雜交在植物育種中起到重要的作用，但是，雜交障礙阻礙基因資源在遠源物種間流動而未能使其應用於植物育種中。雜交障礙基本上分為配子融合前障礙和配子融合後障礙。離體雜交技術可以用來克服配子融合前障礙，如空間和時間的隔離、花期的分離、花粉和雌蕊的不親和性。配子融合後障礙比較複雜，如雜種生存能力差、活力弱和不育，染色體加倍、配拯救和體細胞雜交技術可以用來應對這些障礙。但是，這些技術不能解決染色體消除障礙。染色體消除現象可以在遠源物種間的配子體雜種和體細胞雜種中觀察到，表現為在胚的生長發育過程中一個親本的染色體組隨著有絲分裂的進程完全失，這種現象可以在小麥和玉米的雜種中觀察到。為了克服這種雜交障礙，我們假定在小麥中過量表達玉米的CenH3基因可以促進玉米染色體在小麥中保留，進而克服這種雜交障礙。這種策略如果起作用的話，可以應用到創造遠源雜種，轉移染色體，尤其是轉移迷你染色體並應用於作物的遺傳改良。 / 在本研究中，應用小麥和玉米的雜交系統來驗證CenH3基因是否可以解決染色體消除障礙。通過基因槍介導基因轉化的方法，兩個載體分別被轉化到小麥幼胚中，這兩個載體分別含有YFP標記的玉米CenH3基因，和RFP標記的融合CenH3基因用來表達含有小麥CENH3蛋白loop 1之前以及玉米CENH3蛋白loop 1之後的融合蛋白。通過PCR、RFP熒光標記和熒光原位雜交的方法鑑定了含有融合CENH3蛋白的轉基因小麥，並且這些轉基因小麥是可育和穩定遺傳的植株。但是并没有得到含有玉米CenH3基因的轉基因小麥。因此，含有融合CENH3蛋白的轉基因小麥作為受體應用於同玉米的雜交中。 / 通過體細胞雜交的方法來構建轉基因小麥和玉米的體細胞雜種，並研究該雜種細胞中染色體的行為。通過醋酸地衣紅染色的方法對不同時期含有融合CENH3蛋白小麥和玉米的融合細胞的分析表明該融合CENH3蛋白不能完全消除有絲分裂過程中異常染色體行為的現象。熒光原位雜交分析同樣表明該融合CENH3蛋白可能未能阻止雜種中染色體消除的現象，但是需要更多的結果去驗證該融合CENH3蛋白作用並得出最終結論。 / Hybridization plays an important role in plant breeding, but hybridization barriers block the way of gene flow to use far related genetic resources for breeding. Hybridization barriers are generally classified into pre-zygotic and post-zygotic barriers. Pre-zygotic barriers such as spatial and temporal separation, floral isolation, and pollen-pistil incompatible can be overcome by in vitro hybridization technology. Post-zygotic barriers such as hybrid non-viability, weakness, and sterility are more complicated, and techniques such as chromosome doubling, embryo rescue, and somatic hybridization have been developed to tackle with these problems. However, chromosome eliminations can not be solved by these techniques. Chromosome eliminations are observed in zygotes and somatic hybrids of far related plant species, where chromosomes of one genome can be completely lost in mitosis during the embryo development. Such phenomena were observed in wheat x maize hybridizations. To overcome this kind of barrier, we hypothesize that over expression of maize CenH3 gene in wheat could facilitate maize chromosome adaptation in the hybrids, and thus overcome the hybridization barrier. This strategy, if it works, could be applied to make wide crosses, and transfer chromosomes, especially minichromosome for crop improvements. / In this study, wheat x maize intergenic hybridization system was used to test whether CenH3 gene could be used to solve chromosome eliminations. Two constructs that contained a YFP tagged maize CenH3 gene, and a RFP tagged fusion CenH3 gene of wheat CENH3 before loop 1 and maize CENH3 after loop 1, were delivered to wheat immature embryos respectively by gold particle bombardment approach. Fertile and stable transgenic wheat plants with fusion CENH3 (fusion CENH3 wheat) were produced, and confirmed by PCR, RFP fluorescence detection, and FISH analysis, while transgenic wheat plants with maize CenH3 gene were not obtained. Transgenic wheat plants that expressed the fusion CenH3 gene were used as recipients in hybridizations with maize. / Somatic hybridization between transgenic wheat and maize were performed to investigate chromosome behaviors in the hybrids. Aceto-orcein staining analysis of fusion protoplast of fusion CENH3 wheat and maize at different stages showed that the fusion CENH3 could not completely eliminate abnormal chromosome behaviors during mitosis. FISH analysis also suggested that fusion CENH3 might not prevent chromosome elimination in the hybrids, but more results are needed to make final conclusions. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Li, Jianhui. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 90-97). / Abstracts also in Chinese. / List of Figures --- p.iii / List of Tables --- p.iv / List of Abbreviations --- p.v / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Plant hybridization --- p.1 / Chapter 1.2 --- Hybridization barriers --- p.2 / Chapter 1.3 --- Technologies to overcome hybridization barriers --- p.2 / Chapter 1.4 --- CENH3 and chromosome elimination --- p.3 / Chapter 1.4.1 --- Chromosome elimination --- p.3 / Chapter 1.4.2 --- Centromere and CENH3 --- p.5 / Chapter 1.4.3 --- Hypothesis of CENH3 and chromosome elimination --- p.9 / Chapter 1.5 --- Experiment design --- p.9 / Chapter 1.5.1 --- Wheat transformation --- p.10 / Chapter 1.5.2 --- Somatic hybridization --- p.13 / Chapter 1.5.3 --- Microprotoplast fusion --- p.15 / Chapter 1.6 --- Applications of somatic hybridization --- p.17 / Chapter 1.6.1 --- Plant breeding --- p.17 / Chapter 1.6.2 --- Minichromosome technology --- p.18 / Chapter Chapter 2 --- Materials and methods --- p.23 / Chapter 2.1 --- Genetic transformation of wheat --- p.23 / Chapter 2.1.1 --- Wheat growth and callus induction --- p.23 / Chapter 2.1.2 --- Constructs for wheat transformation --- p.24 / Chapter 2.1.3 --- Plasmid DNA extraction --- p.26 / Chapter 2.1.4 --- Gold particle bombardment and plants regeneration --- p.26 / Chapter 2.1.5 --- Seed setting rate of transgenic wheat plants --- p.27 / Chapter 2.2 --- Identification of transgenic wheat plants --- p.28 / Chapter 2.2.1 --- PCR screening of transgenic plants --- p.28 / Chapter 2.2.2 --- Fluorescence microscopy and imaging --- p.31 / Chapter 2.2.3 --- FISH of transgenic wheat calli --- p.32 / Chapter 2.3 --- Wheat x maize hybridization --- p.33 / Chapter 2.3.1 --- Transgenic maize plants with miniB chromosomes --- p.33 / Chapter 2.3.2 --- Non-transgenic and transgenic wheat growth and wheat x maize hybridization --- p.34 / Chapter 2.3.3 --- Embryo rescue and cytological analysis of hybrids --- p.34 / Chapter 2.4 --- Wheat x maize somatic hybridization --- p.35 / Chapter 2.4.1 --- Wheat callus culture --- p.35 / Chapter 2.4.2 --- Maize callus culture --- p.36 / Chapter 2.4.3 --- Cell suspension cultures --- p.36 / Chapter 2.4.4 --- Protoplast isolation from wheat and maize --- p.37 / Chapter 2.4.5 --- Microprotoplast isolation --- p.41 / Chapter 2.4.6 --- Protoplast fusion, culture and regeneration --- p.43 / Chapter 2.4.7 --- Cytological analysis of fusion protoplasts --- p.45 / Chapter 2.4.8 --- FISH of fusion protoplasts --- p.46 / Chapter Chapter 3 --- Results --- p.47 / Chapter 3.1 --- Transgenic wheat with fusion CenH3 gene --- p.47 / Chapter 3.1.1 --- Generation of transgenic wheat plants --- p.47 / Chapter 3.1.2 --- PCR screening of transgenic wheat --- p.49 / Chapter 3.1.3 --- Dwarf mutant in transgenic wheat --- p.51 / Chapter 3.1.4 --- Localization foreign CENH3 by fluorescent protein tag --- p.54 / Chapter 3.1.5 --- Transgene segregation in T₂ generation and callus induction from T₂ transgenic plants --- p.54 / Chapter 3.1.6 --- Detection of transgene by FISH --- p.57 / Chapter 3.1.7 --- Transgenic plants expressing foreign CENH3 protein have lower seed setting --- p.59 / Chapter 3.2 --- Genetic cross of transgenic wheat x maize --- p.61 / Chapter 3.2.1 --- Embryo rescue of zygotic hybrids --- p.61 / Chapter 3.2.2 --- Cytological examination of rescued events --- p.62 / Chapter 3.3 --- Somatic hybridization of transgenic wheat x maize --- p.64 / Chapter 3.3.1 --- Establishment of wheat suspension cell, protoplast and microprotoplast isolation --- p.64 / Chapter 3.3.2 --- Establishment of maize suspension cell, protoplast and microprotoplast isolation --- p.67 / Chapter 3.3.3 --- Somatic hybridization --- p.70 / Chapter 3.3.4 --- Cytological examinations of somatic hybrids --- p.72 / Chapter Chapter 4 --- Discussion --- p.78 / Chapter 4.1 --- Selectable marker genes for wheat transformation --- p.78 / Chapter 4.2 --- Fragmentation of transformed DNAs --- p.79 / Chapter 4.3 --- Wheat and maize CENH3 --- p.81 / Chapter 4.4 --- Embryo rescue by callus induction --- p.83 / Chapter 4.5 --- Plant microprotoplast isolation --- p.84 / Chapter 4.6 --- Maize protoplast isolation and cell suspension culture --- p.86 / Chapter 4.7 --- Maize B-repeat hybridizes to wheat telomere region --- p.87 / Chapter 4.8 --- Fusion CENH3 could not eliminate abnormal chromosome behaviors in somatic hybrids --- p.88 / Chapter Chapter 5 --- Conclusions --- p.89 / References --- p.90
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_328599 |
| Date | January 2013 |
| Contributors | Li, Jianhui., Chinese University of Hong Kong Graduate School. Division of Life Sciences. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | electronic resource, electronic resource, remote, 1 online resource ([6], vi, 97 leaves) : ill. (some col.) |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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