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Improving the methods for cryopreservation of endangered conifers
Gaiduschová, Daniela
Preservation of plant genetic material is essential for any biological field. Compared to serial in vitro sub-culturing, cryopreservation often represents optimal long-term conservation technique. The crucial factors influencing successful cryopreservation include 1) proper pre-treatment, 2) cryoprotectants, 3) adequate freezing pace and 4) cold-hardening. Finding the appropriate method for cryopreservation of each species is the ultimate goal of research in this field. This work is focused on finding the optimal protocol for cryopreservation of embryogenic cell masses of coniferous species bog pine (Pinus uncinata subsp. uliginosa) and optimizing the existing one for European silver fir (Abies alba), both endangered in the Czech countryside. Cryopreservation design compares effectivity of different pre-treatments and cryoprotectants, determines the role of cold-acclimation step and evaluates resistance of selected conifer cell lines to freezing and desiccation. Post-thaw recovery of Bog pine cell line BE4 was better than cell line BR1, where no living cells were visible using FDA/PI staining. Proliferation rate was higher for European silver fir than for bog pine, especially for cell line II-2-10. Cryoprotective mixture with DMSO (PGD I) proved to be more effective than the mixture with glycerol (PGG) in cryopreservation of European silver fir. PGD I also proved to be optimal cryoprotective mixture for bog pine. In both cases, 0.4M sucrose was used as a pre-treatment and cold-hardening was included.
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Improving the methods for cryopreservation of endangered conifers
Gaiduschová, Daniela
Preservation of plant genetic material is essential for any biological field. Compared to serial in vitro sub-culturing, cryopreservation often represents optimal long-term conservation technique. The crucial factors influencing successful cryopreservation include 1) proper pre-treatment, 2) cryoprotectants, 3) adequate freezing pace and 4) cold-hardening. Finding the appropriate method for cryopreservation of each species is the ultimate goal of research in this field. This work is focused on finding the optimal protocol for cryopreservation of embryogenic cell masses of coniferous species bog pine (Pinus uncinata subsp. uliginosa) and optimizing the existing one for European silver fir (Abies alba), both endangered in the Czech countryside. Cryopreservation design compares effectivity of different pre-treatments and cryoprotectants, determines the role of cold-acclimation step and evaluates resistance of selected conifer cell lines to freezing and desiccation. Post-thaw recovery of Bog pine cell line BE4 was better than cell line BR1, where no living cells were visible using FDA/PI staining. Proliferation rate was higher for European silver fir than for bog pine, especially for cell line II-2-10. Cryoprotective mixture with DMSO (PGD I) proved to be more effective than the mixture with glycerol (PGG) in cryopreservation of European silver fir. PGD I also proved to be optimal cryoprotective mixture for bog pine. In both cases, 0.4M sucrose was used as a pre-treatment and cold-hardening was included.
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Effect of heavy metal ions on Norway spruce embryos
?or?ević, Dragana
Aim of this work was to evaluate effect of copper and arsenic on Norway spruce embryogenic cell masses (ECMs) in different concentrations during 14 days proliferation period but as well during maturation and germination stages. Copper in lower concentrations increased the growth of ECMs while in higher concentrations growth was inhibited. Arsenic in concentrations 50, 250 and 500 uM showed very high toxicity and after 14 days proliferation period all ECMs were dead. In experiments with lower concentrations (10 and 25 uM), control had highest proliferation ratio while arsenic had insignificant difference on growth. Cell line I-1-3 had higher proliferation ratio and produced more somatic embryos during maturation in comparison with cell line III-3-3, respectively. After maturation certain number of abnormally developed cotyledonary somatic embryos was obtained. Norway spruce as a fast growing conifer tree is one of the most widely planted spruces and one of the most economically important species in Europe. The results of this thesis can help to understand effect of two tested metals on somatic embryogenesis but as well can be used for investigation of two tested clones in polluted soils.
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Structural sudy of prolifareation phase of somatic embryogenesis of Norway spruce under the effect of aromatic cytokinines
Kadlecová, Marie ; Albrechtová, Jana (advisor) ; Eliášová, Kateřina (referee)
Somatic embryogenesis (SE) represents potentially very suitable way of plant production - micropropagation - of plants in vitro. The process had been studied for more than thirty years and a lot of knowledge has been gained in the field though new ways based on new knowledge leading to improvement and increase of the effectiveness of cultivation protocols are still under focus in the field. Relatively recent discovery of aromatic cytokinins lead to new knowledge on very positive effects on different morphogenetic and physiological processes during both cultivation in vitro or in vivo treatments. This gave origin to the topic of the present thesis, which focused on study of the effect of meta-topolin (mT), aromatic cytokinin, on proliferation phase of SE on structural level of embryogenic lines of Norway spruce Picea abies (L.) Karst. The aim of the thesis is analysis of structural development of embryogenic lines with the use of anatomical preparations and quantitative stereological methods. The material represented three embryogenic lines: 107 and 34C were obtained from the Forestry and Game Management Research Institute from the lab of Dr. Jana Malá and the line AFO 541, which was used as a standard line. To compare the effect of mT with benzylaminopurine (BAP) on proportion of different...
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Somatic embryogenesis of conifers: description of structural development
Kadlecová, Marie ; Albrechtová, Jana (advisor) ; Konrádová, Hana (referee)
Somatic embryogenesis is a very important tool of in vitro cultivation, therefore, it has recently devoted adequate attention. Stages of somatic embryogenesis involve several consecutive steps: induction of embryogenic culture and its proliferation, maturation of embryo, desiccation, germination and subsequent transfer of embryos into young seedlings. All of these steps are directly influenced by many factors, such as a choice of the original explant, time of cultivation, physical conditions or the composition of the culture medium. There are still emerging new studies describing how the culture conditions and, thus, the quality of embryos could be improved. An important prerequisite for further improving the quality of somatic embryos is good knowledge of aculture protocol, i.e. right concentrations of amended substances in the culture medium, the timing of each step, using the suitable explants etc. Another very important thing is good knowledge of development of all anatomical structures established during embryogenesis. Only then it will get closer to the point when somatic embryos will not arise only in the laboratory, but could be successfully used in practice. The object of this work is to summarize existing knowledge about development of all anatomical structures during somatic...
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Studium vlivu fytohormonů a ektomykorhizních hub při maturaci a konverzi somatických embryí jehličnanů
Filippova, Mariia
This thesis deals with the exploration of the influence of various phytohormones, primarily the influence of abscisic acid on the process of maturation and the influence of auxin and gibberellin on the process of germination of two types of conifers, Abies alba and Picea abies, often used as a material in the discovery of somatic embryogenesis. Each genotype prefers specific concentration ABA. 15 uM and 45 uM is the most effective for the cell lines Abies alba; 15 uM and 30 uM ABA for Picea abies. Auxin (IBA) and gibberellin (GA3) did not support the growth of roots of Abies alba. The interaction between fungal cultures Paxillus involutus and Amanita muscaria and between the embryos during the germination was studied as well. Fungal culture Paxillus involutus had very fast speed of growth on the germination medium. Fungal culture Amanita muscaria was growing very slowly. The effect of fungi on the germination has not been observed.
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Studium konverze somatických embryí smrku v rostliny
Psotová, Martina
This work deals with somatic embryogenesis of Picea abies. Part of this work was to compare the viability of plant growth regulator when used before maturation and without them. The main observations of the effects of regulators focused on the final phase of maturation and phase conversion in the plants. The research was conducted in laboratories Mendel University at the Department of Plant Biology. For conditions in the early stages was artificially maintained darknes phase stable air temperature of 23+-2°C together with artificial air circulation. In the following phases photoperiod was controlled, stable air temperature at 23+-2°C with artificial air circulation in the cultivation room. Effect of growth regulators used before maturation was studiedon plants at the final stage of maturation and during conversion to plants. Slower development of roots and aerial parts of plants in the variant with added growth regulators was observed.
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Strukturální změny somatických embryí jehličnanů během jejich zrání a konverze v rostliny uplatnitelné v krajinářské tvorbě
Pinkasová, Markéta
Somatic embryogenesis is an attractive method of vegetative propagation of conifers. However, in many species of conifers are still problems in growing high quality cotyledonary somatic embryos. This is the reason for low quantity of viable plants. Somatic embryogenesis is an integrated process consisting of several consecutive steps. The right timing of individual steps and appropriate in vitro environment is very important to complete the whole process of somatic embryogenesis successfully. However, in many conifers is still necessary to examine the correct procedures and to create better conditions to achieve successfully grown plants. The aim of this thesis was to study the development of somatic embryos of conifers during the maturation and conversion into viable plants. We focused on the prevalence of abnormal cotyledonary somatic embryos created at the end stage of maturation and whether these somatic embryos have the potential to develop into viable plants. It was further found that if cotyledonary somatic embryos develop in the stage of maturation in a certain way, then it can not be changed the appearance or structure of the somatic embryos, thus encouraging the formation of meristem from which it began to develop a plant. Furthermore, in this work we tested the ability of embryogenic lines of Norway spruce (Picea abies) and silver fir (Abies alba) to start the process of maturation after 3 years of maintenance culture on proliferation medium.
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