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The potential of immature zygotic embryos to produce embryogenic tissue was tested using culture media differed in inorganic salt composition as well as plant growth regulator content. Explants cultured on DCR as well as MLV media gave the highest initiation percentages with maximum values of 8.8% (MLV) to 10.4% (DCR). Plant growth regulators content (standard or reduced) had no profound effect on embryogenic tissue initiation. Somatic embryo maturation as well as germination was dependent on the cell line and salt composition of the medium. Plantlet regeneration occurred in three cell lines out of five tested. During the maturation process profound changes occurred in the internal organisation of somatic embryos such differentiation of root meristem in precotyledonary somatic embryos and formation of provascular strands in cotyledonary somatic embryos.
Suspension cultures have been established from embryogenic tissues of Pinus nigra initiated from immature zygotic embryos. The growth of tissues in liquid medium has been influenced by initial tissue weight used for the establishment of the cultures as well as by genotype. In most of the cases initial tissue weight 0.5 g was insufficient and the cultures showed poor growth and later degeneration. Higher amount of initial tissues (1 or 2.5 g) was more efficient for the establishment and proliferation of somatic embryos in liquid medium. The growth of suspension cultures was also cell line dependent. Somatic embryo maturation in liquid medium was very limited and no plantlet regeneration occurred. Cotyledonary somatic embryos developed and produced emblings when the suspension was plated on filter paper discs and cultured on solid maturation medium. Based on our experiments we can state that the embryogenic tissues are able to grow and proliferate in liquid medium but somatic embryo maturation and plantlet regeneration occur only on solid medium.
Since the first report of somatic embryogenesis in Norway spruce in 1985, the in vitro process has been initiated for a number of conifer species belonging to different genera. The process of somatic embryogenesis involves initiation, proliferation, maturation and plantlets (emblings) regeneration. The initiation of somatic embryogenesis is restricted mostly to juvenile explants, although recently explants taken from adult trees produced embryogenic tissues. The successful initiation, maturation and emblings regeneration are affected by factors as developmental stage of primary explants, genotype, plant growth regulators content in the culture medium, light conditions. Optimisation of mentioned factors resulted in regeneration of emblings capable of growing after transfer to soil.
Embryogenic cell line AN 72 derived from immature hybrid fir Abies alba x A. numidica zygotic embryos was subjected to different maturation treatments. The effect of the carbohydrates sucrose, maltose and glucose (each at 3%, 6% and 9%) or PEG-4000 (5.0%, 7.5% and 10%) combined with different carbohydrate sources was tested. PEG-4000 stimulated somatic embryo maturation of hybrid fir. This stimulatory effect was dependent on the carbohydrate source used. Culture medium with maltose as carbohydrate source combined with PEG-4000 produced the highest number of cotyledonary somatic embryos. Carbohydrates supplied alone (mainly at 6% and 9%) exerted an unfavorable effect, increasing the frequency of abnormally shaped somatic embryos without regeneration capacity. The structural organization of morphologically well-developed cotyledonary somatic embryos was similar to that of zygotic embryos. In abnormal somatic embryos the shoot apical meristem and root meristem were very damaged. Electrophoretic separation of denatured proteins using SDS-PAGE showed differences in the accumulation of low molecular weight storage proteins in somatic embryos. Storage protein accumulation was dependent on the concentration of PEG-4000 and the carbohydrate source.
Benthic and planktonic foraminifers of the hypostratotype of the Paleocene (El Haria Fm., El Kef section, northern Tunisia) are discussed and described. The foraminifers identified have made it possible to distinguish 8 planktonic foraminiferal biozones with 5 subzones within the hypostratotype and to correlate them with their stratigraphic equivalents from Tethyan and epicontinental regions. The hypostratotypes of the Danian, Montian and Landenian are proposed and defined. The hypostratotype of the Paleocene sensu stricto is proposed as the stratotype of marine Paleocene section of the Tethyan region.
Flax suspension cultures have been established from the callus induced from the cotyledons, hypocotyls, and immature zygotic embryos (iZE). The growth of flax suspension culture (expressed as a sedimented cell volume) was compared in both conditioned (by liquid from embryogenic Pinus nigra suspension culture) and nonconditioned media. Conditioning of media significantly increased the growth of the cell lines of hypocotyl and iZE origin; however, it had no promotive effect on embryogenic response of these flax liquid cultures. Formation of embryo-like structures (ELS), confirmed also histologically, has only been found in the cell line derived from iZE and cultivated in non-conditioned MS medium supplemented with 1 mg l⁻¹ 2,4-D. The process of ELS formation in this cell line was accompanied by the expression of the protein(s) with chitinolytic activity and molecular weight approximately of 25 kDa. The relationship between the formation of ELS and secretion of chitinase(s) is discussed.
Embryogenic tissues of Pinus nigra have been cryopreserved using a two step slow-freezing method. In the first experiment, 20 cell lines were included and the effect of the duration of cryostorage (1 h vs. 1 year) on regrowth was compared. After a short-term storage (1 h in liquid nitrogen, LN) out of 20 cell lines tested 15 showed regrowth (75%) with individual frequencies 10–100%. Long term storage (1 year in LN) resulted in regrowth of 14 cell lines (70%) while the individual frequencies reached 10–100%. One year storage had no negative influence on the fresh mass accumulation evaluated 2–3 months after thawing. Another 20 cell lines were included in the second experiment with the aim to study the correlation between cryotolerance and maturation capacity of cell lines. Between maturation capacity and cryotolerance expressed as regrowth frequencies of individual cell lines, no correlation has been found.
Upon stress treatment, isolated microspores of triticale (×Triticosecale Wittm.) were directed towards sporophytic development (androgenesis). We used fluorescence microscopy to study the cell structural reorganization associated with the process. Changes in the developmental pathway coincided with the character of the microtubular cytoskeleton configuration, the number and direction of nuclear divisions, changes in vacuolization, the distribution of mitochondria, ER and starch grains, and the architecture of new cell wall formation. A band of diffused fluorescence surrounding the nucleus was observed before the first symmetric division of microspores. This structure most likely represents a preprophase band (PPB). Successive mitotic divisions within the microspore wall led to the formation of multinucleate or multicellular structures consisting of one or two domains of cells differing in size. They were later released from the sporoderm and continued further development with features typical for a monocotyledonous embryo. The pattern of internal architecture of androgenic structures depended on their developmental phase. Before and after release from the microspore wall, cortical microtubules (MTs) exhibited various configurations without preferential orientation. They formed a denser network in the region opposite to the sporoderm rupture site. Released multicellular structures showed both intensely fluorescing cortical MTs and more dispersed endoplasmic MTs radiating along the cytoplasmic strands from the nuclear region to the cell cortex. Up to globular stage, isotropically expanding cells of androgenic embryos showed a random pattern of MTs. This is the first report that successive events of androgenic development of triticale microspores are associated with MT reorganization. The results support the view that changes in cytoskeleton architecture are critical during induction of androgenesis.
The genes encoding for a cucumber class III chitinase and Nicotiana plumbaginifolia class I glucanase were co-introduced into Slovak potato (Solanum tuberosum L.) cultivar ETA using Agrobacterium tumefaciens. Expression of both genes was driven by wound-inducible polyubiquitin promoter isolated from Slovak potato breeding line 116/86. Analyses showed inducible, peel-specific expression of both transgenes under stress conditions. The effect of transgene expression on fungal susceptibility of transformants was evaluated in vitro and in vivo. Experiments with crude protein extracts isolated from transgenic microtubers showed growth inhibition of Rhizoctonia solani hyphae in the range from 7.3 to 14.2%. In contrast, experiments performed in growth chamber conditions revealed that the polyubiquitin promoter driven transgene expression did not ensure any obvious increase of transgenic potato resistance against Rhizoctonia solani.
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