta and in vitro analyses of proteins secreted by rice leaves (in planta) and seed callus suspension-cultured cells (SCCs; in vitro), respectively, using a combination of biochemical and two-dimensional gel electrophoresis (2-DGE) coupled with liquid chromatography mass spectrometry analyses. Secreted proteins prepared from either leaves or SCCs medium were essentially free from contamination of intracellular proteins as judged by biochemical and Western blot analyses. 2-DGE analyses of secreted proteins collectively identified 222 protein spots with only 6 protein spots common to both in planta and in vitro derived data sets. Data were used to establish high-resolution and high-density 2-D gel reference maps for both in planta and in vitro secreted proteins. Identified proteins belonged to 11 (in planta) and 6 (in vitro) functional classes. Proteins involved in carbon metabolism (33%) and cell wall metabolism having plant defense mechanism (18%) were highly represented in the in planta secreted proteins accounting for 51% of total identified proteins, whereas proteins of cell wall metabolism having plant defense mechanism (64%) were predominant in the in vitro secreted proteins. Interestingly, secreted proteins possessing signal peptides were significantly lower in an in planta (27%) prepared secreted protein population than in vitro (76%) as predicted by SignalP prediction tool, implying the notion that plant might possess yet unidentified secretory pathway(s) in addition to the classical endoplasmic reticulum/Golgi pathway. Taken together, this systematic study provides evidence for (i) significant

  9. Secretion and membrane recycling in plant cells: novel intermediary structures visualized in ultrarapidly frozen sycamore and carrot suspension-culture cells.

    Science.gov (United States)

    Staehelin, L A; Chapman, R L

    1987-05-01

    Freeze-fracture electron microscopy of propane-jet-frozen samples has been employed to investigate vesicle-mediated secretion and membrane recycling events in carrot (Daucus carota L.) and sycamore maple (Acer pseudoplatanus L.) suspension-culture cells. Stabilization of the cells by means of ultrarapid freezing has enabled us to preserve the cells in a turgid state and to visualize new intermediate membrane configurations related to these events. Indeed, many of the observed membrane configurations, such as flattened membrane vesicles with slit-shaped membrane fusion sites and horseshoe-shaped membrane infoldings, appear to result from the action of turgor forces on the plasma membrane. Individual cells exhibited great variations in numbers and types of membrane configurations postulated to be related to secretion and membrane-recycling events. In the majority of cells, the different membrane profiles displayed a patchy distribution, and within each patch the membrane configurations tended to be of the same stage. This result indicates that secretory events are triggered in domains measuring from 0.1 to about 10 μm in diameter. Based on an extensive analysis of the different membrane configurations seen in our samples, we have formulated the following model of vesicle-mediated secretion in plant cells: Fusion of a secretory vesicle with the plasma membrane leads to the formation of a single, narrow-necked pore that increases in diameter up to about 60 nm. During discharge, the vesicle is flattened, forming a disc-shaped structure perpendicular to the plane of the plasma membrane. As the vesicle is flattened, the pore is converted to a slit, the maximum length of which coincides with the diameter of the flattened vesicle. The flattened vesicle then tips over and concomitantly the plasma-membrane slit becomes curved into a horseshoe-shaped configuration as it extends along the outer margins of the tipped-over vesicle. Some coated pits are present interspersed

  10. Effects of cell suspension and cell·free culture filtrate of Pseudomonas aeruginosa in the control of root rot-root kont disease complex of tomato (Lycopersicon esculentum Mill.

    Directory of Open Access Journals (Sweden)

    I. A. Siddiqui

    2013-12-01

    Full Text Available The plant growth-promoting rhizobacterium Pseudomonas aeruginosa strain IE-6 was tested for antagonistic activity towards Meloidogyne javanica, the root-knot nematode and soilbome root-infecting fungi viz., Macrophomina phaseolina, Fusarium solani and Rhizoctonia solani under laboratory and greenhouse conditions. Cell-free culture filtrate of the bacterium caused significant reduction in egg hatching of M.javanica and inhibited radial growth of fungi in vitro. Cell-free culture filtrate also caused lyses in mycelium of F.solani. Under greenhouse conditions, soil drenches with the aqueous cell suspension or cell-free culture resulted in a considerable reduction in nematode population densities in soil and subsequent root-knot development due to M.javanica. In addition to nematode control, rhizobacterium application also inhibited root-infection caused by soilborne root~infecting fungi with significant enhancement of growth of tomato seedlings.

  11. Cotton Life Cycle Inventory & Life Cycle Assessment--A Landmark Benchmark for Cotton Sustainability

    Institute of Scientific and Technical Information of China (English)

    2011-01-01

    Recently, Cotton Incorporated announced the completion of a comprehensive life cycle inventory and life cycJe analysis of cotton products. The endeavor is part of the Cotton Foundation VlSIQN 21 Project and included the participation of the National Cotton Council, Cotton Council International and Cotton Incorporated. The two-year study, managed by PE International,

  12. New Cotton Trade Terms Flashed in China

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    On May 8th, 2006, China Cotton Import Regulations-Cotton Purchase Contract and General Terms (Applicable to Non-Chinese Cotton Trade), short for China Cotton Association Terms (CCAT) was issued and put into practice, which was welcomed by both China and the countries who trade cotton with China.

  13. Cotton Trip in China

    Institute of Scientific and Technical Information of China (English)

    Wang Ting

    2010-01-01

    @@ During their trip in Beijing,the leadership delegation members,Charles Parker,Harrison Ashley(Vice President of NCC Ginner Services),along with Karin Malmstrom(China Director of CCI)shared a time to accept the interview,giving a general introduction about their China trip and the cotton industry in USA.

  14. Biotransformation of artemisinin by Catharanthus roseus and Ginkgo biloba cell suspension cultures%长春花及银杏植物细胞悬浮培养对青蒿素的生物转化研究

    Institute of Scientific and Technical Information of China (English)

    韩健; 戴均贵; 崔亚君; 占纪勋; 郭洪祝; 果德安

    2003-01-01

    Object To investigate the biotransformation of the antimalarial compound artemisinin( Ⅰ ) by Catharanthus roseus and Ginkgo biloba cell suspension cultures. Methods Plant tissue culture technology was employed. The product was isolated on silica gel column chromatography and its structure was elucidated by spectroscopic evidence. Results One product was obtained and its structure was characterized as 3α- hydroxydeoxyartemisinin ( Ⅱ ). Conclusion Both of C. roseus and G. biloba cell suspension cultures can bioconvert artemisinin.%目的对抗疟药物青蒿素(Ⅰ)进行了生物转化研究.方法利用长春花及银杏植物细胞悬浮培养细胞进行生物转化.用硅胶柱色谱进行产物的分离,波谱方法鉴定产物的结构.结果此两种植物悬浮细胞体系均能将青蒿素转化成3α-羟基去氧青蒿素(Ⅱ).结论此两种植物悬浮细胞体系均能有效转化青蒿素.

  15. The rules on suspension cell batch culture of Hedyotis diffusa%白花蛇舌草悬浮细胞分批培养规律的研究

    Institute of Scientific and Technical Information of China (English)

    于瑞莲; 许金国; 顾晓娟

    2012-01-01

    目的 利用白花蛇舌草茎尖的分生组织建立植物悬浮细胞培养系,确定白花蛇舌草悬浮细胞分批培养时的变化规律.方法 通过接种不同量的细胞液确定最适接种量,以细胞干重、蔗糖、铵离子、硝酸盐氮和多糖含量作为检测指标,确定白花蛇舌草悬浮细胞分批培养时的变化规律.结果 白花蛇舌草悬浮细胞分批培养的最适接种量为15%,此时细胞干重达到最大值.在此接种量下,当培养时间达到9d时,pH很快降低到3左右,细胞干重不再增加.培养液中的主要营养成分碳源——蔗糖,在培养开始时,有比较大的降低,在培养后期,细胞干重不再增加时,也不再有大的改变.氮源[N03-]优于[NH4+]先被利用,而且[N0f]的利用速率要远高于[NH4+],达到7.14 μg/(mL·d);细胞液中多糖的生成和细胞的生长属于非偶联型,在培养后期,逐渐大量生成.结论 初步确定白花蛇舌草悬浮细胞分批培养时的变化规律,为以后的培养工艺优化打下了基础.%Purpose Hedyotis diffusa suspension cell was established by the tip of meristem to find out the rules on suspension cell batch culture of Hedyotis diffusa with the time. Methods The optimum inoculum was determined by different inoculation of cell medium. The rules on suspension cell batch culture of Hedyotis diffusa with the time were found by the detection of the amount of cell dry weight, sucrose, ammonium, nitrate and polysaccharide. Results The suspension cell batch culture of Hedyotis diffusa optimum inoculum is 15% .maximum dry cell weight at the same time. At this inoculation,when the incubation time was of 9 days,and pH quickly reduced to about 3 ,the cell dry weight did not increase. The main medium nutrients were carbon -sucrose, a relatively large decreased in the beginning of culture, and the cell dry weight no longer had a big change in the late culture stage. Nitrogen [ NO3-] was used prior to [ NH4+ ] ,and the use

  16. 内生真菌对油樟悬浮细胞培养的影响%Effects of fungal endophytes on cell suspension culture of Cinnamomum longepaniculatum

    Institute of Scientific and Technical Information of China (English)

    魏琴; 谭韵雅; 李群; 游玲; 汪超; 王玉; 廖淋

    2016-01-01

    该文研究了内生真菌YG42、YG71、YY11和YY26发酵液,对油樟悬浮细胞的生长量及挥发性代谢产物积累量的影响。结果表明:4种内生真菌对油樟悬浮细胞的生长均有抑制作用,抑制强度随发酵液添加量的增加而加强。4种内生真菌对油樟悬浮细胞挥发性代谢产物积累总量及1,8-桉叶油素、γ-叶松油烯和α-油松油醇3种油樟油组分物质积累量的影响多表现为低浓度促进高浓度抑制的趋势。其中,1%添加量的YG42和YY26及0.25%添加量的YY11对悬浮细胞总挥发性代谢产物积累的促进作用相当且最强,其积累量分别是空白组的2.00、1.95、2.01倍;0.25%添加量的YG71对1,8-桉叶油素积累的促进作用最强,其积累量为空白组的11.03倍;0.25%添加量的YG71和YY26对α-松油醇积累的促进作用相当且最强,其积累量分别为空白组的1.72和1.81倍;对于γ-松油烯的积累,在空白组中未检测到其峰值,4种真菌诱导子对γ-松油烯的产生有诱导作用,诱导的最大峰面积为0.19,诱导菌是0.25%添加量的YG71。该研究结果为充实内生菌影响香料植物挥发性代谢产物合成理论奠定了基础,也为生产上内生真菌提高油樟油中有用物质组分含量措施的采用提供了依据。%We studied the effects of fungal endophytes YG42, YG72, YY11 and YY26 on cell growth and volatile of secondary metabolites accumulation in suspension cultures of Cinnamomum longepaniculatum. The results showed that four kinds of fungal endophytes had obvious inhibitory effects on C. longepaniculatum cell growth, and the denser the fer-mentation fluid was, the stronger inhibitory effects they had. The trend of the effects that the four kinds of endophytic fungi had on the total volatile of secondary metabolites accumulation and C. longepaniculatum oil component 1,8-cineoleγ-terpinene andα-terpineol accumulation in suspension cultures of C. longepaniculatum was

  17. An improved protocol for the preparation of protoplasts from an established Arabidopsis thaliana cell suspension culture and infection with RNA of turnip yellow mosaic tymovirus: a simple and reliable method.

    Science.gov (United States)

    Schirawski, J; Planchais, S; Haenni, A L

    2000-04-01

    An improved method for preparation of protoplasts of Arabidopsis thaliana cells grown in suspension culture is presented. This method is fast, reliable and can be used for the production of virtually an unlimited number of protoplasts at any time. These protoplasts can be transformed efficiently with RNA from turnip yellow mosaic tymovirus (TYMV) by polyethyleneglycol-mediated transfection. The simple transfection procedure has been optimized at various steps. Replication of TYMV can be monitored routinely by detection of the coat protein in as few as 2 x 10(4) infected protoplasts.

  18. 烟粉虱暴发成因及其治理技术研究%Outbreak Reason of Cotton Whitefly [Bemisia Tabaci(Gennadius)]and its Integrated Pest Measure

    Institute of Scientific and Technical Information of China (English)

    慕卫; 刘峰; 刘海涛

    2003-01-01

    The reason of cotton whitefly [ Bemisia tabaci ( Gennadius) ] happened heavily in China in recent years was analyzed from its biology characteristic, host range, crop culture and environment situation, control measure. At last, integrated pest measure for control cotton whiteflywas clarified.

  19. Callus Induction and Establishment of Cell Suspension Culture System for Thymus vulgaris L.%百里香愈伤组织的诱导及细胞悬浮培养体系的建立

    Institute of Scientific and Technical Information of China (English)

    徐世千; 李晓东; 张建国

    2011-01-01

    The stems and leaves of aseptic seedling of Thymus vulgaris L. Were taken as explants to study the optimum culture conditions of callus induction and proliferation. In addition, the growth characteristics of suspension cell were investigated and the system of thymus cell suspension culture was established preliminarily. The results showed that stem explants were the most suitable for callus induction, the optimal medium for callus induction was MS + NAA 1. 0 mg/L + 6-BA 0. 5 mg/L+ 2,4-D 0. 5 mg/L, the optimal formula for callus proliferation and suspension culture was MS + 6-BA 1. 0 mg/L+ 2,4-D 0. 1 mg/L and MS + 6-BA 0. 5 mg/L+ 2,4-D 0. 1 mg/L respectively. The growth of suspension cells exhibited an S-shaped curve and growth mass reached the maximum after 20 days' culture, the optimal subculture cycle was 18 - 20 d. In the process of suspension culture, the same change trends of pH value and conductivity were showed; firstly decreased then increased and then became stable, and the suspension cell number exhibited the change trend: firstly increased then decreased.%以普通百里香无菌苗的茎段和叶片为材料,对百里香愈伤组织诱导和继代增殖的最佳培养条件进行研究,并对悬浮细胞的生长特性进行监测,初步建立细胞悬浮培养体系.结果表明,茎段是诱导愈伤组织和进行悬浮培养的理想外植体,愈伤组织最佳诱导培养基为MS+NAA 1.0 mg/L+6-BA 0.5 mg/L+2,4-D0.5 mg/L,最佳增殖培养基为MS+6-BA 1.0 mg/L+2,4-D 0.1 mg/L,悬浮培养适宜培养基为MS+6-BA 0.5 mg/L+2,4-D 0.1 mg/L.所建立的悬浮细胞系生长呈“S”型曲线,培养20 d达到最大生长量,最适继代周期为18~20 d,在悬浮培养过程中,培养液的pH和电导率变化趋势均为先下降后上升最后趋于稳定,细胞数量则呈现先上升后下降的趋势.

  20. A simple cost-effective and eco-friendly wet chemical process for the fabrication of superhydrophobic cotton fabrics

    Energy Technology Data Exchange (ETDEWEB)

    Richard, Edna; Lakshmi, R.V.; Aruna, S.T., E-mail: aruna_reddy@nal.res.in; Basu, Bharathibai J.

    2013-07-15

    Superhydrophobic surfaces were created on hydrophilic cotton fabrics by a simple wet chemical process. The fabric was immersed in a colloidal suspension of zinc hydroxide followed by subsequent hydrophobization with stearic acid. The wettability of the modified cotton fabric sample was studied by water contact angle (WCA) and water shedding angle (WSA) measurements. The modified cotton fabrics exhibited superhydrophobicity with a WCA of 151° for 8 μL water droplet and a WSA of 5–10° for 40 μL water droplet. The superhydrophobic cotton sample was also characterized by field emission scanning electron microscopy (FESEM) and energy dispersive X-ray spectroscopy (EDX). The method is simple, eco-friendly and cost-effective and can be applied to large area of cotton fabric materials. It was shown that superhydrophobicity of the fabric was due to the combined effect of surface roughness imparted by zinc hydroxide and the low surface energy of stearic acid.

  1. Cotton Incorporated Documents Industry Gains at ICAC

    Institute of Scientific and Technical Information of China (English)

    2012-01-01

    Those who attack the cotton industry for its perceived impact on the environment will need to have their facts straight, thanks to a major research project undertaken by Cotton Incorporated: a life-cycle assessment (LCA) for cotton.

  2. Cotton in Benin: governance and pest management

    NARCIS (Netherlands)

    Togbe, C.E.

    2013-01-01

    Key words: cotton, synthetic pesticides, neem oil (Azadirachta indica), Beauveria bassiana, Bacillus thuringiensis, field experiment, farmers’ participation   Pests are one of the main factors limiting cotton production worldwide. Most of the pest control strategies in cotton producti

  3. EDITORIAL: Colloidal suspensions Colloidal suspensions

    Science.gov (United States)

    Petukhov, Andrei; Kegel, Willem; van Duijneveldt, Jeroen

    2011-05-01

    Special issue in honour of Henk Lekkerkerker's 65th birthday Professor Henk N W Lekkerkerker is a world-leading authority in the field of experimental and theoretical soft condensed matter. On the occasion of his 65th birthday in the summer of 2011, this special issue celebrates his many contributions to science. Henk Lekkerkerker obtained his undergraduate degree in chemistry at the University of Utrecht (1968) and moved to Calgary where he received his PhD in 1971. He moved to Brussels as a NATO fellow at the Université Libre de Bruxelles and was appointed to an assistant professorship (1974), an associate professorship (1977) and a full professorship (1980) in physical chemistry at the Vrije Universiteit Brussel. In 1985 he returned to The Netherlands to take up a professorship at the Van 't Hoff Laboratory, where he has been ever since. He has received a series of awards during his career, including the Onsager Medal (1999) of the University of Trondheim, the Bakhuys Roozeboom Gold Medal (2003) of the Royal Dutch Academy of Arts and Sciences (KNAW), the ECIS-Rhodia European Colloid and Interface Prize (2003), and the Liquid Matter Prize of the European Physical Society (2008). He was elected a member of KNAW in 1996, was awarded an Academy Chair position in 2005, and has held several visiting lectureships. Henk's work focuses on phase transitions in soft condensed matter, and he has made seminal contributions to both the theoretical and experimental aspects of this field. Here we highlight three major themes running through his work, and a few selected publications. So-called depletion interactions may lead to phase separation in colloid-polymer mixtures, and Henk realised that the partitioning of polymer needs to be taken into account to describe the phase behaviour correctly [1]. Colloidal suspensions can be used as model fluids, with the time- and length-scales involved leading to novel opportunities, notably the direct observation of capillary waves at a

  4. Microarray and suppression subtractive hybridization analyses of gene expression in hybrid poplar (Populus alba × Populus tremula var. glandulosa) cell suspension cultures after exposure to NaCl.

    Science.gov (United States)

    Bae, Eun-Kyung; Lee, Hyoshin; Lee, Jae-Soon; Noh, Eun-Woon; Choi, Young-Im; Lee, Byung-Hyun; Choi, Dong-Woog

    2012-09-01

    The gene expression profiles of hybrid poplar (Populus alba × Populus tremula var. glandulosa) cells in suspension culture after exposure to salinity (NaCl) induced stress were examined by constructing two suppression subtractive hybridization (SSH) libraries. cDNA from non-treated cells was used as a driver and cDNA samples from cell suspension cultures exposed to 150 mM NaCl for 2 or 10 h were used as testers. Randomly selected clones from each SSH library were sequenced and 727 high-quality expressed sequence tags (ESTs) were obtained and analyzed. Four novel ESTs were identified. Between the two libraries, 542 unique SSH clones were selected for placement on a cDNA microarray. In total, 18 differentially expressed genes were identified with 4 and 12 genes being significantly differentially expressed 2 and 10 h after the treatment, respectively. Genes related to metabolism and protein synthesis and several genes whose protein products are implicated in salt or other abiotic stress-related responses were expressed in the salt-stressed cells.

  5. The Structure of Plant Cell Walls: IV. A Structural Comparison of the Wall Hemicellulose of Cell Suspension Cultures of Sycamore (Acer PseudoPlatAnus) and of Red Kidney Bean (Phaseolus Vulgaris).

    Science.gov (United States)

    Wilder, B M; Albersheim, P

    1973-05-01

    The molecular structure and chemical properties of the hemicellulose present in the isolated cell walls of suspension cultures of sycamore (Acer pseudoplatanus) cells has recently been described by Bauer et al. (Plant Physiol. 51: 174-187). The hemicellulose of the sycamore primary cell wall is a xyloglucan. This polymer functions as an important cross-link in the structure of the cell wall; the xyloglucan is hydrogen-bonded to cellulose and covalently attached to the pectic polymers.The present paper describes the structure of a xyloglucan present in the walls and in the extracellular medium of suspension-cultured Red Kidney bean (Phaseolus vulgaris) cells and compares the structure of the bean xyloglucan with the structure of the sycamore xyloglucan. Although some minor differences were found, the basic structure of the xyloglucans in the cell walls of these distantly related species is the same. The structure is based on a repeating heptasaccharide unit which consists of four residues of beta-1, 4-linked glucose and three residues of terminal xylose linked to the 6 position of three of the glucosyl residues.

  6. Effect of Chitinase-Producing Strain V-8 on 3ontrolling Cotton Fusarium Wilt

    Institute of Scientific and Technical Information of China (English)

    2012-01-01

    [Objective] This study aimed to screen endophytic bacteria which is antag- onistic to cotton Fusarium wilt. [Method] Fresh cotton plants collected from cotton- growing areas in Jingzhou City, Hubei Province were used as experimental materials to isolate endophytic bacteria. Through chitinase test and co-culturing both micro-or- ganisms side by side on the same PDA culture plate, antagonistic strains to cotton Fusarium wilt were screened. [Result] A total of 83 bacterial isolates were obtained from cotton plants grown in the fields, six of which were chitinase-productive bacte- ria. Through chitinase test and co-culturing both micro-organisms side by side on the same PDA culture plate, strain V-8 which had the strongest antagonistic effect on Fusarium oxysporum f. sp. vasinfectum was screened. Strain V-8 had a wider anti- fungal spectrum with certain inhibitory effect on all the six important pathogenic fungi including Fusarium oxysporum f. sp niveum; it colonized stably in the rhizospheric soil of cotton, with a colonization density of up to 6.2x10s cfu/g fifty days after inoc- ulation; the relative effect on controlling cotton Fusarium wilt in pot test was 73.2%. The Findings of this study suggested that strain V-8 had great potential for biological control of cotton Fusarium wilt and could be taken as a substantial material for the cloning of chitinase genes. [Conclusion] The results from this study provides bases for the control of cotton fusarium wilt, as well as the exploitation of endophytic bac- teria resources in cotton and the development of novel biological pesticides.

  7. Urinary incontinence - retropubic suspension

    Science.gov (United States)

    ... Marchetti-Krantz (MMK) procedure; Laparoscopic retropubic colposuspension; Needle suspension; Burch colposuspension ... bladder. There are two ways to do retropubic suspension: open surgery or laparoscopic surgery. Either way, surgery ...

  8. Suspension Trauma / Orthostatic Intolerance

    Science.gov (United States)

    ... of Science and Technology Assessment Printer Friendly Version Suspension Trauma/Orthostatic Intolerance Safety and Health Information Bulletin ... information about the hazards of orthostatic intolerance and suspension trauma when using fall arrest systems. This bulletin: ...

  9. Cotton 2K-Management tools for irrigated cotton

    Science.gov (United States)

    The use of simulation models to manage crops was a concept introduced in the 1980’s. For example, the cotton simulation model known as GOSSYM was made available in 1989 and was used by both producers and consultants to manage cotton in real time. More recently, Dr. Avi Marani, Professor Emeritus, Sc...

  10. Removal of methylene blue from aqueous solution using cotton stalk, cotton waste and cotton dust

    Energy Technology Data Exchange (ETDEWEB)

    Ertas, Murat [Department of Forest Industrial Engineering, Faculty of Forestry, Kahramanmaras Sutcu Imam University, 46060 Kahramanmaras (Turkey); Acemioglu, Bilal, E-mail: acemioglu@kilis.edu.tr [Department of Chemistry, Faculty of Science and Arts, Kilis 7 Aralik University, 79000 Kilis (Turkey); Alma, M. Hakki [Department of Forest Industrial Engineering, Faculty of Forestry, Kahramanmaras Sutcu Imam University, 46060 Kahramanmaras (Turkey); Usta, Mustafa [Department of Forest Industrial Engineering, Faculty of Forestry, Karadeniz Technical University, 61080 Trabzon (Turkey)

    2010-11-15

    In this study, cotton stalk (CS), cotton waste (CW) and cotton dust (CD) was used as sorbents to remove methylene blue (MB) from aqueous solution by batch sorption technique. Effects of initial dye concentration, solution pH, solution temperature and sorbent dose on sorption were studied. It was seen that the removal of methylene blue increased with increasing initial dye concentration (from 25 to 100 mg/l), solution pH (from 5 to 10), solution temperature (from 20 to 50 deg. C) and sorbent dose (from 0.25 to 1.50 g/50 ml). The maximum dye removal was reached at 90 min. Sorption isotherms were analyzed by Langmuir and Freundlich models at different temperatures of 20, 30, 40 and 50 deg. C, and the results were discussed in detail. Moreover, the thermodynamics of sorption were also studied. It was found that the values of standard free energy ({Delta}G{sup o}) were positive for cotton stalk and negative for cotton waste and cotton dust. The values of standard enthalpy ({Delta}H{sup o}) and entropy ({Delta}S{sup o}) were found to be positive, and the obtained results were interpreted in detail. The results of this study showed that cotton stalk, cotton waste and cotton dust could be employed as effective and low-cost materials for the removal of dyes from aqueous solution.

  11. Collapsing granular suspensions

    OpenAIRE

    Kadau, D.; Andrade Jr, J. S.; Herrmann, H. J.

    2009-01-01

    A 2D contact dynamics model is proposed as a microscopic description of a collapsing suspension/soil to capture the essential physical processes underlying the dynamics of generation and collapse of the system. Our physical model is compared with real data obtained from in situ measurements performed with a natural collapsing/suspension soil. We show that the shear strength behavior of our collapsing suspension/soil model is very similar to the behavior of this collapsing suspension soil, for...

  12. Cell suspension culture and plant regeneration of a Brazilian plantain, cultivar Terra Cultura de células em suspensão e regeneração de plantas de bananeira cultivar Terra

    Directory of Open Access Journals (Sweden)

    Lucymeire Souza Morais-Lino

    2008-10-01

    Full Text Available The objective of this study was to establish cell suspension culture and plant regeneration via somatic embryogenesis of a Brazilian plantain, cultivar Terra Maranhão, AAB. Immature male flowers were used as explant source for generating highly embryogenic cultures 45 days after inoculation, which were used for establishment of cell suspension culture and multiplication of secondary somatic embryos. Five semisolid culture media were tested for differentiation, maturation, somatic embryos germination and for plant regeneration. An average of 558 plants per one milliliter of 5% SCV (settled cell volume were regenerated in the MS medium, with 11.4 µM indolacetic acid and 2.2 µM 6-benzylaminopurine. Regenerated plants showed a normal development, and no visible somaclonal variation was observed in vitro. It is possible to regenerate plants from cell suspensions of plantain banana cultivar Terra using MS medium supplemented with 11.4 µM of IAA and 2.2 µM of BAP.O objetivo deste trabalho foi estabelecer a cultura de células em suspensão e a regeneração de plantas via embriogênese somática de bananeira cultivar Terra Maranhão, AAB. Flores imaturas masculinas foram utilizadas como fonte de explante para obtenção de culturas altamente embriogênicas 45 dias após a inoculação, as quais foram utilizadas para o estabelecimento de suspensões celulares e multiplicação de embriões somáticos secundários. Cinco meios de cultura semi-sólidos foram testados para a diferenciação, maturação, germinação dos embriões somáticos e para a regeneração de plantas. A média de 558 plantas por mililitro de 5% SCV (volume de células sedimentadas foi regenerada, em meio MS com 11,4 µM de ácido indolacético e 2,2 µM de 6-benzilaminopurina. As plantas regeneradas apresentaram desenvolvimento normal, e não foi observada a ocorrência de variação somaclonal in vitro. É possível a regeneração de plantas a partir de células em suspens

  13. Rapid plant regeneration from cotton(Gossypium hirsutum L.)

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    A simple and rapid regeneration method of cotton (Gossypium hirsutum L.cv.Xinluzao 4) is described.The proper use of phytohormone KT and IBA validly promoted the survival rate of test-tube plants and shortened the period of culture in combination with the techniques of micro-propagation and graft.

  14. Exploring biomedical applications of cotton

    Science.gov (United States)

    The use of cotton as a biomaterial for design of improved wound dressings, and other non-implantable medical textiles will be considered. The research and development of cotton-based wound dressings, which possess a mechanism-based mode of action, has entered a new level of understanding in recent ...

  15. Exploring biomedical ppplications of cotton

    Science.gov (United States)

    The use of cotton as a biomaterial for design of improved wound dressings, and other non-implantable medical textiles will be considered. The research and development of cotton-based wound dressings, which possess a mechanism-based mode of action, has entered a new level of understanding in recent y...

  16. The Spindle Type Cotton Harvester

    Science.gov (United States)

    The spindle type cotton picker was commercialized during the mid 1900’s and is currently produced by two US agricultural equipment manufacturers, John Deere and CaseIH. Picking is the predominate machine harvest method used throughout the US and world. Harvesting efficiency of a spindle type cotton ...

  17. Cotton Textile: Brisk against Bleak

    Institute of Scientific and Technical Information of China (English)

    Dennis K.Zhao

    2009-01-01

    @@ The 6th International cotton and cotton textile conference already scheduled on Sept.8-10 in Xinjiang,China's largest cotton growing area,was called off on a short notice of rascal needle dabbing that had caused a widespread public consternation.But the information that is focused on the leitmotif of "financial crisis and revitalization of textile industry for adjustment,upgrading and innovation"is to be shared,discussed at the upcoming resumed meeting.Cotton textile industry is and will be the most important driver for the global textile and clothing sector as it provides jobs not only for the residents living in the cities,but also for the farmers growing cotton in the poverty-ridden countryside.China and India are the most important players in this sector,for both are the most populous countries in the world...

  18. THE CHANGE OF KINETIK PARAMETERS OF THE WEAK-ASSOCIATED WITH WALL CELL PEROXIDASE IN THE SUSPENSION CULTURE OF POTATO CELLS IN THE BEGINNING OF INFECTION

    Directory of Open Access Journals (Sweden)

    Graskova I.A.

    2006-03-01

    Full Text Available The change in kinetic parameters of extracellular peroxidase of suspension cells of resistant potato variety (Lugovskoi and sensitive variety (Luk,ynovskii in the initial period of infection by 5369 Clavibacter michiganensis subsp. sepedonicus (Spieck. et Kotth. Skapt et Burkh. pathogen was examined. Extracellular peroxidases of resistant and sensitive potato variety without pathogens were shown to be concurrently inhibited. At the beginning of infection enzyme activity was extremely increased due to enhanced affinity to substrate as a result of reducing of competitive inhibiting. In increasing enzyme activity is sensitive potato variant evidently caused by other mechanism.

  19. China Cotton label to be generalized

    Institute of Scientific and Technical Information of China (English)

    2011-01-01

    "China Cotton"authorization press conference was held in Beijing on October 11. China Cotton Association granted authorization to the first four enterprises, allowing them to use the label of China Cotton on their qualified products. Shandong Lanyan Group, Beijing Miantian Textile Co., Ltd are among the fi rst companies authorized to use China Cotton label.

  20. CCI President Participated in China Cotton Summit

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    @@ On May 7-8 the 2010 China Cotton Summit and the International Cotton Fair were held in Sanya, Hainan Province, China. Mr. Wallace L. Darneille, the new president of Cotton Council International (CCI) made a special trip to China to participate in the event and present on the "cotton and textile supply and demand situation in the U.S."

  1. 氮素对印楝愈伤组织和悬浮细胞培养的影响%Effects of Nitrogen on Azadirachta indica A. Juss Callus and Suspension Culture

    Institute of Scientific and Technical Information of China (English)

    韩广建; 李兴林; 别振宇; 张国运

    2015-01-01

    通过改变印楝组织培养基中硝态氮与铵态氮物质的量比,并与培养物的生物量、可溶性蛋白含量、印楝素、柠檬苦素含量相关联,探讨了 MS 培养基中氮素对印楝愈伤组织和悬浮细胞培养的影响.实验结果表明:在印楝的愈伤组织培养过程中,当硝态氮与铵态氮物质的量比为 4:1 时,生物量、可溶性蛋白含量、印楝素以及柠檬苦素含量均达到最大积累量;在悬浮细胞培养的过程中,当硝态氮与铵态氮物质的量比为 3:1 时,可溶性蛋白含量、印楝素以及柠檬苦素含量达到最高,而在 2:1 时生物量达到最大.因此,印楝组织培养的不同阶段应该采用不同形式氮素比例的培养基,只有这样才能获得最佳的目标培养物.%To study the effects of nitrogen in MS medium on neem(Azadirachta indica A. Juss)tissue culture,we deter-mined the biomass,soluble protein content,azadirachtin and limonoid contents of neem callus and suspension cells in the end of their cultures by changing the ratio between nitrate and ammonium. The results indicate,that while culturing callus, the four measurement indicators reached the maximum accumulation when the ratio of nitrate to ammonium was 4:1;while culturing suspension cells,the soluble protein contain,azadirachtin and limonoids contains got their maximum whenthe ratio of nitrate to ammonium was 3:1,but the maximum biomass was obtained when the ratio was 2:1. In conclusion,the ratio between nitrate and ammonium in the MS medium should be corresponded with the objects being cultured at different peri-ods.Only in this way,can we get the best target cultures.

  2. Charm of Cotton Art COTTON USA: Naturally Color Your Life: Cotton & Patchwork Exhibition

    Institute of Scientific and Technical Information of China (English)

    Flora Zhao

    2012-01-01

    The grand opening of Cotton Council International's (CCI) finale event Naturally Color YourLife: Cotton & Patchwork by CO-FFON USA took place in Beijing's 798 Art Bridge Gallery on May 25th, 2012. The exhibition was a perfect marriage of the constant pursuit of traditional patchwork art with the fantastic imagination of modern design.

  3. Effects of culture conditions on biomass and active components of suspension cells of Panax quinquefolium%培养条件对西洋参悬浮细胞生物量和活性成分的影响

    Institute of Scientific and Technical Information of China (English)

    王娟; 高文远; 黄滔; 曹宇

    2009-01-01

    目的:研究主要理化因子对西洋参细胞悬浮培养的影响.方法:利用组织培养技术结合高效液相色谱法和紫外分光光度法,考察接种量、培养基种类、基质pH和光照条件对西洋参悬浮细胞生长,人参皂苷Re,Rb1以及西洋参多糖含量的影响.结果:当接种量为25 g·L~(-1)时,西洋参细胞的干重增殖倍数显著增加;通过考察Ms,SH,B_5 3种培养基对西洋参细胞的影响,结果表明MS培养基最有利于西洋参细胞生长,B_5培养基最有利于人参皂苷和西洋参多糖的合成.3种培养基中西洋参细胞多糖含量均高于栽培西洋参;pH变化对西洋参细胞生长影响不大,pH 6.0时,最有利于人参皂苷Re和西洋参多糖的合成;光照培养显著促进西洋参细胞次生代谢物的合成,但对多糖合成没有太大影响.结论:接种量、培养基种类、基质pH和光照条件对西洋参悬浮细胞生长,人参皂苷Re,Rb_1以及西洋参多糖合成有显著影响.%Objective: To study the effects of inoculum, various media, pH value of medium and illumination conditions on the growth of Panax quinquefolium suspension cells and the synthesis of ginsenosides Re, Rb_1 and polysaccharides. Method: The suspension cells were obtained through tissue culture by manipulation of inoculum, various media, pH value, and illumination conditions. The contents of ginsenosides Re and Rb_1 were determined by HPLC, while the contents of polysaccharide were determined by ultraviolet spectrophotometry. Result: The growth rate of suspension cells was greatly increased when inoculum amount was 25 g·L~(-1). The effect of media MS, SH and B_5 on suspension cells was observed. MS medium was favorable for cells growth, while B_5 medium was favorable for the synthesis of ginsenosides and polysaccharides. The polysaccharide content in three media were higher than that of the cultivations. The pH value showed little influence on the cells growth, medium pH 6.0 enhanced

  4. Association of Phosphatidylinositol Kinase, Phosphatidylinositol Monophosphate Kinase, and Diacylglycerol Kinase with the Cytoskeleton and F-Actin Fractions of Carrot (Daucus carota L.) Cells Grown in Suspension Culture : Response to Cell Wall-Degrading Enzymes.

    Science.gov (United States)

    Tan, Z; Boss, W F

    1992-12-01

    Phosphatidylinositol kinase (PI), phosphatidylinositol monophosphate (PIP) kinase, and diacylglycerol (DAG) kinase activities were detected in the cytoskeletal fraction isolated from microsomes and plasma membranes of carrot (Daucus carota L.) cells grown in suspension culture. The lipid kinase activities were associated with the actin filament fraction (F-actin fraction) isolated from the cytoskeleton. The PI and PIP kinase activity in the F-actin fraction significantly increased after cells were treated with Driselase, a mixture of cell wall-degrading enzymes; however, the DAG kinase activity in the F-actin fraction was unaffected by the Driselase treatment. These data indicate that at least one form of PI, PIP, and DAG kinase preferentially associates with actin filaments and/or actin binding proteins and that cytoskeletal-associated PI and PIP kinase activities can change in response to external stimulation.

  5. Association of Phosphatidylinositol Kinase, Phosphatidylinositol Monophosphate Kinase, and Diacylglycerol Kinase with the Cytoskeleton and F-Actin Fractions of Carrot (Daucus carota L.) Cells Grown in Suspension Culture 1

    Science.gov (United States)

    Tan, Zheng; Boss, Wendy F.

    1992-01-01

    Phosphatidylinositol kinase (PI), phosphatidylinositol monophosphate (PIP) kinase, and diacylglycerol (DAG) kinase activities were detected in the cytoskeletal fraction isolated from microsomes and plasma membranes of carrot (Daucus carota L.) cells grown in suspension culture. The lipid kinase activities were associated with the actin filament fraction (F-actin fraction) isolated from the cytoskeleton. The PI and PIP kinase activity in the F-actin fraction significantly increased after cells were treated with Driselase, a mixture of cell wall-degrading enzymes; however, the DAG kinase activity in the F-actin fraction was unaffected by the Driselase treatment. These data indicate that at least one form of PI, PIP, and DAG kinase preferentially associates with actin filaments and/or actin binding proteins and that cytoskeletal-associated PI and PIP kinase activities can change in response to external stimulation. Images Figure 2 PMID:16653250

  6. Sampling nucleotide diversity in cotton

    Directory of Open Access Journals (Sweden)

    Yu John Z

    2009-10-01

    Full Text Available Abstract Background Cultivated cotton is an annual fiber crop derived mainly from two perennial species, Gossypium hirsutum L. or upland cotton, and G. barbadense L., extra long-staple fiber Pima or Egyptian cotton. These two cultivated species are among five allotetraploid species presumably derived monophyletically between G. arboreum and G. raimondii. Genomic-based approaches have been hindered by the limited variation within species. Yet, population-based methods are being used for genome-wide introgression of novel alleles from G. mustelinum and G. tomentosum into G. hirsutum using combinations of backcrossing, selfing, and inter-mating. Recombinant inbred line populations between genetics standards TM-1, (G. hirsutum × 3-79 (G. barbadense have been developed to allow high-density genetic mapping of traits. Results This paper describes a strategy to efficiently characterize genomic variation (SNPs and indels within and among cotton species. Over 1000 SNPs from 270 loci and 279 indels from 92 loci segregating in G. hirsutum and G. barbadense were genotyped across a standard panel of 24 lines, 16 of which are elite cotton breeding lines and 8 mapping parents of populations from six cotton species. Over 200 loci were genetically mapped in a core mapping population derived from TM-1 and 3-79 and in G. hirsutum breeding germplasm. Conclusion In this research, SNP and indel diversity is characterized for 270 single-copy polymorphic loci in cotton. A strategy for SNP discovery is defined to pre-screen loci for copy number and polymorphism. Our data indicate that the A and D genomes in both diploid and tetraploid cotton remain distinct from each such that paralogs can be distinguished. This research provides mapped DNA markers for intra-specific crosses and introgression of exotic germplasm in cotton.

  7. Optimization of Two-dimensional Electrophoresis for Proteome of Cucumber Suspension Cultured Cells%黄瓜悬浮细胞蛋白质组双向电泳分析技术

    Institute of Scientific and Technical Information of China (English)

    郝宇涵; 范海延; 曲波; 许玉凤; 崔娜; 李楠; 任婧祺

    2012-01-01

    为建立适于黄瓜悬浮细胞蛋白质组分析的双向电泳体系,对黄瓜悬浮细胞蛋白质双向电泳分析所采用的胶条pH范围、样品制备方法、裂解液配方及分离胶浓度等参数进行研究.结果表明,采用pH范围为4~7的IPG胶条,直接裂解后丙酮沉淀法制备黄瓜悬浮细胞蛋白质,裂解液为8 mol/L尿素、2 mol/L硫脲、2% IPG Buffer、4% CHAPS、1%TBP、65 mmol/L DTT、2 mmol/L EDTA、0.001%溴酚蓝和1%鸡尾酒,分离胶浓度为11%,可获得蛋白质点分离清晰的双向电泳图谱.%To obtain suitable protocol for proteome of cucumber suspension cultured cells,we optimized the pH gradient of IPG strip, the method of protein extraction, reagent and concentration of lysis buffer and SDS-PAGE condition. Results showed pH 4~7 IPG strips, the extraction method acetone precipitation after lysis,lysis buffer containing 8 mol/L urea,2 mol/L thiourea,2% IPG Buffer,4% CHAPS, 1% TBP,65 mmol/L DTT,2 mmol/L EDTA,0. 001% bromophenol blue and 1% cocktail and 11% gel concentration were appropriate for the sample preparation of cucumber suspension cultured cells.

  8. Utilization of Cotton DNA Markers in Cotton Breeding

    Institute of Scientific and Technical Information of China (English)

    CANTRELL Roy G; XIAO Jin-hua

    2008-01-01

    @@ Informative,portable,and efficient DNA markers have the potential to accelerate genetic gain in cotton breeding.Discovery and widespread application of DNA markers to cotton has traditionally lagged behind other major crop species.The reasons are well known to ICGI participants.The foundation for widespread development and application of DNA markers has been laid by ICGI and research within the private sector.

  9. Cotton bollworm resistance to Bt transgenic cotton: A case analysis

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    Cotton bollworm (Helicoverpa armigera) is one of the most serious insect pests of cotton. Transgenic cotton expressing Cry toxins derived from a soil bacterium, Bacillus thuringiensis (Bt), has been produced to target this pest. Bt cotton has been widely planted around the world, and this has resulted in efficient control of bollworm populations with reduced use of synthetic insecticides. However, evolution of resistance by this pest threatens the continued success of Bt cotton. To date, no field populations of bollworm have evolved significant levels of resistance; however, several laboratory-selected Cry-resistant strains of H. armigera have been obtained, which suggests that bollworm has the capacity to evolve resistance to Bt. The development of resistance to Bt is of great concern, and there is a vast body of research in this area aimed at ensuring the continued success of Bt cotton. Here, we review studies on the evolution of Bt resistance in H. armigera, focusing on the biochemical and molecular basis of Bt resistance. We also discuss resistance management strategies, and monitoring programs implemented in China, Australia, and India.

  10. In vitro inhibition of pigmentation and fiber development in colored cotton

    Institute of Scientific and Technical Information of China (English)

    Shu-na YUAN; Waqas MALIK; Shui-jin HUA; Noreen BIBI; Xue-de WANG

    2012-01-01

    Colored cotton has naturally pigmented fibers.The mechanism of pigmentation in cotton fiber is not well documented.This experiment was conducted to study the effects cf respiratory chain inhibitors,i.e.,rotenone and thiourea,on pigmentation and fiber development in colored cotton.After 1 d post-anthesis,ovaries were harvested and developing ovules were cultured on the liquid medium containing different concentrations of rotenone and thiourea for 30 d.The results demonstrate that both respiratory inhibitors reduced fiber length and ovule development under ovule culture conditions,and the inhibition efficiency of rotenone was much higher than that of thiourea.Rotenone and thiourea also showed significant effects on fiber pigment (color) development in colored cotton.In green cotton fiber,rotenone advanced fiber pigment development by 7 d at 200 μmol/L,while thiourea inhibited fiber pigmentation at all treatment levels (400,600,800,1000,and 2000 μmol/L).Both respiratory inhibitors,however,had no significant effects on pigmentation of brown cotton fibers.The activities of cytochrome c oxidase (COX) and polyphenol oxidase (PPO) decreased significantly with increasing levels of both respiratory inhibitors.It is suggested that both respiratory inhibitors have important roles in deciphering the mechanism of pigmentation and fiber development in colored cotton.

  11. Collapsing granular suspensions.

    Science.gov (United States)

    Kadau, D; Andrade, J S; Herrmann, H J

    2009-11-01

    A 2D contact dynamics model is proposed as a microscopic description of a collapsing suspension/soil to capture the essential physical processes underlying the dynamics of generation and collapse of the system. Our physical model is compared with real data obtained from in situ measurements performed with a natural collapsing/suspension soil. We show that the shear strength behavior of our collapsing suspension/soil model is very similar to the behavior of this collapsing suspension soil, for both the unperturbed and the perturbed phases of the material.

  12. 7 CFR 28.471 - Below Leaf Grade Cotton.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 2 2010-01-01 2010-01-01 false Below Leaf Grade Cotton. 28.471 Section 28.471... REGULATIONS COTTON CLASSING, TESTING, AND STANDARDS Standards Below Leaf Grade Cotton § 28.471 Below Leaf Grade Cotton. Below leaf grade cotton is American Upland cotton which is lower in leaf grade than...

  13. 7 CFR 28.451 - Below Color Grade Cotton.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 2 2010-01-01 2010-01-01 false Below Color Grade Cotton. 28.451 Section 28.451... REGULATIONS COTTON CLASSING, TESTING, AND STANDARDS Standards Below Color Grade Cotton § 28.451 Below Color Grade Cotton. Below color grade cotton is American Upland cotton which is lower in color grade than...

  14. Suspension trauma; Le traumatisme de suspension

    Energy Technology Data Exchange (ETDEWEB)

    Trudel, S. [Le Centre de sante et de services sociaux du rocher Perce, Chandler, PQ (Canada)

    2010-07-01

    This presentation discussed the precautions that should be taken to avoid falls from wind turbines or transmission towers. Suspension trauma was explained by a medical doctor in terms of physiology and the body's normal circulation and the elements that disturb normal physiology when in suspension. The trauma occurs following a fall, which carries the risk of 1or more disorders, such as massive hemorrhage, high cardiac pulse, and constriction of blood vessels. Nausea, vertigo, cardiac arrhythmia and sweating occur 15 to 20 minutes following the fall. The presentation emphasized the importance of having qualified personnel at the site and wearing proper harnesses and equipment that supports the neck. figs.

  15. Research on School Suspension

    Science.gov (United States)

    Iselin, Anne-Marie

    2010-01-01

    Schools across the nation report increases in the use of punitive disciplinary methods (e.g., suspension). The need for these disciplinary practices to address serious student misconduct is undisputed. What research has questioned is why some students seem to be suspended more often than others, what effects suspension has on students, and whether…

  16. 反应器细胞悬浮培养和微载体培养技术在动物疫苗生产中的应用%Application of Bioreactor Cell Suspension Culture in Animal Vaccine Production

    Institute of Scientific and Technical Information of China (English)

    张韧; 王建超; 陈文庆; 刘华杰; 高飞; 徐舸辰; 林龙飞

    2012-01-01

    介绍了反应器悬浮培养技术在国内外疫苗生产中的研发和应用现状。目前该技术已经在国内口蹄疫疫苗生产中获得成功应用,利用MDCK、Vero等细胞培养生产禽流感疫苗的技术也正在积极研发中。积极推广和应用这一技术将是我国兽用生物制品生产工艺升级换代的必然趋势。%The development and application of cell suspension culture in bioreactorsfor vaccine production were reviewed. at home. developed biological The technique has been successfully developed in foot and mouth disease vaccine (FMDV) production Meanwhile, culturing MDCK or Vero cells to produce avian influenza vaccineis has also being positively in China. To promote and apply this new technique is becoming a trend for upgrading veterinary production technique in China.

  17. Stimulation of de novo synthesis of L-phenylalanine ammonia-lyase in relation to phytoalexin accumulation in Colletotrichum lindemuthianum elicitor-treated cell suspension cultures of french bean (Phaseolus vulgaris).

    Science.gov (United States)

    Dixon, R A; Lamb, C J

    1979-09-03

    (1) The regulation of the accumulation of the isoflavonoid-derived phytoalexin phaseollin in cell suspension cultures of Dwarf French Bean (Phaseolus vulgaris/ has been investigated. (2) An elicitor preparation from cell walls of Colletotrichum lindemuthianum, the causal agent of anthracnose disease of French bean, caused a marked accumulation of phaseollin in the cultures. The elicitor induced phaseollin accumulation to a level of 60% that obtained with the artificial elicitor autoclaved ribonuclease A and was maximally active at a concentration (weight basis) of at least 50 times lower than required for maximal response to ribonuclease. (3) Elicitor preparations from cell walls of Phytophthora megasperma var. sojae, a fungal pathogen of soybean, and Botrytis cinerea, the common grey mould, were much less effective than the C. lindemuthianum wall-released elicitor. (4) There was a marked but transient increase in the extractable activity of phenylalanine ammonia-lyase, the enzyme catalysing the first reaction in the biosynthesis of phaseollin from L-phenylalanine, in response to the elicitor from C. lindemuthianum. (5) Comparative density labelling with 2H from 2H2O indicated that the elicitor stimulates de novo synthesis of phenylalanine ammonie findings provide the basis of a scheme for elicitor induction of phytoalexin accumulation.

  18. Autologous epidermal cell suspension: A promising treatment for chronic wounds.

    Science.gov (United States)

    Zhao, Hongliang; Chen, Yan; Zhang, Cuiping; Fu, Xiaobing

    2016-02-01

    Chronic wounds have become an increasing medical and economic problem of aging societies because they are difficult to manage. Skin grafting is an important treatment method for chronic wounds, which are refractory to conservative therapy. The technique involving epidermal cell suspensions was invented to enable the possibility of treating larger wounds with only a small piece of donor skin. Both uncultured and cultured autologous epidermal cell suspensions can be prepared and survive permanently on the wound bed. A systematic search was conducted of EMBASE, Cochrane Library, PubMed and web of science by using Boolean search terms, from the establishment of the database until May 31, 2014. The bibliographies of all retrieved articles in English were searched. The search terms were: (epithelial cell suspension OR keratinocyte suspension) and chronic and wound. From the included, 6 studies are descriptive interventions and discussed the use of autologous keratinocyte suspension to treat 61 patients' chronic wound. The various methods of preparation of epidermal cell suspension are described. The advantages and shortcomings of different carriers for epidermal cell suspensions are also summarised. Both uncultured and cultured autologous epidermal cell suspensions have been used to treat chronic wounds. Although the limitations of these studies include the small number of patient populations with chronic wounds and many important problems that remain to be solved, autologous epidermal cell suspension is a promising treatment for chronic wounds. Copyright © 2015 Tissue Viability Society. Published by Elsevier Ltd. All rights reserved.

  19. 75 FR 24373 - Cotton Research and Promotion Program: Designation of Cotton-Producing States

    Science.gov (United States)

    2010-05-05

    ... Federal Regulations is sold by the Superintendent of Documents. #0;Prices of new books are listed in the... Service 7 CFR Part 1205 RIN 0581-AC84 Cotton Research and Promotion Program: Designation of Cotton... Marketing Service (AMS) is amending the Cotton Research and Promotion Order (Cotton Order) following...

  20. [Glyphosate-resistant cotton (Gossypium hirsutum L.) Transformed with aroAM12 gene via Agrobacterium tumefaciens].

    Science.gov (United States)

    Xie, Long-Xu; Li, Yun-Feng; Xu, Pei-Lin

    2004-04-01

    A mutant, aroAM12, exhibiting resistance to glyphosate produced in a previous study using the staggered extension process with aroA genes from Salmonella typhimurium and Eschrichia coli. In this paper, we constructed a vector pGRA1300 carrying aroAM12 gene, comprising transit peptide of Arabidopsis EPSPS, under the control of the CaMV35S promoter and used as selectable marker for cotton plant (Gossypium hirsutum L.) transformation. Transgenic cottons with increased resistance to glyphosate were obtained by cotransformtion of hypocotyl segments with Agrobacterium tumefaciens and selected directly on medium containing glyphosate. Regeneration of glyphosate-resistant calli was carried out on a MS basic medium containing 2,4-D 0.1 mg/L, KT 0.1 mg/L, cefotaxime 500 mg/L and glyphosate 60 micromol/L. Globular embryos were induced and then developed by culturing on MSB (MS salts+B(5) vitamins) medium supplemented with asparagine 1 g/L and glutamine 2 g/L, but not containing hormone, for 40 d. The developed plantlets were then removed and cultured on an MS medium. After about 20 d, the deeply-rooted shoots were in soil. PCR analysis showed that the aroAM12 gene was present in all T(0) transgenic plants. The integration of the aroAM12 gene in the genomic DNA of cotton was further confirmed by Southern blot, which showed that the transgenic plants carried one or two copies of the aroAM12 genes. Western blot analysis showed that a 48-kD band of was detected in all T(0) transgenic plants. There was no apparent corelation between copy numbers and the expression level of the aroAM12 gene. Greenhouse screening for glyphosate resistance was performed to test 65 independent T(0) plants by spraying (three times) with an aqueous suspension at a dose corresponding to 9.317 kg/ha of Roundup (once every 5 d). After 15 d, phenotype examination was carried out of the plants in comparison with untransformed control plants. Under these conditions, it was observed that the plants transformed

  1. China International Cotton Conference Concluded in Xinjiang

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    @@ The 2007 China International Cotton Conference was held on June 27-29 in Urumqi, Xinjiang Municipality, China. With the theme "China's Cotton Industry on WTO and It's Implications The Global Market".

  2. Synthesis of Cotton from Tossa Jute Fiber and Comparison with Original Cotton

    Directory of Open Access Journals (Sweden)

    Md. Mizanur Rahman

    2015-01-01

    Full Text Available Cotton fibers were synthesized from tossa jute and characteristics were compared with original cotton by using FTIR and TGA. The FTIR results indicated that the peak intensity of OH group from jute cotton fibers occurred at 3336 cm−1 whereas the peak intensity of original cotton fibers occurred at 3338 cm−1. This indicated that the synthesized cotton fiber properties were very similar to the original cotton fibers. The TGA result showed that maximum rate of mass loss, the onset of decomposition, end of decomposition, and activation energy of synthesized cotton were higher than original cotton. The activation energy of jute cotton fibers was higher than the original cotton fibers.

  3. Serumfree culture of the suspension cell line QB-Tn9-4s of the cabbage looper, Trichoplusia ni, is highly productive for virus replication and recombinant protein expression.

    Science.gov (United States)

    Zheng, Gui-Ling; Zhou, Hong-Xu; Li, Chang-You

    2014-02-12

    Serumfree cultures of insect cells play an important role in the fields of protein engineering, medicine, and biology. In this paper, the suspension cell line QB-Tn9-4s of Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae) was successfully adapted to serumfree Sf-900 III medium and passaged for 52 generations. The adapted QB-Tn9-4s cells grew faster. Their population doubling time shortened from 27.4 hr in serum-containing medium to 24.1 hr, and their maximal density increased by 1.83-fold, reaching 3.50 ×10(6) cells/mL in serumfree culture in T-flasks. The cells readily adapted to spinner culture, with maximum cell density of 4.40 × 10(6) cells/mL in a spinner flask. Although the infection rate of Autographa californica multiple nucleopolyhedrovirus and production of occlusion bodies (OBs) of the adapted QB-Tn9-4s cells were 91.0% and 85.4 OBs/cell, respectively, similar to those of QB-Tn9-4s cells cultured in serum-containing medium and control BTI-Tn5B1-4 cells, their budded virus titer was 4.97 ×10(7) TCID50/mL, significantly higher than those of the latter two. In addition, the expression levels of β-galactosidase at six days postinfection and secreted alkaline phosphatase at seven days postinfection in the adapted QB-Tn9-4s cells reached 2.98 ± 0.15×10(4) IU/mL and 3.34 ± 0.13 IU/mL, respectively, significantly higher than those of QB-Tn9-4s and control BTI-Tn5B1-4 cultured in serum-containing media. The above findings establish a foundation for industrial production of virus and recombinant proteins in QB-Tn9-4s serumfree culture.

  4. Serum-Free Culture of the Suspension Cell Line QB-Tn9-4s of the Cabbage Looper, Trichoplusia ni, is Highly Productive for Virus Replication and Recombinant Protein Expression

    Science.gov (United States)

    Zheng, Gui-Ling; Zhou, Hong-Xu; Li, Chang-You

    2014-01-01

    Serum-free cultures of insect cells play an important role in the fields of protein engineering, medicine, and biology. In this paper, the suspension cell line QB-Tn9-4s of Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae) was successfully adapted to serum-free Sf-900 III medium and passaged for 52 generations. The adapted QB-Tn9-4s cells grew faster. Their population doubling time shortened from 27.4 hr in serum-containing medium to 24.1 hr, and their maximal density increased by 1.83-fold, reaching 3.50 × 106 cells/mL in serum-free culture in T-flasks. The cells readily adapted to spinner culture, with maximum cell density of 4.40 × 106 cells/mL in a spinner flask. Although the infection rate of Autographa californica multiple nucleopolyhedrovirus and production of occlusion bodies (OBs) of the adapted QB-Tn9-4s cells were 91.0% and 85.4 OBs/cell, respectively, similar to those of QB-Tn9-4s cells cultured in serum-containing medium and control BTI-Tn5B1-4 cells, their budded virus titer was 4.97 × 107 TCID50/mL, significantly higher than those of the latter two. In addition, the expression levels of β-galactosidase at six days post-infection and secreted alkaline phosphatase at seven days postinfection in the adapted QB-Tn9-4s cells reached 2.98 ± 0.15×104 IU/mL and 3.34 ± 0.13 IU/mL, respectively, significantly higher than those of QB-Tn9-4s and control BTI-Tn5B1-4 cultured in serum-containing media. The above findings establish a foundation for industrial production of virus and recombinant proteins in QB-Tn9-4s serum-free culture. PMID:25373171

  5. CCI President Participated in China Cotton Summit

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    On May 7-8 the 2010 China Cotton Summit and the International Cotton Fair were held in Sanya,Hainan Province,China.Mr.Wallace L.Darneille, the new president of Cotton Council International(CCI) made a special trip to China to participate in the event and present on the"cotton and textile supply and demand situation in the U.S."

  6. Sucrose metabolizing enzymes in cell suspension cultures of Bauhinia forficata, Curcuma zedoaria and Phaseolus vulgaris Enzimas do metabolismo da sacarose em cultura celular de Bauhinia forficata, Curcuma zedoaria e Phaseolus vulgaris

    Directory of Open Access Journals (Sweden)

    Marcia Ometto de Mello

    2001-09-01

    Full Text Available The objective of this work was to study the activity of sucrose metabolizing enzymes in extracts of cell suspension cultures of Bauhinia forficata Link, Curcuma zedoaria Roscoe and Phaseolus vulgaris L. Invertase pathway was identified in the three studied species. Sucrose synthase pathway was also responsible for sucrose metabolism in Curcuma zedoaria and Phaseolus vulgaris cells. Activity values higher than 300 nmol min-1 mg-1 of protein were found for acid and neutral invertases, UDPglucose pyrophosphorylase and phosphoglucomutase in the cell extract of the three plant species. Sucrose synthase showed low activity in Bauhinia forficata cells. As sucrose concentration in the culture medium decreased, sucrose synthase activity increased in C. zedoaria and P. vulgaris cells. The glycolytic enzymes activity gradually reduced at the end of the culture period, when carbohydrate was limited.O objetivo deste trabalho foi estudar as enzimas do metabolismo da sacarose em culturas de célula em suspensão de Bauhinia forficata Link, Curcuma zedoaria Roscoe e Phaseolus vulgaris L. A via da invertase foi identificada nas três espécies estudadas. A via da sacarose sintase também foi responsável pelo metabolismo da sacarose em células de Curcuma zedoaria e Phaseolus vulgaris. Foram encontradas atividades maiores que 300 nmol min-1 mg-1 de proteína das enzimas invertase ácida e alcalina, UDPglicose pirofosforilase e fosfoglicomutase no extrato celular das três espécies de plantas. A sacarose sintase mostrou atividade baixa nas células de Bauhinia forficata. À medida que a concentração de sacarose no meio de cultura diminuiu, a atividade da sacarose sintase aumentou em células de Curcuma zedoaria e Phaseolus vulgaris. Ao final do período de cultura, quando os carboidratos se tornaram limitantes, as atividades das enzimas glicolíticas reduziram-se gradualmente.

  7. Sequencing the Cotton Genomes-Gossypium spp.

    Institute of Scientific and Technical Information of China (English)

    PATERSON Andrew H

    2008-01-01

    @@ The genomes of most major crops,including cotton,will be fully sequenced in the next fewyears.Cotton is unusual,although not unique,in that we will need to sequence not only cultivated(tetraploid) genotypes but their diploid progenitors,to understand how elite cottons have surpassedthe productivity and quality of their progenitors.

  8. Dielectric permitivity measurement of cotton lint

    Science.gov (United States)

    A technique was developed for making broad band measurements of cotton lint electrical permitivity. The fundamental electrical permitivity value of cotton lint at various densities and moisture contents; is beneficial for the future development of cotton moisture sensors as it provides a...

  9. 7 CFR 1205.308 - Cotton Board.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 10 2010-01-01 2010-01-01 false Cotton Board. 1205.308 Section 1205.308 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (MARKETING AGREEMENTS... Research and Promotion Order Definitions § 1205.308 Cotton Board. Cotton Board means the...

  10. 7 CFR 1205.305 - Upland cotton.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 10 2010-01-01 2010-01-01 false Upland cotton. 1205.305 Section 1205.305 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (MARKETING AGREEMENTS... Research and Promotion Order Definitions § 1205.305 Upland cotton. Upland cotton means all...

  11. Toward cotton molecular breeding: challenges and opportunities

    Science.gov (United States)

    Cotton (Gossypium spp) is the leading natural fiber in the global textile market, but progress in the development and applications of molecular tools to improve cotton lags behind other major crop plants. The slow progress is in part due to cotton's large complex allotetraploid genome of 26 partial...

  12. Characterization of a Cotton Fiber Gene Promoter

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Cotton fibers are unicellular trichomes derived from outer integument cells of the ovule.Our previously study showed that cotton R2R3 MYB transcript factor GaMYB2 could complement the Arabidopsis trichome mutant of glabra1(gl1),suggesting that cotton fiber initiation and Arabidopsis leaf

  13. Greige cotton comber noils for sustainable nonwovens

    Science.gov (United States)

    To increase utilization of cotton in value-added nonwoven products, a study was conducted to examine the feasibility of utilizing cotton textile processing/combing bye-product known as griege cotton comber noils. The study was conducted on a commercial-grade, textile-cum-nonwovens pilot plant and ha...

  14. Bioinspiration and Biomimicry: Possibilities for Cotton Byproducts

    Science.gov (United States)

    The byproducts from cotton gins have commonly been referred to as cotton gin trash or cotton gin waste primarily because the lint and seed were the main focus of the operation and the byproducts were a financial liability that did not have a consistent market. Even though the byproducts were called ...

  15. In vitro morphogenesis and cell suspension culture establishment in Piper solmsianum C. DC. (Piperaceae Morfogênese in vitro e estabelecimento de culturas de suspensão celular em Piper solmsianum C. DC. (Piperaceae

    Directory of Open Access Journals (Sweden)

    Tiago Santana Balbuena

    2009-03-01

    Full Text Available Piper solmsianum is a shrub from Southeast Brazil in which many biologically active compounds were identified. The aim of this work was to establish a cell suspension culture system for this species. With this in mind, petiole and leaf explants obtained from in vitro plantlets were cultured in the presence of different plant growth regulator combinations (IAA, NAA, 2,4-D and BA. Root and indirect shoot adventitious formation, detected by histological analysis, was observed. Besides the different combinations of plant growth regulators, light regime and the supplement of activated charcoal (1.5 mg.l-1 were tested for callus induction and growth. Cultures maintained in light, on a 0.2 mg.l-1 2,4-D and 2 mg.l-1 BA supplemented medium, and in the absence of activated charcoal, showed the highest calli fresh matter increment. From a callus culture, cell suspension cultures were established and their growth and metabolite accumulation studied. The achieved results may be useful for further characterization of the activated secondary metabolites pathways in in vitro systems of P. solmsianum.Piper solmsianum é uma espécie herbácea do sudeste brasileiro onde vários compostos biologicamente ativos já foram identificados. O objetivo deste trabalho foi estabelecer suspensões celulares nesta espécie. Para tanto, foram utilizados explantes de pecíolos e folhas, retirados de plântulas cultivadas in vitro, os quais foram submetidos a diferentes combinações de reguladores de crescimento (AIA, ANA, 2,4-D e BAP. Foi obtida a neo-formação de raízes e brotos, estes últimos através do processo de organogênese indireta evidenciada por estudos histológicos. Para a indução e crescimento dos calos, foram avaliados, além das diferentes combinações de reguladores de crescimento, a suplementação ao meio de cultura de carvão ativado (1,5 mg.l-1 e o regime de luz. Culturas mantidas na luz, em meio de cultura suplementado com 0,2 mg.l-1 2,4-D e 2 mg

  16. Ways of Developing Plants in Interspecific Hybridization of Cotton

    Institute of Scientific and Technical Information of China (English)

    RAKHMANKULOV; S; DAMINOVA; D; RAKHMANKULOV; M

    2008-01-01

    It is known,that there are various barriers to fertilization,development of embryos,and endosperm because of different number of chromosomes in parents in the interspecific hybridization of cotton.Thus the factors providing normal cell fission of a germ and endosperm are necessary.It is necessary to culture embryos in vitro on the artificial environments containing various phytohormones,or to

  17. Identification of Alternaria alternata mycotoxins by LC-SPE-NMR and their cytotoxic effects to soybean (Glycine max) cell suspension culture.

    Science.gov (United States)

    de Souza, Gezimar D; Mithöfer, Axel; Daolio, Cristina; Schneider, Bernd; Rodrigues-Filho, Edson

    2013-02-26

    This present work describes the application of liquid chromatography-solid phase extraction-nuclear magnetic resonance spectroscopy to analyse Alternaria alternata crude extracts. Altenusin (1), alternariol (2), 3'-hydroxyalternariol monomethyl ether (3), and alternariol monomethyl ether (4), were separated and identified. High-resolution mass spectrometry confirmed the proposed structures. The cytotoxic effects of these compounds towards plants were determined using soybean (Glycine max) cell cultures as a model. EC(50) values which range from 0.11 (± 0.02) to 4.69 (± 0.47) μM showed the high cytotoxicity of these compounds.

  18. Identification of Alternaria alternata Mycotoxins by LC-SPE-NMR and Their Cytotoxic Effects to Soybean (Glycine max Cell Suspension Culture

    Directory of Open Access Journals (Sweden)

    Edson Rodrigues-Filho

    2013-02-01

    Full Text Available This present work describes the application of liquid chromatograpy-solid phase extraction-nuclear magnetic resonance spectroscopy to analyse Alternaria alternata crude extracts. Altenusin (1, alternariol (2, 3'-hydroxyalternariol monomethyl ether (3, and alternariol monomethyl ether (4, were separated and identified. High-resolution mass spectrometry confirmed the proposed structures. The cytotoxic effects of these compounds towards plants were determined using soybean (Glycine max cell cultures as a model. EC50 values which range from 0.11 (±0.02 to 4.69 (±0.47 μM showed the high cytotoxicity of these compounds.

  19. Microfluidic Bead Suspension Hopper

    OpenAIRE

    Price, Alexander K.; MacConnell, Andrew B.; Paegel, Brian M.

    2014-01-01

    Many high-throughput analytical platforms, from next-generation DNA sequencing to drug discovery, rely on beads as carriers of molecular diversity. Microfluidic systems are ideally suited to handle and analyze such bead libraries with high precision and at minute volume scales; however, the challenge of introducing bead suspensions into devices before they sediment usually confounds microfluidic handling and analysis. We developed a bead suspension hopper that exploits sedimentation to load b...

  20. The Mystical Suspension

    Directory of Open Access Journals (Sweden)

    Héctor Santiesteban Oliva

    2016-11-01

    Full Text Available Mistical suspension, silence, time, absolute, ontology, ineffability, aletheiaIn the mystical ecstasy there is a sensorial and intellectual suspension when contemplating the absolute, the ontological Being. Silence is not only significant: it is revealing. The greatest expression of experience inner silence . The word is insufficient when the ontological reality is revealed. Revelation or truth , the Greek concept of aletheia, takes on greater significance in that transcendental experience. It is also suspended phenomenological time and remains eternity open.

  1. Primary Studies on Cotton Telomere

    Institute of Scientific and Technical Information of China (English)

    LING Jian; PENG Ren-hai; WANG Kun-bo; WANG Chun-ying; SONG Guo-li; LIU Fang; LI Shao-hui; ZHANG Xiang-di; WANG Yu-hong

    2008-01-01

    @@ The Arabidopsis -type telomere sequence was amplified and cloned using the primers designed from the fragment which contained the telomere sequence in an Arabidopsis BAC.In situ hybridizations with cotton metaphase chromosomes,using the telomere as probe,it indicated that the signals were located at all chromosome ends of 7 diploid and 2 tetraploid cotton species.To identify the signals of FISH,the genome DNA of Xinhai 7,digested by Bal31 kinetics,was used in this study.

  2. Trade Statistics: Cotton Yarn & Fabric in Feb.

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    @@ Cotton is the single most important textile fiber in the world,accounting for nearly 40 percent of total world fiber production.While some 80 countries from around the globe produce cotton,the United States,China,and India together provide over half the world's cotton.This monthly update provides official CNTAC (China National Textile & Apparel Council ) data on China import and export of cotton yarn and cotton fabric,to show a general profile of China's foreign trade in current textile industry.

  3. Chlorogenic acid biosynthesis: characterization of a light-induced microsomal 5-O-(4-coumaroyl)-D-quinate/shikimate 3'-hydroxylase from carrot (Daucus carota L. ) cell suspension cultures

    Energy Technology Data Exchange (ETDEWEB)

    Kuehnl, T.K.; Koch, U.; Heller, W.; Wellmann, E.

    1987-10-01

    Microsomal preparations from carrot (Daucus carota L.) cell suspension cultures catalyze the formation of trans-5-O-caffeoyl-D-quinate (chlorogenate) from trans-5-O-(4-coumaroyl)-D-quinate. trans-5-O-(4-Coumaroyl)shikimate is converted to about the same extent to trans-5-O-caffeoylshikimate. trans-4-O-(4-Coumaroyl)-D-quinate, trans-3-O-(4-coumaroyl)-D-quinate, trans-4-coumarate, and cis-5-O-(4-coumaroyl)-D-quinate do not act as substrates. The reaction is strictly dependent on molecular oxygen and on NADPH as reducing cofactor. NADH and ascorbic acid cannot substitute for NADPH. Cytochrome c, Tetcyclacis, and carbon monoxide inhibit the reaction suggesting a cytochrome P-450-dependent mixed-function monooxygenase. Competition experiments as well as induction and inhibition phenomena indicate that there is only one enzyme species which is responsible for the hydroxylation of the 5-O-(4-coumaric) esters of both D-quinate and shikimate. The activity of this enzyme is greatly increased by in vivo irradiation of the cells with blue/uv light. We conclude that the biosynthesis of the predominant caffeic acid conjugates in carrot cells occurs via the corresponding 4-coumaric acid esters. Thus, in this system, 5-O-(4-coumaroyl)-D-quinate can be seen as the final intermediate in the chlorogenic acid pathway.

  4. The Metabolic Fate of Deoxynivalenol and Its Acetylated Derivatives in a Wheat Suspension Culture: Identification and Detection of DON-15-O-Glucoside, 15-Acetyl-DON-3-O-Glucoside and 15-Acetyl-DON-3-Sulfate

    Directory of Open Access Journals (Sweden)

    Clemens Schmeitzl

    2015-08-01

    Full Text Available Deoxynivalenol (DON is a protein synthesis inhibitor produced by the Fusarium species, which frequently contaminates grains used for human or animal consumption. We treated a wheat suspension culture with DON or one of its acetylated derivatives, 3-acetyl-DON (3-ADON, 15-acetyl-DON (15-ADON and 3,15-diacetyl-DON (3,15-diADON, and monitored the metabolization over a course of 96 h. Supernatant and cell extract samples were analyzed using a tailored LC-MS/MS method for the quantification of DON metabolites. We report the formation of tentatively identified DON-15-O-β-D-glucoside (D15G and of 15-acetyl-DON-3-sulfate (15-ADON3S as novel deoxynivalenol metabolites in wheat. Furthermore, we found that the recently identified 15-acetyl-DON-3-O-β-D-glucoside (15-ADON3G is the major metabolite produced after 15-ADON challenge. 3-ADON treatment led to a higher intracellular content of toxic metabolites after six hours compared to all other treatments. 3-ADON was exclusively metabolized into DON before phase II reactions occurred. In contrast, we found that 15-ADON was directly converted into 15-ADON3G and 15-ADON3S in addition to metabolization into deoxynivalenol-3-O-β-D-glucoside (D3G. This study highlights significant differences in the metabolization of DON and its acetylated derivatives.

  5. Tissue engineering scaffolds electrospun from cotton cellulose.

    Science.gov (United States)

    He, Xu; Cheng, Long; Zhang, Ximu; Xiao, Qiang; Zhang, Wei; Lu, Canhui

    2015-01-22

    Nonwovens of cellulose nanofibers were fabricated by electrospinning of cotton cellulose in its LiCl/DMAc solution. The key factors associated with the electrospinning process, including the intrinsic properties of cellulose solutions, the rotating speed of collector and the applied voltage, were systematically investigated. XRD data indicated the electrospun nanofibers were almost amorphous. When increasing the rotating speed of the collector, preferential alignment of fibers along the drawing direction and improved molecular orientation were revealed by scanning electron microscope and polarized FTIR, respectively. Tensile tests indicated the strength of the nonwovens along the orientation direction could be largely improved when collected at a higher speed. In light of the excellent biocompatibility and biodegradability as well as their unique porous structure, the nonwovens were further assessed as potential tissue engineering scaffolds. Cell culture experiments demonstrated human dental follicle cells could proliferate rapidly not only on the surface but also in the entire scaffold. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. 新疆连作、轮作棉田可培养的土壤微生物区系及活性分析%Dynamics Analysis of Culturable Soil Microflora and Microbial Activity in Continuous and Rotation Cropping Systems of Xinjiang Cotton

    Institute of Scientific and Technical Information of China (English)

    韩剑; 张静文; 徐文修; 罗明; 吴莉莉

    2011-01-01

    Variation of culturable soil microflora and microbial activity were investigated in continuous and rotation cropping cotton field in Xinjiang.The results showed that culturable microbial population gradually decreased with long-term continuous cropping of cotton.Compared with 5 years continous cropping, the total quantity of soil microbes in 6~8 years, 9~12 years and more than 13 years continuous cropping, decreased by 40.2%, 46.7%, 52.4%,respectively.After more than 5 years continuous cropping, the structure of the soil microbial community transformed from rich nutrition bacteria type to lower nutrition fungi type, the ratio ofbacteria to fungi and actinomycetes to fungi decreased significantly.The amount of nitrogen physiological communities such as ammonifying bacteria, nitrobacteria and aerobic nitrogen-fixing bacteria decreased, while denitrifying bacteria increased.Moreover, continuous cropping resulted in soil respiration intensity and cellulolytic activity reducing.Contrary to continuous cropping, under the cotton/melilotus suavena, tomato, spring wheat or com rotation systems were most beneficial for increasing the total quantity of soil micro-organism, improving the capability of soil microbial activity, adjusting the balance of microbial community.Also there was substantial increasement in the number of azotobacteria.The effects of different rotation modes were different, the benefits of cotton-tomato and cotton-melilotus suavena rotation were more obvious.%研究了新疆连作、轮作棉田土壤可培养微生物区系及活性变化.结果表明,棉花多年连作造成土壤中可培养微生物数量减少,连作6~8年、9~12年、大于13年的棉田与连作小于5年的棉田相比,土壤微生物总量分别下降了40.2%,46.7%,52.4%.连作超过5年后,土壤微生物菌群结构逐渐从高肥的"细菌型"向低肥的"真菌型"转化,细菌/真菌(B/F)和放线菌/真菌(A/F)比值均降低,拮抗菌

  7. Cutinase promotes dry esterification of cotton cellulose.

    Science.gov (United States)

    Xiaoman, Zhao; Teresa, Matama; Artur, Ribeiro; Carla, Silva; Jing, Wu; Jiajia, Fu; Artur, Cavaco-Paulo

    2016-01-01

    Cutinase from Thermobifida fusca was used to esterify the hydroxyl groups of cellulose with the fatty acids from triolein. Cutinase and triolein were pre-adsorbed on cotton and the reaction proceeded in a dry state during 48 h at 35°C. The cutinase-catalyzed esterification of the surface of cotton fabric resulted in the linkage of the oleate groups to the glycoside units of cotton cellulose. The superficial modification was confirmed by performing ATR-FTIR on treated cotton samples and by MALDI-TOF analysis of the liquors from the treatment of the esterified cotton with a crude cellulase mixture. Modified cotton fabric also showed a significant increase of hydrophobicity. This work proposes a novel bio-based approach to obtain hydrophobic cotton.

  8. China's Cotton Policy and the Impact of China's WTO Accession and Bt Cotton Adoption on the Chinese and U.S. Cotton Sectors

    OpenAIRE

    Cheng Fang; Bruce A. Babcock

    2003-01-01

    In this paper we provide an analysis of China's cotton policy and develop a framework to quantify the impact of both China's World Trade Organization (WTO) accession and Bt (Bacillus thuringiensis) cotton adoption on Chinese and U.S. cotton sectors. We use a Chinese cotton sector model consisting of supply, demand, price linkages, and textiles output equations. A two-stage framework model provides gross cropping area and total area for cotton and major subsitute crops from nine cotton-produci...

  9. L-Phenylalanine ammonia-lyase from Phaseolus vulgaris. Characterisation and differential induction of multiple forms from elicitor-treated cell suspension cultures.

    Science.gov (United States)

    Bolwell, G P; Bell, J N; Cramer, C L; Schuch, W; Lamb, C J; Dixon, R A

    1985-06-03

    L-Phenylalanine ammonia-lyase (EC 4.3.1.5) has been purified over 200-fold from cell cultures of bean (phaseolus vulgaris L.) exposed to elicitor heat-released from the cell walls of the phytopathogenic fungus Colletotrichum lindemuthianum. Four forms of the enzyme, with identical Mr but differing apparent pI values of 5.4, 5.2, 5.05 and 4.85, were observed following the final chromatofocussing stage of the purification. A preparation (purified 43-fold by ammonium sulphate precipitation, gel-filtration and ion-exchange chromatography) containing all four forms exhibited apparent negative rate cooperativity with respect to substrates. However, the individual forms displayed normal Michaelis-Menten kinetics, with Km values of 0.077 mM, 0.122 mM, 0.256 mM and 0.302 mM in order of decreasing apparent pI value. A preparation purified 200-fold and containing all four forms was used to immunise rabbits for the production of anti-(phenylalanine ammonia-lyase) serum. The antiserum was characterised by: immunotitration experiments; solid phase enzyme-linked immunosorbent assays; comparison of immunoprecipitates of 35S-labelled phenylalanine ammonia-lyase subunits (synthesized both in vivo and in vitro) on both one-dimensional and two-dimensional polyacrylamide gels after immunoprecipitation with the bean antiserum or antisera raised against pea and parsley phenylalanine ammonia-lyase preparations and immune blotting. SDS/polyacrylamide gels and SDS/polyacrylamide gel electrophoresis followed by immune blotting, indicated that the Mr of newly synthesized (in vivo and in vitro) bean phenylalanine ammonia-lyase subunits is 77000; a 70000-Mr form is readily generated as a partial degradation product during purification. Immunoprecipitates of bean phenylalanine ammonia-lyase synthesized both in vivo and in vitro showed the presence of multiple subunit types of identical Mr but differing in pI. Furthermore, treatment of bean cultures with Colletotrichum elicitor resulted in a 10

  10. A comparison between Pseudomonas aureofaciens (chlororaphis and P. fluorescens in biological control of cotton seedling damping-off disease

    Directory of Open Access Journals (Sweden)

    Samaneh Samavat

    2014-07-01

    Full Text Available Due to the importance of the biological control of plant diseases, testing and introducing new biocontrol-active microorganisms is a major concern among plant pathologists. The causal agent of cotton seedling damping-off disease is Rhizoctonia solani. In this regard, we tried to investigate the antagonistic activities of Pseudomonas aureofaciens (chlororaphis 30–84 (phenazine producing wild type and non-phenazine producing mutant strains on R. solani, in comparison with some isolates of P. fluorescent under both in vitro (laboratory and in vivo (greenhouse conditions. In the laboratory experiment, the inhibitory effects of all the bacteria, on the growth of R. solani, were evaluated using the dual culture procedure. Results showed that five isolates of P. fluorescent along with both strains of P. aureofaciens significantly inhibited the growth of R. solani. Effective bacterial antagonists were then evaluated in a greenhouse experiment where cotton seeds were coated with their suspensions and were sown in pasteurised field-soil. The soil had been pre-inoculated with a virulent isolate of R. solani. The efficacy of the bacterial antagonists was evaluated by counting the number of surviving seedlings in different treatments, at 15 and 60 days after sowing, for determining pre- and post-emergence damping-off incidence. According to the results of the greenhouse experiment, at both intervals, two isolates of P. fluorescens along with both strains of P. aureofaciens caused significant increases in the number of healthy seedlings, in comparison with the untreated control, and a commonly used fungicide (carboxin-thiram. The efficacy of phenazine producing a wild type strain of P. aureofaciens was higher than its non-phenazine producing mutant, indicating that phenazine plays an important role in the antagonistic activity of P. aureofaciens. Effective bacterial antagonists were then studied for their antagonistic mechanisms. The results showed that all

  11. 7 CFR 1427.165 - Eligible seed cotton.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 10 2010-01-01 2010-01-01 false Eligible seed cotton. 1427.165 Section 1427.165... OF AGRICULTURE LOANS, PURCHASES, AND OTHER OPERATIONS COTTON Recourse Seed Cotton Loans § 1427.165 Eligible seed cotton. (a) Seed cotton pledged as collateral for a loan must be tendered to CCC by...

  12. 6-Benzyladenine enhancement of cotton

    Science.gov (United States)

    The influence of applied plant growth regulators (PGR) on growth, development and yield in cotton (Gossypium hirsutum L. and Gossypium barbadense L.) has been studied for over half a century. Studies of PGR containing cytokinin alone or in combination with gibbererillins applied at the pinhead squa...

  13. Transgene Stacking in Cotton Improvement

    Institute of Scientific and Technical Information of China (English)

    YANG Ye-hua; WANG Xue-kui; YAO Ming-jing; FAN Yu-peng; GAO Da-yu

    2008-01-01

    @@ To date,more and more transgenic varieties of upland cotton (Gossypium hirsuturn L.) generated with transgenes,which derived from varies of alien species,are playing important role in agricultural production.Stacking of multi-transgenes has a potential for combining all the merits of distinct transgenic lines in a cultivar and possibly makes a significant contribution to cultivar improvement.

  14. Cocoa/Cotton Comparative Genomics

    Science.gov (United States)

    With genome sequence from two members of the Malvaceae family recently made available, we are exploring syntenic relationships, gene content, and evolutionary trajectories between the cacao and cotton genomes. An assembly of cacao (Theobroma cacao) using Illumina and 454 sequence technology yielded ...

  15. Anthraquinone dyes for superhydrophobic cotton.

    Science.gov (United States)

    Salabert, J; Sebastián, R M; Vallribera, A

    2015-09-28

    Water-repellent, self-cleaning and stain resistant textiles are of interest for industrial applications. Anthraquinone reactive dyes were covalently grafted onto cotton fabric surfaces obtaining bright colors with good wash-fastness properties and giving rise to breathable superhydrophobic textiles with self-cleaning properties.

  16. Transgene Stacking in Cotton Improvement

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    To date,more and more transgenic varieties of upland cotton(Gossypium hirsutum L.) generated with transgenes,which derived from varies of alien species,are playing important role in agricultural production.Stacking of multi-transgenes has a potential for combining all the merits of distinct

  17. Future of Cotton in Nonwovens

    Science.gov (United States)

    Although cotton offers several positive attributes, such as absorbency of liquids, dyeability, transportation and dissipation of moisture for wear comfort, static-freedom, sustainability, biodegradability and bioconsumability, and the like, its use in nonwoven products has been minimal. In order to ...

  18. Cottonseed and cotton plant biomass

    Science.gov (United States)

    The cotton plant generates several marketable products as a result of the ginning process. The product that garners the most attention in regards to value and research efforts, is lint with cottonseed being secondary. In addition to lint and cottonseed, the plant material itself has a value that...

  19. Primary Studies on Cotton Telomere

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The Arabidopsis-type telomere sequence was amplified and cloned using the primers designed from the fragment which contained the telomere sequence in an Arabidopsis BAC.In situ hybridizations with cotton metaphase chromosomes,using the telomere as probe,it indicated that the signals

  20. Production of Arbutin through Biotransformation of Exogenous Hydroquinone by Datura stramonium Cell Suspension Cultures%白花曼陀罗细胞悬浮培养生物转化外源氢醌合成熊果苷的研究

    Institute of Scientific and Technical Information of China (English)

    彭春秀; 龚加顺

    2006-01-01

    研究了白花曼陀罗细胞悬浮培养对外源氢醌的糖基化.转化细胞来自白花曼陀罗嫩茎在LS固体培养基上诱导产生的愈伤组织.白花曼陀罗悬浮培养细胞不能分泌熊果苷,但能糖基化外源氢醌合成熊果苷.当氢醌添加量达240 μmol/100mL培养物时,约有93.4%的氢醌转化形成了熊果苷,并应用多种色谱技术进行分离纯化,进行了HPLC分析和结构鉴定.%To investigate the biotransformation of hydroquinone by cell suspension cultures of Datura stramonium. Cultured cells derived from stems of Datura stramonium were maintained in Linsmaiher and Skoog (LS) solid medium. Datura stramonium cells in suspension cultures did not accumulate arbutin (4-hydroxyphenyl-β-D-glucopyranoside) but were able to specifically o-glucosylate exogenous hydroquinone at position 1. In particular, Datura cultures glucosylated ca 93.4% of hydroquinone (240 μmol/100 mL cultures) within 8 days after hydroquinone administered. The arbutin obtained was extracted from the cultures and further purified by silicon Gel column chromatography. The exogenous hydroquinone and arbutin were analyzed by HPLC.

  1. Effect of microbial pretreatment on enzymatic hydrolysis and fermentation of cotton stalks for ethanol production

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Jian; Sharma-Shivappa, Ratna R.; Chinn, Mari [Department of Biological and Agricultural Engineering, Campus Box 7625, North Carolina State University, Raleigh, NC 27695-7625 (United States); Howell, Noura [North Carolina School of Science and Mathematics, Durham, NC 27715 (United States)

    2009-01-15

    The potential of microbial pretreatment of cotton stalks by Phanerochaete chrysosporium to degrade lignin and facilitate fuel ethanol production was investigated under two culture conditions: submerged cultivation (SmC) and solid state (SSC) cultivation. Although microbial pretreatments showed significant lignin degradation (LD) (19.38% and 35.53% for SmC and SSC, respectively), a study on hydrolysis and fermentation of the microbial-pretreated cotton stalks showed no increase in cellulose conversion (10.98% and 3.04% for SmC and SSC pretreated samples, respectively) compared to untreated cotton stalks (17.93%). Solid state cultivation demonstrated better selectivity of 0.82 than 0.70 with submerged pretreatment. Washing of pretreated cotton stalks did not significantly increase cellulose conversion. However, heating and washing remarkably improved (P<0.05) cellulose conversion to 14.94% and 17.81% for SmC and SSC 14 day pretreatment, respectively. Ethanol yields, up to 0.027 g ethanol g{sup -1} initial cotton stalks, were low for all untreated and pretreated samples mainly due to the low cellulose conversion. Although potential and some critical aspects of fungal pretreatment using P. chrysosporium have been explored in this study, additional investigation is still required especially to improve the selectivity for preferential LD and to optimize hydrolysis efficiency. The mechanism of catalytic binding of cellulolytic enzymes to cotton stalks as affected by the presence of fungal mycelia also warrants further study. (author)

  2. Gibberellin overproduction promotes sucrose synthase expression and secondary cell wall deposition in cotton fibers.

    Directory of Open Access Journals (Sweden)

    Wen-Qin Bai

    Full Text Available Bioactive gibberellins (GAs comprise an important class of natural plant growth regulators and play essential roles in cotton fiber development. To date, the molecular base of GAs' functions in fiber development is largely unclear. To address this question, the endogenous bioactive GA levels in cotton developing fibers were elevated by specifically up-regulating GA 20-oxidase and suppressing GA 2-oxidase via transgenic methods. Higher GA levels in transgenic cotton fibers significantly increased micronaire values, 1000-fiber weight, cell wall thickness and cellulose contents of mature fibers. Quantitative RT-PCR and biochemical analysis revealed that the transcription of sucrose synthase gene GhSusA1 and sucrose synthase activities were significantly enhanced in GA overproducing transgenic fibers, compared to the wild-type cotton. In addition, exogenous application of bioactive GA could promote GhSusA1 expression in cultured fibers, as well as in cotton hypocotyls. Our results suggested that bioactive GAs promoted secondary cell wall deposition in cotton fibers by enhancing sucrose synthase expression.

  3. Optimization and influencing factor of electroporation parameters for cells cultured in suspension%悬浮细胞电转染条件的优化及影响因素

    Institute of Scientific and Technical Information of China (English)

    龙潺; 唐雪元

    2008-01-01

    Objective To optimize the electroporation parameters in leukemia cell lines cultured in suspension using green fluorescent protein(GFP)as a reporter gene.Methods The GFP plasmid was transferred into leukemia cell lines K562 by electroporation using differently experimental conditions such as the voltage(200-400 V),the electric capacity(450-1200μF),the state of cell vitality and the serum concentration with buffer solution(0,10%,15%).The electroporation efficiency was evaluated bv flow cytometry and fluorescent microscopy.Results The highest electropration efficiency with leukemia cell lines k562 cultured in suspension was obtained under the condition of voltage 3 10 V,electric eapacity of 1050μF(for pEGFP-C2/K562,67.04%;for pEGFP-C2/BRD7/K562,59.29%).The electmporation efficieney for the cells in logarithmic growth phase Was increaSed highly.The seruln concentration in buffer solution was not related with the electropration efficiency.Conclusion Electroporation is a method with high efficiency to gene transfeetion,and optimizing eleetroporation parameters and controlling the related factors can increase the eleetmporation efficiency.%目的 以绿色荧光蛋白(GFP)为报告基因,优化悬浮培养的白血病细胞的电穿孔转染条件.方法 通过控制电压(200~400 V)、电容(450~1200μF)、细胞状态及缓冲液血清浓度(0%、10%、15%)等转染条件,采用不同条件组合后用电穿孔法将质粒转入悬浮培养的人白血病细胞株K562,通过流式细胞仪和荧光显微镜分析转染率.结果 K562细胞在310 V、1050μF条件下转染率最高,pEGFP-C2/K562为67.04%,pEGFP-C2/BRD7/K562为59.29%.对数生长期细胞电转染率高于生长过老期细胞;而缓冲液中的血清浓度与电转染率无关.结论 电穿孔是一种高效的基因转染法,通过优化转染条件、控制影响因素,可提高转染率.

  4. Analysis of the Cotton E6 Promoter

    Institute of Scientific and Technical Information of China (English)

    WU Aimin; LIU Jinyuan

    2005-01-01

    An E6 gene from sea island cotton (Gossypium barbadense) was expressed specifically in cotton fiber cells to transfer functions to cultivated species for better transgenic engineering. The regulatory activity of the E6 promoter region was then studied by isolating a 614-bp fragment of the 5'-flanking region from upland cotton (Gossypium hirsutum CRI-12) to produce a green fluorescent protein (GFP) reporter construct for analysis of tissue-specific expression in transgenic tobacco seedlings. Fluorescent analyses indicate that the relatively short E6 promoter is sufficient to direct green fluorescent protein expression specifically in the leaf trichomes (hair cells) of the transgenic tobacco plants. As cotton fibers are also unicellular trichomes that differentiate from epidermal cells of developing cotton ovules, the result suggests that the relatively short E6 promoter can serve as a fiber-specific expression promoter for genetic engineering to improve cotton fiber quality.

  5. Digieye Application In Cotton Colour Measurement

    Directory of Open Access Journals (Sweden)

    Matusiak Małgorzata

    2015-06-01

    Full Text Available Colour is one of the most important properties of cotton raw materials. It helps in determining and classifying the quality of fibres according to the Universal Cotton Standards. Organoleptic and instrumental techniques are applied to assess the color of cotton. Worldwide, the colour parameters of cotton are measured by the High Volume Instrument (HVI, which provides information on reflectance (Rd and yellowness (+b that is specific for cotton, but are not the typical and globally recognized colour characteristics. Usually, worldwide, the colour of textile products and other goods is assessed utilizing the spectrophotometer, which provides the colour data that is widely recognized and accepted by the CIE L*a*b* colour space. This paper discusses utilizing the DigiEye system to measure the colour parameters of cotton samples and compares the results with the colour parameters from the HVI.

  6. 75 FR 50847 - Cotton Program Changes for Upland Cotton, Adjusted World Price, and Active Shipping Orders

    Science.gov (United States)

    2010-08-18

    ..., paper, or non-woven cotton fabric, the payment will be calculated on 25 percent of the weight (gross... further processing, for spinning, papermaking, or manufacture of non-woven cotton fabric, 25 percent of... definitions from the regulations for cotton non-recourse loans and loan deficiency payments. It clarifies...

  7. Cotton in Benin: governance and pest management

    OpenAIRE

    Togbe, C.E.

    2013-01-01

    Key words: cotton, synthetic pesticides, neem oil (Azadirachta indica), Beauveria bassiana, Bacillus thuringiensis, field experiment, farmers’ participation   Pests are one of the main factors limiting cotton production worldwide. Most of the pest control strategies in cotton production rely heavily on the application of synthetic pesticides. The recurrent use of synthetic pesticides has large consequences for the environment (air, water, fauna, and flora) and human health. In cott...

  8. Cotton dust-mediated lung epithelial injury.

    OpenAIRE

    Ayars, G H; Altman, L C; O'Neil, C E; Butcher, B T; Chi, E Y

    1986-01-01

    To determine if constituents of cotton plants might play a role in byssinosis by injuring pulmonary epithelium, we added extracts of cotton dust, green bract, and field-dried bract to human A549 and rat type II pneumocytes. Injury was measured as pneumocyte lysis and detachment, and inhibition of protein synthesis. Extracts of cotton dust and field-dried bract produced significant dose- and time-dependent lysis and detachment of both target cells, while green bract extract was less damaging. ...

  9. Enrichment of Breast Cancer Stem Cells by Serum-free Shaking Suspension Culture%无血清摇动悬浮培养富集乳腺癌干细胞

    Institute of Scientific and Technical Information of China (English)

    闫文星; 陈玉丙; 张红梅; 国春龙; 王铁君

    2011-01-01

    Objective To investigate the characteristics of division of breast cancer MCF-7 cells under various conditions so as to develop a rapid and effective method for enrichment of breast cancer stem cells. Methods MCF-7 cells were subjected to static culture in complete medium (group A), shaking culture in complete medium (group B), static culture with cytokine (group C) and shaking culture with cytokine (group D) respectively, for 12, 24 and 36 h, then observed for division under inverted phase contrast microscope, based on which clone formation rate was calculated, and determined for the percentage of CD44+/CD24"/low lymphocyte subsets. Results In groups B and C, rod-like divisions were observed in both about 30% of cells 12 h, and in about 50% and about 60% of cells respectively 24 h, while large cell clones were formed 36 h after culture. However, in group D, rod-like division was observed in about 50% of cells 12 h, and in about 80% of cells 24 h when several cell clones appeared, while the number of cell clones decreased 36 h after culture. The percentage of CD44+/CD24"/km lymphocyte subsets 12 h after culture in group D (8. 05%) was 8 times of those in group B (0. 99%) and 2. 1 times of those in group C (3. 80%). However, the percentage 24 h after culture in group D (15. 24%) was 3 times of those in group B (4. 83%) and 6 times of those in group C (2. 30%). The percentage in group D decreased to 9. 68% 36 h after culture, which was about 9 times of those in groups B (0. 95%) and C (1. 03%). Conclusion The mitotic division of MCF-7 cells was accelerated in shaking culture with cytokine, while the percentage of CD44+/CD24'/low lymphocyte subsets increased rapidly, and stem cell pool increased significantly, indicating serum-free shaking suspension culture a rapid and effective method for enrichment of breast cancer stem cells.%目的 探讨不同培养条件下乳腺癌MCF-7细胞的分裂特点,建立快速、有效的乳腺癌干细胞富集方法.方法 将MCF-7

  10. Light-induced enzyme synthesis in cell suspension cultures of Petroselinum hortense. Demonstration in a heterologous cell-free system of rapid changes in the rate of phenylalanine ammonia-lyase synthesis.

    Science.gov (United States)

    Schröder, J; Betz, B; Hahlbrock, K

    1976-08-16

    The conditions for protein synthesis in vitro with polyribosomes from cell suspension cultures of parsel (Petroselinum hortense) and a wheat-germ extract were investigated. Two different criteria were used as estimated of the translational activity: (a) the total rate of incorporation of [35S]methionine into acid-insoluble material; (b) the ratio of large (molecular weight greater than 25000) to small (molecular weight less than 25000) peptide products. Depending on which of the criteria was employed, the pH optimum and the optimal concentrations for Tris=acetate, magnesium acetate, KCL, methionine and the wheat-germ extract differed considerably. The translational activity of the polyribosomes (both criteria) was effciently protected by 0.1 M Mg2+ against degradation during the isolation procedure. The rate of synthesis of phenylalanine ammonia-lyase in vitro with the polyribosomes was determined by measuring the incorporation rate of L-[35S]methionine into protein which was precipitable by a rabbit antiserum prepared for the purified enzyme. The immunoprecipitate was analyzed by disc gel electrophoresis in the presence of dodecylsulfate and was shown to contain small amounts of the complete enzyme subunits and relatively large amounts of shorter peptides which were also characteristic for the enzyme. The time course of light-induced changes in the rate of phenylalanine ammonia-lyase synthesis in vitro were investigated during a period of 15 h under two different conditions of induction: the cell cultures were irradiated with ultraviolet light eith (A) continuously or (B) for 2.5 h and then returned to darkness. Although the highest rate of enzyme synthesis was observed somewhat later inexperiment A than in experiment B, the periods of time during which the rate of synthesis increased rapidly were limited in both cases to only a few hours. The results obtained in vitro were identical within the limits of the experimental error with theoretical calculations of the

  11. Rheology of organoclay suspension

    CSIR Research Space (South Africa)

    Hato, MJ

    2011-05-01

    Full Text Available -1 Colloid & Polymer Science Volume 289, Number 10, 1119-1125, DOI: 10.1007/s00396-011-2438-4 Rheology of organoclay suspension Mpitloane Joseph Hato, Ke Zhang, Suprakas Sinha Ray and Hyoung Jin Choi Abstract We have studied the rheological...

  12. Alternatives to Student Suspension

    Science.gov (United States)

    Robinett, David

    2012-01-01

    Seven years ago, James A. Garfield High School in East Los Angeles set a school record with 613 student suspensions, out of a total enrollment of 5,000 students. The school, made famous by the 1988 film "Stand and Deliver", was no stranger to the high rates of student discipline all too common within the Los Angeles Unified School…

  13. Viscosity of colloidal suspensions

    Energy Technology Data Exchange (ETDEWEB)

    Cohen, E.G.D. [Rockefeller Univ., New York, NY (United States); Schepper, I.M. de [Delft Univ. of Technology (Netherlands)

    1995-12-31

    Simple expressions are given for the effective Newtonian viscosity as a function of concentration as well as for the effective visco-elastic response as a function of concentration and imposed frequency, of monodisperse neutral colloidal suspensions over the entire fluid range. The basic physical mechanisms underlying these formulae are discussed. The agreement with existing experiments is very good.

  14. Aqueous supercapacitors on conductive cotton

    KAUST Repository

    Pasta, Mauro

    2010-06-01

    Wearable electronics offer the combined advantages of both electronics and fabrics. In this article, we report the fabrication of wearable supercapacitors using cotton fabric as an essential component. Carbon nanotubes are conformally coated onto the cotton fibers, leading to a highly electrically conductive interconnecting network. The porous carbon nanotube coating functions as both active material and current collector in the supercapacitor. Aqueous lithium sulfate is used as the electrolyte in the devices, because it presents no safety concerns for human use. The supercapacitor shows high specific capacitance (~70-80 F·g-1 at 0.1 A·g-1) and cycling stability (negligible decay after 35,000 cycles). The extremely simple design and fabrication process make it applicable for providing power in practical electronic devices. © 2010 Tsinghua University Press and Springer-Verlag Berlin Heidelberg.

  15. QTL Analysis in Tetraploid Cotton

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    QTL analyses were performed in tetraploid cotton.An interspecific F2 population consisting of 69 plants,which was developed from the cross between Gossypium hirsutum L.,cv.Handan 208(characterized as high fiber yield) and G.barbadense L.,cv.Pima 90(characterized as excellent fiber quality),was genotyped with SSR,RAPD,SRAP,and REMAP markers.A 1029-locus linkage map was

  16. Cryonic Suspension and the Law.

    Science.gov (United States)

    Smith, George P.; Hall, Clare

    1987-01-01

    Analyzes three central problems which adversely affect use, development, and perfection of cryonic suspension of individuals: the extent to which a physician may be guilty of malpractice in assisting with a suspension; the need for a recognition of suspension; and the present effect of the law's anachronistic treatment of estate devolution upon a…

  17. Shandong’s Cotton Brocade

    Institute of Scientific and Technical Information of China (English)

    1997-01-01

    SHANDONG Province, also called "Lu," produces traditional hand-woven cotton fabric known as "Lu Jin ("Jin" means brocade in Chinese). Lu Jin has a soft texture and is made in various designs and colors. Although machine-made cotton fabric is easy to buy here, local people, particularly women, prefer this kind of cloth woven in the old style handed down by their ancestors. In the countryside of Southwest Shandong, a girl usually begins learning how to weave cotton brocade as a child and old women are often still busy at the loom. In Jiaxiang County, for example, there are more than 10,400 looms, 74,000 spinning wheels and 90,000 capable weavers, producing 6 million meters of hand-woven fabric annually. Lu Jin is a suitable dowry for local girls. Usually, a girl begins selecting designs and weaving for her dowry two to three years before marriage. When she gets married, she carefully puts the fabric in the cupboards she will bring with

  18. INDUCING RESISTANCE IN COTTON AGAINST COLLETOTRICHUM GOSSYPII VAR. CEPHALOSPORIOIDES WITH ESSENTIAL OILS

    Directory of Open Access Journals (Sweden)

    B. T. Santos

    2016-11-01

    Full Text Available This study aimed to evaluate the potential of essential oils of rosemary (Rosmarinus officinalis, baccharis (Baccharis trimera, lemon grass (Cymbopogon citratus, basil (Ocimum basilicum and eucalyptus (Corymbia citriodora in inducing resistance in cotton plants against C. gossypii var. cephalosporioides. The inductive effect of the essential oils was evaluated in plants growing in pots in the environment, which were treated with 1% essential oil at 47 days of age. 24 hours after elicitor treatment the plants were inoculated with a suspension of 1.5 x 105 conidia mL-1 of C. gossypii var. cephalosporioides. Five evaluations were performed disease and calculated the area under the disease progress curve. All essential oils showed potential for inducing resistance against cotton C. gossypii var. cephalosporioides.

  19. Endothelial cell cytotoxicity of cotton bracts tannin and aqueous cotton bracts extract

    Energy Technology Data Exchange (ETDEWEB)

    Johnson, C.M.; Hanson, M.N.; Rohrbach, M.S.

    1986-04-01

    Using an in vitro cytotoxicity assay based on the release of /sup 51/Cr from cultured porcine thoracic aortic and pulmonary arterial endothelial cells, we have demonstrated that cotton bracts tannin is a potent endothelial cell cytotoxin. It produces dose-dependent lethal injury to both types of endothelial cells with the aortic cells, being somewhat more sensitive to tannin-mediated injury than the pulmonary arterial cells. Cytotoxic injury to the cells was biphasic. During the first 3 hr of exposure to tannin, no lethal injury was detected. However, during this period, profound changes in morphology were observed suggesting sublethal injury to the cells preceded the ultimate toxic damage. Comparison of the cytotoxicity dose curves for aqueous bracts extracts with those for tannin demonstrated that tannin was major cytotoxin present in bracts.

  20. 不同继代时间调控棉花愈伤组织褐化死亡和植株再生效率的研究%Different Subculture Time Affects Two Cell Death Peaks in Tissue Culture and Plantlet Regeneration in Cotton

    Institute of Scientific and Technical Information of China (English)

    罗晓丽; 王志安; 肖娟丽; 张安红; 吴家和

    2013-01-01

      The browning and death of calli would reduce the efficiency of cotton plant regeneration and genetic transformation,and it was pivotal to inhibit the two peaks of browning and death of calli during cotton tissue culture to improve plant regeneration and genetic transformation efficiency. In this study, we analyzed subculture time, browning death degree, regeneration time and regeneration efficiency of calli of " Jihe 713" and " Coker 312" cultivars. The results showed that subculture time was able to regulate two peaks of browning and death of calli. To take an extended subculture time during the first PCD peak, subculture time interval 50 d can significantly reduce callus browning and death, increase the rate of callus embryogenesis and shorten the time of embryogenesis. To Take a shortened time of subculture during the second PCD peak, subculture interval 15d can significantly reduce the degree of browning and death, thereby increasing the plant regeneration rate and reducing the regeneration time of the plants. Regulation browning and death by callus subculture time will significantly improve the efficiency of genetic transformation in two cotton varieties, so as to help the gene function and genomics research in cotton.%  棉花组织培养中愈伤组织褐化常常严重影响植株再生的效率,如何解决棉花组织培养过程中愈伤组织和胚性愈伤组织首次继代的两次褐化死亡高峰,是关系到是否能够大幅度提高棉花植株再生和遗传转化效率的关键因素之一。本研究分析了冀合713和珂字312两品种愈伤组织的继代时间与褐化死亡程度、植株再生时间和再生率,结果表明继代时间对棉花愈伤组织两次褐化死亡高峰存在着显著调控作用。在第一次高峰发生时延长继代时间间隔到50 d,可以显著降低愈伤组织继续褐化死亡,提高胚性愈伤组织的发生率和缩短胚胎发生时间。在第二次高峰发生时

  1. Caging antimicrobial silver nanoparticles inside cotton

    Science.gov (United States)

    In this study, a stable, non-leaching Ag-cotton nanocomposite fiber has been characterized. Siver nanoparticles (Ag NPs) were previously synthesized in the alkali-swollen substructure of cotton fiber; the nano-sized micofibrillar channels allowed diffusion-controlled conditions to produce mono-dispe...

  2. 6-Benzyladenine enhancements of cotton yield

    Science.gov (United States)

    The influence of applied plant growth regulators (PGR) on growth, development and yield in cotton (Gossypium hirsutum L. and Gossypium barbadense L.) has been studied for over half a century. A recent study suggested that cytokinin treatment of young cotton seedlings may enhance overall performanc...

  3. 29 CFR 1910.1043 - Cotton dust.

    Science.gov (United States)

    2010-07-01

    ... of the instrument must have a means of correcting volumes to body temperature saturated with water... 29 Labor 6 2010-07-01 2010-07-01 false Cotton dust. 1910.1043 Section 1910.1043 Labor Regulations...), U.S. Department of Health and Human Services, or designee. Equivalent Instrument means a cotton dust...

  4. Exploring Modifications of Cotton with Biopolymers

    Science.gov (United States)

    Biopolymers including starch, alginate, and chitosan were grafted on to both nonwoven and woven cotton fabrics to examine their hemostatic and antimcrobial properties. The development of cotton-based health care fabrics that promote blood clotting and prevent microbial growth have wide applicability...

  5. China Cotton Situation Report [June 2007

    Institute of Scientific and Technical Information of China (English)

    James H. Zhao

    2007-01-01

    @@ The domestic cotton supply plus import quota released in due time can meet with spinners need in this season as can be assured by the fact that the spring sowing of cotton is finished in May, and summer sowing progresses well on its move.

  6. China International Cotton Conference Concluded in Xinjiang

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The 2007 China International Cotton Conference was held on June 27-29 in Urumqi,Xinjiang Municipality, China.With the theme"China’s Cotton Industry on WTO and It’s Implications The Global Market".the Conference proceeded with three main sessions,one focusing on the

  7. Australia: round module handling and cotton classing

    Science.gov (United States)

    Round modules of seed cotton produced via on-board module building harvesters are the reality of the cotton industry, worldwide. Although round modules have been available to the industry for almost a decade, there is still no consensus on the best method to handle the modules, particularly when th...

  8. The U.S. Cotton Industry.

    Science.gov (United States)

    Starbird, Irving R.; And Others

    This report identifies and describes the structure and performance of the cotton industry, emphasizing the production and marketing of raw cotton. The underlying economic and political forces causing change in the various segments of the industry are also explored. The report provides a single source of economic and statistical information on…

  9. Design of starch coated seed cotton dryers

    Science.gov (United States)

    A model was developed for the design and analysis of a high temperature tunnel dryer, primarily used with a new cotton ginning product, EASIflo ® cottonseed (starch-coated cottonseed). This form of cottonseed has emerged as a viable, value-added product for the cotton ginning industry. Currently, li...

  10. Scouring Process of Natural Color Cotton Products

    Institute of Scientific and Technical Information of China (English)

    XU Wei

    2002-01-01

    In order to improve the absorbency of color cotton products, alkali and pectase scouring processes under different conditions were tested, by comparing the actual results of two different scouring processes. It was considered that the pectase scouring process more suits color cotton products.

  11. Palmer amaranth competition for water in cotton

    Science.gov (United States)

    Palmer amaranth is a troublesome weed in cotton production. Yield losses of 65% have been reported due to season-long Palmer amaranth competition with cotton. To determine if water is a factor in this system, experiments were conducted in 2011, 2012, and 2013 in Citra, FL and in Tifton, GA. In 2011,...

  12. Import and Export for Cotton Textile Shrinking

    Institute of Scientific and Technical Information of China (English)

    2011-01-01

    Recently, the National Development and Reform Committee held a meeting to discuss the preparation work of this year's new cotton storage. The meeting declared clearly the policy for this year's new cotton store up, namely starting from September 1, at the fixed price of CNY 19800 per ton, making the purchase without limitation.

  13. Flame retardant cotton based highloft nonwovens

    Science.gov (United States)

    Flame retardancy has been a serious bottleneck to develop cotton blended very high specific volume bulky High loft fabrics. Alternately, newer approach to produce flame retardant cotton blended High loft fabrics must be employed that retain soft feel characteristics desirable of furnishings. Hence, ...

  14. Antibacterial flame retardant cotton high loft nonwovens

    Science.gov (United States)

    Renewable resources for raw materials and biodegradability of the product at the end of the useful life is entailing a shift from petroleum-based synthetics to agro based natural fibers such as cotton, especially for producing high specific volume high loft nonwovens. Cotton is highly flammable and ...

  15. 77 FR 19925 - Upland Cotton Base Quality

    Science.gov (United States)

    2012-04-03

    ... Commodity Credit Corporation (CCC) upland cotton marketing assistance loan (MAL) regulations to revise... creates technical problems if the loan schedules and base grade specifications are changed. CCC... cotton industry to the USDA Agricultural Marketing Service (AMS). AMS can and does change...

  16. Spectroscopic discernment of seed cotton trash

    Science.gov (United States)

    Detection and identification of foreign material in harvested seed cotton is required for efficient removal by ginning. Trash particles remaining within the cotton fibers can detrimentally impact the quality of resulting textile products. Luminescence has been investigated as a potential tool for su...

  17. Milkweed, stink bugs, and Georgia cotton

    Science.gov (United States)

    In peanut-cotton farmscapes in Georgia, stink bugs, i.e., Nezara viridula (L.)(Say) and Chinavia hilaris (Say), develop in peanut and then disperse at the crop-to-crop interface to feed on fruit in cotton. The main objective of this study was to examine the influence of a habitat of tropical milkwe...

  18. Proteomics Study of Cotton Fiber Cells

    Institute of Scientific and Technical Information of China (English)

    LIU Jin-yuan

    2008-01-01

    @@ A comparative proteomic analysis was applied to explore the mechanism of fiber cell development in cotton.Initially,an efficient protein preparation method was established for proteomic analysis of developing cotton fibers by two-dimensional gel electrophoresis,and a microwave enhanced ink staining technique also was created for fast and sensitive protein quantification in proteomic studies.

  19. cultural

    Directory of Open Access Journals (Sweden)

    Irene Kreutz

    2006-01-01

    Full Text Available Es un estudio cualitativo que adoptó como referencial teorico-motodológico la antropología y la etnografía. Presenta las experiencias vivenciadas por mujeres de una comunidad en el proceso salud-enfermedad, con el objetivo de comprender los determinantes sócio-culturales e históricos de las prácticas de prevención y tratamiento adoptados por el grupo cultural por medio de la entrevista semi-estructurada. Los temas que emergieron fueron: la relación entre la alimentación y lo proceso salud-enfermedad, las relaciones con el sistema de salud oficial y el proceso salud-enfermedad y lo sobrenatural. Los dados revelaron que los moradores de la comunidad investigada tienen un modo particular de explicar sus procedimientos terapéuticos. Consideramos que es papel de los profesionales de la salud en sus prácticas, la adopción de abordajes o enfoques que consideren al individuo en su dimensión sócio-cultural e histórica, considerando la enorme diversidad cultural en nuestro país.

  20. Plasmodesmata in Arabidopsis thaliana suspension cells.

    Science.gov (United States)

    Bayer, E; Thomas, C L; Maule, A J

    2004-06-01

    A current challenge in plant biology is to identify the structural and functional components of plasmodesmata (PDs). The use of plant tissue as a source material for plasmodesmal characterisation has had limited success, so we have explored the frequency and features of PDs occurring in suspension cell cultures of Arabidopsis thaliana. This material has the advantages of homogeneity, quantity, and ease of disruption. Using light and electron microscopy and immunostaining for callose and calreticulin, we showed that suspension cells laid down abundant PDs in division walls, and that vestiges of these structures were retained as half PDs even when the cell-to-cell contacts were disrupted during culture growth. Although callose was a reliable marker for PD distribution, which was deposited in an organised collar around the neck of PDs, it was not abundant in unstressed cells. Calreticulin and the chemical stain 3,3'-dihexyloxacarbocyanine iodide also provided useful markers when monitoring PDs in cell wall preparations by light microscopy. Purified cell walls were shown to be virtually free of contamination from cytoplasmic components, except for the presence of small amounts of cortical endoplasmic reticulum attached to PDs. Hence, clean cell walls from A. thaliana suspension cells provide a valuable resource for a proteomic approach to the analysis of plasmodesmal components.