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Prevention of cellular aging in vitro
Kubovčák, Matej ; Černý, Jan (advisor) ; Ryšánek, David (referee)
This bachelor thesis explores foundational components of the aging process and their importance in cell culture systems. Signaling pathways controlling these processes are discussed, and several attractive targets were identified to be potentially developed as interventions. Aging is described within the evolutionary paradigm to provide a global framework for understanding the various aging processes and their explanation. Several models in nature were found to provide evidence that aging interventions can be successful and could therefore serve as a source of inspiration. Immunological context is provided to underline its importance in vivo, and the immune system-specific longevity mechanisms are elucidated for potential exploitation. Loss of cellular identity is identified as an essential detrimental factor in cell culture and is connected to the foundational aging mechanisms. This thesis can serve as a stepping stone for conducting research on better cultivation methodologies, thereby opening doors to technologies such as cultivated meat and bioartificial organs requiring unprecedented cell expansion. Keywords aging, aging reversal, cellular identity, in vitro cultures, stemness
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The effect of pyrazine derivatives on secondary metabolites content in plant cultures in vitro - III.
Blahnová, Kristýna ; Tůmová, Lenka (advisor) ; Karlíčková, Jana (referee)
One of the possibilities how to increase the production of secondary metabolites (SM) in plant in vitro cultures is the method of elicitation. The aim of this work was to determine the effect of an abiotic elicitor from the class of pyrazine derivatives 1-benzyl-3-(pyrazin-2-yl)urea on the production of flavonolignans of silymarin complex and flavonoid taxifolin in the plant culture Silybum marianum (L.) Gaertn. Pyrazine derivatives are investigated for their herbicidal properties, so elicitation proceeds by the mechanism of stress inducing effect. Tissue cultures were grown on Murashige and Skoog growth medium with the addition of the growth regulator α-naphthylacetic acid. Elicitation was performed on both callus and suspension cultures. The elicitor was used in three different concentrations: c1 = 100.0 mg/100 ml; c2 = 10.0 mg/100 ml; c3 = 1.00 mg/100 ml. Particular samples were taken after 6, 24, 48, 72 and 168 hours of elicitor effect, control samples after 6, 48 and 168 hours. After drying, the callus and suspension tissues were extracted with methanol and the content of the monitored secondary metabolites was determined by HPLC. It was also tested if SM are released into the growth medium. Flavonolignans silybinin A and silybinin B were not detected in any of the analyzed samples. The...
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In vitro cultures of medicinal plants - XVI
Sedláčková, Veronika ; Tůmová, Lenka (advisor) ; Martin, Jan (referee)
Medicinal plant cultures in vitro - XVI The subject of this diploma thesis is the evaluation of secondary metabolites production in Silybum marianum, (L.) Gaertn. cultures in vitro after elicitor treatment. The aim of the study was to find if an abiotic elicitor 5-tert-butyl-N-(4- chlorbenzyl)pyrazine-2-carboxamide increases the flavonolignan production Silybum marianum cultures in vitro. Experiment was carried out in callus and suspension cultures of Silybum marianum using Murashige - Skoog nutrient medium supplemented with 10 mg/l α-naphthylacetic acid. The elicitor was added in the form of solution in three different concentrations (C1 = 3. 292.10-3 mol/l, C2 = 3. 292.10-4 mol/l and C3 = 3. 292.10-5 mol/l) and it was affecting 6, 12, 24, 48, 72 and 168 hours. The content of flavonolignans was determined by HPLC. The maximum flavonolignan production (0. 280 mg.g-1 DW) in callus culture was observed after 24 hours of elicitor application in concentration of C2 = 3. 292.10-4 mol/l, when the highest content of silychristin was detected. The second significant increase in flavonolignan production (0. 271 mg.g-1 DW) in callus culture was noticeable after 12 hours of elicitor treatment in concentration of C3 = 3. 292.10-5 mol/l, when the highest increase in silydianin and silybin B accumulation was found. The...
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The effect of pyrazine derivatives on secondary metabolites content in plant cultures in vitro - III.
Blahnová, Kristýna ; Tůmová, Lenka (advisor) ; Karlíčková, Jana (referee)
One of the possibilities how to increase the production of secondary metabolites (SM) in plant in vitro cultures is the method of elicitation. The aim of this work was to determine the effect of an abiotic elicitor from the class of pyrazine derivatives 1-benzyl-3-(pyrazin-2-yl)urea on the production of flavonolignans of silymarin complex and flavonoid taxifolin in the plant culture Silybum marianum (L.) Gaertn. Pyrazine derivatives are investigated for their herbicidal properties, so elicitation proceeds by the mechanism of stress inducing effect. Tissue cultures were grown on Murashige and Skoog growth medium with the addition of the growth regulator α-naphthylacetic acid. Elicitation was performed on both callus and suspension cultures. The elicitor was used in three different concentrations: c1 = 100.0 mg/100 ml; c2 = 10.0 mg/100 ml; c3 = 1.00 mg/100 ml. Particular samples were taken after 6, 24, 48, 72 and 168 hours of elicitor effect, control samples after 6, 48 and 168 hours. After drying, the callus and suspension tissues were extracted with methanol and the content of the monitored secondary metabolites was determined by HPLC. It was also tested if SM are released into the growth medium. Flavonolignans silybinin A and silybinin B were not detected in any of the analyzed samples. The...
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The effect of methylviologen on secondary metabolites production in in vitro culture of Fagopyrum esculentum, variety Bambi
Vlachová, Veronika ; Tůmová, Lenka (advisor) ; Karlíčková, Jana (referee)
10 ABSTRACT The subject of this study was to evaluate the efect of abiotc elicitor on rutn producton in callus and suspension cultures of buckwheat. The cultvar of buckwheat used for this research was Fagopyrum esculentum Moench var. Bambi, cultvated in Murashige and Skoog nutrient medium with the additon of growth regulator 2,4-dichlorfenoxyacetc acid (2,4-D) in concentraton of 1 mg/l. The elicitor used in this study was a soluton of methylviologen, 1 ml of it was added to the cultures in three diferent concentratons: c1 = 100.0 mg/100 ml, c2 = 10.0 mg/100 ml and c3 = 1.0 mg/100 ml. The elicitor was afectng the cultures for 6, 12, 24, 48, 72 or 168 hours. Afer the defned period of tme, cultures were collected, dried out and stored for further analysis of rutn content. To control samples (without elicitor treatment) 1 ml of ethanol 96% was added and they were collected afer 6, 24, 72 or 168 hours. Releasing of rutn into the nutrient medium was also investgated. Rutn content in each sample of cultures and in each sample of nutrient medium was later determined by HPLC. Any signifcant increase in the producton of rutn was not observed in this study. The maximum amount of rutn detected was 0.1 mg/g DW, thus the lowest quantty detectable, and was found in suspension cultures in three cases, afer the additon of...
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The effect of methylviologen on secondary metabolites production in in vitro culture of Genista tinctoria
Macová, Alena ; Tůmová, Lenka (advisor) ; Kašparová, Marie (referee)
Elicitation is one of possible methods for increasing the secondary metabolites production in plant cell cultures. This paper explores the potential effect of methylviologene on isoflavonoids production in Genista tinctoria L. suspension and callus cultures. Schenk and Hildebrandt nutrient medium was used with the addition of growth regulators 2,4- D in concentration of 5 mg/L and kinetin of 1 mg /L of medium. Elicitor was added in the form of ethanol solution at concentration of 2.19·10-2 mol l-1 , 2.19·10-3 mol l-1 , 2.19·10-4 mol l-1 . Samples were taken after 6, 12, 24, 48, 72 and 168 hours of elicitor exposure. Control samples were collected at 6, 24, 72 and 168 hours. The isoflavonoid content in suspension and callus cultures and in nutrient medium was evaluated by high performance liquid chromatography. The biggest increase of isoflavonoid content in callus culture was reached in daidzein content after 48 hours elicitor treatment in concentration of 2.19·10-3 mol l-1 (8.5 mg/g DW) and 2.19·10-4 mol l-1 (1.6 mg/g DW). The production of genistin, genistein and formononetin was slightly increased or zero compared to controls. Biochanin A was almost absent in the samples. The highest level of isoflavonoids was measured in the suspension culture after 48 hours elicitor application in the...
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The effect of methylviologen on secondary metabolites production in in vitro culture of Fagopyrum esculentum, variety Spačinski
Vaicová, Nicole ; Tůmová, Lenka (advisor) ; Spilková, Jiřina (referee)
Plants are an important source of secondary metabolites, which are a valuable natural substance used in many fields. One way to increase their production is by the elicitation method. In this paper the effect of abiotic elicitor methylviologene in three different concentrations was studied on the rutin production in callus and suspension culture of Fagopyrum esculentum Moench variety Spačinki. The cultivation was carried out on Murashige and Skoog nutrient medium with the addition of a 1 mg/l 2,4- dichlorophenoxyacetic acid as growth regulator. Samples were taken at regular time intervals after 6, 12, 24, 48, 72 and 168 hours of elicitation. The rutin content was analyzed by HPLC. The maximum rutin production (0.3 mg/g DW) was recorded in the callus culture after 48 hours of methylviologene treatment at a concentration of 2.1929.10-4 mol/l. No increase in rutin content after methylviologene elicitation was observed in the suspension culture. The study also included monitoring of the rutin release into nutrient medium, but this was not demonstrated.
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The effect of methylviologen on secondary metabolites production in in vitro culture of Fagopyrum esculentum, variety Pyra
Zajačíková, Pavla ; Tůmová, Lenka (advisor) ; Kašparová, Marie (referee)
Elicitation is one of the methods used for increasing the production of secondary metabolites in vitro cultures. The aim of this work is to evaluate the effect of abiotic elicitor methylviologen (paraquat) on the production of flavonoids in callus and suspension cultures of Fagopyrum esculentum Moench., variety Pyra. The cultures were cultivated in Murashige and Skoog nutrient medium with addition of 2,4-D in concentration of 1 mg/l as a growth regulator. Elicitor was added as a solution in three different concentrations (c1 = 2.1929 · 10-4 mol/l, c2 = 2.1929 · 10-3 mol/l and c3 = 2.1929 · 10-2 mol/l). The effect of elicitation on rutin production was monitored in six time intervals: 6, 12, 24, 48, 72 and 168 hours. The rutin content was determined by HPLC analysis. No rutin was produced in callus and suspension cultures without the presence of elicitor. Even after the elicitation, there was no statistically significant increase in the production of rutin. The maximum rutin content was detected in the suspension culture after 12 hours of elicitor treatment in c2 concentration, the content was 0.1 mg/g DW. The release of rutin into the nutrient medium was also not observed.
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The effect of methylviologen on secondary metabolites production in in vitro culture of Fagopyrum esculentum, variety Bambi
Vlachová, Veronika ; Tůmová, Lenka (advisor) ; Karlíčková, Jana (referee)
10 ABSTRACT The subject of this study was to evaluate the efect of abiotc elicitor on rutn producton in callus and suspension cultures of buckwheat. The cultvar of buckwheat used for this research was Fagopyrum esculentum Moench var. Bambi, cultvated in Murashige and Skoog nutrient medium with the additon of growth regulator 2,4-dichlorfenoxyacetc acid (2,4-D) in concentraton of 1 mg/l. The elicitor used in this study was a soluton of methylviologen, 1 ml of it was added to the cultures in three diferent concentratons: c1 = 100.0 mg/100 ml, c2 = 10.0 mg/100 ml and c3 = 1.0 mg/100 ml. The elicitor was afectng the cultures for 6, 12, 24, 48, 72 or 168 hours. Afer the defned period of tme, cultures were collected, dried out and stored for further analysis of rutn content. To control samples (without elicitor treatment) 1 ml of ethanol 96% was added and they were collected afer 6, 24, 72 or 168 hours. Releasing of rutn into the nutrient medium was also investgated. Rutn content in each sample of cultures and in each sample of nutrient medium was later determined by HPLC. Any signifcant increase in the producton of rutn was not observed in this study. The maximum amount of rutn detected was 0.1 mg/g DW, thus the lowest quantty detectable, and was found in suspension cultures in three cases, afer the additon of...
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In vitro cultures of medicinal plants - XV
Slavík, Marek ; Tůmová, Lenka (advisor) ; Martin, Jan (referee)
The subject of this study is the evaluation of secondary metabolites production in Hypericum perforatum L. cultures in vitro after elicitor treatment. The aim was to find if orthosilicic acid as abiotic elicitor increases the flavonoid and hypericin production in Hypericum perforatum L. cultures in vitro. Experiment was carried out in callus and suspension cultures of H. perforatum using Murashige - Skoog nutrient medium78 supplemented with 10 mg. ml-1 α-naphtylacetic acid as growth regulator. The elicitor was added in the form of solution in 3 different concentrations (C1 = 10.4047∙10-3 mol l-1 , C2 = 10.4047∙10-4 mol l-1 , C3 = 10.4047∙10-5 mol l-1 ), it was affecting 6, 12, 24, 48, 72 and 168 hours. The content of flavonoids and hypericin was determined by HPLC. Secondary metabolites release into nutrient medium was also a part of this study. The increasing flavonoid and hypericin production in callus cultures after elicitor application at any concentrations was not observed. The maximum flavonoid content (0.04 mg g-1 DW) in suspension culture was detected after 72 h of elicitor treatment in concentration of C1 where the maximum hyperoside production was observed. The maximum hypericin production (0.21 mg g-1 DW) in suspension culture was detected after 12 h of elicitor application in...
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