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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 elicitors on the secondary metabolites production in vitro cultures -I.
Damaskinos, Antonios ; Tůmová, Lenka (advisor) ; Martin, Jan (referee)
Active compounds have been always originated from plants. Plants though, were able to produce only very low amounts of them and that was the reason for trying many alternative ways of production, one of them being plant tissue culture cultivation. This method is any fragment of living tissue or organ taken from an intact plant or an already existing explant culture, with the intention of growing an artificial growth medium. Even this method though, is not able to produce large amounts compared to extraction from field plants. Elicitation is considered a possible way to increase the production of secondary metabolites. This method used the plant's own defense system, in order to increase the production of secondary metabolites in vitro. The compound which is used to produce the effect is called elicitor. During our experimental work I used as an elicitor the compound Ethephon (2-Chloroethylphosphonic acid) upon callus and suspension cultures of Hypericum perforatum, with intention to observe its effect on flavonoid production. This experiment was based on three different concentrations and six different withdrawal times, being 6, 12, 24, 72, 168 hours. The maximum effect of elicitor was reached with concentration c1 (1mg/100ml) after 12 hours and with concentration c3 (100mg/100ml) after 72 hours.
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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 abiotic elicitors on secondary metabolites content in plant cultures in vitro - II.
Tomaidesová, Barbora ; Tůmová, Lenka (advisor) ; Karlíčková, Jana (referee)
The Effect of Abiotic Elicitors on the Content of Secondary Metabolites in In vitro Plant Cultures - II. The method of elicitation is used for the increased production of secondary metabolites in in vitro plant cultures. In this study, there the effect of pyridine derivate N-(5-chlorpyridin-2-yl)-4-ethylbenzamide in three concentration - 3.845.10-3 mol/l; 3.845.10-4 mol/l and 3.845.10-5 mol/l for the production of flavonolignans in callus and suspension cultures of Silybum marianum was tested. The evaluation of elicitation effect proceeded after 6, 24, 48, 72 a 168 hours against control samples without the elicitor treatment after 24 and 168 hours. In the same intervals the samples of culture medium were taken and assessed. The cells were cultivated on Murashige and Skoog medium with 10 mg/ml of α-naphtylacetic acid as growth regulator. The obtained and adjusted samples were analysed by the HPLC method. From the parts of silymarin complex were detected silychristin, silybin A, isosylibin A, isosilybin B. Flavonoid taxifolin wasn̕ t detected in any case. In callus culture, there was achieved a maximal production of all detected components of silymarin complex (30.508 μg/g DW) after 168 hours of elicitor treatment in concentration of 3.845.10-4 mol/l, it was about only substance - silychristin. The...
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The effect of abiotic elicitors on secondary metabolites content in plant cultures in vitro - I.
Teplá, Klára ; Tůmová, Lenka (advisor) ; Siatka, Tomáš (referee)
Plants are a source of a wide range of secondary substances, which due to their effects find use in many areas of focus. By a method called elicitation, we can achieve their higher and thus more efficient production. This diploma thesis aimed to determine whether the abiotic elicitor 2-(4-chlorophenyl)-N-(5-chloropyridin-2-yl)acetamide can positively affect the production of the flavonoid hyperoside in callus and suspension culture of Hypericum perforatum L. The elicitor was added to the in vitro cultures in three concentrations: C1 = 3,571.10-3 mol/l; C2 = 3,571.10-4 mol/l and C3 = 3,571.10-5 mol/l. A sample was taken at regular intervals after 6, 24, 48, 72 and 168 hours of elicitor treatment. Control samples were taken after 24 and 168 hours. The content of hyperoside produced was subsequently determined using High Performance Liquid Chromatography. Simultaneously, the amount of hyperoside released into the nutrient media of both plant cultures was also monitored. Maximum hyperoside production was recorded in suspension culture after 6 (17,7 µg/g DW) and 48 hours (3,69 µg/g DW) of elicitor treatment with the lowest concentration of C3 (3,571.10-5 mol/l). The content of hyperoside in the first case was 1770 % higher compared to the control sample. There was a significant release of hyperoside...
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Resynthesis of oilseed rape from Brassica rapa and Brassica oleracea
Klíma, Miroslav ; Hilgert-Delgado, Alois ; Ulvrová, Tereza ; Bryxová, Pavla ; Vítámvás, Pavel ; Prášil, Ilja Tom ; Kosová, Klára
In order to extend the variability of the genofond, so-called resynthesis can be used, when new genotypes are created by crossing selected Brassica rapa and B. oleracea, by early isolation of immature embryos from young eggs and their in vitro fertilization (Hilgert-Delgado et al., 2015). It has been shown that resyntetized genotypes are distinct from rape materials grown at present (Sosnowska et al., 2010), so that they can serve as a source of new variability in breeding programs focusing on cytoplasmic and pollen sterility, resistance to pathogens and tolerance to stresses (Warwick et al., 2009). Representatives of the initial components are characterized by wide variability in terms of morphology and agronomic characteristics, and are a prospective source of heterosis and other traits (Wu et al. The use of resyntetized genotypes for the direct production of rapeseed varieties is not possible due to the poor quality of the seed of these materials. It is mainly the high content of glucosinolates and erucic acid, typical of most of the initial components (Jesske et al., 2013). However, the required properties can be transferred by crossing to conventional breeding materials (Hilgert-Delgado et al., 2017).
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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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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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In vitro cultures of medicinal plants - XIV
Majerová, Jitka ; Tůmová, Lenka (advisor) ; Kašparová, Marie (referee)
62 10 ABSTRACT The object of this study was the influence of abiotic elicitor on the production of rutin in suspension culture of Fagopyrum esculentum Moench. The culture was cultivated in Murashigeho and Skoog nutritive medium with growth regulator: 2,4−dichlorfenoxyacetic acid (1 ml/l). The ultrasound was used as abiotic elicitor (0,1 W/cm3 , 35 kHz) for time period of 1, 2, 3, 4 and 5 min. The samples were taken 0, 6, 12, 24, 48, 72 and 168 hours after elicitation. The kontrol samples (without the influence of ultrasound) were taken 24 and 168 hours after elicitation. The amount of rutin was analyzed by HPLC. Suspension culture of Fagopyrum esculentum Moench. didn't produce any rutin under the influence of ultrasound. No release of rutin into the nutritive medium was observed during this study.
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