scholarly journals Application of Horticultural and Tissue Culture Methods for Ex Situ Conservation of Endangered Primula farinosa L.

2020 ◽  
Vol 89 (1) ◽  
Author(s):  
Ewa Sitek ◽  
Barbara Nowak ◽  
Michał Fecowicz ◽  
Zbigniew Gajewski ◽  
Piotr Dańda ◽  
...  
Keyword(s):  
Tissue Culture ◽  
Culture Conditions ◽  
Ex Situ ◽  
In Vitro Cultivation ◽  
Culture Methods ◽  
Transient Nature ◽  
Micropropagated Plants ◽  
The Difference

Our study aimed at active conservation of the last location of <em>Primula farinosa</em>, an endangered species in Poland, and assessed reproduction by seeds and plant propagation on sterile media in tissue culture conditions. We identified gibberellic acid (GA<sub data-id="subscript-1">3</sub>) as the key factor stimulating germination of <em>P. farinosa</em> seeds. Growing juvenile plants under controlled temperature of 18/16 °C day/night yielded good quality plant material without mycorrhization. In tissue culture, the most favorable medium for shoot propagation was MS supplemented with the lowest tested concentration of indole-3-butyric acid (IBA; 0.05 mg dm<sup data-id="superscript-1">−3</sup>) and 6-benzyl-aminopurine (BAP; 0.1 mg dm<sup data-id="superscript-2">−3</sup>). The rooting ability of shoots was high and comparable for all auxins used. 2C DNA content of seed-derived and micropropagated plants did not indicate any change in the ploidy level during in vitro cultivation. Plants derived from seeds and tissue cultures were compared in a 2-year study. Of all the characteristics compared, only the number of flowers per inflorescence was lower for micropropagated plants when compared with the seed-origin plants in the first year of observation. The difference was of transient nature and was not observed in the second year of the study. Effective protocols for in vivo and in vitro propagation of <em>P. farinosa</em> were developed, which can be used in practical species protection.

Tests by Tissue Culture Methods on the Nature of Immunity Transplanted Skin

1948 ◽  
Vol s3-89 (7) ◽  
pp. 239-252
Author(s):  
P. B. MEDAWAR
Keyword(s):  
Tissue Culture ◽  
Cell Division ◽  
Epidermal Cell ◽  
Cell Suspensions ◽  
Acquired Immunity ◽  
Culture Methods ◽  
Division Frequency

The transplantation of skin from one rabbit to another elicits a reaction that conforms in main outline with that of an actively acquired immunity. The experiments described in this paper were designed to test the hypothesis that the regression of such grafts is secured by the action of antibodies demonstrable in vitro. Skin from adult rabbits has therefore been cultivated in the presence of serum and growing mesenchymal tissues derived solely from rabbits heavily and specifically immunized against it. Immune sera and tissues are without effect on the survival, cell-division frequency and migratory activities of explanted skin, and agglutinins for epidermal cell suspensions are not demonstrable in immune sera. With certain stated qualifications, it has therefore been concluded that the occurrence of free antibodies is not a sufficient explanation of the regression of skin homografts in vivo.

scholarly journals 83ANALYSIS OF RAPID COOLING V. SLOW COOLING COMBINED WITH ICE CRYSTAL SEEDING FOR CRYOPRESERVATION OF PRIMATE EMBRYONIC STEM CELLS

2004 ◽  
Vol 16 (2) ◽  
pp. 163
Author(s):  
S. Baran ◽  
C. Ware
Keyword(s):  
Tissue Culture ◽  
Es Cells ◽  
Embryonic Stem ◽  
Ice Crystal ◽  
Culture Methods ◽  
Slow Cooling ◽  
Rapid Cooling ◽  
Alkaline Phosphatase Staining

Primate embryonic stem (ES) cells have the ability to self-renew indefinitely while maintaining the ability to differentiate. This unique property allows scientists to study the factors necessary for stem cell self-renewal and differentiation in vitro that reflect in vivo processes. Work with primate ES cells is handicapped by the poor survival (1–5%) of rhesus and human ES cells following standard tissue culture methods of rapid cryopreservation. The purpose of this study was to compare and contrast two cryopreservation techniques, slow cooling combined with ice crystal seeding commonly used for mammalian embryos v. rapid cooling commonly used for tissue culture, to find a method for efficient primate ES cell cryopreservation. A combination of trials was run to compare dimethyl sulfoxide (DMSO) v. ethylene glycol as a cryoprotectant, a cooling rate of 0.3°C per minute following ice crystal seeding at −7°C v. placement at −80°C with no seeding, and rapid thaw with step-wise cryoprotectant removal v. one-step sucrose cryoprotectant removal. Cell survival was assessed through a combination of cell surface markers, alkaline phosphatase staining and morphology to look for undifferentiated cells and quantitate survival. All cryopreservations were performed with the same cell density. The survival of the cells with slow embryo-style cooling in DMSO with a step-wise cryoprotectant removal was 64.0% v. 12.8% with rapid cooling.

In vitro analyses of cuticular differentiation at the chromosomal level in Lucilia sericata

Genome ◽  
1988 ◽  
Vol 30 (4) ◽  
pp. 603-607 ◽  
Author(s):  
Manfred G. Schmiemann ◽  
Michael M. Bentley1
Keyword(s):  
Culture Medium ◽  
Polytene Chromosomes ◽  
Culture Conditions ◽  
Lucilia Sericata ◽  
In Vitro Cultivation ◽  
Pupal Development ◽  
Chromosome Conformation ◽  
Tormogen Cell

The large bristles of flies sclerotize and melanize during the pupal phase well before the rest of the cuticle. Each bristle is the product of two cells, the trichogen cell and the tormogen cell. Both their nuclei exhibit analyzable polytene chromosomes. The pupal metathoracic integument of Lucilia sericata has been excised and cultivated in Schneider's Drosophila medium for extended periods of time. Among the differentiations taking place in vitro, the most obvious is the stiffening and coloring of the bristles. The in vitro differentiation very much resembles the in vivo differentiation in its timing and appearance. Explants excised early in pupal development cannot differentiate in vitro, while those excised and cultured at later stages can. Actinomycin D was administered to the culture medium and the incorporation of [3H]uridine was analyzed after long incubations to determine if this process was a reaction of dead cuticle or controlled by living cells. The analysis of chromosome structure and puffing patterns in the five polytene chromosomes of the trichogen cell after in vitro cultivation shows that transcriptional activity is not dependent on the existence of normal chromosome conformation. The degree of pycnotic reaction and puff induction of the nuclei, however, varied under different culture conditions. The capacity of this system for differentiation can be used to study in vitro chromosomal activity during the course of development.Key words: in vitro, polytene chromosomes, sclerotization, cuticle.

The regenerative capacity of the notochordal cell: tissue constructs generated in vitro under hypoxic conditions

2009 ◽  
Vol 10 (6) ◽  
pp. 513-521 ◽  
Author(s):  
W. Mark Erwin ◽  
Facundo Las Heras ◽  
Diana Islam ◽  
Michael G. Fehlings ◽  
Robert D. Inman
Keyword(s):  
Extracellular Matrix ◽  
Tissue Culture ◽  
Sodium Alginate ◽  
Cultured Cells ◽  
3D Culture ◽  
Culture Conditions ◽  
Notochordal Cells ◽  
Collagen Ii

Object The intervertebral disc (IVD) is a highly avascular structure that is occupied by highly specialized cells (nucleus pulposus [NP] cells) that have adapted to survive within an O2 concentration of 2–5%. The object of this study was to investigate the effects of long-term hypoxic and normoxic tissue cultures of nonchondrodystrophic canine notochordal cells—cells that appear to protect the disc NP from degenerative change. Methods The authors obtained notochordal cells from nonchondrodystrophic canines according to their established methods and placed them into monolayer and 3D culture using sodium alginate globules under either hypoxic (3.5% O2) or normoxic (21% O2) conditions. Histological, immunohistochemical, scanning electron microscopy, and histomorphometric methods were used to evaluate the cells within the globules after 5 months in culture. Results Notochordal cells under in vitro hypoxic tissue culture conditions produced a highly complex, organized, 3D cellular construct that was strikingly similar to that observed in vivo. In contrast, traditional normoxic tissue culture conditions resulted in notochordal cells that failed to produce an organized matrix. Hypoxia resulted in a matrix rich in aggrecan and collagen II, whereas normoxic cultured cells did not produce any observable aggrecan or collagen II after 5 months of culture. Conclusions Hypoxia induces notochordal cells to organize a complex 3D cellular/extracellular matrix without an external scaffold other than suspension within sodium alginate. These cells produce an extracellular matrix and large construct that shares exactly the same characteristics as the in vivo condition—robust aggrecan, and type II collagen production. Normoxic tissue culture conditions, however, lead to a failure of these cells to thrive and a lack of extracellular matrix production and significantly smaller cells. The authors suggest that future studies of NP cells and, in particular, notochordal cells should utilize hypoxic tissue culture conditions to derive meaningful, biologically relevant conclusions concerning possible biological/molecular interventions.

The contribution of in vitro technology and cryogenic storage to conservation of indigenous plants

2007 ◽  
Vol 55 (3) ◽  
pp. 345 ◽  
Author(s):  
Eric Bunn ◽  
Shane Turner ◽  
Maggie Panaia ◽  
Kingsley W. Dixon
Keyword(s):  
Tissue Culture ◽  
Mycorrhizal Fungi ◽  
Ex Situ ◽  
Culture Methods ◽  
Germplasm Storage ◽  
Indigenous Plants ◽  
Cryogenic Storage ◽  
Propagation Methods ◽  
Research Initiatives

In vitro culture has enabled a variety of recalcitrant and threatened plant taxa to be micropropagated in the absence of viable conventional propagation methods. Cryogenic storage research has provided alternative protocols for efficient long-term germplasm storage for many plant species. Recent advances in tissue-culture methods such as somatic embryogenesis have enabled the production of >20 000 somatic embryos of a recalcitrant native Australian rush in a few months, far higher than other in vitro methods for these types of plants. Cryogenic protocols are reported for >30 species of Australian vascular plants, seed and numerous mycorrhizal fungi (mainly orchid spp.), greatly extending the range and type of material that can be stored through the application of cryogenic methods. The role of in vitro and cryogenic research initiatives in botanic gardens for plant biodiversity conservation and restoration is discussed, using examples of successful ex situ conservation through tissue-culture and cryogenic-storage research.

scholarly journals MICROPROPOGATION AND ANALYSIS OF PHYTOCHEMICAL PROFILE OF TISSUE CULTURE GROWN PLANTAGO OVATA FORSK.

2017 ◽  
Vol 10 (4) ◽  
pp. 202 ◽  
Author(s):  
Mamta Sharma ◽  
Amita Kumari ◽  
Eshita Mahant
Keyword(s):  
Tissue Culture ◽  
Ecological Factors ◽  
Culture Method ◽  
Culture Conditions ◽  
Culture Technique ◽  
Plantago Ovata ◽  
Tissue Culture Technique ◽  
Grown Plant

Objectives : Plantago ovata is an important medicinal plant of Himalayan region greatly used in herbal dugs manufacturing. The plant is multipurpose and strictly present in the Himalaya. Plantago has many medicinal properties such as antioxidant, anti-inflammatory and hematopoiesis effects and protects the liver and is used for the treatment of cancer. The plant being medicinal possesses complex phytochemicals. The investigation of various Plantago organ (leaves, stem etc) revealed their high potential to produce a wide array of bioactive secondary metabolites. In present study the a new method of micropropagation through tissue culture  was developed for Plantago so as to meet the future demand of plant. Futher a morphological and physiochemical comparison of tissue culture grown plant was done with in vivo grown plants.Methods:  Plantago ovata was grown in -vitro through tissue culture technique using MS media and in-vivo in the nursery area of Shoolini University. In vitro culture of  Plantago ovata forsk. were managed to restrict the ecological factors and to control the culture conditions. Experimental culture parameter including germination and phytochemical constituents of Plantago ovata in vivo and in vitro conditions were observed.Results: The result revealed changes in the concentration of phytochemical constituent’s in tissue culture grown Plantago. Phytochemicals constituents (carbohydrate, tannin, chlorophyll, saponin) was reduced in tissue culture grown plant where as some phytochemicals (phenol, alkaloid, flavanoid, protein, phytosterol) increased in tissue culture grown plant than in vivo plant.  A reduction in morphological trait was found in tissue cultured plant.Conclusion: The developed tissue culture method for the micropropagation of  Plantago ovata can be used as milestone to meet the industrial need in near future.Keywords: Plantago, Tissue Culture Technique, germination, phytochemicals.

scholarly journals Enrichment of Melanoma Stem-Like Cells via Sphere Assays

2021 ◽  
pp. 185-199
Author(s):  
Nabanita Mukherjee ◽  
Karoline A. Lambert ◽  
David A. Norris ◽  
Yiqun G. Shellman
Keyword(s):  
Cell Culture ◽  
Tissue Culture ◽  
Stem Cell ◽  
Three Dimensional ◽  
Culture Conditions ◽  
Low Density ◽  
In Vitro Techniques ◽  
Sphere Culture

AbstractSphere assays are widely used in vitro techniques to enrich and evaluate the stem-like cell behavior of both normal and cancer cells. Utilizing three-dimensional in vitro sphere culture conditions provide a better representation of tumor growth in vivo than the more common monolayer cultures. We describe how to perform primary and secondary sphere assays, used for the enrichment and self-renewability studies of melanoma/melanocyte stem-like cells. Spheres are generated by growing melanoma cells at low density in nonadherent conditions with stem cell media. We provide protocols for preparing inexpensive and versatile polyHEMA-coated plates, setting up primary and secondary sphere assays in almost any tissue culture format and quantification methods using standard inverted microscopy. Our protocol is easily adaptable to laboratories with basic cell culture capabilities, without the need for expensive fluidic instruments.

scholarly journals Epigenomic Insight of Lingonberry: Health Promoting Trait Under Micropropagation

2021 ◽  
Author(s):  
Arindam Sikdar ◽  
Umanath Sharma ◽  
Rajesh Barua ◽  
Abir U. Igamberdiev ◽  
Samir C. Debnath
Keyword(s):  
Tissue Culture ◽  
Secondary Metabolites ◽  
Cytosine Methylation ◽  
Phenolic Content ◽  
Secondary Compounds ◽  
Total Phenolic ◽  
Health Promoting ◽  
Micropropagated Plants

Abstract Epigenetic variation plays a role in developmental gene regulation and responses to the environment. An efficient interaction of zeatin induced cytosine methylation and secondary compounds has been displayed for the first time in tissue-culture shoots of lingonberry (Vaccinium vitis-idaea) in vitro, in vivo and its cutting-cultivar Erntedank. Through MSAP assay, we observed highest methylated sites in leaf regenerants (LC1) from all primer combinations (108 bands), with their highest variation in secondary metabolites. We measured that four tissue-culture plants showed higher methylation bands than cutting propagated donor plants (ED) which exhibited 79 bands of methylation, which is comparatively low. On the other hand, we observed the highest total phenolic content in node culture-derived greenhouse grown plants, NC3 but leaf culture-derived greenhouse grown plants, LC1 represented low phenolic content. Our study showed more methylation in micropropagated plants (NC1, NC2, NC3, LC1) than those derived from cutting propagated ED plants, where methylation was not present. On the contrary, we observed higher secondary metabolites in ED plants but comparatively less in micropropagated shoots (NC1, NC2) and plants (NC3, LC1). Our study displayed that higher methylation sites observed in micropropagated plants possessed less amount of secondary metabolites.

scholarly journals Environmental cystine drives glutamine anaplerosis and sensitizes cells to glutaminase inhibition

2017 ◽  
Author(s):  
Alexander Muir ◽  
Laura V. Danai ◽  
Dan Y. Gui ◽  
Chiara Y. Waingarten ◽  
Matthew G. Vander Heiden
Keyword(s):  
Cell Culture ◽  
Tissue Culture ◽  
Bovine Serum ◽  
Tca Cycle ◽  
Culture Conditions ◽  
Adult Bovine Serum ◽  
Necessary And Sufficient ◽  
Cells Cultured

AbstractMany cancer cell lines depend on extracellular glutamine as a major tri-carboxylic acid (TCA) cycle anaplerotic substrate to support proliferationin vitro. However, recent studies have suggested that some cells that depend on glutamine anaplerosis in culture rely much less on glutamine catabolism to proliferatein vivo, with environmental differences between tumors and cell culture influencing the extent of glutamine catabolism. Here we sought to better understand the environmental differences that cause differential dependence on glutamine for TCA cycle anaplerosis. We find that cells cultured in adult bovine serum, a condition that more closely reflects the nutrients available to cellsin vivo, leads to decreased glutamine catabolism and reliance on glutamine anaplerosis compared to standard tissue culture conditions. By analyzing the nutrient differences between bovine serum and media, we find that levels of a single nutrient, cystine, can account for the differential dependence on glutamine in these different environmental contexts. Further, we show that cystine levels dictate glutamine dependence via the cystine/glutamate antiporter xCT/SLC7A11, and that environmental cystine levels in conjunction withxCT/SLC7A11expression is necessary and sufficient to drive increased glutamine anaplerosis, defining important determinants of glutamine anaplerosis and glutaminase dependence in cancer cells.

scholarly journals Optimization of Culture Conditions (Sucrose, pH, and Photoperiod) forIn VitroRegeneration and Early Detection of Somaclonal Variation in Ginger Lime (Citrus assamensis)

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Jamilah Syafawati Yaacob ◽  
Noraini Mahmad ◽  
Rosna Mat Taha ◽  
Normadiha Mohamed ◽  
Anis Idayu Mad Yussof ◽  
...  
Keyword(s):  
Tissue Culture ◽  
Somaclonal Variation ◽  
Culture Conditions ◽  
Stem Explants ◽  
Ex Vitro ◽  
Root Regeneration ◽  
Stem Leaf ◽  
Full Adaptation

Various explants (stem, leaf, and root) ofCitrus assamensiswere cultured on MS media supplemented with various combinations and concentrations (0.5–2.0 mgL−1) of NAA and BAP. Optimum shoot and root regeneration were obtained from stem cultures supplemented with 1.5 mgL−1NAA and 2.0 mgL−1BAP, respectively. Explant type affects the success of tissue culture of this species, whereby stem explants were observed to be the most responsive. Addition of 30 gL−1sucrose and pH of 5.8 was most optimum forin vitroregeneration of this species. Photoperiod of 16 hours of light and 8 hours of darkness was most optimum for shoot regeneration, but photoperiod of 24 hours of darkness was beneficial for production of callus. The morphology (macro and micro) and anatomy ofin vivoandin vitro/ex vitro Citrus assamensiswere also observed to elucidate any irregularities (or somaclonal variation) that may arise due to tissue culture protocols. Several minor micromorphological and anatomical differences were observed, possibly due to stress of tissue culture, butin vitroplantlets are expected to revert back to normal phenotype following full adaptation to the natural environment.

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