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Imbibed immature zygotic embryos of Gentiana punctata L. were cultured on MS (Murashige and Skoog) medium consisting of 4.5 µM dicamba, 0.54 µM NAA (naphtaleneacetic acid), 8.88 µM BAP (6-benzylaminopurine) and 0.43 mM AS (adenine hemisulfate). The primary response of expiants consisted in thickening of the subcotyledon and hypocotyl root (HR) zone. Cotyledons and the seminal root did not show any response. Ultrastructural analysis of the initial stages of callus formation revealed numerous changes in cells of expiants. Dedifferentiation of the explant tissues was associated with separation of cells resulting from thickening and folding of walls, destruction of plasmodesmata, and enlargement of intercellular spaces. At the same time, the number of lipid bodies decreased and starch appeared. Indicative of changes in 3-day cultures, the first cell divisions were observed to occur in the HR zone, including cells of the primary cortex, endodermis and pericycle. The dividing cells contained small vacuoles, large, centrally located, layered nuclei with vacuolated nucleoli, amyloplasts with starch, lipid bodies, numerous active Golgi structures, mitochondria and rough endoplasmic reticulum. Actively dividing cells formed callus tissue in which three zones of cells could be distinguished after 14 days of culture: (I) outer (starch) layer, (II) middle layer with actively at dividing small cells, and (III) inner layer containing large vacuolated cells. As the result of cell divisions, at about the fourth week of culture the starch zone formed meristematically active centers of small cells, with dense cytoplasm and large amounts of starch. Among them were small cellular complexes consisting of three cells, with the cell wall structure typical for pre-embryos. By the fifth week of culture, numerous globular and early heart-shaped somatic embryos which formed cotyledons were observed, and further mature somatic embryos showing conversion ability.
Goldenrod (Solidago canadensis L.) is an invasive plant species in many countries except North America but a cut-flower species worldwide. There is a need to generate and propagate goldenrod clones efficiently for research and commercial purposes. A callus induction and plantlet regeneration system was developed by studying the influence of explant type and different concentrations of plant growth regulators. The highest callus production from leaf segments was obtained on Murashige and Skoog's medium (MS medium) supplemented with 1.0 mg/L naphthalene acetic acid (NAA) and 1.0 mg/L 6-benzylaminopurine (BA). Adventitious shoots could be regenerated directly from leaf explants without an intermediate callus phase with the highest shoot induction percentage of 87.2%. The largest number of adventitious shoots per leaf explant (3.2) was obtained on MS medium supplemented with 0.4 mg/L NAA and 2.0 mg/L BA. MS medium supplemented with 0.1 mg/L NAA and 1.0 mg/L BA was the best medium for axillary shoot regeneration from nodal segments. The highest root number and longest roots occurred on half-strength MS without the addition of any growth regulator. Rooted plantlets were then transferred to a soil-based growth medium, placed in a greenhouse, and acclimatized with 100% success. All surviving plants grew normally without showing any morphological variation when compared to those grow from seed. This regeneration protocol may be used to produce certain biotypes of goldenrod suitable for genetic transformation, rapid propagation of goldenrod for commercial purposes or for screening fungi and toxins as potential biocontrol agents against this weed.
Tolerance to a new herbicide, pyributycarb, was evaluated both at the plant and cellular levels. Several highly or moderately tolerant strains chosen at the plant level, showed a parallel relation of to tolerance at the cellular level. However, on the whole, correlation between total tolerance indices and survival rates of calli was not significant in 18 out of the 80 studied strains. As a result of somaclonal selection for two herbicides, lines NB-200 and NM-100 were regenerated from the tolerant calli screened with benthiocarb at 200 ppm, and molinate at 100 ppm, respectively. In the R₂ generation, both the lines displaned a stable tolerance both at the plant and cellular levels. Thus the highly tolerant mutant lines were developed from a moderately tolerant line, N-61, via in vitro selection. To achieve a short-cut method in the interspecific genetic exchange, a series of techniques related to cell fusion were established in rice and related species. Two kinds of somatic hybrids between the cultivar Kitaake and tetraploid Oryza species, O. punctata and O. officinalis, were successfully produced. Among the somatic hybrid plants, a wide range of chromosomal variation was observed. Aneuploid plants with a chromosome number around 2n = 72 (hexaploid), which are expected from a symmetric fusion between diploid and tetraploid strains, were obtained showing mixoploidy within a plant. Most of the somatic hybrids were characterized by intermediate features of plant-type showing high sterility, shattering of spikelets and reduced plant height. As an exception, a diploid plant, which was identified by RFLP analysis using the rDNA gene probe, closely resembled Kitaake and produced viable seeds. A tetraploid hybrid plant was also promising for the introduction of economically important characters through the reduction of chromosome numbers by doubled haploids. Gametoclonal variation and gamma radiation was applied to Kitaake. The mutation frequency was prominently increased by gamma ray treatment, especially at high doses of 200 Gy or 300 Gy. In the M₃R₂ or M₄R₃ generations, most of the variants showed unfavourable characters. Most of the mutant characters were governed by single or double recessive genes. Several mutants such as short culm and early flowering time might be used for rice breeding.
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