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Sample records for tissue culture plantlets

  1. Organ and plantlet regeneration of Menyanthes trifoliata through tissue culture

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    Urszula Adamczyk-Rogozińska

    2014-01-01

    Full Text Available The conditions for the regeneration of plants through organogenesis from callus tissues of Menyanthes trifoliata are described. The shoot multiplication rate was affected by basal culture media, the type and concentration of cytokinin and subculture number. The best response was obtained when caulogenic calli were cultured on the modified Schenk and Hildebrandt medium (SH-M containing indole-3-acetic acid (IAA 0,5 mg/l and 6-benzyladenine (BA 1 mg/l or zeatin (2 mg/l. Under these conditions ca 7 shoots (mostly 1 cm or more in length per culture in the 5th and 6th passages could be developed. In older cultures (after 11-12 passages there was a trend for more numerous but shorter shoot formation. All regenerated shoots could be rooted on the SH-M medium supplemented with 0.5 mg/l IAA within 6 weeks; 80% of in vitro rooted plantlets survived their transfer to soil.

  2. EX VITRO ROOTING OF OIL PALM (Elaeis guineensis Jacq. PLANTLETS DERIVED FROM TISSUE CULTURE

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    Sumaryono Sumaryono

    2011-10-01

    Full Text Available Plantlets of oil palm (Elaeis guineensis Jacq. derived from so-matic embryos sometimes do not form well developed-roots. Root formation of unrooted-plantlets can be induced with aux-in during ex vitro acclimatization period to simplify the proce-dure and to reduce seedling production cost. Experiments were conducted using a completely randomized design to determine the effect of different types of auxin, i.e. indole-3-acetic acid (IAA, indole-3-butyric acid (IBA, and 1-naphthalene-acetic acid (NAA at different concentrations, i.e. 0, 2, 4, 8, and 16 mM on root development of oil palm plantlets. The plantlets used were derived from somatic embryos of MK 649 oil palm clone. The basal end of the shoots was dipped in auxin solution for 10 minutes before the shoot was cultured in a small plastic pot containing a mixed growing medium. The cultures were then placed inside a closed transparent plastic tunnel (240 cm x 100 cm x 95 cm for 12 weeks. The results showed that without auxin treatment only 15% of the shoots formed roots. Dipping in auxin solution increased significantly root frequen-cy to more than 50%. The best root formation was found on the shoots treated with 2 mM NAA by which rooting frequency was 80%. Auxin treatments also increased root quality as indi-cated by more number of primary and secondary roots. IAA, IBA, and NAA treatments at all concentrations tested increased significantly shoot height on average by 42% and shoot diame-ter by 30% compared to control treatment, but did not influ-ence root length. The best treatment for inducing roots of oil palm plantlets ex vitro was by dipping the basal end of the plant-lets in 2 mM NAA solution. The result showed that rooting of oil palm plantlets could be successfully conducted ex vitro that would eliminate sterile rooting stage thus simplify the protocol and reduce seedling production time and cost.

  3. Effects of Quantum Flux Density on Photosynthesis and Chloroplast Ultrastructure in Tissue-Cultured Plantlets and Seedlings of Liquidambar styraciflua L. towards Improved Acclimatization and Field Survival 1

    Science.gov (United States)

    Lee, Ni; Wetzstein, Hazel Y.; Sommer, Harry E.

    1985-01-01

    Liquidambar styraciflua L. seedlings and tissue-cultured plantlets were grown under high, medium, or low (315, 155, or 50 microeinsteins per square meter per second photosynthetically active radiation) quantum flux densities. Net photosynthesis, chlorophyll content, and chloroplast ultrastructure of leaves differentiated from these conditions were investigated. Seedling photosynthetic rates at light saturation were positively related to light pretreatments, being 6.44, 4.73, and 2.75 milligrams CO2 per square decimeter per hour for high, medium, and low light, respectively. Cultured plantlets under all light conditions had appreciably higher photosynthetic rates than noncultured seedlings; corresponding rates were 12.14, 13.55, and 11.36 milligrams CO2 per square decimeter per hour. Chlorophyll in seedlings and plantlets was significantly higher in low light-treated plants. Seedling leaves had chloroplasts with abundant starch regardless of light pretreatment. In high light, starch granules were predominant and associated with disrupted granal structure. Low light seedling chloroplasts had smaller starch grains and well-formed grana. In contrast, tissue culture-differentiated leaves were devoid of starch; grana were well organized in higher quantum flux density treatments, but disorganized at low flux densities. Images Figs. 2 to 7 PMID:16664297

  4. Comparative analysis of bioactive N-alkylamides produced by tissue culture raised versus field plantlets of Spilanthes ciliata using LC-Q-TOF (HRMS).

    Science.gov (United States)

    Bhat, Zubair Shanib; Jaladi, Neeharika; Khajuria, Ravi Kant; Shah, Zeeshan Hamid; Arumugam, Neelakantan

    2016-04-01

    Spilanthes ciliata (S. ciliata) is a perennial herb of global importance owing to its luscious source of bioactive fatty acid derived amides known as N-alkylamides. It finds application in skin creams, mouth gels and toothpastes. Despite multifaceted applications, a major limitation associated for its commercial application is the scarcity of contamination free plant source, fluctuations in active metabolites due to variation in extraction procedures, and lack of rapid qualitative method for alkylamide profiling. In the current work, attempts were made to 1) optimize conditions for mass propagation of contamination free plants of S. ciliata by tissue culture using leaf discs as explants, 2) establish an optimum extraction ratio of plant/solvent (w/v) for maximum elution of alkylamides and 3) develop a rapid method for qualitative estimation of alkylamide from in vitro raised plants in comparison with that of the field grown counterpart by using LC-Q-TOF (HRMS). To the best of our knowledge, this is the first qualitative report on alkylamide profile of micropropagated whole plant of Spilanthes. The correlation pattern reported in this study may form the basis for using tissue culture raised plantlets of S. ciliata as potential source of bioactive alkylamides on industrial scale. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Comparative Digital Gene Expression Analysis of Tissue-Cultured Plantlets of Highly Resistant and Susceptible Banana Cultivarsin Response to Fusarium oxysporum

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    Yuqing Niu

    2018-01-01

    Full Text Available Banana Fusarium wilt caused by Fusarium oxysporum f. sp. cubense (Foc is one of the most destructive soil-borne diseases. In this study, young tissue-cultured plantlets of banana (Musa spp. AAA cultivars differing in Foc susceptibility were used to reveal their differential responses to this pathogen using digital gene expression (DGE. Data were evaluated by various bioinformatic tools (Venn diagrams, gene ontology (GO annotation and Kyoto encyclopedia of genes and genomes (KEGG pathway analyses and immunofluorescence labelling method to support the identification of gene candidates determining the resistance of banana against Foc. Interestingly, we have identified MaWRKY50 as an important gene involved in both constitutive and induced resistance. We also identified new genes involved in the resistance of banana to Foc, including several other transcription factors (TFs, pathogenesis-related (PR genes and some genes related to the plant cell wall biosynthesis or degradation (e.g., pectinesterases, β-glucosidases, xyloglucan endotransglucosylase/hydrolase and endoglucanase. The resistant banana cultivar shows activation of PR-3 and PR-4 genes as well as formation of different constitutive cell barriers to restrict spreading of the pathogen. These data suggest new mechanisms of banana resistance to Foc.

  6. Enhancement of Diosgenin Production in Plantlet and Cell Cultures ...

    African Journals Online (AJOL)

    Enhancement of Diosgenin Production in Plantlet and Cell Cultures of Dioscorea zingiberensis by Palmarumycin C13 from the Endophytic fungus, Berkleasmium sp. Dzf12. Y Mou, K Zhou, D Xu, R Yu, J Li, C Yin, L Zhou ...

  7. Effect of applied synthetic auxin on root growth in plantlet propagation by cuttage and tissue culture; Sashiki to soshiki baiyo ni okeru gosei auxin rui no shiyo koka

    Energy Technology Data Exchange (ETDEWEB)

    Shoji, K.; Yoshihara, T. [Central Research Institute of Electric Power Industry, Tokyo (Japan)

    1996-12-01

    The effect of synthetic plant hormone 4-C1-IAA and TFIBA on root growth in plantlet propagation was clarified by the cuttage and the issue culture of strawberry seedling production. A periwinkle, vine, and azalea are the effect of 4-C1-IAA on root growth, and a promotion effect was recognized for rooting and root elongation. The concentration of 4-C1-IAA in which the growth promotion effect of a root most appears varies depending on the species of a plant. The concentration of a periwinkle was 20 ppm, and that of an azalea was 2000 ppm. The growth promotion effect of a root in 4-C1-IAA and TFIBA was compared with IBA for an azalea. The result showed that 4-C1-IAA is the same in the effect as IBA and that TFIBA is higher than for IBA. The growth of a vine`s terminal bud was promoted by the effect of TFIBA on root growth, and the callus occurring when IBA was treated was not formed. The rooting of a strawberry was promoted by the effect of TFIBA on root growth. The combined use of TFIBA and BA promotes the growth of a side bud and forms a multi-bud plant. However, rooting was inhibited. The callus caused by the effect of BA on root growth could be suppressed through the combined use with TFIBA. 6 refs., 7 figs., 2 tabs.

  8. Differential alkaloid profile in Uncaria tomentosa micropropagated plantlets and root cultures.

    Science.gov (United States)

    Luna-Palencia, Gabriela R; Huerta-Heredia, Ariana A; Cerda-García-Rojas, Carlos M; Ramos-Valdivia, Ana C

    2013-05-01

    The alkaloids of Uncaria tomentosa micropropagated plantlets and root cultures were isolated and identified by NMR and mass spectrometry. Plantlets yielded pteropodine (1), isopteropodine (2), mitraphylline (3), isomitraphylline (4), uncarine F (5), speciophylline (6), rhynchophylline (7) and isorhynchophylline (8). In plantlets growing under continuous light, tetracyclic alkaloids 7 and 8 decreased from 20 ± 1.8 at 2 months to 2.2 ± 0.33 mg/g dry wt at 6 months, while the pentacyclic alkaloids 1-4 increased from 7.7 ± 1.4 to 15 ± 0.05 mg/g dry wt, supporting their biogenetic conversion. Micropropagated plantlets produced four times more alkaloids (27.6 ± 3.1 mg/g dry wt) than greenhouse plants. Plantlet roots yielded 3, 4, 8 and the glucoindole alkaloids 3α-dihydrocadambine (9) and dolichantoside (10), the last one not previously found in Uncaria.

  9. Large-Scale Plantlet Conversion and Ex Vitro Transplantation Efficiency of Siberian Ginseng by Bioreactor Culture

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    Jingli Yang

    2013-01-01

    Full Text Available To achieve large-scale low-cost ex vitro acclimatization of Siberian ginseng plants, heart- and torpedo-shaped secondary somatic embryos (SEs induced from germinated SEs on agar medium were collected and then inoculated to 10-l bubble column bioreactor, respectively. For plantlet conversion, inoculation of torpedo-shaped secondary SEs was more effective than heart-shaped SEs. TS2 (culture of torpedo-shaped SEs in a bioreactor with a 2-week subculture interval plantlets had a higher root number and leaf number and larger leaf area than did HS3 (culture of heart-shaped SEs in a bioreactor with a 3-week subculture interval and HS2 (culture of heart-shaped SEs in a bioreactor with a 2-week subculture interval plantlets. Of these converted plants, TS2 plantlets had higher survival rate (83.7% and growth characteristics after transplantation in a simple shed covered with a 50% sunshade net only for 6 months. TS2 plantlets also showed significantly lower H2O2 content and significantly increased superoxide dismutase (SOD, glutathione peroxidase (GPX, and glutathione transferase (GST expression levels as compared to HS2 plants when exposure to ex vitro conditions.

  10. In Vitro Selection of Peanut Somatic Embryos on Medium Containing Culture Filtrate of Sclerotium rolfsii and Plantlet Regeneration

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    YUSNITA

    2005-06-01

    Full Text Available Attempts to identify somaclonal variants of peanut with resistance to Sclerotium stem rot disease due to infection of S. rolfsii were conducted. The objectives of this study were to develop in vitro selection method using culture filtrates of S. rolfsii, identify culture filtrate-insensitive somatic embryo (SE of peanut after in vitro selection and regenerate peanut R0 lines originated from culture filtrate-insensitive SE. To achieve these objectives, peanut embryogenic tissues were cultured on selective medium containing various concentrations of S. rolfsii culture filtrates and sublethal concentration of the filtrates. Medium containing sublethal level of S. rolfsii culture filtrates was used to identify culture filtrate-insensitive SE of peanut. Subsequently, the selected SEs were germinated, plantlets were regenerated and preliminary tested against S. rolfsii. Results of the experiments showed that addition of S. rolfsii culture filtrates into medium for inducing peanut somatic embryos drastically reduced their growth and proliferation. S. rolfsii culture filtrates at 10% concentration has significantly reduced the number of proliferated SE per explant. However, sublethal level was achieved at 30% of culture filtrates concentration. Responses of five peanut cultivars against 30% of culture filtrates were similar, indicating they were similar in their susceptibility against S. rolfsii. A number of culture filtrate-insensitive SE were identified after culturing 1500 clumps of embryogenic tissue of peanut cv. Kelinci for three consecutive passages on medium containing 30% of culture filtrates. Germination of selected SE and regeneration of plantlet from culture filtrate-insensitive SE resulted in 50 peanut R0 lines. These lines have been grown in the plastic house and produced normal seeds for further evaluation. Results of S. rolfsii inoculation indicated the existence of chimera for insensitivity against S. rolfsii.

  11. [Asepsis sowing and tissue culture of Bletilla striata].

    Science.gov (United States)

    Zeng, Songjun; Huang, Xiangli; Chen, Zhilin; Chen, Jiantong; Duan, Jun

    2004-09-01

    The asepsis sowing and tissue culture of Bletilla striata were studied. The results indicated that the embyro culture had highest sprouting percent and plantlets percent when their embryos were mature. The optimal medium for the embryo culture was 1/2 MS. Adding 10% coconut juice can promote embyros sprouting and plantlets formation, 1% active carbon can improve plantlets growing. The best medium for the top of plantlet on culture and multiplication was 1/2 MS + 6-BA 0.5 mg/L + NAA 0.2 mg/L. The best medium of rooting was 1/2 MS + NAA 0.5 mg/L, and 10% banana juice can improve rooting of plantlets.

  12. Regeneration of plantlets from unpollinated ovary cultures of ...

    African Journals Online (AJOL)

    Analysis of variance (ANOVA) has shown that genotypes, types of media, concentrations of 2,4-dichlorophenoxy acetic acid (2,4-D) and kinetin (KIN) and durations of cold pretreatment at 4°C significantly (P≤0.05) affected direct formation of embryonic tissues independently. Stage II of wheat spikes, MS medium containing ...

  13. The plant tissue culture

    International Nuclear Information System (INIS)

    Crocomo, O.J.; Sharp, W.R.

    1973-01-01

    Progress in the field of plant tissue culture at the Plant Biochemistry Sector, Centro de Energia na Agricultura (CENA), Piracicaba, S.P., Brazil, pertains to the simplification of development in 'Phaseolus vulgaris' by dividing the organism into its component organs, tissues, and cells and the maintenance of these components on defined culture media 'in vitro'. This achievement has set the stage for probing the basis for the stability of the differentiated states and/or the reentry of mature differentiated cells into the mitotic cell cycle and their subsequent redifferentiation. Data from such studies at the cytological and biochemical level have been invaluable in the elucidation of the control mechanisms responsible for expression of the cellular phenotype. Unlimited possibilities exist for the application of tissue culture in the vegetative propagation of 'Phaseolus' and other important cultivars in providing genocopies or a large scale and/or readily obtaining plantlets from haploid cell lines or from protoplast (wall-less cells) hybridization products following genetic manipulation. These tools are being applied in this laboratory for the development and selection of high protein synthesizing 'Phaseolus' cultivars

  14. Recovery of Green Plantlets from Albino Shoot Primordia Derived from Anther Culture of Indica Rice (Oryza sativa L.).

    Science.gov (United States)

    Mohiuddin, Abul Kashem Md; Karim, Nilufer Hye; Sultana, Shahanaz; Ferdous, Zannatul

    2011-05-01

    A simple method was developed to permit albino plant regeneration from anther culture of Hobigonj Boro (Hbj B) IV and Hbj B VI, two local varieties of aromatic indica rice from Bangladesh. Three crucial factors were identified for the albino shoot primordia to change into green plantlets in culture; components of M10 induction medium, callus size (range 0.2-0.4 cm long) and height of shoot primordia (range 2-3 mm). Immediate transfer of shoot primordia (2-3 mm) from M10 medium to regeneration medium followed by continuous incubation under fluorescent light (100-lux, 25±1°C) triggered albino shoot primordia to turn green in 2-3 days. Callus size did not show any effect on the change. Albino plantlets derived from anther callus cultured in KA, KB, KC, KD and KE media did not recover in both the varieties. Transfer of albino shoot primordia shorter or longer than 2-3 mm from the above 5 cultures to regeneration medium did not cause the shoot primordia to turn green. 100% albino shoot primordia initiated from Hbj B VI and 79% from Hbj B IV in M10 medium changed to green plantlets upon transfer to regeneration medium. Subsequent culture and subculture of green plantlets showed rapid formation of many new green plantlets.

  15. Silver Nitrate and Different Culture Vessels Influence High Frequency Microrhizome Induction In Vitro and Enhancement Growth of Turmeric Plantlet During Ex Vitro Acclimatization

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    Dikash Singh THINGBAIJAM

    2012-11-01

    Full Text Available Eleven cultivars of C. longa var. Lakadong were collected from Manipur having different topography. Curcumin content in different cultivars has been analyzed by using UV-Visible Spectrophotometer (100 Bio-Carry Spectrophotometer. The curcuminoids content were analyzed and quantified for identification of best quality cultivar. Thoubal Cultivar with highest curcumin content (9.44% was subjected for tissue culture technique using different culture vessels and silver nitrate for rapid multiplication and scaling up of microrhizome production. High multiplication rate of 27.400.47 were obtained in Murashige and Skoogs medium supplemented with 3% sucrose + 1 mg L-1 ?-napthalene acetic acid, 4 mg L-1 6-benzyl-amino-purine and 11 ?M silver nitrate. Effect of different culture vessels and silver nitrate were studied for microrhizome and multiple shoots formation. Relatively higher rate of shoots along with microrhizome (17.50.32 can be seen in Growtek which was grown without any plant growth regulator. Growtek was used for scaling up of microrhizome production in vitro and utmost microrhizome was produced in liquid Murashige and Skoogs medium supplemented with 8% sucrose, 1 mg L-1 ?-napthalene acetic acid, 4 mg L-1 6-benzyl-amino-purine and 11 ?M silver nitrate (36.250.27. Addition of silver nitrate in the medium resulted in improvement of microrhizome induction in vitro. Higher concentration of silver nitrate (33, 44, 66, 88 ?M negatively affected the microrhizome and shoot multiplication and shows inhibition of tissue response completely. Analysis of in vitro derived plantlets during acclimatization shows that the exogenous applied of silver nitrate shows superior growth as compared to control. 90-95% of plantlets with and 75-80% plantlets without silver nitrate treatment were successfully established under ex vitro acclimatization. The protocol could be utilized for large scale production of true-to-type plantlets and as alternative method to step

  16. Acclimatization and leaf anatomy of micropropagated fig plantlets

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    Chrystiane Fráguas Chirinéa

    2012-12-01

    Full Text Available The survival of micropropagated plants during and after acclimatization is a limiting process to plant establishment. There is little information on how the anatomy of vegetative organs of Ficus carica can be affected by culture conditions and acclimatization. The present research aimed to study the effects of time on culture medium and substrates during the acclimatization of fig tree plantlets produced in vitro, characterizing some leaf anatomy aspects of plantlets cultured in vitro and of fig trees produced in field. Plantlets previously multiplied in vitro were separated and transferred into Wood Plant Medium (WPM where they were kept for 0, 15, 30, 45 and 60 days. Different substrates were tested and studies on leaf anatomy were performed in order to compare among plantlets grown in vitro, plantlets under 20, 40 and 60 days of acclimatization, and field grown plants. Keeping plantlets for 30 days in WPM allowed better development in Plantmax during acclimatization. Field grown plants presented higher number of stomata, greater epicuticular wax thickness and greater leaf tissue production compared to in vitro ones. The leaf tissues of in vitro plantlets show little differentiation and have great stomata number compared with acclimatized plants, which reduce the number of stomata during the acclimatization process.

  17. Silver Nitrate and Different Culture Vessels Influence High Frequency Microrhizome Induction In Vitro and Enhancement Growth of Turmeric Plantlet During Ex Vitro Acclimatization

    Directory of Open Access Journals (Sweden)

    Dikash Singh THINGBAIJAM

    2012-11-01

    Full Text Available Eleven cultivars of C. longa var. Lakadong were collected from Manipur having different topography. Curcumin content in different cultivars has been analyzed by using UV-Visible Spectrophotometer (100 Bio-Carry Spectrophotometer. The curcuminoids content were analyzed and quantified for identification of best quality cultivar. Thoubal Cultivar with highest curcumin content (9.44% was subjected for tissue culture technique using different culture vessels and silver nitrate for rapid multiplication and scaling up of microrhizome production. High multiplication rate of 27.40�0.47 were obtained in Murashige and Skoog�s medium supplemented with 3% sucrose + 1 mg L-1 ?-napthalene acetic acid, 4 mg L-1 6-benzyl-amino-purine and 11 ?M silver nitrate. Effect of different culture vessels and silver nitrate were studied for microrhizome and multiple shoots formation. Relatively higher rate of shoots along with microrhizome (17.5�0.32 can be seen in Growtek which was grown without any plant growth regulator. Growtek was used for scaling up of microrhizome production in vitro and utmost microrhizome was produced in liquid Murashige and Skoog�s medium supplemented with 8% sucrose, 1 mg L-1 ?-napthalene acetic acid, 4 mg L-1 6-benzyl-amino-purine and 11 ?M silver nitrate (36.25�0.27. Addition of silver nitrate in the medium resulted in improvement of microrhizome induction in vitro. Higher concentration of silver nitrate (33, 44, 66, 88 ?M negatively affected the microrhizome and shoot multiplication and shows inhibition of tissue response completely. Analysis of in vitro derived plantlets during acclimatization shows that the exogenous applied of silver nitrate shows superior growth as compared to control. 90-95% of plantlets with and 75-80% plantlets without silver nitrate treatment were successfully established under ex vitro acclimatization. The protocol could be utilized for large scale production of true-to-type plantlets and as alternative

  18. Recovery of Green Plantlets from Albino Shoot Primordia Derived from Anther Culture of Indica Rice (Oryza sativa L.)

    OpenAIRE

    Mohiuddin, Abul Kashem Md.; Karim, Nilufer Hye; Sultana, Shahanaz; Ferdous, Zannatul

    2011-01-01

    A simple method was developed to permit albino plant regeneration from anther culture of Hobigonj Boro (Hbj B) IV and Hbj B VI, two local varieties of aromatic indica rice from Bangladesh. Three crucial factors were identified for the albino shoot primordia to change into green plantlets in culture; components of M10 induction medium, callus size (range 0.2–0.4 cm long) and height of shoot primordia (range 2–3 mm). Immediate transfer of shoot primordia (2–3 mm) from M10 medium to regeneration...

  19. Comparative in vitro culture of white and green ash from seed to plantlet production

    Science.gov (United States)

    J. W. Van Sambeek; John E. Preece; Nadia E. Navarrete-Tindall

    2002-01-01

    In vitro procedures have already been reported for white ash (Fraxinus americana L.) to establish cut dormant seeds, force axillary shoot proliferation, and induce rapid rooting to produce clonal plantlets (Preece et al., 1987, Navarrete et al., 1989, Preece et al., 1989, Preece et al., 1995). Hypothetically, a production cycle from seed to...

  20. The basic design and requirement for plant tissue culture laboratory in MINT

    International Nuclear Information System (INIS)

    Azraf Azman; Rosli Darmawan; Rusli Ibrahim; Mohd Nazir Basiran; Azhar Mohamad; Mohamed Najli Mohamed Yasin; Shuhaimi Shamsuddin

    2005-01-01

    The production of multiple species plantlets involves a relatively complex process and it is a highly specialized operation. Tissue culture technology is rapidly becoming a commercialized method for propagating new cultivars, rare species and difficult-to-propagate plant. Not only are skills and knowledge essential but the laboratory itself also plays an important role to ensure the successful growth of the plantlets. To produce quality plantlets, plant tissue culture laboratories should fulfill the basic requirements. The laboratory should have proper building and layout which comprise of media preparation and washing room, sterilization or autoclave room, transfer room and culture or growth room. The scope of this paper is to compare these fundamental requirements with the plant tissue culture laboratory in MINT. All the basic needs and differences will be discussed and the proposal for corrective actions will be presented. (Author)

  1. Plant tissue culture techniques

    Directory of Open Access Journals (Sweden)

    Rolf Dieter Illg

    1991-01-01

    Full Text Available Plant cell and tissue culture in a simple fashion refers to techniques which utilize either single plant cells, groups of unorganized cells (callus or organized tissues or organs put in culture, under controlled sterile conditions.

  2. Mass propagation of in vitro plantlets and extraction of bioactive compounds from Eurycoma Longifolia Jack (Tongkat Ali) using cell and organ cultures

    International Nuclear Information System (INIS)

    Sobri Hussein; Rusli Ibrahim

    2006-01-01

    Eurycoma longifolia Jack or better known in Malaysia as Tongkat Ali is an important herbal plant that owes its popularity locally and worldwide to its aphrodisiac claim and has been sought after as an essential component for the treatment of anti-malarial, anti-ulcer, anti-tumour promoting and anti-parasitic agent and also as health supplements. Quassinoids and canthin-6-one alkaloids contained in Tongkat Ali roots are the main substances having active functions for human body. Propagation is commonly done through seeds or some vegetative means such as cuttings of stems and root, grafting or layering. However, propagation by conventional method is inadequate to meet the demand due to low viability, low germination rate, delayed rooting and long life cycle. Tissue culture has been successfully applied for mass production of plantlets and extraction of many useful secondary metabolites, including pharmaceuticals, pigments and other fine chemicals. Tongkat Ali active substances have also been derived through cell culture technology but its processes need to be optimized before the product can be commercialized. Cell and organ cultures have been proven to be an efficient alternative method for biomass production due to its fast growth and stable metabolite production. In view of its potential commercial value as a plantation crop as well as to conserve its germplasm, micropropagation technique using cell culture system is an attractive method for regeneration and mass propagation. (Author)

  3. Plant Tissue Culture

    Indian Academy of Sciences (India)

    Admin

    The success of plant biotechnology relies on the fundamental techniques of plant tissue culture. Understanding basic biol- ogy of plants is a prerequisite for proper utilization of the plant system or parts thereof. Plant tissue culture helps in providing a basic understanding of physical and chemical requirements of cell, tissue, ...

  4. Plantlet regeneration potential from seedling explants of vitegnus (Vitex agnus castus).

    Science.gov (United States)

    Chamandoosti, F

    2007-11-15

    In this research a simple and repeatable method for regeneration of a important medicinal plant (Vitex agnus castus) described. Different seedling explants such as hypocotyl, cotyledon, root and apical meristem were cultured in MS basal media with different kinds and concentrations of PGRs. Root and apical meristem explants were the only explants that have regeneration whole plantlets potential. It was interesting that regeneration whole plantlets from root and apical meristem explants have different developmental pathways. Whole plantlets from apical meristem explants regenerated by passing phase callusing whereas regeneration whole plantlets from root was direct and without phase callusing. This subject implies that we can have many manipulation possibilities in order to different objects of tissue culture by selecting different explants in vitegnus.

  5. Tissue culture and neurotoxicology

    NARCIS (Netherlands)

    Hooisma, J.

    1982-01-01

    Application of tissue culture in neurotoxicology may serve two purposes. First, they may be used to unravel the mechanism of action of neurotoxic compounds and secondly, they may be used for the screening of neurotoxic agents. Studies belonging to the first group can be subdivided into those aiming

  6. Plant Tissue Culture

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 17; Issue 8. Plant Tissue Culture - Historical Developments and Applied Aspects. H R Dagla. General Article Volume 17 Issue 8 August 2012 pp 759-767. Fulltext. Click here to view fulltext PDF. Permanent link:

  7. Plant Tissue Culture

    Indian Academy of Sciences (India)

    Admin

    closely linked with the discovery and characterization of plant hormones, and has facilitated our understanding of plant growth and development. Furthermore, the ability to grow plant cells and tissues in culture and to control their development forms the basis of many practical applications in agriculture, horticulture indus-.

  8. Plant Tissue Culture Studies.

    Science.gov (United States)

    Smith, Robert Alan

    Plant tissue culture has developed into a valid botanical discipline and is considered a key area of biotechnology, but it has not been a key component of the science curriculum because of the expensive and technical nature of research in this area. This manual presents a number of activities that are relatively easy to prepare and perform. The…

  9. Evaluation on the Effects of Culture Medium on Regeneration of Oil Palm Plantlets from Immature Embryos (IE)

    International Nuclear Information System (INIS)

    Wan Nur Syuhada, W.S.; Rasid, O.A.; Parveez, G.K.A

    2016-01-01

    Oil palm (Elaeis guineensis) tissue culture is slow, inefficient and genotype dependent. Therefore, efforts to improve the regeneration system are essential and thereby on-going. Immature embryos (IE) have been reported to be more responsive to in vitro culture as compared to other explant types. The objective of this study is to evaluate the most efficient culture media for the regeneration of whole plants from IE. IE were cultured on three media, namely N6, MY3 and MS +Y3. Initial observation showed that calli were induced as early as two weeks after culture. They started to produce friable calli around three to four months later. IE were shown to respond differently to the tested culture media. This initial result will be useful to identify the most responsive culture media for IE as explant. (author)

  10. In vitro growth and organogenesis of Prosopis farcta plantlets (Fabaceae, Mimosoideae) in culture medium supplemented with various concentrations of Ca++ and Na+.

    Science.gov (United States)

    Stambouli, S; Bouzid, S; Dutuit, P; Harzallah-Skhiri, Fethia

    2012-03-01

    The objective of this study was to vary the mineral composition of the culture medium of Prosopis farcta seedlings per addition of Na+ and Ca++ ions with the aim to identify the culture media which support the growth and/or the expression of the in vitro plant organogenesis. The Na+ and Ca++ ions were added in the culture medium in various concentrations by taking the Gamborg medium, in which macroelements were diluted 10 times, as the basic one. After two months of culture, parameters relating to the vegetative development of plant seedlings and to the various expressions of organogenesis were measured. Weak concentrations in sodium and calcium ions as well as a weak concentration in Ca++ (0.1 mM) with 50 mM in Na+ support the best vegetative development of the plantlets. The most important percentage of plant seedlings presenting a bud initiation was obtained on a medium containing 0.1 mM of Na+ and 50 mM of Ca++. Our study defined several media likely to support in vitro development of Prosopis farcta plantlets allowing the selection of salt tolerant plants or cellular lines. Some other media were chosen for improving micropropagation of the species without adding growth substances.

  11. Improvement of potato tolerance to salinity using tissue culture techniques and irradiation with in vitro selection

    International Nuclear Information System (INIS)

    Al-Safadi, B.; Arabi, M. I. E.

    2005-06-01

    A mutation breeding program was conducted to improve potato (Solanum tuberosum) tolerance to salinity. In vitro cultured explants from potato cvs. Draga, Diamant, Spunta were irradiated with gamma doses 25, 30, and 35 Gy.Growing shoots were cut and re-cultured every 2 weeks until the 4th generation (MV 4 ) to make sure no chimeral tissues still existed in the mutant material. Plantlets were subsequently propagated to obtain enough explants for in vitro selection pressure. Around 3000 plantlets from the three cultivars were subjected to selection pressure. MV 4 explants were cultured on MS medium supplemented with the NaCl in varying concentrations ranging between 50 to 200 mM. Surviving plantlets were propagated and re-cultured on a similar medium to insure their tolerance to salinity. Tolerant plantlets were acclimatized and transferred to pots and grown under glasshouse conditions. Plants were later subjected to another selection pressure, by irrigating them using water containing NaCl in concentrations ranging between 50-250 mM in addition to controls irrigated with normal water. Cultivar Spunta produced the highest number of tolerant plants. Four plants of Spunta appeared to be tolerant to salinity whereas only one plant from Diamant and was tolerant and no plants from cultivar Draga were tolerant. Mutant plants varied in number of produced minitubers from 8 - 14. Also, weight of these minitubers varied from less than 1 to 31 grams. (author)

  12. THE BIOTECHNOLOGY OF EMBRYOGENIC CELL LINES OBTAINING AND PLANTLETS OF CONIFEROUS SPECIES IN SIBERIA IN CULTURE IN VITRO

    Directory of Open Access Journals (Sweden)

    Tretiakova I.

    2012-08-01

    Full Text Available Experiments of culturing the immature isolated embryos and megagamethophytes of Siberian coniferous species were carried out on different modified media: ½ LV medium for Pinus sibirica and Pinus pumila, MSG and AI media (patent № 2431651 for Larix sibirica and Larix gmelinii, DCR medium for Picea ajanensis. For induction of embryogenic callus every species needs the optimized medium supplemented with L-glutamine, casein hydrolysate, ascorbic acid and hormones with different concentrations and their different proportions. The active proliferation of embryonal masses is observed on the same medium with reduced concentration of cytokinins. The proliferation of embryonal masses was significantly improved when they were subcultured after dispersing in liquid medium. The somatic embryos from embryonal masses are matured on basal medium with ABA (60-120 mM and PEG. In spite of species specificity the embryogenesis of morphogenic structures had the same scheme: elongation and asymmetric division of somatic cells, formation of initial cells and embryonal tubes, development of globular, torpedo and bipolar somatic embryos, embryos maturation and germination. However, not all donor-plants of coniferous species can form the embryogenic cell lines and somatic embryos. The active development of embryonal masses and formation of somatic embryos are observed from zygotic embryo of hybrid seeds of P. sibirica and L. sibirica. The obtained embryogenic lines were characterized by different proliferative activity. During 10 months cultivation the value of embryonal masses in different lines was 140-570 g. The number of somatic embryos varies from 210 to 410 per 100 mg of callus fresh weight. Decreasing proliferation activity did not observed during 24-45 months cultivation. However, development of somatic embryos in long cultivated lines decreased. Maturation of somatic embryos and development of plantlets were established in L. sibirica and P. pumila 50

  13. Propagation of Aquilaria malaccensis seedlings through tissue culture techniques

    International Nuclear Information System (INIS)

    Salahbiah Abdul Majid; Zaiton Ahmad; Mohd Rafaie Abdul Salam; Nurhayati Irwan; Affrida Abu Hassan; Rusli Ibrahim

    2010-01-01

    Aquilaria malaccensis or karas is the principal source of gaharu resin, which is used in many cultures for incense, perfumes and traditional medicines. The species is mainly propagated conventionally through seeds, cuttings and graftings. Propagation by seeds is usually a reliable method for other forest species, but for karas, this technique is inadequate to meet the current demand of seedling supplies. This is principally due to its low seed viability, low germination rate, delayed rooting of seedlings, long life-cycle and rare seed production. Tissue culture has several advantages over conventional propagation, especially for obtaining large number of uniform and high-yielding plantlets or clones. This paper presents the current progress on mass-propagation of Aquilaria malaccensis seedlings through tissue culture technique at Nuclear Malaysia. (author)

  14. The use of tissue culture techniques with irradiation to improve potato resistance to late blight

    International Nuclear Information System (INIS)

    Al-Safadi, B.; Arabi, M.I.E.

    2004-01-01

    A mutation breeding program was conducted to improve potato (Solanum tuberosum) resistance to late blight disease caused by Phytophthora infestans. In vitro cultured explants from potato cvs. Draga, Diamant, Spunta were irradiated with gamma ray doses 25, 30, and 35 Gy. Growing shoots were cut and re-cultured every 2 weeks until the 4 t h generation (MV 4 ) to make sure no chimeral tissues still existed in the mutant material. Plantlets were subsequently propagated to obtain enough explants for in vitro selection pressure. Around 3000 plantlets from the three cultivars were subjected to selection pressure using co-culture technique. MV 4 explants were incubated in jars, containing MS medium, with mycelia of P. infestans. Surviving plantlets were propagated and re-incubated with the pathogen for three consecutive generations. Resistant plantlets were acclimatized and transferred to pots and grown under glasshouse conditions. Plants were later inoculated, at the adult stage, with sporangial suspension. Cultivar Draga produced the highest number of resistant plants. Ten plants of Draga appeared to be resistant to late blight whereas only one plant from each of the other 2 cultivars was resistant. Mutant plants varied in number of produced minitubers from 13 to 70, Also, weight of these minitubers varied from less than 1 to 35 grams. Selected mutant lines will undergo further testing under field conditions for P. infestans resistance and other agronomic characteristics. (author)

  15. Tobacco clones derived from tissue culture with supersensitivity to ozone

    International Nuclear Information System (INIS)

    Sun, E.J.; Kang, H.W.

    2003-01-01

    New tobacco clones supersensitive to ozone were obtained from tissue culture. - At least two supersensitive tobacco somaclones were obtained from tissue culture (TC) , when this approach was used to asexually propagate Bel-W3 tobacco indicator plants. These somaclones can detect as low as 30 ppb ozone for a 4-h exposure duration both within CSTR exposure chambers and in ambient air. Comparison of the injury index and their coefficient of variance showed that the TC plantlets usually have more uniform performance in response to ozone in addition to their higher sensitivity. A quick regeneration procedure was established to preserve the supersensitive germplasm immediately when it was found. The TC plantlets will flower and produce seed similar to seed-grown tobacco. The TC approach proved to be a better propagation system for valuable indicator plant species. The mechanism that causes the variation and the possible difference in their genome from seed-grown tobacco is still unknown. Further studies are needed in the future to determine if factors in the TC system may be responsible for the sensitivity difference

  16. Demonstration of the economic feasibility of plant tissue culture for jojoba (Simmondsia chinensis) and Euphorbia spp

    Energy Technology Data Exchange (ETDEWEB)

    Sluis, C.

    1980-09-01

    The economic feasibility of plant tissue culture was demonstrated as applied to two plants: jojoba (Simmondsia chinensis) and Euphorbia spp. The gopher weed (Euphorbia lathyris) was selected as the species of Euphorbia to research due to the interest in this plant as a potential source of hydrocarbon-like compounds. High yield female selections of jojoba were chosen from native stands and were researched to determine the economic feasibility of mass producing these plants via a tissue culture micropropagation program. The female jojoba selection was successfully mass produced through tissue culture. Modifications in initiation techniques, as well as in multiplication media and rooting parameters, were necessary to apply the tissue culture system, which had been developed for juvenile seedling tissue, to mature jojobas. Since prior attempts at transfer of tissue cultured plantlets were unsuccessful, transfer research was a major part of the project and has resulted in a system for transfer of rooted jojoba plantlets to soil. Euphorbia lathyris was successfully cultured using shoot tip cultures. Media and procedures were established for culture initiation, multiplication of shoots, callus induction and growth, and root initiation. Well-developed root systems were not attained and root initiation percentages should be increased if the system is to become commercially feasible.

  17. Tissue culture of Sophora tonkinensis Gapnep. and its quality evaluation.

    Science.gov (United States)

    Kun-Hua, Wei; Lin-Xuan, Li; Yong-Cai, Huang; Mei-Ying, Wang; Cui, Li; Jian-Hua, Miao

    2013-10-01

    Sophora tonkinensis Gapnep. is an important rare medicinal plant in China. There were only a few papers on the rapid propagation of S. tonkinensis through in vitro tissue culture, and still no report focuses on the quality analysis of in vitro tissue culture plantlets. The different concentrations of 6-benzylaminopurine (BAP), kinetin (KT), and indole-3-acetic acid (IAA) were used to establish and screen the optimal rapid propagation technology of S. tonkinensis by orthogonal test; the different concentrations of a-naphthalene acetic acid (NAA), indole-3-butyric acid (IBA), and ABT rooting power (ABT) were used to screen the optimal rooting technology. For quality evaluation of tissue culture plants, three different sites were chose to finish planting experiment. The leaf characteristics, radix ex rhizoma yield, and contents of matrine and oxymatrine were evaluated, respectively, to provide evidence of high yield and good qualities of tissue culture plants. A large number of buds could be induced directly from epicotyl and hypocotyl explants on the Murashige and Skoog (MS) medium supplemented with 1.5 mg/l BAP, 0.5 mg/l IAA, and 0.5 mg/l KT; the best root induction medium was solid MS medium at half the macronutrient concentration supplemented with 1.0 mg/l NAA, 0.4 mg/l IBA, and 0.1 mg/l ABT. The rooting rate was 98%. All tissue culture plants showed normal leaf characteristics. Tissue culture plants from two sites possessed higher radix ex rhizoma yield and overall productivity of matrine and oxymatrine than those of seed plants. Tissue culture is a rapid, effective, and convenient propagation method for S. tonkinensis, and the quality of S. tonkinensis tissue culture plants meets the requirement of quality standard of China Pharmacopoeia (edition 2010), the crude drug from S. tonkinensis tissue culture plants will be suitable for substituting the crude drug from seed plants.

  18. Seismomorphogenesis: a novel approach to acclimatization of tissue culture regenerated plants.

    Science.gov (United States)

    Sarmast, Mostafa Khoshhal; Salehi, Hassan; Khosh-Khui, Morteza

    2014-12-01

    Plantlets under in vitro conditions transferred to ex vivo conditions are exposed to biotic and abiotic stresses. Furthermore, in vitro regenerated plants are typically frail and sometimes difficult to handle subsequently increasing their risk to damage and disease; hence acclimatization of these plantlets is the most important step in tissue culture techniques. An experiment was conducted under in vitro conditions to study the effects of shaking duration (twice daily at 6:00 a.m. and 9:00 p.m. for 2, 4, 8, and 16 min at 250 rpm for 14 days) on Sansevieria trifasciata L. as a model plant. Results showed that shaking improved handling, total plant height, and leaf characteristics of the model plant. Forty-eight hours after 14 days of shaking treatments with increasing shaking time, leaf length decreased but proline content of leaf increased. However, 6 months after starting the experiment different results were observed. In explants that received 16 min of shaking treatment, leaf length and area and photosynthesis rate were increased compared with control plantlets. Six months after starting the experiment, control plantlets had 12.5 % mortality; however, no mortality was observed in other treated explants. The results demonstrated that shaking improved the explants' root length and number and as a simple, cost-effective, and non-chemical novel approach may be substituted for other prevalent acclimatization techniques used for tissue culture regenerated plantlets. Further studies with sensitive plants are needed to establish this hypothesis.

  19. Impact of LED irradiance on plant photosynthesis and action spectrum of plantlet

    Science.gov (United States)

    Naznin, Most Tahera; Lefsrud, Mark G.

    2014-09-01

    Light emitting diodes (LEDs) can be selected to target the wavelengths absorbed by plantlets, enabling the users to customize the wavelengths of light required for maximum production. The primary purpose of this experiment was to test the effect of different ratios of red to blue LEDs on tomato plantlets photosynthetic action spectrum. Four light treatments including: red LED (100%) and three ratios of red (661 nm) to blue (449 nm) light (5:1, 10:1 and 19:1) at 60 umol m-2 s-1 for this study. The tomato plantlets cultured without blue light showed a three and half-fold decrease in photosynthesis rate. The highest photosynthetic action spectrum was observed at 10:1 but was not significantly difference from the 5:1 and the lowest action spectrum was observed at 100% red LED light. The tomato plantlets grown without the blue light showed a single-fold increase in plantlet height but were not significantly different from the 10:1 red to blue LED light. This research will allow for improved selection of LED lighting for plant tissue culture.

  20. Recloning of regenerated plantlets from elite oil palm ( Elaeis ...

    African Journals Online (AJOL)

    Plant regeneration in oil palm cv. Tenera via somatic embryogenesis was conducted using regenerated plantlets as an explant source. Explants from different positions – apex, middle and basal segments of regenerated plantlets – were cultured in N6 medium supplemented with 100, 120 and 140 mg/L 2 ...

  1. Culture of insect tissues

    International Nuclear Information System (INIS)

    Cestari, A.N.; Simoes, L.C.G.

    1978-01-01

    Several aspects are discussed related to the behavior of politenic chromosomes from Rhyncosciara salivary glands kept in culture during different periods of time, without interference of insect hormones. Nucleic acid-and protein synthesis in isolated nuclei and chromosomes are also investigated. Autoradiographic techniques and radioactive precursors for nucleic acids and proteins are used in the research. (M.A.) [pt

  2. Salinity-Induced Callus Browning and Re-Differentiation, Root Formation by Plantlets and Anatomical Structures of Plantlet Leaves in Two Malus Species

    International Nuclear Information System (INIS)

    Gou, W.; Zheng, P.; Zheng, P.; Wang, K.; Zhang, L.; Akram, N. A.

    2016-01-01

    Apple (Malus domestica L.) is widely grown in northern China. However, soil salinization has become one of the most severe factors limiting apple productivity in some regions including the Loess Plateau. In our study, the regeneration system of both rootstock Rehd (Malus robusta Rehd) and scion Fuji (Malus domestica Borkh. cv. Fuji) was established In vitro. The two Malus species were cultured on the MS medium containing 0 or 150 mM NaCl to examine salt-induced effects on callus browning and re-differentiation, root formation of plantlets and anatomical structures of plantlet leaves at 15 days old callus and plantlet stages. Salt stress caused a marked increase in callus browning rate, while a decrease in re-differentiation rate, rooting rate, root number and length in both species. Additionally, anatomical structures of plantlet leave showed salt-induced damage such as reduced palisade tissue and intracellular chloroplast, incomplete development of xylem and severe damage of the phloem tissue. Salt stress also caused a few adaptive structural features in leaves including increased thickness of upper and lower epidermis, elevated proportion of spongy tissue and formation of lignified vessels. The responses of the two Malus species did not differ significantly at the differentiation stage. However, they were more sensitive to salinity at the callus stage than those at the plantlet stage in each species. Therefore, callus stage has been found to be more suitable for evaluating responses of the two apple species to salt stress. The Fuji and Rehd could be treated as a good scion/rootstock combination of apple to adapt to soil salinity based on their similar degree of salt stress-tolerance. (author)

  3. Application of tissue culture to cashew ( Anacardium occidentale L ...

    African Journals Online (AJOL)

    Summary of the previous works on the in vitro culture of cashew is highlighted with emphasis on the critical factors that influence the explants response and plantlet regeneration. The recalcitrant nature of cashew has been attributed to the limited success recorded so far in the in vitro culture of the crop and abnormal ...

  4. History of plant tissue culture.

    Science.gov (United States)

    Thorpe, Trevor

    2012-01-01

    Plant tissue culture, or the aseptic culture of cells, tissues, organs, and their components under defined physical and chemical conditions in vitro, is an important tool in both basic and applied studies as well as in commercial application. It owes its origin to the ideas of the German scientist, Haberlandt, at the beginning of the twentieth century. The early studies led to root cultures, embryo cultures, and the first true callus/tissue cultures. The period between the 1940s and the 1960s was marked by the development of new techniques and the improvement of those that were already in use. It was the availability of these techniques that led to the application of tissue culture to five broad areas, namely, cell behavior (including cytology, nutrition, metabolism, morphogenesis, embryogenesis, and pathology), plant modification and improvement, pathogen-free plants and germplasm storage, clonal propagation, and product (mainly secondary metabolite) formation, starting in the mid-1960s. The 1990s saw continued expansion in the application of the in vitro technologies to an increasing number of plant species. Cell cultures have remained an important tool in the study of basic areas of plant biology and biochemistry and have assumed major significance in studies in molecular biology and agricultural biotechnology in the twenty-first century. The historical development of these in vitro technologies and their applications is the focus of this chapter.

  5. Development of a vinasse culture medium for plant tissue culture

    International Nuclear Information System (INIS)

    Silva, A.L.L.D.; Gollo, L.

    2014-01-01

    Vinasse is the main pollutant (effluent) obtained from the distillation of sugarcane in the production of fuel alcohol. However, this residue is rich in nutrients that are required by plants. We developed a new culture medium using vinasse for the In vitro propagation of an orchid. The vinasse was treated (decanted and filtered), and the nutrients were determined and quantified. Different formulations using vinasse were tested for an In vitro culture. The vinasse dilutions demonstrated a good buffering effect. The ideal vinasse dilution for media formulation was 2.5%. The best KC formulations with vinasse were KCV1 and KCV5. Compared to KC medium, these formulations demonstrated similar results for In vitro multiplication, with the exception of protocorm-like body number, which was inferior in the vinasse formulations. Conversely, for In vitro elongation and rooting, these vinasse media were superior to KC medium. KC medium promotes a low rooting rate (8%) compared to 68 and 100% obtained by KCV1 and KCV5, respectively. Moreover, plantlets cultured on KC medium become protocorm-like body clusters, which impeded the acclimatization of these explants. Plantlets elongated and rooted on KCV1 and KCV5 were successfully acclimatized with a 91% survival rate for both KC vinasse formulations. This study shows the great potential of this technology as a rational alternative to vinasse disposal and adds value to what is currently considered a waste product. (author)

  6. Aroma production by tissue cultures.

    Science.gov (United States)

    Hrazdina, G

    2006-02-22

    Although plant tissue cultures have been in use for the past hundred years, adapting them for the production of aroma compounds started only in the 1970s. The use of tissue cultures in aroma production has its advantages, because plant cells, unlike whole plants, are not limited to geographic locations or the seasons. Cell mass can be doubled relatively rapidly and can be induced for the production of compounds in a coordinated manner. Compounds can be isolated from cells or the medium with relative ease. Therefore, it would seem to be ideal to use plant cell cultures for the production of aroma compounds. Cell cultures, however, also have some problems. The production of aroma compounds or their precursors is in relatively low amounts, and thus this production method is expensive. Additional expenses are the cost of the medium and the purification of the compounds for food use. Also, cell cultures can only be used effectively in systems for which the biochemical pathway of the aroma compounds is known. In this paper the results of experiments for the use of tissue cultures in the production of vanilla, raspberry, strawberry garlic, and onion aromas is discussed.

  7. Epigenetics in plant tissue culture

    NARCIS (Netherlands)

    Smulders, M.J.M.; Klerk, de G.J.M.

    2011-01-01

    Plants produced vegetatively in tissue culture may differ from the plants from which they have been derived. Two major classes of off-types occur: genetic ones and epigenetic ones. This review is about epigenetic aberrations. We discuss recent studies that have uncovered epigenetic modifications at

  8. [Preliminary study of Dioscorea bulbifera plantlet microtuber in vitro induction].

    Science.gov (United States)

    Hong, Sen-Rong; Yin, Ming-Hua

    2014-04-01

    In order to provide a theoretical basis for the microtuber factory production and its germplasm resources preservation, in vitro induction of Dioscorea bulbifera plantlet microtubers was studied. Through plant tissue culture technique and single factor experiment method, stems with buds of Dioscorea bulbifera plantlets as explants, the effects of various factors such as sugar, inorganic salt, cultivation mode, activated carbon and physiological state of stems with buds on in vitro induction of Dioscorea bulbifera microtubers were studied. The optimal sugar of Dioscorea bulbifera microtuber in vitro induction was 60 g/L sucrose or 90 g/L white sugar. The best inorganic salt concentration of Dioscorea bulbifera microtuber in vitro induction was MS. The best culture method of Dioscorea bulbifera microtuber in vitro induction was solid-liquid double layer culture. Activated carbon had a significant effect on Dioscorea bulbifera microtuber in vitro induction, whose optimum concentration was 0.03%. More mature the stem with buds was, the shorter the time of microtuber formation need. This experiment establishes a rapid method of Dioscorea bulbifera microtuber in vitro induction for the first time,which provides a new way for the application of Dioscorea bulbifera microtubers in agricultural production.

  9. Salt Tolerant Mutants in Potato sp. Cara Induced by Gamma Irradiation and Tissue Culture Technique

    International Nuclear Information System (INIS)

    Sharabash, M.T.; Mohamed, A.A.; Ahmed, F.A.; Afifi, A.M.

    2003-01-01

    Sprouts of 2-3 cm length were cut off from potatoes cv. Cara tubers and sterilized. Apical meristemic tips were sown to produce virus free plantlets. After 6-8 weeks, the new plantlets became at 5-7cm heights. Micro-propagation was begun to obtain sufficient number of plantlets. Plantlets obtained from micro-propagation stage were divided into 3 groups to be exposed to 0.0,20 or 40Gy gamma rays. The dose rate was 27.7rad/sec. Irradiated and unirradiated plantlets were trimmed to be with 5-6 nodes and without leaves. They were transplanted onto 1/2MS [1] liquid medium supplemented with sodium chloride at different concentrations, i.e.,0.0, 2000 or 4000 ppm. Healthy plantlets were chosen, and all unfit ones were excluded. The plantlets were sub-cultured six times on the same fresh medium with the same concentrations of NaCI, with insisting to exclude all unfit plantlets. All maintained healthy plantlets, in the sixth vegetative generation (M 1 V 6 ), grown under salinity stress, whatever the concentration of NaCI, were considered as mutant lines

  10. Use of radiation for inducing mutants in potatoes through tissue culture technique

    International Nuclear Information System (INIS)

    Sharabash, M.T.

    1997-01-01

    Meristem-tips obtained from sprouts of potato tubers, cv. 'Diamant' (Solanum tuberosum) were cultured on MS-medium and multiplied into plantlets through micropropagation. After 2-3 weeks, the micropropagated plantlets had 5-6 nodes each. The plantlets were irradiated with 20 to 40 Gy gamma rays at 27.7 rad/sec. Irradiated plantlets were cut into single nodes and cultured on MS-medium supplemented with 2 g NaCl. Salt resistant plantlets were transferred to MS-liquid medium supplemented with 2g NaCl/l 5.2, and microtubers were collected after 6 weeks. Minitubers were produced under the same level of salinity. (author). 2 tabs

  11. Shoot growth in aseptically cultivated daylily and haplopappus plantlets after a 5-day spaceflight.

    Science.gov (United States)

    Levine, H G; Krikorian, A D

    1992-01-01

    Plantlets of daylily (Hemerocallis cv. Autumn Blaze) regenerated from cell suspensions, and 4 clonal populations of Haplopappus gracilis were aseptically cultivated aboard the Shuttle "Discovery" during a 5-day mission within NASA's Plant Growth Unit (PGU) apparatus. Daylily was selected as a representative herbaceous perennial monocotyledon and the haplopappus clones represented an annual dicotyledon. The latter included 4 strains with different physiological and morphological characteristics: two aseptic seedling clones (each generated from a single seedling) and two tissue culture-derived lines. Mean daily growth rates for the primary shoots of all plantlets averaged 4.13 mm day-1 (SD = 2.20) for the flight experiment and 4.68 mm day-1 (SD = 2.59) for the ground control. Comparable growth rates calculated by summing both the primary and secondary shoots for all plantlets were 5.94 mm day-1 (SD = 2.89) for the flight experiment and 6.38 mm day-1 (SD = 3.71) for the control. Statistically significant differences existed between: (1) flight vs control primary shoot growth (the controls growing more than plantlets subjected to spaceflight conditions), (2) the different populations (the daylily gaining more shoot material than any of the haplopappus populations and the haplopappus seedling clones outperforming the tissue culture-derived haplopappus lines), and (3) the individual Plant Growth Chambers contained within the PGU. The data suggest that some spaceflight-associated factor(s) increased the tendency for primary shoot apices to degrade or senesce, resulting in the release of apical dominance and permitting the emergence of axillary branches, which subsequently partially compensated for the reduced primary axis growth. In addition to spaceflight-associated factors, the physiologically diverse nature of the experimental material as well as environmental heterogeneities within the culture apparatus contributed to the variation in growth results. The findings

  12. Shoot growth in aseptically cultivated daylily and haplopappus plantlets after a 5-day spaceflight

    Science.gov (United States)

    Levine, H. G.; Krikorian, A. D.

    1992-01-01

    Plantlets of daylily (Hemerocallis cv. Autumn Blaze) regenerated from cell suspensions, and 4 clonal populations of Haplopappus gracilis were aseptically cultivated aboard the Shuttle "Discovery" during a 5-day mission within NASA's Plant Growth Unit (PGU) apparatus. Daylily was selected as a representative herbaceous perennial monocotyledon and the haplopappus clones represented an annual dicotyledon. The latter included 4 strains with different physiological and morphological characteristics: two aseptic seedling clones (each generated from a single seedling) and two tissue culture-derived lines. Mean daily growth rates for the primary shoots of all plantlets averaged 4.13 mm day-1 (SD = 2.20) for the flight experiment and 4.68 mm day-1 (SD = 2.59) for the ground control. Comparable growth rates calculated by summing both the primary and secondary shoots for all plantlets were 5.94 mm day-1 (SD = 2.89) for the flight experiment and 6.38 mm day-1 (SD = 3.71) for the control. Statistically significant differences existed between: (1) flight vs control primary shoot growth (the controls growing more than plantlets subjected to spaceflight conditions), (2) the different populations (the daylily gaining more shoot material than any of the haplopappus populations and the haplopappus seedling clones outperforming the tissue culture-derived haplopappus lines), and (3) the individual Plant Growth Chambers contained within the PGU. The data suggest that some spaceflight-associated factor(s) increased the tendency for primary shoot apices to degrade or senesce, resulting in the release of apical dominance and permitting the emergence of axillary branches, which subsequently partially compensated for the reduced primary axis growth. In addition to spaceflight-associated factors, the physiologically diverse nature of the experimental material as well as environmental heterogeneities within the culture apparatus contributed to the variation in growth results. The findings

  13. Cytogenetic studies on stevia rebaudiana produced by tissue culture and affected by gamma rays and drought

    International Nuclear Information System (INIS)

    Awad, A.S.A

    2009-01-01

    The present investigation was under taken to carry out in the laboratories of the Natural Products Department, National Center for Radiation Research and Technology, Atomic Energy authority, Nasr city, Cairo, Egypt, to study the effect of gamma radiation doses, osmostress and the combined effects between them on tissue culture, some biochemical analysis and molecular genetic marker in stevia rebaudiana bertoni. The results obtained were: Tissue culture 1- micropropagation media: stevia rebaudiana plantlets cultured on MS medium hormones free for micropropagation.Hormones such as BAP and NAA with different concentrations induced callus formation and give slight growth.Study the effect of gamma radiation, osmostress and the combined effects between them : 1)The effect of gamma radiation on buds survival: Gamma radiation doses (10, 20 and 30 Gy) induced decreasing in bud survival percentage with increasing radiation dose in stevia rebaudiana. The dose 30 Gy was induced 60% mortality.2) Study the effect of gamma radiation on some biochemical analysis: Gamma radiation doses induced increase in the total carbohydrate with doses (20 and 30 Gy) but decreased with dose 10 Gy. Proline contents increased in plantlets with increasing doses . The total protein was increased with doses (10 and 20 Gy), but the dose 30 Gy induced decrease in total protein. Gamma radiation doses induced decreasing in total DNA while, the nucleic acid RNA increased.3) The effect of osmostress on buds survival: The concentrations (40000,50000,60000,70000 and 80000 ppm) from sucrose or sorbitol decreased the bud survival and shoot length in stevia plantlets with increasing sucrose or sorbitol levels. 4) The effect of osmostress on some biochemical analysis: Sucrose and sorbitol concentrations (40000,50000,60000,70000 and 80000 ppm) caused decrease in total carbohydrate.

  14. Requirements of in vitro plantlets produced in a biofactory

    Directory of Open Access Journals (Sweden)

    Ana Rosa Hernández Freire

    2014-10-01

    Full Text Available The objective of the work was establishing the requirements of quality in in vitro sugarcane plantlets in the adaptation stage aimed to determine the nomina l values of the characteristics of substitute quality for this crop. It was used the cultivar C86-51, after t he rooting stage, being the one that was in existence in that period. The percentage of survival and loss was determi ned, coinciding with the transplant stage to the field where the greatest percentage of survival was reached by in vitro plantlets with size from 5 to 7 cm. To develop this work tools of quality like interviews, surveys, ex perts’ method and brain storming applying statisticians that determined the defined characteristics for t he realization of the study. It was recommended to make extensive the results to all the Tissue Culture Plants of the country and to make a diagnosis of quality that allows establishing a procedure for all the crops to marke t.

  15. PLANT REGENERATION THROUGH TISSUE CULTURE OF PEAR ...

    African Journals Online (AJOL)

    AISA

    culture media, calli embryogenic potential and fertile plants regeneration were conserved for more than 12 months. Characteristics of regenerated plants were similar to control. It appears that dissected shoot apex was a new appropriate tool in tissue culture. Key words: Tissue culture, culture medium, callus induction, shoot ...

  16. Production of virus-free orchid Cymbidium aloifolium (L. Sw. by various tissue culture techniques

    Directory of Open Access Journals (Sweden)

    Shreeti Pradhan

    2016-10-01

    Full Text Available Orchids are affected by many viruses resulting in poor growth, yield and quality, and an overall decline in population. Cymbidium mosaic virus (CymMV is one of the common orchid viruses found in Cymbidium species but it infects different orchid genera. In this study Cymbidium aloifolium was propagated in vitro using MS medium at different strength (1.0, ½, and ¼ with or without 0.5 mg/l BAP (6-benzylaminopurine and 0.5 mg/l NAA (Naphthalene acetic acid. To provide disease-free planting material, source plant for in vitro propagation needs to be screened for pathogenic viruses. In the present study, in vivo-grown source (mother plants and tissue culture-derived plants of C. aloifolium were tested for CymMV virus using Double antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA. All the tissue cultured plants were found to be 100% virus-free whereas the in vivo grown source plants were highly affected by CymMV virus (83.33%. The virus-free in vitro plantlets were multiplied in large scale and then acclimatized on earthen pot containing a mixture of cocopeat, litter and clay in the ratio of 3:2:1. Eighty five percent of acclimatized plantlets survived making this method an efficient mass production system for high quality virus-free C. aloifolium for commercial floriculture and germplasm preservation.

  17. Tissue culture of ornamental cacti

    Directory of Open Access Journals (Sweden)

    Eugenio Pérez-Molphe-Balch

    2015-12-01

    Full Text Available Cacti species are plants that are well adapted to growing in arid and semiarid regions where the main problem is water availability. Cacti have developed a series of adaptations to cope with water scarcity, such as reduced leaf surface via morphological modifications including spines, cereous cuticles, extended root systems and stem tissue modifications to increase water storage, and crassulacean acid metabolism to reduce transpiration and water loss. Furthermore, seeds of these plants very often exhibit dormancy, a phenomenon that helps to prevent germination when the availability of water is reduced. In general, cactus species exhibit a low growth rate that makes their rapid propagation difficult. Cacti are much appreciated as ornamental plants due to their great variety and diversity of forms and their beautiful short-life flowers; however, due to difficulties in propagating them rapidly to meet market demand, they are very often over-collected in their natural habitats, which leads to numerous species being threatened, endangered or becoming extinct. Therefore, plant tissue culture techniques may facilitate their propagation over a shorter time period than conventional techniques used for commercial purposes; or may help to recover populations of endangered or threatened species for their re-introduction in the wild; or may also be of value to the preservation and conservation of the genetic resources of this important family. Herein we present the state-of-the-art of tissue culture techniques used for ornamental cacti and selected suggestions for solving a number of the problems faced by members of the Cactaceae family.

  18. In vitro regeneration of hybrid plantlets of cashew (Anacardium ...

    African Journals Online (AJOL)

    Embryos from immature nuts of cashew (Anacardium occidentale L.) were cultured in vitro to regenerate improved hybrid plantlets. Explants (embryo) were excised from developing F1 hybrid immature nuts derived from diallel cross and harvested at 2-, 4-, 6- and 8-weeks after pollination (WAPo) for in vitro culture.

  19. Tissue culture in forestry and agriculture

    Energy Technology Data Exchange (ETDEWEB)

    Henke, R.R.; Hughes, K.W.; Constantin, M.J.; Hollaender, A. (eds.)

    1985-01-01

    This symposium aims to examine the current state-of-the-art in tissue culture technology and to relate this state of technology to practical, applied, and commercial interests. Thus the focus is on embryogenesis in culture: how to recognize it, factors which affect embryogenesis, use of embryogenic systems, etc.; and variability from culture. A special session on woody species again emphasized somatic embryogenesis as a means of rapid propagation. This volume emphasizes tissue culture of forest trees.

  20. Plant Tissue Culture in a Bag.

    Science.gov (United States)

    Beck, Mike

    2000-01-01

    Describes the use of an oven bag as a sterile chamber for culture initiation and tissue transfer. Plant tissue culture is an ideal tool for introducing students to plants, cloning, and experimental design. Includes materials, methods, discussion, and conclusion sections. (SAH)

  1. Walnut tissue culture: research and field applications

    Science.gov (United States)

    2004-01-01

    Vitrotech Biotecnologia Vegetal began researching propagating Juglans regia (English walnut) and various Juglans hybrids by tissue culture in 1993 and has operated on a commercial scale since 1996. Since this time, more than one and a half million walnuts of different species have been propagated and field planted. Tissue cultured...

  2. GROWTH AND ROOTING SYSTEM OF ACACIA MANGIUM OBTAINED BY TISSUE CULTURE

    Directory of Open Access Journals (Sweden)

    SUPRIYANTO

    1991-01-01

    Full Text Available Since 1980/1981, the government of Indonesia through the Ministry of Forestry has started to reforest logged-over, alang-alang, unproductive areas and to convert them to Forest Industry Plantation. The target is 300 000 ha per year. It means, 750 million seedlings should be provided per year (planting distance 2 m x 2 m. The tree species to be planted in forest industry plantation should have shorter life cycle (8 - 10 years, good stem-form, good rooting system, and should be fast growing. Acacia mangium has been selected as one of the important tree species for forest industry plantation due to its growth, quality of fiber wood (pulp and paper industry and rooting system (produce a lot of secondary root and nitrogen fixater (Soebardjo 1986. The reforestation of logged-over Dipterocarp forests in Malaysia with A. mangium has also been considered (Appanah and Weinland 1989. Generally, reforestation with A. mangium is done with seedlings obtained by seed germination. A. mangium produce a lot of seeds but its production is still limited by the season, while the conventional method of vegetative propagation through cuttings gave very low percentage of rooted-cuttings (1% (Umboh and Syamsul Yani 1989. The micropropagation of A. mangium through tissue culture is a promising method. The production of A. mangium plantlets through that method has been done at the Forest Genetic Laboratory, Tropical Forest Biology, SEAMEO BIOTROP (Situmorang 1988, Umboh 1988, Umboh et al. 1989, 1990. These rooted-plantlets (plantlings were first put in the green house (acclimatization before planting in the field. Field tests of some agricultural plants have been done but information on forest trees species is still lacking because the production of plantlings through tissue culture is still limited as there are still problems of their rooting. In fact, the progress of reproducing woody plants by tissue culture has been much slower than with herbaceous plants. The major

  3. Soil water requirements of tissue-cultured Dwarf Cavendish banana ( Musa spp. L)

    Science.gov (United States)

    Shongwe, V. D.; Tumber, R.; Masarirambi, M. T.; Mutukumira, A. N.

    The banana is one of the most important fruit crops in the world. In terms of consumption, the banana fruit is ranked high yet there has not been much research particularly in relation to water requirements for propagules produced by tissue culture. In recent years, tissue culture banana planting material has become increasingly important due to its vigorous growth and high yields. The objective of this study was to investigate optimum soil water requirements of tissue-cultured banana. Dwarf Cavendish tissue-cultured plantlets grown in pots in a greenhouse were subjected to four irrigation regimes at 100% ETm, 85% ETm, 65% ETm, and 40% ETm. Plant parameters measured were leaf number, plant height, pseudo-stem girth, leaf length, leaf width, leaf area, leaf area index, leaf index, leaf colour, and plant vigour. Soil water potential measurements were also made over a three-month period. Differences between irrigating at 100% ETm and 85% ETm were not significantly ( P plant height, and plant height, compared to 65% and 40% ETm treatments. Pseudo-stem girth was highest from the 100% ETm compared to the other treatments. Economic yields of banana may be obtained with irrigation regimes ranging between 100% ETm and 85% ETm.

  4. 21 CFR 876.5885 - Tissue culture media for human ex vivo tissue and cell culture processing applications.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Tissue culture media for human ex vivo tissue and... DEVICES Therapeutic Devices § 876.5885 Tissue culture media for human ex vivo tissue and cell culture processing applications. (a) Identification. Tissue culture media for human ex vivo tissue and cell culture...

  5. Progress in planta transformation without tissue culture

    International Nuclear Information System (INIS)

    Gu Yunhong; Chinese Academy of Sciences, Hefei; Qin Guangyong; Huo Yuping; Yu Zengliang

    2004-01-01

    With the development of planta genetic engineering, more emphases have been laid on convenient and high efficient genetic transformation methods. And transformation without tissue culture is a prospective direction of it. In this paper, traditional transformation methods and the methods of non-tissue culture were summarized. With the exploration and application of Arabidopsis transformation mechanism, with the use of ion beam-mediated transformation invented by Chinese scientists and the development of other transformation methods, transformation methods without tissue culture and planta genetic engineering could be improved rapidly. (authors)

  6. Organelles genome stability of wheat plantlets produced by anther ...

    African Journals Online (AJOL)

    Yomi

    2012-03-15

    Mar 15, 2012 ... Key words: RFLP analysis, wheat plantlets, wheat anther culture, doubled haploids, genetic stability, mitochondria and chloroplast genome. INTRODUCTION. Wheat (Triticum aestivum L.) is one of the most important staple food crops of the family Poaceae. Among the food crops, wheat is a common source ...

  7. Regeneration of plantlets from nodal and shoot tip explants of ...

    African Journals Online (AJOL)

    Anoectochilus elatus Lindley is an endangered terrestrial orchid. A procedure for the regeneration of complete plantlets of A. elatus Lindley through node and shoot tip explants resulted directly in shoots when cultured on a full strength Murashige and Skoog (1962) medium supplemented with cytokinins at different ...

  8. Shoot and plantlet regeneration from meristems of Dioscorea ...

    African Journals Online (AJOL)

    In vitro culture media capable of regenerating moderate to high shoots and/or plantlets from meristems of two yam species – Dioscorea rotundata and Dioscorea alata within comparable duration of 10 weeks as commonly obtained in other monocots and root and tuber crops were investigated. The study comprised 125 ...

  9. Plant tissue culture--an opportunity for the production of nutraceuticals.

    Science.gov (United States)

    Lucchesini, Mariella; Mensuali-Sodi, Anna

    2010-01-01

    This chapter provides a short discussion about the opportunity to cultivate in vitro plant tissue of species which synthesize secondary metabolites of nutraceutical interest. The introduction of species of particular interest in cultivation and domestication, can be an alternative to the harvest of wild species. In vitro culture techniques are a useful tool to improve production and marketing nutraceutical species which allows to make a rapid clonal propagation of plants selected for their active principles. The techniques of tissue culture are described in detail. In particular, it is underlined the necessity to clone selected plants and produce true-type plants when standardized plant products are the main goal. This can be reached by conventional micropropagation protocols culturing plants in vitro through the five culture phases. Another approach consists in applying unconventional systems in the last phase of in vitro culture which permit to develop autotrophy of the explants. Autotrophic growth improves the quality of the multiplied shoots and facilitates the acclimatization of the plantlets.

  10. Antioxidant and phenolic acid profiles of tissue cultured and acclimatized Merwilla plumbea plantlets in relation to the applied cytokinins

    Czech Academy of Sciences Publication Activity Database

    Aremu, A.O.; Grúz, Jiří; Šubrtová, Michaela; Szüčová, Lucie; Doležal, Karel; Bairu, M. W.; Finnie, J. F.; van Staden, J.

    2013-01-01

    Roč. 170, č. 15 (2013), s. 1303-1308 ISSN 0176-1617 Grant - others:GA MŠk(CZ) ED0007/01/01 Program:ED Institutional research plan: CEZ:AV0Z50380511 Keywords : Conservation * Medicinal plants * Micropropagation Subject RIV: EF - Botanics Impact factor: 2.770, year: 2013

  11. Tissue Culture as a Source of Replicates in Nonmodel Plants: Variation in Cold Response in Arabidopsis lyrata ssp. petraea.

    Science.gov (United States)

    Kenta, Tanaka; Edwards, Jessica E M; Butlin, Roger K; Burke, Terry; Quick, W Paul; Urwin, Peter; Davey, Matthew P

    2016-12-07

    While genotype-environment interaction is increasingly receiving attention by ecologists and evolutionary biologists, such studies need genetically homogeneous replicates-a challenging hurdle in outcrossing plants. This could be potentially overcome by using tissue culture techniques. However, plants regenerated from tissue culture may show aberrant phenotypes and "somaclonal" variation. Here, we examined somaclonal variation due to tissue culturing using the response to cold treatment of photosynthetic efficiency (chlorophyll fluorescence measurements for F v /F m , F v '/F m ', and Φ PSII , representing maximum efficiency of photosynthesis for dark- and light-adapted leaves, and the actual electron transport operating efficiency, respectively, which are reliable indicators of photoinhibition and damage to the photosynthetic electron transport system). We compared this to variation among half-sibling seedlings from three different families of Arabidopsis lyrata ssp. petraea Somaclonal variation was limited, and we could detect within-family variation in change in chlorophyll fluorescence due to cold shock successfully with the help of tissue-culture derived replicates. Icelandic and Norwegian families exhibited higher chlorophyll fluorescence, suggesting higher performance after cold shock, than a Swedish family. Although the main effect of tissue culture on F v /F m , F v '/F m ', and Φ PSII was small, there were significant interactions between tissue culture and family, suggesting that the effect of tissue culture is genotype-specific. Tissue-cultured plantlets were less affected by cold treatment than seedlings, but to a different extent in each family. These interactive effects, however, were comparable to, or much smaller than the single effect of family. These results suggest that tissue culture is a useful method for obtaining genetically homogenous replicates for studying genotype-environment interaction related to adaptively-relevant phenotypes, such

  12. The autologus graft of epithelial tissue culture

    Directory of Open Access Journals (Sweden)

    Minaee B

    1999-08-01

    Full Text Available With the intention of research about culture and autologus graft of epithelial tissue we used 4 french Albino Rabbits with an average age of 2 months. After reproduction on the support in EMEM (Eagle's Minimum Essential Medium we used this for graft after 4 weeks. This region which grafted total replaced. After fixation of this sample and passing them through various process, histological sections were prepared. These sections were stained with H & E and masson's trichrome and studied by light microscope. We succeeded in graft. We hope in the near future by using the method of epithelium tissue culture improving to treat burned patients.

  13. Somatic Embryogenesis Cocoa Plantlets

    Directory of Open Access Journals (Sweden)

    Teguh man santoso

    2009-12-01

    Full Text Available AbstractPratylenchus coffeae is a parasitic nematoda that infected the roots of some plants, one of them is coffee. The Infection of Pratylenchus coffeae cause root tissue damage that led to root lession and make root become rotten, it will interfere the ability of roots to absorb water and nutrients in the soil which resulted in the growth of plants. At the moment, control of Pratylenchus coffeae are following integrated pests management (IPM program, which integrated between the use of coffee resistant clone and application of biological agents. Research on biological control was conducted more intensive, at the moment; due to it is friendlier save against environment and cheaper then using chemical nematicides. The research was conducted to know the effects of Micorrhiza Helper Bacteria (MHB,Pseudomonas diminuta and Bacillus subtilis in suppressing the population of P. coffeaeas well as their effect on growth of coffee seedling.  Coffee arabica (Coffea arabica L. seedling one moth old were used in the experiment. The experiment prepared with eight treatments and five  replications, as follows: A (Pseudomonas diminuta with density of 108 cfu / ml, B (Pseudomonas diminuta with density of 2x108 cfu / ml, C (Bacillus subtilis with density of 108 cfu / ml , D (Bacillus subtilis with density 2x108 cfu / ml, E (Carbofuran nematicide 5 g formulation / pot, F (Pseudomonas diminuta and Bacillus subtilis with each density of 108 cfu / ml, K- (Nematoda inoculation but without bacteria and nematicide, K+ (coffee seedling  without any additional treatment. The experiment was conducted for sixteen weeks or about four months. The results of the experiment showed that application of MHB could suppress population of P. coffeae and increase coffee seedling growth significantly. Inoculation of B. subtilis at 108 cfu per seedling suppressed significantly nematoda population of 71.3% compared with untreated seedling but inoculated with nematoda. It was not

  14. Substituted Indoleacetic Acids Tested in Tissue Cultures

    DEFF Research Database (Denmark)

    Engvild, Kjeld Christensen

    1978-01-01

    Monochloro substituted IAA inhibited shoot induction in tobacco tissue cultures about as much as IAA. Dichloro substituted IAA inhibited shoot formation less. Other substituted IAA except 5-fluoro- and 5-bromoindole-3-acetic acid were less active than IAA. Callus growth was quite variable...

  15. Substituted Indoleacetic Acids Tested in Tissue Cultures

    DEFF Research Database (Denmark)

    Engvild, Kjeld Christensen

    1978-01-01

    Monochloro substituted IAA inhibited shoot induction in tobacco tissue cultures about as much as IAA. Dichloro substituted IAA inhibited shoot formation less. Other substituted IAA except 5-fluoro- and 5-bromoindole-3-acetic acid were less active than IAA. Callus growth was quite variable and not...... and not correlated with auxin strength measured in the Avena coleoptile test....

  16. IN VITRO INOCULATION OF ASPARAGUS OFFICINALIS TISSUE CULTURE SHOOTS WITH FUSARIUM PROLIFERA TUM

    Directory of Open Access Journals (Sweden)

    A.K.MoHD OMAR

    1999-01-01

    Full Text Available Artificially inoculated asparagus tissue culture plantlets with a virulent fungus, Fusarium proliferatum showed signs of infection as early as 4 days after inoculat ion. Macroscopic observations revealed presence of early symptoms such as necrotic lesions at the affected area and light microscopic examinations clearly revealed the post-penetration events that took place including the destruction of surrounding cells. However, little is known of the hyphal activity or advancement on the host's surface at the initial stage after inoculation. Scanning electron microscopic examination clearly revealed the hyphal advancement on the surface and the mode of entrance into the host tissues beneath. Four days after inoculation, the fungi proceeded to spread out from the inoculation point onto the host surface which eventually developed into a sparse network of both aerial and non-aerial hyphae. Non-aerial hyphae form a network of mycelium that adheres to the surface and it's movement appeared to be oriented towards the stomata. Hyphal penetration occurs more often through the stomata, natural openings or wounds. In some cases, the hyphae crossed over the stomatal opening w ithout entering the host tissues. At places where the cuticle layer is absent or not well developed the hyphae successfully grew in between the epidermal cells into the tissues beneath.

  17. Induction of mutations in garlic by combined use of gamma-rays and tissue culture

    International Nuclear Information System (INIS)

    Zhen Hai Rou

    1997-01-01

    Callus cultures were initiated from leaf explants of garlic on MS medium supplemented with 1.0 mg/l KIN + 1.0 mg/l IAA + 2.0 mg/l 2,4-D. Plantlets were induced from leaf calli on MS medium with 2.0 mg/l BA + 2.0 mg/l IAA. Bulblets were induced from plantlets on MS medium containing 3.0 mg/l ITA. Callus growth and plantlets induction were remarkably inhibited with irradiation doses of 8 and 10 Gy. It was found that doses of 3 and 5 Gy were suitable to induce variation. Somatic embryos were induced on MS medium supplemented with 2.0 mg/l 2,4-D + 500 mg/l casein, 1000 mg/l yeast extract and 3 to 5% sucrose. (author). 11 tabs

  18. Genetic fidelity in micropropagated plantlets of Ochreinauclea ...

    African Journals Online (AJOL)

    Genetic fidelity in micropropagated plantlets of Ochreinauclea missionis an endemic, threatened and medicinal tree using ISSR markers. M Chandrika, VR Rai. Abstract. Inter simple sequence repeat (ISSR) markers were employed to determine the genetic fidelity of Ochreinauclea missionis plantlets multiplied in vitro by ...

  19. Somatic embryogenesis and plantlet regeneration from protoplast ...

    African Journals Online (AJOL)

    Somatic embryo development was observed either on half strength MS medium PGR-free or with 1 mg/l abscisic acid at 25°C under continuous illumination with fluorescent light. Maturated embryos germinated and then converted to plantlets on half strength MS medium containing 1 mg/l BA after 3 months. The plantlets left ...

  20. Morphogenesis and tissue culture of sweet orange (Citrus sinensis (L.) Osb.): effect of temperature and photosynthetic radiation

    International Nuclear Information System (INIS)

    Duran-Vila, N.; Gogorcena, Y.; Ortega, V.; Ortiz, J.; Navarro, L.

    1992-01-01

    Both incubation temperature and photosynthetic radiation affected morphogenesis, callus culture and plantlet culture of sweet orange (Citrus sinensis) cultured in vitro. Bud culture from nodal stem segments, regeneration of shoots and buds from internode stem segments and induction of primary callus were near optimal at incubation temperatures between 21–30°C. The optimal temperature for root formation was 27°C with temperatures above and below being clearly deleterious. Incubation in the dark or under low photosynthetic photon flux density (PPFD) was beneficial for callus induction and growth and also favored the production of rooted plantlets from bud cultures. Incubation in the dark improved considerably the regeneration of shoots and buds from internode segments and the recovery of whole plants. No off-types, as determined by protein and isoenzyme analysis, were observed among plantlets recovered from bud cultures or from regeneration of shoots from internode stem segments

  1. Radiation induced variation in potato for tolerance to salinity using tissue culture technique

    International Nuclear Information System (INIS)

    Sharabash, M.T.

    2001-01-01

    Meristem-tips of potato (Solanum tuberosum) cv. 'Diamant', obtained from tuber sprouts, were cultured on MS medium, and multiplied into plantlets through micropropagation. To induce variation for salt tolerance, the obtained plantlets were irradiated with 0, 20, and 40 Gy gamma rays at 27.7 rad/sec. Irradiated plantlets were cut into single nodes and cultured on MS medium, supplemented with 2000 and 4000 ppm NaCI. Salt tolerant plantlets were transferred for tuberization on MS liquid medium supplemented with the same concentration of NaCI. Micro-tubers, collected after 6 weeks of culture, had fresh weight between 0.03 to 0.3 g. Mini-tubers were obtained by planting micro-tubers in 25 cm pots under insect proof greenhouse. Mini-tuber number per plant ranged from 3 to 6, and the mini-tuber weight ranged from 0.5-3.0 g, depending upon the treatment. Further studies are in progress to produce conventional tubers under salinity stress from the promising variants, specially those tolerant to 4000 ppm, and to assure the stability of the obtained variants. (author)

  2. Tissue culture as a plant production technique for horticultural crops ...

    African Journals Online (AJOL)

    Over 100 years ago, Haberlandt envisioned the concept of plant tissue culture and provided the groundwork for the cultivation of plant cells, tissues and organs in culture. Initially plant tissue cultures arose as a research tool and focused on attempts to culture and study the development of small, isolated cells and segments ...

  3. Pathogen propagation in cultured three-dimensional tissue mass

    Science.gov (United States)

    Goodwin, Thomas J. (Inventor); Spaulding, Glenn F. (Inventor); Wolf, David A. (Inventor)

    2000-01-01

    A process for propagating a pathogen in a three-dimensional tissue mass cultured at microgravity conditions in a culture vessel containing culture media and a culture matrix is provided. The three-dimensional tissue mass is inoculated with a pathogen and pathogen replication in the cells of the tissue mass achieved.

  4. Gastrointestinal Epithelial Organoid Cultures from Postsurgical Tissues.

    Science.gov (United States)

    Hahn, Soojung; Yoo, Jongman

    2017-08-17

    An organoid is a cellular structure three-dimensionally (3D) cultured from self-organizing stem cells in vitro, which has a cell population, architectures, and organ specific functions like the originating organs. Recent advances in the 3D culture of isolated intestinal crypts or gastric glands have enabled the generation of human gastrointestinal epithelial organoids. Gastrointestinal organoids recapitulate the human in vivo physiology because of all the intestinal epithelial cell types that differentiated and proliferated from tissue resident stem cells. Thus far, gastrointestinal organoids have been extensively used for generating gastrointestinal disease models. This protocol describes the method of isolating a gland or crypt using stomach or colon tissue after surgery and establishing them into gastroids or colonoids.

  5. Genetic programming based models in plant tissue culture: An addendum to traditional statistical approach.

    Science.gov (United States)

    Mridula, Meenu R; Nair, Ashalatha S; Kumar, K Satheesh

    2018-02-01

    In this paper, we compared the efficacy of observation based modeling approach using a genetic algorithm with the regular statistical analysis as an alternative methodology in plant research. Preliminary experimental data on in vitro rooting was taken for this study with an aim to understand the effect of charcoal and naphthalene acetic acid (NAA) on successful rooting and also to optimize the two variables for maximum result. Observation-based modelling, as well as traditional approach, could identify NAA as a critical factor in rooting of the plantlets under the experimental conditions employed. Symbolic regression analysis using the software deployed here optimised the treatments studied and was successful in identifying the complex non-linear interaction among the variables, with minimalistic preliminary data. The presence of charcoal in the culture medium has a significant impact on root generation by reducing basal callus mass formation. Such an approach is advantageous for establishing in vitro culture protocols as these models will have significant potential for saving time and expenditure in plant tissue culture laboratories, and it further reduces the need for specialised background.

  6. Rose (Rosa hybrida L.) tissue culture mutagenesis for new mutants generation

    International Nuclear Information System (INIS)

    Salahbiah Abdul Majid; Rusli Ibrahim

    2004-01-01

    Tissue culture technique can be used to obtain complete regeneration of plant cells from shoots, rots, flowers, axillary buds and other parts of the plant. In this study, axillary buds from stem cuttings of Cutting Red, Christine Dior and Mini Rose varieties were used as the stating explants. Murashige and Skoog (1962) media supplemented with 6-Benzylaminopurine (BAP, at 4.44 - 8.88μM/l), Napthaleneacetic acid (NAA at 0.54μM/l),, nad 3% sucrose were used for plantlet initiation and regeneration. Cultured axillary buds were exposed to gamma ray (0.250 Gy/s) at 0, 15, 25, 35, 45, 55, 65 and 75 Gy for radiosensitivity test. From the dose respond curve, LD 5 0 the value for cutting red variety was 25 Gy, Christion Dior 30 Gy and Mini Rose 38 Gy, yet 22% of Mini Rose samples survived at 65 Gy and another 10% at 70 Gy. Screening of M3 plants of irradiated cultured shoots, 2 colour variations were obtained at 40 Gy for Cutting Red variety, while 3 colour variations for Mini Rose at 20 Gy. When 6 varieties of Fragrance Rose were irradiated at 40 Gy, 1 colour variation was obtained from 99 screened plants. This study suggests that the dose range of 20 to 45 can be considered for rose mutagenesis study to produce mutants. (Author)

  7. Application of 3D printing to prototype and develop novel plant tissue culture systems.

    Science.gov (United States)

    Shukla, Mukund R; Singh, Amritpal S; Piunno, Kevin; Saxena, Praveen K; Jones, A Maxwell P

    2017-01-01

    Due to the complex process of designing and manufacturing new plant tissue culture vessels through conventional means there have been limited efforts to innovate improved designs. Further, development and availability of low cost, energy efficient LEDs of various spectra has made it a promising light source for plant growth in controlled environments. However, direct replacement of conventional lighting sources with LEDs does not address problems with uniformity, spectral control, or the challenges in conducting statistically valid experiments to assess the effects of light. Prototyping using 3D printing and LED based light sources could help overcome these limitations and lead to improved culture systems. A modular culture vessel design in which the fluence rate and spectrum of light are independently controlled was designed, prototyped using 3D printing, and evaluated for plant growth. This design is compatible with semi-solid and liquid based culture systems. Observations on morphology, chlorophyll content, and chlorophyll fluorescence based stress parameters from in vitro plants cultured under different light spectra with similar overall fluence rate indicated different responses in Nicotiana tabacum and Artemisia annua plantlets. This experiment validates the utility of 3D printing to design and test functional vessels and demonstrated that optimal light spectra for in vitro plant growth is species-specific. 3D printing was successfully used to prototype novel culture vessels with independently controlled variable fluence rate/spectra LED lighting. This system addresses several limitations associated with current lighting systems, providing more uniform lighting and allowing proper replication/randomization for experimental plant biology while increasing energy efficiency. A complete procedure including the design and prototyping of a culture vessel using 3D printing, commercial scale injection molding of the prototype, and conducting a properly replicated

  8. Environmental scanning electron microscopy of the surface of normal and vitrified leaves of Gypsophila paniculata (Babies Breath) cultured in vitro.

    Science.gov (United States)

    Gribble, K; Sarafis, V; Nailon, J; Holford, P; Uwins, P

    1996-06-01

    Leaf surfaces of non-tissue-cultured, vitrified and non-vitrified plantlets of Gypsophila paniculata (Babies Breath) were examined using an environmental scanning electron microscope. Non-tissue-cultured plants had a complete epidermal surface, recessed stomata and wax present on the leaf surface. The surface of tissue-cultured plantlets appeared similar to non-tissue-cultured plants excepting stomata were slightly protruding and less wax appeared to be present. In both non-tissue-cultured and tissue-cultured plants stomata were found both opened and closed and were observed closing. In contrast vitrified plantlets had abnormal, malformed stomata which appeared non-functional. The ventral surfaces of leaves seemed more normal than the dorsal, this may be due to the former receiving more light. Additionally, discontinuities were found in the epidermis. Often epidermal holes were found in association with stomatal apertures. It is suggested that the main cause of desiccation of vitrified G. paniculata plantlets ex vitro is due to loss of water from the discontinuity in epidermis and not because of non-functional stomata. Liquid water could be seen through the epidermal holes indicating that at least some of the extra water in vitrified plantlets is contained in the intercellular spaces.

  9. Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System.

    Science.gov (United States)

    Kruithof, Boudewijn P T; Lieber, Samuel C; Kruithof-de Julio, Marianna; Gaussin, Vincian; Goumans, Marie José

    2015-10-19

    Heart valve disease is a major burden in the Western world and no effective treatment is available. This is mainly due to a lack of knowledge of the molecular, cellular and mechanical mechanisms underlying the maintenance and/or loss of the valvular structure. Current models used to study valvular biology include in vitro cultures of valvular endothelial and interstitial cells. Although, in vitro culturing models provide both cellular and molecular mechanisms, the mechanisms involved in the 3D-organization of the valve remain unclear. While in vivo models have provided insight into the molecular mechanisms underlying valvular development, insight into adult valvular biology is still elusive. In order to be able to study the regulation of the valvular 3D-organization on tissue, cellular and molecular levels, we have developed the Miniature Tissue Culture System. In this ex vivo flow model the mitral or the aortic valve is cultured in its natural position in the heart. The natural configuration and composition of the leaflet are maintained allowing the most natural response of the valvular cells to stimuli. The valves remain viable and are responsive to changing environmental conditions. This MTCS may provide advantages on studying questions including but not limited to, how does the 3D organization affect valvular biology, what factors affect 3D organization of the valve, and which network of signaling pathways regulates the 3D organization of the valve.

  10. Effect of radiation-degraded chitosan on growth promotion of flower plant in tissue culture

    Energy Technology Data Exchange (ETDEWEB)

    Luan, Le Quang; Ha, Vo Thi Thu; Hai, Le; Hien, Nguyen Quoc [Vietnam Atomic Energy Commission, Nuclear Research Institute, Dalat (Viet Nam); Nagasawa, Naotsugu; Yoshii, Fumio; Kume, Tamikazu [Japan Atomic Energy Research Inst., Takasaki, Gunma (Japan). Takasaki Radiation Chemistry Research Establishment

    2002-03-01

    Radiation is a useful tool for degradation of polysaccharides, such as starch, carrageenan, alginate and chitin/chitosan. The viscosity molecular weight (Mw) of chitosan with 80% degree of deacetylation was reduced to 1.5 x 10{sup 5} by irradiation of 50kGy in solid phase. The solution of 10% of chitosan with Mw ca. 15 x 10{sup 5} was then irradiated at doses ranging 10-250kGy for further degradation and the products were supplemented into cultural media for testing of plant growth promotion effect. The results indicated that irradiated chitosan showed a strong growth-promotion effect on the increase of the length of shoot, the length of root and fresh biomass for flower plants namely Limonium latifolium, Eustoma grandiflorum and Chrysanthemum morifolium in tissue culture. The growth-promotion effect was obtained by the treatments with 50ppm of chitosan irradiated at the doses of 75-100kGy in 10% solution. The suitable concentrations of chitosan irradiated at 100kGy are ca. 100ppm for C. morifolium, 30ppm for E. grandiflorum and 40ppm for L. latifolium. In addition, our study also indicated that the survival ratio of transferred flower plantlets treated with irradiated chitosan was improved after acclimatizing for 30 days in the greenhouse. Accordingly, it is concluded that degraded chitosan obtained by radiation degradation technique is effective as a plant growth promoter as well as irradiated alginate. (author)

  11. Clonal multiplication of Cymbidiums through tissue culture of the shoot meristem

    Energy Technology Data Exchange (ETDEWEB)

    Wimber, Donald E.

    1963-09-01

    The propagation of clonal varieties of some orchids is at times exasperatingly slow and occasionally an almost futile effort. Clonal multiplication is generally confined to dlvidlng mature plants and to starting plants from pseudobulbs. There is, of course, the specialized technique for obtaining Phalaenopsis plantlets from the aseptic culture of inflorescence nodes, but this is basically the same thing as propagating plants from pseudobulbs. In certain cases it is highly desirable to rapidly multiply certain clones of orchids. Awarded varieties could thereby be dispersed with great rapidity where now it may take decades for some clones to became fairly common. Commercial flower production would be very much enhanced if certain desirable clones could be multiplied ad infinitum within a short time. Orchid flower production could then be placed more on a par with many of the other cut flowers and the clonal peculiarities of some fo the current hybrids could be pampered instead of ignored. This paper describes a tissue culture method for the rapid propagation of Cymbidium clones.

  12. Genetic uniformity of sungkai (Peronema canescens Jack regenerated from tissue culture

    Directory of Open Access Journals (Sweden)

    MARIA IMELDA

    2007-01-01

    Full Text Available Sungkai or jati sebrang (Peronema canescens Jack is one of the industrial timber estate species native to Indonesia, which is commonly chosen for reforestation and as raw materials for the furniture and handicraft industry. In order to provide this planting material in large and sustainable quantities, a technique for in vitro propagation of sungkai through adventitious shoot proliferation is needed and has been successfully developed at the Research Centre for Biotechnology, LIPI. Since tissue culture method is prone to genetic variations, it is important to assess the genetic uniformity of sungkai planting materials derived from this in vitro method at an early stage. In this research, early detection of genetic uniformity was done by morphological observation of the regenerant plants and RAPD analysis using 4 primers namely OPB 5, OPB 9, OPH 11 and OPH 19. Morphological test showed differences in leaf shape, stem diameter and plant height among plantlets originating from Kalbar, Kaltim, Jambi and Cibinong. However, RAPD analysis with PCR showed that all planting materials were genetically uniform among those originating from the same or different places.

  13. Effect of Different Concentrations of Growth Regulators on Gardenia jasminoides cv. Veitchii Micropropagation by Tissue Culture Technique

    Directory of Open Access Journals (Sweden)

    G. R. Abdullah

    2003-01-01

    Full Text Available Micropropagation techniques were set up for Gardenia jasminoides c.v. veitchi. Many plantlets were obtained by culturing shoot cuttings in MS nutrient media, 30 g/L Sucrose, 7 g/L Agar Agar, and different concentrations of BAP and IAA. The best concentration was 1mg /L BAP with 0.5 mg/L IAA. This concentration gave the best sprout growth suitable for rooting in primary and secondary culture by reculturing the stuck cutting every 6 weeks and for many times. We also obtained a high rooting percentage up to 98 % of natural rooting in rooting media different from propagation media by reducing mineral salt concentration to half, Sucrose to 20gm/L, and 2gm/L active charcoal, and 1mg/L IAA. Plantlets were transferred to greenhous and subjected for hardening. This technique gave 22 plantlets from one cutting in one year.

  14. Nanotechnology, Cell Culture and Tissue Engineering

    Directory of Open Access Journals (Sweden)

    Kazutoshi Haraguchi

    2011-01-01

    Full Text Available We have fabricated new types of polymer hydrogels and polymer nanocomposites, i.e., nanocomposite gels (NC gels and soft, polymer nanocomposites (M-NCs: solid, with novel organic/inorganic network structures. Both NC gels and M-NCs were synthesized by in-situ free-radical polymerization in the presence of exfoliated clay platelets in aqueous systems and were obtained in various forms such as film, sheet, tube, coating, etc. and sizes with a wide range of clay contents. Here, disk-like inorganic clay nanoparticles act as multi-functional crosslinkers to form new types of network systems. Both NC gels and M-NCs have extraordinary optical and mechanical properties including ultra-high reversible extensibility, as well as a number of new characteristics relating to optical anisotropy, polymer/clay morphology, biocompatibility, stimuli-sensitive surfaces, micro-patterning, etc. For examples, the biological testing of medical devices, comprised of a sensitization test, an irritation test, an intracutaneous test and an in vitro cytotoxicity test,was carried out for NC gels and M-NCs. The safety of NC gels and M-NCs was confirmed in all tests. Also, the interaction of living tissue with NC gel was investigated in vivo by implantation in live goats; neither inflammation nor concrescence occurred around the NC gels. Furthermore, it was found that both N-NC gels consisting of poly(N-isopropylacrylamide(PNIPA/clay network and M-NCs consisting of poly(2-methoxyethyacrylate(PMEA/clay network show characteristic cell culture and subsequent cell detachment on their surfaces, although it was almost impossible to culture cells on conventional, chemically-crosslinked PNIPA hydrogels and chemically crossslinked PMEA, regardless of their crosslinker concentration. Various kinds of cells, such ashumanhepatoma cells (HepG2, normal human dermal fibroblast (NHDF, and human umbilical vein endothelial cells (HUVEC, could be cultured to be confluent on the surfaces of N

  15. Smallholder adoption and economic impacts of tissue culture ...

    African Journals Online (AJOL)

    This study was conducted with an objective of determining the correlates of adoption of tissue culture banana technology and its impacts on household incomes in Kenya. The results show that while some households have opted not to adopt tissue culture banana biotechnology, almost all the adopters are growing tissue ...

  16. Aeroponics for the culture of organisms, tissues and cells.

    Science.gov (United States)

    Weathers, P J; Zobel, R W

    1992-01-01

    Characteristics of aeroponics are discussed. Contrast is made, where appropriate, with hydroponics and aero-hydroponics as applies to research and commercial applications of nutrient mist technology. Topics include whole plants, plant tissue cultures, cell and microbial cultures, and animal tissue cultures with regard to operational considerations (moisture, temperature, minerals, gaseous atmosphere) and design of apparati.

  17. Development of germ-free plants and tissue culture

    Science.gov (United States)

    Venketeswaran, S.

    1973-01-01

    The botanical program is reported for experiments performed at the Lunar Receiving Laboratory. Papers prepared during this program are listed. The studies reported include: tissues cultured on various mediums, nutritional studies, preparation of plant cultures for Apollo 15, and pine tissue cultures.

  18. Qualitative and quantitative analysis of anthraquinone derivatives in rhizomes of tissue culture-raised Rheum emodi Wall. plants.

    Science.gov (United States)

    Malik, Sonia; Sharma, Nandini; Sharma, Upendra K; Singh, Narendra P; Bhushan, Shashi; Sharma, Madhu; Sinha, Arun K; Ahuja, Paramvir S

    2010-06-15

    This paper presents quantification of five anthraquinone derivatives (emodin glycoside, chrysophanol glycoside, emodin, chrysophanol and physcion) in rhizomes of hardened micro-propagated Rheum emodi plants using high-performance liquid chromatography (HPLC). Aseptic shoot cultures were raised using rhizome buds. Shoot multiplication occurred in both agar gelled and liquid Murashige and Skoog (MS) medium supplemented with 10.0 microM 6-benzylaminopurine (BAP) and 5.0 microM indole-3-butyric acid (IBA). Rooted plantlets obtained on plant growth regulator (PGR)-free medium were transferred to soil with 92% survival. HPLC analysis revealed the presence of five anthraquinone derivatives: emodin glycoside, chrysophanol glycoside, emodin, chrysophanol and physcion in rhizomes of tissue culture-raised plants. Only emodin glycoside (1) and chrysophanol glycoside (2) were present in 6-month-old hardened tissue cultured plants. In addition, the other three derivatives (emodin (3), chrysophanol (4) and physcion (5)) were also detected after 9 months. Copyright 2010 Elsevier GmbH. All rights reserved.

  19. In vitro Micropropagation of Cassava Through Low Cost Tissue Culture

    OpenAIRE

    Kwame O. Ogero; Gitonga N. Mburugu; Maina Mwangi; Omwoyo Ombori; Michael Ngugi

    2012-01-01

    Adoption of drought-tolerant crops such as cassava can help alleviate food insecurity in sub-Saharan Africa. However, production is constrained by lack of disease-free planting materials. This can be circumvented through tissue culture but the technology is costly limiting its adoption. There is therefore, need to put in place interventions that will reduce the cost of production hence making tissue culture products affordable. In this research, a low cost protocol for cassava tissue culture ...

  20. Study Progress on Tissue Culture of Maize Mature Embryo

    Science.gov (United States)

    Wang, Hongzhen; Cheng, Jun; Cheng, Yanping; Zhou, Xioafu

    It has been paid more and more attention on maize tissue culture as it is a basic work in maize genetic transformation, especially huge breakthrough has been made in maize tissue culture utilizing mature embryos as explants in the recent years. This paper reviewed the study progress on maize tissue culture and plant regeneration utilizing mature embryos as explants from callus induction, subculture, plant regeneration and browning reduction and so on.

  1. Citrus tissue culture employing vegetative explants.

    Science.gov (United States)

    Chaturvedi, H C; Singh, S K; Sharma, A K; Agnihotri, S

    2001-11-01

    Citrus being a number one fruit of the world due to its high nutritional value, huge production of fruits and fruit products, the citrus industry may be considered a major fruit industry. Though citrus orchard area in India is comparable to USA, the produce is far less, while its export is nil. Biotechnology has played an outstanding role in boosting the citrus industry, e.g., in Spain, which is now the biggest exporter of citrus fruit with the application of micrografting. Amongst the fruit trees, perhaps the maximum tissue culture research has been done in citrus during the past four decades, however, the results of practical value are meagre. The shortfalls in citrus tissue culture research and some advancements made in this direction along with bright prospects are highlighted, restricting the review to vegetative explants only. Whilst utilization of nucellar embryogenesis is limited to rootstocks, the other aspects, like, regeneration and proliferation of shoot meristems measuring 200 microm in length--a global breakthrough--of two commercially important scion species, Citrus aurantifolia and C. sinensis and an important rootstock, C. limonia, improvement of micrografting technique, cloning of the same two scion species as well as some Indian rootstock species, employing nodal stem segments of mature trees, of immense practical value have been elaborated. A rare phenomenon of shift in the morphogenetic pattern of differentiation from shoot bud differentiation to embryoid formation occurred during the long-term culture of stem callus of C. grandis. Stem callus-regenerated plants of C. aurantifolia, C. sinensis and C. grandis showed variation in their ploidy levels and a somaclonal variant of C. sinensis, which produced seedless fruits was isolated. Tailoring of rooting in microshoots to a tap root-like system by changing the inorganic salt composition of the rooting medium, resulting in 100% transplant success, and germplasm preservation through normal growth

  2. Iron induces ferritin synthesis in maize plantlets.

    Science.gov (United States)

    Lobreaux, S; Massenet, O; Briat, J F

    1992-07-01

    The iron-storage protein ferritin has been purified to homogeneity from maize seeds, allowing to determine the sequence of the first 29 NH2-terminal amino acids of its subunit and to raise specific rabbit polyclonal antibodies. Addition of 500 microM Fe-EDTA/75 microM Fe-citrate to hydroponic culture solutions of maize plantlets, previously starved for iron, led to a significant increase of the iron concentration of roots and leaves, albeit root iron was mainly found associated with the apoplast. Immunodetection of ferritin by western blots indicated that this iron treatment induced ferritin protein accumulation in roots and leaves over a period of 3 days. In order to investigate this induction at the ferritin mRNA level, various ferritin cDNA clones were isolated from a cDNA library prepared from poly(A)+ mRNA isolated from roots 48 h after iron treatment. These cDNAs were classified into two groups called FM1 and FM2. Upstream of the sequence encoding the mature ferritin subunit, both of these cDNAs contained an in-frame coding sequence with the characteristics of a transit peptide for plastid targeting. Two members of the FM1 subfamily, both partial at their 5' extremity, were characterized. They are identical, except in their 3' untranslated region: FM1A extends 162 nucleotides beyond the 3' terminus of FM1B. These two mRNAs could arise from the use of two different polyadenylation signals. FM2 is 96% identical to FM1 and contains 45 nucleotides of 5' untranslated region. Northern analyses of root and leaf RNAs, at different times after iron treatment, revealed ferritin mRNA accumulation in response to iron. Ferritin mRNA accumulation was transient and particularly abundant in leaves, reaching a maximum at 24 h. The level of ferritin mRNA in roots was affected to a lesser extent than in leaves.

  3. Crinivirus and begomovirus detection in tomato plantlets and weeds associated to nurseries

    Directory of Open Access Journals (Sweden)

    Ántony Solórzano Morales

    2017-04-01

    Full Text Available The aim of this work was to detect plant infections caused by Tomato chlorosis virus (ToCV and begomovirus in tomato plantlets, and in growing weeds around nursery greenhouses. During one year, starting in April 2008, 168 leaf tissue samples were collected, 90 tomato plantlets and 78 weeds from three different nurseries in Cartago province, Costa Rica. Reverse transcription and real time polymerase chain reaction (qRT-PCR was used to determine that 18,9% of tomato plantlets and 7,7% of weeds were infected with ToCV virus. Begomoviruses were detected using Dot Blot hybridization and non-radioactive probe. Next, hybridization results were confirmed using Rolling Circle Amplification (RCA followed by PCR, using universal primers. None tomato plantlet resulted positive when tested, but there were six weeds infected; in fact, Phytolacca icosandra and Brassica sp. were both coinfected with ToCV virus. These results suggest that ToCV infected tomato plantlets when commercialized, could serve as way of virus introduction to other country regions. Finally, weeds growing around greenhouses have shown to be potential viral sources of ToCV and begomovirus.

  4. Identification of phytochemical components of aloe plantlets by gas ...

    African Journals Online (AJOL)

    After plantlets production, extracts of A. vera plantlet were analyzed by gas chromatography-mass spectrometry (GC-MS). According to the results, 26 phytochemical compounds were identified. Results indicate that these compounds of micropropagated plantlets are similar to the phytochemical compounds identified by ...

  5. Provision of low cost media options for in vitro culture of Celosia sp.

    African Journals Online (AJOL)

    use

    2011-12-14

    Dec 14, 2011 ... (2007) have tried household sugar and tap water to substitution laboratory sucrose and double distilled water used in plant tissue culture. Besides successful in promoting the plantlet regeneration, by using the substitution items provide cost reducing in media culture preparation. This present study aims at ...

  6. Smallholder adoption and economic impacts of tissue culture ...

    African Journals Online (AJOL)

    PRECIOUS

    2009-12-01

    Dec 1, 2009 ... Kenya are yet to realize the full potential of tissue culture banana biotechnology. Key words: Biotechnology, adoption, tissue culture bananas, Kenya. INTRODUCTION. Most sub-Saharan African countries, if not all, are grappling with the challenge of food insecurity. Top of the agenda for world leaders today ...

  7. ENDIVE PLANTLETS FROM FREELY SUSPENDED CELLS AND CELL GROUPS GROWN IN VITRO.

    Science.gov (United States)

    VASIL, I K; HILDEBRANDT, A C; RIKER, A J

    1964-10-02

    Callus tissue derived from mature embryos of the endive, Cichorium endivia Linn. (family Compositae) grows and develops chlorophyll on a completely defined nutrient medium. The tissue breaks up into a thick suspentsion of cells and cell groups in a liquid medium kept in a flask on a shaker. Gradually, many small round masses of tissue, designated here as embryoids, are formed; these become differentiated and organized to form numnerous small plantlets having typical curled and fringed green leaves and roots.

  8. Acclimatization and leaf anatomy of micropropagated fig plantlets Aclimatização e anatomia foliar de plântulas de figo micropropagadas

    Directory of Open Access Journals (Sweden)

    Chrystiane Fráguas Chirinéa

    2012-12-01

    Full Text Available The survival of micropropagated plants during and after acclimatization is a limiting process to plant establishment. There is little information on how the anatomy of vegetative organs of Ficus carica can be affected by culture conditions and acclimatization. The present research aimed to study the effects of time on culture medium and substrates during the acclimatization of fig tree plantlets produced in vitro, characterizing some leaf anatomy aspects of plantlets cultured in vitro and of fig trees produced in field. Plantlets previously multiplied in vitro were separated and transferred into Wood Plant Medium (WPM where they were kept for 0, 15, 30, 45 and 60 days. Different substrates were tested and studies on leaf anatomy were performed in order to compare among plantlets grown in vitro, plantlets under 20, 40 and 60 days of acclimatization, and field grown plants. Keeping plantlets for 30 days in WPM allowed better development in Plantmax during acclimatization. Field grown plants presented higher number of stomata, greater epicuticular wax thickness and greater leaf tissue production compared to in vitro ones. The leaf tissues of in vitro plantlets show little differentiation and have great stomata number compared with acclimatized plants, which reduce the number of stomata during the acclimatization process.A sobrevivência de plantas micropropagadas durante e após a aclimatização é um processo limitante para o estabelecimento de plantas. Poucas informações são encontradas de como a anatomia de órgãos vegetativos de Ficus carica pode ser afetada pelas condições de cultivo e aclimatização. O presente trabalho teve por objetivo estudar os efeitos do tempo em meio de cultura e substratos durante a aclimatização de plântulas de figueira produzidas in vitro e cultivadas no campo. Plântulas multiplicadas in vitro foram separadas e transferidas para meio WPM, onde foram mantidas por 0; 15; 30; 45 e 60 dias. Diferentes

  9. Effect of lunar materials on plant tissue culture.

    Science.gov (United States)

    Walkinshaw, C. H.; Venketeswaran, S.; Baur, P. S.; Croley, T. E.; Scholes, V. E.; Weete, J. D.; Halliwell, R. S.; Hall, R. H.

    1973-01-01

    Lunar material collected during the Apollo 11, 12, 14, and 15 missions has been used to treat 12 species of higher plant tissue cultures. Biochemical and morphological studies have been conducted on several of these species. Tobacco tissue cultures treated with 0.22 g of lunar material exhibited increased greening more complex chloroplasts, less cytoplasmic vacuolation and greater vesiculation. Pine tissue cultures reacted to treatment by an increased deposition of tannin-like materials. The percentage of dry weight and soluble protein was increased in cultures treated with either lunar or terrestrial rock materials.

  10. Micropropagation of herbal plants for mass production of in vitro plantlets

    International Nuclear Information System (INIS)

    Sobri Hussein; Rusli Ibrahim

    2006-01-01

    Malaysia herbal industry is considered to be one of the most dynamic enterprises with annual growth estimated at 20 % a year. The total import value of the medicinal and aromatic plants increased from RM 141 million in 1986 to RM 431 million in 1996. Species that have been identified in terms of current priority are Eurycoma longifolia (Tongkat Ali), Labisia pumila (Kacip Fatimah), Andrographis paniculata (Hempedu Bumi), Morinda citrifolia (Mengkudu), Centella asiatica (Pegaga), Orthosiphon aristatus (Misai Kucing) and Gynura procumbens (Sambung Nyawa). Herbal and medicinal plants have a larger pool of genetic resources for the production of compounds valuable to the industry and human well being such as in pharmaceuticals, nutraceuticals and health food. The use of tissue culture technology has long been considered as an attractive solution to the problem of limited supply of raw materials for the industries. Kacip Fatimah (Labisia pumila) has been well known to increase female hormone, overcome tiredness and regain overall health. Misai Kucing (Orthosiphon aristatus) has proven to be active against kidney stones, high blood pressure, diabetes and gout and Sambung Nyawa (Gynura procumbens) is active against hypertension, diabetes and cancer. Tissue culture protocols had been optimized for the mass production of in vitro plantlets of these three selected herbal plants. (Author)

  11. The role of silicon in plant tissue culture

    OpenAIRE

    Sivanesan, Iyyakkannu; Park, Se Won

    2014-01-01

    Growth and morphogenesis of in vitro cultures of plant cells, tissues, and organs are greatly influenced by the composition of the culture medium. Mineral nutrients are necessary for the growth and development of plants. Several morpho-physiological disorders such as hooked leaves, hyperhydricity, fasciation, and shoot tip necrosis are often associated with the concentration of inorganic nutrient in the tissue culture medium. Silicon (Si) is the most abundant mineral element in the soil. The ...

  12. Basic Techniques in Mammalian Cell Tissue Culture.

    Science.gov (United States)

    Phelan, Katy; May, Kristin M

    2016-11-01

    Cultured mammalian cells are used extensively in cell biology studies. It requires a number of special skills in order to be able to preserve the structure, function, behavior, and biology of the cells in culture. This unit describes the basic skills required to maintain and preserve cell cultures: maintaining aseptic technique, preparing media with the appropriate characteristics, passaging, freezing and storage, recovering frozen stocks, and counting viable cells. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

  13. Application of Hanging Drop Technique for Kidney Tissue Culture.

    Science.gov (United States)

    Wang, Shaohui; Wang, Ximing; Boone, Jasmine; Wie, Jin; Yip, Kay-Pong; Zhang, Jie; Wang, Lei; Liu, Ruisheng

    2017-01-01

    The hanging drop technique is a well-established method used in culture of animal tissues. However, this method has not been used in adult kidney tissue culture yet. This study was to explore the feasibility of using this technique for culturing adult kidney cortex to study the time course of RNA viability in the tubules and vasculature, as well as the tissue structural integrity. In each Petri dish with the plate covered with sterile buffer, a section of mouse renal cortex was cultured within a drop of DMEM culture medium on the inner surface of the lip facing downward. The tissue were then harvested at each specific time points for Real-time PCR analysis and histological studies. The results showed that the mRNA level of most Na+ related transporters and cotransporters were stably maintained within 6 hours in culture, and that the mRNA level of most receptors found in the vasculature and glomeruli were stably maintained for up to 9 days in culture. Paraffin sections of the cultured renal cortex indicated that the tubules began to lose tubular integrity after 6 hours, but the glomeruli and vasculatures were still recognizable up to 9 days in culture. We concluded that adult kidney tissue culture by hanging drop method can be used to study gene expressions in vasculature and glomeruli. © 2017 The Author(s). Published by S. Karger AG, Basel.

  14. Ex vivo culture of patient tissue & examination of gene delivery.

    LENUS (Irish Health Repository)

    Rajendran, Simon

    2012-01-31

    This video describes the use of patient tissue as an ex vivo model for the study of gene delivery. Fresh patient tissue obtained at the time of surgery is sliced and maintained in culture. The ex vivo model system allows for the physical delivery of genes into intact patient tissue and gene expression is analysed by bioluminescence imaging using the IVIS detection system. The bioluminescent detection system demonstrates rapid and accurate quantification of gene expression within individual slices without the need for tissue sacrifice. This slice tissue culture system may be used in a variety of tissue types including normal and malignant tissue and allows us to study the effects of the heterogeneous nature of intact tissue and the high degree of variability between individual patients. This model system could be used in certain situations as an alternative to animal models and as a complementary preclinical mode prior to entering clinical trial.

  15. Effects of ionizing radiation on plant tissue cultures

    International Nuclear Information System (INIS)

    Hell, K.G.

    1978-01-01

    A short review is done of the biological effects of ionizing radiations on plant tissues kept in culture, from the work of Gladys King, in 1949, with X-ray irradiated tobacco. The role of plant hormones is discussed in the processes of growth inhibition and growth restoration of irradiated tissues, as well as morphogenesis. Radioresistance of cells kept in culture and the use of ionizing radiations as mutagens are also commented. Some aspects of the biological effects of ionizing radiations that need to be investigated are discussed, and the problem of genome instability of plant tissues kept in culture is pointed out. (M.A.) [pt

  16. Using Tissue Culture To Investigate Plant Cell Differentiation and Dedifferentiation.

    Science.gov (United States)

    Bozzone, Donna M.

    1997-01-01

    Describes an experimental project that uses plant tissue culture techniques to examine cell differentiation in the carrot. Allows students to gain experience in some important techniques and to explore fundamental questions about cell differentiation. (DDR)

  17. Cell/Tissue Culture Radiation Exposure Facility, Phase I

    Data.gov (United States)

    National Aeronautics and Space Administration — We propose to develop a Cell/Tissue Culture Radiation Exposure Facility (CTC-REF) to enable radiobiologists to investigate the real-time radiation effects on...

  18. Low technology tissue culture materials for initiation and ...

    African Journals Online (AJOL)

    Putting into account energy, labour and capital investments, the cost dropped from approximately US $ 1.5 to 1.0 per plantlet. Contamination was not observed when the media and equipment were sterilised using a pressure cooker instead of an autoclave. Use of plastic syringes instead of glass cylinders and micropipettes, ...

  19. Tissue-Culture Method of Cloning Rubber Plants

    Science.gov (United States)

    Ball, E. A.

    1983-01-01

    Guayule plant, a high-yield rubber plant cloned by tissue-culture method to produce multiple new plants that mature quickly. By adjusting culture medium, excised shoot tip produces up to 50 identical guayule plants. Varying concentration of cytokinin, single excised tip produces either 1 or several (up to 50) new plants.

  20. Laboratory Workflow Analysis of Culture of Periprosthetic Tissues in Blood Culture Bottles.

    Science.gov (United States)

    Peel, Trisha N; Sedarski, John A; Dylla, Brenda L; Shannon, Samantha K; Amirahmadi, Fazlollaah; Hughes, John G; Cheng, Allen C; Patel, Robin

    2017-09-01

    Culture of periprosthetic tissue specimens in blood culture bottles is more sensitive than conventional techniques, but the impact on laboratory workflow has yet to be addressed. Herein, we examined the impact of culture of periprosthetic tissues in blood culture bottles on laboratory workflow and cost. The workflow was process mapped, decision tree models were constructed using probabilities of positive and negative cultures drawn from our published study (T. N. Peel, B. L. Dylla, J. G. Hughes, D. T. Lynch, K. E. Greenwood-Quaintance, A. C. Cheng, J. N. Mandrekar, and R. Patel, mBio 7:e01776-15, 2016, https://doi.org/10.1128/mBio.01776-15), and the processing times and resource costs from the laboratory staff time viewpoint were used to compare periprosthetic tissues culture processes using conventional techniques with culture in blood culture bottles. Sensitivity analysis was performed using various rates of positive cultures. Annualized labor savings were estimated based on salary costs from the U.S. Labor Bureau for Laboratory staff. The model demonstrated a 60.1% reduction in mean total staff time with the adoption of tissue inoculation into blood culture bottles compared to conventional techniques (mean ± standard deviation, 30.7 ± 27.6 versus 77.0 ± 35.3 h per month, respectively; P < 0.001). The estimated annualized labor cost savings of culture using blood culture bottles was $10,876.83 (±$337.16). Sensitivity analysis was performed using various rates of culture positivity (5 to 50%). Culture in blood culture bottles was cost-effective, based on the estimated labor cost savings of $2,132.71 for each percent increase in test accuracy. In conclusion, culture of periprosthetic tissue in blood culture bottles is not only more accurate than but is also cost-saving compared to conventional culture methods. Copyright © 2017 American Society for Microbiology.

  1. Biotechnological applications of tissue culture to forest tree improvement.

    Science.gov (United States)

    Thorpe, T A

    1983-01-01

    Plant tissue culture techniques are of tremendous potential value to forest tree improvement. The technology is envisaged as playing a complementary role to traditional methods through exploiting spontaneous or induced genetic and epigenetic variability in culture, by use of haploidy and by the use of protoplasts. Haploids and protoplasts will aid in shortening breeding cycles and allow for unconventional crosses respectively. Clonal propagation is an integral part of any tree improvement program, and in addition can play an independent role in reforestation, clonal orchard establishment and in energy foresting. The goals, problems and limitations of these applications of tissue culture technology to forest tree improvement are indicated and assessed.

  2. [Issues of large scale tissue culture of medicinal plant].

    Science.gov (United States)

    Lv, Dong-Mei; Yuan, Yuan; Zhan, Zhi-Lai

    2014-09-01

    In order to increase the yield and quality of the medicinal plant and enhance the competitive power of industry of medicinal plant in our country, this paper analyzed the status, problem and countermeasure of the tissue culture of medicinal plant on large scale. Although the biotechnology is one of the most efficient and promising means in production of medicinal plant, it still has problems such as stability of the material, safety of the transgenic medicinal plant and optimization of cultured condition. Establishing perfect evaluation system according to the characteristic of the medicinal plant is the key measures to assure the sustainable development of the tissue culture of medicinal plant on large scale.

  3. (BAP) on meristem culture for virus free seed production of some ...

    African Journals Online (AJOL)

    Hiru

    2013-05-01

    May 1, 2013 ... tubers for ware potato production: Influence of size and plant spacing. Asian Potato J. 3:14-17. Sanavy SAMM, Moieni MJ (2003). Effects of different hormone combinations and planting beds on growth of single nodes and plantlets resulted from potato meristem culture. Plant Tissue Cult. 13(2):145-150.

  4. Use of diathermy for weeding heterogeneous tissue cultures.

    Science.gov (United States)

    Marks, R M; Penny, R

    1986-06-01

    Cultures generated from tissues consisting of multiple types of cells are often heterogeneous. Unless the cell type of interest has or can be given some selective growth advantage it may be overgrown by other cells. While developing techniques for the tissue culture of microvascular endothelial cells we evaluated an electrosurgical generator (diathermy) to selectively kill nonendothelial cells. Primary cell cultures were observed at X 100 magnification under phase contrast microscopy and a needle electrode apposed to the cell to be destroyed. A return electrode was constructed by placing a sterile clip in contact with the culture medium. The diathermy power setting controlled the area of lysis. Use of this technique allowed weeding of unwanted cells without damage to endothelial cells, which were able to grow to confluence in pure culture.

  5. Histology, histochemistry and SEM are useful tools to study regeneration processes in plant tissue culture

    Directory of Open Access Journals (Sweden)

    Piotr Żabicki

    2013-04-01

    Full Text Available Tissue cultures in vitroare used for the multiplication of plants via direct and indirect (via callus regeneration. This approach is commonly applied in the protection of endangered species by the introduction of regenerated in vitro plantlets to botanical gardens and to the nature (so called ex situ plant conservation. In vitroconditions, especially the supplementation of tissue culture media with plant growth regulators, cause a somaclonal variation, resulting in genetic differences among regenerated plants. To analyze callus structure, including cell shapes and sizes, cell differentiation (e.g. the presence of xylem vessels and regeneration processes (organogenesis, somatic embryogenesis, the histological, histochemical and SEM techniques are applied. In this study, to obtain regeneration of plants in culture conditions, we have used three Viola species (V. epipsilaLedeb., V. stagnina Kit. and V. uliginosaBesser, indicated to be critically endangered according to Polish Red Book of Plants (Kazmierczakowa & Zarzycki 2001 and two genotypes of a model plant Arabidopsis thaliana(L. Heynh. (Columbia-0 and an insertional cdkg ;2mutant line. An Arabidopsis homozygous cdkg ;2 knock-out originated from a T 3 generation of T-DNA insertional line SALK_090262 (Alonso et al. 2003 and has been selected from a subsequent T 4 generation based on PCR analysis using primers complementary to flanking positions of full-length cDNA of CDKG;2gene product (a clone isolated by Seki et al. 2002. The aims of the study were: 1 to select the most convenient method to obtain regenerated Violaplants with maternal genotype i.e., via direct organogenesis or somatic embryogenesis; 2 to determine the effect of mutation in CDKG;2 gene on the explant response to in vitroconditions, including callus proliferation and regeneration. In three Viola species organogenesis was induced on MS (Murashige and Skoog basal medium supplied with thidiazuron (TDZ in concentrations 0.5 mg

  6. EUPHORBIACEAE - A CRITICAL REVIEW ON PLANT TISSUE CULTURE

    Directory of Open Access Journals (Sweden)

    Rajesh Kondamudi

    2009-05-01

    Full Text Available The members of Euphorbiaceae are valuable source of different kinds of useful products like dyes, edible tubers, oil crops, furniture, agricultural implements, ornamental plants, pharmacological products, rubber, timber and aesthetic items. Micropropagation is an alternative mean of propagation that can be employed in conservation of the flora in relatively shorter time. Tissue culture is useful for multiplying and conserving the species, which are difficult to regenerate by conservation methods and save them from extinction. Cryopreservation of germplasm would help in maintaining the genetic diversity of the endangered population. Improved cell and tissue culture technologies would help in producing the active compounds in vitro with better productivities without cutting down the natural resources. There is sufficient progress at research level to suggest that the tissue culture of Euphorbiaceae can and should be further developed. This review emphasizes the in vitro manipulation and remarkable achievements with biotechnology in this family made during the last six decades.

  7. Yield improvement strategies for the production of secondary metabolites in plant tissue culture: silymarin from Silybum marianum tissue culture.

    Science.gov (United States)

    AbouZid, S

    2014-01-01

    Plant cell culture can be a potential source for the production of important secondary metabolites. This technology bears many advantages over conventional agricultural methods. The main problem to arrive at a cost-effective process is the low productivity. This is mainly due to lack of differentiation in the cultured cells. Many approaches have been used to maximise the yield of secondary metabolites produced by cultured plant cells. Among these approaches: choosing a plant with a high biosynthetic capacity, obtaining efficient cell line for growth and production of metabolite of interest, manipulating culture conditions, elicitation, metabolic engineering and organ culture. This article gives an overview of the various approaches used to maximise the production of pharmaceutically important secondary metabolites in plant cell cultures. Examples of using these different approaches are shown for the production of silymarin from Silybum marianum tissue culture.

  8. Identification of Stevioside Using Tissue Culture-Derived Stevia ( Leaves

    Directory of Open Access Journals (Sweden)

    Ziaul Karim Md.

    2015-01-01

    Full Text Available Stevioside is a natural sweetener from Stevia leaf, which is 300 times sweeter than sugar. It helps to reduce blood sugar levels dramatically and thus can be of benefit to diabetic people. Tissue culture is a very potential modern technology that can be used in large-scale disease-free stevia production throughout the year. We successfully produced stevia plant through in vitro culture for identification of stevioside in this experiment. The present study describes a potential method for identification of stevioside from tissue culture-derived stevia leaf. Stevioside in the sample was identified using HPLC by measuring the retention time. The percentage of stevioside content in the leaf samples was found to be 9.6%. This identification method can be used for commercial production and industrialization of stevia through in vitro culture across the world.

  9. Methods of epithelial tissue culture in albino rabbit skin

    Directory of Open Access Journals (Sweden)

    Anarluki J

    1998-05-01

    Full Text Available With the intention of research of various methods of epithelial tissue culture we've studied five French Albino rabbits with an average of 8 weeks. In order to evaluate and control growth and proliferation of autologus cultured tissue samples were obtained on 1st, 5th and 8th days. After fixation of these samples and passing them through various processes, histologic sections were prepared. These sections were stained with H-E and studied by light microscope, we succeeded in developing the original donor surface by 18 times.

  10. The Role of Silicon in Plant Tissue Culture

    Directory of Open Access Journals (Sweden)

    Iyyakkannu eSivanesan

    2014-10-01

    Full Text Available Growth and morphogenesis of in vitro cultures of plant cells, tissues and organs are greatly influenced by the composition of the culture medium. Mineral nutrients are necessary for the growth and development of plants. Several morpho-physiological disorders such as hooked leaves, hyperhydricity, fasciation and shoot tip necrosis are often associated with the concentration of inorganic nutrient in the tissue culture medium. Silicon (Si is the most abundant mineral element in the soil. The application of Si has been demonstrated to be beneficial for growth, development and yield of various plants and to alleviate various stresses including nutrient imbalance. Addition of Si to the tissue culture medium improves organogenesis, embryogenesis, growth traits, morphological, anatomical and physiological characteristics of leaves, enhances tolerance to low temperature and salinity, protects cells and against metal toxicity, prevents oxidative phenolic browning and reduces the incidence of hyperhydricity in various plants. Therefore, Si possesses considerable potential for application in a wide range of plant tissue culture studies such as cryopreservation, organogenesis, micropropagation, somatic embryogenesis and secondary metabolites production.

  11. The role of silicon in plant tissue culture.

    Science.gov (United States)

    Sivanesan, Iyyakkannu; Park, Se Won

    2014-01-01

    Growth and morphogenesis of in vitro cultures of plant cells, tissues, and organs are greatly influenced by the composition of the culture medium. Mineral nutrients are necessary for the growth and development of plants. Several morpho-physiological disorders such as hooked leaves, hyperhydricity, fasciation, and shoot tip necrosis are often associated with the concentration of inorganic nutrient in the tissue culture medium. Silicon (Si) is the most abundant mineral element in the soil. The application of Si has been demonstrated to be beneficial for growth, development and yield of various plants and to alleviate various stresses including nutrient imbalance. Addition of Si to the tissue culture medium improves organogenesis, embryogenesis, growth traits, morphological, anatomical, and physiological characteristics of leaves, enhances tolerance to low temperature and salinity, protects cells and against metal toxicity, prevents oxidative phenolic browning and reduces the incidence of hyperhydricity in various plants. Therefore, Si possesses considerable potential for application in a wide range of plant tissue culture studies such as cryopreservation, organogenesis, micropropagation, somatic embryogenesis and secondary metabolites production.

  12. Micromolded Gelatin Hydrogels for Extended Culture of Engineered Cardiac Tissues

    Science.gov (United States)

    McCain, Megan L.; Agarwal, Ashutosh; Nesmith, Haley W.; Nesmith, Alexander P.; Parker, Kevin Kit

    2014-01-01

    Defining the chronic cardiotoxic effects of drugs during preclinical screening is hindered by the relatively short lifetime of functional cardiac tissues in vitro, which are traditionally cultured on synthetic materials that do not recapitulate the cardiac microenvironment. Because collagen is the primary extracellular matrix protein in the heart, we hypothesized that micromolded gelatin hydrogel substrates tuned to mimic the elastic modulus of the heart would extend the lifetime of engineered cardiac tissues by better matching the native chemical and mechanical microenvironment. To measure tissue stress, we used tape casting, micromolding, and laser engraving to fabricate gelatin hydrogel muscular thin film cantilevers. Neonatal rat cardiac myocytes adhered to gelatin hydrogels and formed aligned tissues as defined by the microgrooves. Cardiac tissues could be cultured for over three weeks without declines in contractile stress. Myocytes on gelatin had higher spare respiratory capacity compared to those on fibronectin-coated PDMS, suggesting that improved metabolic function could be contributing to extended culture lifetime. Lastly, human induced pluripotent stem cell-derived cardiac myocytes adhered to micromolded gelatin surfaces and formed aligned tissues that remained functional for four weeks, highlighting their potential for human-relevant chronic studies. PMID:24731714

  13. Bridging the gap between cell culture and live tissue

    Directory of Open Access Journals (Sweden)

    Stefan Przyborski

    2017-11-01

    Full Text Available Traditional in vitro two-dimensional (2-D culture systems only partly imitate the physiological and biochemical features of cells in their original tissue. In vivo, in organs and tissues, cells are surrounded by a three-dimensional (3-D organization of supporting matrix and neighbouring cells, and a gradient of chemical and mechanical signals. Furthermore, the presence of blood flow and mechanical movement provides a dynamic environment (Jong et al., 2011. In contrast, traditional in vitro culture, carried out on 2-D plastic or glass substrates, typically provides a static environment, which, however is the base of the present understanding of many biological processes, tissue homeostasis as well as disease. It is clear that this is not an exact representation of what is happening in vivo and the microenvironment provided by in vitro cell culture models are significantly different and can cause deviations in cell response and behaviour from those distinctive of in vivo tissues. In order to translate the present basic knowledge in cell control, cell repair and regeneration from the laboratory bench to the clinical application, we need a better understanding of the cell and tissue interactions. This implies a detailed comprehension of the natural tissue environment, with its organization and local signals, in order to more closely mimic what happens in vivo, developing more physiological models for efficient in vitro systems. In particular, it is imperative to understand the role of the environmental cues which can be mainly divided into those of a chemical and mechanical nature.

  14. The use of animal tissues alongside human tissue: Cultural and ethical considerations.

    Science.gov (United States)

    Kaw, Anu; Jones, D Gareth; Zhang, Ming

    2016-01-01

    Teaching and research facilities often use cadaveric material alongside animal tissues, although there appear to be differences in the way we handle, treat, and dispose of human cadaveric material compared to animal tissue. This study sought to analyze cultural and ethical considerations and provides policy recommendations on the use of animal tissues alongside human tissue. The status of human and animal remains and the respect because of human and animal tissues were compared and analyzed from ethical, legal, and cultural perspectives. The use of animal organs and tissues is carried out within the context of understanding human anatomy and function. Consequently, the interests of human donors are to be pre-eminent in any policies that are enunciated, so that if any donors find the presence of animal remains unacceptable, the latter should not be employed. The major differences appear to lie in differences in our perceptions of their respective intrinsic and instrumental values. Animals are considered to have lesser intrinsic value and greater instrumental value than humans. These differences stem from the role played by culture and ethical considerations, and are manifested in the resulting legal frameworks. In light of this discussion, six policy recommendations are proposed, encompassing the nature of consent, respect for animal tissues as well as human remains, and appropriate separation of both sets of tissues in preparation and display. © 2015 Wiley Periodicals, Inc.

  15. A Method to Preclude Moisture Condensation in Plated Tissue Cultures

    Science.gov (United States)

    Alex M. Diner

    1992-01-01

    Excessive condensate normally accumulates in in vitro-illuminated petri dishes containing plant tissue cultures, causing avariety of problems. A dark-colored rubber net-mesh placed over the petri dishes prevented such condensation, even when charcoal-supplemented media are used under high light intensity in a growth chamber.

  16. Local Sugars Alternatives for Tissue Culture of Dendrobium Hybrid ...

    African Journals Online (AJOL)

    Nafiisah

    In developing countries, commercial exploitation of tissue culture technology is limited by high cost of production. ... cost alternative sugars for the in vitro micropropagation of Dendrobium cv. sonia was investigated. ... local growers are unable to meet the hotel demands (The president of the Republic of Mauritius, 2008).

  17. Banana Musa tissue culture plants enhanced by endophytic fungi

    African Journals Online (AJOL)

    Mo

    Merging biotechnology with biological control: Banana Musa tissue culture plants enhanced by endophytic fungi. T. Dubois, C. S. Gold, D. Coyne, P. Paparu, E. Mukwaba, S. Athman, S. Kapinduand E. Adipala1. International Institute of Tropical Agriculture, Southern and Eastern Africa Regional Centre, Namulonge. P.O. Box ...

  18. Evaluation of nutritional properties of tissue cultured sorghum ...

    African Journals Online (AJOL)

    Tissue culture techniques are commonly used in plants as an efficient way to propagate and store valuable genotypes. ... 1 and El Gardam) were developed at the Jomo Kenyatta University of Agriculture and Technology towards improvement for water stress tolerance for improved food production in the ASALs in Kenya.

  19. Assessing the determinants of tissue culture banana adoption in ...

    African Journals Online (AJOL)

    In this study cross-section data was used to analyze the effect of farmers' demographic, socioeconomic and institutional setting, market access and physical attributes on the probability and intensity of tissue culture banana (TCB) adoption. The study was carried out between July 2011 and November 2011. Both descriptive ...

  20. Plant Tissue Culture - Historical Developments and Applied Aspects

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 17; Issue 8. Plant Tissue Culture - Historical Developments and Applied Aspects. H R Dagla. General Article Volume 17 Issue 8 August 2012 pp 759-767. Fulltext. Click here to view fulltext PDF. Permanent link:

  1. Anthemideae: advances in tissue culture, genetics and transgenic ...

    African Journals Online (AJOL)

    Anthemideae: advances in tissue culture, genetics and transgenic biotechnology. Jaime A Teixeira da Silva. Abstract. Members of the Anthemideae include important floricultural (cut-flower) and ornamental (pot and garden) crops, as well as plants of medicinal and ethno-pharmacological interest. Despite the use of many of ...

  2. [Tissue culture of medicinal plant and abscisic acid].

    Science.gov (United States)

    Fang, Hui-Yong; Zhu, Hong; Yao, Jian-Xun; Jia, Cai-Feng; Shan, Gao-Wei; Li, Min-Hui

    2013-01-01

    Abscisic acid (ABA) plays a key role in many physiological processes of plants, and it was also applied to fields of medicinal plant biotechnology. The article presents a review of some recent application of ABA in enhancing the production of secondary metabolites of medicinal plants, improving the in vitro conservation in medicinal plant tissue culture system.

  3. Oxidative stress in recalcitrant tissue cultures of grapevine.

    Science.gov (United States)

    Benson, E E; Roubelakis-Angelakis, K A

    1994-03-01

    Thiobarbituric acid reactive substances (TBARS), and fluorescent compounds with spectral characteristics typical of products associated with oxidative stress in senescent and aging plant and animal cells, were detected in tissue cultures of the recalcitrant grapevine Vitis vinifera L. cultivar, Sultanina. These compounds increased during the early stages of dedifferentiation (callogenesis) of nodal stem explants. Catalase activity was not detected in the original explant, but was induced during callogenic dedifferentiation. Conversely, superoxide dismutase activity was detectable in the original explant, but diminished during the first week of callus induction. Transfer to callus induction medium promoted a large increase in the sulfhydryl content of nodal tissues. TBARS and fluorescent products accumulated in Sultanina callus during long-term culture (over 6 months). The possibility that oxidative stress may contribute to culture recalcitrance in this vine is discussed.

  4. The role of activated charcoal in plant tissue culture.

    Science.gov (United States)

    Thomas, T Dennis

    2008-01-01

    Activated charcoal has a very fine network of pores with large inner surface area on which many substances can be adsorbed. Activated charcoal is often used in tissue culture to improve cell growth and development. It plays a critical role in micropropagation, orchid seed germination, somatic embryogenesis, anther culture, synthetic seed production, protoplast culture, rooting, stem elongation, bulb formation etc. The promotary effects of AC on morphogenesis may be mainly due to its irreversible adsorption of inhibitory compounds in the culture medium and substancially decreasing the toxic metabolites, phenolic exudation and brown exudate accumulation. In addition to this activated charcoal is involved in a number of stimulatory and inhibitory activities including the release of substances naturally present in AC which promote growth, alteration and darkening of culture media, and adsorption of vitamins, metal ions and plant growth regulators, including abscisic acid and gaseous ethylene. The effect of AC on growth regulator uptake is still unclear but some workers believe that AC may gradually release certain adsorbed products, such as nutrients and growth regulators which become available to plants. This review focuses on the various roles of activated charcoal in plant tissue culture and the recent developments in this area.

  5. Variations on metabolic activities of legume tissues through radiation in tissue culture

    International Nuclear Information System (INIS)

    Batra, Amla

    1977-01-01

    Cell cultures from Arachis hypogaea L. cultivated in a modified medium developed by Murashige and Skoog (1962) showed vigorous qrowth after radiation treatment. Investigations on the effect of various sugars on the chlorophyll formation and growth of the irradiated tissues showed that sucrose was superior to maltose, glucose or fructose as a carbon source. Lactose and mannitol supported growth and development of chlorophyll to a less degree. On prolonging the cultures on a sugar free medium, the tissues failed to regain either growth or chlorophyll content. (author)

  6. In vitro germination, protocorm formation and plantlet development of mature versus immature seeds from several Ophrys species (Orchidaceae).

    Science.gov (United States)

    Kitsaki, C K; Zygouraki, S; Ziobora, M; Kintzios, S

    2004-11-01

    We investigated the effect of genotype, seed maturity and culture medium on the in vitro germination and development of protocorms and plantlets from seeds of 13 different Ophrys species (O. apifera, O. attica, O. cornuta, O. delfinensis, O. ferrum-equinum, O. lutea, O. mammosa, O. speculum, O. spruneri, O. umbilicata, O. argolica, O. irricolor and O. tenthredinifera) collected in Greece, some of which are endemic to this country. Mature seeds (10 months after collection) and immature seeds (2 months after anthesis) were cultured in a coconut milk-enriched or a pineapple-enriched medium (CEM or PEM, respectively). The highest percentage of callogenesis (96%) was observed in immature seeds of O. delphinensis in the CEM, while the highest percentage of protocorm formation (52%) was observed in mature seeds of O. spuneri in the CEM. Protocorm formation was significantly lower in immature seeds than in mature seeds in both culture media. Eventually almost all of the transferred protocorms developed to plantlets, which later formed minitubers. PEM appeared to be the most suitable for the development of minitubers from plantlets. All of the factors investigated--as well as their interactions--significantly affected callogenesis and protocorm formation. The results are discussed with the perspective of applying an improved protocol for in vitro seed germination and plantlet formation in several under-utilized Ophrys species.

  7. Improved Diagnosis of Prosthetic Joint Infection by Culturing Periprosthetic Tissue Specimens in Blood Culture Bottles

    Directory of Open Access Journals (Sweden)

    Trisha N. Peel

    2016-01-01

    Full Text Available Despite known low sensitivity, culture of periprosthetic tissue specimens on agars and in broths is routine. Culture of periprosthetic tissue samples in blood culture bottles (BCBs is potentially more convenient, but it has been evaluated in a limited way and has not been widely adopted. The aim of this study was to compare the sensitivity and specificity of inoculation of periprosthetic tissue specimens into blood culture bottles with standard agar and thioglycolate broth culture, applying Bayesian latent class modeling (LCM in addition to applying the Infectious Diseases Society of America (IDSA criteria for prosthetic joint infection. This prospective cohort study was conducted over a 9-month period (August 2013 to April 2014 at the Mayo Clinic, Rochester, MN, and included all consecutive patients undergoing revision arthroplasty. Overall, 369 subjects were studied; 117 (32% met IDSA criteria for prosthetic joint infection, and 82% had late chronic infection. Applying LCM, inoculation of tissues into BCBs was associated with a 47% improvement in sensitivity compared to the sensitivity of conventional agar and broth cultures (92.1 versus 62.6%, respectively; this magnitude of change was similar when IDSA criteria were applied (60.7 versus 44.4%, respectively; P = 0.003. The time to microorganism detection was shorter with BCBs than with standard media (P < 0.0001, with aerobic and anaerobic BCBs yielding positive results within a median of 21 and 23 h, respectively. Results of our study demonstrate that the semiautomated method of periprosthetic tissue culture in blood culture bottles is more sensitive than and as specific as agar and thioglycolate broth cultures and yields results faster.

  8. Mineralization and growth of cultured embryonic skeletal tissue in microgravity

    Science.gov (United States)

    Klement, B. J.; Spooner, B. S.

    1999-01-01

    Microgravity provides a unique environment in which to study normal and pathological phenomenon. Very few studies have been done to examine the effects of microgravity on developing skeletal tissue such as growth plate formation and maintenance, elongation of bone primordia, or the mineralization of growth plate cartilage. Embryonic mouse premetatarsal triads were cultured on three space shuttle flights to study cartilage growth, differentiation, and mineralization, in a microgravity environment. The premetatarsal triads that were cultured in microgravity all formed cartilage rods and grew in length. However, the premetatarsal cartilage rods cultured in microgravity grew less in length than the ground control cartilage rods. Terminal chondrocyte differentiation also occurred during culture in microgravity, as well as in the ground controls, and the matrix around the hypertrophied chondrocytes was capable of mineralizing in both groups. The same percentage of premetatarsals mineralized in the microgravity cultures as mineralized in the ground control cultures. In addition, the sizes of the mineralized areas between the two groups were very similar. However, the amount of 45Ca incorporated into the mineralized areas was significantly lower in the microgravity cultures, suggesting that the composition or density of the mineralized regions was compromised in microgravity. There was no significant difference in the amount of 45Ca liberated from prelabeled explants in microgravity or in the ground controls.

  9. Rice improvement involving altered flower structure more suitable to cross-pollination, using in vitro culture in combination with mutagenesis

    International Nuclear Information System (INIS)

    Min, S.K.

    1998-01-01

    Anther and somatic tissue culture in combination with mutagenesis were carried out to evaluate the efficiency of different mutagenic treatments of various in vitro culture materials, and to obtain some promising variants for rice improvement. Results indicated that in japonica rice radiation treatment of dry seeds and young panicles influenced the percentage of green plantlets regeneration from anther culture. Both treatments increased significantly the percentage of regenerated green plant lets in comparison with the control

  10. Hydrodynamic effects on cells in agitated tissue culture reactors

    Science.gov (United States)

    Cherry, R. S.; Papoutsakis, E. T.

    1986-01-01

    The mechanisms by which hydrodynamic forces can affect cells grown on microcarrier beads in agitated cell culture reactors were investigated by analyzing the motion of microcarriers relative to the surrounding fluid, to each other, and to moving or stationary solid surfaces. It was found that harmful effects on cell cultures that have been previously attributed to shear can be better explained as the effects of turbulence (of a size scale comparable to the microcarriers or the spacing between them) or collisions. The primary mechanisms of cell damage involve direct interaction between microcarriers and turbulent eddies, collisions between microcarriers in turbulent flow, and collisions against the impeller or other solid surfaces. The implications of these analytical results for the design of tissue culture reactors are discussed.

  11. Addressing the instability of DNA nanostructures in tissue culture.

    Science.gov (United States)

    Hahn, Jaeseung; Wickham, Shelley F J; Shih, William M; Perrault, Steven D

    2014-09-23

    DNA nanotechnology is an advanced technique that could contribute diagnostic, therapeutic, and biomedical research devices to nanomedicine. Although such devices are often developed and demonstrated using in vitro tissue culture models, these conditions may not be compatible with DNA nanostructure integrity and function. The purpose of this study was to characterize the sensitivity of 3D DNA nanostructures produced via the origami method to the in vitro tissue culture environment and identify solutions to prevent loss of nanostructure integrity. We examined whether the physiological cation concentrations of cell culture medium and the nucleases present in fetal bovine serum (FBS) used as a medium supplement result in denaturation and digestion, respectively. DNA nanostructure denaturation due to cation depletion was design- and time-dependent, with one of four tested designs remaining intact after 24 h at 37 °C. Adjustment of medium by addition of MgSO4 prevented denaturation. Digestion of nanostructures by FBS nucleases in Mg(2+)-adjusted medium did not appear design-dependent and became significant within 24 h and when medium was supplemented with greater than 5% FBS. We estimated that medium supplemented with 10% FBS contains greater than 256 U/L equivalent of DNase I activity in digestion of DNA nanostructures. Heat inactivation at 75 °C and inclusion of actin protein in medium inactivated and inhibited nuclease activity, respectively. We examined the impact of medium adjustments on cell growth, viability, and phenotype. Adjustment of Mg(2+) to 6 mM did not appear to have a detrimental impact on cells. Heat inactivation was found to be incompatible with in vitro tissue culture, whereas inclusion of actin had no observable effect on growth and viability. In two in vitro assays, immune cell activation and nanoparticle endocytosis, we show that using conditions compatible with cell phenotype and nanostructure integrity is critical for obtaining reliable

  12. Synthesis of the flavour precursor, alliin, in garlic tissue cultures.

    Science.gov (United States)

    Hughes, J; Tregova, A; Tomsett, A B; Jones, M G; Cosstick, R; Collin, H A

    2005-01-01

    The path of synthesis of alkyl cysteine sulphoxides, or flavour precursors, in the Alliums is still speculative. There are two proposed routes for alliin biosynthesis, one is from serine and allyl thiol while the other is from glutathione and an allyl source via gamma glutamyl peptides. The routes have been investigated by exposing undifferentiated callus cultures of garlic and onion to potential pathway intermediates. After a period of incubation of 2 days the callus was extracted, and analysed for flavour precursors and related compounds by HPLC. Standards of alliin, isoallin and propiin were synthesised and their identity confirmed by HPLC and NMR. Putative intermediates selected included the amino acids serine and cysteine, as well as more complex intermediates such as allylthiol, allyl cysteine and glutathione. Both garlic and onion tissue cultures were able to synthesize alliin following incubation with allylthiol, and cysteine conjugates such as allyl cysteine. The ability of the tissue cultures to form alliin from intermediates was compatible with the proposed routes of synthesis of alliin.

  13. Design of a miniature tissue culture system to culture mouse heart valves.

    Science.gov (United States)

    Lieber, Samuel C; Kruithof, Boudewijn P T; Aubry, Nadine; Vatner, Stephen F; Gaussin, Vinciane

    2010-03-01

    Valvular heart disease is a leading cause of morbidity and mortality in adults but little is known about the underlying etiology. A better understanding of the genetic and hemodynamic mechanisms involved in growth and remodeling of heart valves during physiological and pathological conditions is needed for a better understanding of valvular heart disease. Here, we report the design of a miniature tissue culture system (MTCS) that allows the culture of mitral valves from perinatal to adult mice. The design of the MTCS is novel in that fine positioning and cannulation can be conducted with hearts of different sizes (perinatal to adult). Perfusion of the heart and hence, culture of the mitral valve in its natural position, occurs in a hydraulically sealed culture bath environment. Using the MTCS, we successfully cultured the mitral valve of adult mouse hearts for 3 days. Histological analysis indicated that the cultured valves remained viable and their extracellular matrix organization was similar to age-matched native valves. Gene expression could also be modified in cultured valves by perfusion with medium containing beta-galactosidase-expressing adenovirus. Thus, the MTCS is a new tool to study the genetic and hemodynamic mechanisms underlying the three-dimensional organization of the heart valves, which could provide insights in the pathology of valvular heart disease and be used in animal models for the development of tissue-engineered heart valves.

  14. Identification of phytochemical components of aloe plantlets by gas ...

    African Journals Online (AJOL)

    MRT Pack 20 DVDs

    2013-12-04

    Dec 4, 2013 ... So, we can use this method (tissue culture) instead of aloe cultivation which is limited in some regions of the world. Key words: Aloe medicinal plant, phytochemical components, micropropagation, tissue culture, gas chromatography-mass spectrometry (GC-MS) analysis. INTRODUCTION. Aloe vera is a ...

  15. Cell Migration in Tissues: Explant Culture and Live Imaging.

    Science.gov (United States)

    Staneva, Ralitza; Barbazan, Jorge; Simon, Anthony; Vignjevic, Danijela Matic; Krndija, Denis

    2018-01-01

    Cell migration is a process that ensures correct cell localization and function in development and homeostasis. In disease such as cancer, cells acquire an upregulated migratory capacity that leads to their dissemination throughout the body. Live imaging of cell migration allows for better understanding of cell behaviors in development, adult tissue homeostasis and disease. We have optimized live imaging procedures to track cell migration in adult murine tissue explants derived from: (1) healthy gut; (2) primary intestinal carcinoma; and (3) the liver, a common metastatic site. To track epithelial cell migration in the gut, we generated an inducible fluorescent reporter mouse, enabling us to visualize and track individual cells in unperturbed gut epithelium. To image intratumoral cancer cells, we use a spontaneous intestinal cancer model based on the activation of Notch1 and deletion of p53 in the mouse intestinal epithelium, which gives rise to aggressive carcinoma. Interaction of cancer cells with a metastatic niche, the mouse liver, is addressed using a liver colonization model. In summary, we describe a method for long-term 3D imaging of tissue explants by two-photon excitation microscopy. Explant culturing and imaging can help understand dynamic behavior of cells in homeostasis and disease, and would be applicable to various tissues.

  16. 21 CFR 864.2220 - Synthetic cell and tissue culture media and components.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Synthetic cell and tissue culture media and... Products § 864.2220 Synthetic cell and tissue culture media and components. (a) Identification. Synthetic cell and tissue culture media and components are substances that are composed entirely of defined...

  17. Plant cell tissue culture: A potential source of chemicals

    Energy Technology Data Exchange (ETDEWEB)

    Scott, C.D.; Dougall, D.K.

    1987-08-01

    Higher plants produce many industrially important products. Among these are drugs and medicinal chemicals, essential oils and flavors, vegetable oils and fats, fine and specialty chemicals, and even some commodity chemicals. Although, currently, whole-plant extraction is the primary means of harvesting these materials, the advent of plant cell tissue culture could be a much more effective method of producing many types of phytochemicals. The use of immobilized plant cells in an advanced bioreactor configuration with excretion of the product into the reactor medium may represent the most straightforward way of commercializing such techniques for lower-value chemicals. Important research and development opportunities in this area include screening for plant cultures for nonmedical, lower-value chemicals; understanding and controlling plant cell physiology and biochemistry; optimizing effective immobilization methods; developing more efficient bioreactor concepts; and perfecting product extraction and purification techniques. 62 refs., 2 figs.

  18. Media Compositions for Three Dimensional Mammalian Tissue Growth Under Microgravity Culture Conditions

    Science.gov (United States)

    Goodwin, Thomas J. (Inventor)

    1998-01-01

    Normal mammalian tissue and the culturing process has been developed for the three groups of organ, structural and blood tissue. The cells are grown in vitro under microgravity culture conditions and form three dimensional cells aggregates with normal cell function. The microgravity culture conditions may be microgravity or simulated microgravity created in a horizontal rotating wall culture vessel.

  19. Media Compositions for Three-Dimensional Mammalian Tissue Growth under Microgravity Culture Conditions

    Science.gov (United States)

    Goodwin, Thomas J. (Inventor)

    1998-01-01

    Normal mammalian tissue and the culturing process has been developed for the three groups of organ, structural and blood tissue.The cells are grown in vitro under microgravity culture conditions and form three dimensional cells aggregates with normal cell function. The microgravity culture conditions may be microgravity or simulated microgravity created in a horizontal rotating wall culture vessel.

  20. Biomaterials and Culture Technologies for Regenerative Therapy of Liver Tissue.

    Science.gov (United States)

    Perez, Roman A; Jung, Cho-Rok; Kim, Hae-Won

    2017-01-01

    Regenerative approach has emerged to substitute the current extracorporeal technologies for the treatment of diseased and damaged liver tissue. This is based on the use of biomaterials that modulate the responses of hepatic cells through the unique matrix properties tuned to recapitulate regenerative functions. Cells in liver preserve their phenotype or differentiate through the interactions with extracellular matrix molecules. Therefore, the intrinsic properties of the engineered biomaterials, such as stiffness and surface topography, need to be tailored to induce appropriate cellular functions. The matrix physical stimuli can be combined with biochemical cues, such as immobilized functional groups or the delivered actions of signaling molecules. Furthermore, the external modulation of cells, through cocultures with nonparenchymal cells (e.g., endothelial cells) that can signal bioactive molecules, is another promising avenue to regenerate liver tissue. This review disseminates the recent approaches of regenerating liver tissue, with a focus on the development of biomaterials and the related culture technologies. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Molecular and biochemical characterization in Rauvolfia tetraphylla plantlets grown from synthetic seeds following in vitro cold storage.

    Science.gov (United States)

    Faisal, Mohammad; Alatar, Abdularhaman A; Hegazy, Ahmad K

    2013-01-01

    Synseed technology is one of the most important applications of plant biotechnology for in vitro conservation and regeneration of medicinal and aromatic plants. In the present investigation, synseeds of Rauvolfia tetraphylla were produced using in vitro-proliferated shoots upon complexation of 3 % sodium alginate and 100 mM CaCl(2). The encapsulated buds were stored at 4, 8, 12, and 16 °C and high conversion was observed in synseeds stored at 4 °C for 4 weeks. The effect of different medium strength on in vitro conversion response of synseed was evaluated and the maximum conversion (80.6 %) into plantlets was recorded on half-strength woody plant medium supplemented with 7.5 μM 6-benzyladenine and 2.5 μM α-naphthalene acetic acid after 8 weeks of culture. Plantlets with well-developed shoot and roots were hardened and successfully transplanted in field condition. After 4 weeks of transfer to ex vitro conditions, the performance of synseed-derived plantlets was evaluated on the basis of some physiological and biochemical parameters and compared with the in vivo-grown plants. Short-term storage of synthetic seeds at low temperature had no negative impact on physiological and biochemical profile of the plants that survived the storage process. Furthermore, clonal fidelity of synseed-derived plantlets was also assessed and compared with mother plant using rapid amplified polymorphic DNA and inter-simple sequence repeats analysis. No changes in molecular profiles were found among the regenerated plantlets and comparable to mother plant, which confirm the genetic stability among clones. This synseed protocol could be useful for in vitro clonal multiplication, conservation, and short-term storage and exchange of germplasm of this antihypertensive drug-producing plant.

  2. Discarded human fetal tissue and cell cultures for transplantation research

    International Nuclear Information System (INIS)

    Hay, R.J.; Phillips, T.; Thompson, A.; Vilner, L.; Cleland, M.; Tchaw-ren Chen; Zabrenetzky, V.

    1999-01-01

    A feasibility study has been performed to explore the utility of various tissues from discarded human abortuses for transplantation and related research. Specifically, aborted fetuses plus parental blood samples and all relevant clinical data were obtained through a local hospital complex. Whenever possible, pancreas, skin and skeletal muscle, heart, liver, kidney, cartilage and lung tissues were removed, dissociated and subfractionated for cryopreservation, characterization and cultivation trials in vitro. Existing protocols for these manipulations were compared and improved upon as required. Clonal culture, cell aggregate maintenance techniques and use of feeder cell populations have been utilized where appropriate to develop quantitative comparative data. Histological and biochemical assays were applied both to evaluate separation/cultivation methods and to identify optimal culture conditions for maintaining functional cells. Immunochemical and molecular biological procedures were applied to study expression of Major Histocompatibility Vomplex (MHC) class 1 and 11 molecules on cell lines derived. Tissue and cell culture populations were examined for infections with bacteria, ftingi, mycoplasma, HIV, CMV, hepatitis B and other viruses. Only 1% of the abortuses tested were virally infected. Cytogenetic analyses confin-ned the normal diploid status in the vast majority (>98%) of lines tested. A total of over 250 abortuses have been obtained and processed. Only 25 were found to be contaminated with bacteria or fungi and unsuitable for further cultivation trials. A total of over 200 cell populations were isolated, characterized and cryopreserved for further study. Included were kidney, lung, liver and epidermal epithelia: cartilage-derived cells from the spine and epiphyses plus myogenic myoblasts. Selected lines have been immortalized using HPV I 6E6/E7 sequences. Epithelia from the liver and pancreas and cardiac myocytes were the most problematic in that initial

  3. Methods for the Organogenesis of Skeletal Muscle in Tissue Culture

    Science.gov (United States)

    Vandenburgh, Herman; Shansky, Janet; DelTatto, Michael; Chromiak, Joseph

    1997-01-01

    Skeletal muscle structure is regulated by many factors, including nutrition, hormones, electrical activity, and tension. The muscle cells are subjected to both passive and active mechanical forces at all stages of development and these forces play important but poorly understood roles in regulating muscle organogenesis and growth. For example, during embryogenesis, the rapidly growing skeleton places large passive mechanical forces on the attached muscle tissue. These forces not only help to organize the proliferating mononucleated myoblasts into the oriented, multinucleated myofibers of a functional muscle but also tightly couple the growth rate of muscle to that of bone. Postnatally, the actively contracting, innervated muscle fibers are subjected to different patterns of active and passive tensions which regulate longitudinal and cross sectional myofiber growth. These mechanically-induced organogenic processes have been difficult to study under normal tissue culture conditions, resulting in the development of numerous methods and specialized equipment to simulate the in vivo mechanical environment.These techniques have led to the "engineering" of bioartificial muscles (organoids) which display many of the characteristics of in vivo muscle including parallel arrays of postmitotic fibers organized into fascicle-like structures with tendon-like ends. They are contractile, express adult isoforms of contractile proteins, perform directed work, and can be maintained in culture for long periods. The in vivo-like characteristics and durability of these muscle organoids make them useful for long term in vitro studies on mechanotransduction mechanisms and on muscle atrophy induced by decreased tension. In this report, we described a simple method for generating muscle organoids from either primary embrionic avain or neonatal rodent myoblasts.

  4. Tissue culture and mutagenesis of rain lily (zephyranthes)

    International Nuclear Information System (INIS)

    Mohd Nazir Basiran; Zaiton Ahmad; Shakinah Salleh; Shuhaimi Shamsudin; Aiza Shaliha Jamaludin

    2004-01-01

    There are three varieties of Zephyranthes used widely in landscaping due to their robust growth and attractive flowers in pink, yellow and white. Both in vivo and in vitro mutagenesis are an effective approach to increase the flower colour variations of Zephyranthes. In vitro propagation for the three varieties was attempted by using the induction medium developed by Sachar and Kapoor in 1959. The medium contains I ma of each indole 3-acetic acid (IAA), indole 3-butyric acid (IBA) and kinetin. Following surface sterilization of bulb scales, 17.8%, 10.5% and 10.7% of pink, white and yellow varieties respectively, were able to form small bulblets on the induction media. Further development of these bulblets into plantlets was also achieved on the same medium. Work is now being carried out to improve the efficiency of bulblet regeneration. Mutagenesis of Zephyranthes was initiated from bulbs of the pink varieties to develop new varieties with attractive combinations of flower colour and forms, shelf life and growth habits. These bulbs were irradiated using a gamma cell with a 60 Co source. Three variants with different flower colour and morphology have been achieved so far and are now being propagated in the nursery. (Author)

  5. Bone morphogenetic protein-induced cartilage development in tissue culture

    Energy Technology Data Exchange (ETDEWEB)

    Sato, K.; Urist, M.R.

    1984-03-01

    Outgrowths of mesenchyme-type cells from explants of allogeneic rat muscle onto a substratum of bone matrix containing bone morphogenetic protein (BMP) differentiate into cartilage. When BMP is chemically extracted from the bone matrix, the explanted cells develop only into fibrous tissue. When exogenous bovine BMP is introduced into the culture medium, either as a microsuspension or as a layer of particles between the matrix and the muscle cell tissue, cartilage develops at the interface between the matrix and the mesenchymal cell outgrowth. The chondrogenetic response is induced by as little as 2 micrograms of BMP; the optimum dose is 10 micrograms/40 mg (wet weight) of explant. The endogenous BMP equivalent for a comparable chondrogenetic response is about 0.6 micrograms/mg of allogeneic matrix. The minimum time for transfer of BMP to mesenchymal cell receptors is 1.0 hour, adequate time is 2.5 hours, and optimum time is approximately 5.0 hours. Measured in terms of incorporation of /sup 3/H-thymidine into DNA and of /sup 35/S sulfate into glycosaminoglycan, there is a latent period of one to three days preceeding the differentiation of mesenchyme-type cells into cartilage. During this latent period BMP-modulated mesenchymal cells disaggregate, migrate, reaggregate, and proliferate on new surfaces and constitute the morphogenetic phase of bone development. By the fourth day cells simultaneously undergo mitotic division, synthesize extracellular cartilage matrix, and establish the cytodifferentiation phase of development.

  6. The use of tissue culture techniques to detect irradiated vegetables

    International Nuclear Information System (INIS)

    Al-Safadi, B.; Sharabi, N.E.; Nabulsi, I

    2001-01-01

    the ability of two tissue culture methods, callus and vegetable growth induction, to detect irradiated vegetables was evaluated. Potato tubers, carrot roots, garlic cloves and onion bulbs were subjected to various gamma radiation doses (0, 25, 100, 150, 250, 500, 750, and 1000 Gy). Irradiated vegetables were cultured in vitro and in vivo (pots). Gamma irradiation significantly reduced callus-forming ability especially in carrot and potato where no callus was observed in doses higher than 50 Gy. Length of shoots and roots growing from irradiated garlic and onion explants was considerably reduced starting from the 25 Gy dose. No roots were formed on garlic explants at any irradiation dose. Garlic leaves growing from irradiated explants were spotted with purple to brown spots. The intensity of these spots increased as gamma ray dosage increased. In the pot experiment, potato plant appeared in the control only. On the contrary, a complete sprouting of garlic and onion was seen in all irradiation treatments. It was not possible to distinguish between the various irradiation treatments and the control 3 days after planting in pots. The two in vitro techniques, tested in our study, may effectively be used to detect irradiated vegetables and estimate the range of doses used. The callus formation method is more useful for potato and carrot, since regeneration of shoots in vitro from these two plants takes along time, making this method unpractical. The other technique is very useful in the case of onion and garlic since it is rapid. The two techniques can be used with most of the vegetables that can be cultured in vitro. (Author)

  7. Human epithelial tissue culture study on restorative materials.

    Science.gov (United States)

    Forster, András; Ungvári, Krisztina; Györgyey, Ágnes; Kukovecz, Ákos; Turzó, Kinga; Nagy, Katalin

    2014-01-01

    Health condition of the gingival tissues contacting the surfaces of fixed prostheses is a result of multiple etiologic factors. The aim of the investigation discussed here was to evaluate the attachment and proliferation rate of cultured human epithelial cells on three commonly used restorative materials under in vitro conditions. Morphological and chemical structure of polished lithium-disilicate (IPS e.max Press, Ivoclar Vivadent AG, Germany), yttrium modified zirconium dioxide (5-TEC ICE Zirkon Translucent, Zirkonzahn GmbH Srl, Germany) and cobalt chromium alloy (Remanium star, Dentaurum GmbH & Co. KG, Germany) discs were examined by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS) and atomic force microscopy (AFM). Human epithelial cells harvested and cultured from one donor, were applied to investigate cell attachment (24h observation) and proliferation (72h observation) via dimethylthiazol-diphenyl tetrazolium bromide (MTT) and AlamarBlue(®) (AB) assays on control surface (cell-culture plate) and on the restorative materials (n=3×20 specimens/material). SEM and AFM revealed typical morphology and roughness features for the materials. Zirconia presented significantly higher Ra value. EDS confirmed typical elements on the investigated restorative materials: lithium-disilicate (Si, O); Zirconia (Zi, Y, O); CoCr (Co, Cr, W). All surfaces except CoCr exhibited significant cell proliferation according to MTT and AB assays after 72h compared to 24h. Among the restorative materials, CoCr samples showed the highest cell attachment as indicated by MTT assay. AB results showed that attachment and proliferation of human epithelial cells is supported more on lithium-disilicate. Both assays indicated the lowest value for zirconia. The results indicate that the restorative materials examined are equally suitable for subgingival restorations. Lithium-disilicate exhibited the best biocompatibility. The examined materials are indicated for use

  8. High frequency induction of somatic embryos and plantlet ...

    African Journals Online (AJOL)

    High frequency induction of somatic embryos and plantlet regeneration from nodal explants of Hygrophila spinosa T. Anders. A Varshney, A Shahzad, M Anis. Abstract. An efficient protocol is described for the rapid in vitro plant regeneration of a medicinally important plant, Hygrophila spinosa through direct somatic ...

  9. Eucalyptus plantlet growth in relation to foliar application with ...

    African Journals Online (AJOL)

    In one experiment at greenhouse, condition was established to determine the effects of complete fertilizers on growth of plantlet Eucalyptus camaldulensis in Southeast of Iran. This experiment was conducted in the Agricultural Research Center, University of Zabol, in autumn year of 2008. This study was designed as a ...

  10. High frequency callus induction and plantlet regeneration from ...

    African Journals Online (AJOL)

    Most of the nodal segment derived calli got differentiated into roots rather shoots. Comparative callusing and shoot regeneration from different explants revealed that root segments are the best explant for in vitro studies in P. kurroa. The rooted plantlets were acclimatized to the external environment through hardening and ...

  11. Mass micropropagation of pineapple tissue culture using bioreactor technology

    International Nuclear Information System (INIS)

    Irwan Syafri; Amir Hamzah Harun; Rusli Ibrahim

    2005-01-01

    Pineapple (ananas comosus) is the most important fruit in terms of revenue earner in this country. The export of the canned pineapple is about 2 million standard cases annually valued at RM 60 million, while the export of fresh pineapple is about 40,000 tonnes worth about RM 10 million. The industry for canning is however, an ailing industry with production on the decline since the 70s. Scaling up the pineapple propagation using in vitro methods seems to be possible solutions for the lack of planting material. Temporary immersion system (TIS) has been described by Teisson and Alvard (1995) for plant tissue culture propagation. This system, also known as RITA, has been successfully used with embryogenic tissues of banana (Alvard et al 1993), coffee (Berthouly 1991), rubber (Etienne et al 1993) and sugarcane (Lorenzo et al 1998). In this study, the system has been set up with a potential capacity of 3 manifolds with 10 RITA each, to multiply meristem explants at different immersion periods. The system was compared with the conventional micropropagation system on solid medium. Both systems were treated with MS media containing 2.5 mg/l BAP and 0.1 NAA. In TIS the shoots were able to multiplied faster in comparison with solid media. The multiplication rates were increased up to 1:3 to 1:5 compared to normal propagation on solid media. The results show that TIS not only increase the propagation rates of pineapple but could also be adapted to reduce implementation costs to establish low-cost propagation systems. (Author)

  12. Embryonic myocardium shows increased longevity as a functional tissue when cultured in the presence of a noncardiac tissue layer.

    Science.gov (United States)

    Eisenberg, Leonard M; Eisenberg, Carol A

    2006-04-01

    A major aim of regenerative medicine is the construction of bioengineered organs and tissue for transplantation into human patients; yet living tissue is dynamic, and thus arranging cellular and extracellular constituents into an architecture resembling normal adult organs may not be sufficient to maintain tissue stability. In this study, we used cultures of embryonic chick heart tissue as a model to explore how newly formed cardiac tissue constructs can sustain their morphological structure and functional capabilities over extended periods. During the initial days of incubation, embryonic cardiac explants will thrive as beating three-dimensional tissue aggregates. However, within the first week of culture, cardiac aggregates lose their contractile function and flatten. After 2 weeks of incubation, the cardiac cells will have spread out into a homogeneous monolayer and dedifferentiated to a noncardiac phenotype. In contrast, when the embryonic heart tissue was co-cultured with a noncardiac cell layer obtained from adult bone marrow, the cardiac aggregates maintained their contractile function, three-dimensional tissue morphology, and myocyte phenotype for a full month of incubation. The capacity of this noncardiac cell layer to sustain the phenotype and morphology of the cardiac explants was partially replicated by treatment of the heart tissue with conditioned media from bone marrow cells. These findings are discussed in regard to the importance of adjacent cell layers for facilitating organogenesis in the developing embryo and having potential utility in producing stable bioengineered tissue constructs.

  13. [Application of plant tissue culture in field of Chinese medicine resources].

    Science.gov (United States)

    Wang, Juan; Li, Jin-Xin; Li, Jian-Li; Gao, Wen-Yuan

    2017-06-01

    Plant tissue culture technology has been widely used in the field of traditional Chinese medicine(TCM) resources with its unique advantages, playing an important role in the protection of TCM resources. In this review, some applications of plant tissue culture were summarized, including production of active compounds by using plant tissue culture, genetic diversity analysis, Dao-di herbs, elicitor application, biosynthesis and transgenic plants. Through the above researches will promote the further development of plant tissue culture technology, making it play a greater role in the field of TCM resources. Copyright© by the Chinese Pharmaceutical Association.

  14. Micro fluidic System for Culturing and Monitoring of Neuronal Cells and Tissue

    DEFF Research Database (Denmark)

    Bakmand, Tanya; Waagepetersen, Helle S.

    The aim of this Ph.D. project was to combine experience within cell and tissue culturing, electrochemistry and microfabrication in order to develop an in vivo-like fluidic culturing platform, challenging the traditional culturing methods. The first goal was to develope a fluidic system for cultur...

  15. Enhancement of Diosgenin Production in Plantlet and Cell Cultures ...

    African Journals Online (AJOL)

    HSCCC),. Sephadex LH-20 chromatography and preparative .... phase of acetonitrile-water (90:10, v/v) at a flow rate of 1 mL/min at 30 ºC, and an LC ..... Spirobisnaphthalenes from fungi and their biological activities. Mini-Rev Med Chem 2010; 10: ...

  16. Regeneration of plantlets from unpollinated ovary cultures of ...

    African Journals Online (AJOL)

    Tsega

    2013-09-25

    % ethanol for 1 min, follo- wed by 20% sodium hypochlorite of the original stock 5.25% and four drops of Tween 20 for 10 min, and then rinsed with sterile double distilled water. Unpollinated ovaries were excised aseptically.

  17. NMR-based metabolomics of mammalian cell and tissue cultures

    International Nuclear Information System (INIS)

    Aranibar, Nelly; Borys, Michael; Mackin, Nancy A.; Ly, Van; Abu-Absi, Nicholas; Abu-Absi, Susan; Niemitz, Matthias; Schilling, Bernhard; Li, Zheng Jian; Brock, Barry; Russell, Reb J.; Tymiak, Adrienne; Reily, Michael D.

    2011-01-01

    NMR spectroscopy was used to evaluate growth media and the cellular metabolome in two systems of interest to biomedical research. The first of these was a Chinese hamster ovary cell line engineered to express a recombinant protein. Here, NMR spectroscopy and a quantum mechanical total line shape analysis were utilized to quantify 30 metabolites such as amino acids, Krebs cycle intermediates, activated sugars, cofactors, and others in both media and cell extracts. The impact of bioreactor scale and addition of anti-apoptotic agents to the media on the extracellular and intracellular metabolome indicated changes in metabolic pathways of energy utilization. These results shed light into culture parameters that can be manipulated to optimize growth and protein production. Second, metabolomic analysis was performed on the superfusion media in a common model used for drug metabolism and toxicology studies, in vitro liver slices. In this study, it is demonstrated that two of the 48 standard media components, choline and histidine are depleted at a faster rate than many other nutrients. Augmenting the starting media with extra choline and histidine improves the long-term liver slice viability as measured by higher tissues levels of lactate dehydrogenase (LDH), glutathione and ATP, as well as lower LDH levels in the media at time points out to 94 h after initiation of incubation. In both models, media components and cellular metabolites are measured over time and correlated with currently accepted endpoint measures.

  18. Flowering of Woody Bamboo in Tissue Culture Systems

    Directory of Open Access Journals (Sweden)

    Jin-Ling Yuan

    2017-09-01

    Full Text Available Flowering and subsequent seed set are not only normal activities in the life of most plants, but constitute the very reason for their existence. Woody bamboos can take a long time to flower, even over 100 years. This makes it difficult to breed bamboo, since flowering time cannot be predicted and passing through each generation takes too long. Another unique characteristic of woody bamboo is that a bamboo stand will often flower synchronously, both disrupting the supply chain within the bamboo industry and affecting local ecology. Therefore, an understanding of the mechanism that initiates bamboo flowering is important not only for biology research, but also for the bamboo industry. Induction of flowering in vitro is an effective way to both shorten the flowering period and control the flowering time, and has been shown for several species of bamboo. The use of controlled tissue culture systems allows investigation into the mechanism of bamboo flowering and facilitates selective breeding. Here, after a brief introduction of flowering in bamboo, we review the research on in vitro flowering of bamboo, including our current understanding of the effects of plant growth regulators and medium components on flower induction and how in vitro bamboo flowers can be used in research.

  19. Cloning of medicinal plants through tissue culture--a review.

    Science.gov (United States)

    Chaturvedi, H C; Jain, Madhu; Kidwai, N R

    2007-11-01

    In order to have standardized formulations, the chemical constituents from plants and their parts are required to be uniform both qualitatively and quantitatively. Furthermore, an ever increasing demand of uniform medicinal plants based medicines warrants their mass cloning through plant tissue culture strategy. A good number of medicinal plants have been reported to regenerate in vitro from their various parts, but a critical evaluation of such reports reveals that only a few complete medicinal plants have been regenerated and still fewer have actually been grown in soil, while their micropropagation on a mass scale has rarely been achieved, particularly in those medicinal plants where conventional propagation is inadequate, like, the mass clonal propagation of Dioscorea floribunda leading to its successful field trials. Such facts make it imperative to document the factual position of micropropagation of medicinal plants bringing out the advancements made along with the short falls, in this important area. The present review deals with the futuristic view on the said subject restricted to higher plants.

  20. Gamma radiation effects on growth and pattern of protein synthesis organ and tissue cultures of sweet potato (Ipomoea L. Lam)

    International Nuclear Information System (INIS)

    El-Fiki, A.A.; Moustafa, M.A.M.; Sayed, A.I.H.

    2004-01-01

    Buds, shoot and leaf explants from sweet potato cvs. Mabruka and abees were exposed to different gamma radiation doses. Shoot and leaf explants were irradiated with doses (o,10, 20, 30 and Gy) and cultured on MS medium supplemented with 2, 4- D + KIN to induce the callus. Growths of produced calli were lower than the control. Suvival calli were transferred to regeneration medium. Irradiated shoot callus only was able to differentiate to roots and the No. of root / calluses were decreased with increasing gamma radiation dose. The all irradiated calli failed to regenerate to plants. However, buds were irradiated with doses; 10, 20, 30, 40, 50, 60, 70 and 80 Gy. Gamma radiation doses caused the death of 6-75 % explants, also shoot length was decreased to 0.5-4.3 cm. The study of gamma radiation on the some chemical alterations in plantlets exhibited that, ion accumulation (Na, K, Ca and Mg) was decreased. Also, carbohydrates (total carbohydrate and reducing sugars) were decreased. Phenolic commpounds in irradiated plantlets were increased

  1. Short Communication: Effect of mulching materials on mini tuber production of potato from in vitro plantlets

    Directory of Open Access Journals (Sweden)

    D.A.N. MAJUMDER

    2016-06-01

    Full Text Available Abstract. Majumder DAN, Nath SC, Kabir MA, Majumder S. 2016. Effect of mulching materials on mini tuber production of potato from in vitro plantlets. Nusantara Bioscience 8: 123-127. This paper describes the effectiveness of organic and plastic mulching for potato mini tuber production in Bangladesh. The field experiment was carried out during the Rabi season of 2013-2014 to 2014-2015 with virus free in vitro cultured plantlets of var. Diamant. The mulching materials of water hyacinth mulch (WHM, rice straw mulch (RSM and black polythene mulch (BPM were compared to no-mulching (control to find out suitable mulching material (s for obtaining higher tuber yield. During the whole production period morphological characters, yield characters as well as of soil temperature and soil moisture were assessed. The results showed that WHM (5.28 t/ac and RSM (4.59 t/ac had a positive effect on increased the proportion of tuber size above 28 mm and on increasing of tuber yields by 54.0% to 77.2% compared with control (2.98 t/ac. Higher soil temperatures were recorded with plastic mulch caused lower potato tuber yield (3.04 t/ac while WHM and RSM decreased soil temperatures and increased the moisture percentage. The mulching of mini tubers had negative effect on tubers quality in regards of scab, green tuber and weed biomass.

  2. Procedure for identifying and prevent risks in in vitro sugarcane plantlets marketing

    Directory of Open Access Journals (Sweden)

    Ana Rosa Hernández Freire

    2014-10-01

    Full Text Available Seed Banks and agricultural farms benefit greatly from plant in vitro Culture techniques, for their rapid propagation processes and the production of desease-free plantlets. However, the production and commercialization process can fail and it is not as successful l as it was supposed to. The objective of this study is to identify potential failure modes at the acclimatiztion stage of plantlets to ex vitro conditions at Estación Territorial de Investigaciones de la Caña de Azúcar de Villa Clara (ETICA. A methodology of Failure Mode and Effects Analysis (FMEA was applied. A value of 80 or higher was determined to be the Risk Priority Number (RPN and the more relevant failure modes were defined a t the acclimatization and commercialization stages and ground rules and assumptions were established in order to prevent and control failure modes of the process. The study and implementation of the methodology to other processes at ETICA is strongly recommended.

  3. THE EFFICIENCY OF SHOOT AND PLANTLET FORMATION OF Cephaelis ipecacuanha AFTER THREE SUBCULTURES IN VITRO

    Directory of Open Access Journals (Sweden)

    Osmar Alves Lameira

    1994-12-01

    Full Text Available Multiple adventitious shoot formed from internodal segments of Cephaelis ipecacuanha cultured 25 days on Gamborg basal medium (GAMBORG et al., 1968 supplemented with 6.66mM 6-benzylaminopurine there was a maximum of nine shoots per segment and an average of five shoots per segment formed. The presence of gibberellic acid in the subculture media promoted shoot elongation in all treatments. The shoots attained 3cm in height and rooting of 100% after 35 days of culturing upon Murashige and Skoog's basal medium (MS, added with 4.92mM indole-3-butyric acid, 0.87m gibberellic acid and 0.1% activated charcoal. Further growth was accelerated after the transfer to 1/2 MS without growth regulators. Rooted plantlets transferred to potting soil could be successfully established.

  4. Somatic embryogenesis and plantlet regeneration from protoplast ...

    African Journals Online (AJOL)

    Administrator

    2010-05-30

    May 30, 2010 ... (Isa et al., 1990; Brodelius and Nilsson, 1980; Karamian and Ebrahimzadeh, 2001; Karamian, 2007). In this experiment, the original medium was frequently replaced with a medium with lower osmotic pressure. Protoplast culture is usually successful when medium changes are frequently performed to allow ...

  5. Advances in tissue engineering through stem cell-based co-culture.

    Science.gov (United States)

    Paschos, Nikolaos K; Brown, Wendy E; Eswaramoorthy, Rajalakshmanan; Hu, Jerry C; Athanasiou, Kyriacos A

    2015-05-01

    Stem cells are the future in tissue engineering and regeneration. In a co-culture, stem cells not only provide a target cell source with multipotent differentiation capacity, but can also act as assisting cells that promote tissue homeostasis, metabolism, growth and repair. Their incorporation into co-culture systems seems to be important in the creation of complex tissues or organs. In this review, critical aspects of stem cell use in co-culture systems are discussed. Direct and indirect co-culture methodologies used in tissue engineering are described, along with various characteristics of cellular interactions in these systems. Direct cell-cell contact, cell-extracellular matrix interaction and signalling via soluble factors are presented. The advantages of stem cell co-culture strategies and their applications in tissue engineering and regenerative medicine are portrayed through specific examples for several tissues, including orthopaedic soft tissues, bone, heart, vasculature, lung, kidney, liver and nerve. A concise review of the progress and the lessons learned are provided, with a focus on recent developments and their implications. It is hoped that knowledge developed from one tissue can be translated to other tissues. Finally, we address challenges in tissue engineering and regenerative medicine that can potentially be overcome via employing strategies for stem cell co-culture use. Copyright © 2014 John Wiley & Sons, Ltd.

  6. Comparative study on Allium schoenoprasum cultivated plant and Allium schoenoprasum tissue culture organs antioxidant status.

    Science.gov (United States)

    Stajner, D; Popović, B M; Calić-Dragosavac, D; Malenčić, D; Zdravković-Korać, S

    2011-11-01

    This study was designed to examine Allium schoenoprasum tissue culture organs antioxidant and scavenging activity and to make a comparison between Allium schoenoprasum cultivated plant and Allium schoenoprasum tissue culture organs antioxidant activity. This study reports the results on the root, stalk and leaf antioxidant enzyme activities (superoxide dismutase, catalase, guaiacol peroxidase and glutathione peroxidase), reduced glutathione quantity, flavonoids and soluble protein contents and quantities of malonyldialdehyde and ·OH radical. In Allium schoenoprasum tissue culture organs the total antioxidant capacity was determined by the FRAP method and scavenger activity by the DPPH method. The present results indicated that the crude extract of Allium schoenoprasum tissue culture exhibited antioxidant and scavenging abilities in all investigated plant parts, especially in the roots. According to our results, the tissue culture plants exhibited the highest activities in the roots in contrast to the cultivated plants where highest activities were observed in the leaves. Copyright © 2011 John Wiley & Sons, Ltd.

  7. Rice improvement, involving altered flower structure more suitable to cross-pollination, using in vitro culture in combination with mutagenesis

    International Nuclear Information System (INIS)

    Min, S.K.

    1998-01-01

    Anther and somatic tissue culture in combination with mutagenesis were carried out to evaluate the efficiency of different mutagenic treatments of various in vitro culture materials, and to obtain some promising variants for rice improvement. Results indicated that in japonica rice radiation treatment of dry seeds and young panicles influenced the percentage of green plantlets regeneration from anther culture. Both treatments increased significantly the percentage of regenerated green plantlets in comparison with the control. Irradiation with 30 Gy of rice callus increased also the percentage of regenerated green plantlets. For indica rice, the combination of the suitable dose of gamma rays irradiation on seeds and an improved medium, increased the percentage of callus induction. This approach made it possible to use anther culture in indica rice breeding. Somatic tissue cultures combined with radiation-induced mutagenesis led to the development of a number of promising mutants including some new cytoplasm-nucleus interacting male-sterile lines with almost 100% stigma exertion. Their development would be of practical significance for increasing the genetic diversity for production of hybrid rice. (author)

  8. Origination of asexual plantlets in three species of Crassulaceae.

    Science.gov (United States)

    Guo, Jiansheng; Liu, Hailiang; He, Yangyang; Cui, Xianghuan; Du, Xiling; Zhu, Jian

    2015-03-01

    During asexual plant reproduction, cells from different organs can be reprogrammed to produce new individuals, a process that requires the coordination of cell cycle reactivation with the acquisition of other cellular morphological characteristics. However, the factors that influence the variety of asexual reproduction have not yet been determined. Here, we report on plantlet formation in Kalanchoe daigremontiana, Graptopetalum paraguayense, and Crassula portulacea (Crassulaceae) and analyse the effect of initiating cells on asexual reproduction in these three species. Additionally, the roles of WUSCHEL (WUS) and CUP-SHAPED COTYLEDON 1 (CUC1) in the asexual reproduction of these species were analysed through qRT-PCR. Our results indicated that pre-existing stem cell-like cells at the sites of asexual reproduction were responsible for the formation of plantlets. These cells were arrested in different phases of the cell cycle and showed different cell morphological characteristics and cell counts. The accumulation of auxin and cytokinin at the sites of asexual plantlet formation indicated their important functions, particularly for cell cycle reactivation. These differences may influence the pattern and complexity of asexual reproduction in these Crassulaceae species. Additionally, the dynamic expression levels of CUC1 and WUS may indicate that CUC1 functions in the formation of callus and shoot meristems; whereas, WUS was only associated with shoot induction.

  9. Pre-metatarsal skeletal development in tissue culture at unit- and microgravity

    Science.gov (United States)

    Klement, B. J.; Spooner, B. S.

    1994-01-01

    Explant organ culture was used to demonstrate that isolated embryonic mouse pre-metatarsal mesenchyme is capable of undergoing a series of differentiative and morphogenetic developmental events. Mesenchyme differentiation into chondrocytes, and concurrent morphogenetic patterning of the cartilage tissue, and terminal chondrocyte differentiation with subsequent matrix mineralization show that cultured tissue closely parallels in vivo development. Whole mount alizarin red staining of the cultured tissue demonstrates that the extracellular matrix around the hypertrophied chondrocytes is competent to support mineralization. Intensely stained mineralized bands are similar to those formed in pre-metatarsals developing in vivo. We have adapted the culture strategy for experimentation in a reduced gravity environment on the Space Shuttle. Spaceflight culture of pre-metatarsals, which have already initiated chondrogenesis and morphogenetic patterning, results in an increase in cartilage rod size and maintenance of rod shape, compared to controls. Older pre-metatarsal tissue, already terminally differentiated to hypertrophied cartilage, maintained rod structure and cartilage phenotype during spaceflight culture.

  10. Research progress in plant mutation by combining ion beam irradiations and tissue culture

    International Nuclear Information System (INIS)

    Zhou Linbin; Li Wenjian; Qu Ying; Li Ping

    2007-01-01

    About a new mutation breeding method which combines plant tissue culture technique with heavy ion beam irradiations were discussed in this paper with the principles, operation steps, molecular mechanisms, etc. The mutation method developed a few advantages coming from plant tissue culture, which can produce offspring by asexual ways. Meanwhile, using this method, the study of biological effects of high energy particles with different linear energy transfer values on plant tissues or cells can be explored and optimized in theory or practice. (authors)

  11. Revision washout decreases implant capsule tissue culture positivity: a multicenter study.

    Science.gov (United States)

    Henry, Gerard D; Carson, Culley C; Wilson, Steven K; Wiygul, Jeremy; Tornehl, Chris; Cleves, Mario A; Simmons, Caroline J; Donatucci, Craig F

    2008-01-01

    Positive cultures, visible biofilm and confocal micrography confirm bacterial presence on clinically uninfected inflatable penile prostheses at revision surgery. Salvage irrigation has been proved to rescue patients with clinically infected inflatable penile prostheses. Similar washout at revision for noninfectious reasons significantly lowers subsequent infection rates. We investigated a larger series of patients for positive culture rates and evaluated implant capsule tissue culture rates before and after revision washout. At 4 institutions a total of 148 patients with inflatable penile prostheses underwent revision surgery for noninfectious reasons between June 2001 and September 2005. Swab cultures of the fluid around the pump and visible biofilm were obtained. Also, in 65 patients a wedge of tissue from the capsule that forms around the pump was cultured. After implant removal revision washout of the implant spaces was performed and a second wedge of tissue was cultured. Of the 148 patients 97 (66%) had positive bacterial swab cultures of the fluid around the pump or biofilm. A total of 124 isolates were cultured. Of the 65 implant capsule tissue cultures obtained before washout 28 (43%) were positive for bacteria, while 16 (25%) obtained after revision washout were positive. Positive cultures and visible bacterial biofilm are present on clinically uninfected inflatable penile prostheses at revision surgery in most patients. Revision washout appears to decrease the bacterial load on implant capsule tissue at revision surgery of inflatable penile prostheses for noninfectious reasons.

  12. one step method of plantlet regeneration in trichosanthes dioica roxb

    African Journals Online (AJOL)

    User

    2011-01-03

    Jan 3, 2011 ... Tissue Culture Laboratory, Department of Botany, Patna University, Patna, India. E-mail: .... The inclusion of liquid endosperm from immature corn (Netien et al,. 1951), tomato juice (Nitsch, 1951; Straus and La Rue,. 1954), immature fruits and seeds ... Technology, SERC div., New Delhi for the financial sup-.

  13. The potential of prolonged tissue culture to reduce stress generation and retraction in engineered heart valve tissues.

    Science.gov (United States)

    van Vlimmeren, Marijke A A; Driessen-Mol, Anita; Oomens, Cees W J; Baaijens, Frank P T

    2013-03-01

    In tissue-engineered (TE) heart valves, cell-mediated processes cause tissue compaction during culture and leaflet retraction at time of implantation. We have quantified and correlated stress generation, compaction, retraction, and tissue quality during a prolonged culture period of 8 weeks. Polyglycolic acid/poly-4-hydroxybutyrate strips were seeded with vascular-derived cells and cultured for 4-8 weeks. Compaction in width, generated force, and stress was measured during culture. Retraction in length, generated force, and stress was measured after release of constraints at weeks 4, 6, and 8. Further, the amount of DNA, glycosaminoglycans (GAGs), collagen, and collagen cross-links was assessed. During culture, compaction and force generation increased to, respectively, 63.9% ± 0.8% and 43.7 ± 4.3 mN at week 4, after which they remained stable. Stress generation reached 27.7 ± 3.2 kPa at week 4, after which it decreased to ∼8.5 kPa. At release of constraints, tissue retraction was 44.0% ± 3.7% at week 4 and decreased to 29.2% ± 2.8% and 26.1% ± 2.2% at, respectively, 6 and 8 weeks. Generated force (8-16 mN) was lower at week 6 than at weeks 4 and 8. Generated stress decreased from 11.8 ± 0.9 kPa at week 4 to 1.4 ± 0.3 and 2.4 ± 0.4 kPa at, respectively, weeks 6 and 8. The amount of GAGs increased at weeks 6 and 8 compared to week 4 and correlated to the reduced stress and retraction. In summary, prolonged culture resulted in decreased stress generation and retraction, likely as a result of the increased amount of GAGs. These results demonstrate the potential of prolonged tissue culture in developing functional, nonretracting, TE heart valves.

  14. Influence of growth regulators on somatic embryogenesis, plantlet regeneration, and post-transplant survival of Echinochloa frumentacea.

    Science.gov (United States)

    Sankhla, A; Davis, T D; Sankhla, D; Sankhla, N; Upadhyaya, A; Joshi, S

    1992-07-01

    After placement on Murashige and Skoog's basal medium supplemented with 3-5 mg/l 2,4-D, immature inflorescence expiants of Echinochloa frumentacea gave rise to three distinct types of callus: a) loosely arranged and soft; b) compact and translucent; c) compact, sticky and mucilaginous. Somatic embryo formation occurred in type 'b' callus in about 18-24 d. Callus types 'a' and 'c' did not produce somatic embryos. The highest percentage of cultures exhibiting somatic embryogenesis occurred on the medium containing 5 mg/l 2,4-D and 0.5 mg/l kinetin. Somatic embryos also formed directly on the inflorescence (without intervening callus formation) in about 15% of the expiants placed on this medium. The addition of paclobutrazol or uniconazole (0.25 or 1 mg/l) to the medium had no influence on the percentage of cultures exhibiting direct somatic embryogenesis, but paclobutrazol slightly increased the mean number of somatic embryos per culture. Many of the callus-derived somatic embryos germinated when subcultured on basal MS medium supplemented with kinetin. Addition of paclobutrazol or uniconazole to the culture medium at 0.25 or 1 mg/l decreased somatic embryo germination and shoot elongation but increased root length and leaf width. Both paclobutrazol and uniconazole increased survival of the plantlets following transplanting to soil. Increased post-transplant survival was accompanied by reduced water loss from plantlets produced on culture media containing triazoles.

  15. Plant tissue culture study on two different races of purslane ...

    African Journals Online (AJOL)

    This study was performed on two races of purslane; agronomic purslane and wild one. All the explants were cultured on MS basal medium supplement with 30 g/l sucrose, 8 g/l agar and different plant growth regulator. Petioles, shoot tips and leaves of wild purslane and also leaves of agronomic purslane were cultured in ...

  16. Biomaterials in co-culture systems: towards optimizing tissue integration and cell signaling within scaffolds.

    Science.gov (United States)

    Battiston, Kyle G; Cheung, Jane W C; Jain, Devika; Santerre, J Paul

    2014-05-01

    Most natural tissues consist of multi-cellular systems made up of two or more cell types. However, some of these tissues may not regenerate themselves following tissue injury or disease without some form of intervention, such as from the use of tissue engineered constructs. Recent studies have increasingly used co-cultures in tissue engineering applications as these systems better model the natural tissues, both physically and biologically. This review aims to identify the challenges of using co-culture systems and to highlight different approaches with respect to the use of biomaterials in the use of such systems. The application of co-culture systems to stimulate a desired biological response and examples of studies within particular tissue engineering disciplines are summarized. A description of different analytical co-culture systems is also discussed and the role of biomaterials in the future of co-culture research are elaborated on. Understanding the complex cell-cell and cell-biomaterial interactions involved in co-culture systems will ultimately lead the field towards biomaterial concepts and designs with specific biochemical, electrical, and mechanical characteristics that are tailored towards the needs of distinct co-culture systems. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. Equine ovarian tissue viability after cryopreservation and in vitro culture

    Science.gov (United States)

    The efficiency of several cryoprotective agents were compared using both slow-freezing and vitrification methods. Results indicate that the viability of ovarian tissue cells increases when DMSO (slow-freezing) and ethylene glycol (vitrification) are used....

  18. Growth of Igbo-Ora virus in some tissue cultures.

    Science.gov (United States)

    Olaleye, O D; Omilabu, S A; Baba, S S

    1990-08-01

    VERO, MRC5, MDCK, and MA104 cells were tested for their ability to support the growth of Igbo-Ora virus. In VERO and MRC5 cell cultures the virus replicated to high titres causing apparent cytopathic effects (CPE) (cell rounding and complete lysis) and formation of complement fixing antigens. The virus grew to lower infectious titre in MDCK and MA104 cell cultures in which CPE was limited to cell rounding only.

  19. Therapeutically important proteins from in vitro plant tissue culture systems.

    Science.gov (United States)

    Doran, Pauline M

    2013-01-01

    Plant cells cultured in liquid medium in bioreactors are now being used commercially to produce biopharmaceutical proteins. The emergence of in vitro plant cell culture as a production vehicle reflects the importance of key biosafety and biocontainment concerns affecting the competitiveness of alternative systems such as mammalian cell culture and agriculture. Food plant species are particularly attractive as hosts for in vitro protein production: the risk of transgene escape and food chain contamination is eliminated using containment facilities, while regulatory approval for oral delivery of drugs may be easier than if non-edible species were used. As in whole plants, proteolysis in cultured plant cells can lead to significant degradation of foreign proteins after synthesis; however, substantial progress has been made to counter the destructive effects of proteases in plant systems. Although protein secretion into the culture medium is advantageous for product recovery and purification, measures are often required to minimise extracellular protease activity and product losses due to irreversible surface adsorption. Disposable plastic bioreactors, which are being used increasingly in mammalian cell bioprocessing, are also being adopted for plant cell culture to allow rapid scale-up and generation of saleable product. This review examines a range of technical and regulatory issues affecting the choice of industrial production platform for foreign proteins, and assesses progress in the development of in vitro plant systems for biopharmaceutical production.

  20. Efforts to accelerate domestication of winged bean (Psophocarpus tetragonolobus (L.) DC.) by means of induced mutations and tissue culture

    NARCIS (Netherlands)

    Klu, G.Y.P.

    1996-01-01

    This thesis describes mutation breeding and tissue culture techniques developed for accelerated domestication of winged bean ( Psophocarpustetragonolobus (L.) DC.). The tissue culture techniques, which are the first steps towards genetic transformation of

  1. A method for establishing human primary gastric epithelial cell culture from fresh surgical gastric tissues.

    Science.gov (United States)

    Aziz, Faisal; Yang, Xuesong; Wen, Qingping; Yan, Qiu

    2015-08-01

    At present, biopsy specimens, cancer cell lines and tissues obtained by gastric surgery are used in the study and analysis of gastric cancer, including the molecular mechanisms and proteomics. However, fibroblasts and other tissue components may interfere with these techniques. Therefore, the present study aimed to develop a procedure for the isolation of viable human gastric epithelial cells from gastric surgical tissues. A method was developed to culture human gastric epithelial cells using fresh, surgically excised tissues and was evaluated using immunocytochemistry, periodic acid-Schiff (PAS) staining and cell viability assays. Low cell growth was observed surrounding the gastric tissue on the seventh day of tissue explant culture. Cell growth subsequently increased, and at 12 days post-explant a high number of pure epithelial cells were detected. The gastric cancer cells exhibited rapid growth with a doubling time of 13-52 h, as compared to normal cells, which had a doubling time of 20-53 h. Immunocytochemical analyses of primary gastric cells revealed positive staining for cytokeratin 18 and 19, which indicated that the culture was comprised of pure epithelial cells and contained no fibroblasts. Furthermore, PAS staining demonstrated that the cultured gastric cells produced neutral mucin. Granulin and carbohydrate antigen 724 staining confirmed the purity of gastric cancer and normal cells in culture. This method of cell culture indicated that the gastric cells in primary culture consisted of mucin-secreting gastric epithelial cells, which may be useful for the study of gastric infection with Helicobacter pylori and gastric cancer.

  2. Long term organ culture of human prostate tissue in a NASA-designed rotating wall bioreactor

    Science.gov (United States)

    Margolis, L.; Hatfill, S.; Chuaqui, R.; Vocke, C.; Emmert-Buck, M.; Linehan, W. M.; Duray, P. H.

    1999-01-01

    PURPOSE: To maintain ex vivo integral prostatic tissue including intact stromal and ductal elements using the NASA-designed Rotating Wall Vessel (RWV) which maintains colocalized cells in an environment that promotes both three-dimensional cellular interactions together with the uniform mass transfer of nutrients and metabolic wastes. MATERIALS AND METHODS: Samples of normal prostate were obtained as a byproduct of transurethral prostatectomy or needle biopsy. Prostatic tissue dissected into small 1 x 1 mm. blocks was cultured in the Rotating Wall Vessel (RWV) Bioreactor for various time periods and analyzed using histological, immunochemical, and total cell RNA assays. RESULTS: We report the long term maintenance of benign explanted human prostate tissue grown in simple culture medium, under the simulated microgravity conditions afforded by the RWV bioreactor. Mesenchymal stromal elements including blood vessels and architecturally preserved tubuloglandular acini were maintained for a minimum of 28 days. Cytokeratins, vimentin and TGF-beta2 receptor and ligand were preserved through the entire culture period as revealed by immunocytochemistry. Prostatic acid phosphatase (PAP) was continuously expressed during the culture period, although somewhat decreased. Prostatic specific antigen (PSA) and its transcript were down regulated over time of culture. Prostatic carcinoma cells from the TSU cell line were able to invade RWV-cultured benign prostate tissue explants. CONCLUSIONS: The RWV bioreactor represents an additional new technology for culturing prostate tissue for further investigations concerning the basic physiology and pathobiology of this clinically important tissue.

  3. Variation in bioactive principles of Artemisia amygdalina Decne. in wild and tissue culture regenerants.

    Science.gov (United States)

    Rasool, Rafia; Ganai, Bashir Ahmad; Akbar, Seema; Kamili, Azra Nahaid; Dar, Muhammad Younus; Masood, Akbar

    2013-05-01

    Wild and tissue culture raised regenerants of Artemisia amygdalina, a critically endangered and endemic plant of Kashmir and North West Frontier Provinces of Pakistan were screened for the amount of bioactive principles and in particular antimalarial compound artemesinin. Phytochemical screening of extracts revealed the presence of terpenes, alkaloids, phenolics, tannins (polyphenolics), cardiac glycosides and steroids in wild (aerial, inflorescence) and tissue culture regenerants (in vitro grown plant, callus and green house acclimatized plants). HPLC of Artemisia amygdalina revealed the presence of artemesinin in petroleum ether extracts of wild aerial part, tissue culture raised plant and green house acclimatized plants. Acetonitrile and water in 70:30 ratios at flow rate of 1ml/min was standardised as mobile phase. Retention time for standard chromatogram was 6.7. Wild inflorescences and callus does not produce artemesinin. This is the first report of phytochemical screening and artemesinin estimation of wild and tissue culture raised regenerants of Artemisia amygdalina.

  4. How-To-Do-It: Using Cauliflower to Demonstrate Plant Tissue Culture.

    Science.gov (United States)

    Haldeman, Janice H.; Ellis, Jane P.

    1988-01-01

    Presents techniques used for disinfestation of plant material, preparation of equipment and media, and laboratory procedures for tissue culture using cauliflower. Details methods for preparing solutions and plant propagation by cloning. (CW)

  5. Substrate specific hydrolysis of aromatic and aromatic-aliphatic esters in orchid tissue cultures

    Directory of Open Access Journals (Sweden)

    Agnieszka Mironowicz

    2014-01-01

    Full Text Available We found that tissue cultures of higher plants were able, similarly as microorganisms, to transform low-molecular-weight chemical compounds. In tissue cultures of orchids (Cymbidium 'Saint Pierre' and Dendrobium phalaenopsis acetates of phenols and aromatic-aliphatic alcohols were hydrolyzed, whereas methyl esters of aromatic and aromatic-aliphatic acids did not undergo this reaction. Acetates of racemic aromatic-aliphatic alcohols were hydrolyzed with distinct enantiospecificity.

  6. Study on the propagation of some sugar cane clones derived from tissue culture

    Energy Technology Data Exchange (ETDEWEB)

    Chagvardieff, P.; Mauboussin, J.C.; Weil, J.

    1983-01-01

    The study of the propagation by cuttings of sugar-cane clones derived from tissue cultures showed that the emergence of one bud setts was variable. A definition of parameters for this germination is proposed: germination vigour, germination capacity, shooting vigour, shooting capacity. Variants optained, among them germination or shooting vigour, were always improved when compared with the control; this might result from a cell rejuvenation induced by the tissue culture. 11 references.

  7. In vitro propagation of plant virus using different forms of plant tissue culture and modes of culture operation.

    Science.gov (United States)

    Shih, Sharon M-H; Doran, Pauline M

    2009-09-10

    Plant virus accumulation was investigated in vitro using three different forms of plant tissue culture. Suspended cells, hairy roots and shooty teratomas of Nicotiana benthamiana were infected with tobacco mosaic virus (TMV) using the same initial virus:biomass ratio. Viral infection did not affect tissue growth or morphology in any of the three culture systems. Average maximum virus concentrations in hairy roots and shooty teratomas were similar and about an order of magnitude higher than in suspended cells. Hairy roots were considered the preferred host because of their morphological stability in liquid medium and relative ease of culture. The average maximum virus concentration in the hairy roots was 0.82+/-0.14 mg g(-1) dry weight; viral coat protein represented a maximum of approximately 6% of total soluble protein in the biomass. Virus accumulation in hairy roots was investigated further using different modes of semi-continuous culture operation aimed at prolonging the root growth phase and providing nutrient supplementation; however, virus concentrations in the roots were not enhanced compared with simple batch culture. The relative infectivity of virus in the biomass declined by 80-90% during all the cultures tested, irrespective of the form of plant tissue used or mode of culture operation. Hairy root cultures inoculated with a transgenic TMV-based vector in batch culture accumulated green fluorescent protein (GFP); however, maximum GFP concentrations in the biomass were relatively low at 39 microg g(-1) dry weight, probably due to genetic instability of the vector. This work highlights the advantages of using hairy roots for in vitro propagation of TMV compared with shooty teratomas and suspended plant cells, and demonstrates that batch root culture is more effective than semi-continuous operations for accumulation of high virus concentrations in the biomass.

  8. ELIMINATION OF CVB ( FROM A RANGE OF CHRYSANTHEMUM VARIETIES BY APICAL MERISTEM CULTURE FOLLOWING ANTIVIRAL AGENT AND HEAT TREATMENTS

    Directory of Open Access Journals (Sweden)

    KURNIAWAN BUDIARTO

    2011-09-01

    Full Text Available CVB elimination for retaining healthy protocols from infected chrysanthemum plant wasinvestigated through combined treatment of meristem culture with synthetic antiviral ribavirinor thermotherapy under conditions. The biological materials used for the experimentconstituted of six commercial varieties: Dewi Sartika, Saraswati, Yellow Fiji, White Puma,Yellow Puma and White Reagent. Tissue culture initiation was conducted through plantletestablishment using MS supplemented with IAA. Ribavirin was added in media with theconcentration of 40 mg/l on cv. Dewi Sartika, Saraswati and Yellow Fiji. Parallel with this step,heat treatment with different durations (1, 2, and 3 weeks was also conducted on the plantletson White Puma, Yellow Puma and White Reagent. Meristem culture was done followingthe chemo- and thermotherapy. The experiment resumed the failure of single treatment ofmeristem culture in eliminating CVB from the infected chrysanthemum plantlets. Under heattreatment, percentage of virus-free plantlets increased along with the duration ofthermotherapy, though the survival rate of plantlets decreased in lengthened heat treatment.The best results regarding virus free plant percentage were obtained when meristem culture wasapplied following ribavirin or three weeks of heat treatment.

  9. Organoid culture systems for prostate epithelial and cancer tissue

    NARCIS (Netherlands)

    Drost, Jarno; Karthaus, Wouter R.|info:eu-repo/dai/nl/37034958X; Gao, Dong; Driehuis, Else; Sawyers, Charles L.; Chen, Yu; Clevers, Hans|info:eu-repo/dai/nl/07164282X

    2016-01-01

    This protocol describes a strategy for the generation of 3D prostate organoid cultures from healthy mouse and human prostate cells (either bulk or FACS-sorted single luminal and basal cells), metastatic prostate cancer lesions and circulating tumor cells. Organoids derived from healthy material

  10. Tissue culture as a plant production technique for horticultural crops

    African Journals Online (AJOL)

    STORAGESEVER

    2009-08-18

    Aug 18, 2009 ... Recovery of regenerants from transformed cells. - Cell culture .... methods. Micropropagation techniques. Micropropagation is a simple concept. The basic pro- tocols were well established by the 1960s and a whole research field and ... the environment are naturally contaminated on their sur- faces (and ...

  11. Rat fetal ventral mesencephalon grown as solid tissue cultures

    DEFF Research Database (Denmark)

    Höglinger, G U; Sautter, J; Meyer, Morten

    1998-01-01

    in vitro (DIV) in the presence or absence (controls) of BDNF [100 ng/ml]. The dopamine content in the culture medium, analyzed by HPLC, was significantly higher (4-5 fold) in the BDNF group at DIV 8 and DIV 12 compared to the corresponding control levels (40 pg/ml). The number of tyrosine hydroxylase...

  12. Tissue culture in Pinus caribaea Mor. var. Hondurensis barr. and ...

    African Journals Online (AJOL)

    SERVER

    2008-03-18

    Mar 18, 2008 ... and Harada (1979), L-glutamine is very important for somatic embryogenesis. They reached this conclusion after comparing the performance of individual amino acids in carrot cultures and they discovered that carrot somatic embryos were best promoted by L-glutamine. Secondly, the concentrations of the ...

  13. Organoid culture systems for prostate epithelial and cancer tissue

    NARCIS (Netherlands)

    Drost, Jarno; Karthaus, Wouter R; Gao, Dong; Driehuis, Else; Sawyers, Charles L; Chen, Yu; Clevers, Hans

    This protocol describes a strategy for the generation of 3D prostate organoid cultures from healthy mouse and human prostate cells (either bulk or FACS-sorted single luminal and basal cells), metastatic prostate cancer lesions and circulating tumor cells. Organoids derived from healthy material

  14. Plant Regeneration Through Tissue Culture Of Pear Millet ...

    African Journals Online (AJOL)

    1. 1. 2,5), MS(5) and N6(1.100.25) culture media, calli embryogenic potential and fertile plants regeneration were conserved for more than 12 months. Characteristics of regenerated plants were similar to control. It appears that dissected shoot ...

  15. Tissue cultures from adult human postmortem subcortical brain areas

    NARCIS (Netherlands)

    Verwer, R. W. H.; Dubelaar, E. J. G.; Hermens, W. T. J. M. C.; Swaab, D. F.

    2002-01-01

    Animal models used to study human aging and neurodegeneration do not display all symptoms of these processes as they are found in humans. Recently, we have shown that many cells in neocortical slices from adult human postmortem brain may survive for extensive periods in vitro. Such cultures may

  16. Callus formation using in vitro tissue culture technique in cultivated ...

    African Journals Online (AJOL)

    Abhishek

    2013-07-24

    Jul 24, 2013 ... cotyledon, coteledonary node and hypocotyl measuring 4 to 5 mm obtained from asceptically grown seedlings were inoculated on the surface of different culture medium. Murashige and Skoog (MS) salts supplemented with B5 vitamins was used as basal medium and fortified with different concentrations of ...

  17. [Research progress of co-culture system for constructing vascularized tissue engineered bone].

    Science.gov (United States)

    Fu, Weili; Xiang, Zhou

    2014-02-01

    To review the research progress of the co-culture system for constructing vascularized tissue engineered bone. The recent literature concerning the co-culture system for constructing vascularized tissue engineered bone was reviewed, including the selection of osteogenic and endothelial lineages, the design and surface modification of scaffolds, the models and dimensions of the co-culture system, the mechanism, the culture conditions, and their application progress. The construction of vascularized tissue engineered bone is the prerequisite for their survival and further clinical application in vivo. Mesenchymal stem cells (owning the excellent osteogenic potential) and endothelial progenitor cells (capable of directional differentiation into endothelial cell) are considered as attractive cell types for the co-culture system to construct vascularized tissue engineered bone. The culture conditions need to be further optimized. Furthermore, how to achieve the clinical goals of minimal invasion and autologous transplantation also need to be further studied. The strategy of the co-culture system for constructing vascularized tissue engineered bone would have a very broad prospects for clinical application in future.

  18. Co-culture systems-based strategies for articular cartilage tissue engineering.

    Science.gov (United States)

    Zhang, Yu; Guo, Weimin; Wang, Mingjie; Hao, Chunxiang; Lu, Liang; Gao, Shuang; Zhang, Xueliang; Li, Xu; Chen, Mingxue; Li, Penghao; Jiang, Peng; Lu, Shibi; Liu, Shuyun; Guo, Quanyi

    2018-03-01

    Cartilage engineering facilitates repair and regeneration of damaged cartilage using engineered tissue that restores the functional properties of the impaired joint. The seed cells used most frequently in tissue engineering, are chondrocytes and mesenchymal stem cells. Seed cells activity plays a key role in the regeneration of functional cartilage tissue. However, seed cells undergo undesirable changes after in vitro processing procedures, such as degeneration of cartilage cells and induced hypertrophy of mesenchymal stem cells, which hinder cartilage tissue engineering. Compared to monoculture, which does not mimic the in vivo cellular environment, co-culture technology provides a more realistic microenvironment in terms of various physical, chemical, and biological factors. Co-culture technology is used in cartilage tissue engineering to overcome obstacles related to the degeneration of seed cells, and shows promise for cartilage regeneration and repair. In this review, we focus first on existing co-culture systems for cartilage tissue engineering and related fields, and discuss the conditions and mechanisms thereof. This is followed by methods for optimizing seed cell co-culture conditions to generate functional neo-cartilage tissue, which will lead to a new era in cartilage tissue engineering. © 2017 Wiley Periodicals, Inc.

  19. Low melting point agarose beads as a standard method for plantlet regeneration from protoplasts within the Cichorium genus.

    Science.gov (United States)

    Deryckere, Dieter; Eeckhaut, Tom; Van Huylenbroeck, Johan; Van Bockstaele, Erik

    2012-12-01

    A standard method has been developed with which we are able to fully regenerate protoplasts of different Cichorium species. For the first time, endive protoplasts have been regenerated into plantlets. Protoplast regeneration is essential for somatic hybridizations. In this study, a standard method for plantlet regeneration from Cichorium protoplasts was developed. We evaluated the effect of the low melting point agarose (LMPA) bead technique on the regeneration capacity of protoplasts of seven C. intybus and four C. endivia genotypes. The LMPA bead technique was more efficient than culture in liquid or solid medium and allowed us to obtain plating efficiencies up to 4.9 % in C. intybus genotypes and efficiencies of up to 0.7 % in C. endivia genotypes. Moreover, the LMPA bead technique offers great advantages over liquid and solid culture systems: the media can be readily refreshed, protoplasts can be monitored separately, and microcalli can easily be removed from the beads. This increased efficiency was observed for all of the 11 Cichorium genotypes tested. Shoot formation was induced more efficiently when using 0.5 mg l(-1) indole-3-acetic acid-enriched medium (up to 87.5 % of the protoplast-derived calli started shoot development) compared to 1-naphthaleneacetic acid-enriched medium. The LMPA bead technique optimized in this study enabled for the first time the full plantlet regeneration from protoplasts of C. endivia genotypes and increased the protoplast regenerating ability in other Cichorium species. This fine-tuned LMPA bead technique can therefore be applied for protoplast regeneration after protoplast fusions of the genus Cichorium.

  20. Quantitation of ranaviruses in cell culture and tissue samples

    DEFF Research Database (Denmark)

    Holopainen, Riikka; Honkanen, Jarno; Jensen, Britt Bang

    2011-01-01

    – epithelioma papulosum cyprini (EPC) and bluegill fry (BF-2) – were infected with four of the isolates (EHNV, ECV, FV3 and DFV), and the viral quantity was determined from seven time points during the first three days after infection. The qPCR was also used to determine the viral load in tissue samples from...... pike (Esox lucius) fry challenged experimentally with EHNV....

  1. Pattern matching and adaptive image segmentation applied to plant reproduction by tissue culture

    Science.gov (United States)

    Vazquez Rueda, Martin G.; Hahn, Federico

    1999-03-01

    This paper shows the results obtained in a system vision applied to plant reproduction by tissue culture using adaptive image segmentation and pattern matching algorithms, this analysis improves the number of tissue obtained and minimize errors, the image features of tissue are considered join to statistical analysis to determine the best match and results. Tests make on potato plants are used to present comparative results with original images processed with adaptive segmentation algorithm and non adaptive algorithms and pattern matching.

  2. Researches about selecting resistant melon types to fusarium oxyporum f. sp.melonis race 1,2 by using tissue culture and mutation techniques

    International Nuclear Information System (INIS)

    2009-01-01

    breeding was quickly understood. In vitro techniques for crop improvement first consisted of micropropagation and plant regeneration, and then in vitro methods were also found to be useful for eliminating disease and selecting for resistant cells or explants. In vitro selection using specific chemical compounds and pathogens is another useful aspect of tissue culture. More recently, in vitro techniques were combined to mutation induction for generating genetic variation, including novel disease resistant mutants. Mutation induction can be caused by chemical or physical mutagens that alter the structure of the DNA. Treatment of in vitro tissues with physical or chemical mutagens may increase the frequency of genetic variation considerably. The physical mutagens most commonly used are X-rays, gamma rays and UV light, whereas Ethyl Methane- Sulphonate (EMS) is the chemical mutagen most used in crop improvement. Irradiation treatments may be a suitable choice of mutagen for a number of reasons because the application is fast and, in contrast to chemical mutagens, there is no risk that residues remain in the medium. In this research we are going to determine the resistant cells, which will come from irradiated and non-irradiated explants by using races of F. oxysporum f. sp. melonis culture filtrates in vitro conditions. If we will get successful results by this method we will be able to develop new melon cultivars, which will be resistant to F. oxysporum f. sp. melonis. The present work was performed with in vitro plantlets obtained from seeds of melon cv. Yuva which is an important commercial cultivar in Turkey. According to our observations filtrate and gamma ray treatment have an important effect on tolerant plantlet and callus formation. In this research, we show a method for mass-selection of melon mutants resistant to Fusarium wilt. In vitro selection of resistant cells, which are come from irradiated and non-irradiated explants, is done using culture filtrates of

  3. Culture methods of allograft musculoskeletal tissue samples in Australian bacteriology laboratories.

    Science.gov (United States)

    Varettas, Kerry

    2013-12-01

    Samples of allograft musculoskeletal tissue are cultured by bacteriology laboratories to determine the presence of bacteria and fungi. In Australia, this testing is performed by 6 TGA-licensed clinical bacteriology laboratories with samples received from 10 tissue banks. Culture methods of swab and tissue samples employ a combination of solid agar and/or broth media to enhance micro-organism growth and maximise recovery. All six Australian laboratories receive Amies transport swabs and, except for one laboratory, a corresponding biopsy sample for testing. Three of the 6 laboratories culture at least one allograft sample directly onto solid agar. Only one laboratory did not use a broth culture for any sample received. An international literature review found that a similar combination of musculoskeletal tissue samples were cultured onto solid agar and/or broth media. Although variations of allograft musculoskeletal tissue samples, culture media and methods are used in Australian and international bacteriology laboratories, validation studies and method evaluations have challenged and supported their use in recovering fungi and aerobic and anaerobic bacteria.

  4. Viability of post acclimatized plantlets of Robusta coffee (Coffea canephora after storage

    Directory of Open Access Journals (Sweden)

    Teguh Iman Santoso

    2011-08-01

    Full Text Available This research related to the storage method of planting materials in the form of post acclimatized plantlets of Robusta coffee multiplied by somatic embryogenesis using plastic film that wraped the whole of plantlets. This information is important to support the delivery of clonal planting materials to distribution points, especially Robusta coffee plantlets viability based on condition of the container, storage period and density of plantlets. The research was conducted at Kaliwining Experimental Station of Indonesian Coffee and Cocoa Research Institute, located at 45 m asl. D rainfall type (Schmidt—Ferguson classification. The first experiment determind the effect of container condition and storage duration on viability of Robusta coffee plantlets. Each experimental unit contained 100 plantlets and each treatment was repeated three times with completely randomized design in factorial. The first factor was condition of storage container, i.e. airtight and non air tight. The second factor was storage period levels: 0, 5, 10, 15 and 20 days. The storage container was cardboard volume 11 dm3. The second experiment was conducted for the optimization of storage volume and storage period. Each treatment using 100 plantlets was repeated three times in completly randomized design with factorial. The first factor was storage volume of 7 dm3 and 11 dm3, the second factor was storage period levels: 0, 5, 10, 15 and 20 days. The results indicated that the maximum store period was obtained in an airtight storage treatment with 10 days, 96.3% plantlets viability, 1% fallen leaves, 3.3% water loss and not significantly different to control. For packing 100 plantlets with height 8—10 cm and leaf number 4—6 can use the volume of container store up to 7 dm3, which showed no significant difference to container volume 11 dm3 in the percentage of viability, the percentage of fallen leaves, loss of water. Key words : Robusta coffee, plantlets, storage

  5. TCUP: A novel hAT transposon active in maize tissue culture

    Directory of Open Access Journals (Sweden)

    Alan eSmith

    2012-01-01

    Full Text Available Transposable elements are capable of inducing heritable de novo genetic variation. The sequences capable of reactivation, and environmental factors that induce mobilization, remain poorly defined even in well-studied genomes such as maize. We treated maize tissue culture with the demethylating agent 5-aza-2-deoxcytidine and examined long-term tissue culture lines to discover silenced transposable elements that have the potential to induce heritable genetic variation. Through these screens we have identified a novel low copy number hAT transposon, Tissue Culture Up-Regulated (TCUP, which is transcribed at high levels in long-term maize Black Mexican Sweet (BMS tissue culture and up-regulated in response to treatment with 5-aza-2-deoxycytidine. Analysis of the TIGR Maize Gene Index revealed that this element is the most frequently represented EST from the BMS cell culture library and is not represented in other tissue libraries, which is the basis for its name. A full-length sequence was assembled in inbred B73 that contains the putative functional motifs required for autonomous movement of a hAT transposon. Transposon display detected movement of TCUP in two long-term tissue cultured cell lines of the genotype Hi-II AxB and BMS. This research implicates TCUP as a transposon that is capable of reactivation and which may also be particularly sensitive to the stress of the tissue culture environment. Our findings are consistent with the hypothesis that epigenetic alterations potentiate genomic responses to stress during clonal propagation of plants.

  6. Single molecule microscopy in 3D cell cultures and tissues.

    Science.gov (United States)

    Lauer, Florian M; Kaemmerer, Elke; Meckel, Tobias

    2014-12-15

    From the onset of the first microscopic visualization of single fluorescent molecules in living cells at the beginning of this century, to the present, almost routine application of single molecule microscopy, the method has well-proven its ability to contribute unmatched detailed insight into the heterogeneous and dynamic molecular world life is composed of. Except for investigations on bacteria and yeast, almost the entire story of success is based on studies on adherent mammalian 2D cell cultures. However, despite this continuous progress, the technique was not able to keep pace with the move of the cell biology community to adapt 3D cell culture models for basic research, regenerative medicine, or drug development and screening. In this review, we will summarize the progress, which only recently allowed for the application of single molecule microscopy to 3D cell systems and give an overview of the technical advances that led to it. While initially posing a challenge, we finally conclude that relevant 3D cell models will become an integral part of the on-going success of single molecule microscopy. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. ELIMINATION OF Cucumber Mozaic Virus (CMV FROM A RANGE OF CHRYSANTHEMUM CULTIVARS THROUGH MERISTEM CULTURE FOLLOWING HEAT TREATMENT

    Directory of Open Access Journals (Sweden)

    Kurniawan Budiarto

    2011-11-01

    Full Text Available Cucumber Mosaic Virus (CMV is one of pathogenic viruses that commonly found and has caused significant losses in many chrysanthemum growers in Indonesia.  Efforts have been made to get healthy plant source through eliminating virus from infected plants and one of the promising methods was the combination of thermotherapy and meristem culture. The research was conducted to find out the effect of meristem culture following heat treatments on the existence of CMV in infected chrysanthemum plantlets. The experiment was carried out in the laboratory of tissue culture and virology at The Indonesian Ornamental Crops Research Institute (IOCRI from August 2007 until June 2008. A complete factorial with ten replications was designed to accomplish the combination of two factors.  The first factor was three chrysanthemum cultivars, namely Stroika, Dewi Sartika and White Fiji, while the second dealt with the durations of heat treatment i.e. one, two and three weeks.  The results showed that plantlet survival decreased, yet improved plantlet performance by faster bud initiation with lengthened heat duration.  The percentage of virus-free planlets also increased along with the duration of treatment and three weeks heat treatment followed by meristem culture effectively eliminated CMV from infected planlets.

  8. Pathogen and biological contamination management in plant tissue culture: phytopathogens, vitro pathogens, and vitro pests.

    Science.gov (United States)

    Cassells, Alan C

    2012-01-01

    The ability to establish and grow plant cell, organ, and tissue cultures has been widely exploited for basic and applied research, and for the commercial production of plants (micro-propagation). Regardless of whether the application is for research or commerce, it is essential that the cultures be established in vitro free of biological contamination and be maintained as aseptic cultures during manipulation, growth, and storage. The risks from microbial contamination are spurious experimental results due to the effects of latent contaminants or losses of valuable experimental or commercial cultures. Much of the emphasis in culture contamination management historically focussed on the elimination of phytopathogens and the maintenance of cultures free from laboratory contamination by environmental bacteria, fungi (collectively referred to as "vitro pathogens", i.e. pathogens or environmental micro-organisms which cause culture losses), and micro-arthropods ("vitro pests"). Microbial contamination of plant tissue cultures is due to the high nutrient availability in the almost universally used Murashige and Skoog (Physiol Plant 15:473-497, 1962) basal medium or variants of it. In recent years, it has been shown that many plants, especially perennials, are at least locally endophytically colonized intercellularly by bacteria. The latter, and intracellular pathogenic bacteria and viruses/viroids, may pass latently into culture and be spread horizontally and vertically in cultures. Growth of some potentially cultivable endophytes may be suppressed by the high salt and sugar content of the Murashige and Skoog basal medium and suboptimal temperatures for their growth in plant tissue growth rooms. The management of contamination in tissue culture involves three stages: disease screening (syn. disease indexing) of the stock plants with disease and endophyte elimination where detected; establishment and pathogen and contaminant screening of established initial cultures

  9. Fabrication of a thermoresponsive cell culture dish: a key technology for cell sheet tissue engineering

    Directory of Open Access Journals (Sweden)

    Jun Kobayashi and Teruo Okano

    2010-01-01

    Full Text Available This article reviews the properties and characterization of an intelligent thermoresponsive surface, which is a key technology for cell sheet-based tissue engineering. Intelligent thermoresponsive surfaces grafted with poly(N-isopropylacrylamide exhibit hydrophilic/hydrophobic alteration in response to temperature change. Cultured cells are harvested on thermoresponsive cell culture dishes by decreasing the temperature without the use of digestive enzymes or chelating agents. Our group has developed cell sheet-based tissue engineering for therapeutic uses with single layer or multilayered cell sheets, which were recovered from the thermoresponsive cell culture dish. Using surface derivation techniques, we developed a new generation of thermoresponsive cell culture dishes to improve culture conditions. We also designed a new methodology for constructing well-defined organs using microfabrication techniques.

  10. Effect of induced mutagenesis in rice tissue culture

    International Nuclear Information System (INIS)

    Maddumage, R.

    1994-01-01

    The influence of chemical mutagens and ionising radiation on growth, regenerative capacity of rice callus culture and the effect o9f mutagens on frequency and spectrum of mutant regenerants, derived from calli and determination of approximate semi-lethal dose of each mutagen on rice calli was studied. Intact mature de-husked grains and pieces of primordial particles of four varieties were used as explants in the experiment. Organogenesis was induced using MS media supplemented with agar. After thirty days calluses were subjected to varying concentrations/dosage of mutagens. The effect of mutagens on growth of callus was stimulative in low concentration/doses at short exposure, but in higher concentration/doses at longer exposure it was oppressive. In x-radiation treatment all the studied doses showed only stimulative effect on growth. The effect of mutagenic treatment on regenerative capacity was negative. No specificity was found even between two chemical mutagens of their action on studied characters

  11. Oil palm (Elaeis guineensis Jacq.) tissue culture ESTs: identifying genes associated with callogenesis and embryogenesis.

    Science.gov (United States)

    Low, Eng-Ti L; Alias, Halimah; Boon, Soo-Heong; Shariff, Elyana M; Tan, Chi-Yee A; Ooi, Leslie Cl; Cheah, Suan-Choo; Raha, Abdul-Rahim; Wan, Kiew-Lian; Singh, Rajinder

    2008-05-29

    Oil palm (Elaeis guineensis Jacq.) is one of the most important oil bearing crops in the world. However, genetic improvement of oil palm through conventional breeding is extremely slow and costly, as the breeding cycle can take up to 10 years. This has brought about interest in vegetative propagation of oil palm. Since the introduction of oil palm tissue culture in the 1970s, clonal propagation has proven to be useful, not only in producing uniform planting materials, but also in the development of the genetic engineering programme. Despite considerable progress in improving the tissue culture techniques, the callusing and embryogenesis rates from proliferating callus cultures remain very low. Thus, understanding the gene diversity and expression profiles in oil palm tissue culture is critical in increasing the efficiency of these processes. A total of 12 standard cDNA libraries, representing three main developmental stages in oil palm tissue culture, were generated in this study. Random sequencing of clones from these cDNA libraries generated 17,599 expressed sequence tags (ESTs). The ESTs were analysed, annotated and assembled to generate 9,584 putative unigenes distributed in 3,268 consensi and 6,316 singletons. These unigenes were assigned putative functions based on similarity and gene ontology annotations. Cluster analysis, which surveyed the relatedness of each library based on the abundance of ESTs in each consensus, revealed that lipid transfer proteins were highly expressed in embryogenic tissues. A glutathione S-transferase was found to be highly expressed in non-embryogenic callus. Further analysis of the unigenes identified 648 non-redundant simple sequence repeats and 211 putative full-length open reading frames. This study has provided an overview of genes expressed during oil palm tissue culture. Candidate genes with expression that are modulated during tissue culture were identified. However, in order to confirm whether these genes are suitable as

  12. Oil palm (Elaeis guineensis Jacq. tissue culture ESTs: Identifying genes associated with callogenesis and embryogenesis

    Directory of Open Access Journals (Sweden)

    Ooi Leslie CL

    2008-05-01

    Full Text Available Abstract Background Oil palm (Elaeis guineensis Jacq. is one of the most important oil bearing crops in the world. However, genetic improvement of oil palm through conventional breeding is extremely slow and costly, as the breeding cycle can take up to 10 years. This has brought about interest in vegetative propagation of oil palm. Since the introduction of oil palm tissue culture in the 1970s, clonal propagation has proven to be useful, not only in producing uniform planting materials, but also in the development of the genetic engineering programme. Despite considerable progress in improving the tissue culture techniques, the callusing and embryogenesis rates from proliferating callus cultures remain very low. Thus, understanding the gene diversity and expression profiles in oil palm tissue culture is critical in increasing the efficiency of these processes. Results A total of 12 standard cDNA libraries, representing three main developmental stages in oil palm tissue culture, were generated in this study. Random sequencing of clones from these cDNA libraries generated 17,599 expressed sequence tags (ESTs. The ESTs were analysed, annotated and assembled to generate 9,584 putative unigenes distributed in 3,268 consensi and 6,316 singletons. These unigenes were assigned putative functions based on similarity and gene ontology annotations. Cluster analysis, which surveyed the relatedness of each library based on the abundance of ESTs in each consensus, revealed that lipid transfer proteins were highly expressed in embryogenic tissues. A glutathione S-transferase was found to be highly expressed in non-embryogenic callus. Further analysis of the unigenes identified 648 non-redundant simple sequence repeats and 211 putative full-length open reading frames. Conclusion This study has provided an overview of genes expressed during oil palm tissue culture. Candidate genes with expression that are modulated during tissue culture were identified. However

  13. RAPD and phytochemical analysis of Thymus moroderi plantlets after cryopreservation.

    Science.gov (United States)

    Marco-Medina, Ana; Casas, José Luis

    2013-01-01

    Cryopreservation is at present the most reliable strategy to preserve plant germplasm. When aromatic plants are the object of conservation it is necessary to assess not only the genetic but also the phytochemical stability to ensure that plant material maintains its qualities after storage. In this work we present molecular and phytochemical stability data related to a previously described vitrification-based cryopreservation protocol for Thymus moroderi Pau ex Martínez. RAPD markers have been used to assess the genetic stability of T. moroderi explants and revealed 0.34 percent of variation in the cryopreserved material studied. Phytochemical data collected from GC-MS analysis of dichloromethane extracts from cryopreserved plantlets rendered a profile in which 1,8-cineole (14.5 percent), camphor (5.9 percent) and borneol (5.2 percent) were the major components. Both data confirmed the suitability of the cryopreservation protocol applied.

  14. Effects of chronic gamma irradiation on adventitious plantlet formation of Saintpaulia ionantha (African violet) detached leaves

    International Nuclear Information System (INIS)

    Arunee Wongpiyasatid; Peeranuch Jompuk; Katarat Chusreeaeom; Thanya Taychasinpitak

    2007-01-01

    Formation of adventitious plantlets on unrootedly detached leaves of two African violet (Saintpaulia ionantha) cultivars, pink and violet flowers, chronically gamma-irradiated in gamma room at The Gamma Irradiation Service and Nuclear Technology Research Center, Kasetsart University was compared. Detached leaves were immediately planted after detachment in plastic trays containing peat moss, 18 leaves per treatment with 3 replications. Three dose rates (rad/h) with 3 doses (rad)/dose rate, were applied to the irradiated samples while the controls were placed outside the gamma room. Three months after irradiation, the number of survived leaves, the number of leaves producing adventitious plantlets and the number of plantlets per leaf were recorded. After that, the young plantlets were transferred to the new pots for further observation on plant growth and mutation characters. The results revealed that the number of survived leaves, the number of leaves producing adventitious plantlets and the number of plantlets per leaf varied slightly with radiation doses but were not significantly different at different dose rates. Radiosensitivity was noticed to be higher in pink flower cultivar than the violet one. M 1 V 1 plantlets will be followed up for growth and mutation character observations

  15. Over-expression of KdSOC1 gene affected plantlet morphogenesis in Kalanchoe daigremontiana.

    Science.gov (United States)

    Zhu, Chen; Wang, Li; Chen, Jinhua; Liu, Chenglan; Zeng, Huiming; Wang, Huafang

    2017-07-17

    Kalanchoe daigremontiana reproduces asexually by producing plantlets along the leaf margin. The aim of this study was to identify the function of the SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 gene in Kalanchoe daigremontiana (KdSOC1) during plantlet morphogenesis. In this study, KdSOC1 gene expression was detected at stem cell niche during in vitro somatic embryogenesis and plantlet morphogenesis. Disrupting endogenous auxin transportation suppressed the KdSOC1 gene response. Knockdown of the KdSOC1 gene caused a defect in cotyledon formation during the early heart stage of somatic embryogenesis. Over-expression (OE) of the KdSOC1 gene resulted in asymmetric plantlet distribution, a reduced number of plantlets, thicker leaves, and thicker vascular fibers. Higher KdPIN1 gene expression and auxin content were found in OE plant compared to those of wild-type plant leaves, which indicated possible KdSOC1 gene role in affecting auxin distribution and accumulation. KdSOC1 gene OE in DR5-GUS Arabidopsis reporting lines resulted in an abnormal auxin response pattern during different stages of somatic embryogenesis. In summary, the KdSOC1 gene OE might alter auxin distribution and accumulation along leaf margin to initiate plantlet formation and distribution, which is crucial for plasticity during plantlet formation under various environmental conditions.

  16. Project on production of mutants by irradiation of in vitro cultured tissues of coconut and banana and their mass propagation by the tissue culture technique

    International Nuclear Information System (INIS)

    Guzman, E.V. de

    1975-01-01

    Fruit pulp tissue, ovary segments with or without ovules and sections from shoot tips of banana were used for studies on growth stimulating or morphogenetic effects of irradiation. Irradiation at 0.1-1.0 kR tended to induce faster callus growth in the otherwise slow-growing cultures. The physical condition and composition of the culture media especially with respect to growth regulators were studied, as were techniques to overcome discoloration of explants, the best choice of plant tissue for explant, and radiation effects on growth and morphogenesis. Due to the difficulty of callus induction with coconut, only the effects of irradiation on embryos cultured in vitro were studied. They were irradiated at various stages of development, i.e. during the early and final stage of liquid culture, and several days after transfer to a solid medium. Adverse effects of irradiation became evident only during the subsequent growth in solid, during the latter stage of which morphological changes were observed. Whereas irradiation of the liquid as well as solid media up to 50 kR had no adverse effect; survival and development became adversely affected at a dose of 1 kR

  17. Tissue culture system using a PANDA ring resonator and wavelength router for hydroponic plant.

    Science.gov (United States)

    Kamoldilok, Surachart; Suwanpayak, Nathaporn; Suttirak, Saisudawan; Yupapin, Preecha P

    2012-06-01

    A novel system of nanofluidics trapping and delivery, which is known as a tissue culture system is proposed. By using the intense optical pulse(i.e., a soliton pulse) and a system constructed by a liquid core waveguide, the optical vortices (gradient optical fields/wells) can be generated, where the trapping tools in the same way as the optical tweezers in the PANDA ring resonator can be formed. By controlling the suitable parameters, the intense optical vortices can be generated within the PANDA ring resonator, in which the nanofluidics can be trapped and moved (transported) dynamically within the Tissue culture system(a wavelength router), which can be used for tissue culture and delivery in the hydroponic plant system.

  18. Plants regenerated from tissue culture contain stable epigenome changes in rice.

    Science.gov (United States)

    Stroud, Hume; Ding, Bo; Simon, Stacey A; Feng, Suhua; Bellizzi, Maria; Pellegrini, Matteo; Wang, Guo-Liang; Meyers, Blake C; Jacobsen, Steven E

    2013-03-19

    Most transgenic crops are produced through tissue culture. The impact of utilizing such methods on the plant epigenome is poorly understood. Here we generated whole-genome, single-nucleotide resolution maps of DNA methylation in several regenerated rice lines. We found that all tested regenerated plants had significant losses of methylation compared to non-regenerated plants. Loss of methylation was largely stable across generations, and certain sites in the genome were particularly susceptible to loss of methylation. Loss of methylation at promoters was associated with deregulated expression of protein-coding genes. Analyses of callus and untransformed plants regenerated from callus indicated that loss of methylation is stochastically induced at the tissue culture step. These changes in methylation may explain a component of somaclonal variation, a phenomenon in which plants derived from tissue culture manifest phenotypic variability. DOI:http://dx.doi.org/10.7554/eLife.00354.001.

  19. The response of human nasal and bronchial organotypic tissue cultures to repeated whole cigarette smoke exposure.

    Science.gov (United States)

    Talikka, Marja; Kostadinova, Radina; Xiang, Yang; Mathis, Carole; Sewer, Alain; Majeed, Shoaib; Kuehn, Diana; Frentzel, Stefan; Merg, Celine; Geertz, Marcel; Martin, Florian; Ivanov, Nikolai V; Peitsch, Manuel C; Hoeng, Julia

    2014-01-01

    Exposure to cigarette smoke (CS) is linked to the development of respiratory diseases, and there is a need to understand the mechanisms whereby CS causes damage. Although animal models have provided valuable insights into smoking-related respiratory tract damage, modern toxicity testing calls for reliable in vitro models as alternatives for animal experimentation. We report on a repeated whole mainstream CS exposure of nasal and bronchial organotypic tissue cultures that mimic the morphological, physiological, and molecular attributes of the human respiratory tract. Despite the similar cellular staining and cytokine secretion in both tissue types, the transcriptomic analyses in the context of biological network models identified similar and diverse biological processes that were impacted by CS-exposed nasal and bronchial cultures. Our results demonstrate that nasal and bronchial tissue cultures are appropriate in vitro models for the assessment of CS-induced adverse effects in the respiratory system and promising alternative to animal experimentation. © The Author(s) 2014.

  20. Fractals in tissue engineering: toward biomimetic cell-culture matrices, microsystems and microstructured implants.

    Science.gov (United States)

    Díaz Lantada, Andrés; Pareja Sánchez, Beatriz; Gómez Murillo, Cristina; Urbieta Sotillo, Javier

    2013-09-01

    Tissue engineering is a rapidly evolving field in which the complexity of biomaterials and biostructures, with typically non-Euclidean or fractal-like geometries, has to be adequately taken into account for the promotion of enhanced and even personalized diagnostic and therapeutic solutions. This study covers the main applications of fractals in the field of tissue engineering, including their advantages for modeling biological processes and cell-culture procedures, but specially focusing on their benefits for describing the complex geometries and structures of biomaterials (both natural and synthetic), many of which have potential uses for the development of cell culture microsystems, scaffolds for tissue repair and implants for tissue repair in general. We also explore the main supporting design, simulation and manufacturing technologies, as well as the most remarkable difficulties and limitations linked to the generalized use of fractals in engineering design, and also detail some current solution proposals and future directions.

  1. Consistent and heritable alterations of DNA methylation are induced by tissue culture in maize.

    Science.gov (United States)

    Stelpflug, Scott C; Eichten, Steven R; Hermanson, Peter J; Springer, Nathan M; Kaeppler, Shawn M

    2014-09-01

    Plants regenerated from tissue culture and their progenies are expected to be identical clones, but often display heritable molecular and phenotypic variation. We characterized DNA methylation patterns in callus, primary regenerants, and regenerant-derived progenies of maize using immunoprecipitation of methylated DNA (meDIP) to assess the genome-wide frequency, pattern, and heritability of DNA methylation changes. Although genome-wide DNA methylation levels remained similar following tissue culture, numerous regions exhibited altered DNA methylation levels. Hypomethylation events were observed more frequently than hypermethylation following tissue culture. Many of the hypomethylation events occur at the same genomic sites across independent regenerants and cell lines. The DNA methylation changes were often heritable in progenies produced from self-pollination of primary regenerants. Methylation changes were enriched in regions upstream of genes and loss of DNA methylation at promoters was associated with altered expression at a subset of loci. Differentially methylated regions (DMRs) found in tissue culture regenerants overlap with the position of naturally occurring DMRs more often than expected by chance with 8% of tissue culture hypomethylated DMRs overlapping with DMRs identified by profiling natural variation, consistent with the hypotheses that genomic stresses similar to those causing somaclonal variation may also occur in nature, and that certain loci are particularly susceptible to epigenetic change in response to these stresses. The consistency of methylation changes across regenerants from independent cultures suggests a mechanistic response to the culture environment as opposed to an overall loss of fidelity in the maintenance of epigenetic states. Copyright © 2014 by the Genetics Society of America.

  2. Usefulness of fibroblast culture for testing of cattle tissues polluted with heavy metals

    International Nuclear Information System (INIS)

    Weglarz, L.; Drozdz, M.Wa.; Wardas, M.; Kula, B.; Pawlaczyk-Szpilowa, M.

    1990-01-01

    Cattle tissues (liver, kidney, brain, and lung) that had been polluted with heavy metals were tested for their ability to alter fibroblast culture growth, cellular protein and DNA content, and fibroblast DNA synthesis. At 72 hr of incubation a significant increase in cellular DNA and [14C]thymidine incorporation was noted in the primary cultures as well as in the subcultures compared to controls. Fibroblast cultures also displayed growth inhibition and reduction in protein content. The measurement of basic biochemical parameters of the fibroblast culture may represent a sensitive means of assessing rapidly the activity of heavy metals deposited in the tissues of cattle as a result of their grazing on polluted soil

  3. Current status and future prospects for cultured limbal tissue transplants in Australia and New Zealand.

    Science.gov (United States)

    Harkin, Damien G; Apel, Andrew J; Di Girolamo, Nick; Watson, Stephanie; Brown, Karl; Daniell, Mark D; McGhee, J Jane; McGhee, Charles N J

    2013-04-01

    Cultured limbal tissue transplants have become widely used over the last decade as a treatment for limbal stem cell deficiency (LSCD). While the number of patients afflicted with LSCD in Australia and New Zealand is considered to be relatively low, the impact of this disease on quality of life is so severe that the potential efficacy of cultured transplants has necessitated investigation. We presently review the basic biology and experimental strategies associated with the use of cultured limbal tissue transplants in Australia and New Zealand. In doing so, we aim to encourage informed discussion on the issues required to advance the use of cultured limbal transplants in Australia and New Zealand. Moreover, we propose that a collaborative network could be established to maintain access to the technology in conjunction with a number of other existing and emerging treatments for eye diseases. © 2012 The Authors. Clinical and Experimental Ophthalmology © 2012 Royal Australian and New Zealand College of Ophthalmologists.

  4. Identification of Stevioside Using Tissue Culture-Derived Stevia (Stevia rebaudiana) Leaves

    Science.gov (United States)

    Karim, Md. Ziaul; Uesugi, Daisuke; Nakayama, Noriyuki; Hossain, M. Monzur; Ishihara, Kohji; Hamada, Hiroki

    2015-01-01

    Stevioside is a natural sweetener from Stevia leaf, which is 300 times sweeter than sugar. It helps to reduce blood sugar levels dramatically and thus can be of benefit to diabetic people. Tissue culture is a very potential modern technology that can be used in large-scale disease-free stevia production throughout the year. We successfully produced stevia plant through in vitro culture for identification of stevioside in this experiment. The present study describes a potential method for identification of stevioside from tissue culture-derived stevia leaf. Stevioside in the sample was identified using HPLC by measuring the retention time. The percentage of stevioside content in the leaf samples was found to be 9.6%. This identification method can be used for commercial production and industrialization of stevia through in vitro culture across the world. PMID:28008268

  5. Multifunctional sensing membrane-based platform for tissue or cell culturing and monitoring

    DEFF Research Database (Denmark)

    2014-01-01

    The present application discloses a water-permeable sensor membrane comprising i) a first layer of a conductive material defining at least one electrode and having a thickness of 0.1-,000 [mu]m; ii) a second layer of a nanostructure material build on the first layer; and iii) a third, topmost......, layer of a conducting polymer material defining at least one electrode and having a thickness of 0.001-1.0 [mu]m. The application also discloses a tissue or cell culture sample monitoring assembly comprising a sensor assembly and a tissue sample or a cell culture sample arranged on top of the third...

  6. Structure and component alteration of rabbit Achilles tendon in tissue culture.

    Science.gov (United States)

    Hosaka, Yoshinao; Ueda, Hiromi; Yamasaki, Tadatsugu; Suzuki, Daisuke; Matsuda, Naoya; Takehana, Kazushige

    2005-12-01

    The aim of this study was to investigate alterations of cultured tendon tissues to determine whether tissue culture is a useful method for biological analyses of the tendon. Tendon tissues for tissue culture were isolated from Achilles tendons of rabbits. The tendon segments were placed one segment per well and incubated in growth medium consisting of Dullbecco's modified Eagle's medium supplemented with 5% fetal bovine serum at 37 degrees C in a humidified atmosphere with 5% CO(2) for various periods. The alignment of collagen fibrils was preserved for 48 h, but tendon structure has disintegrated at 96 h. Alcian blue staining and gelatine zymography revealed that proteoglycan markedly diminished and that matrix metalloproteinase (MMPs) activity was upregulated sharply at 72 and 96 h. The ratio of collagen fibrils with large diameter had increased and the mean diameter and mass average diameter value had reached maximum at 48 h. The values then decreased and mean diameters at 72 and 96 h were significantly different from that at 48 h. At 96 h, the ratio of collagen fibrils with small diameters had increased and collagen fibrils with large diameters had disappeared. These findings indicate that structural alteration is possible to be induced by disintegration of collagen fibrils and disappearance of glycosaminoglycans from extracellular matrix (ECM), subsequent of upregulation of MMPs activity. Although the study period is limited, the tissue culture method is available for investigating cell-ECM interaction in tendons.

  7. Production of immunoglobulins in gingival tissue explant cultures from juvenile periodontitis patients

    International Nuclear Information System (INIS)

    Hall, E.R.; Falkler, W.A. Jr.; Suzuki, J.B.

    1990-01-01

    B lymphocytes and plasma cells are histologically observed in granulomatous periodontal tissues of juvenile periodontitis (JP) patients. Local immune processes may participate in protective or immunopathologic roles in the pathogenesis of this disease. An in vitro explant culture system was utilized to demonstrate the production of immunoglobulins by diseased JP tissues. Immunodiffusion studies using goat anti-human gamma, alpha, or mu chain serum revealed IgG to be the major immunoglobulin present in 92% of the day 1 supernatant fluids (SF) of the 47 JP gingival tissue explant cultures. IgA was present in 15% of the SF; however, no IgM was detected. Staph Protein A isolated 14C-labeled IgG from the SF, when allowed to react with goat anti-human gamma chain serum, formed lines of precipitation. Positive autoradiographs confirmed the biosynthesis of IgG by the explant cultures. The in vitro gingival tissue explant culture system described provides a useful model for the study of localized immunoglobulins produced by diseased tissues of JP patients

  8. Production of immunoglobulins in gingival tissue explant cultures from juvenile periodontitis patients

    Energy Technology Data Exchange (ETDEWEB)

    Hall, E.R.; Falkler, W.A. Jr.; Suzuki, J.B. (Univ. of Maryland Dental School, Baltimore (USA))

    1990-10-01

    B lymphocytes and plasma cells are histologically observed in granulomatous periodontal tissues of juvenile periodontitis (JP) patients. Local immune processes may participate in protective or immunopathologic roles in the pathogenesis of this disease. An in vitro explant culture system was utilized to demonstrate the production of immunoglobulins by diseased JP tissues. Immunodiffusion studies using goat anti-human gamma, alpha, or mu chain serum revealed IgG to be the major immunoglobulin present in 92% of the day 1 supernatant fluids (SF) of the 47 JP gingival tissue explant cultures. IgA was present in 15% of the SF; however, no IgM was detected. Staph Protein A isolated 14C-labeled IgG from the SF, when allowed to react with goat anti-human gamma chain serum, formed lines of precipitation. Positive autoradiographs confirmed the biosynthesis of IgG by the explant cultures. The in vitro gingival tissue explant culture system described provides a useful model for the study of localized immunoglobulins produced by diseased tissues of JP patients.

  9. Identification of Stevioside Using Tissue Culture-Derived Stevia (Stevia rebaudiana) Leaves

    OpenAIRE

    Karim, Md. Ziaul; Uesugi, Daisuke; Nakayama, Noriyuki; Hossain, M. Monzur; Ishihara, Kohji; Hamada, Hiroki

    2016-01-01

    Stevioside is a natural sweetener from Stevia leaf, which is 300 times sweeter than sugar. It helps to reduce blood sugar levels dramatically and thus can be of benefit to diabetic people. Tissue culture is a very potential modern technology that can be used in large-scale disease-free stevia production throughout the year. We successfully produced stevia plant through in vitro culture for identification of stevioside in this experiment. The present study describes a potential method for iden...

  10. Culture of three-dimensional tissue model and its application in bystander-effect research

    International Nuclear Information System (INIS)

    Wu Ruqun; Xu An; Wu Lijun; Hu Burong

    2012-01-01

    Compared with the cultured monolayer (2D) cells, three-dimensional (3D) tissue could be more similar to the environment in vivo including the physical support, chemical factors, cell-cell and cell-matrix interaction and so on. With the development of three-dimensional cell culture techniques (TDCC), 3D tissue is widely used in the areas of bystander effect research. This review focuses on introducing the TDCC method and its application in bystander-effect research. First, the development process of 3D tissue culture method was introduced. Secondly, the induction of radiation induced bystander effects both in 2D cell and 3D tissue and its mechanisms were reviewed. Finally, because heavy ion (carbon ion beam) has been developed as a useful tool to cure solid cancer, and the 3D tissue model is an ideal material to study the damages on body after being irradiated and to understand the underlying mechanisms, future study about heavy ion radiation inducing bystander effect in 3D tissue was discussed. (authors)

  11. Detection of genotoxicity in the marine environment: A preliminary feasibility study using primary mussel tissue culture

    Energy Technology Data Exchange (ETDEWEB)

    Cornet, Michel [UMR 5805 EPOC ' Environnements et Paleoenvironnements Oceaniques' , Universite Bordeaux 1, CNRS, Avenue des Facultes, 33405 Talence Cedex (France)]. E-mail: m.cornet@epoc.u-bordeaux1.fr

    2007-08-15

    The purpose of this study was to evaluate the feasibility and potential usefulness of primary cultures of somatic tissues from adult mussel by means of sister chromatid exchange induction (SCE). This research is an initial pilot study carried out with mussel mantle tissue using seawater artificially contaminated with cadmium and polluted seawater from the port of Arcachon. With regard to cadmium concentration, mean SCE numbers showed a progressive increase from 1.07 {+-} 0.18 per diploid cell in controls (i.e. cultures without contaminant) to 2.91 {+-} 0.42 per diploid cell for the highest concentration, 10{sup -4} M. With regard to the medium prepared with seawater from the port of Arcachon, the mean SCE number reached a value of 5.85 {+-} 0.85 per diploid cell. The analysis of SCEs induced by cadmium showed DNA responses even at the lowest concentration (i.e. 10{sup -7} M). The study demonstrates the feasibility of the sister chromatid exchange (SCE) approach based upon primary mussel tissue culture, for the genotoxicity testing of contaminated seawater. Highlights from this procedure are (1) the presence of an active cell proliferation, (2) the use whole-water samples, (3) the possibility of culturing without serum, (4) the absence of cell dissociation before culturing and (5) a cellular proliferation which can be obtained in cultures carried out in a medium containing seawater whose salinity is comprise between 28 and 35 per mille.

  12. Biolistic-mediated transformation protocols for maize and pearl millet using pre-cultured immature zygotic embryos and embryogenic tissue

    CSIR Research Space (South Africa)

    O'Kennedy, MM

    2011-01-01

    Full Text Available transgenic food crop products, but biolistic-mediated transformation are still valid for proof of concept and functional genomics applications. Biolistic-mediated transformation and the production of transgenic plantlets via somatic embryogenesis of two maize...

  13. Generation and multiplication of plantlets from callus derived from Haplopappus gracilus (Nutt.) Gray and their karyotype analysis

    Science.gov (United States)

    Kann, R. P.; O'Connor, S. A.; Levine, H. G.; Krikorian, A. D.

    1991-01-01

    Unopened flower heads of Haplopappus gracilis (2n = 4) provided primary explants for callus production and subsequent induction of organized growth. Callus was initiated from small (3-5 mm in length) floral buds with benzylaminopurine (BAP) (44.4 micromoles; 10 mg/l) and naphthalene acetic acid (NAA) (0.54 micromole; 0.1 mg/l). Lowering the BAP level to 4.44 micromoles (1 mg/l) but maintaining the NAA level, gave rise to organized but highly compressed shoot growing points from an otherwise undifferentiated callus mass. Shoots selected from such cultures were maintainable and could be proliferated by growing 1-1.5-cm stem tip cuttings on Murashige and Skoog basal medium (solidified with agar) containing 0.444 micromole (0.1 mg/l) BAP and 0.054 micromole (0.01 mg/l) NAA. The stem tip multiplication rates obtainable by these means permit reliable strategies for shoot multiplication or production of rooted plantlets. Prolonged subculture and maintenance of shoots on growth regulator-free medium leads to in vitro flowering and greatly reduces rooting capacity. Karyotype analysis of chromosomes from root tip cells at metaphase and chromosome measurements show that karyologically uniform plantlets (based on chromosome number and morphology) can be obtained.

  14. Participation of cob tissue in the transport of medium components into maize kernels cultured in vitro

    International Nuclear Information System (INIS)

    Felker, F.C.

    1990-01-01

    Maize (Zea mays L.) kernels cultured in vitro while still attached to cob pieces have been used as a model system to study the physiology of kernel development. In this study, the role of the cob tissue in uptake of medium components into kernels was examined. Cob tissue was essential for in vitro kernel growth, and better growth occurred with larger cob/kernel ratios. A symplastically transported fluorescent dye readily permeated the endosperm when supplied in the medium, while an apoplastic dye did not. Slicing the cob tissue to disrupt vascular connections, but not apoplastic continuity, greatly reduced [ 14 C]sucrose uptake into kernels. [ 14 C]Sucrose uptake by cob and kernel tissue was reduced 31% and 68%, respectively, by 5 mM PCMBS. L-[ 14 C]glucose was absorbed much more slowly than D-[ 14 C]glucose. These and other results indicate that phloem loading of sugars occurs in the cob tissue. Passage of medium components through the symplast cob tissue may be a prerequisite for uptake into the kernel. Simple diffusion from the medium to the kernels is unlikely. Therefore, the ability of substances to be transported into cob tissue cells should be considered in formulating culture medium

  15. Using the "Kalanchoe daigremontiana" Plant To Show the Effects of Photoperiodism on Plantlet Formation.

    Science.gov (United States)

    Hershey, David R.

    2002-01-01

    Describes an activity demonstrating the importance of photoperiod on plant development. Uses the plant devil's backbone for the experiment and studies the details of photoperiodic requirement for plantlet formation. (Contains 12 references.) (YDS)

  16. Rate of re-infection of tissue culture-derived Latin American and ...

    African Journals Online (AJOL)

    Jane

    2010-12-20

    Dec 20, 2010 ... Rate of re-infection of tissue culture-derived Latin. American and East and Southern African cassava genotypes by mosaic disease. E. B. Okorogri1, V. O. Adetimirin1, G. Ssemakula2*, B. Odu2 and A. G. O. Dixon2. 1Department of Agronomy, University of Ibadan, Oyo State, Nigeria. 2International Institute of ...

  17. Simple and high yielding method for preparing tissue specific extracellular matrix coatings for cell culture.

    Science.gov (United States)

    DeQuach, Jessica A; Mezzano, Valeria; Miglani, Amar; Lange, Stephan; Keller, Gordon M; Sheikh, Farah; Christman, Karen L

    2010-09-27

    The native extracellular matrix (ECM) consists of a highly complex, tissue-specific network of proteins and polysaccharides, which help regulate many cellular functions. Despite the complex nature of the ECM, in vitro cell-based studies traditionally assess cell behavior on single ECM component substrates, which do not adequately mimic the in vivo extracellular milieu. We present a simple approach for developing naturally derived ECM coatings for cell culture that provide important tissue-specific cues unlike traditional cell culture coatings, thereby enabling the maturation of committed C2C12 skeletal myoblast progenitors and human embryonic stem cells differentiated into cardiomyocytes. Here we show that natural muscle-specific coatings can (i) be derived from decellularized, solubilized adult porcine muscle, (ii) contain a complex mixture of ECM components including polysaccharides, (iii) adsorb onto tissue culture plastic and (iv) promote cell maturation of committed muscle progenitor and stem cells. This versatile method can create tissue-specific ECM coatings, which offer a promising platform for cell culture to more closely mimic the mature in vivo ECM microenvironment.

  18. Cost-effective nutrient sources for tissue culture of cassava (Manihot ...

    African Journals Online (AJOL)

    shawgi ali

    2012-08-16

    Aug 16, 2012 ... Key words: Tissue culture, cassava, nutrient sources, micropropagation, low cost medium, acclimatization. INTRODUCTION. Cassava ... This situation is exacerbated by lack of healthy planting materials. Farmers often ... a channel for transmission of systemic infections from one generation to other, leading.

  19. Efficient one-step tissue culture protocol for propagation of endemic ...

    African Journals Online (AJOL)

    Efficient one-step tissue culture protocol for propagation of endemic plant, Lilium martagon var. cattaniae Vis. M Skorić, S Živković, J Savić, B Šiler, A Sabovljević, S Todorović, D Grubišić ...

  20. [Comparative study on alkaloids of tissue-culture seedling and wild plant of Dendrobium huoshanense ].

    Science.gov (United States)

    Chen, Nai-dong; Gao, Feng; Lin, Xin; Jin, Hui

    2014-06-01

    To compare the composition and content of alkaloid of Dendrobium huoshanense tissue-culture seedling and wild plant. A comparative evaluation on the quality was carried out by HPLC and TLC methods including the composition and the content of alkaloids. Remarkable variation existed in the two kinds of Dendrobium huoshanense. For the tissue-culture plant, only two alkaloids were checked out by both HPLC and TLC while four alkaloids were observed in the wild plant. The alkaloid content of tissue-culture seedling and wild plant was(0. 29 ± 0. 11)%o and(0. 43 ± 0. 15) %o,respectively. Distinguished difference is observed in both composition and content of alkaloids from the annual shoots of different provenances of Dendrobium huoshanense. It suggested that the quality of tissue-culture seedling of Dendrobium huoshanense might be inconsistent with the wild plant. Furthermore, the established alkaloids-knock-out HPLC method would provide a new research tool on quality control of Chinese medicinal materials which contain unknown alkaloids.

  1. Tissue culture-induced alteration in cytosine methylation in new rice ...

    African Journals Online (AJOL)

    Zizania DNA introgression could induce a large number of genetic and epigenetic changes of the new rice recombinant inbred lines genome. In this present study, we employed inter-simple sequence repeat (ISSR) to further study the genetic and epigenetic changes that are induced by tissue culture. Changes induced by ...

  2. Cost-effective nutrient sources for tissue culture of cassava ( Manihot ...

    African Journals Online (AJOL)

    Application of tissue culture technology is constrained by high costs making seedlings unaffordable. The objective of this study was to evaluate the possibility of using locally available fertilizers as alternative nutrient sources for cassava micropropagation. A Low Cost Medium (LCM) whereby the conventional sources of four ...

  3. Mass Spectrometry-Based Proteomics in Molecular Diagnostics: Discovery of Cancer Biomarkers Using Tissue Culture

    Science.gov (United States)

    Paul, Debasish; Kumar, Avinash; Gajbhiye, Akshada; Santra, Manas K.; Srikanth, Rapole

    2013-01-01

    Accurate diagnosis and proper monitoring of cancer patients remain a key obstacle for successful cancer treatment and prevention. Therein comes the need for biomarker discovery, which is crucial to the current oncological and other clinical practices having the potential to impact the diagnosis and prognosis. In fact, most of the biomarkers have been discovered utilizing the proteomics-based approaches. Although high-throughput mass spectrometry-based proteomic approaches like SILAC, 2D-DIGE, and iTRAQ are filling up the pitfalls of the conventional techniques, still serum proteomics importunately poses hurdle in overcoming a wide range of protein concentrations, and also the availability of patient tissue samples is a limitation for the biomarker discovery. Thus, researchers have looked for alternatives, and profiling of candidate biomarkers through tissue culture of tumor cell lines comes up as a promising option. It is a rich source of tumor cell-derived proteins, thereby, representing a wide array of potential biomarkers. Interestingly, most of the clinical biomarkers in use today (CA 125, CA 15.3, CA 19.9, and PSA) were discovered through tissue culture-based system and tissue extracts. This paper tries to emphasize the tissue culture-based discovery of candidate biomarkers through various mass spectrometry-based proteomic approaches. PMID:23586059

  4. Mass Spectrometry-Based Proteomics in Molecular Diagnostics: Discovery of Cancer Biomarkers Using Tissue Culture

    Directory of Open Access Journals (Sweden)

    Debasish Paul

    2013-01-01

    Full Text Available Accurate diagnosis and proper monitoring of cancer patients remain a key obstacle for successful cancer treatment and prevention. Therein comes the need for biomarker discovery, which is crucial to the current oncological and other clinical practices having the potential to impact the diagnosis and prognosis. In fact, most of the biomarkers have been discovered utilizing the proteomics-based approaches. Although high-throughput mass spectrometry-based proteomic approaches like SILAC, 2D-DIGE, and iTRAQ are filling up the pitfalls of the conventional techniques, still serum proteomics importunately poses hurdle in overcoming a wide range of protein concentrations, and also the availability of patient tissue samples is a limitation for the biomarker discovery. Thus, researchers have looked for alternatives, and profiling of candidate biomarkers through tissue culture of tumor cell lines comes up as a promising option. It is a rich source of tumor cell-derived proteins, thereby, representing a wide array of potential biomarkers. Interestingly, most of the clinical biomarkers in use today (CA 125, CA 15.3, CA 19.9, and PSA were discovered through tissue culture-based system and tissue extracts. This paper tries to emphasize the tissue culture-based discovery of candidate biomarkers through various mass spectrometry-based proteomic approaches.

  5. Smooth muscle myosin regulation by serum and cell density in cultured rat lung connective tissue cells.

    Science.gov (United States)

    Babij, P; Zhao, J; White, S; Woodcock-Mitchell, J; Mitchell, J; Absher, M; Baldor, L; Periasamy, M; Low, R B

    1993-08-01

    RNA and protein analyses were used to detect expression of SM1 and SM2 smooth muscle myosin heavy chain (MHC) in cultured adult rat lung connective tissue cells (RL-90). Smooth muscle MHC mRNA expression in confluent cells grown in 10% serum was approximately 50% of the level in adult stomach. Similar results were obtained in cells cultured at low density (25% confluency) in 1% serum. However, in low-density cultures transferred to 10% serum for 24 h, the level of MHC mRNA decreased to approximately 20% of that in adult stomach. Smooth muscle alpha-actin showed a pattern of expression similar to that for smooth muscle MHC. Expression of nonmuscle MHC-A mRNA was higher in all culture conditions compared to stomach. MHC-A mRNA expression was less in low-density cultures in low serum and increased when low-density cultures were transferred to 10% serum for 24 h. MHC-B mRNA expression was less in low- vs. high-density cultures. In contrast to MHC-A, however, MHC-B mRNA expression in low-density cultures was higher in low serum. Immunofluorescence and immunoblotting with SM1-specific antibody demonstrated the presence of the SM1 protein isoform as well as reactivity to a protein band migrating slightly faster than SM2. These results demonstrate that cultured rat lung connective tissue cells express smooth muscle MHC and that expression is modulated by culture conditions.

  6. Ex-vivo Potential of Cadaveric and Fresh Limbal Tissues to Regenerate Cultured Epithelium

    Directory of Open Access Journals (Sweden)

    Vemuganti Geeta

    2004-01-01

    Full Text Available Purpose: To evaluate and compare the ex-vivo growth potential and formation of cultured corneal epithelium from residual corneo-limbal rings obtained from the operating room after penetrating keratoplasty, and fresh limbal tissues from patients undergoing routine cataract surgery. Methods: With the approval of the Institutional Review Board and informed consent from patients, 1-2mm of limbal tissues from 15 patients and 31 tissues from the cadaveric limbal ring preserved in MK medium (16 tissues and Optisol (15 tissues were used for the study. Donor data included age, time lapse between death and collection, collection and preservation and preservation and culture. Tiny bits of the limbal tissue were explanted on the de-epithelialised human amniotic membrane prepared following standard guidelines, and cultured using Human Corneal Epithelial cell medium. Radial growth from the explant was observed and measured by phase contrast microscopy over 2-4 weeks. After adequate confluent growth, whole mount preparation of the membrane was made and stained with haematoxylin and eosin. Part of the membrane was fixed in formalin and processed for routine histologic examination. The sections were stained with haematoxylin and eosin. Results: Forty-six tissues were evaluated from 42 eyes (15 from patients, 31 from cadaveric eyes with a mean age of 55.3 years ± 21.23 years (range 18 years - 110 years. The growth pattern observed was similar in all the positive cases with clusters of cells budding from the explant over 24- 72 hours, and subsequent formation of a monolayer over the next 2-3 weeks. The stained whole mount preparation showed a radial growth of cells around explants with diameter ranging from 5 to 16mm. Histologic evaluation of the membrane confirmed the growth of 2-3 cell-layered epithelium over the amniotic membrane. Cultivated epithelium around explant cell cultures was observed in 100% (15/15 of limbal tissue obtained from patients, as against

  7. Tissue culture characteristics of maize (Zea mays L.) haploid coleoptile sections.

    Science.gov (United States)

    Jiang, L; Jing, G X; Li, X Y; Wang, X Q; Xing, Z; Deng, P K; Zhao, R G

    2015-12-08

    Doubled haploid (DH) technology, which is used for rapidly purifying genetic resources, is a key technology in modern maize breeding. The present study evaluated the tissue culture characteristics of maize haploid coleoptile sections, in order to provide a new way of haploid doubling. With 20 combinations of haploid coleoptile sections, obtained by hybridization within Reid, Tangsipingtou, and Term-tropical groups, as explants, we analyzed the induction and differentiation rate of callus, observed the number of root tip chromosomes in regenerated plants, and analyzed the pollen fertility. In addition, we used 47 SSR markers to analyze the genotypes of regenerated plants. The Reid and Tangsipingtou groups had significantly higher induction rates of haploid coleoptile callus compared to the Term-tropical group. Fifteen haploid plants were obtained which had 10 chromosomes in the root tips as assessed by I-KI staining. It was also noticed that the pollen of pollinated anthers were partially fertile. The haploid plants had genetic stability and showed no variation. The Reid and Tangsipingtou groups had good culture characteristics of haploid coleoptile sections, while the Term-tropical group had poor culture characteristics. Genotypes of haploid plants generated by tissue culture were evidenced to come from recombinant types of parents. Thus, this study established a tissue culture system of maize haploid coleoptile.

  8. Effect of adipose tissue processing procedures in culture result: a study preliminary

    Directory of Open Access Journals (Sweden)

    Jeanne A. Pawitan

    2011-02-01

    Full Text Available Background: There are various methods of processing adipose tissue before culture, depending on the adipose tissue samples. The aim of this study is to compare several modifications of culturing and sub-culturing procedures of adipose tissue to fit the condition in our laboratory.Method: This is a descriptive study that was done in the Immunology and Endocrinology Integrated Laboratory, University of Indonesia, from  October 2009 to April 2010. Three adipose tissue processing procedures, various amount of seeding and two subculture methods were compared in term of cell yield and time needed. In the first procedure, collagenase-1 digestion was done in 30minutes, cell seeding were 24,000 and 36,000 per flask; in the second procedure, collagenase-1 digestion was done in 60minutes, cell seeding were 24,000, 48,000, and 72,000 per flask; and in the third procedure, the adipose tissue remnants from the first  procedure were again digested for another 45 minutes, cell seeding were 74,000, and 148,000 per flask. Difference in subculture methods were the presence or absence of washing step.Result: Procedure 1 yielded the lowest amount of cell, and after culture, the cells grew very slow, and was contaminated before harvest of primary culture. Procedure-2 and -3 succeeded to yield primary cultures. Some of the cultures were contaminated, so that further subculture was not  applicable, and only one tissue processing procedure (procedure 2: 60 minute collagenase-1 digestion, without lysis buffer, cell seeding 48,000 and 72,000 could complete the three subcultures. Though some of the procedures could not be completed, final result could be concluded.Conclusion: In this preliminary study, 60 minute colagenase-1 digestion with intermittent shaking every 5 minutes and cell seeding around 50,000 or more, followed by subculture method without washing step gave the best result. (Med J Indones 2011; 20:15-9Keywords: collagenase-1, primary culture, subculture

  9. Design and validation of a biomechanical bioreactor for cartilage tissue culture.

    Science.gov (United States)

    Correia, V; Panadero, J A; Ribeiro, C; Sencadas, V; Rocha, J G; Gomez Ribelles, J L; Lanceros-Méndez, S

    2016-04-01

    Specific tissues, such as cartilage, undergo mechanical solicitation under their normal performance in human body. In this sense, it seems necessary that proper tissue engineering strategies of these tissues should incorporate mechanical solicitations during cell culture, in order to properly evaluate the influence of the mechanical stimulus. This work reports on a user-friendly bioreactor suitable for applying controlled mechanical stimulation--amplitude and frequency--to three-dimensional scaffolds. Its design and main components are described, as well as its operation characteristics. The modular design allows easy cleaning and operating under laminar hood. Different protocols for the sterilization of the hermetic enclosure are tested and ensure lack of observable contaminations, complying with the requirements to be used for cell culture. The cell viability study was performed with KUM5 cells.

  10. tissue culture

    African Journals Online (AJOL)

    ONOS

    2010-07-05

    Jul 5, 2010 ... Spinach (Spinacia oleracea L.) is an important vegetable crop of which dioecy in nature has made cultivar improvement difficult using .... hormonal treatments at callous formation stage in all cultivars. Treatment. Means of callous ... hormones in the medium (Molvig and Rose, 1994), as shown in our present ...

  11. Whole genome characterization of non-tissue culture adapted HRSV strains in severely infected children

    Directory of Open Access Journals (Sweden)

    Kumaria Rajni

    2011-07-01

    Full Text Available Abstract Background Human respiratory syncytial virus (HRSV is the most important virus causing lower respiratory infection in young children. The complete genetic characterization of RSV clinical strains is a prerequisite for understanding HRSV infection in the clinical context. Current information about the genetic structure of the HRSV genome has largely been obtained using tissue culture adapted viruses. During tissue culture adaptation genetic changes can be introduced into the virus genome, which may obscure subtle variations in the genetic structure of different RSV strains. Methods In this study we describe a novel Sanger sequencing strategy which allowed the complete genetic characterisation of 14 clinical HRSV strains. The viruses were sequenced directly in the nasal washes of severely hospitalized children, and without prior passage of the viruses in tissue culture. Results The analysis of nucleotide sequences suggested that vRNA length is a variable factor among primary strains, while the phylogenetic analysis suggests selective pressure for change. The G gene showed the greatest sequence variation (2-6.4%, while small hydrophobic protein and matrix genes were completely conserved across all clinical strains studied. A number of sequence changes in the F, L, M2-1 and M2-2 genes were observed that have not been described in laboratory isolates. The gene junction regions showed more sequence variability, and in particular the intergenic regions showed a highest level of sequence variation. Although the clinical strains grew slower than the HRSVA2 virus isolate in tissue culture, the HRSVA2 isolate and clinical strains formed similar virus structures such as virus filaments and inclusion bodies in infected cells; supporting the clinical relevance of these virus structures. Conclusion This is the first report to describe the complete genetic characterization of HRSV clinical strains that have been sequenced directly from clinical

  12. Development and growth of plantlets of Pinus contorta regenerated from adventitious buds

    Energy Technology Data Exchange (ETDEWEB)

    Flygh, G.; Groenroos, R.; Arnold, S. von [Swedish Univ. of Agricultural Sciences, Uppsala (Sweden). Dept. of Forest Genetics; Hoegberg, K.A. [The Association for Forest Tree Breeding, Svaloev (Sweden)

    1998-11-01

    Before micropropagation techniques can be applied to a particular species, it is crucial to optimize the method and to determine how the micropropagated plants grow in the field. Adventitious shoots developed on embryos of Pinus contorta Dougl. ex Loud. after a 2 h pulse treatment with 250 {mu}M N6-benzyladenine. The time to first subculture after the pulse treatment influenced the yield of adventitious shoots. On average, 68% of the adventitious shoots had developed roots 12 weeks after treatment with 1.25 mM indole-3-butyric acid for 6 h. The auxin treatment stimulated early rooting (i.e. within 6 weeks) but had no effect on late rooting (i.e. after 6 weeks). The size of the plantlets was of importance for the survival when potted. All plantlets with a distinct stem elongated during the first growth period while some without did not. The relative height growth rate of plantlets was similar to that of seedlings. In the field the increase of height was similar for plantlets and seedlings. Plagiotropy was higher for the plantlets than for the seedlings (35 and 10% respectively). We concluded that most plantlets of P. contorta elongate normally and have a similar gross morphology to seedlings 23 refs, 8 figs, 4 tabs

  13. The effects of varying concentrations of growth regulators benzyladenine and naphthalene acetic acid and gamma irradiation on the gross plantlet morphology of Garcinia mangostana L

    International Nuclear Information System (INIS)

    Bonsol, Ma. Fatima Nona M.; Lasiste, Jade Marie Edenvirg F.; Quinio, Ma. Franchesca S.

    2004-02-01

    Mangosteen (Garcinia mangostana L.) is regarded as one of the best flavored fruits in the world. Though this fruit yields high income and has great potential as an export, productivity in the Philippines is still limited due to its slow and difficult conventional propagation. To increase the plants' productivity, growth regulators can be added, the seeds can be exposed to irradiation to cause genetic mutations, and micropropagation techniques can be used. This study seeks to determine the effects of these three variables on the general morphological characteristics of the plantlets grown both in vitro and in vivo. The first set of mangosteen seeds was inoculated and grown in Murashige and Skoog (MS) basal culture medium with different treatment combinations of growth regulators, naphthalene acetic acid (NAA) and benzyladenine (BA). Treatment concentrations ranged from 0 parts per million (ppm) to 6 ppm. The treatment that best induced stem, leaf and root formation, and produced plantlets with long stems and roots was MS with 6 ppm NAA combined with 6 ppm BA. A second set of seeds was then exposed to varying levels of gamma radiation and propagated in vivo and in vitro. For in vivo, it was observed that the length of stem of the plantlet decreases with increasing radiation dose; length of leaves of the mangosteen plantlets was best noted in seeds exposed to 10 Gy gamma rays. In in vitro, the two treatment concentrations that best helped seeds to develop callus, which were 0 ppm BA with 4 pmm NAA and 4 ppm BA with 6 ppm NAA, were added to MS basal medium. Seeds exposed to 5 Gy and grown in basal medium with 0 ppm BA and 4 ppm NAA were observed to have developed the most callus. (Author)

  14. Exploring plant tissue culture in Withania somnifera (L.) Dunal: in vitro propagation and secondary metabolite production.

    Science.gov (United States)

    Shasmita; Rai, Manoj K; Naik, Soumendra K

    2017-12-26

    Withania somnifera (L.) Dunal (family: Solanaceae), commonly known as "Indian Ginseng", is a medicinally and industrially important plant of the Indian subcontinent and other warmer parts of the world. The plant has multi-use medicinal potential and has been listed among 36 important cultivated medicinal plants of India that are in high demand for trade due to its pharmaceutical uses. The medicinal importance of this plant is mainly due to the presence of different types of steroidal lactones- withanolides in the roots and leaves. Owing to low seed viability and poor germination, the conventional propagation of W. somnifera falls short to cater its commercial demands particularly for secondary metabolite production. Therefore, there is a great need to develop different biotechnological approaches through tissue and organ culture for seasonal independent production of plants in large scale which will provide sufficient raw materials of uniform quality for pharmaceutical purposes. During past years, a number of in vitro plant regeneration protocols via organogenesis and somatic embryogenesis and in vitro conservation through synthetic seed based encapsulation technology have been developed for W. somnifera. Several attempts have also been made to standardize the protocol of secondary metabolite production via tissue/organ cultures, cell suspension cultures, and Agrobacterium rhizogenes-mediated transformed hairy root cultures. Employment of plant tissue culture based techniques would provide means for rapid propagation and conservation of this plant species and also provide scope for enhanced production of different bioactive secondary metabolites. The present review provides a comprehensive report on research activities conducted in the area of tissue culture and secondary metabolite production in W. somnifera during the past years. It also discusses the unexplored areas which might be taken into consideration for future research so that the medicinal properties and

  15. Metabolomics reveals the heterogeneous secretome of two entomopathogenic fungi to ex vivo cultured insect tissues.

    Directory of Open Access Journals (Sweden)

    Charissa de Bekker

    Full Text Available Fungal entomopathogens rely on cellular heterogeneity during the different stages of insect host infection. Their pathogenicity is exhibited through the secretion of secondary metabolites, which implies that the infection life history of this group of environmentally important fungi can be revealed using metabolomics. Here metabolomic analysis in combination with ex vivo insect tissue culturing shows that two generalist isolates of the genus Metarhizium and Beauveria, commonly used as biological pesticides, employ significantly different arrays of secondary metabolites during infectious and saprophytic growth. It also reveals that both fungi exhibit tissue specific strategies by a distinguishable metabolite secretion on the insect tissues tested in this study. In addition to showing the important heterogeneous nature of these two entomopathogens, this study also resulted in the discovery of several novel destruxins and beauverolides that have not been described before, most likely because previous surveys did not use insect tissues as a culturing system. While Beauveria secreted these cyclic depsipeptides when encountering live insect tissues, Metarhizium employed them primarily on dead tissue. This implies that, while these fungi employ comparable strategies when it comes to entomopathogenesis, there are most certainly significant differences at the molecular level that deserve to be studied.

  16. Assay of anticancer drugs in tissue culture: cell cultures of biopsies from human astrocytoma.

    Science.gov (United States)

    Morgan, D; Freshney, R I; Darling, J L; Thomas, D G; Celik, F

    1983-02-01

    A method has been developed for measuring the drug sensitivity of human gliomas in short-term culture, using scintillation counting or autofluorography. Cell cultures prepared from malignant astrocytomas were treated with anticancer drugs whilst in exponential growth in microtitration plates. After drug treatment and a recovery period, residual viability was measured by [3H] leucine incorporation followed by scintillation counting or by [35S] methionine incorporation and autofluorography in situ. In 5 glioma cell lines tested against 6 drugs, the microtitration method correlated well with monolayer cloning. Although replicate samples of the same tumour showed little variation in chemosensitivity, there was marked variation between the chemosensitivities of cultures derived from the tumours of different patients. However, as variability between replicates was apparent during drug exposure or shortly after, it is important to allow the assay to run as long as possible after drug removal. It is hoped that this assay may provide the basis of a method for the prediction of in vivo chemosensitivity or the screening of potential chemotherapeutic drugs.

  17. Interaction of Ulocladium atrum, a Potential Biological Control Agent, with Botrytis cinerea and Grapevine Plantlets

    Directory of Open Access Journals (Sweden)

    Sébastien Ronseaux

    2013-09-01

    Full Text Available The effectiveness of biological control agent, Ulocladium atrum (isolates U13 and U16 in protecting Vitis vinifera L. cv. Chardonnay against gray mold disease caused by Botrytis cinerea, and simulation of the foliar defense responses was investigated. A degraded mycelium structure during cultural assay on potato dextrose agar revealed that U. atrum isolates U13 and U16 were both antagonistic to B. cinerea, mainly when isolates were inoculated two days before Botrytis. Under in vitro conditions, foliar application of U. atrum protected grapevine leaves against gray mold disease. An increase in chitinase activity was induced by the presence of U. atrum isolates indicating that the biological control agents triggered plant defense mechanisms. Moreover, U13 has the potential to colonize the grapevine plantlets and to improve their growth. The ability of U. atrum isolates to exhibit an antagonistic effect against B. cinerea in addition to their aptitude to induce plant resistance and to promote grapevine growth may explain a part of their biological activity. Hence, this study suggests that U. atrum provides a suitable biocontrol agent against gray mold in grapevines.

  18. Evaluating the Different Levels of Diaminozied on the Five Mini-Tuber Potato Cultivars produced by Tissue Culture under Greenhouse Condition

    Directory of Open Access Journals (Sweden)

    M Soleimani Agdam

    2013-11-01

    Full Text Available To evaluate the effect of different levels of diaminozied on the potato cultivars mini-tubers produced by tissue culture under greenhouse condition a factorial an experiment on the base of completely randomized design in three replications was performed at the Ardabil Villkige Company Greenhouse in 2010. The first factor consisted of four levels of diaminozied, as plant growth regulator, (0, 40, 80 and 120 mg.L-1 and the second factor of five cultivars of virus-free potato (Satina, Savalan, Marfona, Agria and Cesear. Virus-free plantlets were transferred to isolated greenhouse and planted in 20×20×15 cm in plastic pots beds made by 1:1 volume of Biolan peat moss and perlite. During growth period traits like plant height, leaf and stem number per plant, mini-tuber weight and number per plant and square meter, and mini-tuber size were average measured. The analysis of variance showed significant differences among different levels of diaminozied for leaf number per plant, mini-tuber weight and its number per square meter, tuber average size in all cultivars under study. The interaction of diaminozied by cultivars showed significant difference as to leaf number per plant, mini-tuber weight, its number per square meter and average tuber size. Cesear and Marfona cultivars produced higher mini-tuber weight and tuber number per square meter by applying 40 mg.L-1 diaminozied. The correlation was significant and positive between mini-tuber number per square meter with mini-tuber weight per square meter and root length. It was also significant and positive between mini-tuber weight per square meter with plant height and average mini-tuber size. The results of multi regression analysis indicated that mini-tuber weight per square meter, average tuber size, plant height and root length were effective on changes of the mini-tuber number. The highest positive direct effect on number of mini-tuber per square meter was due to the plant height and the less

  19. Diolistic labeling of neuronal cultures and intact tissue using a hand-held gene gun.

    Science.gov (United States)

    O'Brien, John A; Lummis, Sarah C R

    2006-01-01

    Diolistic labeling is a highly efficient method for introducing dyes into cells using biolistic techniques. The use of lipophilic carbocyanine dyes, combined with particle-mediated biolistic delivery using a hand-held gene gun, allows non-toxic labeling of multiple cells in both living and fixed tissue. The technique is rapid (labeled cells can be visualized in minutes) and technically undemanding. Here, we provide a detailed protocol for diolistic labeling of cultured human embryonic kidney 293 cells and whole brain using a hand-held gene gun. There are four major steps: (i) coating gold microcarriers with one or more dyes; (ii) transferring the microcarriers into a cartridge to make a bullet; (iii) preparation of cells or intact tissue; and (iv) firing the microcarriers into cells or tissue. The method can be readily adapted to other cell types and tissues. This protocol can be completed in less than 1 h.

  20. Studies on the reaction in tissue culture of tomato genotypes under biotic stress

    Directory of Open Access Journals (Sweden)

    Ewa Hanus-Fajerska

    2014-01-01

    Full Text Available Plant regeneration in vitro from virus-infected somatic tomato (Lycopersicon sp. tissue was performed. Regeneration experiments were started after the determination of virus presence, using enzyme-linked immunosorbent assay, in leaves used as a source of explants. Leaf explants infected with selected strains of tomato mosaic Tobamovirus or cucumber mosaic Cucumovirus respectively, were cultured on a standarised MS agar medium to induce adventitious shoots, which were afterwards excised, rooted in vitro and cultured to plants. Explants were also screened for their ability to produce callus. Diverse effects of viral infection, ranging from stimulation to inhibition of callus formation and of morphogenesis rate, were observed. The health condition of the tissue proved to affect regeneration potential of Lycopersicon esculentum, whereas wild accesions did not react in that case so distinctly. In cultivated tomato was encountered the decline in competence to reproduce shoots adventitiously in infected tissue. There was also relationship between donor plant health condition and adventitious root formation in regenerated shoots. Experiments with short-term cultures of L. esculenum reveled also that a certain number of shoots regenerated from diseased tissue can be virus-free.

  1. RPE in perfusion tissue culture and its response to laser application. Preliminary report.

    Science.gov (United States)

    Framme, Carsten; Kobuch, Karin; Eckert, Elfriede; Monzer, Jan; Roider, Johann

    2002-01-01

    To study the effects of conventional laser application on the retinal pigment epithelium (RPE) in a perfusion tissue culture model of porcine retinal pigment epithelium without overlying neurosensory retina. RPE with underlying choroid was prepared from enucleated porcine eyes and fixed in a holding ring (Minusheet). Specimens were then placed in two-compartment tissue culture containers (MinuCell & Minutissue, Bad Abbach, Germany) and were cultured during continuous perfusion with culture medium at both sides of the entire specimen, the upper RPE and the lower choroid (12 specimens out of 6 eyes). Cultures were kept for 1, 3, 7 and 14 days and were examined histologically. Laser treatment was performed on each tissue ring by application of 3 x 3 laser burns one day after culture began (argon ion laser, wavelength: 514 nm, pulse duration: 100 ms; spot size: 200 microm) using different energy levels (400-1,000 mW); (16 specimens out of 8 eyes). During laser treatment a marked lightening of the RPE with centrifugal spreading was observed. Using higher levels of energy, a contraction of the RPE towards the center of the laser spot was noticed. One day after laser photocoagulation histology revealed destruction of RPE; within 3-7 days of culture, migration and proliferation of neighboring cells was observed in several lesions. After 7 days the initial defect of the irradiated area was covered with dome shaped RPE cells and after 14 days multilayered RPE cells were showing ongoing proliferation. However, there were also cases without proliferation after laser treatment. The non-treated, continuously perfused RPE showed regular appearance in histological sections: during the first 7 days of culture, light microscopy revealed a normal matrix with a well-differentiated RPE monolayer. Subsequently proliferation even without treatment was observed and after 14 days the RPE became multilayered. It was possible to study the early healing response to the effect of laser

  2. Floral induction in tissue culture: a system for the analysis of LEAFY-dependent gene regulation.

    Science.gov (United States)

    Wagner, Doris; Wellmer, Frank; Dilks, Kieran; William, Dilusha; Smith, Michael R; Kumar, Prakash P; Riechmann, José Luis; Greenland, Andrew J; Meyerowitz, Elliot M

    2004-07-01

    We have developed a versatile floral induction system that is based on ectopic overexpression of the transcription factor LEAFY (LFY) in callus. During shoot regeneration, flowers or floral organs are formed directly from root explants without prior formation of rosette leaves. Morphological and reporter gene analyses show that leaf-like structures are converted to floral organs in response to LFY activity. Thus, increased levels of LFY activity are sufficient to bypass normal vegetative development and to direct formation of flowers in tissue culture. We found that about half of the cultured cells respond to inducible LFY activity with a rapid upregulation of the known direct target gene of LFY, APETALA1 (AP1). This dramatic increase in the number of LFY-responsive cells compared to whole plants suggested that the tissue culture system could greatly facilitate the analysis of LFY-dependent gene regulation by genomic approaches. To test this, we monitored the gene expression changes that occur in tissue culture after activation of LFY using a flower-specific cDNA microarray. Induction of known LFY target genes was readily detected in these experiments. In addition, several other genes were identified that had not been implicated in signaling downstream of LFY before. Thus, the floral induction system is suitable for the detection of low abundance transcripts whose expression is controlled in an LFY-dependent manner.

  3. Transcriptomic comparisons between cultured human adipose tissue-derived pericytes and mesenchymal stromal cells

    Directory of Open Access Journals (Sweden)

    Lindolfo da Silva Meirelles

    2016-03-01

    Full Text Available Mesenchymal stromal cells (MSCs, sometimes called mesenchymal stem cells, are cultured cells able to give rise to mature mesenchymal cells such as adipocytes, osteoblasts, and chondrocytes, and to secrete a wide range of trophic and immunomodulatory molecules. Evidence indicates that pericytes, cells that surround and maintain physical connections with endothelial cells in blood vessels, can give rise to MSCs (da Silva Meirelles et al., 2008 [1]; Caplan and Correa, 2011 [2]. We have compared the transcriptomes of highly purified, human adipose tissue pericytes subjected to culture-expansion in pericyte medium or MSC medium, with that of human adipose tissue MSCs isolated with traditional methods to test the hypothesis that their transcriptomes are similar (da Silva Meirelles et al., 2015 [3]. Here, we provide further information and analyses of microarray data from three pericyte populations cultured in pericyte medium, three pericyte populations cultured in MSC medium, and three adipose tissue MSC populations deposited in the Gene Expression Omnibus under accession number GSE67747. Keywords: Mesenchymal stromal cells, Mesenchymal stem cells, Pericytes, Microarrays

  4. [Effect of activated charcoal on rooting in tissue culture seedling of Begonia fimbristipula on Dinghushan Mountain].

    Science.gov (United States)

    Chen, Xiong-wei; Shao, Ling; Liang, Lian; Pan, Zhen-tao

    2012-09-01

    To study the effect of different plant growth substance and activated charcoal on rooting in culture seedling of Begonia fimbristpula on Dinghushan mountain. Tissue culture single factor experiment method was used. NAA 0. 3 mg/L + IBA 0. 2 mg/L preferably induction adventitious bud clump with corm to take rooting, but the number of adventitious root were less, short and small, callow shoot more germination. 300 mg/L activated carbon obviously increased radicate quality and inhibited fine buds point differentiation, root number up to 15.5 institia, root length range was 2.0-5.1 cm, root system developed. Tissue culture seedlings were higher, corn and leaf were good quality, strong growth. Took root of seedling cultivation with bulb for bush in the form of scattered bud planted to peat soil: perlite (3:1) mixed in matrix, after the transplant survival rate reached 100%, plant form seedlings fast, grew exuberant. MS with sucrose 30 g/L + NAA 0.3 mg/L + IBA 0.2 mg/L + activated carbon 300 mg/L + carrageenan 7.0 g/L as the tissue culture seedling of Begonia fimbristipula radicate system, is rapid propagation and preserve local unique plant in an effective way.

  5. SIS with tissue-cultured allogenic cartilages patch tracheoplasty in a rabbit model for tracheal defect.

    Science.gov (United States)

    Zhang, Longfang; Liu, Zhi; Cui, Pengcheng; Zhao, Daqing; Chen, Wenxian

    2007-06-01

    In the rabbit model, small intestinal submucosa (SIS) compounded with tissue-cultured allogenic cartilages appeared to be an efficacious method for the patch repair of partial circumferential tracheal defects instead of autologous grafts. SIS appears to be a safe and promising means of facilitating neovascularization and tissue regeneration. The long-term use of SIS and tissue-cultured allogenic cartilages warrants further investigation. Tracheal defect reparation remains a challenging surgical problem that can require reconstruction using autologous grafts or artificial stents. This study was performed to evaluate the efficacy of SIS, a biocompatible, acellular matrix, compounded with different tissue-cultured allogenic cartilages, in the repair of a critical-size tracheal defect. A full-thickness defect (4 x 8 mm) was created in tracheal rings four to six in adult rabbits. A piece of 8-ply SIS sandwiched in thyroid cartilage, auricular cartilage, or without cartilage, respectively (designated experiment 1, 2, or 3, respectively), was sutured to the edges of the defect with interrupted 4-0 polypropylene sutures. In control animals, the defect was closed with lamina praetrachealis. All animals were followed until signs of dyspnea became apparent or for 4 or 12 weeks. After follow-up and euthanasia, the trachea was harvested and prepared for histologic evaluation using conventional techniques. All animals tolerated the procedure well but two animals in group 1 (n=5), three in group 2 (n=5), and one in group 3 (n=5) had stridor after operation and expired within 1 month. Histologically, neovascularization of the patch was noted with moderate inflammation. The surface of the SIS patch was covered with a lining of ciliated epithelial cells. The tissue-cultured allogenic cartilages degraded to some extent.

  6. A Protocol for Rapid, Measurable Plant Tissue Culture Using Stem Disc Meristem Micropropagation of Garlic ("Allium Sativum L.")

    Science.gov (United States)

    Peat, Gerry; Jones, Meriel

    2012-01-01

    Plant tissue culture is becoming an important technique for the mass propagation of plants. Problems with existing techniques, such as slow growth and contamination, have restricted the practical work in plant tissue culture carried out in schools. The new protocol using garlic meristematic stem discs explained in this article addresses many of…

  7. Antiandrogenic actions of medroxyprogesterone acetate on epithelial cells within normal human breast tissues cultured ex vivo.

    Science.gov (United States)

    Ochnik, Aleksandra M; Moore, Nicole L; Jankovic-Karasoulos, Tanja; Bianco-Miotto, Tina; Ryan, Natalie K; Thomas, Mervyn R; Birrell, Stephen N; Butler, Lisa M; Tilley, Wayne D; Hickey, Theresa E

    2014-01-01

    Medroxyprogesterone acetate (MPA), a component of combined estrogen-progestin therapy (EPT), has been associated with increased breast cancer risk in EPT users. MPA can bind to the androgen receptor (AR), and AR signaling inhibits cell growth in breast tissues. Therefore, the aim of this study was to investigate the potential of MPA to disrupt AR signaling in an ex vivo culture model of normal human breast tissue. Histologically normal breast tissues from women undergoing breast surgical operation were cultured in the presence or in the absence of the native AR ligand 5α-dihydrotestosterone (DHT), MPA, or the AR antagonist bicalutamide. Ki67, bromodeoxyuridine, B-cell CLL/lymphoma 2 (BCL2), AR, estrogen receptor α, and progesterone receptor were detected by immunohistochemistry. DHT inhibited the proliferation of breast epithelial cells in an AR-dependent manner within tissues from postmenopausal women, and MPA significantly antagonized this androgenic effect. These hormonal responses were not commonly observed in cultured tissues from premenopausal women. In tissues from postmenopausal women, DHT either induced or repressed BCL2 expression, and the antiandrogenic effect of MPA on BCL2 was variable. MPA significantly opposed the positive effect of DHT on AR stabilization, but these hormones had no significant effect on estrogen receptor α or progesterone receptor levels. In a subset of postmenopausal women, MPA exerts an antiandrogenic effect on breast epithelial cells that is associated with increased proliferation and destabilization of AR protein. This activity may contribute mechanistically to the increased risk of breast cancer in women taking MPA-containing EPT.

  8. Advanced cell culture technology for generation of in vivo-like tissue models

    Directory of Open Access Journals (Sweden)

    Stefan Przyborski

    2017-06-01

    Full Text Available Human tissues are mostly composed of different cell types, that are often highly organised in relation to each other. Often cells are arranged in distinct layers that enable signalling and cell-to-cell interactions. Here we describe the application of scaffold-based technology, that can be used to create advanced organotypic 3D models of various tissue types that more closely resemble in vivo-like conditions (Knight et al., 2011. The scaffold comprises a highly porous polystyrene material, engineered into a 200 micron thick membrane that is presented in various ways including multi-welled plates and well inserts, for use with conventional culture plasticware and medium perfusion systems. This technology has been applied to generate numerous unique types of co-culture model. For example: 1 a full thickness human skin construct comprising dermal fibroblasts and keratinocytes, raised to the air-liquid interface to induce cornification of the upper layers (Fig.1 (Hill et al., 2015; 2 a neuron-glial co-culture to enable the study of neurite outgrowth interacting with astroglial cells to model and investigate the glial scar found in spinal cord injury (Clarke et al., 2016; 3 formation of a sub-mucosa consisting of a polarised simple epithelium, layer of ECM proteins simulating the basement membrane, and underlying stromal tissues (e.g. intestinal mucosa. These organotypic models demonstrate the versatility of scaffold membranes and the creation of advanced in vivo-like tissue models. Creating a layered arrangement more closely simulates the true anatomy and organisation of cells within many tissue types. The addition of different cell types in a temporal and spatial fashion can be used to study inter-cellular relationships and create more physiologically relevant in vivo-like cell-based assays. Methods that are relatively straightforward to use and that recreate the organised structure of real tissues will become valuable research tools for use in

  9. Biodynamic imaging for phenotypic profiling of three-dimensional tissue culture

    Science.gov (United States)

    Sun, Hao; Merrill, Daniel; An, Ran; Turek, John; Matei, Daniela; Nolte, David D.

    2017-01-01

    Three-dimensional (3-D) tissue culture represents a more biologically relevant environment for testing new drugs compared to conventional two-dimensional cancer cell culture models. Biodynamic imaging is a high-content 3-D optical imaging technology based on low-coherence interferometry and digital holography that uses dynamic speckle as high-content image contrast to probe deep inside 3-D tissue. Speckle contrast is shown to be a scaling function of the acquisition time relative to the persistence time of intracellular transport and hence provides a measure of cellular activity. Cellular responses of 3-D multicellular spheroids to paclitaxel are compared among three different growth techniques: rotating bioreactor (BR), hanging-drop (HD), and nonadherent (U-bottom, UB) plate spheroids, compared with ex vivo living tissues. HD spheroids have the most homogeneous tissue, whereas BR spheroids display large sample-to-sample variability as well as spatial heterogeneity. The responses of BR-grown tumor spheroids to paclitaxel are more similar to those of ex vivo biopsies than the responses of spheroids grown using HD or plate methods. The rate of mitosis inhibition by application of taxol is measured through tissue dynamics spectroscopic imaging, demonstrating the ability to monitor antimitotic chemotherapy. These results illustrate the potential use of low-coherence digital holography for 3-D pharmaceutical screening applications.

  10. Characterization of cytoskeletal and junctional proteins expressed by cells cultured from human arachnoid granulation tissue

    Directory of Open Access Journals (Sweden)

    Mehta Bhavya C

    2005-10-01

    Full Text Available Abstract Background The arachnoid granulations (AGs are projections of the arachnoid membrane into the dural venous sinuses. They function, along with the extracranial lymphatics, to circulate the cerebrospinal fluid (CSF to the systemic venous circulation. Disruption of normal CSF dynamics may result in increased intracranial pressures causing many problems including headaches and visual loss, as in idiopathic intracranial hypertension and hydrocephalus. To study the role of AGs in CSF egress, we have grown cells from human AG tissue in vitro and have characterized their expression of those cytoskeletal and junctional proteins that may function in the regulation of CSF outflow. Methods Human AG tissue was obtained at autopsy, and explanted to cell culture dishes coated with fibronectin. Typically, cells migrated from the explanted tissue after 7–10 days in vitro. Second or third passage cells were seeded onto fibronectin-coated coverslips at confluent densities and grown to confluency for 7–10 days. Arachnoidal cells were tested using immunocytochemical methods for the expression of several common cytoskeletal and junctional proteins. Second and third passage cultures were also labeled with the common endothelial markers CD-31 or VE-cadherin (CD144 and their expression was quantified using flow cytometry analysis. Results Confluent cultures of arachnoidal cells expressed the intermediate filament protein vimentin. Cytokeratin intermediate filaments were expressed variably in a subpopulation of cells. The cultures also expressed the junctional proteins connexin43, desmoplakin 1 and 2, E-cadherin, and zonula occludens-1. Flow cytometry analysis indicated that second and third passage cultures failed to express the endothelial cell markers CD31 or VE-cadherin in significant quantities, thereby showing that these cultures did not consist of endothelial cells from the venous sinus wall. Conclusion To our knowledge, this is the first report of

  11. Tissue culture and associated biotechnological interventions for the improvement of coconut (Cocos nucifera L.): a review.

    Science.gov (United States)

    Nguyen, Quang Thien; Bandupriya, H D Dharshani; López-Villalobos, Arturo; Sisunandar, S; Foale, Mike; Adkins, Steve W

    2015-11-01

    The present review discusses not only advances in coconut tissue culture and associated biotechnological interventions but also future research directions toward the resilience of this important palm crop. Coconut (Cocos nucifera L.) is commonly known as the 'tree of life'. Every component of the palm can be used to produce items of value and many can be converted into industrial products. Coconut cultivation faces a number of acute problems that reduce its productivity and competitiveness. These problems include various biotic and abiotic challenges as well as an unstable market for its traditional oil-based products. Around 10 million small-holder farmers cultivate coconut palms worldwide on c. 12 million hectares of land, and many more people own a few coconut palms that contribute to their livelihoods. Inefficiency in the production of seedlings for replanting remains an issue; however, tissue culture and other biotechnological interventions are expected to provide pragmatic solutions. Over the past 60 years, much research has been directed towards developing and improving protocols for (i) embryo culture; (ii) clonal propagation via somatic embryogenesis; (iii) homozygote production via anther culture; (iv) germplasm conservation via cryopreservation; and (v) genetic transformation. Recently other advances have revealed possible new ways to improve these protocols. Although effective embryo culture and cryopreservation are now possible, the limited frequency of conversion of somatic embryos to ex vitro seedlings still prevents the large-scale clonal propagation of coconut. This review illustrates how our knowledge of tissue culture and associated biotechnological interventions in coconut has so far developed. Further improvement of protocols and their application to a wider range of germplasm will continue to open up new horizons for the collection, conservation, breeding and productivity of coconut.

  12. Low cost options for tissue culture technology in developing countries. Proceedings of a technical meeting

    International Nuclear Information System (INIS)

    2004-02-01

    Tissue culture technology is used for the production of doubled haploids, cryopreservation, propagating new plant varieties, conserving rare and endangered plants, difficult-to-propagate plants, and to produce secondary metabolites and transgenic plants. The production of high quality planting material of crop plants and fruit trees, propagated from vegetative parts, has created new opportunities in global trading, benefited growers, farmers, and nursery owners, and improved rural employment. However, there are still major opportunities to produce and distribute high quality planting material, e.g. crops like banana, date palm, cassava, pineapple, plantain, potato, sugarcane, sweet potato, yams, ornamentals, fruit and forest trees. The main advantage of tissue culture technology lies in the production of high quality and uniform planting material that can be multiplied on a year-round basis under disease-free conditions anywhere irrespective of the season and weather. However, the technology is capital, labor and energy intensive. Although, labor is cheap in many developing countries, the resources of trained personnel and equipment are often not readily available. In addition, energy, particularly electricity, and clean water are costly. The energy requirements for tissue culture technology depend on day temperature, day-length and relative humidity, and they have to be controlled during the process of propagation. Individual plant species also differ in their growth requirements. Hence, it is necessary to have low cost options for weaning, hardening of micropropagated plants and finally growing them in the field. This publication describes options for reducing costs to establish and operate tissue culture facilities and primarily focus on plant micropropagation. It includes papers on the basics of tissue culture technology, low cost options for the design of laboratories, use of culture media and containers, energy and labor saving, integration and adoption of

  13. Functional enhancement of chitosan and nanoparticles in cell culture, tissue engineering, and pharmaceutical applications

    Directory of Open Access Journals (Sweden)

    Wenjuan eGao

    2012-08-01

    Full Text Available Abstract: As a biomaterial, chitosan has been widely used in tissue engineering, wound healing, drug delivery, and other biomedical applications. It can be formulated in a variety of forms, such as powder, film, sphere, gel and fiber. These features make chitosan an almost ideal biomaterial in cell culture applications, and cell cultures arguably constitute the most practical way to evaluate biocompatibility and biotoxicity. The advantages of cell cultures are that they can be performed under totally controlled environments, allow high throughput functional screening, and are less costly, as compared to other assessment methods. Chitosan can also be modified into multilayer composite by combining with other polymers and moieties to alter the properties of chitosan for particular biomedical applications. This review briefly depicts and discusses applications of chitosan and nanoparticles in cell culture, in particular, the effects of chitosan and nanoparticles on cell adhesion, cell survival, and the underlying molecular mechanisms: both stimulatory and inhibitory influences are discussed. Our aim is to update the current status of how nanoparticles can be utilized to modify the properties of chitosan to advance the art of tissue engineering by using cell cultures.

  14. Hormonal control of root development on epiphyllous plantlets of Bryophyllum (Kalancho?) marnierianum: role of auxin and ethylene

    OpenAIRE

    Kulka, Richard G.

    2008-01-01

    Epiphyllous plantlets develop on leaves of Bryophyllum marnierianum when they are excised from the plant. Shortly after leaf excision, plantlet shoots develop from primordia located near the leaf margin. After the shoots have enlarged for several days, roots appear at their base. In this investigation, factors regulating plantlet root development were studied. The auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA) abolished root formation without markedly affecting shoot growth. This ...

  15. Three-dimensional hydrogel cell culture systems for modeling neural tissue

    Science.gov (United States)

    Frampton, John

    Two-dimensional (2-D) neural cell culture systems have served as physiological models for understanding the cellular and molecular events that underlie responses to physical and chemical stimuli, control sensory and motor function, and lead to the development of neurological diseases. However, the development of three-dimensional (3-D) cell culture systems will be essential for the advancement of experimental research in a variety of fields including tissue engineering, chemical transport and delivery, cell growth, and cell-cell communication. In 3-D cell culture, cells are provided with an environment similar to tissue, in which they are surrounded on all sides by other cells, structural molecules and adhesion ligands. Cells grown in 3-D culture systems display morphologies and functions more similar to those observed in vivo, and can be cultured in such a way as to recapitulate the structural organization and biological properties of tissue. This thesis describes a hydrogel-based culture system, capable of supporting the growth and function of several neural cell types in 3-D. Alginate hydrogels were characterized in terms of their biomechanical and biochemical properties and were functionalized by covalent attachment of whole proteins and peptide epitopes. Methods were developed for rapid cross-linking of alginate hydrogels, thus permitting the incorporation of cells into 3-D scaffolds without adversely affecting cell viability or function. A variety of neural cell types were tested including astrocytes, microglia, and neurons. Cells remained viable and functional for longer than two weeks in culture and displayed process outgrowth in 3-D. Cell constructs were created that varied in cell density, type and organization, providing experimental flexibility for studying cell interactions and behavior. In one set of experiments, 3-D glial-endothelial cell co-cultures were used to model blood-brain barrier (BBB) structure and function. This co-culture system was

  16. [Extraction and analysis of chemical components of essential oil in Thymus vulgaris of tissue culture].

    Science.gov (United States)

    Li, Xiao-Dong; Yang, Li; Xu, Shi-Qian; Li, Jian-Guo; Cheng, Zhi-Hui; Dang, Jian-Zhang

    2011-10-01

    To extract the essential oils from the Seedlings, the Aseptic Seedlings and the Tissue Culture Seedlings of Thymus vulgaris and analyze their chemical components and the relative contents. The essential oils were extracted by steam distillation, the chemical components and the relative contents were identified and analyzed by gas chromatography-mass spectrometry (GC/MS) and peak area normalization method. The main chemical components of essential oil in these three samples had no significant difference, they all contained the main components of essential oil in Thymus vulgaris: Thymol, Carvacrol, o-Cymene, gamma-Terpinene, Caryophyllene et al. and only had a slight difference in the relative content. This study provides important theoretical foundation and data reference for further study on production of essential oil in thyme by tissue culture technology.

  17. Formation of Stomach Tissue by Organoid Culture Using Mouse Embryonic Stem Cells.

    Science.gov (United States)

    Noguchi, Taka-Aki K; Kurisaki, Akira

    2017-01-01

    In this chapter, we describe a method for the induction of stomach organoids from mouse embryonic stem (ES) cells. We used an embryoid body-based differentiation method to induce gastric primordial epithelium covered with mesenchyme and further differentiate it in Matrigel by 3D culture. The differentiated organoid contains both corpus- and antrum-specific mature gastric tissue cells. This protocol may be useful for a variety of studies in developmental biology and disease modeling of the stomach.

  18. Relation of arginine-lysine antagonism to herpes simplex growth in tissue culture.

    Science.gov (United States)

    Griffith, R S; DeLong, D C; Nelson, J D

    1981-01-01

    In the studies conducted, arginine deficiency suppressed herpes simplex virus replication in tissue culture. Lysine, an analog of arginine, as an antimetabolite, antagonized the viral growth-promoting action of arginine. The in vitro data may be the basis for the observation that patients prone to herpetic lesions and other related viral infections, particularly during periods of stress, should abstain from arginine excess and may also require supplemental lysine in their diet.

  19. Analysis of laser-induced fluorescence spectra of in vitro plant tissue cultures

    Science.gov (United States)

    Muñoz-Muñoz, Ana Celia; Gutiérrez-Pulido, Humberto; Rodríguez-Domínguez, José Manuel; Gutiérrez-Mora, Antonia; Rodríguez-Garay, Benjamín; Cervantes-Martínez, Jesús

    2007-04-01

    We demonstrate the effectiveness of laser-induced fluorescence (LIF) for monitoring the development and stress detection of in vitro tissue cultures in a nondestructive and noninvasive way. The changes in LIF spectra caused by the induction of organogenesis, the increase of the F690/F740 ratio as a result of the stress originated in the organogenic explants due to shoot emergence, and the relationship between fluorescence spectra and shoot development were detected by LIF through closed containers of Saintpaulia ionantha.

  20. Stimulatory effect of serum from diabetic patients on insulin release from mouse pancreatic islets maintained in tissue culture

    DEFF Research Database (Denmark)

    Eff, C; Deckert, T; Andersson, A

    1981-01-01

    Islets of Langerhans from NMRI-mice were kept for one week in tissue culture in medium supplemented with human serum obtained from either normal healthy subjects or newly diagnosed juvenile diabetic patients before insulin treatment. Islets cultured in diabetic serum released more insulin than...... islets cultured in normal serum, whether tissue culture medium 199 with 5.5-8.3 mmol/l glucose and 10% serum, or culture medium RPMI 1640 with 11 mmol/l glucose and 0.5% serum were used. Islets kept for one week in culture with diabetic serum did not show any decrease in DNA content or glucose induced...... insulin secretion and biosynthesis. It is concluded that serum from newly diagnosed insulin-dependent diabetic patients stimulates insulin release from isolated mouse islets kept in tissue culture. The underlying mechanism is unknown....

  1. Biodynamic Doppler imaging of subcellular motion inside 3D living tissue culture and biopsies (Conference Presentation)

    Science.gov (United States)

    Nolte, David D.

    2016-03-01

    Biodynamic imaging is an emerging 3D optical imaging technology that probes up to 1 mm deep inside three-dimensional living tissue using short-coherence dynamic light scattering to measure the intracellular motions of cells inside their natural microenvironments. Biodynamic imaging is label-free and non-invasive. The information content of biodynamic imaging is captured through tissue dynamics spectroscopy that displays the changes in the Doppler signatures from intracellular constituents in response to applied compounds. The affected dynamic intracellular mechanisms include organelle transport, membrane undulations, cytoskeletal restructuring, strain at cellular adhesions, cytokinesis, mitosis, exo- and endo-cytosis among others. The development of 3D high-content assays such as biodynamic profiling can become a critical new tool for assessing efficacy of drugs and the suitability of specific types of tissue growth for drug discovery and development. The use of biodynamic profiling to predict clinical outcome of living biopsies to cancer therapeutics can be developed into a phenotypic companion diagnostic, as well as a new tool for therapy selection in personalized medicine. This invited talk will present an overview of the optical, physical and physiological processes involved in biodynamic imaging. Several different biodynamic imaging modalities include motility contrast imaging (MCI), tissue-dynamics spectroscopy (TDS) and tissue-dynamics imaging (TDI). A wide range of potential applications will be described that include process monitoring for 3D tissue culture, drug discovery and development, cancer therapy selection, embryo assessment for in-vitro fertilization and artificial reproductive technologies, among others.

  2. Vegetative propagation of adult Eucalyptus grandis X urophylla and comparison of growth between micropropagated plantlets and rooted cuttings.

    Science.gov (United States)

    Yang, J C; Chung, J D; Chen, Z Z

    1995-12-01

    Methods for the production of micropropagated plantlets and rooted cuttings were developed and used to vegetatively multiply adult Eucalyptus grandis X urophylla. Rooting success was less than 5% when cuttings excised from twigs of 3-year-old trees were used. The rooted cuttings were grown in the greenhouse as explant- or cutting-donors and maintained at a height of 30 to 100 cm by trimming back periodically. Good rooting success (95%) of cuttings was obtained for epicormic shoots produced from donor plants after trimming 5 times. Explants of both apical and axillary buds taken from the donor plants produced multiple shoots when cultured in vitro. In vitro multiple shoot production was optimal on MS medium containing 0.1 mg/l BA and 0.01 mg/l NAA averaging 13.7 shoots per explant in a 40-day culture period. Shoot elongation was accelerated on a modified MS medium containing half strength potassium nitrate and sucrose. Elongated shoots excised at approximately 1.5 cm in length were successfully rooted on media with NAA or IBA concentrations ranging from 0.1 to 10 mg/l. Root formation was optimal on medium consisting of full strength MS basal macro elements and vitamins, half strength micro elements, 1% sucrose and supplemented with 0.3 mg/l IBA. In the field test, no significant differences were found in tree height and DBH between micropropagated plantlets and rooted cuttings at 1 and 3 years old, with the exception at 2 years old. A considerable difference arose between the 2 types of vegetative propagules in physiological response to flowering, caused by dissimilar degrees of rejuvenation.

  3. Direct plantlet inoculation with soil or insect-associated fungi may control cabbage root fly maggots.

    Science.gov (United States)

    Razinger, Jaka; Lutz, Matthias; Schroers, Hans-Josef; Palmisano, Marilena; Wohler, Christian; Urek, Gregor; Grunder, Jürg

    2014-07-01

    A potential Delia radicum biological control strategy involving cauliflower plantlet inoculation with various fungi was investigated in a series of laboratory and glasshouse experiments. In addition to entomopathogenic fungi, fungi with a high rhizosphere competence and fungi with the ability to survive as saprotrophs in soil were tested. The following fungal species were evaluated in the experiments: Trichoderma atroviride, T. koningiopsis, T. gamsii, Beauveria bassiana, Metharhizium anisopliae, M. brunneum and Clonostachys solani. A commercial carbosulfan-based insecticide was used as a positive control. Additionally, two commercial products, one based on B. bassiana (Naturalis) and one on Bacillus thuringiensis (Delfin) were used as reference biocontrol agents. The aims were (i) to assess the pathogenicity of the selected fungal isolates to Delia radicum, (ii) to evaluate the fungal isolates' rhizosphere competence, with the emphasis on the persistence of the original inoculum on the growing roots, (iii) to assess possible endophytic plant tissue colonization, and (iv) to evaluate potential plant growth stimulating effects of the added inoculi. Significant pathogenicity of tested fungi against Delia radicum was confirmed in in vitro and glasshouse experiments. All tested fungi persisted on cauliflower rhizoplane. More importantly, the added fungi were found on thoroughly washed roots outside the original point of inoculation. This provided us with evidence that our tested fungi could be transferred via or grow with the elongating roots. In addition to colonizing the rhizoplane, some fungi were found inside the plant root or stem tissue, thus exhibiting endophytic characteristics. The importance of fungal ecology as a criterion in appropriate biological control agent selection is discussed. Copyright © 2014 Elsevier Inc. All rights reserved.

  4. Tissue culture methods for the clonal propagation and genetic improvement of Spanish red cedar (Cedrela odorata).

    Science.gov (United States)

    Peña-Ramírez, Yuri; Juárez-Gómez, Juan; González-Rodríguez, José Antonio; Robert, Manuel L

    2012-01-01

    The choice of a method to culture red cedar tissues depends on the final objectives pursued. If homogeneous clonal material is required for experimental purposes, the easiest way is to generate the lines through adventitious shoot induction from seedlings germinated from seeds. If the objective is to generate high yielding material for plantation purposes, the choice will be the same method but starting from mature vegetative tissues from selected elite plants. Most of the process are the same, but the initial steps are less efficient and much more elaborate. If the purpose is to generate lines with new genetic characteristics through somaclonal variation, mutagenesis, or genetic transformation, somatic embryogenesis will be required. No single method in its present form is suitable for all purposes. Eventually, the efficient production of somatic embryos from rejuvenated shoots collected from mature selected plants is the ideal way to culture this species, but for the time being we have to choose one or the other. In this chapter, we present a grafting procedure to rejuvenate and maintain mother plants in the greenhouse and the in vitro culture systems we have developed for the production of Cedrela odorata propagules using explants from both young seedlings and mature tissues from selected old trees. Using a modified TY17 medium and the BioMINT(®) temporary immersion system, we obtained high multiplication and ex vitro transplantation rates for efficient large-scale propagation of this species.

  5. Comparison of tumour age response to radiation for cells derived from tissue culture or solid tumours

    International Nuclear Information System (INIS)

    Keng, P.C.; Siemann, D.W.; Rochester Univ., NY; Rochester Univ., NY; Wheeler, K.T.

    1984-01-01

    Direct comparison of the cell age response of 9L and KHT tumour cells derived either from tissue culture or solid tumours was achieved. Cells from dissociated KHT and 9L tumours (the latter implanted either subcutaneously or intracerebrally) and cells from tissue culture were separated into homogenous sized populations by centrifugal elutriation. In both tumour models these homogeneous sized populations correspond to populations enriched at different stages of the cell cycle. The survival of these elutriated cell populations was measured after a single dose of Cs-137 gamma rays. For cells isolated from 9L solid tumours, there was little variation in radiosensitivity throughout the cell cycle; however, a very small but significant increase in resistance was found in late G 1 cells. This lack of a large variation in radiosensitivity through the cell cycle for 9L cells from solid tumours also was seen in 9L cells growing in monolayer tissue culture. When similar experiments were performed using the KHT sarcoma tumour model, the results showed that KHT cells in vitro exhibited a fairly conventional increase in radioresistance in both mid G 1 and late S. However, the cell age response of KHT cells from solid tumours was different; particularly in the late S and G 2 + M phases. (author)

  6. Toxicity and oxidative stress of canine mesenchymal stromal cells from adipose tissue in different culture passages

    Directory of Open Access Journals (Sweden)

    Arícia Gomes Sprada

    2015-12-01

    Full Text Available Abstract: Stem cells in regenerative therapy have received attention from researchers in recent decades. The culture of these cells allows studies about their behavior and metabolism. Thus, cell culture is the basis for cell therapy and tissue engineering researches. A major concern regarding the use of cultivated stem cell in human or veterinary clinical routine is the risk of carcinogenesis. Cellular activities require a balanced redox state. However, when there is an imbalance in this state, oxidative stress occurs. Oxidative stress contributes to cytotoxicity, which may result in cell death or genomic alterations, favoring the development of cancer cells. The aim of this study was to determine whether there are differences in the behavior of cultured mesenchymal stem cells from canine adipose tissue according to its site of collection (omentum and subcutaneous evaluating the rate of proliferation, viability, level of oxidative stress and cytotoxicity over six passages. For this experiment, two samples of adipose tissue from subcutaneous and omentum where taken from a female dog corpse, 13 years old, Pitbull. The results showed greater levels of oxidative stress in the first and last passages of both groups, favoring cytotoxicity and cell death.

  7. Application of Tissue Culture and Transformation Techniques in Model Species Brachypodium distachyon.

    Science.gov (United States)

    Sogutmaz Ozdemir, Bahar; Budak, Hikmet

    2018-01-01

    Brachypodium distachyon has recently emerged as a model plant species for the grass family (Poaceae) that includes major cereal crops and forage grasses. One of the important traits of a model species is its capacity to be transformed and ease of growing both in tissue culture and in greenhouse conditions. Hence, plant transformation technology is crucial for improvements in agricultural studies, both for the study of new genes and in the production of new transgenic plant species. In this chapter, we review an efficient tissue culture and two different transformation systems for Brachypodium using most commonly preferred gene transfer techniques in plant species, microprojectile bombardment method (biolistics) and Agrobacterium-mediated transformation.In plant transformation studies, frequently used explant materials are immature embryos due to their higher transformation efficiencies and regeneration capacity. However, mature embryos are available throughout the year in contrast to immature embryos. We explain a tissue culture protocol for Brachypodium using mature embryos with the selected inbred lines from our collection. Embryogenic calluses obtained from mature embryos are used to transform Brachypodium with both plant transformation techniques that are revised according to previously studied protocols applied in the grasses, such as applying vacuum infiltration, different wounding effects, modification in inoculation and cocultivation steps or optimization of bombardment parameters.

  8. Air exposure induced characteristics of dry eye in conjunctival tissue culture.

    Directory of Open Access Journals (Sweden)

    Hui Lin

    Full Text Available There are several animal models illustrating dry eye pathophysiology. Current study would like to establish an ex vivo tissue culture model for characterizing dry eye. Human conjunctival explants were cultured under airlift or submerged conditions for up to 2 weeks, and only airlifted conjunctival cultures underwent increased epithelial stratification. Starting on day 4, the suprabasal cells displayed decreased K19 expression whereas K10 keratin became evident in airlift group. Pax6 nuclear expression attenuated already at 2 days, while its perinuclear and cytoplasmic expression gradually increased. MUC5AC and MUC19 expression dramatically decreased whereas the full thickness MUC4 and MUC16 expression pattern disappeared soon after initiating the airlift condition. Real time PCR showed K16, K10 and MUC16 gene up-regulated while K19, MUC5AC, MUC19 and MUC4 down-regulated on day 8 and day 14. On day 2 was the appearance of apoptotic epithelial and stromal cells appeared. The Wnt signaling pathway was transiently activated from day 2 to day 10. The inflammatory mediators IL-1β, TNF-α, and MMP-9 were detected in the conditioned media after 6 to 8 days. In conclusion, airlifted conjunctival tissue cultures demonstrated Wnt signaling pathway activation, coupled with squamous metaplasia, mucin pattern alteration, apoptosis and upregulation of proinflammatory cytokine expression. These changes mimic the pathohistological alterations described in dry eye. This correspondence suggests that insight into the pathophysiology of dry eye may be aided through the use of airlifted conjunctival tissue cultures.

  9. Culture Environment-Induced Pluripotency of SACK-Expanded Tissue Stem Cells

    Directory of Open Access Journals (Sweden)

    Jean-François Paré

    2011-01-01

    Full Text Available Previous efforts to improve the efficiency of cellular reprogramming for the generation of induced pluripotent stem cells (iPSCs have focused mainly on transcription factors and small molecule combinations. Here, we report the results of our focus instead on the phenotype of the cells targeted for reprogramming. We find that adult mouse pancreatic tissue stem cells derived by the method of suppression of asymmetric cell kinetics (SACK acquire increased potency simply by culture under conditions for the production and maintenance of pluripotent stem cells. Moreover, supplementation with the SACK agent xanthine, which promotes symmetric self-renewal, significantly increases the efficiency and degree of acquisition of pluripotency properties. In transplantation analyses, clonal reprogrammed pancreatic stem cells produce slow-growing tumors with tissue derivative of all three embryonic germ layers. This acquisition of pluripotency, without transduction with exogenous transcription factors, supports the concept that tissue stem cells are predisposed to cellular reprogramming, particularly when symmetrically self-renewing.

  10. Neurite outgrowth in cultured mouse pelvic ganglia - Effects of neurotrophins and bladder tissue.

    Science.gov (United States)

    Ekman, Mari; Zhu, Baoyi; Swärd, Karl; Uvelius, Bengt

    2017-07-01

    Neurotrophic factors regulate survival and growth of neurons. The urinary bladder is innervated via both sympathetic and parasympathetic neurons located in the major pelvic ganglion. The aim of the present study was to characterize the effects of the neurotrophins nerve growth factor (NGF), brain derived neurotrophic factor (BDNF) and neurotrophin 3 (NT-3) on the sprouting rate of sympathetic and parasympathetic neurites from the female mouse ganglion. The pelvic ganglion was dissected out and attached to a petri dish and cultured in vitro. All three factors (BDNF, NT-3 and NGF) stimulated neurite outgrowth of both sympathetic and parasympathetic neurites although BDNF and NT-3 had a higher stimulatory effect on parasympathetic ganglion cells. The neurotrophin receptors TrkA, TrkB and TrkC were all expressed in neurons of the ganglia. Co-culture of ganglia with urinary bladder tissue, but not diaphragm tissue, increased the sprouting rate of neurites. Active forms of BDNF and NT-3 were detected in urinary bladder tissue using western blotting whereas tissue from the diaphragm expressed NGF. Neurite outgrowth from the pelvic ganglion was inhibited by a TrkB receptor antagonist. We therefore suggest that the urinary bladder releases trophic factors, including BDNF and NT-3, which regulate neurite outgrowth via activation of neuronal Trk-receptors. These findings could influence future strategies for developing pharmaceuticals to improve re-innervation due to bladder pathologies. Copyright © 2017. Published by Elsevier B.V.

  11. An evolutionary view of plant tissue culture: somaclonal variation and selection.

    Science.gov (United States)

    Wang, Qin-Mei; Wang, Li

    2012-09-01

    Plants regenerated from in vitro cultures possess an array of genetic and epigenetic changes. This phenomenon is known as 'somaclonal variation' and the frequency of somaclonal variation (SV) is usually elevated far beyond that expected in nature. Initially, the relationship between time in culture and detected SV was found to support the widespread belief that SV accumulates with culture age. However, a few studies indicated that older cultures yielded regenerants with less SV. What leads to this seemed contradiction? In this article, we have proposed a novel in vitro callus selection hypothesis, differentiation bottleneck (D-bottleneck) and dedifferentiation bottleneck (Dd-bottleneck), which consider natural selection theory to be fit for cell population in vitro. The results of multiplication races between the cells with the true-to-type phenotype and the deleterious cells determine the increase/decrease of SV frequencies in calli or regenerants as in vitro culture time goes on. The possibility of interpreting the complex situation of time-related SV by the evolutionary theory is discussed in this paper. In addition, the SV threshold, space-determined hypothesis and D-bottleneck are proposed to interpret the loss of the regenerability through a long period of plant tissue culture (PTC).

  12. Packaging of Post Acclimatized Somatic Embryogenesis Cocoa Plantlet (Theobroma cacao L.

    Directory of Open Access Journals (Sweden)

    Soedarsianto Soedarsianto

    2009-05-01

    Full Text Available Clonal plants that produced by somatic embryogenesis technique is one of the best choice to produce supperior clonal cacao (Theobroma cacao L. planting materials. The somatic embryogenesis technique is a possible way for massive propagation, the outcome is true to type plants, the architecture similarity that the seedlings but there is not segregation like seedlings plants. At present mass production started of plantlets production until post-acclimatized plantlets of somatic embryogenesis cocoa was done at Indonesian Coffee and Cocoa Research Institute. Distribution system of the planting materials to whole areas in form of as up-rooted post-acclimatized plantlet. Some problems identified to reduce probability of decreasing viability of up-rooted post-acclimatized plantlets and one of them is extreme internal water deficit. This research investigate of the influece storage condition (air tight and non-air tight and box storage (mica plastic and cardboardbox. The first experiment result show, there is no significant different between mica plastic and cardboard box usage for storage of post-acclimatized cocoa pantlet. Viability of up-rooted post acclimatized cocoa plantlet influenced exactly by air tight and non-air tight storage condition. Air tight storage condition have better viability of up-rooted post acclimatised (81,58% than non-air tight storage condition (65,00%. Leaf sanasence on air tight storage condition (10,33% lower than non-air tight storage (32,58%. There is not significantly on volume storage per plantlet between 4.416 cm3 and 12.600 cm3. Relationship between fallen leaves and cocoa planlet viability follow negative linear correlation y = -1,4719x + 104,88 (R2 = 0,9772. The second experiment treatment showed that maximal storage periode of post cclimatized cocoa plantlet just until 6 days stored (97% and not significant with 3 days one. Viability of post acclimatized cocoa plantlet decreased after 6 days storage period

  13. Impact assessment of repeated exposure of organotypic 3D bronchial and nasal tissue culture models to whole cigarette smoke.

    Science.gov (United States)

    Kuehn, Diana; Majeed, Shoaib; Guedj, Emmanuel; Dulize, Remi; Baumer, Karine; Iskandar, Anita; Boue, Stephanie; Martin, Florian; Kostadinova, Radina; Mathis, Carole; Ivanov, Nikolai V; Frentzel, Stefan; Hoeng, Julia; Peitsch, Manuel C

    2015-02-12

    Cigarette smoke (CS) has a major impact on lung biology and may result in the development of lung diseases such as chronic obstructive pulmonary disease or lung cancer. To understand the underlying mechanisms of disease development, it would be important to examine the impact of CS exposure directly on lung tissues. However, this approach is difficult to implement in epidemiological studies because lung tissue sampling is complex and invasive. Alternatively, tissue culture models can facilitate the assessment of exposure impacts on the lung tissue. Submerged 2D cell cultures, such as normal human bronchial epithelial (NHBE) cell cultures, have traditionally been used for this purpose. However, they cannot be exposed directly to smoke in a similar manner to the in vivo exposure situation. Recently developed 3D tissue culture models better reflect the in vivo situation because they can be cultured at the air-liquid interface (ALI). Their basal sides are immersed in the culture medium; whereas, their apical sides are exposed to air. Moreover, organotypic tissue cultures that contain different type of cells, better represent the physiology of the tissue in vivo. In this work, the utilization of an in vitro exposure system to expose human organotypic bronchial and nasal tissue models to mainstream CS is demonstrated. Ciliary beating frequency and the activity of cytochrome P450s (CYP) 1A1/1B1 were measured to assess functional impacts of CS on the tissues. Furthermore, to examine CS-induced alterations at the molecular level, gene expression profiles were generated from the tissues following exposure. A slight increase in CYP1A1/1B1 activity was observed in CS-exposed tissues compared with air-exposed tissues. A network-and transcriptomics-based systems biology approach was sufficiently robust to demonstrate CS-induced alterations of xenobiotic metabolism that were similar to those observed in the bronchial and nasal epithelial cells obtained from smokers.

  14. Selection of seed lots of Pinus taeda L. for tissue culture

    Directory of Open Access Journals (Sweden)

    Diego Pascoal Golle

    2014-06-01

    Full Text Available The aim of this work was to identify the fungi genera associated with three Pinus taeda L. seed lots and to assess the sanitary and physiological quality of these lots for use as selection criteria for tissue culture and evaluate the in vitro establishment of explants from seminal origin in different nutritive media. It was possible to discriminate the lots on the sanitary and physiological quality, as well as to establish in vitro plants of Pinus taeda from cotyledonary nodes obtained from aseptic seed germination of a selected lot by the sanitary and physiological quality higher. The nutritive media MS, ½ MS and WPM were equally suitable for this purpose. For the sanitary analysis the fungal genera Fusarium, Penicillium and Trichoderma were those of the highest sensitivity. For the physiological evaluation were important the variables: abnormal seedlings, strong normal seedlings; length, fresh and dry weight of strong normal seedlings. The analyzes were favorable to choose lots of seeds for in vitro culture and all culture media were adequate for the establishment of this species in tissue culture.

  15. Optimization of an Efficient Non-Tissue Culture Transformation Method for Brassica Juncea

    International Nuclear Information System (INIS)

    Naeem, I.; Munir, I.; Iqbal, A.; Ullah, F.

    2016-01-01

    The major hurdles in successful in vitro transformation of Brassica juncea through standard tissue culture (STC) method are: culture contamination, somaclonal variations, and lack of expertise. Moreover, the current STC method is time consuming and needs continuous electricity. In the present study, the in planta transformation method through floral dip with or without vacuum infiltration was optimized for successful transformation of B. juncea. The B. juncea CV RAYA Anmol was used for transformation through Agrobacterium tumefaciens strain GV3101 harboring the binary vector plasmid pBinGlyBar4-EADcT. Based on the resistance reaction to the herbicide Basta, 20 and 40 resistant seedlings were obtained from 2000 seed germinated from the plants transformed through floral dip and vacuum infiltration methods, respectively. The PCR analyses further confirmed the presence of transgene in 3 floral dipped plants without vacuum infiltration and 17 floral dipped plants with vacuum infiltration, giving the transformation frequencies of 1.5*10/sup -3/ and 8.5*10/sup -3/, respectively. This method, which avoids tissue culture, will reduce the somaclonal variation accompanying prolonged culture of cells in a dedifferentiated state, will facilitate functional genomics and improvement of Brassica juncea with novel desirable traits while reducing time and expense. (author)

  16. The structure of tissue on cell culture-extracted thyroglobulin is independent of its iodine content.

    Science.gov (United States)

    Delain, E; Aouani, A; Vignal, A; Couture-Tosi, E; Hovsépian, S; Fayet, G

    1987-02-01

    The major protein synthesized in vitro by the ovine thyroid cell line OVNIS 6H is the prothyroid hormone thyroglobulin. Purified from serum-free cell culture media using sucrose gradient centrifugation, the thyroglobulin dimer was analysed for iodine content and observed by electron microscopy. In their usual medium, the OVNIS 6H cells produce a very poorly iodinated thyroglobulin containing 0.05 I atom per molecule. When cultured with methimazole or propylthiouracil, two inhibitors of iodide organification, less than 0.007 I atom/molecules was found. These molecules purified from cell cultures were compared to those purified from ovine thyroid tissue containing 26 I atoms/mol. Despite large differences in iodine content, the three preparations all consist of 19 S thyroglobulin dimers with the classical ovoidal shape. The variability in size measurements remains in a 2% range for all thyroglobulin types. Consequently, no real significant variation can be found between the highly iodinated thyroglobulin isolated from tissue, and the poorly or non-iodinated thyroglobulins isolated from cells cultured with or without methimazole or propylthiouracil.

  17. Insights into the multifaceted application of microscopic techniques in plant tissue culture systems.

    Science.gov (United States)

    Moyo, Mack; Aremu, Adeyemi O; Van Staden, Johannes

    2015-10-01

    Microscopic techniques remain an integral tool which has allowed for the better understanding and manipulation of in vitro plant culture systems. The recent advancements will inevitably help to unlock the long-standing mysteries of fundamental biological mechanisms of plant cells. Beyond the classical applications in micropropagation aimed at the conservation of endangered and elite commercial genotypes, plant cell, tissue and organ cultures have become a platform for elucidating a myriad of fundamental physiological and developmental processes. In conjunction with microscopic techniques, in vitro culture technology has been at the centre of important breakthroughs in plant growth and development. Applications of microscopy and plant tissue culture have included elucidation of growth and development processes, detection of in vitro-induced physiological disorders as well as subcellular localization using fluorescent protein probes. Light and electron microscopy have been widely used in confirming the bipolarity of somatic embryos during somatic embryogenesis. The technique highlights basic anatomical, structural and histological evidence for in vitro-induced physiological disorders during plant growth and development. In this review, we discuss some significant biological insights in plant growth and development, breakthroughs and limitations of various microscopic applications and the exciting possibilities offered by emergent in vivo live imaging and fluorescent protein engineering technologies.

  18. Removal of viruses from Lebanese fig varieties using tissue culture and thermotherapy

    Directory of Open Access Journals (Sweden)

    Lamis CHALAK

    2015-12-01

    Full Text Available Two Lebanese fig accessions of local varieties (Biadi and Aswad, infected by Fig leaf mottle-associated virus 1 (FLMaV-1, Fig leaf mottle-associated virus 2 (FLMaV-2 and Fig mosaic virus (FMV, were subjected to tissue culture and thermotherapy for producing virus-free plant material. The virus status of all progeny explants was assayed by RT-PCR using viruses-specific primers. The shoot tip culture technique was reliable for elimination of from 60 to 100% of fig viruses. However, stem cutting culture coupled with thermotherapy was the most effective for shoot regeneration (40% of reactive explants, while elimination of the three viruses was possible even though with lower rates of removal (from zero to 81% were achieved. This study has indicated that FLMaV-2 is more susceptible to thermotherapy than FLMaV-1 and FMV.

  19. Effects of micronutrients (Cu, Zn, Mn, and Fe on the growth of Spathoglottis plicata plantlets

    Directory of Open Access Journals (Sweden)

    Zaliyatun Akhma Mat Yasin

    2017-05-01

    Full Text Available The micropropagation of valuable orchid species such as Spathoglottis plicata could help in their conservation and increase their propagation rate. The objective of this study was to investigate the effects of micronutrients (CuSO 4 , ZnSO 4 , MnSO 4 and Fe-EDTA on the growth of S. plicata plantlets. Plantlets of uniform height (1.5 cm were transferred to a half-strength MS media supplemented with vitamin B5 and different concentrations of selected micronutrients (copper, zinc, manganese and iron. The highest production of soluble protein content (38.98 mg/g of fresh weight, FW was recorded when plantlets were treated with 25 μM MnSO 4 . Spathoglottis plicata plantlets formed the highest amount of chlorophyll (22.32 mg/g FW when the growth media were supplemented with 75 μM Fe-EDTA. A total of 25 μM Fe-EDTA induced the production of up to 19.78 mg/g FW of carbohydrates in S. plicata plantlets. Furthermore, we demonstrate that different concentrations of micronutrients had different effects on the activities of several enzymes, such as peroxidase, catalase, polyphenol oxidase and nitrate reductase.

  20. Assessment of mint (Mentha spp. species for large-scale production of plantlets by micropropagation

    Directory of Open Access Journals (Sweden)

    Hugo Teixeira Gomes

    2015-10-01

    Full Text Available Species of the genus Mentha produce essential oils which are widely used in pharmaceutical and cosmetic industries. Current study evaluates the potential for in vitro propagation and estimates mass production of plantlets of Mentha species. Nine species (M. piperita, M. suaveolens, M. canadensis, M. longiflora, M. aquatica, M. arvensis, Mentha x gracilis, M. gracilis and M. spicata were propagated with five successive 30-day subcultures in MS medium supplemented with NAA (0.05 µM and BAP (4.4 µM. Shoots were rooted in MS with IBA, IAA or NAA (0.0; 0.25; 0.5; 2.5 or 5.0 µM. The rooted plantlets were finally acclimatized in a greenhouse. Studied species increased in multiplication rates between 4.2 and 9.0-fold per month. M. piperita, M. longiflora, M. arvensis, M. x gracilis and M. gracilis showed the greatest potential for plantlet production since the estimated production varied between 6,000 and 27,000 plantlets after five 30-days subcultures. The addition of auxin to the medium did not influence root induction. However, IAA at a concentration of 5 µM provided the best results for root length and fresh weight, with averages 11.1 cm and 0.16 g, respectively. Survival of plantlets reached 100% during the greenhouse acclimatization process.

  1. Review of vascularised bone tissue-engineering strategies with a focus on co-culture systems.

    Science.gov (United States)

    Liu, Yuchun; Chan, Jerry K Y; Teoh, Swee-Hin

    2015-02-01

    Poor angiogenesis within tissue-engineered grafts has been identified as a main challenge limiting the clinical introduction of bone tissue-engineering (BTE) approaches for the repair of large bone defects. Thick BTE grafts often exhibit poor cellular viability particularly at the core, leading to graft failure and lack of integration with host tissues. Various BTE approaches have been explored for improving vascularisation in tissue-engineered constructs and are briefly discussed in this review. Recent investigations relating to co-culture systems of endothelial and osteoblast-like cells have shown evidence of BTE efficacy in increasing vascularization in thick constructs. This review provides an overview of key concepts related to bone formation and then focuses on the current state of engineered vascularized co-culture systems using bone repair as a model. It will also address key questions regarding the generation of clinically relevant vascularized bone constructs as well as potential directions and considerations for research with the objective of pursuing engineered co-culture systems in other disciplines of vascularized regenerative medicine. The final objective is to generate serious and functional long-lasting vessels for sustainable angiogenesis that will enable enhanced cellular survival within thick voluminous bone grafts, thereby aiding in bone formation and remodelling in the long term. However, more evidence about the quality of blood vessels formed and its associated functional improvement in bone formation as well as a mechanistic understanding of their interactions are necessary for designing better therapeutic strategies for translation to clinical settings. Copyright © 2012 John Wiley & Sons, Ltd.

  2. Protein and Glycoprotein Patterns Related to Morphogenesis in Mammillaria gracillis Pfeiff. Tissue Culture

    Directory of Open Access Journals (Sweden)

    Biljana Balen

    2002-01-01

    Full Text Available As plants with Crassulacean Acid Metabolism (CAM, cacti are highly affected by artificial environmental conditions in tissue culture. Plants of Mammillaria gracillis Pfeiff. (Cactaceae propagated in vitro produced callus spontaneously. This habituated callus regenerated normal and hyperhydric shoots without the addition of growth regulators. In order to compare habituated callus with the tumorous one, cactus cells were transformed with two strains of Agrobacterium tumefaciens: the wild strain B6S3 (tumour line TW and the rooty mutant GV3101 (tumour line TR. Gene expression in cactus plants, habituated callus, regenerated shoots and two tumour lines was analysed at the level of cellular and extracellular protein and glycoprotein profiles. Proteins were separated by SDS-polyacrylamide gel electrophoresis and 2-D PAGE electrophoresis and silver stained. Concavalin A-peroxidase staining detected glycoproteins with D-manose in their glycan component on protein blots. Developmentally specific protein patterns of Mammillaria gracillis tissue lines were detected. The 2-D PAGE electrophoresis revealed some tissue specific protein groups. The cellular glycoprotein of 42 kDa detected by ConA was highly expressed in undifferentiated tissues (habituated callus, TW and TR tumours and in hyperhydric regenerants. Tumours produced extracellular proteins of 33, 23 and 22 kDa. The N glycosylation of cellular and extracellular proteins was related to specific developmental stage of cactus tissue.

  3. Study of the agroindustrial alterations induced by the irradiated tissue culture in sugar cane, variety NA 56-79

    International Nuclear Information System (INIS)

    Figueiredo Junior, O.

    1991-01-01

    The use of plant tissue culture and the application of gamma radiation as mutation inducing agents, in the sugar cane plant, variety NA 5679, are studied. The variation in the contents of brix, pol, fiber, purity, extraction, phosphorus, nitrogen, reducing sugars as well as the morphological characteristics are analysed. The 'callus' obtained by the tissue culture were irradiated with 20, 40, and 60 Gy doses. The statistical analysis indicated that the method of tissue culture may, eventually, increase the contents of the technological parameters and the dosages of gamma radiation were not efficient for such purpose. (M.A.C.)

  4. Use of slow-release fertilizer on the production of sweet potatoes plantlets in tray

    Directory of Open Access Journals (Sweden)

    Amarílis Beraldo Rós

    2013-12-01

    Full Text Available The production of plantlets in containers generally requires the use of fertilizers. The aim of this study was to evaluate the production of sweet potato in styrofoam trays using slow-release fertilizer. The experiment was carried out, under a screen-protected nursery, in a factorial scheme 5x5, with five doses of slow-release fertilizer NPK 19-06-10 (0, 50, 100, 150 e 200 g per 25 kg de substrate and five times of plantlets permanence in tray (14, 28, 42, 56 and 70 days after planting the cuttings. The number and dry matter of roots and leaves were evaluated. The number of roots was not influenced by fertilizer addition. In general, there is not damage to plantlets growth until the highest dose used. Therefore, the fertilizer addition increases the sweet potato plants growth and the dose of 200 g per 25 kg of substrate is responsible for the best results.

  5. Arbuscular mycorrhizal inoculation enhances survival rates and growth of Micropropagated plantlets of Echinacea pallida.

    Science.gov (United States)

    Lata, Hemant; De Andrade, Zita; Schaneberg, Brian; Bedir, Ebru; Khan, Ikhlas; Moraes, Rita

    2003-07-01

    In an attempt to induce positive effects on the acclimatization of in vitro propagated Echinacea pallida, four arbuscular mycorrhizal (AM) fungi, Glomus mosseae, Gigaspora ramisporophora, Scutellospora fulgida and Entrophospora colombiana were selected to aid the soil adaptation process. Fungal inocula affected the survival of E. pallida plantlets ranging from 83 % to 92 %, depending on the AM species and also contrasting with 58 % survival of the non-inoculated plantlets. Growth and development were faster in mycorrhizal treated plantlets than in non-treated ones, especially among those treated with Glomus mossae and Scutellospora fulgida. The presence of well-formed arbuscules and vesicles in Echinacea infested roots was confirmed by microscopic examinations in addition to 90 % success in the survival rate of vigorous plants indicated that mycorrhization is a valuable tool to overcome Echinacea acclimatization shock.

  6. Changes in adipose tissue stromal-vascular cells in primary culture due to porcine sera

    International Nuclear Information System (INIS)

    Jewell, D.E.; Hausman, G.J.

    1986-01-01

    This study was conducted to determine the response of rat stromal-vascular cells to pig sea. Sera were collected from unselected contemporary (lean) and high backfat thickness selected (obese) pigs. Sera from obese pigs were collected either by exsanguination or cannulation. sera from lean pigs during the growing phase (45 kg) and the fattening phase (100-110 kg) were collected. Stromal-vascular cells derived rom rat inguinal tissue were cultured on either 25 cm 2 flasks, collagen-coated coverslips or petri dishes. Cell proliferation was measured by [ 3 H]-thymidine incorporation during the fourth day of culture. Coverslip cultures were used for histochemical analysis. Petri dish cultures were used for analysis of Sn-glycerol-3-phosphate dehydrogenase (GPDH) activity. All cells were plated for 24 hours in media containing 10 fetal bovine sera. Test media contained 2.5, 5.0, 10.0% sera. Sera from obese pigs increased GPDH activity and fat cell production when compared to the lean controls. The increased concentration of sera increased esterase activity and lipid as measured with oil red O. The sera from obese pigs collected at slaughter stimulated more fat cell production than obese sera collected by cannulation. These studies show there are adipogenic factors in obese pigs sera which promote fat cell development in primary cell culture

  7. Chemical And Physiological Studies On Drought Stress Tolerance Of Irradiated Communis Pear Using Tissue Culture

    International Nuclear Information System (INIS)

    Zaied, N.S.; Ragab, E.A.

    2007-01-01

    The rooted in vitro irradiated pear rootstocks (Pyrus communis) were subjected to drought stress by using different concentrations of mannitol (20, 40, 60, 80 and 100 gm/l), polyethylene glycol (PEG) at concentrations 2, 4, 6, 8 and 10 % to culture medium and also agar at concentrations 6, 8, 10, 12 and 14 gm/l to study their effects on tissue culture and chemical analysis and their tolerance to drought stress. The obtained results showed that the number of shoots, shoot length and number of leaves were higher at 20 and 40 gm/l mannitol. Increasing mannitol concentration enhanced the increase of chlorophyll b, reducing sugars, total indoles and total phenols up to the highest level at 100 gm/l. Adding PEG at concentration 2% to the culture medium encouraged significant increases in the number of shoots and number of leaves and increase chlorophyll a, and non-reducing sugars as well as significant decrease in number of shoots, shoots length, number of leaves, root length and number of roots with increasing agar concentrations to the culture medium. However, decreasing agar concentration in the culture medium induced increase in chlorophyll A and non-reducing sugar

  8. Frozen and fresh ovarian tissue require different culture media to promote in vitro development of bovine preantral follicles.

    Science.gov (United States)

    Castro, Simone Vieira; Carvalho, Adeline Andrade; Silva, Cleidson Manoel Gomes; Santos, Francielli Weber; Campello, Cláudio Cabral; de Figueiredo, José Ricardo; Rodrigues, Ana Paula Ribeiro

    2014-10-01

    The aim of this study was to evaluate the efficiency of different media in the in vitro culture of bovine preantral follicles that were used either fresh or following slow freezing treatment. Frozen and fresh noncultured or cultured ovarian fragments were processed for histological, viability, and cell proliferation analyses. For cryopreservation, a solution containing 1.5 M ethylene glycol was frozen in a programmable biological freezer. After thawing, a portion of the samples was destined for frozen controls. The remainder were cultured in vitro for 5 days in three media: α-MEM, McCoy, or M199. Samples from these culture media were collected on days 1 and 5 for quantification of reactive oxygen species (ROS) and for hormonal assays. In fresh-cultured tissues, the percentage of morphologically normal follicles was significantly higher when cultured in M199 compared to that in the other media. In frozen-cultured tissues, McCoy medium was significantly superior to the other media, and was the only treatment that helped in maintaining the viability similar to fresh and frozen controls. Upon quantification of the nucleolus organizer region, we observed greater proliferation of granulosa cells in the frozen-cultured tissues with McCoy medium, and lesser proliferation in fresh-cultured tissues only with α-MEM. In frozen-cultured tissues, ROS levels were highest at day 1 and progressively reduced during culture, independent of the media used. In conclusion, under the conditions used in this study, the M199 and McCoy media are recommended for the culture of follicles derived from fresh and frozen ovarian tissues, respectively.

  9. Improved tissue culture conditions for the emerging C4model Panicum hallii.

    Science.gov (United States)

    Grant, Joshua N; Burris, Jason N; Stewart, C Neal; Lenaghan, Scott C

    2017-04-27

    Panicum hallii Vasey (Hall's panicgrass) is a compact, perennial C 4 grass in the family Poaceae, which has potential to enable bioenergy research for switchgrass (Panicum virgatum L.). Unlike P. hallii, switchgrass has a large genome, allopolyploidy, self-incompatibility, a long life cycle, and large stature-all suboptimal traits for rapid genetics research. Herein we improved tissue culture methodologies for two inbred P. hallii populations: FIL2 and HAL2, to enable further development of P. hallii as a model C 4 plant. The optimal seed-derived callus induction medium was determined to be Murashige and Skoog (MS) medium supplemented with 40 mg L -1 L-cysteine, 300 mg L -1 L-proline, 3% sucrose, 1 g L -1 casein hydrolysate, 3 mg L -1 2,4-dichlorophenoxyacetic acid (2,4-D), and 45 μg L -1 6-benzylaminopurine (BAP), which resulted in callus induction of 51 ± 29% for FIL2 and 81 ± 19% for HAL2. The optimal inflorescence-derived callus induction was observed on MP medium (MS medium supplemented with 2 g L -1 L-proline, 3% maltose, 5 mg L -1 2,4-D, and 500 μg L -1 BAP), resulting in callus induction of 100 ± 0.0% for FIL2 and 84 ± 2.4% for HAL2. Shoot regeneration rates of 11.5 ± 0.8 shoots/gram for FIL2 and 11.3 ± 0.6 shoots/gram for HAL2 were achieved using seed-induced callus, whereas shoot regeneration rates of 26.2 ± 2.6 shoots/gram for FIL2 and 29.3 ± 3.6 shoots/gram for HAL2 were achieved from inflorescence-induced callus. Further, cell suspension cultures of P. hallii were established from seed-derived callus, providing faster generation of callus tissue compared with culture using solidified media (1.41-fold increase for FIL2 and 3.00-fold increase for HAL2). Aside from abbreviated tissue culture times from callus induction to plant regeneration for HAL2, we noted no apparent differences between FIL2 and HAL2 populations in tissue culture performance. For both populations, the cell suspension cultures

  10. Enhancing plant regeneration in tissue culture: a molecular approach through manipulation of cytokinin sensitivity.

    Science.gov (United States)

    Hill, Kristine; Schaller, G Eric

    2013-10-01

    Micropropagation is used for commercial purposes worldwide, but the capacity to undergo somatic organogenesis and plant regeneration varies greatly among species. The plant hormones auxin and cytokinin are critical for plant regeneration in tissue culture, with cytokinin playing an instrumental role in shoot organogenesis. Type-B response regulators govern the transcriptional output in response to cytokinin and are required for plant regeneration. In our paper published in Plant Physiology, we explored the functional redundancy among the 11 type-B Arabidopsis response regulators (ARRs). Interestingly, we discovered that the enhanced expression of one family member, ARR10, induced hypersensitivity to cytokinin in multiple assays, including callus greening and shoot induction of explants. Here we 1) discuss the hormone dependence for in vitro plant regeneration, 2) how manipulation of the cytokinin response has been used to enhance plant regeneration, and 3) the potential of the ARR10 transgene as a tool to increase the regeneration capacity of agriculturally important crop plants. The efficacy of ARR10 for enhancing plant regeneration likely arises from its ability to transcriptionally regulate key cytokinin responsive genes combined with an enhanced protein stability of ARR10 compared with other type-B ARRs. By increasing the capacity of key tissues and cell types to respond to cytokinin, ARR10, or other type-B response regulators with similar properties, could be used as a tool to combat the recalcitrance of some crop species to tissue culture techniques.

  11. Progress of tissue culture and genetic transformation research in pigeon pea [Cajanus cajan (L.) Millsp.].

    Science.gov (United States)

    Krishna, Gaurav; Reddy, P Sairam; Ramteke, P W; Bhattacharya, P S

    2010-10-01

    Pigeon pea [Cajanus cajan (L.) Millsp.] (Family: Fabaceae) is an important legume crop cultivated across 50 countries in Asia, Africa, and the Americas; and ranks fifth in area among pulses after soybean, common bean, peanut, and chickpea. It is consumed as a major source of protein (21%) to the human population in many developing countries. In India, it is the second important food legume contributing to 80% of the global production. Several biotic and abiotic stresses are posing a big threat to its production and productivity. Attempts to address these problems through conventional breeding methods have met with partial success. This paper reviews the chronological progress made in tissue culture through organogenesis and somatic embryogenesis, including the influence of factors such as genotypes, explant sources, and culture media including the supplementation of plant growth regulators. Comprehensive lists of morphogenetic pathways involved in in vitro regeneration through organogenesis and somatic embryogenesis using different explant tissues of diverse pigeon pea genotypes are presented. Similarly, the establishment of protocols for the production of transgenics via particle bombardment and Agrobacterium-mediated transformation using different explant tissues, Agrobacterium strains, Ti plasmids, and plant selectable markers, as well as their interactions on transformation efficiency have been discussed. Future research thrusts on the use of different promoters and stacking of genes for various biotic and abiotic stresses in pigeon pea are suggested.

  12. Rapid detection of Mannheimia haemolytica in lung tissues of sheep and from bacterial culture

    Directory of Open Access Journals (Sweden)

    Jyoti Kumar

    2015-09-01

    Full Text Available Aim: This study was aimed to detect Mannheimia haemolytica in lung tissues of sheep and from a bacterial culture. Introduction: M. haemolytica is one of the most important and well-established etiological agents of pneumonia in sheep and other ruminants throughout the world. Accurate diagnosis of M. haemolytica primarily relies on bacteriological examination, biochemical characteristics and, biotyping and serotyping of the isolates. In an effort to facilitate rapid M. haemolytica detection, polymerase chain reaction assay targeting Pasteurella haemolytica serotype-1 specific antigens (PHSSA, Rpt2 and 12S ribosomal RNA (rRNA genes were used to detect M. haemolytica directly from lung tissues and from bacterial culture. Materials and Methods: A total of 12 archived lung tissues from sheep that died of pneumonia on an organized farm were used. A multiplex polymerase chain reaction (mPCR based on two-amplicons targeted PHSSA and Rpt2 genes of M. haemolytica were used for identification of M. haemolytica isolates in culture from the lung samples. All the 12 lung tissue samples were tested for the presence M. haemolytica by PHSSA and Rpt2 genes based PCR and its confirmation by sequencing of the amplicons. Results: All the 12 lung tissue samples tested for the presence of PHSSA and Rpt2 genes of M. haemolytica by mPCR were found to be positive. Amplification of 12S rRNA gene fragment as internal amplification control was obtained with each mPCR reaction performed from DNA extracted directly from lung tissue samples. All the M. haemolytica were also positive for mPCR. No amplified DNA bands were observed for negative control reactions. All the three nucleotide sequences were deposited in NCBI GenBank (Accession No. KJ534629, KJ534630 and KJ534631. Sequencing of the amplified products revealed the identity of 99-100%, with published sequence of PHSSA and Rpt2 genes of M. haemolytica available in the NCBI database. Sheep specific mitochondrial 12S r

  13. Mutagenic treatments towards increasing the frequency of day-neutral mutations and standardization of procedures for tissue culture, in potato

    International Nuclear Information System (INIS)

    Upadhya, M.D.; Chandra, R.; Abraham, M.J.

    1976-01-01

    Various chemical mutagens and gamma radiation have been used on single dormant eyes and true seeds with a view to finding effective mutagenic treatment for the induction of day-length neutral mutants in potato using an effective screening technique for the isolation of day-length neutral mutants. Sodium meta bisulphite (SMS) was found to be an efficient mutagen in inducing mutations for this trait in true seeds although the same concentrations, when used for treating the single tuber eyes proved lethal. Pre-soaking the seeds for 24 hrs prior to treatment with 0.0025M SMS gave highest frequency of the mutants followed by 48 hrs presoaking, indicating a sensitive stage during the cell cycle in true seeds. Other mutagen treatments gave different frequencies of mutations. The highest frequency of day-length neutral mutants was observed when seeds irradiated with 40 Kr of gamma radiation were treated with 0.05M hydrazinium dichloride solution. Screening procedures have also been standardised with the development of synethetic media for the isolation of biochemical mutants at the true seed level. Initial efforts have yielded mutants resistant to LD 100 doses of ethionine. Another aspect of the study was to develop a proper potato callus culture technique. A medium has been developed to produce and maintain callus from potato leaf strips. Efforts on the regeneration of shoot and roots from callus, have so far lead to differentiation of callus to form roots. The ultimate aim of these studies is to develop plantlets from single cell which would form the units of mutation induction and isolation. (author)

  14. Effects of the architecture of tissue engineering scaffolds on cell seeding and culturing.

    Science.gov (United States)

    Melchels, Ferry P W; Barradas, Ana M C; van Blitterswijk, Clemens A; de Boer, Jan; Feijen, Jan; Grijpma, Dirk W

    2010-11-01

    The advance of rapid prototyping techniques has significantly improved control over the pore network architecture of tissue engineering scaffolds. In this work, we have assessed the influence of scaffold pore architecture on cell seeding and static culturing, by comparing a computer designed gyroid architecture fabricated by stereolithography with a random pore architecture resulting from salt leaching. The scaffold types showed comparable porosity and pore size values, but the gyroid type showed a more than 10-fold higher permeability due to the absence of size-limiting pore interconnections. The higher permeability significantly improved the wetting properties of the hydrophobic scaffolds and increased the settling speed of cells upon static seeding of immortalised mesenchymal stem cells. After dynamic seeding followed by 5 days of static culture gyroid scaffolds showed large cell populations in the centre of the scaffold, while salt-leached scaffolds were covered with a cell sheet on the outside and no cells were found in the scaffold centre. It was shown that interconnectivity of the pores and permeability of the scaffold prolonged the time of static culture before overgrowth of cells at the scaffold periphery occurred. Furthermore, novel scaffold designs are proposed to further improve the transport of oxygen and nutrients throughout the scaffolds and to create tissue engineering grafts with a designed, pre-fabricated vasculature. Copyright © 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  15. An electrochemical approach to monitor pH change in agar media during plant tissue culture.

    Science.gov (United States)

    Wang, Min; Ha, Yang

    2007-05-15

    In this work, metal oxide microelectrodes were developed to monitor pH change in agar media during plant tissue culture. An antimony wire was produced by a new approach "capillary melt method". The surface of the obtained antimony wire was oxidized in a potassium nitrate melt to fabricate an antimony oxide film for pH sensing. Characterization results show that the oxide layer grown on the wire surface consists of Sb(2)O(3) crystal phase. The sensing response, open-circuit potential, of the electrode has a good linear relationship (R(2)=1.00) with pH value of the test solution. Adding organic compounds into the test media would not affect the linear relationship, although the slope of the lines varied with different ingredients added. The antimony oxide electrodes were employed to continuously monitor pH change of agar culture media during a 2-week plant tissue culture of Dendrobium candidum. The antimony oxide electrode fabricated this way has the advantages of low cost, easy fabrication, fast response, and almost no contamination introduced into the system. It would be suitable for in situ and continuous pH measurement in many bio applications.

  16. Detection, isolation, and preliminary characterization of bacteria contaminating plant tissue cultures

    Directory of Open Access Journals (Sweden)

    Monika Kałużna

    2014-01-01

    Full Text Available In order to limit the contamination problem in plant tissue cultures experiments on selection of media suitable for detection and isolation of bacteria contaminating plant tissue explants, and preliminary characterization of isolates were made. In the first experiment aiming at detection of bacteria in plant explants four strains representing genera most often occurring at our survey of plant tissue cultures, and earlier isolated and identified (Bacillus, Methylobacterium, Pseudomonas and Xanthomonas were streaked on five bacteriological media (NA, King B, K, R2A and 523 and on the medium used for plant culture initiation – ½ MS with milk albumin (IM. All strains grew on all media but on K and IM at the slowest rate and on 523 medium at the fastest. The IM medium proved to be useful for immediate bacteria detection at the initial stage of culture. In the second experiment, aiming at characterization of isolates on the basis of colony growth and morphology 14 strains (Agrobacterium, Bacillus, Curtobacterium, Flavobacterium, Lactobacillus, Methylobacterium – 2 strains Mycobacterium, Paenibacillus, Plantibacterium, Pseudomonas, Stenotrophomonas, Xanthomonas, and species Serratia marcescens were streaked on five microbiological media: KB, NBY, YDC, YNA and YPGA. All strains grew on all those media but at different rates. The only exception was the strain of Lactobacillus spp., which did not grow on King B medium. This medium allowed the detection of such characteristic traits as fluorescence (Pseudomonas and secretion of inclusions (Stenotrophomonas. The third experiment was focussed on assessment of the sensitivity of detection of specific bacteria in pure cultures and in plant tis- sue cultures using standard PCR and BIO-PCR techniques with genus specific primers and 2 methods of DNA isolation. Results showed that the use of Genomic Mini kit enabled an increase of the sensitivity by 100 times as compared to extraction of DNA by boiling

  17. Standardized production of Phyllanthus tenellus Roxb. by plant tissue culture Produção padronizada de Phyllanthus tenellus Roxb. por cultura de tecidos vegetais

    Directory of Open Access Journals (Sweden)

    Cristiane Pimentel Victório

    2010-06-01

    Full Text Available Exigencies as ethic plant raw material are part of the needs of modern phytotherapy. Micropropagation offers opportunities to obtain mass propagation of superior genotypes in short time. This study aimed to develop a protocol of direct and indirect organogenesis of Phyllanthus tenellus Roxb. Nodal segments from plantlets obtained by in vitro germination were subcultured in modified Murashige and Skoog medium added with different plant growth regulators: IAA (indole-3-acetic acid, IBA (indole-3-butyric acid, GA3 (3-giberelic acid and KIN (kinetin. The highest proliferation rate was obtained using the combinations: IBA, KIN + GA3 (3.5 mg L-1 and IBA + KIN (2.4 mg L-1. Rooting was intensified after 40 days, reaching 100% for all media with indole-3-butyric acid. Addition of 2,4 dichlorophenoxyacetic acid (2,4D provided the best results for production of friable calli. Acclimatization was 100% effective for plantlets cultured in control medium, with decrease in survival rate in grown plantlets from media added with growth regulators.Exigências como matérias-primas vegetais fidedignas fazem parte das necessidades da fitoterapia moderna. A propagação in vitro permite a obtenção em massa de genótipos superiores, em curto espaço de tempo. Este estudo teve como objetivo desenvolver um protocolo de organogênese direta e indireta de Phyllanthus tenellus Roxb. Segmentos nodais de plântulas obtidas por germinação in vitro foram introduzidos em meio Murashige e Skoog adicionado de diferentes reguladores de crescimento vegetal: AIA (ácido indol-3-acético, AIB (ácido indol-3-butírico, AG3 (ácido giberélico-3 e CIN (cinetina. A maior taxa de proliferação foi obtida utilizando as combinações: AIB, CIN + AG3 (3,5 mg L-1 e AIB + CIN (2,4 mg L-1. A rizogênese foi intensificada após 40 dias de cultivo, atingindo 100% para todos os meios contendo AIB. A adição de ácido 2,4 diclorofenoxiacético (2,4D proporcionou os melhores resultados para

  18. In vitro differentiation of rat spermatogonia into round spermatids in tissue culture.

    Science.gov (United States)

    Reda, A; Hou, M; Winton, T R; Chapin, R E; Söder, O; Stukenborg, J-B

    2016-09-01

    Do the organ culture conditions, previously defined for in vitro murine male germ cell differentiation, also result in differentiation of rat spermatogonia into post-meiotic germ cells exhibiting specific markers for haploid germ cells? We demonstrated the differentiation of rat spermatogonia into post-meiotic cells in vitro, with emphasis on exhibiting, protein markers described for round spermatids. Full spermatogenesis in vitro from immature germ cells using an organ culture technique in mice was first reported 5 years ago. However, no studies reporting the differentiation of rat spermatogonia into post-meiotic germ cells exhibiting the characteristic protein expression profile or into functional sperm have been reported. Organ culture of testicular fragments of 5 days postpartum (dpp) neonatal rats was performed for up to 52 days. Evaluation of microscopic morphology, testosterone levels, mRNA and protein expression as measured by RT-qPCR and immunostaining were conducted to monitor germ cell differentiation in vitro. Potential effects of melatonin, Glutamax® medium, retinoic acid and the presence of epidydimal fat tissue on the spermatogenic process were evaluated. A minimum of three biological replicates were performed for all experiments presented in this study. One-way ANOVA, ANOVA on ranks and student's t-test were applied to perform the statistical analysis. Male germ cells, present in testicular tissue pieces grown from 5 dpp rats, exhibited positive protein expression for Acrosin and Crem (cAMP (cyclic adenosine mono phosphate) response element modulator) after 52 days of culture in vitro. Intra-testicular testosterone production could be observed after 3 days of culture, while when epididymal fat tissue was added, spontaneous contractility of cultured seminiferous tubules could be observed after 21 days. However, no supportive effect of the supplementation with any factor or the co-culturing with epididymal fat tissue on germ cell differentiation in

  19. Using organotypic (raft) epithelial tissue cultures for the biosynthesis and isolation of infectious human papillomaviruses.

    Science.gov (United States)

    Ozbun, Michelle A; Patterson, Nicole A

    2014-08-01

    Papillomaviruses have a strict tropism for epithelial cells, and they are fully reliant on cellular differentiation for completion of their life cycles, resulting in the production of progeny virions. Thus, a permissive environment for full viral replication in vitro-wherein virion morphogenesis occurs under cooperative viral and cellular cues-requires the cultivation of epithelium. Presented in the first section of this unit is a protocol to grow differentiating epithelial tissues that mimic many important morphological and biochemical aspects of normal skin. The technique involves growing epidermal cells atop a dermal equivalent consisting of live fibroblasts and a collagen lattice. Epithelial stratification and differentiation ensues when the keratinocyte-dermal equivalent is placed at the air-liquid interface. The apparent floating nature of the cell-matrix in this method led to the nickname "raft" cultures. The general technique can be applied to normal low passage keratinocytes, to cells stably transfected with papillomavirus genes or genomes, or keratinocytes established from neoplastic lesions. However, infectious papillomavirus particles have only been isolated from organotypic epithelial cultures initiated with cells that maintain oncogenic human papillomavirus genomes in an extrachomosomal replicative form. The second section of this unit is dedicated to a virion isolation method that minimizes aerosol and skin exposure to these human carcinogens. Although the focus of the protocols is on the growth of tissues that yields infectious papillomavirus progeny, this culture system facilitates the investigation of these fastidious viruses during their complex replicative cycles, and raft tissues can be manipulated and harvested at any point during the process. Importantly, a single-step virus growth cycle is achieved in this process, as it is unlikely that progeny virions are released to initiate subsequent rounds of infection. Copyright © 2014 John Wiley

  20. [Action spectra of anthocyanin synthesis in tissue cultures and seedlings of Haplopappus gracilis].

    Science.gov (United States)

    Lackmann, I

    1971-09-01

    The biosynthesis of anthocyanin in tissue cultures and intact seedlings of Haplopappus gracilis is a light-dependent reaction which can be induced by blue light only. Anthocyanin appeared in all organs of the seedling.Wounding of the plant led to an increase in the content of anthocyanin due to increased anthocyanin synthesis in the cotyledons.The action spectra of anthocyanin formation in tissue cultures and intact seedlings have two peaks, one at 438 nm and the other at 372 nm. The limit of activity in the direction of longer wavelengths lies between 474 and 493 nm. Red light of short and long wavelength is ineffective in the induction of pigment synthesis. The photoreceptor of the light reaction is supposed to be a yellow pigment (flavoprotein or carotinoid). In contrast to the intact plants, isolated cotyledons and wounded seedlings are able to form anthocyanin not only in the blue region but also during irradiation with red light of high intensity. The action spectrum of anthocyanin synthesis in the isolated cotyledons has a marked maximum at about 440 nm and a second one at about 660 nm. A little activity can be observed throughout the visible spectrum. The pigment synthesis induced by red light can be completely suppressed by DCMU, an inhibitor of photosynthesis. This indicates that in the case of the activity in the red light caused by wounding chlorophyll serves as photoreceptor.The anthocyanin synthesis in tissue cultures and seedlings could not be influenced by low energy radiation in the red or in the far red region, even after induction of anthocyanin synthesis by blue light of high intensity. Therefore it seems that the phytochrome system is not involved in anthocyanin synthesis in Haplopappus gracilis.

  1. Three-dimensional hydrogel cultures for modeling changes in tissue impedance around microfabricated neural probes

    Science.gov (United States)

    Frampton, J. P.; Hynd, M. R.; Williams, J. C.; Shuler, M. L.; Shain, W.

    2007-12-01

    One limitation to the use of neuroprosthestic devices for chronic application, in the treatment of disease, is the reactive cell responses that occur surrounding the device after insertion. These cell and tissue responses result in increases in device impedance and failure of the device to interact with target populations of neurons. However, few tools are available to assess which components of the reactive response contribute most to changes in tissue impedance. An in vitro culture system has been developed that is capable of assessing individual components of the reactive response. The system utilizes alginate cell encapsulation to construct three-dimensional architectures that approach the cell densities found in rat cortex. The system was constructed around neuroNexus acute probes with on-board circuitry capable of monitoring the electrical properties of the surrounding tissue. This study demonstrates the utility of the system by demonstrating that differences in cell density within the three-dimensional alginate constructs result in differences in resistance and capacitance as measured by electrochemical impedance spectroscopy. We propose that this system can be used to model components of the reactive responses in brain tissue, and that the measurements recorded in vitro are comparable to measurements recorded in vivo.

  2. Unusual 4-hydroxybenzaldehyde synthase activity from tissue cultures of the vanilla orchid Vanilla planifolia.

    Science.gov (United States)

    Podstolski, Andrzej; Havkin-Frenkel, Daphna; Malinowski, Jacek; Blount, Jack W; Kourteva, Galina; Dixon, Richard A

    2002-11-01

    Tissue cultures of the vanilla orchid, Vanilla planifolia, produce the flavor compound vanillin (4-hydroxy-3-methoxybenzaldehyde) and vanillin precursors such as 4-hydroxybenzaldehyde. A constitutively expressed enzyme activity catalyzing chain shortening of a hydroxycinnamic acid, believed to be the first reaction specific for formation of vanilla flavor compounds, was identified in these cultures. The enzyme converts 4-coumaric acid non-oxidatively to 4-hydroxybenzaldehyde in the presence of a thiol reagent but with no co-factor requirement. Several forms of this 4-hydroxybenzaldehyde synthase (4HBS) were resolved and partially purified by a combination of hydrophobic interaction, ion exchange and gel filtration chromatography. These forms appear to be interconvertible. The unusual properties of the 4HBS, and its appearance in different protein fractions, raise questions as to its physiological role in vanillin biosynthesis in vivo.

  3. Effect of interferon on the development of Trypanosoma cruzi in tissue culture "Vero" cells

    Directory of Open Access Journals (Sweden)

    R. R. Golgher

    1980-01-01

    Full Text Available Results are presented on the effects of interferon on the intracellular stages of T. cruzi in tissue culture "Vero" cells. Interferon was obtained by infecting monolayers of human amniotic cells with inactivated Newcastle disease virus. Interferon has not affected the cell infection by T. cruzi culture infective stages and neither has it prevented the transformation of amastigote into trypomastigote stages.Interferon obtido através da infecção de células amnióticas humanas por vírus inativado da doença de Newcastle foi incapaz de influir sobre a infectividade de formas de cultura do T. cruzi para células "Vero" de cultura de tecido. A transformação amastigota-tripomastigota também não foi afetada pelo interferon.

  4. Study on rapid propagation of Zanhuang Chinese jujube by tissue culture

    International Nuclear Information System (INIS)

    Li Yun; Wang Yu; Tian Yanting

    2002-01-01

    Zanhuang jujube is a very precious and rare variety of Chinese jujube. Its development was restricted by the under-developed propagate technique in history. The rapid propagation by tissue culture was studied and the optimum media were screened out. Through studying the condition of initial, proliferating, acclimatizing and rooting culture, 4 media, MS +6-BA 0.5 mg/L+IBA 0.1 mg/L, MS+6-BA 1.5 mg/L+IBA 0.1-0.2 mg/L, MS+KT 0.5 mg/L+NAA 0.2 mg/L and 1/2 MS+IBA 0.6 mg/L+NAA 0.2-0.3 mg/L were selected respectively

  5. Target detect system in 3D using vision apply on plant reproduction by tissue culture

    Science.gov (United States)

    Vazquez Rueda, Martin G.; Hahn, Federico

    2001-03-01

    This paper presents the preliminary results for a system in tree dimension that use a system vision to manipulate plants in a tissue culture process. The system is able to estimate the position of the plant in the work area, first calculate the position and send information to the mechanical system, and recalculate the position again, and if it is necessary, repositioning the mechanical system, using an neural system to improve the location of the plant. The system use only the system vision to sense the position and control loop using a neural system to detect the target and positioning the mechanical system, the results are compared with an open loop system.

  6. Improving tolerance to Fusarium oxysporum f. sp. melonis in melon using tissue culture and mutation techniques

    International Nuclear Information System (INIS)

    Kantoglu, Y.; Secer, E.; Tutluer, I.; Kunter, B.; Peskircioglu, H.; Sagel, Z.; Erzurum, K.

    2010-01-01

    Fusarium wilt is a vascular disease of the Cucurbitaceae family, especially in muskmelon (Cucumis melo L.), caused by the soil fungus Fusarium oxysporum f. sp. melonis (FOM). This pathogen persists in the soil for extended periods of time, and the only effective control is the use of resistant cultivars. During the last three decades, tissue culture techniques have been utilised in crop improvement to generate changes in the genetic material of plants via in vitro somaclonal variations (by organogenesis or somatic embryogenesis) and induced mutagenesis. More recently, researchers have been using in vitro techniques to investigate the effects of fungal culture filtrates or toxins on susceptible and resistant genotypes of different plant species or cultivars to assess disease resistance. This method is effectively used for cucumber and melon. There are various in vitro culture techniques that can be used for cucumber (Malepszy, 1988). In this chapter, we show a method for mass-selection of melon mutants resistant to Fusarium wilt. In vitro selection of resistant cells, from both irradiated and non- irradiated explants, is performed using culture filtrates of different FOM races. This research could lead to the development of new melon cultivars resistant to Fusarium wilt. (author)

  7. Application of Synthetic Polymeric Scaffolds in Breast Cancer 3D Tissue Cultures and Animal Tumor Models

    Directory of Open Access Journals (Sweden)

    Girdhari Rijal

    2017-01-01

    Full Text Available Preparation of three-dimensional (3D porous scaffolds from synthetic polymers is a challenge to most laboratories conducting biomedical research. Here, we present a handy and cost-effective method to fabricate polymeric hydrogel and porous scaffolds using poly(lactic-co-glycolic acid (PLGA or polycaprolactone (PCL. Breast cancer cells grown on 3D polymeric scaffolds exhibited distinct survival, morphology, and proliferation compared to those on 2D polymeric surfaces. Mammary epithelial cells cultured on PLGA- or PCL-coated slides expressed extracellular matrix (ECM proteins and their receptors. Estrogen receptor- (ER- positive T47D breast cancer cells are less sensitive to 4-hydroxytamoxifen (4-HT treatment when cultured on the 3D porous scaffolds than in 2D cultures. Finally, cancer cell-laden polymeric scaffolds support consistent tumor formation in animals and biomarker expression as seen in human native tumors. Our data suggest that the porous synthetic polymer scaffolds satisfy the basic requirements for 3D tissue cultures both in vitro and in vivo. The scaffolding technology has appealing potentials to be applied in anticancer drug screening for a better control of the progression of human cancers.

  8. Ultra high content image analysis and phenotype profiling of 3D cultured micro-tissues.

    Directory of Open Access Journals (Sweden)

    Zi Di

    Full Text Available In many situations, 3D cell cultures mimic the natural organization of tissues more closely than 2D cultures. Conventional methods for phenotyping such 3D cultures use either single or multiple simple parameters based on morphology and fluorescence staining intensity. However, due to their simplicity many details are not taken into account which limits system-level study of phenotype characteristics. Here, we have developed a new image analysis platform to automatically profile 3D cell phenotypes with 598 parameters including morphology, topology, and texture parameters such as wavelet and image moments. As proof of concept, we analyzed mouse breast cancer cells (4T1 cells in a 384-well plate format following exposure to a diverse set of compounds at different concentrations. The result showed concentration dependent phenotypic trajectories for different biologically active compounds that could be used to classify compounds based on their biological target. To demonstrate the wider applicability of our method, we analyzed the phenotypes of a collection of 44 human breast cancer cell lines cultured in 3D and showed that our method correctly distinguished basal-A, basal-B, luminal and ERBB2+ cell lines in a supervised nearest neighbor classification method.

  9. Induction of embryogenic callus and plantlet regeneration from young leaves of high yielding mature oil palm

    Directory of Open Access Journals (Sweden)

    Yeedum, I.

    2004-09-01

    Full Text Available Callus induction and plantlet regeneration from young leaves of high-yielding mature oil palm were carried out using 10-year and 20-year-old trees from Thepa Research Station, Faculty of Natural Resources,Prince of Songkla University, Hat Yai, and Trang Agricultural College, respectively. Culture media used in this experiment were Murashige and Skoog (1962 and Oil Palm supplemented with various concentrations of α-naphthaleneacetic acid (NAA or 2,4- dichlorophenoxy acetic acid (2,4-D or dicamba (Di and antioxidants.Young leaves from 6th to 11st frond were excised, sterilized, cut into 5x5 mm pieces and cultured in the dark at 26±4ºC or 28±0.5ºC for 3 months. The results revealed that MS medium with 200 mg/l ascorbic acid (As and 1 mg/l Di (MS-AsDi gave the highest callus induction percentage (7.93 after culture for 3 months at 28±0.5ºC. Leaf segments from 6th - 8th frond yielded callus forming percentage at 10% (averaged from 1, 2.5 and 5 mg/l Di containing MS medium. Ascorbic acid as an antioxidant at concentration of 200 mg/l supplemented in MS medium in the presence of 2.5 mg/l Di produced the highest callus induction percentage (11.2 and number of nodules (7.06. A high percentage of embryogenic callus formation (66.67 was obtained when the calli were transferred to the same medium component supplemented with 0.5 mg/l Di and 1,000 mg/l casein hydrolysate (CH (MS-AsDiCH. Haustorial-staged embryos were observed to be isolated as an individual embryo and germinated on MS medium without plant growth regulator (MS-free. Development of root could be classified into two distinct types, fibrous and tap root.

  10. Tissue culture on a chip: Developmental biology applications of self-organized capillary networks in microfluidic devices.

    Science.gov (United States)

    Miura, Takashi; Yokokawa, Ryuji

    2016-08-01

    Organ culture systems are used to elucidate the mechanisms of pattern formation in developmental biology. Various organ culture techniques have been used, but the lack of microcirculation in such cultures impedes the long-term maintenance of larger tissues. Recent advances in microfluidic devices now enable us to utilize self-organized perfusable capillary networks in organ cultures. In this review, we will overview past approaches to organ culture and current technical advances in microfluidic devices, and discuss possible applications of microfluidics towards the study of developmental biology. © 2016 Japanese Society of Developmental Biologists.

  11. Finite element study of scaffold architecture design and culture conditions for tissue engineering.

    Science.gov (United States)

    Olivares, Andy L; Marsal, Elia; Planell, Josep A; Lacroix, Damien

    2009-10-01

    Tissue engineering scaffolds provide temporary mechanical support for tissue regeneration and transfer global mechanical load to mechanical stimuli to cells through its architecture. In this study the interactions between scaffold pore morphology, mechanical stimuli developed at the cell microscopic level, and culture conditions applied at the macroscopic scale are studied on two regular scaffold structures. Gyroid and hexagonal scaffolds of 55% and 70% porosity were modeled in a finite element analysis and were submitted to an inlet fluid flow or compressive strain. A mechanoregulation theory based on scaffold shear strain and fluid shear stress was applied for determining the influence of each structures on the mechanical stimuli on initial conditions. Results indicate that the distribution of shear stress induced by fluid perfusion is very dependent on pore distribution within the scaffold. Gyroid architectures provide a better accessibility of the fluid than hexagonal structures. Based on the mechanoregulation theory, the differentiation process in these structures was more sensitive to inlet fluid flow than axial strain of the scaffold. This study provides a computational approach to determine the mechanical stimuli at the cellular level when cells are cultured in a bioreactor and to relate mechanical stimuli with cell differentiation.

  12. Pleurotus Spent Mushroom Compost as Green Supplementary Nutrient in Tissue Culture

    Directory of Open Access Journals (Sweden)

    Tay Chia Chay

    2016-01-01

    Full Text Available This study investigates the effect of Pleurotus spent mushroom compost as green supplementary nutrient in tissue culture. Different concentration of Pleurotus spent mushroom compost in powder (SMC and activated carbon (SMCAC were added into Murashige and Skoog media (MS with 0.1 mg/L 6-benzylaminopurine (BAP. The shoot regeneration of Clinacantus nutans plant such as number of shoots, number of leaves and length of leaves were recorded for four weeks. Collected data was analyzed using one way analysis of variance (ANOVA and Tukey test through the SPSS Statistics 21 software. The 0.1 g/L SMC recorded the highest leaves numbers. Meanwhile, 0.2 g/L SMC showed highest performance in length of shoot. The SMCAC showed a negative response in number of leaves and shoot as well as length of shoot. In summary, the SMC showed significant performance in number of leave and shoot length but less effective on number of shoot. In contradictory, the SMCAC reported poor performance in shoot regeneration but contribute in absorption of nutrient from environment and storage of the nutrient asfunction of biochar. Therefore, the SMC has a high potential as a green supplementary nutrient for tissue culture. The application of this material has contributes into the green technology via convert waste to product, which is in-line with zero waste concept.

  13. Application of plant cell and tissue culture for the production of phytochemicals in medicinal plants.

    Science.gov (United States)

    Pant, Bijaya

    2014-01-01

    Approximately 80% of the world inhabitants depend on the medicinal plants in the form of traditional formulations for their primary health care system well as in the treatment of a number of diseases since the ancient time. Many commercially used drugs have come from the information of indigenous knowledge of plants and their folk uses. Linking of the indigenous knowledge of medicinal plants to modern research activities provides a new reliable approach, for the discovery of novel drugs much more effectively than with random collection. Increase in population and increasing demand of plant products along with illegal trade are causing depletion of medicinal plants and many are threatened in natural habitat. Plant tissue culture technique has proved potential alternative for the production of desirable bioactive components from plants, to produce the enough amounts of plant material that is needed and for the conservation of threatened species. Different plant tissue culture systems have been extensively studied to improve and enhance the production of plant chemicals in various medicinal plants.

  14. Optimized FISH methods for visualizing RNA localization properties in Drosophila and human tissues and cultured cells.

    Science.gov (United States)

    Diot, Cédric; Chin, Ashley; Lécuyer, Eric

    2017-08-15

    Eukaryotic gene expression is orchestrated by a large number of regulatory steps to modulate the synthesis, maturation and fate of various families of protein-coding and non-coding RNA molecules. Defining the subcellular localization properties of an RNA molecule is thus of considerable importance for gleaning its function(s) and for elucidating post-transcriptional gene regulation pathways. For decades, fluorescent In Situ hybridization (FISH) has constituted the gold-standard technique for assessing RNA expression and distribution properties in cultured cells, tissue specimens, and whole mount organisms. Recently, several attempts aimed at advancing multiplex RNA-FISH experiments have been published. However, these procedures are both financially demanding and technically challenging, while their full potential remains unexploited. Here we describe an optimized RNA-FISH method employing the Tyramide Signal Amplification system that robustly enhances resolution and sensitivity needed for exploring RNA localization in Drosophila embryos, tissues and commonly cultured human and insect cell lines. Methodological details and key parameters are outlined for high-throughput analyses conducted in 96-well plate format. Copyright © 2017. Published by Elsevier Inc.

  15. Ultrastructural and physiological responses of potato (Solanum tuberosum L.) plantlets to gradient saline stress.

    Science.gov (United States)

    Gao, Hui-Juan; Yang, Hong-Yu; Bai, Jiang-Ping; Liang, Xin-Yue; Lou, Yan; Zhang, Jun-Lian; Wang, Di; Zhang, Jin-Lin; Niu, Shu-Qi; Chen, Ying-Long

    2014-01-01

    Salinity is one of the major abiotic stresses that impacts plant growth and reduces the productivity of field crops. Compared to field plants, test tube plantlets offer a direct and fast approach to investigate the mechanism of salt tolerance. Here we examined the ultrastructural and physiological responses of potato (Solanum tuberosum L. c.v. "Longshu No. 3") plantlets to gradient saline stress (0, 25, 50, 100, and 200 mM NaCl) with two consequent observations (2 and 6 weeks, respectively). The results showed that, with the increase of external NaCl concentration and the duration of treatments, (1) the number of chloroplasts and cell intercellular spaces markedly decreased, (2) cell walls were thickened and even ruptured, (3) mesophyll cells and chloroplasts were gradually damaged to a complete disorganization containing more starch, (4) leaf Na and Cl contents increased while leaf K content decreased, (5) leaf proline content and the activities of catalase (CAT) and superoxide dismutase (SOD) increased significantly, and (6) leaf malondialdehyde (MDA) content increased significantly and stomatal area and chlorophyll content decline were also detected. Severe salt stress (200 mM NaCl) inhibited plantlet growth. These results indicated that potato plantlets adapt to salt stress to some extent through accumulating osmoprotectants, such as proline, increasing the activities of antioxidant enzymes, such as CAT and SOD. The outcomes of this study provide ultrastructural and physiological insights into characterizing potential damages induced by salt stress for selecting salt-tolerant potato cultivars.

  16. Ultrastructural and physiological responses of potato (Solanum tuberosum L.) plantlets to gradient saline stress

    Science.gov (United States)

    Gao, Hui-Juan; Yang, Hong-Yu; Bai, Jiang-Ping; Liang, Xin-Yue; Lou, Yan; Zhang, Jun-Lian; Wang, Di; Zhang, Jin-Lin; Niu, Shu-Qi; Chen, Ying-Long

    2015-01-01

    Salinity is one of the major abiotic stresses that impacts plant growth and reduces the productivity of field crops. Compared to field plants, test tube plantlets offer a direct and fast approach to investigate the mechanism of salt tolerance. Here we examined the ultrastructural and physiological responses of potato (Solanum tuberosum L. c.v. “Longshu No. 3”) plantlets to gradient saline stress (0, 25, 50, 100, and 200 mM NaCl) with two consequent observations (2 and 6 weeks, respectively). The results showed that, with the increase of external NaCl concentration and the duration of treatments, (1) the number of chloroplasts and cell intercellular spaces markedly decreased, (2) cell walls were thickened and even ruptured, (3) mesophyll cells and chloroplasts were gradually damaged to a complete disorganization containing more starch, (4) leaf Na and Cl contents increased while leaf K content decreased, (5) leaf proline content and the activities of catalase (CAT) and superoxide dismutase (SOD) increased significantly, and (6) leaf malondialdehyde (MDA) content increased significantly and stomatal area and chlorophyll content decline were also detected. Severe salt stress (200 mM NaCl) inhibited plantlet growth. These results indicated that potato plantlets adapt to salt stress to some extent through accumulating osmoprotectants, such as proline, increasing the activities of antioxidant enzymes, such as CAT and SOD. The outcomes of this study provide ultrastructural and physiological insights into characterizing potential damages induced by salt stress for selecting salt-tolerant potato cultivars. PMID:25628634

  17. SIGNAL MEDIATORS AT INDUCTION OF HEAT RESISTANCE OF WHEAT PLANTLETS BY SHORT-TERM HEATING.

    Science.gov (United States)

    Karpets, Yu V; Kolupaev, Yu E; Yastreb, T O

    2015-01-01

    The effects of functional interplay of calcium ions, reactive oxygen species (ROS) and nitric oxide (NO) in the cells of wheat plantlets roots (Triticum aestivum L.) at the induction of their heat resistance by a short-term influence of hyperthermia (heating at the temperature of 42 degrees C during 1 minute) have been investigated. The transitional increase of NO and H2O2 content, invoked by heating, was suppressed by the treatment of plantlets with the antagonists of calcium EGTA (chelator of exocellular calcium), lanthanum chloride (blocker of calcium channels of various types) and neomycin (inhibitor of phosphatidylinositol-dependent phospholipase C). The rise of hydrogen peroxide content, caused by hardening, was partially suppressed by the action of inhibitors of nitrate reductase (sodium wolframate) and NO-synthase (N(G)-nitro-L-arginine methyl ester--L-NAME), and the increasing of nitric oxide content was suppressed by the treatment of plants with the antioxidant ionol and with the scavenger of hydrogen peroxide (dimethylthiourea). These compounds and antagonists of calcium also partially removed the effect of the rise of plantlets' heat resistance, invoked by hardening heating. The conclusion on calcium's role in the activation of enzymatic systems, generating reactive oxygen species and nitric oxide, and on the functional interplay of these signal mediators at the induction of heat resistance of plantlets by hardening heating is made.

  18. Ultrastructural and physiological responses of potato (Solanum tuberosum L. plantlets to gradient saline stress

    Directory of Open Access Journals (Sweden)

    Hui-Juan eGao

    2015-01-01

    Full Text Available Salinity is one of the major abiotic stresses that impacts plant growth and reduces the productivity of field crops. Compared to field plants, test tube plantlets offer a direct and fast approach to investigate the mechanism of salt tolerance. Here we examined the ultrastructural and physiological responses of potato (Solanum tuberosum L. c.v. ‘Longshu No. 3’ plantlets to gradient saline stress (0, 25, 50, 100 and 200 mM NaCl with two consequent observations (two and six weeks, respectively. The results showed that, with the increase of external NaCl concentration and the duration of treatments, (1 the number of chloroplasts and cell intercellular spaces markedly decreased, (2 cell walls were thickened and even ruptured, (3 mesophyll cells and chloroplasts were gradually damaged to a complete disorganization containing more starch, (4 leaf Na and Cl contents increased while leaf K content decreased, (5 leaf proline content and the activities of catalase (CAT and superoxide dismutase (SOD increased significantly, and (6 leaf malondialdehyde (MDA content increased significantly and stomatal area and chlorophyll content decline were also detected. Severe salt stress (200 mM NaCl inhibited plantlet growth. These results indicated that potato plantlets adapt to salt stress to some extent through accumulating osmoprotectants, such as proline, increasing the activities of antioxidant enzymes, such as CAT and SOD. The outcomes of this study provide ultrastructural and physiological insights into characterizing potential damages induced by salt stress for selecting salt-tolerant potato cultivars.

  19. Pertumbuhan Plantlet Anggrek Cattleya sp. dengan Perlakuan Benzyl Amino Purine pada Media Dasar Pupuk Daun Modifikasi

    Directory of Open Access Journals (Sweden)

    HESTIN YUSWANTI

    2015-09-01

    Full Text Available Growth of Plantlets of Cattleya Orchid on The Fooliar Fertilizer-Based Medium added with Benzyl Amino Purine. The aim of the current research was to investigate the appropriate concentrationof plant growth regulator BAP on the growth of Cattleya plantlet. The experiment was utilized a Randomized Completely Design with five treatments and six replications. The basal medium used was modification of foliar fertilizer of Growmore (trade mark with addition of fish emulsion, Vitamin B1 and active charcoal. BAP concentration used as treatment were 0 ppm, 0.5 ppm, 1.0 ppm, 1.5 ppm and 2.0 ppm. Variables observed were height, leaf number, root number, root length, fresh weight and dry weight of plantlets. The result showed that concentration of 1 ppm BAP resulted in the highest value for plantlet height (5.67 cm, leaf number (4.67, root length (2.07 cm, fresh weight (0.36 g and dry weight (0.043 g.

  20. The gene expression profile of non-cultured, highly purified human adipose tissue pericytes: Transcriptomic evidence that pericytes are stem cells in human adipose tissue

    Energy Technology Data Exchange (ETDEWEB)

    Silva Meirelles, Lindolfo da, E-mail: lindolfomeirelles@gmail.com [Center for Cell-Based Therapy (CEPID/FAPESP), Regional Center for Hemotherapy of Ribeirão Preto, University of São Paulo, Rua Tenente Catão Roxo 2501, 14051-140 Ribeirão Preto, SP (Brazil); Laboratory for Stem Cells and Tissue Engineering, PPGBioSaúde, Lutheran University of Brazil, Av. Farroupilha 8001, 92425-900 Canoas, RS (Brazil); Deus Wagatsuma, Virgínia Mara de; Malta, Tathiane Maistro; Bonini Palma, Patrícia Viana [Center for Cell-Based Therapy (CEPID/FAPESP), Regional Center for Hemotherapy of Ribeirão Preto, University of São Paulo, Rua Tenente Catão Roxo 2501, 14051-140 Ribeirão Preto, SP (Brazil); Araújo, Amélia Goes; Panepucci, Rodrigo Alexandre [Laboratory of Large-Scale Functional Biology (LLSFBio), Regional Center for Hemotherapy of Ribeirão Preto, University of São Paulo, Rua Tenente Catão Roxo 2501, 14051-140 Ribeirão Preto, SP (Brazil); and others

    2016-12-10

    Pericytes (PCs) are a subset of perivascular cells that can give rise to mesenchymal stromal cells (MSCs) when culture-expanded, and are postulated to give rise to MSC-like cells during tissue repair in vivo. PCs have been suggested to behave as stem cells (SCs) in situ in animal models, although evidence for this role in humans is lacking. Here, we analyzed the transcriptomes of highly purified, non-cultured adipose tissue (AT)-derived PCs (ATPCs) to detect gene expression changes that occur as they acquire MSC characteristics in vitro, and evaluated the hypothesis that human ATPCs exhibit a gene expression profile compatible with an AT SC phenotype. The results showed ATPCs are non-proliferative and express genes characteristic not only of PCs, but also of AT stem/progenitor cells. Additional analyses defined a gene expression signature for ATPCs, and revealed putative novel ATPC markers. Almost all AT stem/progenitor cell genes differentially expressed by ATPCs were not expressed by ATMSCs or culture-expanded ATPCs. Genes expressed by ATMSCs but not by ATPCs were also identified. These findings strengthen the hypothesis that PCs are SCs in vascularized tissues, highlight gene expression changes they undergo as they assume an MSC phenotype, and provide new insights into PC biology. - Highlights: • Non-cultured adipose tissue-derived human pericytes (ncATPCs) exhibit a distinctive gene expression signature. • ncATPCs express key adipose tissue stem cell genes previously described in vivo in mice. • ncATPCs express message for anti-proliferative and antiangiogenic molecules. • Most ncATPC-specific transcripts are absent in culture-expanded pericytes or ATMSCs • Gene expression changes ncATPCs undergo as they acquire a cultured ATMSC phenotype are pointed out.

  1. Withania somnifera: Advances and Implementation of Molecular and Tissue Culture Techniques to Enhance Its Application

    Directory of Open Access Journals (Sweden)

    Vibha Pandey

    2017-08-01

    Full Text Available Withania somnifera, commonly known as Ashwagandha an important medicinal plant largely used in Ayurvedic and indigenous medicine for over 3,000 years. Being a medicinal plant, dried powder, crude extract as well as purified metabolies of the plant has shown promising therapeutic properties. Withanolides are the principal metabolites, responsible for the medicinal properties of the plant. Availability and amount of particular withanolides differ with tissue type and chemotype and its importance leads to identification characterization of several genes/ enzymes related to withanolide biosynthetic pathway. The modulation in withanolides can be achieved by controlling the environmental conditions like, different tissue culture techniques, altered media compositions, use of elicitors, etc. Among all the in vitro techniques, hairy root culture proved its importance at industrial scale, which also gets benefits due to more accumulation (amount and number of withanolides in roots tissues of W. somnifera. Use of media compostion and elicitors further enhances the amount of withanolides in hairy roots. Another important modern day technique used for accumulation of desired secondary metabolites is modulating the gene expression by altering environmental conditions (use of different media composition, elicitors, etc. or through genetic enginnering. Knowing the significance of the gene and the key enzymatic step of the pathway, modulation in withanolide contents can be achieved upto required amount in therapeutic industry. To accomplish maximum productivity through genetic enginnering different means of Withania transformation methods have been developed to obtain maximum transformation efficiency. These standardized transformation procedues have been used to overexpress/silence desired gene in W. somnifera to understand the outcome and succeed with enhanced metabolic production for the ultimate benefit of human race.

  2. Withania somnifera: Advances and Implementation of Molecular and Tissue Culture Techniques to Enhance Its Application

    Science.gov (United States)

    Pandey, Vibha; Ansari, Waquar Akhter; Misra, Pratibha; Atri, Neelam

    2017-01-01

    Withania somnifera, commonly known as Ashwagandha an important medicinal plant largely used in Ayurvedic and indigenous medicine for over 3,000 years. Being a medicinal plant, dried powder, crude extract as well as purified metabolies of the plant has shown promising therapeutic properties. Withanolides are the principal metabolites, responsible for the medicinal properties of the plant. Availability and amount of particular withanolides differ with tissue type and chemotype and its importance leads to identification characterization of several genes/ enzymes related to withanolide biosynthetic pathway. The modulation in withanolides can be achieved by controlling the environmental conditions like, different tissue culture techniques, altered media compositions, use of elicitors, etc. Among all the in vitro techniques, hairy root culture proved its importance at industrial scale, which also gets benefits due to more accumulation (amount and number) of withanolides in roots tissues of W. somnifera. Use of media compostion and elicitors further enhances the amount of withanolides in hairy roots. Another important modern day technique used for accumulation of desired secondary metabolites is modulating the gene expression by altering environmental conditions (use of different media composition, elicitors, etc.) or through genetic enginnering. Knowing the significance of the gene and the key enzymatic step of the pathway, modulation in withanolide contents can be achieved upto required amount in therapeutic industry. To accomplish maximum productivity through genetic enginnering different means of Withania transformation methods have been developed to obtain maximum transformation efficiency. These standardized transformation procedues have been used to overexpress/silence desired gene in W. somnifera to understand the outcome and succeed with enhanced metabolic production for the ultimate benefit of human race. PMID:28848589

  3. Transcriptomic comparison of primary bovine horn core carcinoma culture and parental tissue at early stage

    Directory of Open Access Journals (Sweden)

    Sharadindu Shil

    2017-01-01

    Full Text Available Aim: Squamous cell carcinoma or SCC of horn in bovines (bovine horn core carcinoma frequently observed in Bos indicus affecting almost 1% of cattle population. Freshly isolated primary epithelial cells may be closely related to the malignant epithelial cells of the tumor. Comparison of gene expression in between horn’s SCC tissue and its early passage primary culture using next generation sequencing was the aim of this study. Materials and Methods: Whole transcriptome sequencing of horn’s SCC tissue and its early passage cells using Ion Torrent PGM were done. Comparative expression and analysis of different genes and pathways related to cancer and biological processes associated with malignancy, proliferating capacity, differentiation, apoptosis, senescence, adhesion, cohesion, migration, invasion, angiogenesis, and metabolic pathways were identified. Results: Up-regulated genes in SCC of horn’s early passage cells were involved in transporter activity, catalytic activity, nucleic acid binding transcription factor activity, biogenesis, cellular processes, biological regulation and localization and the down-regulated genes mainly were involved in focal adhesion, extracellular matrix receptor interaction and spliceosome activity. Conclusion: The experiment revealed similar transcriptomic nature of horn’s SCC tissue and its early passage cells.

  4. Picroside I and Picroside II from Tissue Cultures of Picrorhiza kurroa

    Science.gov (United States)

    Ganeshkumar, Yamjala; Ramarao, Ajmera; Veeresham, Ciddi

    2017-01-01

    Background: Picrorhiza kurroa (PK) belongs to Scrophulariaceae family and is a representative endemic, medicinal herb, widely distributed throughout the higher altitudes of alpine Himalayas from west to east, between 3000 and 4500 m above mean sea level. Objective: The objective of the present study is to assess the production of picroside I and picroside II from tissue cultures of PK. Materials and Methods: Auxiliary shoot tips of PK were incubated in Murashige and Skoog medium supplemented with indole-3-butyric acid and kinetin phytohormones. The callus produced was collected at different time intervals and was processed for extraction of picroside I and picroside II followed by thin layer chromatography and high-performance liquid chromatography HPLC analysis. Results: The maximum growth index was found to be 5.109 ± 0.159 at 16-week-old callus culture. The estimation of picroside-I and picroside-II was carried out by (HPLC) analysis; quantity of secondary metabolite found to be 16.37 ± 0.0007 mg/g for PK-I and 6.34 ± 0.0012 mg/g for PK-II. Conclusion: This is the first attempt to produce the Picroside-I and II in large amount by the tissue culture technique. It can be observed that the method of callus culture can be used in production of secondary metabolites Picroside-I and II from PK SUMMARY Picrorhiza kurroa is a high value medicinal herb due to rich source of hepatoprotective metabolites, Picroside-I and Picroside-II. The medicinal importance of P. kurroa is due to its pharmacological properties like hepatoprotective, antioxidant (particularly in liver), antiallergic and antiasthamatic, anticancer activity particularly in liver and immunomodulatory. Shoot apices which were produced a good response was inoculated on selected medium i.e., on MS medium containing 2, 4 D (mg/l) + KN (1mg/l) for induction of callus. The initiation of callus was observed after 4weeks and it was light green and fragile Maximum growth was observed with 3% w/v of sucrose

  5. Graft of autologous fibroblasts in gingival tissue in vivo after culture in vitro. Preliminary study on rats.

    Science.gov (United States)

    Simain-Sato, F; Lahmouzi, J; Heinen, E; Defresne, M P; De Pauw-Gillet, M C; Grisar, T; Legros, J J; Legrand, R

    1999-08-01

    Several grafting techniques and guided tissue regeneration techniques (GTR) have been well-developed in periodontal surgery. However, these techniques could induce pain and side effects, such as a gingival recession during the healing period following the therapy. The graft of a small autologous connective tissue, using non-invasive surgical techniques could yield several benefits for the patients. Our preliminary study explores the feasibility of collecting healthy gingival tissues, culturing them in vitro to amplify rat gingival fibroblasts (RGF) and inoculating the obtained cells into autologous rat gingival tissues in vivo. Gingival tissues samples were cultured as explants as described by Freshney et al. and Adolphe. Confluent cells surrounding explants were detached after 7 d of culture from Petri dishes using 0.05% trypsin and designated "first transferred cells" (T1). At the third passage (T3), cells cultured as monolayer were either examined under microscopy--phase contrast, scanning, or transmission electron--or numerated after trypan blue exclusion test. Autologous RGF labelled with fluorochrome were inoculated at the vestibular and palatine site of gingival tissue close to the superior incisors. In this preliminary study, 12 Wistar rats were used; for each, 2 biopsies were dissected and fixed for phase contrast or fluorescence microscopy. On d 1, 3 and 7 after injection in rat gingival tissues, fluorochrome-labelled cells could be detected in all these.

  6. Pre-irradiation of tissue culture flasks leads to diminished stem and progenitor cell production in long-term bone marrow cultures

    International Nuclear Information System (INIS)

    Rooney, P.; Wright, E.G.

    1993-01-01

    Empty plastic tissue culture flasks were exposed to X-irradiation doses of 0.3-10.0 Gy, prior to the establishment of long-term bone marrow cultures. During the course of a 10 week culture period, all irradiated plastic flasks exhibited a dramatic decrease in the number of both haemopoietic stem cells and myeloid progenitor cells, in the non-adherent layer, when compared with controls. This decrease was not due to a decrease in the number of non-adherent cells produced. Histological examination of non-adherent cells showed an increase in mature granulocytic cells with few blast cells. Morphologically, the adherent layers of irradiated flasks demonstrated a delay in appearance or absence of fat cell production. X-irradiation of glass tissue culture flasks had no deleterious effect. (author)

  7. An RNA-Seq Analysis of Grape Plantlets Grown in vitro Reveals Different Responses to Blue, Green, Red LED Light, and White Fluorescent Light.

    Science.gov (United States)

    Li, Chun-Xia; Xu, Zhi-Gang; Dong, Rui-Qi; Chang, Sheng-Xin; Wang, Lian-Zhen; Khalil-Ur-Rehman, Muhammad; Tao, Jian-Min

    2017-01-01

    Using an RNA sequencing (RNA-seq) approach, we analyzed the differentially expressed genes (DEGs) and physiological behaviors of "Manicure Finger" grape plantlets grown in vitro under white, blue, green, and red light. A total of 670, 1601, and 746 DEGs were identified in plants exposed to blue, green, and red light, respectively, compared to the control (white light). By comparing the gene expression patterns with the growth and physiological responses of the grape plantlets, we were able to link the responses of the plants to light of different spectral wavelengths and the expression of particular sets of genes. Exposure to red and green light primarily triggered responses associated with the shade-avoidance syndrome (SAS), such as enhanced elongation of stems, reduced investment in leaf growth, and decreased chlorophyll levels accompanied by the expression of genes encoding histone H3, auxin repressed protein, xyloglucan endotransglycosylase/hydrolase, the ELIP protein, and microtubule proteins. Furthermore, specific light treatments were associated with the expression of a large number of genes, including those involved in the glucan metabolic pathway and the starch and sucrose metabolic pathways; these genes were up/down-regulated in ways that may explain the increase in the starch, sucrose, and total sugar contents in the plants. Moreover, the enhanced root growth and up-regulation of the expression of defense genes accompanied with SAS after exposure to red and green light may be related to the addition of 30 g/L sucrose to the culture medium of plantlets grown in vitro . In contrast, blue light induced the up-regulation of genes related to microtubules, serine carboxypeptidase, chlorophyll synthesis, and sugar degradation and the down-regulation of auxin-repressed protein as well as a large number of resistance-related genes that may promote leaf growth, improve chlorophyll synthesis and chloroplast development, increase the ratio of chlorophyll a (chla

  8. Isolation and propagation of mutations in dahlia by in vitro culture

    International Nuclear Information System (INIS)

    Asahira, T.; Yamagata, H.; Inagaki, M.; Osuga, S.

    1975-01-01

    The present study was undertaken to search for a successful method for in vitro culture of mutated tissues in dahlia. Preceding the objective, the features of induced mutations and the effects of cutting propagation in dahlia were investigated, and the tissues easily regenerating plantlets in vitro were searched following the examination of effective condition of medium. Induction of mutations: Tuberous roots of two cultivars, 'Kosei' and 'Sunlight', were irradiated with 1,000 - 2,000 R of X-rays. Chlorophyll and flower-color mutations were successfully induced in both cultivars, but the frequency differed with genotypic constitution. The maximum frequency was observed at leaves and shoots on or from the fourth to fifth nodes from the base of plant. The use of M 1 tuberous roots seemed a way for isolating mutations though not so much efficient. Tissue culture: In vitro cultured basal parts of ray florets, exactly the ovary, differentiated shoots. No shoot formation occurred in receptacle and leaf cultures, while roots were differentiated in leaf culture. Supplements of auxin and adenine to the medium besides cytokinin appeared to be necessary for inducing shoots. It is a serious problem in the tissue culture of dahlia that a large number of explants are endogenously comtaminated with bacteria. Taking into consideration low rates of surviving and regenerating explants, it seems difficult at present for dahlia to conclude whether or not the tissue culture may become efficient in mutation breeding as compared with cutting propagation. (author)

  9. Organotypic tissue culture of adult rodent retina followed by particle-mediated acute gene transfer in vitro.

    Directory of Open Access Journals (Sweden)

    Satoru Moritoh

    Full Text Available BACKGROUND: Organotypic tissue culture of adult rodent retina with an acute gene transfer that enables the efficient introduction of variable transgenes would greatly facilitate studies into retinas of adult rodents as animal models. However, it has been a difficult challenge to culture adult rodent retina. The purpose of this present study was to develop organotypic tissue culture of adult rodent retina followed by particle-mediated acute gene transfer in vitro. METHODOLOGY/PRINCIPAL FINDINGS: We established an interphase organotypic tissue culture for adult rat retinas (>P35 of age which was optimized from that used for adult rabbit retinas. We implemented three optimizations: a greater volume of Ames' medium (>26 mL per retina, a higher speed (constant 55 rpm of agitation by rotary shaker, and a greater concentration (10% of horse serum in the medium. We also successfully applied this method to adult mouse retina (>P35 of age. The organotypic tissue culture allowed us to keep adult rodent retina morphologically and structurally intact for at least 4 days. However, mouse retinas showed less viability after 4-day culture. Electrophysiologically, ganglion cells in cultured rat retina were able to generate action potentials, but exhibited less reliable light responses. After transfection of EGFP plasmids by particle-mediated acute gene transfer, we observed EGFP-expressing retinal ganglion cells as early as 1 day of culture. We also introduced polarized-targeting fusion proteins such as PSD95-GFP and melanopsin-EYFP (hOPN4-EYFP into rat retinal ganglion cells. These fusion proteins were successfully transferred into appropriate locations on individual retinal neurons. CONCLUSIONS/SIGNIFICANCE: This organotypic culture method is largely applicable to rat retinas, but it can be also applied to mouse retinas with a caveat regarding cell viability. This method is quite flexible for use in acute gene transfection in adult rodent retina, replacing

  10. A fluid dynamics approach to bioreactor design for cell and tissue culture.

    Science.gov (United States)

    Dusting, Jonathan; Sheridan, John; Hourigan, Kerry

    2006-08-20

    The problem of controlling cylindrical tank bioreactor conditions for cell and tissue culture purposes has been considered from a flow dynamics perspective. Simple laminar flows in the vortex breakdown region are proposed as being a suitable alternative to turbulent spinner flask flows and horizontally oriented rotational flows. Vortex breakdown flows have been measured using three-dimensional Stereoscopic particle image velocimetry, and non-dimensionalized velocity and stress distributions are presented. Regions of locally high principal stress occur in the vicinity of the impeller and the lower sidewall. Topological changes in the vortex breakdown region caused by an increase in Reynolds number are reflected in a redistribution of the peak stress regions. The inclusion of submerged scaffold models adds complexity to the flow, although vortex breakdown may still occur. Relatively large stresses occur along the edge of disks jutting into the boundary of the vortex breakdown region. Copyright 2006 Wiley Periodicals, Inc.

  11. Advanced tissue culture used by Twyfords to build up jojoba clones

    Energy Technology Data Exchange (ETDEWEB)

    1983-01-01

    Twyford Plant Laboratories Ltd. in the UK, using their own advanced methods of plant tissue culture, have built up a bank of 30 different male and female clones of jojoba, the arid land crop whose seeds produced a liquid wax which - amongst other uses - can be substituted for sperm whale oil. The technique involves growing microscopic parts of a parent plant on a medium containing all the necessary growth hormones, salts, vitamins and other nutrients. Growth takes place under artificial light in an all-electric controlled, air-conditioned environment. No other method is so successful for rapidly multiplying plants, particularly those that do not breed true from seed. These include most fruits and some flowers and vegetables.

  12. DEVELOPMENT OF PRIMARY CELL CULTURE FROM TAIL EPIDERMAL TISSUE OF KOI CARP (Cyprinus carpio koi)

    OpenAIRE

    Lila Gardenia; Isti Koesharyani

    2014-01-01

    Primary cell culture from tail epidermal tissue of koi carp (Cyprinus carpio koi) was developed. Cells were grown in Leibovits-15 medium supplemented with 20% fetal bovine serum and antibiotics (Penicillin/Streptomycin and Kanamycin). Cell growth was observed in a range of incubation temperature (17oC±2oC, 22oC±2oC, 27oC±2oC, and 32oC±2oC) in order to determine the optimum temperature. The cells were able to grow at a range of temperature between 17oC to 32oC with optimal growth at 22oC. Prim...

  13. Plant regeneration from petiole segments of some species in tissue culture

    Directory of Open Access Journals (Sweden)

    Krystyna Klimaszewska

    2013-12-01

    Full Text Available The regeneration ability of 21 plant species belonging to 14 families was tested. The method of tissue culture in vitro was applied, on basic MS medium with an addition of growth regulators from the auxin and cytokinin groups. From among the investigated plant groups Peperomia scandens and Caladium × hortulanum were capable of plant regeneration, Passiilora coerulea regenerated shoots, Hedera helix, Begonia glabra, Coleus blumei, Fuchsia hybrida, Passiflora suberosa and Peperomia eburnea formed callus and roots, Kalanchoe blossfeldiana, Pelargonium grandiflorum, P. peltatum, P. radula, Coleus shirensis and Magnolia soulangeana produced callus, Philodendron scandens, Rhododendron smirnovii, Hibiscus rosa-sinensis, Coprosma baueri, Cestrum purpureum and Solanum rantonnetii did not exhibit any regeneration reactions.

  14. [Solubility of metal components into tissue culture medium from dental amalgams (author's transl)].

    Science.gov (United States)

    Kawahara, H; Yamada, T; Nakamura, M; Tomoda, T; Kobayashi, H; Saijo, A; Kawata, Y; Hikari, S

    1981-10-01

    Solubility of metal components into tissue culture medium, YLH, from various dental amalgams including high copper, conventional and copper amalgam was measured with atomic absorption spectrometry. The results obtained were as follows.: 1. Mercury solubility was found much in all the dental amalgams after one day extraction. This was followed by gradual increase in solubility in the high copper amalgams until after seven days. On the otherhand, initial high solubility was maintained in conventional and copper amalgams through the whole experimental period. 2. Higher solubility of silver and copper was recorded in the high copper amalgams. 3. Zinc was only found in Dispersalloy and copper amalgam. 4. Tin was unable to be measured in all the amalgams examined. It was considered that the present results could shed a light on the mechanism for cytotoxicity yielding of the dental amalgams.

  15. Tissue culture of adult larch as a tool for breeding purposes

    Energy Technology Data Exchange (ETDEWEB)

    Ewald, D.; Kretzschmar, U. [Federal Research Centre of Forestry and Forest Products, Waldsieversdorf (Germany). Inst. for Forest Tree Biology

    1995-12-31

    Aimed at the identical reproduction of genotypes which are considered superior different methods were tested to establish and to propagate tissue cultures from old larch trees (L. decidua, L. kaempferi, L. sukaczewii, L. gmelinii, L. eurolepis). Serial subcultures without phytohormones (shoot tip propagation) led to the establishment of clone lines. After ten subcultures propagation velocity, shoot morphology and rooting behavior were similar to juvenile plant material. Serial subcultures which included a cytokinin induction led to the formation of adventitious shoot clusters (adventitious bud propagation). Adventitious shoots derived from male flowers of one L. kaempferi clone could be propagated via shoot tip propagation. Micrografting of meristems in vitro resulted in a regained rooting capacity of green cuttings from micrografts. Combining these in vitro techniques offers now the possibility to propagate selected mature larch trees for different breeding purposes. 23 refs, 5 figs, 2 tabs

  16. Diagnostic utility of melanin production by fungi: Study on tissue sections and culture smears with Masson-Fontana stain

    Directory of Open Access Journals (Sweden)

    Challa Sundaram

    2014-01-01

    Full Text Available Background: Dematiaceous fungi appear brown in tissue section due to melanin in their cell walls. When the brown color is not seen on routine H and E and culture is not available, differentiation of dematiaceous fungi from other fungi is difficult on morphology alone. Aims and Objective: To study if melanin production by dematiaceous fungi can help differentiate them from other types of fungi. Materials and Methods: Fifty tissue sections of various fungal infections and 13 smears from cultures of different species of fungi were stained with Masson Fontana stain to assess melanin production. The tissue sections included biopsies from 26 culture-proven fungi and 24 biopsies of filamentous fungi diagnosed on morphology alone with no culture confirmation. Results: All culture-proven dematiaceous fungi and Zygomycetes showed strong positivity in sections and culture smears. Aspergillus sp showed variable positivity and intensity. Cryptococcus neoformans showed strong positivity in tissue sections and culture smears. Tissue sections of septate filamentous fungi (9/15, Zygomycetes (4/5, and fungi with both hyphal and yeast morphology (4/4 showed positivity for melanin. The septate filamentous fungi negative for melanin were from biopsy samples of fungal sinusitis including both allergic and invasive fungal sinusitis and colonizing fungal balls. Conclusion: Melanin is produced by both dematiaceous and non-dematiaceous fungi. Masson-Fontana stain cannot reliably differentiate dematiaceous fungi from other filamentous fungi like Aspergillus sp; however, absence of melanin in the hyphae may be used to rule out dematiaceous fungi from other filamentous fungi. In the differential diagnosis of yeast fungi, Cryptococcus sp can be differentiated from Candida sp by Masson-Fontana stain in tissue sections.

  17. Cryopreservation of chayote (Sechium edule JACQ. SW.) zygotic embryos and shoot-tips from in vitro plantlets.

    Science.gov (United States)

    Abdelnour-Esquivel, Ana; Engelmann, Florent

    2002-01-01

    This paper presents the development of cryopreservation protocols for zygotic embryos and apices of chayote (Sechium edule Jacq. Sw.), a tropical plant species with recalcitrant seeds. Zygotic embryos of two cultivars, Ccocro negro (CN) and Claudio (Cl) could withstand cryopreservation, with survival percentages of 10 and 30 %, after desiccation to 23 and 19 % moisture content (fresh weight basis), respectively. Apices sampled on in vitro plantlets of cultivars Cl, 13 and JM were successfully cryopreserved using a vitrification technique. Optimal conditions included the culture of mother-plants for 22 days on medium containing 0.3 M sucrose, culture of excised apices on the same medium for 1 day, loading of apices for 20 min with 2M glycerol + 0.4M glycerol, treatment with a series of diluted PVS2 solution (60 % PVS2 followed by 80 % PVS2 solution for 15 min (cultivar Cocoro Blanco [CB]) or 30 min (cultivars CN and Cl) at each concentration), rapid freezing and thawing, washing of shoot-tips with a 1.2 M sucrose solution, followed by recovery on media with progressively decreasing sucrose concentrations until the standard concentration of 0.1 M was reached. The highest survival percentages achieved ranged between 17 and 38 %, depending on the cultivar.

  18. Steroid-inducible BABY BOOM system for development of fertile Arabidopsis thaliana plants after prolonged tissue culture.

    Science.gov (United States)

    Lutz, Kerry A; Martin, Carla; Khairzada, Sahar; Maliga, Pal

    2015-10-01

    We describe a steroid-inducible BABY BOOM system that improves plant regeneration in Arabidopsis leaf cultures and yields fertile plants. Regeneration of Arabidopsis thaliana plants for extended periods of time in tissue culture may result in sterile plants. We report here a novel approach for A. thaliana regeneration using a regulated system to induce embryogenic cultures from leaf tissue. The system is based on BABY BOOM (BBM), a transcription factor that turns on genes involved in embryogenesis. We transformed the nucleus of A. thaliana plants with BBM:GR, a gene in which the BBM coding region is fused with the glucocorticoid receptor (GR) steroid-binding domain. In the absence of the synthetic steroid dexamethasone (DEX), the BBM:GR fusion protein is localized in the cytoplasm. Only when DEX is included in the culture medium does the BBM transcription factor enter the nucleus and turn on genes involved in embryogenesis. BBM:GR plant lines show prolific shoot regeneration from leaf pieces on media containing DEX. Removal of DEX from the culture media allowed for flowering and seed formation. Therefore, use of BBM:GR leaf tissue for regeneration of plants for extended periods of time in tissue culture will facilitate the recovery of fertile plants.

  19. Studies on the use of gamma irradiation and tissue culture in improving brassica napus

    International Nuclear Information System (INIS)

    Khedr, E.K.A.

    2012-01-01

    The objectives of this study were to:1- Studying the effect of different doses of gamma rays on some growth and yield component traits of three Brassica napus cultivars (Serow6, Serow4 and Pactol) during four consecutive generations aiming to create new genotypes characterized with high yielding traits. 2- Studying the effect of different doses of gamma rays on in vitro biotechnology technique (tissue culture) used in improving Brassica napus. Seeds of three Brassica napus cultivars were irradiated with different gamma ray doses then sown for four consecutive seasons. Data were collected and recorded to clarify the effect gamma irradiation on some yield component traits which were days to flowering , plant height, number of main branches per plant, number of secondary branches per plant, number of pods per plant, number of seeds per pod, weight of 1000-seed, weight of grain yield/plant and oil content of seeds). Results showed that high doses of gamma radiation had enhanced all of the studied traits for each of the three tested cultivars (except the plant height trait for Serow6 and Pactol cultivars). Seven new mutant lines were selected for their superiority in one or more of the studied yield component traits. Regarding the effect of gamma rays on tissue culture techniques, the applied gamma radiation doses did not affect the percentage of seed germination of the three studied cultivars, whereas the percentage of callus induction decreased by increasing the dose of gamma rays for each of the three cultivars and in both types of explants (hypocotyl and cotyledons) used in this experiment.

  20. Standard Operating Procedure (SOP) for Rapid and Efficient Production of Stevia Tissue Culture Seedlings

    International Nuclear Information System (INIS)

    Norazlina Noordin; Peng, C.S.; Rusli Ibrahim

    2015-01-01

    Stevia rebaudiana Bertoni is a non-caloric natural sweetener which is 300 times sweeter than cane sugar. Extracts from stevia leaves has vast application in food and beverages based industries, can be added to tea and coffee, cooked or baked goods, processed foods and confectionary goods. Recently, stevia attained awareness owing to its natural, non-caloric sweetness by diet/ health conscious and diabetic persons (Arpita et al., 2011). This natural sweetener has high commercial value in global market, it was estimated that global market value for stevia is be around USD11 billion by year 2015. Although stevia is being largely popularized in Malaysia and other countries but large-scale propagation procedures for the continuous supply of planting materials in commercial plantation has yet to be established, optimized and standardized. Furthermore, propagation through stevia seeds is often very difficult due to self-incompatibility which results in sterile seeds (Sakaguchi et al., 1982). Tissue culture is the only rapid process for the mass propagation of stevia and there have been few reports of in vitro growth of stevia (Miyagaya et al., 1986) and in vitro micropropagation from shoot tip and leaf (Uddin et al., 2006). Hence, study was carried out to establish a suitable protocol for in vitro propagation of S. rebaudiana Bertoni that can be further up-scaled for mass propagation of stevia seedlings. The established Standard Operating Procedure (SOP) will ensure rapid and efficient production of stevia tissue culture seedlings for continuous supply of planting materials for commercial stevia plantations in Malaysia. Preparation of growth medium, multiplication of shoots, rooting of plant lets and hardening of ex-vitro rooted plant lets is discussed in this paper. (author)

  1. COMPARISON OF CULTURE OF SYNOVIAL FLUID, PERIPROSTHETIC TISSUE AND PROSTHESIS SONICATE FOR THE DIAGNOSIS OF KNEE PROSTHESIS INFECTION

    Directory of Open Access Journals (Sweden)

    Andrej Trampuž

    2003-03-01

    Full Text Available Background. Synovial fluid and periprosthetic tissue specimens are the standard specimens cultured for the diagnosis of prosthetic joint infection (PJI. We hypothesize that ultrasonication of the explanted prosthesis may improve diagnosis of PJI by dislodging biofilm bacteria from the prosthesis surface and improve the sensitivity and specificity of diagnosis of PJI.Methods. Included were patients undergoing knee prosthesis exchange for septic or biomechanical failure and have not received antimicrobial therapy in the last 2 weeks prior specimen collection. Cultures of synovial fluid and periprosthetic tissue specimens were performed per the usual clinical practice. Additionally, explanted joint components were sonicated for 5 minutes at frequency 40 kHz in sterile Ringer’s solution; aliquots of 0.5 ml sonicate were plated onto five aerobic and five anaerobic blood agar plates, and incubated at 37 °C and examined for the next seven days. The number and identity of each colony morphology was recorded.Results. 35 patients undergoing knee replacement have been studied (24 for aseptic biomechanical failure and 11 for suspected PJI. In patients with PJI, coagulase-negative staphylococci (7 cases, Corynebacterium spp. (2 cases, Staphylococcus aureus (1 case, and viridans group streptococcus (1 case were recovered. Culture sensitivity and specificity were for synovial fluid 88% and 100%, for periprosthetic tissue 83% and 81%, and for explant sonicate 91% and 100%, respectively. In sonicate cultures higher numbers of microorganisms than in periprosthetic tissue cultures were consistently detected.Conclusions. Using synovial fluid, periprosthetic tissue, and explant sonicate cultures, 12%, 17% and 9% of PJI were missed, respectively. Explant sonicate cultures were the most sensitive with respect to the diagnosis of PJI, indicating that explant ultrasonication may improve bacterial recovery. In sonicate cultures, infecting organisms were detected in

  2. Shear and mixing effects on cells in agitated microcarrier tissue culture reactors

    Science.gov (United States)

    Cherry, Robert S.; Papoutsakis, E. Terry

    1987-01-01

    Tissue cells are known to be sensitive to mechanical stresses imposed on them by agitation in bioreactors. The amount of agitation provided in a microcarrier or suspension bioreactor should be only enough to provide effective homogeneity. Three distinct flow regions can be identified in the reactor: bulk turbulent flow, bulk laminar flow and boundary-layer flows. Possible mechanisms of cell damage are examined by analyzing the motion of microcarriers or free cells relative to the surrounding fluid, to each other and to moving or stationary solid surfaces. The primary mechanisms of cell damage appear to result from: (1) direct interaction between microcarriers and turbulent eddies; (2) collisions between microcarriers in turbulent flow; and (3) collisions against the impeller or other stationary surfaces. If the smallest eddies of turbulent flow are of the same size as the microcarrier beads, they may cause high shear stresses on the cells. Eddies the size of the average interbead spacing may cause bead-bead collisions which damage cells. The severity of the collisions increases when the eddies are also of the same size as the beads. Impeller collisions occur when beads cannot avoid the impeller leading edge as it advances through the liquid. The implications of the results of this analysis on the design and operation of tissue culture reactors are discussed.

  3. Tissue plasminogen activator inhibits NMDA-receptor-mediated increases in calcium levels in cultured hippocampal neurons

    Directory of Open Access Journals (Sweden)

    Samuel D Robinson

    2015-10-01

    Full Text Available NMDA receptors (NMDARs play a critical role in neurotransmission, acting as essential mediators of many forms of synaptic plasticity, and also modulating aspects of development, synaptic transmission and cell death. NMDAR-induced responses are dependent on a range of factors including subunit composition and receptor location. Tissue-type plasminogen activator (tPA is a serine protease that has been reported to interact with NMDARs and modulate NMDAR activity. In this study we report that tPA inhibits NMDAR-mediated changes in intracellular calcium levels in cultures of primary hippocampal neurons stimulated by low (5 μM but not high (50 μM concentrations of NMDA. tPA also inhibited changes in calcium levels stimulated by presynaptic release of glutamate following treatment with bicucculine/4-AP. Inhibition was dependent on the proteolytic activity of tPA but was unaffected by α2-antiplasmin, an inhibitor of the tPA substrate plasmin, and RAP, a pan-ligand blocker of the low-density lipoprotein receptor, two proteins previously reported to modulate NMDAR activity. These findings suggest that tPA can modulate changes in intracellular calcium levels in a subset of NMDARs expressed in cultured embryonic hippocampal neurons through a mechanism that involves the proteolytic activity of tPA and synaptic NMDARs.

  4. Neoteric trends in tissue culture-mediated biotechnology of Indian ipecac [Tylophora indica (Burm. f.) Merrill].

    Science.gov (United States)

    Gantait, Saikat; Kundu, Suprabuddha

    2017-07-01

    Tylophora indica (Burm. f.) Merrill, an ethno-pharmacologically important perennial climber of Asclepiadaceae, is commonly known as Antamul or Indian ipecac. It is essentially accredited for its medicinal properties owing to its wide range of alkaloids in the form of bioactive secondary metabolites, such as tylophorine, tylophorinine, and tylophorinidine. Accelerated mass propagation of Tylophora is challenging because of its reduced seed germination frequency that consequently headed the pursuit for efficient protocols on in vitro propagation for the large-scale regeneration, conservation as well as sustainable supply of quality propagules. Ample tissue culture-mediated biotechnological investigations have been carried out on this medicinal plant till date and several micropropagation protocols have been standardized as well. The present review compares between several typical methods as well as factors, involving on direct and indirect organogenesis of Tylophora along with various up-to-date and modified techniques such as somatic embryogenesis, protoplast culture, synthetic seed production, genetic transformation, and in vitro interventions for the secondary metabolite production that have been reported in last two decades. This compilation will allow assessing the achievements and trends of Tylophora research so far, as well as will advance the research more rapidly, since many aspects, basic and applied, have yet to be explored.

  5. Plant tissue culture of fast-growing trees for phytoremediation research.

    Science.gov (United States)

    Couselo, José Luis; Corredoira, Elena; Vieitez, Ana M; Ballester, Antonio

    2012-01-01

    The ability of plants to remove pollutants from the environment is currently used in a simple and low-cost cleaning technology known as phytoremediation. Unfortunately, little is known about the metabolic pathways involved in the transformation of xenobiotic compounds and the ability of certain plants to tolerate, detoxify, and store high concentrations of heavy metals. Plant cell and tissue culture is considered an important tool for fundamental studies that provide information about the plant-contaminant relationships, help to predict plant responses to environmental contaminants, and improve the design of plants with enhanced characteristics for phytoremediation. Callus, cell suspensions, hairy roots, and shoot multiplication cultures are used to study the interactions between plants and pollutants under aseptic conditions. Many plant species have an inherent ability to accumulate/metabolize a variety of pollutants, but they normally produce little biomass. However, fast-growing trees are excellent candidates for phytoremediation because of their rapid growth, extensive root system, and high water uptake. This chapter outlines the in vitro plant production of both somaclonal variants and transgenic plants of Populus spp. that exhibit high tolerance to heavy metals.

  6. Development of a refined tenocyte differentiation culture technique for tendon tissue engineering.

    Science.gov (United States)

    Qiu, Yiwei; Wang, Xiao; Zhang, Yaonan; Rout, Raj; Carr, Andrew J; Zhu, Liwei; Xia, Zhidao; Sabokbar, Afsie

    2013-01-01

    We have established that human tenocytes can differentiate in the absence of exogenous fetal bovine serum (FBS) but in the presence of insulin-like growth factor-1 (IGF-1) and transforming growth factor-β3 (TGF-β3). The extent of tenocyte differentiation was assessed by examining cell survival, collagen synthesis, cell morphology and expression of tenocyte differentiation markers such as scleraxis (Scx), tenomodulin (Tnmd), collagen type I (Col-I) and decorin (Dcn). Our results indicate that 50 ng/ml IGF-1 and 10 ng/ml TGF-β3 (in the absence of FBS) were capable of maintaining in vitro human tenocyte survival in 14-day cultures. The extent of collagen synthesis and messenger ribonucleic acid expression of Scx, Tnmd, Col-I and Dcn were significantly upregulated in response to IGF-1 and TGF-β3. These findings have shown for the first time that human tenocytes can be maintained in long-term culture, in serum-free conditions, making this approach a suitable one for the purpose of tendon tissue engineering. Copyright © 2012 S. Karger AG, Basel.

  7. Culture of equine bone marrow mononuclear fraction and adipose tissue-derived stromal vascular fraction cells in different media

    Directory of Open Access Journals (Sweden)

    Gesiane Ribeiro

    2013-12-01

    Full Text Available The objective of this study was to evaluate the culture of equine bone marrow mononuclear fraction and adipose tissue - derived stromal vascular fraction cells in two different cell culture media. Five adult horses were submitted to bone marrow aspiration from the sternum, and then from the adipose tissue of the gluteal region near the base of the tail. Mononuclear fraction and stromal vascular fraction were isolated from the samples and cultivated in DMEM medium supplemented with 10% fetal bovine serum or in AIM-V medium. The cultures were observed once a week with an inverted microscope, to perform a qualitative analysis of the morphology of the cells as well as the general appearance of the cell culture. Colony-forming units (CFU were counted on days 5, 15 and 25 of cell culture. During the first week of culture, differences were observed between the samples from the same source maintained in different culture media. The number of colonies was significantly higher in samples of bone marrow in relation to samples of adipose tissue.

  8. Responses of macropropagated Musa plantlets to transient moisture ...

    African Journals Online (AJOL)

    Moisture stress causes serious devastating effects on crops ranging from retarded growth, tissue desiccation, and severe yield drop to outright death. In this study, the efficacy of six nursery substrates formulated in varying volume ratios of composted plantain fruit/bunch wastes (PFW), sawdust (SD), poultry manure (PM) and ...

  9. High frequency callus induction and plantlet regeneration from ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-05-04

    May 4, 2009 ... The identification of genetically superior strains of medicinal and aromatic plants is a high priority. However, there is no report on the identification of high content strains of P. kurroa neither from the natural habitat nor induced through in vitro cell cultures. Although conditions for micropropagation has been ...

  10. [Investigations on the relation between differentiation and the composition of soluble protein of tissue cultures and leaves of daucus carota].

    Science.gov (United States)

    Neumann, K H; Pauler, B

    1969-12-01

    The correlations between differentiation, the amino acid composition of total protein and of soluble protein, and the disc electrophoretic distribution of soluble protein of carrot tissue cultures and of carrot plants (leaves) were studied. In spite of pronounced and characteristic changes in the electrophoretic distribution of the components of soluble protein in various developmental stages of both bioassays, no significant differences in the amino acid composition of protein were observed. With progressive development of whole carrot plants and of carrot tissue cultures, the number of protein bands on disc electropherogramms increased.

  11. Hormonal control of root development on epiphyllous plantlets of Bryophyllum (Kalanchoe) marnierianum: role of auxin and ethylene.

    Science.gov (United States)

    Kulka, Richard G

    2008-01-01

    Epiphyllous plantlets develop on leaves of Bryophyllum marnierianum when they are excised from the plant. Shortly after leaf excision, plantlet shoots develop from primordia located near the leaf margin. After the shoots have enlarged for several days, roots appear at their base. In this investigation, factors regulating plantlet root development were studied. The auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA) abolished root formation without markedly affecting shoot growth. This suggested that auxin transport from the plantlet shoot induces root development. Excision of plantlet apical buds inhibits root development. Application of indole-3-acetic acid (IAA) in lanolin at the site of the apical buds restores root outgrowth. Naphthalene acetic acid (NAA), a synthetic auxin, reverses TIBA inhibition of plantlet root emergence on leaf explants. Both of these observations support the hypothesis that auxin, produced by the plantlet, induces root development. Exogenous ethylene causes precocious root development several days before that of a control without hormone. Ethylene treatment cannot bypass the TIBA block of root formation. Therefore, ethylene does not act downstream of auxin in root induction. However, ethylene amplifies the effects of low concentrations of NAA, which in the absence of ethylene do not induce roots. Ag(2)S(2)O(3), an ethylene blocker, and CoCl(2), an ethylene synthesis inhibitor, do not abolish plantlet root development. It is therefore unlikely that ethylene is essential for root formation. Taken together, the experiments suggest that roots develop when auxin transport from the shoot reaches a certain threshold. Ethylene may augment this effect by lowering the threshold and may come into play when the parent leaf senesces.

  12. Prolonged hypoxic culture and trypsinization increase the pro-angiogenic potential of human adipose tissue-derived stem cells

    DEFF Research Database (Denmark)

    Rasmussen, Jeppe Grøndahl; Frøbert, Ole; Pilgaard, Linda

    2011-01-01

    Transplantation of mesenchymal stromal cells (MSC), including adipose tissue-derived stem cells (ASC), is a promising option in the treatment of vascular disease. Short-term hypoxic culture of MSC augments secretion of anti-apoptotic and angiogenic cytokines. We hypothesized that prolonged hypoxic...... (1% and 5% oxygen) culture and trypsinization would augment ASC expression of anti-apoptotic and angiogenic cytokines and increase the angiogenic potential of ASC-conditioned media....

  13. Inhibition of phenylpropanoid biosynthesis in Artemisia annua L.: a novel approach to reduce oxidative browning in plant tissue culture.

    Directory of Open Access Journals (Sweden)

    Andrew Maxwell Phineas Jones

    Full Text Available Oxidative browning is a common and often severe problem in plant tissue culture systems caused by the accumulation and oxidation of phenolic compounds. The current study was conducted to investigate a novel preventative approach to address this problem by inhibiting the activity of the phenylalanine ammonia lyase enzyme (PAL, thereby reducing the biosynthesis of phenolic compounds. This was accomplished by incorporating 2-aminoindane-2-phosphonic acid (AIP, a competitive PAL inhibitor, into culture media of Artemisia annua as a model system. Addition of AIP into culture media resulted in significant reductions in visual tissue browning, a reduction in total phenol content, as well as absorbance and autoflourescence of tissue extracts. Reduced tissue browning was accompanied with a significant increase in growth on cytokinin based medium. Microscopic observations demonstrated that phenolic compounds accumulated in discrete cells and that these cells were more prevalent in brown tissue. These cells were highly plasmolyzed and often ruptured during examination, demonstrating a mechanism in which phenolics are released into media in this system. These data indicate that inhibiting phenylpropanoid biosynthesis with AIP is an effective approach to reduce tissue browning in A. annua. Additional experiments with Ulmus americana and Acer saccharum indicate this approach is effective in many species and it could have a wide application in systems where oxidative browning restricts the development of biotechnologies.

  14. Inhibition of phenylpropanoid biosynthesis in Artemisia annua L.: a novel approach to reduce oxidative browning in plant tissue culture.

    Science.gov (United States)

    Jones, Andrew Maxwell Phineas; Saxena, Praveen Kumar

    2013-01-01

    Oxidative browning is a common and often severe problem in plant tissue culture systems caused by the accumulation and oxidation of phenolic compounds. The current study was conducted to investigate a novel preventative approach to address this problem by inhibiting the activity of the phenylalanine ammonia lyase enzyme (PAL), thereby reducing the biosynthesis of phenolic compounds. This was accomplished by incorporating 2-aminoindane-2-phosphonic acid (AIP), a competitive PAL inhibitor, into culture media of Artemisia annua as a model system. Addition of AIP into culture media resulted in significant reductions in visual tissue browning, a reduction in total phenol content, as well as absorbance and autoflourescence of tissue extracts. Reduced tissue browning was accompanied with a significant increase in growth on cytokinin based medium. Microscopic observations demonstrated that phenolic compounds accumulated in discrete cells and that these cells were more prevalent in brown tissue. These cells were highly plasmolyzed and often ruptured during examination, demonstrating a mechanism in which phenolics are released into media in this system. These data indicate that inhibiting phenylpropanoid biosynthesis with AIP is an effective approach to reduce tissue browning in A. annua. Additional experiments with Ulmus americana and Acer saccharum indicate this approach is effective in many species and it could have a wide application in systems where oxidative browning restricts the development of biotechnologies.

  15. EFFECTS INDUCED BY DIETHYL SULPHATE ON SOME CYTOGENETICAL PARAMETERS AND LENGTH GROWTH OF HEMP PLANTLETS

    Directory of Open Access Journals (Sweden)

    Elena Truta

    2007-08-01

    Full Text Available The hemp seeds were treated with diethyl sulphate, in four concentrations (0.1%, 0.25%, 0.5%, and 1% and in two variants of alkylant exposure (3 and 6 hours. The length growth of plantlets, mitotic index and frequency of chromosomal aberrations were the analyzed parameters. Significant modifications were obtained at the level of parameters in variants treated with DES, comparatively with control.

  16. Key KdSOC1 gene expression profiles during plantlet morphogenesis under hormone, photoperiod, and drought treatments.

    Science.gov (United States)

    Liu, C; Zhu, C; Zeng, H M

    2016-02-11

    Kalanchoe daigremontiana utilizes plantlet formation between its zigzag leaf margins as its method of asexual reproduction. In this study, K. daigremontiana SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (KdSOC1), a key intermediate in the transition from vegetative to asexual growth, was cloned. Furthermore, its expression profiles during plantlet formation under different environmental and hormone induction conditions were analyzed. The full-KdSOC1 cDNA sequence length was 1410 bp with 70% shared homology with Carya cathayensis SOC1. The conserved domain search of KdSOC1 showed the absence of I and C domains, which might indicate novel biological functions in K. daigremontiana. The full-KdSOC1 promoter sequence was 1401 bp long and contained multiple-hormone-responsive cis-acting elements. Hormone induction assays showed that gibberellins and salicylic acid mainly regulated KdSOC1 expression. The swift change from low to high KdSOC1 expression levels during long-day induction was accompanied by the rapid emergence of plantlets. Drought stress stimulated KdSOC1 expression in leaves both with and without plantlet formation. Together, the results suggested that KdSOC1 was closely involved in environmental stimulation signal perception and the transduction of K. daigremontiana plantlet formation. Therefore, future identification of KdSOC1 functions might reveal key information that will help elucidate the transition network between embryogenesis and organogenesis during plantlet formation.

  17. Characterization of the Embryogenic Tissue of the Norway Spruce Including a Transition Layer between the Tissue and the Culture Medium by Magnetic Resonance Imaging

    Science.gov (United States)

    Kořínek, R.; Mikulka, J.; Hřib, J.; Hudec, J.; Havel, L.; Bartušek, K.

    2017-02-01

    The paper describes the visualization of the cells (ESEs) and mucilage (ECMSN) in an embryogenic tissue via magnetic resonance imaging (MRI) relaxometry measurement combined with the subsequent multi-parametric segmentation. The computed relaxometry maps T1 and T2 show a thin layer (transition layer) between the culture medium and the embryogenic tissue. The ESEs, mucilage, and transition layer differ in their relaxation times T1 and T2; thus, these times can be used to characterize the individual parts within the embryogenic tissue. The observed mean values of the relaxation times T1 and T2 of the ESEs, mucilage, and transition layer are as follows: 1469 ± 324 and 53 ± 10 ms, 1784 ± 124 and 74 ± 8 ms, 929 ± 164 and 32 ± 4.7 ms, respectively. The multi-parametric segmentation exploiting the T1 and T2 relaxation times as a classifier shows the distribution of the ESEs and mucilage within the embryogenic tissue. The discussed T1 and T2 indicators can be utilized to characterize both the growth-related changes in an embryogenic tissue and the effect of biotic/abiotic stresses, thus potentially becoming a distinctive indicator of the state of any examined embryogenic tissue.

  18. Comparative physiological and proteomic analyses of poplar (Populus yunnanensis plantlets exposed to high temperature and drought.

    Directory of Open Access Journals (Sweden)

    Xiong Li

    Full Text Available Plantlets of Populus yunnanensis Dode were examined in a greenhouse for 48 h to analyze their physiological and proteomic responses to sustained heat, drought, and combined heat and drought. Compared with the application of a single stress, simultaneous treatment with both stresses damaged the plantlets more heavily. The plantlets experienced two apparent response stages under sustained heat and drought. During the first stage, malondialdehyde and reactive oxygen species (ROS contents were induced by heat, but many protective substances, including antioxidant enzymes, proline, abscisic acid (ABA, dehydrin, and small heat shock proteins (sHSPs, were also stimulated. The plants thus actively defended themselves against stress and exhibited few pathological morphological features, most likely because a new cellular homeostasis was established through the collaborative operation of physiological and proteomic responses. During the second stage, ROS homeostasis was overwhelmed by substantial ROS production and a sharp decline in antioxidant enzyme activities, while the synthesis of some protective elements, such as proline and ABA, was suppressed. As a result, photosynthetic levels in P. yunnanensis decreased sharply and buds began to die, despite continued accumulation of sHSPs and dehydrin. This study supplies important information about the effects of extreme abiotic environments on woody plants.

  19. Varietal Evaluation of Potato Microtuber and Plantlet in Seed Tuber Production

    Directory of Open Access Journals (Sweden)

    Md. Sadek Hossain

    2017-01-01

    Full Text Available Diamant, Asterix, and Granola varieties differed significantly in foliage coverage, plant height, and yield. They produced lower graded minituber (67.62%, 78.16% ha−1, and 66.27% of Asterix, Granola, and Diamant varieties, resp. as per seed rule of the National Seed Board of Bangladesh, while foliage coverage (74.38% was the maximum in Diamant. Microtuber in field condition showed the maximum survivability, plant height, foliage coverage, number of stems plant−1, and SPAD value as well as yield of minituber compared to plantlet. On the contrary, microtuber derived plants of the three varieties gave the maximum yield (20.49 t ha−1, 19.12 t/ha−1, and 19.98 t ha−1 of Asterix, Granola, and Diamant varieties, resp. and it was the minimum in plants of plantlets derived from all varieties (9.50 t ha−1, 7.88 t ha−1, and 9.70 t ha−1 of Asterix, Granola, and Diamant varieties, resp.. Microtuber derived plants produced a minimum percentage of <28 mm size of minituber compared to plantlet derived plants in case of all varieties.

  20. Signal mediators at induction of heat resistance of wheat plantlets by short-term heating

    Directory of Open Access Journals (Sweden)

    Yu. V. Karpets

    2015-12-01

    Full Text Available The effects of functional interplay of calcium ions, reactive oxygen species (ROS and nitric oxide (NO in the cells of wheat plantlets roots (Triticum aestivum L. at the induction of their heat resistance by a short-term influence of hyperthermia (heating at the temperature of 42 °С during 1 minute have been investigated. The transitional increase of NO and H2O2 content, invoked by heating, was suppressed by the treatment of plantlets with the antagonists of calcium EGTA (chelator of exocellular calcium, lanthanum chloride (blocker of calcium channels of various types and neomycin (inhibitor of phosphatidylinositol-dependent phospholipase C. The rise of hydrogen peroxide content, caused by hardening, was partially suppressed by the action of inhibitors of nitrate reductase (sodium wolframate and NO-synthase (NG-nitro-L-arginine methyl ester – L-NAME, and the increasing of nitric oxide content was suppressed by the treatment of plants with the antioxidant ionol and with the scavenger of hydrogen peroxide (dimethylthiourea. These compounds and antagonists of calcium also partially removed the effect of the rise of plantlets’ heat resistance, invoked by hardening heating. The conclusion on calcium’s role in the activation of enzymatic systems, generating reactive oxygen species and nitric oxide, and on the functional interplay of these signal mediators at the induction of heat resistance of plantlets by hardening heating is made.

  1. [Regulating role of various amino acids in development of apoptosis in organotypic culture of the nervous and lymphoid tissue].

    Science.gov (United States)

    Chalisova, N I; Pennijainen, V A; Haase, G

    2002-05-01

    The effect of aminoacids L-arginin, L-lysine, L-asparagin was investigated in organotypic tissue culture of brain cortex and spleen of 1-day old rats. The aminoacids in concentrations 0.05 and 0.1 ng/ml are active, inducing a less intensive growth zone as compared to the control, excluding the effect of asparagin on lymphoid tissue. Method of fluorescent staining shows a negative correlation between growth zone size and apoptotic cell number. The nerve and lymphoid tissue express apoptosis in response to aminoacids.

  2. The gene expression profile of non-cultured, highly purified human adipose tissue pericytes: Transcriptomic evidence that pericytes are stem cells in human adipose tissue.

    Science.gov (United States)

    da Silva Meirelles, Lindolfo; de Deus Wagatsuma, Virgínia Mara; Malta, Tathiane Maistro; Bonini Palma, Patrícia Viana; Araújo, Amélia Goes; Panepucci, Rodrigo Alexandre; Silva, Wilson Araújo; Kashima, Simone; Covas, Dimas Tadeu

    2016-12-10

    Pericytes (PCs) are a subset of perivascular cells that can give rise to mesenchymal stromal cells (MSCs) when culture-expanded, and are postulated to give rise to MSC-like cells during tissue repair in vivo. PCs have been suggested to behave as stem cells (SCs) in situ in animal models, although evidence for this role in humans is lacking. Here, we analyzed the transcriptomes of highly purified, non-cultured adipose tissue (AT)-derived PCs (ATPCs) to detect gene expression changes that occur as they acquire MSC characteristics in vitro, and evaluated the hypothesis that human ATPCs exhibit a gene expression profile compatible with an AT SC phenotype. The results showed ATPCs are non-proliferative and express genes characteristic not only of PCs, but also of AT stem/progenitor cells. Additional analyses defined a gene expression signature for ATPCs, and revealed putative novel ATPC markers. Almost all AT stem/progenitor cell genes differentially expressed by ATPCs were not expressed by ATMSCs or culture-expanded ATPCs. Genes expressed by ATMSCs but not by ATPCs were also identified. These findings strengthen the hypothesis that PCs are SCs in vascularized tissues, highlight gene expression changes they undergo as they assume an MSC phenotype, and provide new insights into PC biology. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Biomimetic synthesis of antimicrobial silver nanoparticles using in vitro-propagated plantlets of a medicinally important endangered species: Phlomis bracteosa

    Directory of Open Access Journals (Sweden)

    Anjum S

    2016-04-01

    Full Text Available Sumaira Anjum, Bilal Haider Abbasi Department of Biotechnology, Quaid-i-Azam University, Islamabad, Pakistan Abstract: In vitro-derived cultures of plants offer a great potential for rapid biosynthesis of chemical-free antimicrobial silver nanoparticles (AgNPs by enhancing their phytochemical reducing potential. Here, we developed an efficient protocol for in vitro micropropagation of a high-value endangered medicinal plant species, Phlomis bracteosa, in order to explore its biogenic potential in biomimetic synthesis of antimicrobial AgNPs. Murashige and Skoog medium supplemented with 2.0 mg/L thidiazuron was found to be more efficient in inducing optimum in vitro shoot regeneration (78%±4.09%, and 2.0 mg/L indole-3-butyric acid was used for maximum root induction (86%±4.457%. Antimicrobial AgNPs were successfully synthesized by using aqueous extract (rich in total phenolics and flavonoids content of in vitro derived plantlets of P. bracteosa. Ultraviolet–visible spectroscopy of synthesized AgNPs showed characteristic surface plasmon band in the range of 420–429 nm. The crystallinity, size, and shape of the AgNPs were characterized by X-ray diffraction and scanning electron microscopy. Face-centered cubic AgNPs of almost uniform spherical size (22.41 nm were synthesized within a short time (1 hour at room temperature. Fourier-transform infrared spectroscopy revealed that the polyphenols were mainly responsible for reduction and capping of synthesized AgNPs. Energy dispersive X-ray analysis further endorsed the presence of elemental silver in synthesized AgNPs. These biosynthesized AgNPs displayed significantly higher bactericidal activity against multiple drug-resistant human pathogens. The present work highlighted the potent role of in vitro-derived plantlets of P. bracteosa for feasible biosynthesis of antimicrobial AgNPs, which can be used as nanomedicines in many biomedical applications. Keywords: silver nanoparticles, Phlomis

  4. Structural interaction between GFP-labeled diazotrophic endophytic bacterium Herbaspirillum seropedicae RAM10 and pineapple plantlets 'Vitória'

    Directory of Open Access Journals (Sweden)

    Lílian Estrela Borges Baldotto

    2011-03-01

    Full Text Available The events involved in the structural interaction between the diazotrophic endophytic bacterium Herbaspirillum seropedicae, strain RAM10, labeled with green fluorescent protein, and pineapple plantlets 'Vitória' were evaluated by means of bright-field and fluorescence microscopy, combined with scanning electron microscopy for 28 days after inoculation. After 6 hours of inoculation, H. seropedicae was already adhered to the roots, colonizing mainly root hair surface and bases, followed by epidermal cell wall junctions. Bacteria adherence in the initial periods occurred mainly in the form of solitary cells and small aggregates with pleomorphic cells. Bacteria infection of root tissue occurred through the cavities caused by the disruption of epidermal cells during the emergence of lateral roots and the endophytic establishment by the colonization of intercellular spaces of the cortical parenchyma. Moreover, within 1 day after inoculation the bacteria were colonizing the shoots. In this region, the preferred sites of epiphytic colonization were epidermal cell wall junctions, peltate scutiform trichomes and non-glandular trichomes. Subsequently, the bacteria occupied the outer periclinal walls of epidermal cells and stomata. The penetration into the shoot occurred passively through stoma aperture followed by the endophytic establishment on the substomatal chambers and spread to the intercellular spaces of spongy chlorenchyma. After 21 days of inoculation, bacterial biofilm were seen at the root hair base and on epidermal cell wall surface of root and leaf, also confirming the epiphytic nature of H. seropedicae.

  5. Glucocorticoids affect 24 h clock genes expression in human adipose tissue explant cultures.

    Directory of Open Access Journals (Sweden)

    Purificación Gómez-Abellán

    Full Text Available to examine firstly whether CLOCK exhibits a circadian expression in human visceral (V and subcutaneous (S adipose tissue (AT in vitro as compared with BMAL1 and PER2, and secondly to investigate the possible effect of the glucocorticoid analogue dexamethasone (DEX on positive and negative clock genes expression.VAT and SAT biopsies were obtained from morbid obese women (body mass index ≥ 40 kg/m(2 (n = 6. In order to investigate rhythmic expression pattern of clock genes and the effect of DEX on CLOCK, PER2 and BMAL1 expression, control AT (without DEX and AT explants treated with DEX (2 hours were cultured during 24 h and gene expression was analyzed at the following times: 10:00 h, 14:00 h, 18:00 h, 22:00 h, 02:00 h and 06:00 h, using qRT-PCR.CLOCK, BMAL1 and PER2 expression exhibited circadian patterns in both VAT and SAT explants that were adjusted to a typical 24 h sinusoidal curve. PER2 expression (negative element was in antiphase with respect to CLOCK and in phase with BMAL1 expression (both positive elements in the SAT (situation not present in VAT. A marked effect of DEX exposure on both positive and negative clock genes expression patterns was observed. Indeed, DEX treatment modified the rhythmicity pattern towards altered patterns with a period lower than 24 hours in all genes and in both tissues.24 h patterns in CLOCK and BMAL1 (positive clock elements and PER2 (negative element mRNA levels were observed in human adipose explants. These patterns were altered by dexamethasone exposure.

  6. Glucocorticoids affect 24 h clock genes expression in human adipose tissue explant cultures.

    Science.gov (United States)

    Gómez-Abellán, Purificación; Díez-Noguera, Antoni; Madrid, Juan A; Luján, Juan A; Ordovás, José M; Garaulet, Marta

    2012-01-01

    to examine firstly whether CLOCK exhibits a circadian expression in human visceral (V) and subcutaneous (S) adipose tissue (AT) in vitro as compared with BMAL1 and PER2, and secondly to investigate the possible effect of the glucocorticoid analogue dexamethasone (DEX) on positive and negative clock genes expression. VAT and SAT biopsies were obtained from morbid obese women (body mass index ≥ 40 kg/m(2)) (n = 6). In order to investigate rhythmic expression pattern of clock genes and the effect of DEX on CLOCK, PER2 and BMAL1 expression, control AT (without DEX) and AT explants treated with DEX (2 hours) were cultured during 24 h and gene expression was analyzed at the following times: 10:00 h, 14:00 h, 18:00 h, 22:00 h, 02:00 h and 06:00 h, using qRT-PCR. CLOCK, BMAL1 and PER2 expression exhibited circadian patterns in both VAT and SAT explants that were adjusted to a typical 24 h sinusoidal curve. PER2 expression (negative element) was in antiphase with respect to CLOCK and in phase with BMAL1 expression (both positive elements) in the SAT (situation not present in VAT). A marked effect of DEX exposure on both positive and negative clock genes expression patterns was observed. Indeed, DEX treatment modified the rhythmicity pattern towards altered patterns with a period lower than 24 hours in all genes and in both tissues. 24 h patterns in CLOCK and BMAL1 (positive clock elements) and PER2 (negative element) mRNA levels were observed in human adipose explants. These patterns were altered by dexamethasone exposure.

  7. Oxygen tension and formation of cervical-like tissue in two-dimensional and three-dimensional culture.

    Science.gov (United States)

    House, Michael; Daniel, Jennifer; Elstad, Kirigin; Socrate, Simona; Kaplan, David L

    2012-03-01

    Cervical dysfunction contributes to a significant number of preterm births and is a common cause of morbidity and mortality in newborn infants. Cervical dysfunction is related to weakened load bearing properties of the collagen-rich cervical stroma. However, the mechanisms responsible for cervical collagen changes during pregnancy are not well defined. It is known that blood flow and oxygen tension significantly increase in reproductive tissues during pregnancy. To examine the effect of oxygen tension, a key mediator of tissue homeostasis, on the formation of cervical-like tissue in vitro, we grew primary human cervical cells in both two-dimensional (2D) and three-dimensional (3D) culture systems at 5% and 20% oxygen. Immunofluorescence studies revealed a stable fibroblast phenotype across six passages in all subjects studied (n=5). In 2D culture for 2 weeks, 20% oxygen was associated with significantly increased collagen gene expression (p<0.01), increased tissue wet weight (p<0.01), and increased collagen concentration (p=0.046). 3D cultures could be followed for significantly longer time frames than 2D cultures (12 weeks vs. 2 weeks). In contrast to 2D cultures, 20% oxygen in 3D cultures was associated with decreased collagen concentration (p<0.01) and unchanged collagen gene expression, which is similar to cervical collagen changes seen during pregnancy. We infer that 3D culture is more relevant for studying cervical collagen changes in vitro. The data suggest that increased oxygen tension may be related to significant cervical collagen changes seen in pregnancy.

  8. A 3D epithelial-mesenchymal co-culture model of human bronchial tissue recapitulates multiple features of airway tissue remodeling by TGF-β1 treatment.

    Science.gov (United States)

    Ishikawa, Shinkichi; Ishimori, Kanae; Ito, Shigeaki

    2017-11-22

    The collagen gel contraction assay measures gel size to assess the contraction of cells embedded in collagen gel matrices. Using the assay with lung fibroblasts is useful in studying the lung tissue remodeling process in wound healing and disease development. However, the involvement of bronchial epithelial cells in this process should also be investigated. We applied a layer of mucociliary differentiated bronchial epithelial cells onto collagen gel matrices with lung fibroblasts. This co-culture model enables direct contact between epithelial and mesenchymal cells. We stimulated the culture with transforming growth factor (TGF) β1 as an inducer of tissue remodeling for 21 days, and measured gel size, histological changes, and expression of factors related to extracellular matrix homeostasis. TGF-β1 exerted a concentration-dependent effect on collagen gel contraction and on contractile myofibroblasts in the mesenchymal collagen layer. TGF-β1 also induced expression of the mesenchymal marker vimentin in the basal layer of the epithelium, suggesting the induction of epithelial-mesenchymal transition. In addition, the expression of various genes encoding extracellular matrix proteins was upregulated. Fibrotic tenascin-C accumulated in the sub-epithelial region of the co-culture model. Our findings indicate that TGF-β1 can affect both epithelial and mesenchymal cells, and induce gel contraction and structural changes. Our novel in vitro co-culture model will be a useful tool for investigating the roles of epithelial cells, fibroblasts, and their interactions in the airway remodeling process.

  9. Investigation of the in vitro culture process for skeletal-tissue-engineered constructs using computational fluid dynamics and experimental methods.

    Science.gov (United States)

    Hossain, Md Shakhawath; Chen, X B; Bergstrom, D J

    2012-12-01

    The in vitro culture process via bioreactors is critical to create tissue-engineered constructs (TECs) to repair or replace the damaged tissues/organs in various engineered applications. In the past, the TEC culture process was typically treated as a black box and performed on the basis of trial and error. Recently, computational fluid dynamics (CFD) has demonstrated its potential to analyze the fluid flow inside and around the TECs, therefore, being able to provide insight into the culture process, such as information on the velocity field and shear stress distribution that can significantly affect such cellular activities as cell viability and proliferation during the culture process. This paper briefly reviews the CFD and experimental methods used to investigate the in vitro culture process of skeletal-type TECs in bioreactors, where mechanical deformation of the TEC can be ignored. Specifically, this paper presents CFD modeling approaches for the analysis of the velocity and shear stress fields, mass transfer, and cell growth during the culture process and also describes various particle image velocimetry (PIV) based experimental methods to measure the velocity and shear stress in the in vitro culture process. Some key issues and challenges are also identified and discussed along with recommendations for future research.

  10. Characterization of aldehyde dehydrogenase isozymes in ovarian cancer tissues and sphere cultures

    International Nuclear Information System (INIS)

    Saw, Yu-Ting; Thompson, David; Vasiliou, Vasilis; Berkowitz, Ross S; Ng, Shu-Wing; Yang, Junzheng; Ng, Shu-Kay; Liu, Shubai; Singh, Surendra; Singh, Margit; Welch, William R; Tsuda, Hiroshi; Fong, Wing-Ping

    2012-01-01

    Aldehyde dehydrogenases belong to a superfamily of detoxifying enzymes that protect cells from carcinogenic aldehydes. Of the superfamily, ALDH1A1 has gained most attention because current studies have shown that its expression is associated with human cancer stem cells. However, ALDH1A1 is only one of the 19 human ALDH subfamilies currently known. The purpose of the present study was to determine if the expression and activities of other major ALDH isozymes are associated with human ovarian cancer and ovarian cancer sphere cultures. Immunohistochemistry was used to delineate ALDH isozyme localization in clinical ovarian tissues. Western Blot analyses were performed on lysates prepared from cancer cell lines and ovarian cancer spheres to confirm the immunohistochemistry findings. Quantitative reverse transcription-polymerase chain reactions were used to measure the mRNA expression levels. The Aldefluor® assay was used to measure ALDH activity in cancer cells from the four tumor subtypes. Immunohistochemical staining showed significant overexpression of ALDH1A3, ALDH3A2, and ALDH7A1 isozymes in ovarian tumors relative to normal ovarian tissues. The expression and activity of ALDH1A1 is tumor type-dependent, as seen from immunohistochemisty, Western blot analysis, and the Aldefluor® assay. The expression was elevated in the mucinous and endometrioid ovarian epithelial tumors than in serous and clear cell tumors. In some serous and most clear cell tumors, ALDH1A1 expression was found in the stromal fibroblasts. RNA expression of all studied ALDH isozymes also showed higher expression in endometrioid and mucinous tumors than in the serous and clear cell subtypes. The expression of ALDH enzymes showed tumor type-dependent induction in ovarian cancer cells growing as sphere suspensions in serum-free medium. The results of our study indicate that ALDH enzyme expression and activity may be associated with specific cell types in ovarian tumor tissues and vary according to

  11. Direct long-term effect of hydrocortisone on insulin and glucagon release from mouse pancreatic islets in tissue culture

    DEFF Research Database (Denmark)

    Brunstedt, J; Nielsen, Jens Høiriis

    1981-01-01

    The effects of glucocorticoids on the pancreatic endocrine function was studied in isolated mouse pancreatic islets maintained in tissue culture for 1 to 3 weeks. Following culture for 2 week without corticoid supplement acute experiments with hydrocortisone showed no significant effect...... on the glucose-induced insulin release at 10(-8) to 10(-5) mol/l hydrocortisone. When, however, the islets were cultured in the presence of hydrocortisone, there was an increased insulin release to the medium in a dose-dependent manner, with the maximal effect at 10(-7) mol/l hydrocortisone. The release...... of glucagon to the medium was not affected to the same degree, but showed a slight inhibition at increasing concentrations of hydrocortisone. Short-term experiments after the culture period showed that islets cultured for 3 weeks in the presence of 10(-7) to 10(-5) mol/l hydrocortisone had an enhanced insulin...

  12. Cytoenzymology and 3H-thymidine uptake of retro-ocular connective tissue cultures in experimental endocrino-exophthalmos.

    Science.gov (United States)

    Vaida, E; Petrescu, R; Ghinea, E; Stefaneanu, L

    1976-01-01

    The in vitro retro-ocular connective tissue cultures from guinea pigs with endocrine exophthalmos were studied before and after retro-ocular treatment with cortisol and hyaluronidase. Both cortisol and hyaluronidase inhibited the cell proliferation, the cytoenzymic activities of oxydoreductases, the 3H-thymidine uptake, the number of mitoses and the protein content of cultivated cells.

  13. Evaluation of viability and proliferative activity of human urothelial cells cultured onto xenogenic tissue-engineered extracellular matrices.

    LENUS (Irish Health Repository)

    Davis, Niall F

    2011-04-01

    To evaluate the viability and proliferative activity of human urothelial cells (HUCs) cultured on tissue-engineered extracellular matrix scaffolds and to assess the potential of extracellular matrixes to support the growth of HUCs in their expected in vivo urine environment.

  14. Prolonged hypoxic culture and trypsinization increase the pro-angiogenic potential of human adipose tissue-derived stem cells

    DEFF Research Database (Denmark)

    Rasmussen, Jeppe Grøndahl; Frøbert, Ole; Pilgaard, Linda

    2011-01-01

    Transplantation of mesenchymal stromal cells (MSC), including adipose tissue-derived stem cells (ASC), is a promising option in the treatment of vascular disease. Short-term hypoxic culture of MSC augments secretion of anti-apoptotic and angiogenic cytokines. We hypothesized that prolonged hypoxic...

  15. Root system architecture in Arabidopsis grown in culture is regulated by sucrose uptake in the aerial tissues.

    Science.gov (United States)

    Macgregor, Dana R; Deak, Karen I; Ingram, Paul A; Malamy, Jocelyn E

    2008-10-01

    This article presents a detailed model for the regulation of lateral root formation in Arabidopsis thaliana seedlings grown in culture. We demonstrate that direct contact between the aerial tissues and sucrose in the growth media is necessary and sufficient to promote emergence of lateral root primordia from the parent root. Mild osmotic stress is perceived by the root, which then sends an abscisic acid-dependent signal that causes a decrease in the permeability of aerial tissues; this reduces uptake of sucrose from the culture media, which leads to a repression of lateral root formation. Osmotic repression of lateral root formation in culture can be overcome by mutations that cause the cuticle of a plant's aerial tissues to become more permeable. Indeed, we report here that the previously described lateral root development2 mutant overcomes osmotic repression of lateral root formation because of a point mutation in Long Chain Acyl-CoA Synthetase2, a gene essential for cutin biosynthesis. Together, our findings (1) impact the interpretation of experiments that use Arabidopsis grown in culture to study root system architecture; (2) identify sucrose as an unexpected regulator of lateral root formation; (3) demonstrate mechanisms by which roots communicate information to aerial tissues and receive information in turn; and (4) provide insights into the regulatory pathways that allow plants to be developmentally plastic while preserving the essential balance between aboveground and belowground organs.

  16. Characterization of connective tissue growth factor expression in primary cultures of human tubular epithelial cells: modulation by hypoxia

    NARCIS (Netherlands)

    Kroening, Sven; Neubauer, Emily; Wullich, Bernd; Aten, Jan; Goppelt-Struebe, Margarete

    2010-01-01

    Kroening S, Neubauer E, Wullich B, Aten J, Goppelt-Struebe M. Characterization of connective tissue growth factor expression in primary cultures of human tubular epithelial cells: modulation by hypoxia. Am J Physiol Renal Physiol 298:F796-F806, 2010. First published December 23, 2009;

  17. Comparison of Biocompatibility and Adsorption Properties of Different Plastics for Advanced Microfluidic Cell and Tissue Culture Models

    NARCIS (Netherlands)

    van Midwoud, Paul M.; Janse, Arnout; Merema, M.T.; Groothuis, Geny M. M.; Verpoorte, Elisabeth

    2012-01-01

    Microfluidic technology is providing new routes toward advanced cell and tissue culture models to better understand human biology and disease. Many advanced devices have been made from poly(dimethylsiloxane) (PDMS) to enable experiments, for example, to study drug metabolism by use of precision cut

  18. Porous PEOT/PBT scaffolds for bone tissue engineering: preparation, characterization, and in vitro bone marrow cell culturing

    NARCIS (Netherlands)

    Claase, M.B.; Grijpma, Dirk W.; Mendes, S.C.; Mendes, Sandra C.; de Bruijn, Joost Dick; Feijen, Jan

    2003-01-01

    The preparation, characterization, and in vitro bone marrow cell culturing on porous PEOT/PBT copolymer scaffolds are described. These scaffolds are meant for use in bone tissue engineering. Previous research has shown that PEOT/PBT copolymers showed in vivo degradation, calcification, and bone

  19. Gamma irradiation as activator of antioxidant activity and essential oil contents in lavender (Lavandula multifida) plantlets

    International Nuclear Information System (INIS)

    El-Naggar, H.A.; Atallah, R.K.; Aly, A.A.; Maraei, R.W.

    2012-01-01

    This study was conducted to evaluate the stimulation effect of γ-irradiation on the chemical composition of essential oils, total phenolic compounds, flavonoid contents and antioxidant activities in lavender plantlets (Lavandula multifida) at three multiplication stages. Lavender plantlets were irradiated using different γ- irradiation dose levels (0.0, 5, 15, 30, 45, 60 and 75 Gy). After irradiation; plantlets were sectioned to start the multiplication stage (three subcultures). Increasing irradiation dose levels at multiplication stages significantly increased the total phenolic content and reached to the maximum increment at the dose level of 75 Gy (26.88 g/100 g DW) in zero time stage in comparison with the untreated plantlets (7.250 g/100 g DW). The highest content of flavonoids (21.50 g/100 g DW) was detected at dose level of 75 Gy at zero time stage (M0). The highest applied irradiation dose of 75 Gy gave the highest reducing power activity compared with control at zero time stage (M0). Scavenging activity by DPPH was increased gradually by increasing irradiation dose levels in all multiplication stages until the high dose of 75 Gy which gave the maximum scavenging activity (91.05%) in zero time stage. Also, there was a significant increase in antioxidant activity on linoleic acid system with increasing the dose of γ-irradiation level. The application of γ-irradiation at dose level of 15 Gy and M3 stage produced the highest value of essential oil content (0.12%), followed by 5 Gy treatments (0.082%). The most increased volatile oil compounds by γ-irradiation were; limonene which increased from 4.87% to 5.37% at 0.0 and 5 Gy, respectively and linalool increased from 86.07% to 91.5% at 0.0 and 15 Gy respectively. The present study suggests that γ-irradiation led to increase antioxidant activities of lavender plantlets by increasing the availability of free polyphenolic compounds and also the content of volatile oil. This shows that lavender plants may be

  20. Inhibitory effect of progesterone on cervical tissue formation in a three-dimensional culture system with human cervical fibroblasts.

    Science.gov (United States)

    House, Michael; Tadesse-Telila, Serkalem; Norwitz, Errol R; Socrate, Simona; Kaplan, David L

    2014-01-01

    Progesterone supplementation is recommended to prevent preterm birth in women with a short cervix, but the mechanism is unclear. We hypothesize that progesterone acts by altering the composition of the cervical extracellular matrix (ECM). We tested this hypothesis using human cervical fibroblasts in both two-dimensional (2D) and three-dimensional (3D) cultures. For 2D culture, cells were seeded in 6-well plates and cultured with media supplemented with estradiol (10(-8) M), progesterone (10(-7) or 10(-6) M), and vehicle. For 3D culture, the cells were cultured on a porous silk protein scaffold system. Progesterone and estrogen receptors were documented by immunohistochemistry and Western blot analysis. In both 2D and 3D cultures, decreased collagen synthesis was seen with increased progesterone concentration. Three-dimensional cultures could be maintained significantly longer than 2D cultures, and the morphology of 3D cultures appeared similar to native cervical tissue. Thus, further studies were performed in 3D culture. To determine the effect of progesterone concentration, the 3D scaffolds were cultured with estradiol (10(-8) M) and five conditions: vehicle; 10(-9), 10(-8), or 10(-7) M progesterone; or 10(-7) M progesterone plus 10(-6) M mifepristone. The highest progesterone concentration correlated with the least amount of collagen synthesis. Collagen synthesis progressively increased as progesterone concentration decreased. This effect was partially antagonized by mifepristone, suggesting the mechanism is mediated by the progesterone receptor. This hormonally responsive 3D culture system supports the hypothesis that progesterone has a direct effect on remodeling cervical ECM during pregnancy. The 3D culture system could be useful for studying the mechanism of progesterone effects on the cervix.

  1. Inhibitory Effect of Progesterone on Cervical Tissue Formation in a Three-Dimensional Culture System with Human Cervical Fibroblasts1

    Science.gov (United States)

    House, Michael; Tadesse-Telila, Serkalem; Norwitz, Errol R.; Socrate, Simona; Kaplan, David L.

    2013-01-01

    ABSTRACT Progesterone supplementation is recommended to prevent preterm birth in women with a short cervix, but the mechanism is unclear. We hypothesize that progesterone acts by altering the composition of the cervical extracellular matrix (ECM). We tested this hypothesis using human cervical fibroblasts in both two-dimensional (2D) and three-dimensional (3D) cultures. For 2D culture, cells were seeded in 6-well plates and cultured with media supplemented with estradiol (10−8 M), progesterone (10−7 or 10−6 M), and vehicle. For 3D culture, the cells were cultured on a porous silk protein scaffold system. Progesterone and estrogen receptors were documented by immunohistochemistry and Western blot analysis. In both 2D and 3D cultures, decreased collagen synthesis was seen with increased progesterone concentration. Three-dimensional cultures could be maintained significantly longer than 2D cultures, and the morphology of 3D cultures appeared similar to native cervical tissue. Thus, further studies were performed in 3D culture. To determine the effect of progesterone concentration, the 3D scaffolds were cultured with estradiol (10−8 M) and five conditions: vehicle; 10−9, 10−8, or 10−7 M progesterone; or 10−7 M progesterone plus 10−6 M mifepristone. The highest progesterone concentration correlated with the least amount of collagen synthesis. Collagen synthesis progressively increased as progesterone concentration decreased. This effect was partially antagonized by mifepristone, suggesting the mechanism is mediated by the progesterone receptor. This hormonally responsive 3D culture system supports the hypothesis that progesterone has a direct effect on remodeling cervical ECM during pregnancy. The 3D culture system could be useful for studying the mechanism of progesterone effects on the cervix. PMID:24285720

  2. Evaluation of Reference Genes for Quantitative Real-Time PCR in Oil Palm Elite Planting Materials Propagated by Tissue Culture

    Science.gov (United States)

    Chan, Pek-Lan; Rose, Ray J.; Abdul Murad, Abdul Munir; Zainal, Zamri; Leslie Low, Eng-Ti; Ooi, Leslie Cheng-Li; Ooi, Siew-Eng; Yahya, Suzaini; Singh, Rajinder

    2014-01-01

    Background The somatic embryogenesis tissue culture process has been utilized to propagate high yielding oil palm. Due to the low callogenesis and embryogenesis rates, molecular studies were initiated to identify genes regulating the process, and their expression levels are usually quantified using reverse transcription quantitative real-time PCR (RT-qPCR). With the recent release of oil palm genome sequences, it is crucial to establish a proper strategy for gene analysis using RT-qPCR. Selection of the most suitable reference genes should be performed for accurate quantification of gene expression levels. Results In this study, eight candidate reference genes selected from cDNA microarray study and literature review were evaluated comprehensively across 26 tissue culture samples using RT-qPCR. These samples were collected from two tissue culture lines and media treatments, which consisted of leaf explants cultures, callus and embryoids from consecutive developmental stages. Three statistical algorithms (geNorm, NormFinder and BestKeeper) confirmed that the expression stability of novel reference genes (pOP-EA01332, PD00380 and PD00569) outperformed classical housekeeping genes (GAPDH, NAD5, TUBULIN, UBIQUITIN and ACTIN). PD00380 and PD00569 were identified as the most stably expressed genes in total samples, MA2 and MA8 tissue culture lines. Their applicability to validate the expression profiles of a putative ethylene-responsive transcription factor 3-like gene demonstrated the importance of using the geometric mean of two genes for normalization. Conclusions Systematic selection of the most stably expressed reference genes for RT-qPCR was established in oil palm tissue culture samples. PD00380 and PD00569 were selected for accurate and reliable normalization of gene expression data from RT-qPCR. These data will be valuable to the research associated with the tissue culture process. Also, the method described here will facilitate the selection of appropriate

  3. Evaluation of reference genes for quantitative real-time PCR in oil palm elite planting materials propagated by tissue culture.

    Science.gov (United States)

    Chan, Pek-Lan; Rose, Ray J; Abdul Murad, Abdul Munir; Zainal, Zamri; Low, Eng-Ti Leslie; Ooi, Leslie Cheng-Li; Ooi, Siew-Eng; Yahya, Suzaini; Singh, Rajinder

    2014-01-01

    The somatic embryogenesis tissue culture process has been utilized to propagate high yielding oil palm. Due to the low callogenesis and embryogenesis rates, molecular studies were initiated to identify genes regulating the process, and their expression levels are usually quantified using reverse transcription quantitative real-time PCR (RT-qPCR). With the recent release of oil palm genome sequences, it is crucial to establish a proper strategy for gene analysis using RT-qPCR. Selection of the most suitable reference genes should be performed for accurate quantification of gene expression levels. In this study, eight candidate reference genes selected from cDNA microarray study and literature review were evaluated comprehensively across 26 tissue culture samples using RT-qPCR. These samples were collected from two tissue culture lines and media treatments, which consisted of leaf explants cultures, callus and embryoids from consecutive developmental stages. Three statistical algorithms (geNorm, NormFinder and BestKeeper) confirmed that the expression stability of novel reference genes (pOP-EA01332, PD00380 and PD00569) outperformed classical housekeeping genes (GAPDH, NAD5, TUBULIN, UBIQUITIN and ACTIN). PD00380 and PD00569 were identified as the most stably expressed genes in total samples, MA2 and MA8 tissue culture lines. Their applicability to validate the expression profiles of a putative ethylene-responsive transcription factor 3-like gene demonstrated the importance of using the geometric mean of two genes for normalization. Systematic selection of the most stably expressed reference genes for RT-qPCR was established in oil palm tissue culture samples. PD00380 and PD00569 were selected for accurate and reliable normalization of gene expression data from RT-qPCR. These data will be valuable to the research associated with the tissue culture process. Also, the method described here will facilitate the selection of appropriate reference genes in other oil palm

  4. Evaluation of reference genes for quantitative real-time PCR in oil palm elite planting materials propagated by tissue culture.

    Directory of Open Access Journals (Sweden)

    Pek-Lan Chan

    Full Text Available BACKGROUND: The somatic embryogenesis tissue culture process has been utilized to propagate high yielding oil palm. Due to the low callogenesis and embryogenesis rates, molecular studies were initiated to identify genes regulating the process, and their expression levels are usually quantified using reverse transcription quantitative real-time PCR (RT-qPCR. With the recent release of oil palm genome sequences, it is crucial to establish a proper strategy for gene analysis using RT-qPCR. Selection of the most suitable reference genes should be performed for accurate quantification of gene expression levels. RESULTS: In this study, eight candidate reference genes selected from cDNA microarray study and literature review were evaluated comprehensively across 26 tissue culture samples using RT-qPCR. These samples were collected from two tissue culture lines and media treatments, which consisted of leaf explants cultures, callus and embryoids from consecutive developmental stages. Three statistical algorithms (geNorm, NormFinder and BestKeeper confirmed that the expression stability of novel reference genes (pOP-EA01332, PD00380 and PD00569 outperformed classical housekeeping genes (GAPDH, NAD5, TUBULIN, UBIQUITIN and ACTIN. PD00380 and PD00569 were identified as the most stably expressed genes in total samples, MA2 and MA8 tissue culture lines. Their applicability to validate the expression profiles of a putative ethylene-responsive transcription factor 3-like gene demonstrated the importance of using the geometric mean of two genes for normalization. CONCLUSIONS: Systematic selection of the most stably expressed reference genes for RT-qPCR was established in oil palm tissue culture samples. PD00380 and PD00569 were selected for accurate and reliable normalization of gene expression data from RT-qPCR. These data will be valuable to the research associated with the tissue culture process. Also, the method described here will facilitate the selection

  5. Comparison of regeneration potentials in tissue cultures of primitive and cultivated tomato species (Lycopersicon sp.

    Directory of Open Access Journals (Sweden)

    M. Lech

    2014-01-01

    Full Text Available Regeneration capacities of two tomato cultivars: Potentat and Rutgers, and of three accessions of wild tomato species: Lycopersicon peruvianum PI 128650, L. peruvianum var. dentatum PI 128655 and L. glandulosum were studied using an universal medium suitable for regeneration of those plants from leaf pieces in tissue culture. Fragments of leaf blades were taken from plants raised in greenhouse conditions and placed on a modified MS medium containing 0.3 mg/l IAA and 3.0 mg/l BAP solidified with 1% agar. The explants were transferred every 4-5 weeks on fresh medium of the same composition. It was shown that all the three primitive tomato species revealed much higher multiplication coefficients than the two cultivars. Appropriate values were: 11 - for L. glandulosum, 8 - for L. peruvianum, 7 - for L. peruvianum var. dentatum, 4 - for L. esculentum cv. Potentat and 2 - cv. Rutgers. Completely regenerated plants were obtained from all the tested species, but organogenesis occurred almost two weeks earlier in wild tomatoes than in the culitivated varieties of L. esculentum.

  6. The application of cell cultures, body fluids and tissues in oncoproteomics

    Directory of Open Access Journals (Sweden)

    Kamila Duś-Szachniewicz

    2014-11-01

    Full Text Available Mass spectrometry (MS-based proteomics is a rapidly developing technology for the large scale analysis of proteins, their interactions and subcellular localization. In recent years proteomics has attracted much attention in medicine. Since a single biomarker might not have sufficient sensitivity and specificity in clinical practice, the identification of biomarker panels that comprise several proteins would improve the detection and clinical management of cancer patients. Additionally, the characteristics of protein profiles of most severe human malignancies certainly contribute to the understanding of the biology of cancer and fill the gap in our knowledge of carcinogenesis. This knowledge also is likely to result in the discovery of novel potential cancer markers and targets for molecular therapeutics. It is believed that the novel biomarkers will help in the development of personalized therapy tailored to the individual patient and will thereby reduce the mortality rate from cancer. In this review, the use of different types of human clinical samples (cell cultures, tissues and body fluids in oncoproteomics is explained and the latest advances in mass spectrometry-based proteomics biomarker discovery are discussed.

  7. DEVELOPMENT OF PRIMARY CELL CULTURE FROM TAIL EPIDERMAL TISSUE OF KOI CARP (Cyprinus carpio koi

    Directory of Open Access Journals (Sweden)

    Lila Gardenia

    2014-06-01

    Full Text Available Primary cell culture from tail epidermal tissue of koi carp (Cyprinus carpio koi was developed. Cells were grown in Leibovits-15 medium supplemented with 20% fetal bovine serum and antibiotics (Penicillin/Streptomycin and Kanamycin. Cell growth was observed in a range of incubation temperature (17oC±2oC, 22oC±2oC, 27oC±2oC, and 32oC±2oC in order to determine the optimum temperature. The cells were able to grow at a range of temperature between 17oC to 32oC with optimal growth at 22oC. Primary cells infected with koi herpes virus produced typical cytopathic effects characterized by severe vacuolation and deformation of nuclei, which is consistent with those of previous reports. Artificial injection experiment by using supernatant koi herpes virus SKBM-1 isolate revealed that it could cause 90% mortality in infected fish within two weeks. PCR test with Sph I-5 specific primers carried out with DNA template from supernatant virus, pellet cell, and gills of infected fish showed positive results in all samples (molecular weight of DNA target 290 bp. The cells were found to be susceptible to koi herpes virus and can be used for virus propagation.

  8. Plant cell, tissue and organ culture: the most flexible foundations for plant metabolic engineering applications.

    Science.gov (United States)

    Ogita, Shinjiro

    2015-05-01

    Significant advances in plant cell, tissue and organ culture (PCTOC) have been made in the last five decades. PCTOC is now thought to be the underlying technique for understanding general or specific biological functions of the plant kingdom, and it is one of the most flexible foundations for morphological, physiological and molecular biological applications of plants. Furthermore, the recent advances in the field of information technology (IT) have enabled access to a large amount of information regarding all aspects of plant biology. For example, sequencing information is stored in mega repositories such as the National Center for Biotechnology Information (NCBI), which can be easily accessed by researchers worldwide. To date, the PCTOC and IT combination strategy for regulation of target plant metabolism and the utilization of bioactive plant metabolites for commercial purposes is essential. In this review, the advantages and the limitations of these methodologies, especially regarding the production of bioactive plant secondary metabolites and metabolic engineering in target plants are discussed mainly from the phenotypic view point.

  9. Cell therapy, 3D culture systems and tissue engineering for cardiac regeneration.

    Science.gov (United States)

    Emmert, Maximilian Y; Hitchcock, Robert W; Hoerstrup, Simon P

    2014-04-01

    Ischemic Heart Disease (IHD) still represents the "Number One Killer" worldwide accounting for the death of numerous patients. However the capacity for self-regeneration of the adult heart is very limited and the loss of cardiomyocytes in the infarcted heart leads to continuous adverse cardiac-remodeling which often leads to heart-failure (HF). The concept of regenerative medicine comprising cell-based therapies, bio-engineering technologies and hybrid solutions has been proposed as a promising next-generation approach to address IHD and HF. Numerous strategies are under investigation evaluating the potential of regenerative medicine on the failing myocardium including classical cell-therapy concepts, three-dimensional culture techniques and tissue-engineering approaches. While most of these regenerative strategies have shown great potential in experimental studies, the translation into a clinical setting has either been limited or too rapid leaving many key questions unanswered. This review summarizes the current state-of-the-art, important challenges and future research directions as to regenerative approaches addressing IHD and resulting HF. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. Tissue culture and regeneration of an antimalarial plant, Artemisia sieberi Besser

    Directory of Open Access Journals (Sweden)

    A. Sharafi

    2014-10-01

    Full Text Available WHO recommends artemisinin-based combination therapies (ACTs as the most effective choice to treat malaria. For developing transgenic plants with high accumulation of artemisinin (by introducing genes encoding enzymes which regulate the biosynthetic pathway of artemisinin, an efficient protocol for tissue culture and plant regeneration is necessary. In the present study, leaf explants of Artemisia sieberi were cultivated in Murashige & Skoog based medium supplemented by combination of different plant growth regulators including 6-benzyl-aminopurine (BA, α-naphthalene-acetic acid (NAA, indole-3-acetic acid (IAA, picloram (Pic and 2,4-dichlorophenoxyacetic acid (2,4-D. The highest frequency of shoot induction was obtained on MS medium supplemented with 2 mg/L BA plus 0.05 mg/L NAA (95% regeneration and MS medium supplemented with 2 mg/L BA plus 0.5 mg/L IAA (85% regeneration. Rooting was obtained on MS medium supplemented with 0.05 mg/L NAA. The present study has revealed a simple, reliable, rapid and high efficient regeneration system for A. sieberi Besser as a source of artemisinin in short period via adventitious shoot induction procedure.

  11. Tissue culture-induced genetic and epigenetic variation in triticale (× Triticosecale spp. Wittmack ex A. Camus 1927) regenerants.

    Science.gov (United States)

    Machczyńska, Joanna; Zimny, Janusz; Bednarek, Piotr Tomasz

    2015-10-01

    Plant regeneration via in vitro culture can induce genetic and epigenetic variation; however, the extent of such changes in triticale is not yet understood. In the present study, metAFLP, a variation of methylation-sensitive amplified fragment length polymorphism analysis, was used to investigate tissue culture-induced variation in triticale regenerants derived from four distinct genotypes using androgenesis and somatic embryogenesis. The metAFLP technique enabled identification of both sequence and DNA methylation pattern changes in a single experiment. Moreover, it was possible to quantify subtle effects such as sequence variation, demethylation, and de novo methylation, which affected 19, 5.5, 4.5% of sites, respectively. Comparison of variation in different genotypes and with different in vitro regeneration approaches demonstrated that both the culture technique and genetic background of donor plants affected tissue culture-induced variation. The results showed that the metAFLP approach could be used for quantification of tissue culture-induced variation and provided direct evidence that in vitro plant regeneration could cause genetic and epigenetic variation.

  12. Lipid-mediated glial cell line-derived neurotrophic factor gene transfer to cultured porcine ventral mesencephalic tissue

    DEFF Research Database (Denmark)

    Bauer, Matthias; Meyer, Morten; Brevig, Thomas

    2002-01-01

    Transplantation of dopaminergic ventral mesencephalic (VM) tissue into the basal ganglia of patients with Parkinson's disease (PD) shows at best moderate symptomatic relief in some of the treated cases. Experimental animal studies and clinical trials with allogenic and xenogenic pig-derived VM......-mediated transfer of the gene for human glial cell line-derived neurotrophic factor (GDNF) to embryonic (E27/28) porcine VM tissue kept as organotypic explant cultures. Treatment of the developing VM with two mitogens, basic fibroblast growth factor and epidermal growth factor, prior to transfection significantly...... increased transfection yields. Expression of human GDNF via an episomal vector could be detected by in situ hybridization and by the measuring of GDNF protein secreted into the culture medium. When compared to mock-transfected controls, VM tissue expressing recombinant GDNF contained significantly higher...

  13. An assessment of genetic fidelity of in vitro grown plantlets of rose ...

    African Journals Online (AJOL)

    A simple and routine method for the analysis of somaclonal variation among tissue culture derived rose plants is a prerequisite for precise monitoring of quality control during rapid mass micropropagation. Random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) molecular marker techniques ...

  14. Discrimination and similarity evaluation of tissue-cultured and wild Dendrobium species using Fourier transform infrared spectroscopy

    Science.gov (United States)

    Chen, Nai-dong; Chen, Han; Li, Jun; Sang, Mang-mang; Ding, Shen; Yu, Hao

    2015-04-01

    The FTIR method was applied to evaluate the similarity of tissue-cultured and wild Dendrobium huoshanense C.Z. Tang et S.J. Cheng, Dendrobium officinale Kimura et Migo and Dendrobium moniliforme (Linn.) Sw and discriminate different Dendrobium species, especially D. huoshanense and its main goldbrick Dendrobium henanense J.L. Lu et L.X. Gao. Despite the general pattern of the IR spectra, different intensities, shapes and peak positions were found in the IR spectra of these samples, especially in the range of 1800-600 cm-1, which could be used to discriminate them. The methanol, aqueous extracting procedure and the second derivative transformation obviously enlarged the tiny spectral differences among these samples. The similarity evaluation based on the IR spectra and the second derivative IR spectrum revealed that the similarity of the methanol extracts between tissue-cultured and wild Dendrobiums might be lower than that between different Dendrobium species. The similarities of the powders and aqueous extracts between tissue-cultured and wild Dendrobiums were higher than those between different Dendrobium species. The further principal component analysis showed that the first three components explained 99.7%, 87.7% and 85.1% of data variance for powder, methanol extract and aqueous extract, respectively, demonstrating a good discrimination between samples. Our research suggested that the variations of secondary metabolites between different origins of the investigated Dendrobiums might be higher than what we had supposed. Tissue culture techniques were widely used in the conversation of rare and endangered medicinal amedica, however, our study suggested that the chemical constituents of tissue-cultured plants might be quite different from their wild correspondences.

  15. Skin equivalent tissue-engineered construct: co-cultured fibroblasts/ keratinocytes on 3D matrices of sericin hope cocoons.

    Science.gov (United States)

    Nayak, Sunita; Dey, Sancharika; Kundu, Subhas C

    2013-01-01

    The development of effective and alternative tissue-engineered skin replacements to autografts, allografts and xenografts has became a clinical requirement due to the problems related to source of donor tissue and the perceived risk of disease transmission. In the present study 3D tissue engineered construct of sericin is developed using co-culture of keratinocytes on the upper surface of the fabricated matrices and with fibroblasts on lower surface. Sericin is obtained from "Sericin Hope" silkworm of Bombyx mori mutant and is extracted from cocoons by autoclave. Porous sericin matrices are prepared by freeze dried method using genipin as crosslinker. The matrices are characterized biochemically and biophysically. The cell proliferation and viability of co-cultured fibroblasts and keratinocytes on matrices for at least 28 days are observed by live/dead assay, Alamar blue assay, and by dual fluorescent staining. The growth of the fibroblasts and keratinocytes in co-culture is correlated with the expression level of TGF-β, b-FGF and IL-8 in the cultured supernatants by enzyme-linked immunosorbent assay. The histological analysis further demonstrates a multi-layered stratified epidermal layer of uninhibited keratinocytes in co-cultured constructs. Presence of involucrin, collagen IV and the fibroblast surface protein in immuno-histochemical stained sections of co-cultured matrices indicates the significance of paracrine signaling between keratinocytes and fibroblasts in the expression of extracellular matrix protein for dermal repair. No significant amount of pro inflammatory cytokines (TNF-α, IL-1β and nitric oxide) production are evidenced when macrophages grown on the sericin matrices. The results all together depict the potentiality of sericin 3D matrices as skin equivalent tissue engineered construct in wound repair.

  16. Skin equivalent tissue-engineered construct: co-cultured fibroblasts/ keratinocytes on 3D matrices of sericin hope cocoons.

    Directory of Open Access Journals (Sweden)

    Sunita Nayak

    Full Text Available The development of effective and alternative tissue-engineered skin replacements to autografts, allografts and xenografts has became a clinical requirement due to the problems related to source of donor tissue and the perceived risk of disease transmission. In the present study 3D tissue engineered construct of sericin is developed using co-culture of keratinocytes on the upper surface of the fabricated matrices and with fibroblasts on lower surface. Sericin is obtained from "Sericin Hope" silkworm of Bombyx mori mutant and is extracted from cocoons by autoclave. Porous sericin matrices are prepared by freeze dried method using genipin as crosslinker. The matrices are characterized biochemically and biophysically. The cell proliferation and viability of co-cultured fibroblasts and keratinocytes on matrices for at least 28 days are observed by live/dead assay, Alamar blue assay, and by dual fluorescent staining. The growth of the fibroblasts and keratinocytes in co-culture is correlated with the expression level of TGF-β, b-FGF and IL-8 in the cultured supernatants by enzyme-linked immunosorbent assay. The histological analysis further demonstrates a multi-layered stratified epidermal layer of uninhibited keratinocytes in co-cultured constructs. Presence of involucrin, collagen IV and the fibroblast surface protein in immuno-histochemical stained sections of co-cultured matrices indicates the significance of paracrine signaling between keratinocytes and fibroblasts in the expression of extracellular matrix protein for dermal repair. No significant amount of pro inflammatory cytokines (TNF-α, IL-1β and nitric oxide production are evidenced when macrophages grown on the sericin matrices. The results all together depict the potentiality of sericin 3D matrices as skin equivalent tissue engineered construct in wound repair.

  17. Tooth Tissue Engineering: The Importance of Blood Products as a Supplement in Tissue Culture Medium for Human Pulp Dental Stem Cells.

    Science.gov (United States)

    Pisciolaro, Ricardo Luiz; Duailibi, Monica Talarico; Novo, Neil Ferreira; Juliano, Yara; Pallos, Debora; Yelick, Pamela Crotty; Vacanti, Joseph Phillip; Ferreira, Lydia Masako; Duailibi, Silvio Eduardo

    2015-11-01

    One of the goals in using cells for tissue engineering (TE) and cell therapy consists of optimizing the medium for cell culture. The present study compares three different blood product supplements for improved cell proliferation and protection against DNA damage in cultured human dental pulp stem cells for tooth TE applications. Human cells from dental pulp were first characterized as adult stem cells (ectomesenchymal mixed origin) by flow cytometry. Next, four different cell culture conditions were tested: I, supplement-free; II, supplemented with fetal bovine serum; III, allogeneic human serum; and IV, autologous human serum. Cultured cells were then characterized for cell proliferation, mineralized nodule formation, and colony-forming units (CFU) capability. After 28 days in culture, the comet assay was performed to assess possible damage in cellular DNA. Our results revealed that Protocol IV achieved higher cell proliferation than Protocol I (p = 0.0112). Protocols II and III resulted in higher cell proliferation than Protocol I, but no statistical differences were found relative to Protocol IV. The comet assay revealed less cell damage in cells cultured using Protocol IV as compared to Protocols II and III. The damage percentage observed on Protocol II was significantly higher than all other protocols. CFUs capability was highest using Protocol IV (p = 0.0018) and III, respectively, and the highest degree of mineralization was observed using Protocol IV as compared to Protocols II and III. Protocol IV resulted in significantly improved cell proliferation, and no cell damage was observed. These results demonstrate that human blood product supplements can be used as feasible supplements for culturing adult human dental stem cells.

  18. Condensed tannins in the tissue culture of sainfoin (Onobrychis viciifolia Scop.) and birdsfoot trefoil (Lotus corniculatus L.).

    Science.gov (United States)

    Lees, G L

    1986-08-01

    Two forage legumes, birdsfoot trefoil (Lotus corniculatus L.) and sainfoin (Onobrychis viciifolia Scop.), containing condensed tannins in their leaves and stems were used as source material to study condensed tannins in tissue culture. More protoplasts were isolated from mesophyll tissue of a low tannin-containing strain of birdsfoot trefoil than from a high tannin-containing strain, but more tannin-filled protoplasts were observed in the latter. Growth rates of leaf explant-derived callus tissue were greater for the high-tannin than for the low-tannin strain. In sainfoin, callus cultures from leaf explants produced numerous tannin-filled cells by 21 days. Explants from sainfoin cotyledons and roots, tissues which normally do not contain tannins, also formed callus with tannin-filled cells in 21 days but in almost every case, a cytokinin was required for tannin formation to occur. The occurrence of tannin-filled cells in callus from root and cotyledon explants was variable and genotype specific. These results show that endogenous tannins can affect protoplast isolation and possibly callus growth in birds-foot trefoil, and that the formation of condensed tannins in sainfoin callus culture can be influenced by a growth regulator.

  19. Recent progress in the understanding of tissue culture-induced genome level changes in plants and potential applications.

    Science.gov (United States)

    Neelakandan, Anjanasree K; Wang, Kan

    2012-04-01

    In vitro cell and tissue-based systems have tremendous potential in fundamental research and for commercial applications such as clonal propagation, genetic engineering and production of valuable metabolites. Since the invention of plant cell and tissue culture techniques more than half a century ago, scientists have been trying to understand the morphological, physiological, biochemical and molecular changes associated with tissue culture responses. Establishment of de novo developmental cell fate in vitro is governed by factors such as genetic make-up, stress and plant growth regulators. In vitro culture is believed to destabilize the genetic and epigenetic program of intact plant tissue and can lead to chromosomal and DNA sequence variations, methylation changes, transposon activation, and generation of somaclonal variants. In this review, we discuss the current status of understanding the genomic and epigenomic changes that take place under in vitro conditions. It is hoped that a precise and comprehensive knowledge of the molecular basis of these variations and acquisition of developmental cell fate would help to devise strategies to improve the totipotency and embryogenic capability in recalcitrant species and genotypes, and to address bottlenecks associated with clonal propagation. © Springer-Verlag 2011

  20. The effects of gamma irradiation on the growth and cytology of carrot (Dacus Carota L.) tissue culture

    International Nuclear Information System (INIS)

    Al-Safady, B.; Simon, P.W.

    1992-01-01

    Carrot (Dacus Carota L.) tissue suspension cultures were subjected to 0.5-40 krad of gamma irradiation. Callus fresh weight was significantly increased and dry weight slightly increased by low doses (0.5 and 1 krad). Cells size increased at all doses. Cell number and colony-forming ability decreased. There was a negative correlation between radiation dose and mitotic index, and a positive correlation between dose and mitotic abnormalities (multipolar separations, lagging chromosomes, and bridges). Frequency of prophase was increased, and ana phase and telophase were reduced. Tissue culture conditions increased the incidence of aneuploidy and polyploidy in comparison to carrot root tips. Aneuploidy in tissue cultures was decreased and polyploidy was increased by gamma irradiation. Gamma irradiation stimulated shoot formation at 1 krad, and inhibited shoot formation at high doses (20-40 krads). The frequency of abnormal plants (albinos, and plants with no roots or deformation of leaves) regenerated from carrot cell cultures was increased by gamma irradiation. (authors). 30 refs., 3 figs

  1. Dynamic culture of a thermosensitive collagen hydrogel as an extracellular matrix improves the construction of tissue-engineered peripheral nerve.

    Science.gov (United States)

    Huang, Lanfeng; Li, Rui; Liu, Wanguo; Dai, Jin; Du, Zhenwu; Wang, Xiaonan; Ma, Jianchao; Zhao, Jinsong

    2014-07-15

    Tissue engineering technologies offer new treatment strategies for the repair of peripheral nerve injury, but cell loss between seeding and adhesion to the scaffold remains inevitable. A thermosensitive collagen hydrogel was used as an extracellular matrix in this study and combined with bone marrow mesenchymal stem cells to construct tissue-engineered peripheral nerve composites in vitro. Dynamic culture was performed at an oscillating frequency of 0.5 Hz and 35° swing angle above and below the horizontal plane. The results demonstrated that bone marrow mesenchymal stem cells formed membrane-like structures around the poly-L-lactic acid scaffolds and exhibited regular alignment on the composite surface. Collagen was used to fill in the pores, and seeded cells adhered onto the poly-L-lactic acid fibers. The DNA content of the bone marrow mesenchymal stem cells was higher in the composites constructed with a thermosensitive collagen hydrogel compared with that in collagen I scaffold controls. The cellular DNA content was also higher in the thermosensitive collagen hydrogel composites constructed with the thermosensitive collagen hydrogel in dynamic culture than that in static culture. These results indicate that tissue-engineered composites formed with thermosensitive collagen hydrogel in dynamic culture can maintain larger numbers of seeded cells by avoiding cell loss during the initial adhesion stage. Moreover, seeded cells were distributed throughout the material.

  2. Transcriptome analysis reveals in vitro cultured Withania somnifera leaf and root tissues as a promising source for targeted withanolide biosynthesis.

    Science.gov (United States)

    Senthil, Kalaiselvi; Jayakodi, Murukarthick; Thirugnanasambantham, Pankajavalli; Lee, Sang Choon; Duraisamy, Pradeepa; Purushotham, Preethi M; Rajasekaran, Kalaiselvi; Nancy Charles, Shobana; Mariam Roy, Irene; Nagappan, Arul Kumar; Kim, Gon Sup; Lee, Yun Sun; Natesan, Senthil; Min, Tae-Sun; Yang, Tae Jin

    2015-01-22

    The production of metabolites via in vitro culture is promoted by the availability of fully defined metabolic pathways. Withanolides, the major bioactive phytochemicals of Withania somnifera, have been well studied for their pharmacological activities. However, only a few attempts have been made to identify key candidate genes involved in withanolide biosynthesis. Understanding the steps involved in withanolide biosynthesis is essential for metabolic engineering of this plant to increase withanolide production. Transcriptome sequencing was performed on in vitro adventitious root and leaf tissues using the Illumina platform. We obtained a total of 177,156 assembled transcripts with an average unigene length of 1,033 bp. About 13% of the transcripts were unique to in vitro adventitious roots but no unique transcripts were observed in in vitro-grown leaves. A putative withanolide biosynthetic pathway was deduced by mapping the assembled transcripts to the KEGG database, and the expression of candidate withanolide biosynthesis genes -were validated by qRT PCR. The accumulation pattern of withaferin A and withanolide A varied according to the type of tissue and the culture period. Further, we demonstrated that in vitro leaf extracts exhibit anticancer activity against human gastric adenocarcinoma cell lines at sub G1 phase. We report here a validated large-scale transcriptome data set and the potential biological activity of in vitro cultures of W. somnifera. This study provides important information to enhance tissue-specific expression and accumulation of secondary metabolites, paving the way for industrialization of in vitro cultures of W. somnifera.

  3. GENETIC VARIABILITY OF CULTURED PLANT TISSUES UNDER NORMAL CONDITIONS AND UNDER STRESS

    Directory of Open Access Journals (Sweden)

    Dolgikh Yu.I.

    2012-08-01

    Full Text Available The genetic variability induced by in vitro conditions known as somaclonal variation is of practical interest due to its potential uses in plant breeding but, on the other hand, if clonal propagation or transformation is main goal, it becomes an unwelcome phenomenon. Thus, it is important to know frequency, the genomic distribution, the mechanisms and factors influencing somaclonal variation. We studied variability of PCR-based DNA markers of cultured tissues and regenerated plants of maize and bread wheat. The original A188 line of maize and the somaclones obtained were tested using 38 RAPD and 10 ISSR primers. None of the A188 plants showed variation in the RAPD and ISSR spectra for any of the primers used. However, the PCR spectra obtained from the somaclones demonstrated some variations, i.e., 22 RAPD primers and 6 ISSR primers differentiated at least one somaclonal variant from the progenitor line. Six SCAR markers were developed based on several RAPD and ISSR fragments. The inheritance of these SCAR markers was verified in the selfing progeny of each somaclone in the R1–R4 generations and in the hybrids, with A188 as the parental line in the F1 and F2 generations. These markers were sequenced and bioinformatic searches were performed to understand the molecular events that may underlie the variability observed in the somaclones. All changes were found in noncoding sequences and were induced by different molecular events, such as the insertion of long terminal repeat transposon, precise miniature inverted repeat transposable element (MITE excision, microdeletion, recombination, and a change in the pool of mitochondrial DNA. In two groups of independently produced somaclones, the same features (morphological, molecular were variable, which confirms the theory of ‘hot spots’ occurring in the genome. The presence of the same molecular markers in the somaclones and in different non-somaclonal maize variants suggests that in some cases

  4. Genetic improvement of cocoa for resistance to swollen shoot disease through mutation breeding and in vitro culture techniques

    International Nuclear Information System (INIS)

    Adu-Ampomah, Y.; Novak, F.J.

    1991-01-01

    Various tissues (vegetative buds, pollen and selfed beans) of three cocoa types were irradiated with gamma rays with the view to inducing mutants resistant to cocoa swollen shoot virus (CSSV). To save time and space in cocoa mutation breeding, certain aspects of the tissue culture techniques in cocoa were also investigated. Efforts were made to delimit some of the factors crucial to producing plants via cocoa tissues in vitro. Use of concentrations of 2-4 mg/L of BAP coupled with weekly subculturing and light durations between 16-20 hours at 2000 lx greatly enhanced differentiation, plantlet growth and plant production. Indications are that the possibility of using in vitro techniques for mutation breeding in cocoa could be exploited. 5 refs, 1 fig., 6 tabs

  5. Excitatory synapse in the rat hippocampus in tissue culture and effects of aniracetam.

    Science.gov (United States)

    Ozawa, S; Iino, M; Abe, M

    1991-10-01

    Excitatory synaptic connections between rat hippocampal neurons were established in tissue culture. The electrophysiological and pharmacological properties of these synapses were studied with the use of the tight-seal whole-cell recording technique. The excitatory postsynaptic current (EPSC) in a dissociated CA1 neuron evoked by stimulation of an explant from the CA3/CA4 region of the hippocampus had two distinct components in Mg(2+)-free medium. The fast component was abolished by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (2 microM), whereas the slow component was abolished by the N-methyl-D-aspartate (NMDA) receptor antagonist D-2-amino-5-phosphonovalerate (D-APV) (50 microM). In solution containing 1 mM Mg2+, the peak amplitude of the fast component was almost linearly related to the membrane potential. In contrast, the conductance change underlying the slow component of the EPSC was voltage-dependent with a region of negative-slope conductance in the range of -80 to -20 mV. A nootropic drug, aniracetam, increased both the amplitude and duration of the fast component of the EPSC in a concentration-dependent manner in the range of 0.1-5 mM, whereas it had no potentiating effect on the slow component. Aniracetam (0.1-5 mM) similarly increased current responses of the postsynaptic neuron to alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA). Current responses to quisqualate and glutamate in the presence of D-APV were also potentiated by aniracetam. However, neither NMDA- nor kainate-induced current was potentiated by 1 mM aniracetam.

  6. Ontogenetically-regulated male sterility in tissue culture - induced and spontaneous sorghum mutants

    Directory of Open Access Journals (Sweden)

    Elkonin L.A.

    2003-01-01

    Full Text Available Variability of male fertility expression in the AS-1 line, a somaclonal variant obtained from tissue culture of CMS-plant, and in the progeny of revenant '124-1' obtained from fertile tiller, which developed on CMS-plant transferred from the field to the greenhouse, was investigated. Both revertants were characterized by similar expression of male fertility during plant ontogenesis: the panicle on the main tiller was almost completely sterile whereas formation of fertile pollen grains and seed set were observed on the panicles of the shoot tillers. A clear basipetal gradient of male fertility was manifested on all panicles: the base had significantly higher per cent of fertile pollen grains in comparison with the middle part, while in the top the anthers were either absent or had few sterile pollen grains. Such an ontogenetically-regulated restoration of male fertility was controlled by nuclear genes and could be transferred through the pollen in crosses with progenitor CMS-line. Growing of AS-1 plants in the growth chambers simultaneously under a long (16/8 and a short (12/12 daylength conditions demonstrated that differences of fertility level in different tillers was not caused by change of photoperiod during plant ontogenesis and functioning of photoperiod-sensitive fertility restoring gene. Whereas, the ontogenetically-regulated expression of male fertility in both revenants was temperature-dependent and was clearly manifested under relatively cool conditions during 2-week period before the beginning of anthesis of the first panicle (average daily temperature 21°C. The increase of the average daily temperature by 2-3 С resulted in sharp increase of male fertility level. Possibility of using AS-1 line in a new "two-line system" of hybrid seed production, which require only two lines (sterile mutant and fertility restorer, is discussed.

  7. Cultured Human Epidermis Combined With Meshed Skin Autografts Accelerates Epithelialization and Granulation Tissue Formation in a Rat Model.

    Science.gov (United States)

    Sakamoto, Michiharu; Morimoto, Naoki; Inoie, Masukazu; Takahagi, Miki; Ogino, Shuichi; Jinno, Chizuru; Suzuki, Shigehiko

    2017-06-01

    As the take rate of cultured epidermal autografts in burn wound treatment is variable, widely expanded meshed auto skin grafts are often used in combination with cultured epidermal autograft to increase the take rate and achieve definitive wound coverage. However, a long time (3-4 weeks) required to prepare a cultured epidermis sheet is a disadvantage. Allogeneic cultured epidermis can be prepared in advance and cryopreserved to be used in combination with auto meshed skin grafts for treating third-degree burns. Nevertheless, the human cultured epidermis (hCE) has not been proved to accelerate wound healing after meshed skin grafting. Here, we investigated the effect of hCE on wound healing in a rat model of meshed skin grafting. Human cultured epidermis was prepared from human neonatal foreskin and assessed by the release of growth factors into the culture medium using enzyme-linked immunosorbent assay. Skin wounds were inflicted on male F344 rats and treated by the application of widely meshed (6:1 ratio) autogenous skin grafts with or without hCE (n = 8 rats per group). Wound area, neoepithelium length, granulation tissue formation, and neovascularization were evaluated on day 7 postgrafting. Human cultured epidermis secreted IL-1α, Basic fibroblast growth factor, platelet-derived growth factor-AA, TGF-α, TGF-β1, and vascular endothelial growth factor in vitro. In rats, hCE accelerated wound closure (P = 0.003), neoepithelium growth (P = 0.019), and granulation tissue formation (P = 0.043), and increased the number of capillaries (P = 0.0003) and gross neovascularization area (P = 0.008) compared with the control group. The application of hCE with meshed grafts promoted wound closure, possibly via secretion of growth factors critical for cell proliferation and migration, suggesting that hCE can enhance the healing effect of widely expanded skin autografts.

  8. Tissue culture-induced genetic and epigenetic alterations in rice pure-lines, F1 hybrids and polyploids.

    Science.gov (United States)

    Wang, Xiaoran; Wu, Rui; Lin, Xiuyun; Bai, Yan; Song, Congdi; Yu, Xiaoming; Xu, Chunming; Zhao, Na; Dong, Yuzhu; Liu, Bao

    2013-05-05

    Genetic and epigenetic alterations can be invoked by plant tissue culture, which may result in heritable changes in phenotypes, a phenomenon collectively termed somaclonal variation. Although extensive studies have been conducted on the molecular nature and spectrum of tissue culture-induced genomic alterations, the issue of whether and to what extent distinct plant genotypes, e.g., pure-lines, hybrids and polyploids, may respond differentially to the tissue culture condition remains poorly understood. We investigated tissue culture-induced genetic and epigenetic alterations in a set of rice genotypes including two pure-lines (different subspecies), a pair of reciprocal F1 hybrids parented by the two pure-lines, and a pair of reciprocal tetraploids resulted from the hybrids. Using two molecular markers, amplified fragment length polymorphism (AFLP) and methylation-sensitive amplified polymorphism (MSAP), both genetic and DNA methylation alterations were detected in calli and regenerants from all six genotypes, but genetic alteration is more prominent than epigenetic alteration. While significant genotypic difference was observed in frequencies of both types of alterations, only genetic alteration showed distinctive features among the three types of genomes, with one hybrid (N/9) being exceptionally labile. Surprisingly, difference in genetic alteration frequencies between the pair of reciprocal F1 hybrids is much greater than that between the two pure-line subspecies. Difference also exists in the pair of reciprocal tetraploids, but is to a less extent than that between the hybrids. The steady-state transcript abundance of genes involved in DNA repair and DNA methylation was significantly altered in both calli and regenerants, and some of which were correlated with the genetic and/or epigenetic alterations. Our results, based on molecular marker analysis of ca. 1,000 genomic loci, document that genetic alteration is the major cause of somaclonal variation in rice

  9. Longitudinal Claudin Gene Expression Analyses in Canine Mammary Tissues and Thereof Derived Primary Cultures and Cell Lines

    Directory of Open Access Journals (Sweden)

    Susanne C. Hammer

    2016-09-01

    Full Text Available Human and canine mammary tumours show partial claudin expression deregulations. Further, claudins have been used for directed therapeutic approaches. However, the development of claudin targeting approaches requires stable claudin expressing cell lines. This study reports the establishment and characterisation of canine mammary tissue derived cell lines, analysing longitudinally the claudin-1, -3, -4 and -7 expressions in original tissue samples, primary cultures and developed cell lines. Primary cultures were derived from 17 canine mammary tissues: healthy, lobular hyperplasia, simple adenoma, complex adenoma, simple tubular carcinoma, complex carcinoma, carcinoma arising in a benign mixed tumour and benign mixed tissue. Cultivation was performed, if possible, until passage 30. Claudin mRNA and protein expressions were analysed by PCR, QuantiGene Plex Assay, immunocytochemistry and immunofluorescence. Further, cytokeratin expression was analysed immunocytochemically. Cultivation resulted in 11 established cell lines, eight showing epithelial character. In five of the early passages the claudin expressions decreased compared to the original tissues. In general, claudin expressions were diminished during cultivation. Three cell lines kept longitudinally claudin, as well as epithelial marker expressions, representing valuable tools for the development of claudin targeted anti-tumour therapies.

  10. An assessment of the cultural capabilities of Trifolium repens L. (white clover) and Onobrychis viciifolia Scop. (sainfoin) mesophyll protoplasts.

    Science.gov (United States)

    Ahuja, P S; Lu, D Y; Cocking, E C; Davey, M R

    1983-10-01

    Mesophyll protoplasts isolated from white clover and sainfoin divided to form callus under similar cultural conditions. White clover protoplasts showed varietal differences in their plating efficiency. Sainfoin tissues regenerated readily by forming shoots, but induction of morphogenesis in white clover was only achieved after testing several media and culture sequences. Many of the white clover shoots were abnormal in being fused together to form green plate-like structures, but the latter still developed into plantlets while attached to the parent callus. The ability to isolate, culture, and regenerate mesophyll protoplasts of these two forage legumes is discussed in relation to future attempts to produce somatic hybrids between high tannin containing bloat-safe sainfoin and other major forage legumes such as alfalfa, white clover, and red clover.

  11. EFFECT OF PERMEABLE VESSEL CLOSURE AND GELLING AGENT ON REDUCTION OF HYPERHYDRICITY IN IN VITRO CULTURE OF CARNATION

    Directory of Open Access Journals (Sweden)

    B. Winarto

    2016-10-01

    Full Text Available Hyperhydricity, an abnormal morphological appearance and physiologicalfunction, is an important problem in carnation tissue culture. The problem causes premature flowering, high occurrence of abnormal shoots, difficulty in transferring hyperhydric plantlets to soil, and low survival rate of plantlets. High relative humidity and the water potential are considered as the key factors involved in the abnormality. Furthermore, permeable culture vessel and gelling agent were assured to be high potential treatment to eliminate it. Objective of this research was to reduce  hyperhydricity in regenerants of carnation using different permeable vessel closures and gelling agents and to assess the multiplication and  acclimatization abilities of recovered shoots. Experiment was arranged in randomized complete block design with four replications. First factor was different types of closure, i.e. cotton wool, plastic wrap, parafilm and aluminium foil, while second one was gelling agents, i.e. bacto agar, phytagel, swallow agar, and Type 900 agar. The recovered shoots were then multiplied, rooted, and acclimatized. The results showed that hyperhydricity was successfully reduced by applying permeable closure (cotton wool and plastic wrap in combination with Type 900 agar. The combination of plastic wrap and Type 900 agar was the most appropriate treatment in reducing hyperhydricity and producing good quality shoots. The treatment reduced the problem down to 23% of total condition of hyperhydricity (100% and increased leaf chlorophyll content from 0.0883 to 0.1288 mg mg-1. The plastic wrap was easily applied and cheaper material compared to cotton wool. The recovered shoots were able to produce 1-3 healthy axillary shoots and easily rooted on half-strength MS. The recovered plantlets were simply acclimatized with survival rate up to 100% on kossas peat + soil (1:1, v/v and flowered 4-5 months after acclimatization with decreasing in number and size of flower.

  12. Spheroid culture as a tool for creating 3D complex tissues

    NARCIS (Netherlands)

    Fennema, E.M.; Rivron, N.C.; Rouwkema, Jeroen; van Blitterswijk, Clemens; de Boer, Jan

    2013-01-01

    3D cell culture methods confer a high degree of clinical and biological relevance to in vitro models. This is specifically the case with the spheroid culture, where a small aggregate of cells grows free of foreign materials. In spheroid cultures, cells secrete the extracellular matrix (ECM) in which

  13. [The modulating role of essential and non-essential amino acids in organotypic tissue culture in rats of different ages].

    Science.gov (United States)

    Chalisova, N I; Penniiaĭnen, V A

    2003-05-01

    The effect of amino acids L-lysin, L-asparagin, L-arginin, L-glutamate was investigated in organotypic tissue culture of spleen, liver and brain cortex of rats at different age. The amino acids in concentration 0.05 ng/ml are active inducing a less intensive growth zone, as compared to control, in 1-day and in older rats--an intensive growth zone, as compared to control in 21-day rats. The data obtained suggest a modulating role of amino acids in the tissues at different stages of maturation.

  14. Growth of plant tissue cultures in simulated lunar soil: Implications for a lunar base Controlled Ecological Life Support System (CELSS)

    Science.gov (United States)

    Venketeswaran, S.

    1987-01-01

    Experiments to determine whether plant tissue cultures can be grown in the presence of simulated lunar soil (SLS) and the effect of simulated lunar soil on the growth and morphogenesis of such cultures, as well as the effect upon the germination of seeds and the development of seedlings were carried out . Preliminary results on seed germination and seedling growth of rice and calli growth of winged bean and soybean indicate that there is no toxicity or inhibition caused by SLS. SLS can be used as a support medium with supplements of certain major and micro elements.

  15. Adherence of coagulase-negative staphylococci to plastic tissue culture plates: a quantitative model for the adherence of staphylococci to medical devices.

    OpenAIRE

    Christensen, G D; Simpson, W A; Younger, J J; Baddour, L M; Barrett, F F; Melton, D M; Beachey, E H

    1985-01-01

    The adherence of coagulase-negative staphylococci to smooth surfaces was assayed by measuring the optical densities of stained bacterial films adherent to the floors of plastic tissue culture plates. The optical densities correlated with the weight of the adherent bacterial film (r = 0.906; P less than 0.01). The measurements also agreed with visual assessments of bacterial adherence to culture tubes, microtiter plates, and tissue culture plates. Selected clinical strains were passed through ...

  16. New method for the study of Amaryllidaceae alkaloid biosynthesis using biotransformation of deuterium-labeled precursor in tissue cultures

    International Nuclear Information System (INIS)

    Tahchy, A. E.; Boisbrun, M.; Chretien, F.; Henry, M.; Chapleur, Y.; Laurain-Mattar, D.; Ptak, A.; Dupire, F.

    2010-01-01

    Biotransformation of deuterated-4'-O-methylnorbelladine into alkaloids galanthamine and lycorine in tissue cultures of Leucojum aestivum was demonstrated using HPLC coupled to mass spectrometry. GC-MS screening was also carried to investigate other native and deuterated alkaloids. A total of six labeled alkaloids were identified indicating that 4'-O-methyl-d3-norbelladine is incorporated into three different groups of Amaryllidaceae alkaloids that are biosynthesized by three modes of intramolecular oxidative phenol coupling. (authors)

  17. Screening Test of Greenhouse Seeding Exercise Matrix for Tissue Culture Seeding of Dendrobium Officinale Kimura et Migo

    Directory of Open Access Journals (Sweden)

    Zhou Yuan

    2015-01-01

    Full Text Available The Dendrobium officinale Kimura et Migo has a high demand on planting matrix, while its tissue culture seeding has much more demands on planting matrix. To find out a seeding exercise matrix to enhance the survival rate of tissue culture seeding of Dendrobium officinale Kimura et Migo more efficiently, this article carries out a screening test of greenhouse seeding exercise matrix material for tissue culture seeding of Dendrobium officinale Kimura et Migo. The test adopts full random test design, mainly for screening test of five matrix materials, namely pine bark, camphor tree bark, fern root, peanut shell and longan bark. Compare the impact of prepared seeding exercise matrix on the survival rate and growth trend (including plant height, growth rate and bud growth rate. The test result shows that: The seeding exercise matrix prepared by fern root is the most efficient, and the survival rate, plant height, growth rate and bud growth rate have achieved 100%, 4.5cm, 43.67% and 54.33% respectively. The main reason may be that the seeding exercise matrix C prepared by fern root is fairly loose and has a great water permeability, which is conducive to the growth of Dendrobium officinale Kimura et Migo.

  18. ASSESSMENT OF MOLECULAR GENETIC STABILITY BETWEEN LONG-TERM CRYOPRESERVED AND TISSUE CULTURED WASABI (Wasabia japonica) PLANTS.

    Science.gov (United States)

    Maki, S; Hirai, Y; Niino, T; Matsumoto, T

    2015-01-01

    Maintaining the genetic integrity in long-term tissue cultured and cryopreserved plants is important for the conservation of plant genetic resources. In this study, the genetic stability of cryopreserved wasabi shoot tips stored for 10 years at -150 degree C was visualized using Amplified Fragment Length Polymorphism (AFLP) and Methylation Sensitive Amplified Polymorphism (MSAP). The study included plants derived from cryopreserved shoot tips after 10.5 years storage at -150 degree C (LN10yr), after 2 h storage at -196 degree C (LN2hr), cryopreservation controls (No LN cooling (TC)) and non-treated controls without LN cooling (LC). The donor plants for LN2hr, TC and LC were also maintained in vitro at 20 degree C for the same period. Neither technique detected genetic variations in either control or cryopreserved plants. Some mutations were noted in plants maintained in tissue culture for 10 years. Comparison of genome stability for TC and LN2hr plants showed only a minor change in DNA. However, when comparing the LC and Ln10yr, many differences were found. We conclude that cryopreservation is a superior conservation method compared to tissue culture in maintaining genetic stability for a long-term storage of wasabi germplasm.

  19. Royal Jelly Prevents Osteoporosis in Rats: Beneficial Effects in Ovariectomy Model and in Bone Tissue Culture Model

    Directory of Open Access Journals (Sweden)

    Saburo Hidaka

    2006-01-01

    Full Text Available Royal jelly (RJ has been used worldwide for many years as medical products, health foods and cosmetics. Since RJ contains testosterone and has steroid hormone-type activities, we hypothesized that it may have beneficial effects on osteoporosis. We used both an ovariectomized rat model and a tissue culture model. Rats were divided into eight groups as follows: sham-operated (Sham, ovariectomized (OVX, OVX given 0.5% (w/w raw RJ, OVX given 2.0% (w/w RJ, OVX given 0.5% (w/w protease-treated RJ (pRJ, OVX given 2.0% (w/w pRJ, OVX given 17β-estradiol and OVX given its vehicle, respectively. The Ovariectomy decreased tibial bone mineral density (BMD by 24%. Administration of 17β-estradiol to OVX rats recovered the tibial BMD decrease by 100%. Administration of 2.0% (w/w RJ and 0.5–2.0% (w/w pRJ to OVX rats recovered it by 85% or more. These results indicate that both RJ and pRJ are almost as effective as 17β-estradiol in preventing the development of bone loss induced by ovariectomy in rats. In tissue culture models, both RJ and pRJ increased calcium contents in femoral-diaphyseal and femoral-metaphyseal tissue cultures obtained from normal male rats. However, in a mouse marrow culture model, they neither inhibited the parathyroid hormone (PTH-induced calcium loss nor affected the formation of osteoclast-like cells induced by PTH in mouse marrow culture system. Therefore, our results suggest that both RJ and pRJ may prevent osteoporosis by enhancing intestinal calcium absorption, but not by directly antagonizing the action of PTH.

  20. Evaluation of the effects of titanium dioxide nanoparticles on cultured Rana catesbeiana tailfin tissue.

    Science.gov (United States)

    Hammond, S Austin; Carew, Amanda C; Helbing, Caren C

    2013-01-01

    Nanoparticles (NPs), materials that have one dimension less than 100 nm, are used in manufacturing, health, and food products, and consumer products including cosmetics, clothing, and household appliances. Their utility to industry is derived from their high surface-area-to-volume ratios and physico-chemical properties distinct from their bulk counterparts, but the near-certainty that NPs will be released into the environment raises the possibility that they could present health risks to humans and wildlife. The thyroid hormones (THs), thyroxine, and 3,3',5-triiodothyronine (T3), are involved in development and metabolism in vertebrates including humans and frogs. Many of the processes of anuran metamorphosis are analogous to human post-embryonic development and disruption of TH action can have drastic effects. These shared features make the metamorphosis of anurans an excellent model for screening for endocrine disrupting chemicals (EDCs). We used the cultured tailfin (C-fin) assay to examine the exposure effects of 0.1-10 nM (~8-800 ng/L) of three types of ~20 nm TiO2 NPs (P25, M212, M262) and micron-sized TiO2 (μ TiO2) ±10 nM T3. The actual Ti levels were 40.9-64.7% of the nominal value. Real-time quantitative polymerase chain reaction (QPCR) was used to measure the relative amounts of mRNA transcripts encoding TH-responsive THs receptors (thra and thrb) and Rana larval keratin type I (rlk1), as well as the cellular stress-responsive heat shock protein 30 kDa (hsp30), superoxide dismutase (sod), and catalase (cat). The levels of the TH-responsive transcripts were largely unaffected by any form of TiO2. Some significant effects on stress-related transcripts were observed upon exposure to micron-sized TiO2, P25, and M212 while no effect was observed with M262 exposure. Therefore, the risk of adversely affecting amphibian tissue by disrupting TH-signaling or inducing cellular stress is low for these compounds relative to other previously-tested NPs.

  1. Evaluation of the effects of titanium dioxide nanoparticles on cultured Rana catesbeiana tailfin tissue

    Directory of Open Access Journals (Sweden)

    S. Austin eHammond

    2013-11-01

    Full Text Available Nanoparticles (NPs, materials that have one dimension less than 100 nm, are used in manufacturing, health and food products, and consumer products including cosmetics, clothing and household appliances. Their utility to industry is derived from their high surface-area-to-volume ratios and physico-chemical properties distinct from their bulk counterparts, but the near-certainty that NPs will be released into the environment raises the possibility that they could present health risks to humans and wildlife. The thyroid hormones (THs, thyroxine and 3,3’,5-triiodothyronine (T3, are involved in development and metabolism in vertebrates including humans and frogs. Many of the processes of anuran metamorphosis are analogous to human post-embryonic development and disruption of TH action can have drastic effects. These shared features make the metamorphosis of anurans an excellent model for screening for endocrine disrupting chemicals (EDCs. We used the cultured tailfin (C-fin assay to examine the exposure effects of 0.1-10 nM (~8-800 ng/L of three types of ~20 nm TiO2 NPs (P25, M212, M262 and micron-sized TiO2 (μTiO2 ±10 nM T3. The actual Ti levels were 40.9 – 64.7% of the nominal value. Real-time quantitative polymerase chain reaction (QPCR was used to measure the relative amounts of mRNA transcripts encoding TH-responsive thyroid hormone receptors (thra and thrb and Rana larval keratin type I (rlk1, as well as the cellular stress-responsive heat shock protein 30 kDa (hsp30, superoxide dismutase (sod, and catalase (cat. The levels of the TH-responsive transcripts were largely unaffected by any form of TiO2. Some significant effects on stress-related transcripts were observed upon exposure to micron-sized TiO2, P25 and M212 while no effect was observed with M262 exposure. Therefore the risk of adversely affecting amphibian tissue by disrupting TH-signalling or inducing cellular stress is low for these compounds relative to other previously

  2. 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.

  3. Diagnostic accuracy of morphologic identification of filamentous fungi in paraffin embedded tissue sections: Correlation of histological and culture diagnosis

    Directory of Open Access Journals (Sweden)

    Sundaram Challa

    2014-01-01

    Full Text Available Aims and Objectives: The aim was to investigate the correlation between histological and culture diagnosis of filamentous fungi. Materials and Methods: Tissue sections from biopsy samples stained with Hematoxylin and Eosin and special stains from samples of chronic invasive/noninvasive sinusitis and intracranial space occupying lesions during 2005-2011 diagnosed to have infection due to filamentous fungi were reviewed. The histopathology and culture diagnoses were analyzed for correlation and discrepancy. Results: There were 125 samples positive for filamentous fungi on biopsy. Of these 76 (60.8% were submitted for culture and fungi grew in 30 (39.97% samples. There was a positive correlation between histological and culture diagnosis in 25 (83.33% samples that included Aspergillus species (16/19, Zygomycetes species (8/10 and dematiaceous fungi (1/1. The negative yield of fungi was more in Zygomycetes species (20/30 when compared to Aspergillus species (25/44. There was a discrepancy in diagnosis in 5/30 (16.67% samples which included probable dual infection in two, and dematiaceous fungi being interpreted as Aspergillus species in three samples. Conclusion: Histopathology plays a major role in the diagnosis of infection due to filamentous fungi, especially when cultures are not submitted or negative. The discrepancy between histological and culture diagnosis was either due to dematiaceous fungi being interpreted as Aspergillus species or probable dual infection.

  4. Profound improvements of isolated microspores culture techniques ...

    African Journals Online (AJOL)

    We studied the effects of sampling stages, physical conditions (like temperature), culture conditions, embryo long-distance transportation methodology and plantlet regeneration on isolated microspores from donor plants in field. Results indicated that if microspores were sampled in bud stage instead of blooming stage to ...

  5. Modulation of cell differentiation in bone tissue engineering constructs cultured in a bioreactor.

    NARCIS (Netherlands)

    Holtorf, H.L.; Jansen, J.A.; Mikos, A.G.

    2006-01-01

    In summary, many factors can influence the osteoblastic differentiation of marrow stromal cells when cultivated on three-dimensional tissue engineering scaffolds. In creating ideal bone tissue engineering constructs consisting of a combination of a scaffold, cells, and bioactive factors; a flow

  6. Modified tissue culture medium for corneal storage I. Investigation of the effect of antibiotics on bacterial contamination.

    Science.gov (United States)

    Liesegang, T J; Robinson, N; Jones, D B

    1984-04-01

    A series of experiments were performed to determine the effect of modified tissue culture medium (MTCM), temperature, time, and antibiotics on the replication of Staphylococcus epidermidis and Pseudomonas aeruginosa. A known inoculum of the organisms (usually 10(6)/mL) was introduced, and the recovery of viable organisms was compared by different methods of culturing, including techniques of diluting, inactivating, or removing the antibiotic from the system. The invalidity of techniques that failed to remove the antibiotic from the system was substantiated. The newly developed antibiotic removal device was superior to all other culturing techniques. Penicillin G potassium was ineffective in the MTCM system, and gentamicin reagent solution was not uniformly effective against a gentamicin-sensitive strain of P aeruginosa. Placement of the MTCM at room temperature for two hours enhanced the antibacterial activity of gentamicin, although organisms may still be recovered in small numbers.

  7. Effects of cyclic compression on the mechanical properties and calcification process of immature chick bone tissue in culture.

    Science.gov (United States)

    Maeda, Eijiro; Nakagaki, Masashi; Ichikawa, Katsuhisa; Nagayama, Kazuaki; Matsumoto, Takeo

    2017-06-01

    Contribution of mechanical loading to tissue growth during both the development and post-natal maturation is of a particular interest, as its understanding would be important to strategies in bone tissue engineering and regenerative medicine. The present study has been performed to investigate how immature bone responds to mechanical loading using an ex vivo culture system. A slice of the tibia, with the thickness of 3 mm, was obtained from 0-day-old chick. For the ex vivo culture experiment in conjunction with cyclic compressive loading, we developed a custom-made, bioreactor system where both the load and the deformation applied to the specimen was recorded. Cyclic compression, with an amplitude of 0.3 N corresponding to 1 to 2% compressive strain, was applied to immature bone specimen during a 3-day culture period at an overall loading rate 3-4 cycles/min, in the presence of β-glycerol phosphate and dexamethasone in culture medium. The stress-strain relationship was obtained at the beginning and the end of the culture experiment. In addition, analyses for alkaline phosphate release, cell viability and tissue calcification were also performed. It was exhibited that elastic moduli of bone slices were significantly elevated at the end of the 3-day culture in the presence of cyclic compression, which was a similar phenomenon to significant elevation of the elastic moduli of bone tissue by the maturation from 0-day old to 3-day old. By contrast, no significant changes in the moduli were observed in the absence of cyclic compression or in deactivated, cell-free samples. The increases in the moduli were coincided with the increase in calcified area in the bone samples. It was confirmed that immature bone can respond to compressive loading in vitro and demonstrate the growth of bone matrix, similar to natural, in vivo maturation. The elevation of the elastic moduli was attributable to the increased calcified area and the realignment of collagen fibers parallel to

  8. Effects of cyclic compression on the mechanical properties and calcification process of immature chick bone tissue in culture

    Directory of Open Access Journals (Sweden)

    Eijiro Maeda

    2017-06-01

    Full Text Available Contribution of mechanical loading to tissue growth during both the development and post-natal maturation is of a particular interest, as its understanding would be important to strategies in bone tissue engineering and regenerative medicine. The present study has been performed to investigate how immature bone responds to mechanical loading using an ex vivo culture system. A slice of the tibia, with the thickness of 3 mm, was obtained from 0-day-old chick. For the ex vivo culture experiment in conjunction with cyclic compressive loading, we developed a custom-made, bioreactor system where both the load and the deformation applied to the specimen was recorded. Cyclic compression, with an amplitude of 0.3 N corresponding to 1 to 2% compressive strain, was applied to immature bone specimen during a 3-day culture period at an overall loading rate 3–4 cycles/min, in the presence of β-glycerol phosphate and dexamethasone in culture medium. The stress-strain relationship was obtained at the beginning and the end of the culture experiment. In addition, analyses for alkaline phosphate release, cell viability and tissue calcification were also performed. It was exhibited that elastic moduli of bone slices were significantly elevated at the end of the 3-day culture in the presence of cyclic compression, which was a similar phenomenon to significant elevation of the elastic moduli of bone tissue by the maturation from 0-day old to 3-day old. By contrast, no significant changes in the moduli were observed in the absence of cyclic compression or in deactivated, cell-free samples. The increases in the moduli were coincided with the increase in calcified area in the bone samples. It was confirmed that immature bone can respond to compressive loading in vitro and demonstrate the growth of bone matrix, similar to natural, in vivo maturation. The elevation of the elastic moduli was attributable to the increased calcified area and the realignment of collagen

  9. Characteristics of micro-propagated banana (Musa spp.) cultures ...

    African Journals Online (AJOL)

    Administrator

    2011-05-23

    May 23, 2011 ... was conducted to assess the effect of NaCl and PEG separately as well as in combination on plant micro- propagation efficiency of banana (Musa spp.) cv., Basrai. In this experiment, 4-weeks old plantlets of the 3rd sub- culture with well propagation on MS2b nutrient were sub- cultured on three differentially ...

  10. Culture of human vascular endothelial cells on a positively charged polystyrene surface, primaria: comparison with fibronectin-coated tissue culture grade polystyrene.

    Science.gov (United States)

    Klein-Soyer, C; Hemmendinger, S; Cazenave, J P

    1989-03-01

    Two culture surfaces, fibronectin-coated tissue culture grade polystyrene and a surface-modified polystyrene called Primaria (Falcon), were compared. The morphological (contact inhibition and cobblestone aspect), biological (production of von Willebrand factor and prostacyclin) and physiological (growth activity, non-thombogenicity and regeneration after mechanical injury) properties of human endothelial cells were studied. Adhesion and growth of endothelial cells at low and clonal density were identical on both substrates and the biological properties were preserved. Regeneration of injured endothelium was less easy to study on Primaria polystyrene because the extracellular matrix was damaged during the lesion process. Nevertheless, Primaria polystyrene can easily be substituted for fibronectin coating in growth experiments, especially at very low seeding density.

  11. Aseptic germination and rapid propagation by tissue culture from space returned seeds of Rehmannia glutinosa f. hueiching ensis (Chan et Sehih) Hsiao

    International Nuclear Information System (INIS)

    Li Mingjun; Zhang Xiaoli; Wang Fengjuan; Zhang Nan; Li Xiang; Deng Li; Sun Ruibin

    2009-01-01

    Aseptic germination of Rehmannia glutinosa f. hueichingensis (Chan et Sehih) Hsiao seeds space returned and rapid propagation of plantlets were studied. The results were as follows: (1) When the sterilization time of 0.05% HgCl 2 was 5 s, both the germination rate and the germination energy of the seeds reached the maximum, which were 68.33% and 53.33% respectively; (2) The best aseptic germination medium of the seeds was 1/4MS, the germination rate and the germination energy of the seeds were 76.67% and 65.08%; (3) The optimum rapid propagation medium of the plantlets was MS + 6-BA 1mg/L + NAA 0.002 mg/L, after grew in the medium for 28 d, height of the plantlets increased 7.85 cm, the propagation coefficient could achieve 6.56. (authors)

  12. Tissue engineering approaches to develop decellularized tendon matrices functionalized with progenitor cells cultured under undifferentiated and tenogenic conditions

    Directory of Open Access Journals (Sweden)

    Daniele D’Arrigo

    2017-11-01

    Full Text Available Tendon ruptures and retractions with an extensive tissue loss represent a major clinical problem and a great challenge in surgical reconstruction. Traditional approaches consist in autologous or allogeneic grafts, which still have some drawbacks. Hence, tissue engineering strategies aimed at developing functionalized tendon grafts. In this context, the use of xenogeneic tissues represents a promising perspective to obtain decellularized tendon grafts. This study is focused on the identification of suitable culture conditions for the generation of reseeded and functional decellularized constructs to be used as tendon grafts. Equine superficial digital flexor tendons were decellularized, reseeded with mesenchymal stem cells (MSCs from bone marrow and statically cultured in two different culture media to maintain undifferentiated cells (U-MSCs or to induce a terminal tenogenic differentiation (T-MSCs for 24 hours, 7 and 14 days. Cell viability, proliferation, morphology as well as matrix deposition and type I and III collagen production were assessed by means of histological, immunohistochemical and semi-quantitative analyses. Results showed that cell viability was not affected by any culture conditions and active proliferation was maintained 14 days after reseeding. However, seeded MSCs were not able to penetrate within the dense matrix of the decellularized tendons. Nevertheless, U-MSCs synthesized a greater amount of extracellular matrix rich in type I collagen compared to T-MSCs. In spite of the inability to deeply colonize the decellularized matrix in vitro, reseeding tendon matrices with U-MSCs could represent a suitable method for the functionalization of biological constructs, considering also any potential chemoattractant capability of the newly deposed extracellular matrix to recruit resident cells. This bioengineering approach can be exploited to produce functionalized tendon constructs for the substitution of large tendon defects.

  13. Absence of maternal cell contamination in mesenchymal stromal cell cultures derived from equine umbilical cord tissue

    Czech Academy of Sciences Publication Activity Database

    Vacková, Irena; Czerneková, V.; Tománek, M.; Navrátil, J.; Moško, Tibor; Nováková, Z.

    2014-01-01

    Roč. 35, č. 8 (2014), s. 655-657 ISSN 0143-4004 Institutional support: RVO:68378041 Keywords : maternal cell contamination * mesenchymal stromal cells * umbilical cord tissue Subject RIV: FH - Neurology Impact factor: 2.710, year: 2014

  14. Organotypic three-dimensional culture model of mesenchymal and epithelial cells to examine tissue fusion events.

    Science.gov (United States)

    Tissue fusion during early mammalian development requires coordination of multiple cell types, the extracellular matrix, and complex signaling pathways. Fusion events during processes including heart development, neural tube closure, and palatal fusion are dependent on signaling ...

  15. Distribution of PLGA-modified nanoparticles in 3D cell culture models of hypo-vascularized tumor tissue.

    Science.gov (United States)

    Sims, Lee B; Huss, Maya K; Frieboes, Hermann B; Steinbach-Rankins, Jill M

    2017-10-05

    Advanced stage cancer treatments are often invasive and painful-typically comprised of surgery, chemotherapy, and/or radiation treatment. Low transport efficiency during systemic chemotherapy may require high chemotherapeutic doses to effectively target cancerous tissue, resulting in systemic toxicity. Nanotherapeutic platforms have been proposed as an alternative to more safely and effectively deliver therapeutic agents directly to tumor sites. However, cellular internalization and tumor penetration are often diametrically opposed, with limited access to tumor regions distal from vasculature, due to irregular tissue morphologies. To address these transport challenges, nanoparticles (NPs) are often surface-modified with ligands to enhance transport and longevity after localized or systemic administration. Here, we evaluate stealth polyethylene-glycol (PEG), cell-penetrating (MPG), and CPP-stealth (MPG/PEG) poly(lactic-co-glycolic-acid) (PLGA) NP co-treatment strategies in 3D cell culture representing hypo-vascularized tissue. Smaller, more regularly-shaped avascular tissue was generated using the hanging drop (HD) method, while more irregularly-shaped masses were formed with the liquid overlay (LO) technique. To compare NP distribution differences within the same type of tissue as a function of different cancer types, we selected HeLa, cervical epithelial adenocarcinoma cells; CaSki, cervical epidermoid carcinoma cells; and SiHa, grade II cervical squamous cell carcinoma cells. In HD tumors, enhanced distribution relative to unmodified NPs was measured for MPG and PEG NPs in HeLa, and for all modified NPs in SiHa spheroids. In LO tumors, the greatest distribution was observed for MPG and MPG/PEG NPs in HeLa, and for PEG and MPG/PEG NPs in SiHa spheroids. Pre-clinical evaluation of PLGA-modified NP distribution into hypo-vascularized tumor tissue may benefit from considering tissue morphology in addition to cancer type.

  16. binding characteristics of 125I-labelled tetanus toxin to primary tissue cultures from mouse embryonic CNS

    International Nuclear Information System (INIS)

    Dimpfel, W.; Habermann, E.

    1977-01-01

    The interaction of 125 I-labelled tetanus toxin with cells in tissue cultures derived from embryonic CNS has been studied. The optimum toxin binding occurs about 2 to 3 weeks after transfer of the cells to culture conditions. The amount of label bound per culture was doubled at this time in comparison to the fourth day after inoculation. The amount of toxin bound depended on the concentration applied. It reached its maximum 8 h after application then decreased slowly. Low amounts of radioactivity were still detectable 97 h after washing off the unbound toxin. Up to 80% of the label could be replaced by simultaneous application of 'cold' toxin. Fixation of the toxin was higher at 4 0 C than at 37 0 C. Preincubation of the cultures with neuraminidase prevented about 75% of the binding. The presence of cytochalasin B led to a small but reproducible decrease of binding, whereas colchicine had no measurable effect. The radioactive ( 125 I) material was identified by a double-isotope technique in disc gel electrophoresis before and after reductive cleavage of its disulphide bonds. In every test it was indistinguishable from 131 I-labelled toxin added as standard. These results largely parallel those obtained with synaptosomes and other systems. They suggest that gangliosides might be the acceptor molecules, and that the culture system will be suitable for studying the actions of this toxin in vitro. (author)

  17. Cytokeratin expression of engrafted three-dimensional culture tissues using epithelial cells derived from porcine periodontal ligaments.

    Science.gov (United States)

    Yamada, Rie; Kitajima, Kayoko; Arai, Kyoko; Igarashi, Masaru

    2014-09-01

    This study investigated the differentiation and proliferation of epithelial cells derived from periodontal ligaments after three-dimensional culture using collagen gel with fibroblasts in vitro and in vivo. Epithelial cells and fibroblasts were derived from porcine periodontal ligaments. Epithelial cells were labeled using a fluorescent red membrane marker (PKH-26GL) and were seeded onto collagen gel with fibroblasts, followed by incubation in an air-liquid interface for 7 days. Three-dimensional cultures were grafted onto the backs of nude mice and removed at 1, 7, and 14 days after surgery (in vivo model). Unfixed sections (5 μm) were used to detect the presence of red fluorescent cells. Paraffin sections were analyzed histologically and immunohistochemically. Specimens were compared with three-dimensional culture tissues at 8, 14 and 21 days (in vitro model). Grafted three-dimensional cultures formed a stratified epithelial structure similar to skin in vivo. Epithelial cells were sequenced in basal-layer-like structures at 14 days in vivo. Immunohistochemical findings showed that the expression of cytokeratin was detected in the epithelial layer in in vitro and in vivo models. Ck8 + 18 + 19 was expressed in the upper epithelial layer in the in vitro model at 14 and 21 days, but not in vivo. Involucrin was expressed in the certified layers in vitro at 14 days, but not in vivo. Laminin was detected at the dermo-epidermal junction in vivo at 7 and 14 days, but not in vitro. These results suggest that differentiation of three-dimensional culture tissues differs in vivo and in vitro. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  18. Development of tissue-culture methods for the in-vitro evaluation of polysaccharide wound management products

    Energy Technology Data Exchange (ETDEWEB)

    Spyratou, L.

    1987-01-01

    The use of normal human fibroblasts, mouse connective tissue fibroblasts (L929), and guinea-pig epidermal cells in culture, as possible systems for evaluating wound management products was investigated. This necessitated the development of a biological assay system for comparing the effects of wound management products on the skin cell lines. Polysaccharide based wound management products were selected for this study. Published tissue-culture assay methods were found to be inadequate, hence a new biological assay system was developed and validated. The haemacytometer chamber, the Coulter counter and the incorporation of /sup 3/(H)-thymidine as methods for assessing growth were compared. It was found that final cell growth in control culture was dependent on the frequency of change of media, on substrate area, and on initial cell number, which at confluency was found to be related to the final yield of cells by equation y = 1420 * 10/sup (/minus/0.004x)/. Representative monosaccharides and polysaccharides were tested in addition to the wound management products.

  19. Irradiation effect on in vitro organogenesis, callus growth and plantlet development of Gerbera jamesonii Efeito da irradiação na organogênese in vitro, crescimento de calos e desenvolvimento de plântulas de gerbera

    Directory of Open Access Journals (Sweden)

    Nor A Hasbullah

    2012-06-01

    Full Text Available The present work was carried out to study the effects of gamma irradiation on in vitro growth of explants, callus and the formation of shoots and plantlets. Irradiation is known to exhibit or inhibit the differentiation of cells and growth of plants in vitro, which helps in producing new plant varieties. Gamma irradiation is one of the physical mutagens that are widely used for mutation breeding. A gradual decline was observed in the number of shoots regenerated from irradiated petiole explants compared to control. Numbers of shoots regenerated from irradiated petiole explant cultured on Murashige & Skoog medium supplemented with 2.0 mg L-1 BAP and 0.5 mg L-1 NAA was reduced to 6.6±0.9 from 7.5±0.4 (control when explants were exposed to 20 Gray of irradiation dose. Similar observation was reported on effects of gamma irradiation on in vitro propagated plantlets. Gradual decline was observed based on plant height as the dose of gamma irradiation increased. A significant decline was observed in the fresh weight of irradiated callus compared to control. In this case, growth responses of callus were strongly influenced by the radiation dose. The fresh weight of callus was reduced to 76.4±2.2% compared to 89.7±0.5% of control when callus tissues were exposed to 20 Gy.O presente trabalho foi realizado para estudar os efeitos da radiação gama no crescimento in vitro de explantes de calos, e a formação de brotos e mudas. A irradiação é conhecida por induzir ou inibir a diferenciação de células e o crescimento das plantas in vitro, o que ajuda na produção de novas variedades vegetais. Radiação gama é um dos agentes mutagenicos que são amplamente utilizados para o melhoramento através da mutação. Um declínio gradual foi observado no número de brotos regenerados a partir de explantes de pecíolos irradiados comparado com o controle. O número de brotações regeneradas de explantes de pecíolos irradiados, cultivados em meio

  20. Desenvolvimento de mudas de bromélia (Neoregelia cruenta (R. Graham L. B. Smith cultivadas em diferentes substartos e adubação foliar Development of plantlets of bromeliad (Neoregelia cruenta cultivated in different substrates and leaf fertilizers

    Directory of Open Access Journals (Sweden)

    Clarissa Alves Ferreira

    2007-06-01

    extraction. This situation is also a consequence of the reduced technical information regarding its propagation and development. One of the limitations is the lack of knowledge regarding the type of substrate and adequate fertilizer for its cultivation. The objective of this study was to evaluate the development of Neoregelia cultivated in different substrates and leaf fertilizers through the variables height, leaf number and fresh and dry matter of shoots and roots. The plantlets used were produced by tissue culture. After a period of pre-acclimatization, the plantlets were transferred to a green house without nebulization. The substrates consisted of different proportions of soil, sand and carbonized rice husk and a commercial substrate composed of vermiculite. Leaf fertilizers were applied to the substrates combining urea and sucrose in intervals of fifteen days. The results showed that the interactions of the substrates and the sucrose and urea dosages had no effect on height and leaf number. The use of sucrose also showed no influence on plant development. The commercial substrate composed of vermiculite, independent of the leaf fertilizer application, showed the best results for plant height and leaf number. The use of urea presented a crescent linear effect during the evaluated period.

  1. Trichomonas gallinae, in comparison to Tetratrichomonas gallinarum, induces distinctive cytopathogenic effects in tissue cultures.

    Science.gov (United States)

    Amin, Aziza; Bilic, Ivana; Berger, Evelyn; Hess, Michael

    2012-05-25

    In the present study the interaction of three genetically different clonal cultures of Trichomonas gallinae and Tetratrichomonas gallinarum with a permanent chicken liver (LMH) and a permanent quail fibroblast (QT35) cell culture was studied. Proliferation of T. gallinae cells was associated with a disintegration of the cell monolayer. The initial lesions on the LMH monolayer consisted of a progressive accumulation of the flagellate, forming clumps attached to the monolayer. A prolonged incubation time was characterized by appearance of holes in the cell monolayer with accumulation of trichomonads at their periphery. According to the severeness of the monolayer disruption differences among three tested T. gallinae clones were noticed. Furthermore, filtrates obtained either from axenic cultures of T. gallinae or from infected cell cultures produced a cytopathogenic effect similar to the protozoal cells, on both types of cell cultures. However, the destructive effect of the flagellates and their cell-free filtrates was much more pronounced on the LMH monolayer in comparison with the QT35 cells. Furthermore, freshly seeded LMH and QT35 cells suspended in cell-free filtrates of T. gallinae were unable to form a confluent monolayer. In comparison to T. gallinae, clonal cultures of T. gallinarum or their cell-free filtrates produced no effect on both types of monolayers. Interestingly, the cell-free filtrates obtained from both trichomonad species had an effect on the viability of both cell cultures. However, the cytotoxic effect of T. gallinarum filtrates was less severe than that recorded by T. gallinae. Consequently, for the first time a destruction of specified monolayers induced by T. gallinae-free filtrates could be demonstrated. Copyright © 2011 Elsevier B.V. All rights reserved.

  2. How Age of Transplants from In Vitro Derived Potato Plantlets Affects Crop Growth and Seed Tuber Yield After Field Transplanting

    NARCIS (Netherlands)

    Lommen, W.J.M.

    2015-01-01

    In vitro produced plantlets are used in potato seed systems for production of minitubers under protected conditions or for production of transplants to be transplanted to the field. Three field experiments were carried out to analyse how transplant age (Age) affected the field performance. In the

  3. Regeneration and acclimatization of salt-tolerant arachis hypogaea plants through tissue culture

    International Nuclear Information System (INIS)

    Ghauri, E.G.

    2006-01-01

    Excised embryos of Arachis hypogaea were cultured on Murashige and Skoog's medium (MS medium) supplemented with different combinations of growth hormones. The highest frequency of callus proliferation (80%) was recorded on MS medium mixed with 1.0 mg/1 of 2,4-D and 0.5 mg/1 of BAP. These cultures were treated with 0.65 mg/l of trans-4-hydroxy-L-proline (HyP) a:1d various concentrations (0.1-0.5%) of NaCl. In all cases the presence of salt reduced the fresh mass of callus. Shoot regeneration in the cultures took place when transferred to MS medium supplemented with 1.0 mg/1 of kinetin (Kin) and 0.5 mg/1 of 6-benzyl aminopurine (BAP). Percentage of shoot regeneration decreased with the increase of NaCl (0.1- 0.5%) in the shoot regeneration medium. Root formation in these cultures took place when the cultures were nurtured on MS medium free of growth hormones. Regeneration, hardening and acclimatization of the salt tolerant plants was conducted. (author)

  4. Use of tissue culture cell lines to evaluate HIV antiviral resistance.

    Science.gov (United States)

    Krowicka, Halina; Robinson, James E; Clark, Rebecca; Hager, Shannon; Broyles, Stephanie; Pincus, Seth H

    2008-07-01

    Most current assays of HIV antiviral resistance are based on either sequencing of viral genes (genotypic assays) or amplification and insertion of these genes into standardized virus backbones and culture. These latter are called phenotypic assays. But the only generally accepted phenotypic assay is based upon culture of intact patient virus, performed in phytohemagglutinin-activated peripheral blood mononuclear cells (PHA blasts) in the presence of differing drug concentrations. However, PHA blast culture is difficult and not always reproducible. Therefore we have sought cell lines that may produce more predictable results, yet faithfully mirror results in PHA blasts. We have compared 10 different cell lines for receptor and coreceptor expression, growth of laboratory-adapted strains of HIV, growth by direct inoculation of PBMC from infected patients, and in assays of antiviral drug effects. One of these cell lines, C8166-R5, is statistically not inferior to CD8-depleted PHA blasts for culturing HIV from the peripheral blood cells of patients. The effective concentrations of antiviral drugs of all classes were similar when assayed in C8166-R5 or PHA blasts. Known drug-resistant isolates grown in C8166-R5 demonstrated the predicted effects. We followed a patient longitudinally and demonstrated that resistance testing in C8166-R5 was predictive of clinical outcome. These experiments represent the first steps in developing a clinically useful phenotypic drug resistance assay based upon culturing the patient's own virus.

  5. The use of Citrus tissue culture for mutation breeding. Effects of plant growth substances and gamma irradiation on embryogenesis

    International Nuclear Information System (INIS)

    Kochba, J.; Spiegel-Roy, P.

    1976-01-01

    An embryogenic callus subcultured from unfertilized ovules of the 'Shamouti' orange (Citrus sinensis) was established and is used for mutation-breeding. The callus is habituated and lines of differing embryogenic potential were established. The effect of growth substances and of gamma-irradiation on embryogenesis were studied. Auxins and cytokinins inhibited embryogenesis while inhibitors of auxin synthesis and a cytokinin antagonist significantly stimulate embryogenesis in an embryogenic line. A non-embryogenic callus line did not respond to these treatments. Stimulation of embryogenesis was observed when callus but not when the medium was irradiated. Age of callus prior to subculture and irradiation intensities modify irradiation induced embryogenesis by changing optimal dose range and radiosensitivity of the callus. Addition of IAA to unirradiated medium resulted in increased embryogenesis and greatly stimulated plantlet development in certain combinations of irradiation dose and IAA concentration. (author)

  6. Growth of plant tissue cultures in simulated lunar soil: Implications for a lunar base CELSS (Controlled Ecological Life Support System)

    Science.gov (United States)

    Venketeswaran, S.

    1988-01-01

    Experiments were carried out on plant tissue cultures, seed germination, seedling development and plants grown on Simulated Lunar Soil to evaluate the potential of future development of lunar based agriculture. The studies done to determine the effect of the placement of SLS on tissue cultures showed no adverse effect of SLS on tissue cultures. Although statistically insignificant, SLS in suspension showed a comparatively higher growth rate. Observations indicate the SLS, itself cannot support calli growth but was able to show a positive effect on growth rate of calli when supplemented with MS salts. This positive effect related to nutritive value of the SLS was found to have improved at high pH levels, than at the recommended low pH levels for standard media. Results from seed germination indicated that there is neither inhibitory, toxicity nor stimulatory effect of SLS, even though SLS contains high amounts of aluminum compounds compared to earth soil. Analysis of seeding development and growth data showed significant reduction in growth rate indicating that, SLS was a poor growth medium for plant life. This was confirmed by the studies done with embryos and direct plant growth on SLS. Further observations attributed this poor quality of SLS is due to it's lack of essential mineral elements needed for plant growth. By changing the pH of the soil, to more basic conditions, the quality of SLS for plant growth could be improved up to a significant level. Also it was found that the quality of SLS could be improved by almost twice, by external supply of major mineral elements, directly to SLS.

  7. Use of gel zymography to examine matrix metalloproteinase (gelatinase) expression in brain tissue or in primary glial cultures.

    Science.gov (United States)

    Frankowski, Harald; Gu, Yu-Huan; Heo, Ji Hoe; Milner, Richard; Del Zoppo, Gregory J

    2012-01-01

    Glia synthesize, package, and secrete several species of matrix proteases, including the gelatinases (pro-)MMP-2 and (pro-)MMP-9. In appropriate settings (e.g., experimental ischemia), these MMPs can be assayed from cerebral tissues or from astrocytes and microglia in culture by enzymatic substrate-dependent assays and by gelatin-based zymography. We describe the methodologies for the sensitive quantitative development of the inactive and active forms of both MMP-2 and MMP-9 from tissues and cells, by means of lysis of the collagen substrate in collagen-impregnated gel electropheresis by the zymogen and active gelatinases. These methodologies are a refinement of those used commonly, with instructions to increase sensitivity. Serious and often overlooked issues regarding sources of sample contamination and elements confounding the MMP band development and their interpretation are discussed.

  8. Differential Proteomic Analysis of Human Placenta-Derived Mesenchymal Stem Cells Cultured on Normal Tissue Culture Surface and Hyaluronan-Coated Surface

    Directory of Open Access Journals (Sweden)

    Tzyy Yue Wong

    2016-01-01

    Full Text Available Our previous results showed that hyaluronan (HA preserved human placenta-derived mesenchymal stem cells (PDMSC in a slow cell cycling mode similar to quiescence, the pristine state of stem cells in vivo, and HA was found to prevent murine adipose-derived mesenchymal stem cells from senescence. Here, stable isotope labeling by amino acid in cell culture (SILAC proteomic profiling was used to evaluate the effects of HA on aging phenomenon in stem cells, comparing (1 old and young passage PDMSC cultured on normal tissue culture surface (TCS; (2 old passage on HA-coated surface (CHA compared to TCS; (3 old and young passage on CHA. The results indicated that senescence-associated protein transgelin (TAGLN was upregulated in old TCS. Protein CYR61, reportedly senescence-related, was downregulated in old CHA compared to old TCS. The SIRT1-interacting Nicotinamide phosphoribosyltransferase (NAMPT increased by 2.23-fold in old CHA compared to old TCS, and is 0.48-fold lower in old TCS compared to young TCS. Results also indicated that components of endoplasmic reticulum associated degradation (ERAD pathway were upregulated in old CHA compared to old TCS cells, potentially for overcoming stress to maintain cell function and suppress senescence. Our data points to pathways that may be targeted by HA to maintain stem cells youth.

  9. Basic procedures for epigenetic analysis in plant cell and tissue culture.

    Science.gov (United States)

    Rodríguez, José L; Pascual, Jesús; Viejo, Marcos; Valledor, Luis; Meijón, Mónica; Hasbún, Rodrigo; Yrei, Norma Yague; Santamaría, María E; Pérez, Marta; Fernández Fraga, Mario; Berdasco, María; Rodríguez Fernández, Roberto; Cañal, María J

    2012-01-01

    In vitro culture is one of the most studied techniques, and it is used to study many developmental processes, especially in forestry species, because of growth timing and easy manipulation. Epigenetics has been shown as an important influence on many research analyses such as cancer in mammals and developmental processes in plants such as flowering, but regarding in vitro culture, techniques to study DNA methylation or chromatin modifications were mainly limited to identify somaclonal variation of the micropropagated material. Because in vitro culture is not only a way to generate plant material but also a bunch of differentially induced developmental processes, an approach of techniques and some research carried out to study the different changes regarding DNA methylation and chromatin and translational modifications that take place during these processes is reviewed.

  10. A multifunctional 3D co-culture system for studies of mammary tissue morphogenesis and stem cell biology.

    Directory of Open Access Journals (Sweden)

    Jonathan J Campbell

    Full Text Available Studies on the stem cell niche and the efficacy of cancer therapeutics require complex multicellular structures and interactions between different cell types and extracellular matrix (ECM in three dimensional (3D space. We have engineered a 3D in vitro model of mammary gland that encompasses a defined, porous collagen/hyaluronic acid (HA scaffold forming a physiologically relevant foundation for epithelial and adipocyte co-culture. Polarized ductal and acinar structures form within this scaffold recapitulating normal tissue morphology in the absence of reconstituted basement membrane (rBM hydrogel. Furthermore, organoid developmental outcome can be controlled by the ratio of collagen to HA, with a higher HA concentration favouring acinar morphological development. Importantly, this culture system recapitulates the stem cell niche as primary mammary stem cells form complex organoids, emphasising the utility of this approach for developmental and tumorigenic studies using genetically altered animals or human biopsy material, and for screening cancer therapeutics for personalised medicine.

  11. Negative-ion beam surface modification of tissue-culture polystyrene dishes for changing hydrophilic and cell-attachment properties

    International Nuclear Information System (INIS)

    Tsuji, H.; Satoh, H.; Ikeda, S.; Ikemura, S.; Gotoh, Y.; Ishikawa, J.

    1999-01-01

    Negative-silver-ion implantation into tissue-culture polystyrene (TCPS) dishes was investigated and it was found to modify hydrophilic and cell attachment properties of the dishes. Negative-ion implantation has an advantage of being almost free of surface charging, and is a suitable method for implantation into insulators such as polymers. Negative silver ions are used due to the antibacterial property of silver. Ag-implanted TCPS dishes had a contact angle larger than the normal value of 66 deg. of unimplanted dishes. The contact angle of water had a strong dependence on the ion energy rather than the dose. As a cell-culture experiment, human umbilical vascular endothelial cell (HUVEC) was used in unimplanted and Ag-implanted TCPS dishes, the implantation removed the cell-attachment property of the surface. In implantation with a mask with a striped pattern, most attached cells of HUVEC were in the unimplanted region aligned along a stripe direction

  12. Quantification of cell surface receptor expression in live tissue culture media using a dual-tracer stain and rinse approach

    Science.gov (United States)

    Xu, Xiaochun; Sinha, Lagnojita; Singh, Aparna; Yang, Cynthia; Xiang, Jialing; Tichauer, Kenneth M.

    2015-03-01

    Immunofluorescence staining is a robust way to visualize the distribution of targeted biomolecules invasively in in fixed tissues and tissue culture. Despite the fact that these methods has been a well-established method in fixed tissue imaging for over 70 years, quantification of receptor concentration still simply assumes that the signal from the targeted fluorescent marker after incubation and sufficient rinsing is directly proportional to the concentration of targeted biomolecules, thus neglecting the experimental inconsistencies in incubation and rinsing procedures and assuming no, nonspecific binding of the fluorescent markers. This work presents the first imaging approach capable of quantifying the concentration of cell surface receptor on cancer cells grown in vitro based on compartment modeling in a nondestructive way. The approach utilizes a dual-tracer protocol where any non-specific retention or variability in incubation and rinsing of a receptor-targeted imaging agent is corrected by simultaneously imaging the retention of a chemically similar, "untargeted" imaging agent. Various different compartment models were used to analyze the data in order to find the optimal procedure for extracting estimates of epidermal growth factor receptor (EGFR) concentration (a receptor overexpressed in many cancers and a key target for emerging molecular therapies) in tissue cultures with varying concentrations of human glioma cells (U251). Preliminary results demonstrated a need to model nonspecific binding of both the targeted and untargeted imaging agents used. The approach could be used to carry out the first repeated measures of cell surface receptor dynamics during 3D tumor mass development, in addition to the receptor response to therapies.

  13. Human endothelin subtype A receptor enhancement during tissue culture via de novo transcription

    DEFF Research Database (Denmark)

    Hansen-Schwartz, Jacob; Nordström, Carl-Henrik; Edvinsson, Lars

    2002-01-01

    pharmacological methods and molecular biological techniques. RESULTS: After organ culture of the cerebral arteries, both the sensitivity to and potency of ET were enhanced (maximal response, 152 +/- 9%; -log (50% effective concentration), 10.3 +/- 0.3), in comparison with data for fresh cerebral arteries...... of human cerebral arteries to change their sensitivity to ET. METHODS: Human cerebral arteries were obtained from patients undergoing intracranial tumor surgery. The vessels were divided into segments and subjected to organ culture for 48 hours. The vessels were then examined by using in vitro...

  14. Design of a microscopic electrical impedance tomography system for 3D continuous non-destructive monitoring of tissue culture.

    Science.gov (United States)

    Lee, Eun Jung; Wi, Hun; McEwan, Alistair Lee; Farooq, Adnan; Sohal, Harsh; Woo, Eung Je; Seo, Jin Keun; Oh, Tong In

    2014-10-06

    Non-destructive continuous monitoring of regenerative tissue is required throughout the entire period of in vitro tissue culture. Microscopic electrical impedance tomography (micro-EIT) has the potential to monitor the physiological state of tissues by forming three-dimensional images of impedance changes in a non-destructive and label-free manner. We developed a new micro-EIT system and report on simulation and experimental results of its macroscopic model. We propose a new micro-EIT system design using a cuboid sample container with separate current-driving and voltage sensing electrodes. The top is open for sample manipulations. We used nine gold-coated solid electrodes on each of two opposing sides of the container to produce multiple linearly independent internal current density distributions. The 360 voltage sensing electrodes were placed on the other sides and base to measure induced voltages. Instead of using an inverse solver with the least squares method, we used a projected image reconstruction algorithm based on a logarithm formulation to produce projected images. We intended to improve the quality and spatial resolution of the images by increasing the number of voltage measurements subject to a few injected current patterns. We evaluated the performance of the micro-EIT system with a macroscopic physical phantom. The signal-to-noise ratio of the developed micro-EIT system was 66 dB. Crosstalk was in the range of -110.8 to -90.04 dB. Three-dimensional images with consistent quality were reconstructed from physical phantom data over the entire domain. From numerical and experimental results, we estimate that at least 20 × 40 electrodes with 120 μm spacing are required to monitor the complex shape of ingrowth neotissue inside a scaffold with 300 μm pore. The experimental results showed that the new micro-EIT system with a reduced set of injection current patterns and a large number of voltage sensing electrodes can be potentially used for tissue

  15. Establishment of a primary hepatocyte culture from the small Indian mongoose (Herpestes auropunctatus) and distribution of mercury in liver tissue.

    Science.gov (United States)

    Horai, Sawako; Yanagi, Kumiko; Kaname, Tadashi; Yamamoto, Masatatsu; Watanabe, Izumi; Ogura, Go; Abe, Shintaro; Tanabe, Shinsuke; Furukawa, Tatsuhiko

    2014-11-01

    The present study established a primary hepatocyte culture for the small Indian mongoose (Herpestes auropunctatus). To determine the suitable medium for growing the primary hepatic cells of this species, we compared the condition of cells cultured in three media that are frequently used for mammalian cell culture: Dulbecco's Modified Eagle's Medium, RPMI-1640, and William's E. Of these, William's E medium was best suited for culturing the hepatic cells of this species. Using periodic acid-Schiff staining and ultrastructural observations, we demonstrated the cells collected from mongoose livers were hepatocytes. To evaluate the distribution of mercury (Hg) in the liver tissue, we carried out autometallography staining. Most of the Hg compounds were found in the central region of hepatic lobules. Smooth endoplasmic reticulum, which plays a role inxenobiotic metabolism, lipid/cholesterol metabolism, and the digestion and detoxification of lipophilic substances is grown in this area. This suggested that Hg colocalized with smooth endoplasmic reticulum. The results of the present study could be useful to identify the detoxification systems of wildlife with high Hg content in the body, and to evaluate the susceptibility of wildlife to Hg toxicity.

  16. Effects of auxins on the production of steroidal alkaloids in rapidly proliferating tissue and cell cultures of Solanum lyratum.

    Science.gov (United States)

    Kuo, Chung-Io; Chao, Chi-Hsein; Lu, Mei-Kuang

    2012-01-01

    Solanum lyratum, a rare species, is used to treat cancer, tumours and warts. Plant cell and tissue culture of S. lyratum, producing steroidal alkaloids, could be useful supplements to natural sources. To study the production of solanine, solanidine and solasodine by adding auxin-type phytohormones including indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) to cell and callus cultures of S. lyratum. Methanolic extracts were made from callus and cell cultures of S. lyratumand and analysed using RP C₁₈ HPLC with UV detection. 2,4-D-induced calli from roots led to a significant enhancement in solanine production with a value of 4.13 mg/g dry weight (DW). The maximal solanidine and solasodine levels of 6.26 and 7.69 mg/g DW were respectively obtained with IBA- and IAA-treated S. lyratum cells at concentrations of 1 and 5 mg/L. Auxins were found to be useful phytohormones for the production of steroidal alkaloids. The callus and cell culture system developed is simple and can hence be a method of production of steroidal alkaloids in S. lyratum and other Solanaceae species. Copyright © 2011 John Wiley & Sons, Ltd.

  17. Addressing Challenges to Enhance the Bioactives of Withania somnifera through Organ, Tissue, and Cell Culture Based Approaches

    Science.gov (United States)

    Singh, Pritika; Guleri, Rupam; Angurala, Amrita; Kaur, Kuldeep; Kaur, Kulwinder; Kaul, Sunil C.; Wadhwa, Renu

    2017-01-01

    Withania somnifera is a highly valued medicinal plant in traditional home medicine and is known for a wide range of bioactivities. Its commercial cultivation is adversely affected by poor seed viability and germination. Infestation by various pests and pathogens, survival under unfavourable environmental conditions, narrow genetic base, and meager information regarding biosynthesis of secondary metabolites are some of the other existing challenges in the crop. Biotechnological interventions through organ, tissue, and cell culture provide promising options for addressing some of these issues. In vitro propagation facilitates conservation and sustainable utilization of the existing germplasms and broadening the genetic base. It would also provide means for efficient and rapid mass propagation of elite chemotypes and generating uniform plant material round the year for experimentation and industrial applications. The potential of in vitro cell/organ cultures for the production of therapeutically valuable compounds and their large-scale production in bioreactors has received significant attention in recent years. In vitro culture system further provides distinct advantage for studying various cellular and molecular processes leading to secondary metabolite accumulation and their regulation. Engineering plants through genetic transformation and development of hairy root culture system are powerful strategies for modulation of secondary metabolites. The present review highlights the developments and sketches current scenario in this field. PMID:28299323

  18. Addressing Challenges to Enhance the Bioactives ofWithania somniferathrough Organ, Tissue, and Cell Culture Based Approaches.

    Science.gov (United States)

    Singh, Pritika; Guleri, Rupam; Angurala, Amrita; Kaur, Kuldeep; Kaur, Kulwinder; Kaul, Sunil C; Wadhwa, Renu; Pati, Pratap Kumar

    2017-01-01

    Withania somnifera is a highly valued medicinal plant in traditional home medicine and is known for a wide range of bioactivities. Its commercial cultivation is adversely affected by poor seed viability and germination. Infestation by various pests and pathogens, survival under unfavourable environmental conditions, narrow genetic base, and meager information regarding biosynthesis of secondary metabolites are some of the other existing challenges in the crop. Biotechnological interventions through organ, tissue, and cell culture provide promising options for addressing some of these issues. In vitro propagation facilitates conservation and sustainable utilization of the existing germplasms and broadening the genetic base. It would also provide means for efficient and rapid mass propagation of elite chemotypes and generating uniform plant material round the year for experimentation and industrial applications. The potential of in vitro cell/organ cultures for the production of therapeutically valuable compounds and their large-scale production in bioreactors has received significant attention in recent years. In vitro culture system further provides distinct advantage for studying various cellular and molecular processes leading to secondary metabolite accumulation and their regulation. Engineering plants through genetic transformation and development of hairy root culture system are powerful strategies for modulation of secondary metabolites. The present review highlights the developments and sketches current scenario in this field.

  19. Glucocorticoids affect 24 h clock genes expression in human adipose tissue explant cultures

    Science.gov (United States)

    To examine firstly whether CLOCK exhibits a circadian expression in human visceral (V) and subcutaneous (S) adipose tissue (AT) in vitro as compared with BMAL1 and PER2, and secondly to investigate the possible effect of the glucocorticoid analogue dexamethasone (DEX) on positive and negative clock ...

  20. Social and cultural issues in human tissue use in South Africa | de ...

    African Journals Online (AJOL)

    'One frequently encounters references in lay and professional writings of the ever expanding markets for human tissue where the body is reduced to a “source of raw material for saleable products” ... we are witnessing the global expansion of a “human body shop” …'1 ...