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Cytotoxic potential of melamine was evaluated with the use of two in vitro models i.e. cell cultures of rat hepatoma (line FaO) and rat skeletal muscle (line L6). The cultures were exposed for 24, 48, and 72 h to melamine at eight concentrations, ranged from 0.01 to 10 mM. Four different assays were applied in which various biochemical endpoints were assessed: mitochondrial activity - MTT reduction assay, proliferation - Commassie Brilliant Blue (CBB) dye binding assay, lysosomal activity - neutral red uptake (NRU) assay, and membrane integrity - LDH release assay. Effective concentrations (EC₂₀, EC₅₀, EC₈₀) were calculated from concentration-response curves, and then they were averaged over three independently conducted experiments. It was found that MTT assay was the most sensitive to this compound. After 48 h exposure EC₅₀ values (mM, mean ± SD) for FaO and L6 cells were 6.4 ± 0.62, and 8.2 ± 1.51, respectively. The inhibition of lysosomal activity measured by NRU assay, and damage of plasma membrane measured by LDH assay were detected in L6 (but not in FaO) cells; however, the effects took place after longer (72 h) exposure. At that time EC₅₀ values were 5.2 mM and 9.2 mM for NRU and LDH, respectively. In spite of the low cytotoxicity of melamine, more studies are needed for hazard identification and characterization of the compound.
In vitro embryogenic callus is a critical factor for genetic transformation of rice, especially for indica varieties. In this study, we investigated the relationship between polyamines, including putrescine (Put), spermidine (Spd) and spermine (Spm), and callus browning, and we studied the effect of exogenous Put on callus regeneration and on the content of endogenous polyamines. In addition, the expression levels of arginine decarboxylase gene (Adc1) and S-adenosylmethionine decarboxylase gene (Samdc) in embryogenic callus were studied by quantitative Real-time PCR analysis. The results showed that the contents of endogenous Put and Spd in the browning callus were significantly lower than those in normal callus. Exogenous Put could effectively improve the growing state of callus of indica rice and enhance the development of embryogenic callus. The content of endogenous polyamines in embryogenic callus, especially Spd and Spm, was increased after addition of exogenous Put. Additionally, exogenous Put also had an obvious impact on the expression levels of Adc1 but partial effect on the expression levels of Samdc gene. This study could increase the knowledge of both embryogenic callus induction and polyamine catabolism in callus in indica rice.
Isatis concstricta Davis (an endemic plant of Turkey) suffers from low propagation rates under natural conditions and is threatened due to fast unplanned urbanisation. The study compared the effects of variants of BA + NAA on shoot regeneration on leaf and petiole explants excised from one week old in vitro regenerated seedlings. MS medium containing 1 mg L–1 BA + 1 mg L–1 NAA induced maximum proliferation on petiole and leaf explants with 13.33 and 12.75 shoots per explant repectively. However, leaf explant induced shoots were sturdy and healthier compared to petiole explant induced shoots. These shoots were rooted on MS medium containing 0.5 mg L–1 IBA and the plants were acclimatized in peat moss and sand (v/v). They grew to flowering under ex vitro conditions. This system of regeneration is advantageous for conventional propagation and the results will help in establishment of a powerful and meaningful micropropagation system for I. constricta.
Steroidal saponins isolated from many plant species belonging to Monocotyledones display potent cytotoxic activity towards many human tumor cells. We examined the cytotoxic effects of crude Paris quadrifolia extract for the first time, testing isolated saponin-rich fractions against four different human cell lines using the [(3-(4,5- dimethylthiazol-2-yl)]-2,5-diphenyltetrazolium bromide (MTT) assay. Cytotoxic activity was tested against human promyelocytic leukemia (HL-60) cells, human cervical adenocarcinoma (HeLa) cells and human breast cancer (MDA-MB-468) cells. Human skin fibroblasts were used as non-neoplastic control cells. Our results show significant activity of the weakly water-soluble solid residue and butanolic fraction against HL-60 and HeLa cells. The solid residue exerted cytotoxicity against all tested cell lines.
This paper is the first published report describing micropropagation of Carlina onopordifolia, using shoot tip and hypocotyl explants. The explants were excised from 10-day-old seedlings and transferred to proliferation medium supplemented with 6-benzylaminopurine (BA; 1.0 or 3.0 mg l-1), kinetin (Kn; 1.0 or 3.0 mg l-1) or zeatin (ZEA; 1.0 or 3.0 mg l-1) in combination with naphthaleneacetic acid (NAA; 0.1 mg l-1). The shoot tips were significantly better than hypocotyls as initial material for shoot regeneration. For shoot multiplication, MS medium supplemented with BA proved superior to the other cytokinins tested. Medium supplemented with 1.0 mg l-1 BA gave the highest shoot propagation frequency (66.9%) and number of shoots per explant (2.5). Single shoots were separated from each other and rooted on MS supplemented with IBA for the whole period of culture, with longor short-pulse IBA application. The highest rooting frequency (84.8%) and root number (18.8) were for shortpulse (1 min) 1000 mg l-1 IBA solution. The higher IBA concentration stimulated callus formation and the development of short roots. The shoots were transferred to MS medium without growth regulators. Survival was highest (54.4%) for the plants from the short-pulse 100 mg l-1 IBA treatment. After 8 weeks of acclimatization the plantlets were removed to field conditions and grew normally.
Roots of Codonopsis pilosula (Franch.) Nannf. are among the most popular Chinese herbal medicines, exhibiting various beneficial activities which support immunity and stress resistance. The plant shows high intraspecific genetic variation. There is a need for effective vegetative propagation methods yielding high and sustainable quality. Here we report a micropropagation method using axillary shoot proliferation. Nodal segments from aseptically germinated plants were inoculated on modified MS media enriched with different concentrations of cytokinins: benzyladenine, kinetin (1, 4, 10 or 20 μM) or thidiazuron (1, 4 or 8 μM), with or without the auxin NAA (1 μM). Axillary bud break was initiated most efficiently on media with 1 or 4 μM BA and 1μM NAA. Shoot number increased markedly in subsequent cycles of harvesting and transfer to fresh 1 μM BA and NAA medium, leading to the maximum 69 shoots (mean 38.16±4.35) from a single nodal explant in the fourth harvest. The shoots were successfully (>98% efficiency) rooted in MS medium containing high sucrose (60 g/L) and 5 μM IAA, and acclimatized to soil cultivation with a survival rate of 90%. These results can be used to establish a simple and commercially viable protocol for mass propagation of C. pilosula for plantations or breeding.
For breeding purposes, the number of neo-tetraploids of apple cultivars have been derived by in vitro technique. The first attempts at rooting and acclimatization of tetraploid shoots failed. The aim of the study was to develop an effective method for rooting microcuttings of apple neo-tetraploids. In the first stage of the study, in vitro rooting method was optimized for the shoots of diploid donor cultivars. Shoots were rooted on Murashige and Skoog [1962] (MS) medium with a reduced content of nitrogen, in the presence of auxins alone or in combination (indole-3-butyric acid – IBA, 1-naphthaleneacetic acid – NAA and indole- -3-acetic acid – IAA) with addition of putrescine, arginine or ornithine. The compounds were applied continuously for 25 days (one-step rooting system) or for seven days with subsequent transplanting shoots onto a medium without these compounds (two-step rooting method). Tetraploid microcuttings of the cultivars ‘Free Redstar’, ‘Gala Must’, ‘Pinova’ and ‘Redchief’ were evaluated for rooting on the selected medium considered optimal for their diploid counterparts. The shoots of all diploid apple scion cultivars had low rooting capacity. IBA alone poorly stimulated root formation. Significant improvement of rooting to 60–80% was achieved through the application of auxins, 2.5 µM IBA or 1.3 µM NAA combined with 5 µM IAA and 50 µM putrescine in the two-step rooting system with darkness and increased temperature of 26°C during seven-day induction phase. The replacement of benzyladenine (BA) by meta-Topolin (m-T) in the last multiplication subculture influenced positively shoot acclimatization. Tetraploids had comparable or slightly lower rooting and acclimatization ability compared to their diploid counterparts.
Zbadano aktywność mutagenną wybranych formulacji herbicydów i regulatorów wzrostu oraz ich substancji biologicznie czynnych. Do oceny zastosowano model mutacji mitochondrialnych u Saccharomyces cerevisiae, mutacji barwnikowych u Chlorella vulgaris oraz test mikrojądrowy w komórkach szpiku kostnego myszy.
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