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Somatic embryos were induced from immature zygotic embryos of Arabidopsis thaliana cultured on Gamborg basal medium (B5) supplemented with dichlorophenoxyacetic acid (2,4-D) at 5 µM l-1. The somatic embryos were of unicellular origin. The sequence of divisions and the orientation of newly formed walls resembled the Onagrad pattern of early embryogenesis. Histological studies revealed no connection between the somatic embryos and explant tissue. In contrast to zygotic embryos, in late heart-shaped somatic embryos both sieve and tracheary elements were present. The sieve elements that formed in somatic embryos were characterized by larger plates than normal sieve elements observed in seedlings. Typical features of the tracheary elements in somatic embryos were irregular shape and thickening of the secondary walls.
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.
On the basis of EC6 medium, specific conditions were established to improve its properties as a nutrient medium for isolated, immature zygotic embryos of white clover. The highest frequency of embryo development occurred on medium with 117 mM sucrose concentration and 30% (v/v) coconut water for globular-stage (23.3%) and heart-stage embryos (78.3%). The development of globular-stage embryos was abnormal, however. The embryos callused after two weeks of culture. From the callus produced, plantlets and then plants were regenerated. A double-layer culture system, with the top layer having higher osmolarity than the bottom layer, enabled proembryos smaller than 60 µm to be cultured. For in ovulo embryo culture, Nitsch medium supplemented with 10% (v/v) cucumber juice proved most suitable. On this medium, approximately 13% of the ovules containing few-celled embryos germinated and produced seedlings.
A method for micropropagation of the protected species Gentiana pneumonanthe L. by somatic embryogenesis (SE) was elaborated. For SE induction, secondary explants of leaves and apical meristems from in vitro cultures were successfully used. Tests of callus induction were carried out on ten media containing BA (0.04-8.0 µM) and Picloram or 2,4-D (4.0-8.0 µM). The greatest amounts of embryogenic callus developed on media containing 2,4-D and also when the incubation period in darkness was prolonged. Embryos matured on media with reduced content of auxins [Picloram or 2,4-D (0.8 µM or 0.08 µM) and BA (0.8 µM)] and germinated on hormone-free media. Cytometric analysis of the callus and plants obtained from somatic embryos showed that DNA content differed from that of the mother plants, but acclimatized marsh gentians contained the same amount of DNA as donor plants.
The association of two opposing reproductive processes - sexual and asexual - in the course of evolution has given rise to seed multiplication. Genetic heterogeneity, based mainly on the different origins of embryos, appears to be one of the most important traits of seeds. Seeds may contain zygotic embryos, which arise by means of the sexual process (meiosis and fertilization). This mechanism is the main source of gene recombination, which determines genetic variation. However, somatic embryos developed without fertilization also may give rise to a new generation of plants. In connection with the problem of genetic heterogeneity of seeds, special attention has been paid to a new category, embryoidogeny, which seems to be a specialized form of vegetative propagation. The origins of embryos may differ, as these arise by means of monozygotic cleavage, nucellar, or integumentary embryoidogeny. Other apomictic embryos may be formed by means of gametophytic apomixis (diplospory or apospory, followed by diploid parthenogenesis or apogamety). Though these embryos are of different origins, they all are matroclinous offspring. Based on the origin of the embryos, several types of genetic heterogeneity of seeds can be distinguished, such as seeds with sexual (zygotic) embryos, seeds with hemigamous chimaerous embryos, seeds with parthenogenetic embryos, seeds with somatic embryos (embryoids), and seeds that contain sexual (zygotic) and parthenogenetic embryos and embryoids. This produces clones with different heredities in the offspring: clones of the parental organism (on the basis of gametophytic apomixis, nucellar and integumentary embryoidogeny) and clones of the new daughter organism on the basis of the zygotic embryo, including the zygote (monozygotic cleavage embryoidogeny, which is the source of monozygotic twins, triplets, etc.). Embryogenetic traits that determine the heterogeneity of the population should be taken into account together with other morphological traits of seed structure. The homeostasis of a species or population is to a large extent provided by the seed bank in the soil. Seeds stored in the soil represent an embryonal population of species.
‘Milam’ (a purported hybrid of Citrus jambhiri Lush) + ‘Femminello’ lemon (Citrus limon L. Burm. f.) allotetraploid somatic hybrids were used as pollen parents in interploid crosses with diploid ‘Femminello’ lemon to achieve mal secco tolerance in different populations of seedless triploid lemon types with good fruit quality. A total of 137 plantlets were obtained and subjected to screening experiments, in order to distinguish zygotic embryos from nucellars. Here we report on and discuss the results obtained with three techniques: flow cytometry, isozyme analysis and ISSR-PCR (the inter-simple sequence repeats-polymerase chain reaction). ISSR-PCR resulted to be a very efficient and reliable technique for the identification of zygotic plantlets.
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