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Saffron, made from the dried stigmas of Crocus sativus L., contains pigments and valuable aromatic compounds, and can be used in medicine and as a spice. Nowadays its production is lower than demand. Tissue culture presents an alternative biochemical tool which can be used to produce stigma-like structure (SLS) in vitro. In this study, the origin and induction of SLS formation was investigated in ovary and style explants of floral buds on MS medium supplemented with 1-naphthalene acetic acid (NAA) and 6-benzlaminopurine (BAP). SLS were directly originated through meristematic cells or indirectly in the form of colorless globular structures from parenchyma tissue. The colorless globular structures initially were conical and pale yellow color at the sharp ends; subsequently they matured into trumpet-like red stigmas with or without finger-like papillae at the margins. Light and electron microscopic observations of ultra- and semithin sections of different developmental stages of SLS showed that these structures possess two kinds of cells: (1) small cells close to parenchyma tissues and (2) large cells oriented towards the peripheral area and apparently originated from the small ones. Our results suggest that the SLS originated from internal parenchyma tissues.
In vitro organogenesis was studied using Citrus limon L. Burm cv. ‘Primofiore’ leaf explants. The purpose of the present study was to optimize conditions for callogenesis and organogenesis of C. limon. Explants of C. limon were cultured on 16 different media supplemented with various combinations of plant growth regulators, both auxins and cytokinins, such as 6-benzylaminopurine (BAP), naphthalene acetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), and kinetin. The best shoot induction was obtained when the leaf explants were cultured on Murashige & Tucker media supplemented with 3.5 mg·L-1 BAP. Histological investigation revealed most likely the initial phase of development of leaf explants during in vitro regeneration of C. limon. t
Helianthus tuberosus is economically important species. To improve characters of this energetic plant via genetic modification, production of callus tissue and plant regeneration are the first steps. A new, potentially energetic cultivar Albik was used in this study to test callus induction and regeneration. Callus was produced on leaves, petioles, apical meristems and stems from field-harvested plants but was totally non-morphogenic. Its induction started in the cortex and vascular bundles as confirmed by histological analysis. The surface of heterogeneous callus was partially covered with a membranous extracellular matrix surface network visible in scanning and transmission electron microscopies. The results clearly indicate that: (i) the morphogenic capacity of callus in topinambur is genotype dependent, (ii) cv. Albik of H. tuberosus proved recalcitrant in in vitro regeneration, and (iii) extracellular matrix surface network is not a morphogenic marker in this cultivar.
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There are limited published data on in vitro reproduction of Fagus sylvatica L. (European beech). This study was aimed to determine the efficiency of induction of somatic embryogenesis or organogenesis of beech from different types of explants in various culture conditions. Explants derived from immature, fresh seeds (collected in 2011 and 2013) and from mature seeds, stored at –10ºC and some stratified at 3ºC, were placed on induction media with various combinations of plant growth regulators: zeatin, 2,4-dichlorophenoxyacetic acid (2,4-D) and/or benzyladenine (BA). Initial cultures were kept in darkness or weak light (white fluorescent or blue-red LED). Limited success has been achieved in initiation of somatic embryogenesis. We obtained friable, yellow-white callus with characteristic PEM-like structures (cPEM-ls, from embryonic axes or fragments of immature embryos with embryonic axes), which may be an early developmental stage of embryogenic callus of Fagus sylvatica. This type of callus regenerated from explants incubated in darkness, mainly on WPM medium with addition of 6.8 μM zeatin or WPM and MSG media with 9.1 μM 2,4-D and 2.2 μM BA. The highest frequency of regeneration of callus with cPEM-ls was 5%. Instead, we succeeded to induce organogenesis from both immature and mature zygotic embryos and from embryonic axes. The best results were obtained for mature zygotic embryos incubated on ½WPM medium (half-strength Woody Plant Medium) with 9.1 μM 2,4-D and 2.2 μM BA. Adventitious buds were regenerated on up to 15% of the explants. The induced buds developed into shoots, enabling us to establish tissue cultures of beech. Induction of organogenesis from the tested explants was more efficient than induction of somatic embryogenesis.
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