Phang Tsui-Hung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 146-162). / Abstracts in English and Chinese. / Thesis committee --- p.i / Statement --- p.ii / Abstract --- p.iii / Acknowledgement --- p.vi / Abbreviations --- p.viii / Table of contents --- p.xii / List of figures --- p.xviii / List of tables --- p.xx / Chapter 1. --- Literature Review --- p.1 / Chapter 1.1 --- Salinity as a global problem --- p.1 / Chapter 1.2 --- Formation of saline soil --- p.1 / Chapter 1.3 --- Urgent need to reclaim saline lands --- p.2 / Chapter 1.4 --- Cellular routes for Na+ uptake --- p.2 / Chapter 1.4.1 --- Carriers involved in K+ and Na+ uptake --- p.2 / Chapter 1.4.2 --- Channels involved in K+ and Na+ uptake --- p.4 / Chapter 1.5 --- Adverse effects of high salinity --- p.5 / Chapter 1.5.1 --- Hyperosmotic stress --- p.5 / Chapter 1.5.2 --- Ionic stress --- p.6 / Chapter 1.5.2.1 --- Deficiency of K+ --- p.6 / Chapter 1.5.2.2 --- Perturbation of calcium balance --- p.7 / Chapter 1.5.3 --- Toxicity of specific ions --- p.7 / Chapter 1.5.4 --- Oxidative stress --- p.10 / Chapter 1.6 --- Mechanisms of salt stress adaptation in plants --- p.11 / Chapter 1.6.1. --- Maintenance of ion homeostasis --- p.12 / Chapter 1.6.1.1 --- Regulation of cytosolic Na+ concentration --- p.12 / Chapter 1.6.1.2 --- SOS signal transduction pathway --- p.15 / Chapter 1.6.2 --- Dehydration stress adaptation --- p.17 / Chapter 1.6.2.1 --- Aquaporins ´ؤ water channel proteins --- p.17 / Chapter 1.6.2.2 --- Osmotic adjustment --- p.20 / Chapter 1.6.2.2.1 --- Genetic engineering of glycinebetaine biosynthesis --- p.23 / Chapter 1.6.2.2.2 --- Genetic engineering of mannitol biosynthesis --- p.27 / Chapter 1.6.3 --- Morphological and structural adaptation --- p.28 / Chapter 1.6.4 --- Restoration of oxidative balance --- p.29 / Chapter 1.6.5 --- Other metabolic adaptation --- p.31 / Chapter 1.6.5.1 --- Induction of Crassulacean acid (CAM) metabolism --- p.34 / Chapter 1.6.5.2 --- Coenzyme A biosynthesis --- p.34 / Chapter 1.7 --- Soybean as a target for studying salt tolerance --- p.36 / Chapter 1.7.1 --- Economical importance of soybean --- p.36 / Chapter 1.7.2 --- Reasons for studying salt stress physiology in soybeans --- p.38 / Chapter 1.7.3 --- Salt tolerant soybean in China --- p.39 / Chapter 1.7.4 --- Exploring salt tolerant crops by genetic engineering --- p.41 / Chapter 1.8 --- Significance of this project --- p.47 / Chapter 2. --- Materials and methods --- p.48 / Chapter 2.1 --- Materials --- p.48 / Chapter 2.1.1 --- Plant materials used --- p.48 / Chapter 2.1.2 --- Bacteria strains and plasmid vectors --- p.48 / Chapter 2.1.3 --- Growth media for soybean --- p.48 / Chapter 2.1.4 --- Equipment and facilities used --- p.48 / Chapter 2.1.5 --- Primers used --- p.48 / Chapter 2.1.6 --- Chemicals and reagents used --- p.49 / Chapter 2.1.7 --- Solutions used --- p.49 / Chapter 2.1.8 --- Commercial kits used --- p.49 / Chapter 2.1.9 --- Growth and treatment condition --- p.49 / Chapter 2.1.9.1 --- Characterization of salt tolerance of Wenfeng7 --- p.49 / Chapter 2.1.9.2 --- Samples for subtractive library preparations --- p.50 / Chapter 2.1.9.3 --- Samples for slot blot and northern blot analyses --- p.50 / Chapter 2.1.9.4 --- Samples for gene expression pattern analysis --- p.50 / Chapter 2.2. --- Methods --- p.52 / Chapter 2.2.1 --- Total RNA extraction --- p.52 / Chapter 2.2.2 --- Construction of subtractive libraries --- p.53 / Chapter 2.2.3 --- Cloning of salt-stress inducible genes --- p.53 / Chapter 2.2.3.1 --- Preparation of pBluescript II KS(+) T-vector for cloning --- p.53 / Chapter 2.2.3.2 --- Ligation of candidate DNA fragments with T-vector --- p.53 / Chapter 2.2.3.3 --- Transformation --- p.54 / Chapter 2.2.3.4 --- PCR screening --- p.54 / Chapter 2.2.4 --- Preparation of recombinant plasmid for sequencing --- p.55 / Chapter 2.2.5 --- Sequencing of differentially expressed genes --- p.55 / Chapter 2.2.6 --- Homology search of differentially expressed genes --- p.56 / Chapter 2.2.7 --- Expression pattern analysis --- p.56 / Chapter 2.2.7.1 --- Preparation of single-stranded DIG-labeled PCR probes --- p.56 / Chapter 2.2.7.2 --- Preparation of cRNA DIG-labeled probes --- p.57 / Chapter 2.2.7.3 --- Testing the concentration of DIG-labeled probes --- p.58 / Chapter 2.2.7.4 --- Slot blot --- p.58 / Chapter 2.2.7.5 --- Northern blot --- p.59 / Chapter 2.2.7.6 --- Hybridization --- p.60 / Chapter 2.2.7.7 --- Stringency washed --- p.60 / Chapter 2.2.7.8 --- Chemiluminescent detection --- p.60 / Chapter 3. --- Results --- p.61 / Chapter 3.1 --- Characterization of salt tolerance of Wenfeng7 --- p.61 / Chapter 3.2 --- Identification of salt-stress induced genes from Wenfeng7 --- p.70 / Chapter 3.2.1 --- Screening subtractive libraries of Wenfeng 7 for salt inducible genes --- p.70 / Chapter 3.2.1.1 --- Results of homology search for salt inducible genes --- p.71 / Chapter 3.2.1.2 --- Northern blot showing the salt inducibility of selected clones --- p.72 / Chapter 3.3 --- Genes expression pattern of selected salt inducible genes --- p.104 / Chapter 3.3.1 --- Expression of genes related to dehydration adjustment --- p.104 / Chapter 3.3.1.1 --- Dehydration responsive protein RD22 (Clone no.: HML806) --- p.104 / Chapter 3.3.1.2 --- Beta-amylase (Clone no.: HML767) --- p.104 / Chapter 3.3.2 --- Expression of genes related to structural modification --- p.105 / Chapter 3.3.3 --- Expression of genes related to metabolic adaptation --- p.105 / Chapter 3.3.3.1 --- Subgroup 1: Gene related to protein synthesis --- p.105 / Chapter 3.3.3.1.1 --- Translational initiation factor nsp45 (Clone no.: HML1042) --- p.105 / Chapter 3.3.3.1.2 --- Seed maturation protein PM37 (Clone no.: HML931) --- p.106 / Chapter 3.3.3.2 --- Subgroup 2: Genes related to phosphate metabolism (Clone no.: HML1000) --- p.107 / Chapter 3.3.3.3 --- Subgroup 3: Genes related to storage and mobilization of nutrient resources --- p.107 / Chapter 3.3.3.3.1 --- Vegetative storage protein A (Clone no.: HML762) --- p.107 / Chapter 3.3.3.3.2 --- Cysteine proteinase (Clone no.: HML928) --- p.107 / Chapter 3.3.3.4 --- Subgroup 4: Genes related to senescence --- p.109 / Chapter 3.3.4 --- Expression of genes encoding novel protein (Clone no.: HML782) --- p.109 / Chapter 4. --- Discussion --- p.125 / Chapter 4.1 --- Evaluation of salt tolerance of Wenfeng7 --- p.125 / Chapter 4.2 --- Isolation of salt inducible genes in Wenfeng7 --- p.127 / Chapter 4.2.1 --- Genes associated with dehydration adaptation --- p.129 / Chapter 4.2.1.1 --- Dehydration responsive protein RD22 --- p.129 / Chapter 4.2.1.2 --- Beta-amylase --- p.130 / Chapter 4.2.2 --- Genes associated with structural adaptation --- p.132 / Chapter 4.2.3 --- Genes associated with metabolic adaptation --- p.133 / Chapter 4.2.3.1 --- Subgroup 1: Genes related to protein synthesis --- p.133 / Chapter 4.2.3.2 --- Subgroup 2: Genes related to phosphate metabolism --- p.137 / Chapter 4.2.3.3 --- Subgroup 3: Genes related to storage and mobilization of nutrient resources --- p.138 / Chapter 4.2.3.4 --- Subgroup 4: Genes related to senescence --- p.140 / Chapter 4.2.4 --- Novel genes --- p.142 / Chapter 4.3 --- Brief summary --- p.142 / Chapter 5. --- Conclusion and perspectives --- p.144 / References --- p.146 / Appendix I: Screening for salt tolerant soybeans --- p.163 / Appendix II: Major equipment and facilities used --- p.165 / Appendix III: Major chemicals and reagents used in this research --- p.166 / Appendix IV: Major common solutions used in this research --- p.168 / Appendix V: Commercial kits used in this research --- p.170

by Wong Fuk Ling. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 100-115). / Abstracts in English and Chinese. / Thesis committee --- p.i / Statement --- p.ii / Abstract --- p.iii / Chinese abstract --- p.v / Acknowledgements --- p.vii / Abbreviations --- p.ix / Table of contents --- p.xii / List of figures --- p.xvi / List of tables --- p.xvii / Chapter 1. --- Literature review / Chapter 1.1 --- Soybeans / Chapter 1.1.1 --- Economical importance of soybean --- p.1 / Chapter 1.1.2 --- History and origin of soybean --- p.4 / Chapter 1.1.3 --- Qualitative traits of cultivated and wild soybeans --- p.5 / Chapter 1.1.4 --- Soybean resource in China --- p.6 / Chapter 1.1.5 --- Salt tolerant soybeans in China --- p.6 / Chapter 1.2 --- Salinization as a global problem --- p.7 / Chapter 1.3 --- Toxicity of salt in plants --- p.8 / Chapter 1.4 --- Salt stress signal transduction in plants --- p.10 / Chapter 1.4.1 --- Ionic and osmotic stress signaling / Chapter 1.4.1.1 --- Ca2+ signaling --- p.11 / Chapter 1.4.1.2 --- The SOS pathway --- p.12 / Chapter 1.4.1.3 --- Protein kinase pathways --- p.13 / Chapter 1.4.1.4 --- Phospholipid signaling --- p.14 / Chapter 1.4.1.5 --- ABA signaling --- p.17 / Chapter 1.4.2 --- Detoxification signaling --- p.17 / Chapter 1.4.3 --- signaling to coordinate cell division ana expansion --- p.18 / Chapter 1.5 --- Plant adaptations in plants --- p.18 / Chapter 1.5.1 --- Ion homeostasis --- p.18 / Chapter 1.5.1.1 --- Reduction of Na+ influx into the cells --- p.19 / Chapter 1.5.1.2 --- Extrusion of Na+ out of the cell --- p.19 / Chapter 1.5.1.3 --- Vacuolar compartmentation of Na+ --- p.20 / Chapter 1.5.2 --- Osmotic adjustment --- p.20 / Chapter 1.5.3 --- Antioxidant protection --- p.21 / Chapter 1.5.4 --- Morphological and structural modification --- p.21 / Chapter 1.6 --- The relationship of salt stress and phosphorus deficiency --- p.22 / Chapter 1.7 --- The importance of phosphorus in plants --- p.25 / Chapter 1.8 --- The role of purple acid phosphatase (PAP) in plants --- p.25 / Chapter 1.9 --- PAPs in soybean --- p.27 / Chapter 1.10 --- Hypothesis and significance of this project --- p.27 / Chapter 2. --- Materials and methods / Chapter 2.1 --- Materials / Chapter 2.1.1 --- Plant materials --- p.29 / Chapter 2.1.2 --- The clones used in this work --- p.30 / Chapter 2.1.3 --- Growth media for soybeans --- p.31 / Chapter 2.1.4 --- Equipment and facilities --- p.31 / Chapter 2.1.5 --- Primers --- p.31 / Chapter 2.1.6 --- Chemicals and reagents --- p.31 / Chapter 2.1.7 --- Solutions --- p.32 / Chapter 2.1.8 --- Commercial kits --- p.32 / Chapter 2.1.9 --- Software --- p.32 / Chapter 2.2. --- Methods / Chapter 2.2.1 --- Growth and salt treatment condition / Chapter 2.2.1.1 --- Establishment a collection of typical salt tolerant and sensitive soybean varieties --- p.33 / Chapter 2.2.1.2 --- Samples for northern analysis of salt inducible genes --- p.33 / Chapter 2.2.1.3 --- Samples for characteristics of GmPAP3 gene --- p.35 / Chapter 2.2.1.4 --- Samples for oxidative stress test --- p.36 / Chapter 2.2.2 --- Total RNA extraction --- p.36 / Chapter 2.2.3 --- Denaturing gel electrophoresis of RNA --- p.38 / Chapter 2.2.4 --- Expression pattern analysis / Chapter 2.2.4.1 --- Preparation of single-stranded DIG-labeled PCR probes --- p.39 / Chapter 2.2.4.2 --- Testing the concentration of DIG-labeled probes --- p.40 / Chapter 2.2.4.3 --- Northern blot --- p.41 / Chapter 2.2.5 --- Soluble ions analysis --- p.42 / Chapter 2.2.6 --- Acid phosphatase activity assays --- p.42 / Chapter 2.2.7 --- Phylogenetic analysis and subcellular localization prediction of GmPAP3 --- p.43 / Chapter 3. --- Results / Chapter 3.1 --- Establishing a collection of typical salt tolerant and sensitive soybean varieties --- p.44 / Chapter 3.2 --- Characterization of salt inducible genes --- p.48 / Chapter 3.3 --- Characterization of GMPAP3 gene --- p.63 / Chapter 3.3.1 --- Phylogenetic studies of the GmPAP3 --- p.64 / Chapter 3.3.2 --- The salt-inducible GmPAP3 gene in soybean encodes a putative mitochondria-located PAP --- p.64 / Chapter 3.3.3 --- Expression of GmPAP3 was induced by NaCl stress but not P deficiency --- p.71 / Chapter 3.3.4 --- Expression of GmPAP3 was induced by oxidative stress --- p.80 / Chapter 4. --- Discussion --- p.82 / Chapter 4.1 --- A collection of typical salt tolerant and sensitive soybean varieties --- p.83 / Chapter 4.2 --- Inducibility of identified salt-inducible gene in different varieties --- p.85 / Chapter 4.2.1 --- The possible roles of identified salt-inducible genes --- p.85 / Chapter 4.2.2 --- Expression profiles of identified salt-inducible genes --- p.89 / Chapter 4.3 --- The novel gene GmPAP3 --- p.92 / Chapter 5. --- Conclusion and perspectives --- p.98 / References --- p.100 / Appendix I: Expression profiles of the salt inducible genes in root tissue of selected varieties --- p.116 / Appendix II: Major equipment and facilities used in this research --- p.124 / Appendix III: Major chemicals and reagents used in this research --- p.125 / Appendix IV: Major common solutions used in this research --- p.127

我們在大豆的耐鹽候選基因中進行了篩選和調查，確定了一個穀胱甘肽-S -轉移酶（Glutathion-S-transferase ）基因（ GmGSTL1 ）具抗鹽特性，其表達量也跟隨鹽處理上調。系統發育分析表明，GmGSTL1 屬於LAMBDA 類，文獻對這類蛋白功能的記載甚少。我們在異源系統，包括煙草BY- 2 細胞和擬南芥，測試其保護細胞/植物對鹽脅迫的功能。結果表示GmGSTL1 轉基因細胞的活性氧積累比對照顯著降低，存活率也有所改善。同樣，轉基因擬南芥在鹽處理壓力下的症狀也得以緩解。為了進一步剖析GmGSTL1 的保護機制，我們在大豆葉片中提取多元酚，並發現兩個候選黃酮（槲皮素，山奈酚）與GmGSTL1 起相互作用。槲皮素的外源性應用同樣可以緩解細胞/植物在鹽脅迫下的症狀，表示槲皮素在功能上與GmGSTL1 相約。 / In a survey of candidate genes located in the salinity tolerance locus of soybean, we identified a putative glutathione-S-transferase (GST) gene (GmGSTL1) which was up-regulated in response to salt treatment. Phylogenetic analyses revealed that this putative GST belongs to the Lambda class, a plant-specific group with unknown functions. We expressed GmGSTL1 in heterologous systems, including tobacco BY-2 cells and Arabidopsis thaliana, to test its ability to protect cell/plant against salinity stress. Compare to the wild type control, we observed a marked reduction of ROS accumulation in transgenic cells under salt treatment, and their survival rate was also improved. Similarly, expression of GmGST1 in transgenic A. thaliana also alleviated stress symptoms under salt treatment. To further address the possible protective mechanisms of GmGSTL1, we identified two candidate flavonoid interactants (quercetin and kaemferol) of the GmGSTL1 protein from soybean leaf extract. Exogenous application of quercetin could reduce salinity-induced ROS accumulation in BY-2 cells and leaf chlorosis in A. thaliana. / Detailed summary in vernacular field only. / Chan, Ching. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 80-104). / Abstracts also in Chinese.

Histone modifications and histone variants are of importance in many biological processes. Whether they play some roles in regulating soybean salinity stress response is unknown. Previously, no study of histone modifications and histone variants in soybean were reported. In this study, I elucidated that in soybean leaves, mono-, di- and tri-methylation at Lysine (K) 4, 27 and 36, and acetylation at Lysine 14, 18 and 23 were present in histone H3. Moreover, H3K27 methylation and H3K36 methylation usually excluded each other. Although H3K79 methylation was not reported in Arabidopsis, they were detected in soybean. In soybean histone H4, Lysine 8 and 12 were acetylated. In addition, the variants of histone H3 and H4 and their modifications were also determined. The variants of histone H3 were different at positions of A31F41S87S90 (histone variant H3.1) and T31Y41H87 L90 (histone variant H3.2), respectively. Lysine 4 and 36 methylation were only detected in histone H3.2, suggesting that histone variant H3.2 might associate with actively transcribing genes. The two variants of histone H4 (H4.1 and H4.2) were different at amino acid 60. Moreover, I also found that the abundance of most of the histone modifications and histone variants did not change under the salinity stress except that H3K79 methylation would be up-regulated by the salinity stress. / In a parallel study, a PHD (plant homeodomain) finger domain containing protein, GmPHD1, was able to decipher the 'code' underlying H3K4 methylation. GmPHD1 was ubiquitously expressed in soybean and its expression increased upon salinity stress. GmPHD1 could bind to histone H3K4 methylation, with the preference to H3K4 dimethylation. It could then recruit several proteins, which were GmGNAT1, GmElongin A, and GmISWI. The interaction between GmPHD1 and GmGNAT1 was regulated by the self-acetylation of GmGNAT1. GmGNAT1 could also acetylate histone H3; GmElongin A was a transcription elongation factor; and GmISWI was a chromatin remodeling protein. Our data also indicated that the GmPHD1 located at the promoter of several soybean salt stress inducible genes. Therefore, the GmPHD1 recruited proteins to remodel the chromatin structure and facilitate the transcription of those salt stress inducible genes. Moreover, GmGNAT1 exhibited the preference to acetylate histone H3K14, therefore representing a kind of histone crosstalk between H3K4 methylation and H3K14 acetylation. / Proteomics studies with 2-DE revealed that salt treatment may affect soybean photosynthesis and chloroplast formation. Comparison between the proteomic profiles of salt tolerant soybean variety (wild type) and salt sensitive soybean variety (cultivated, Union) indicated that protein levels in the detoxification and defense pathway as well as energy metabolism were higher in the wild type soybean, while the process of protein metabolism was less active. In addition, proteomic profiles of the cultivated soybean roots at different developmental stages were also compared to identify proteins related to soybean development. The expression of proteins which play critical roles in detoxification and defense pathways were higher at the seedling stage, especially the proteins which regulated the formation of ROS. / Soybean is an important economic crop and its production can be severely affected by salinity stress. At present, the soybean response to salinity stress is not clear. In my studies, I tried to understand this process from the perspective of proteomics and epigenetics, especially histone modifications. / Wu, Tao. / Advisers: Njai Sai Ming; Lam Hon Ming. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 126-151). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

By immumolabeling and electronmicroscopy, the subcellular localization of GmPAP3 has been proved to be mainly localized in mitochondria, a primary site for ROS production. Ectopic expression of GmPAP3 in transgenic tobacco BY-2 cells mimicked the protective effects exhibited by the antioxidant ascorbic acid by: (1) increase the percentage of cells with active mitochondria; (2) reduce the percentage of dead cells; and (3) lower the accumulation of ROS under NaCl and osmotic stress treatments. However, when ectopically express a truncated GmPAP3 with the mitochondria transit peptide removed, such protective effect was not observed. This provides evidences on the significance of mitochondria localization to the physiological function of GmPAP3. In addition, when GmPAP3 transgenic Arabidopsis thaliana seedlings were subjected to NaCl, osmotic stress, and oxidative stress treatments, the growth performance of the transgenic lines was significantly better than the wild type. To summarize, these studies has demonstrate that the mitochondrial localized GmPAP3 may play a role in stress tolerance by enhancing ROS scavenging. / Mitochondrion is one of the major sites for the production of reactive oxygen species (ROS). Abiotic stresses such as salinity and osmotic stress can cause oxidative damage to organelle membranes due to excess accumulation of ROS. The inducibility of GmPAP3 gene expression by salinity and oxidative stresses and the putative mitochondrial localization of GmPAP3 prompt us to further investigate the possible physiological roles of GmPAP3 under abiotic stress-induced oxidative stress. / My Ph.D. study has been focused on the detailed functional analysis of the GmPAP3 gene. The objectives of my research include: (i) to verified the subcellular localization of GmPAP3; (ii) to investigate the physiological functions of GmPAP3 under NaC1 and osmotic stress in both cellular level and in planta level. and (iii) to examine the significance of mitochondria] localization of GmPAP3 in relationship to its protective roles. / Purple acid phosphatases (PAPs) represent a diverse group of acid phosphatases in animals and plants. While the mammalian PAPs were found to be related to Reactive Oxygen Species (ROS) evolution in important physiological functions, the roles of plant PAPs remain largely unknown. / Recently, we have isolated a novel PAP-like gene (GmPAP3) from soybean that is induced by NaC1 and oxidative stresses. Subcellular localization prediction programs suggested that GmPAP3 may be a novel PAP that localized in mitochondria. Most other PAPs are extracellularly located and membrane localization of PAPs was only verified in a few cases. / by Li, Wing Yen Francisca. / "December 2009." / Adviser: Lam Hon-Ming. / Source: Dissertation Abstracts International, Volume: 72-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 123-134). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Cheng, Chun Chiu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 164-183). / Abstracts in English and Chinese. / Thesis Committee --- p.i / Statement --- p.ii / Abstract --- p.iii / 摘要 --- p.v / Acknowledgements --- p.vi / General Abbreviations --- p.viii / Abbreviations of Chemicals --- p.xi / List of Figures --- p.xv / List of Tables --- p.xvii / Table of Contents --- p.xix / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Salt stress in plants --- p.1 / Chapter 1.2 --- Overview of the molecular basis of salt tolerance in plants --- p.2 / Chapter 1.2.1 --- Stress perception --- p.3 / Chapter 1.2.2 --- Signal transduction --- p.3 / Chapter 1.2.2.1 --- Protein phosphatases --- p.4 / Chapter 1.2.2.2 --- The SOS pathway for ion homeostasis --- p.4 / Chapter 1.2.3 --- DNA and RNA helicases in post-transcriptional control --- p.6 / Chapter 1.2.4 --- ROS scavengers --- p.7 / Chapter 1.2.5 --- Proteases and proteinase inhibitors --- p.8 / Chapter 1.2.6 --- Heat shock proteins (Hsps) --- p.9 / Chapter 1.2.7 --- Highlights on DnaJ/Hsp40 --- p.9 / Chapter 1.3 --- Review on functional genomics of salt stress responses in plants --- p.11 / Chapter 1.3.1 --- Genomics on model organisms --- p.12 / Chapter 1.3.2 --- Transcriptomics for identifying salt stress responsive genes --- p.12 / Chapter 1.3.2.1 --- Multiple stress transcriptome analysis --- p.13 / Chapter 1.3.2.2 --- Genome-wide transcriptome analysis on molecular crosstalk --- p.14 / Chapter 1.3.2.3 --- Tissue specific transcriptome analysis --- p.16 / Chapter 1.3.2.4 --- Comparative transcriptome analysis --- p.17 / Chapter 1.3.2.5 --- Transcriptome analysis of soybean --- p.24 / Chapter 1.3.3 --- Proteomics in plant salt stress studies --- p.26 / Chapter 1.3.4 --- Beyond the transcriptome and proteome --- p.27 / Chapter 1.4 --- Significance of using soybean germplasms for identifying salt stress responsive genes --- p.28 / Chapter 1.5 --- Objectives --- p.29 / Chapter Chapter 2 --- Materials and Methods --- p.30 / Chapter 2.1 --- Materials --- p.30 / Chapter 2.1.1 --- "Plants, bacterial strains，and vectors" --- p.30 / Chapter 2.1.2 --- Enzymes and major chemicals --- p.33 / Chapter 2.1.3 --- Primers --- p.34 / Chapter 2.1.4 --- Commercial kits --- p.34 / Chapter 2.1.5 --- Equipment and facilities --- p.34 / Chapter 2.1.6 --- "Buffer, solution, gel and medium" --- p.34 / Chapter 2.2 --- Methods --- p.35 / Chapter 2.2.1 --- cDNA microarray analysis --- p.35 / Chapter 2.2.1.1 --- Construction of cDNA subtraction libraries --- p.35 / Chapter 2.2.1.2 --- Assembly of cDNA microarray --- p.36 / Chapter 2.2.1.3 --- External control RNA synthesis --- p.39 / Chapter 2.2.1.4 --- Probe labelling and hybridization --- p.40 / Chapter 2.2.1.5 --- Hybridization signal collection --- p.41 / Chapter 2.2.1.6 --- Image analysis --- p.41 / Chapter 2.2.1.7 --- Data analysis --- p.42 / Chapter 2.2.1.8 --- Selection of salt responsive genes using fold difference in expression --- p.45 / Chapter 2.2.1.9 --- DNA sequencing --- p.46 / Chapter 2.2.1.10 --- Real-time PCR analysis --- p.47 / Chapter 2.2.2 --- Growth conditions and treatments of plants --- p.48 / Chapter 2.2.2.1 --- Soybean for microarray hybridization and real-time PCR --- p.48 / Chapter 2.2.2.2 --- Soybean for the study of GmDNJ1 expression under ABA treatment --- p.48 / Chapter 2.2.2.3 --- Wild-type and transgenic Arabidopsis for functional analysis --- p.49 / Chapter 2.2.2.4 --- Wild-type and transgenic rice for functional analysis --- p.49 / Chapter 2.2.3 --- "DNA, RNA, and protein extraction" --- p.50 / Chapter 2.2.3.1 --- Plasmid DNA extraction from E. coli cells --- p.50 / Chapter 2.2.3.2 --- RNA extraction from plant tissues --- p.51 / Chapter 2.2.3.3 --- Soluble protein extraction from plant tissues --- p.51 / Chapter 2.2.4 --- Blot analysis --- p.51 / Chapter 2.2.4.1 --- Northern blot analysis --- p.52 / Chapter 2.2.4.2 --- Western blot analysis --- p.53 / Chapter 2.2.5 --- Subcloning of GmDNJ1 into pGEX-4T-1 --- p.53 / Chapter 2.2.5.1 --- "Restriction digestion, DNA purification and ligation" --- p.53 / Chapter 2.2.5.2 --- Transformation of competent Escherichia coli (DH5a and BL21) --- p.54 / Chapter 2.2.6 --- Luciferase refolding assay --- p.54 / Chapter 2.2.6.1 --- Culture of E. coli strain BL21 (DE3) --- p.54 / Chapter 2.2.6.2 --- Cell lysis --- p.55 / Chapter 2.2.6.3 --- Purification of the GST-GmDNJ1 fusion protein --- p.55 / Chapter 2.2.6.4 --- Quantitation of protein --- p.55 / Chapter 2.2.6.5 --- Luciferase refolding assay --- p.56 / Chapter Chapter 3 --- Results --- p.57 / Chapter 3.1 --- Overview of cDNA microarray analysis --- p.57 / Chapter 3.2 --- Identification of salt responsive genes in subtraction libraries concerning two contrasting soybean germplasms --- p.61 / Chapter 3.3 --- Data processing before selection of salt stress responsive genes --- p.75 / Chapter 3.3.1 --- M-A plots --- p.75 / Chapter 3.3.2 --- Boxplots --- p.76 / Chapter 3.3.3 --- Scatterplots --- p.76 / Chapter 3.4 --- Selection of salt responsive genes using fold difference in expression --- p.77 / Chapter 3.4.1 --- Selection of genes with differential expression between tolerant and sensitive germplasms --- p.77 / Chapter 3.4.2 --- Selection of genes with differential expression between cultivated and wild germplasms --- p.89 / Chapter 3.4.3 --- Data validation by real-time PCR analysis --- p.91 / Chapter 3.5 --- Selection of salt responsive genes using statistical tools --- p.95 / Chapter 3.5.1 --- Quantitative trait analysis for salt responsive genes --- p.95 / Chapter 3.5.2 --- Identification of salt stress correlation genes --- p.100 / Chapter 3.5.3 --- Cluster analyses --- p.104 / Chapter 3.5.3.1 --- Clustering genes --- p.104 / Chapter 3.5.3.2 --- Clustering samples --- p.108 / Chapter 3.5.4 --- Data validation by real-time PCR analysis --- p.111 / Chapter 3.6 --- Summary of cDNA microarray analysis --- p.112 / Chapter 3.7 --- Studies on GmDNJ1 --- p.120 / Chapter 3.7.1 --- Sequence analysis of GmDNJ1 --- p.120 / Chapter 3.7.2 --- GmDNJ1 was induced by salt stress and ABA treatment in soybean (Glycine max) --- p.127 / Chapter 3.7.3 --- Expressing GmDNJ1 in transgenic Arabidopsis (Arabidopsis thaliana) enhances the tolerance to salt stress and dehydration stress --- p.129 / Chapter 3.7.4 --- Expressing GmDNJ1 in transgenic rice (Oryza sativa) enhances the tolerance to salt stress and dehydration stress --- p.135 / Chapter 3.7.5 --- The GmDNJ1 protein can replace DnaJ in the in vitro luciferase refolding assay --- p.141 / Chapter Chapter 4 --- Discussion --- p.145 / Chapter 4.1 --- Overview of expression profiling of the 20 soybean germplasms --- p.145 / Chapter 4.2 --- Identification of salt responsive genes from subtraction libraries --- p.146 / Chapter 4.3 --- Normalization of data from microarray experiments --- p.148 / Chapter 4.4 --- The fold difference analysis --- p.149 / Chapter 4.4.1 --- Response to stress --- p.149 / Chapter 4.4.2 --- Gene expression --- p.150 / Chapter 4.4.3 --- Molecular function --- p.150 / Chapter 4.4.4 --- Metabolic activity --- p.151 / Chapter 4.4.5 --- Cellular component --- p.152 / Chapter 4.4.6 --- Genes with 2.5-fold difference in expression between cultivated and wild germplasms --- p.153 / Chapter 4.5 --- Selection of salt responsive genes using statistical tools --- p.153 / Chapter 4.5.1 --- Quantitative trait analysis --- p.153 / Chapter 4.5.2 --- Cluster analyses --- p.154 / Chapter 4.6 --- Studies on GmDNJ1 --- p.157 / Chapter 4.6.1 --- GmDNJ1 is a good candidate for gene studies --- p.157 / Chapter 4.6.2 --- Sequence analysis of GmDNJ1 suggested it to be a DnaJ/Hsp40 homologue in soybean --- p.158 / Chapter 4.6.3 --- GmDNJ1 was induced by salt stress and ABA treatment --- p.158 / Chapter 4.6.4 --- GmDNJ1 has a higher expression in salt tolerant soybean germplasms over sensitive ones --- p.159 / Chapter 4.6.5 --- Ectopic expression of GmDNJ1 enhanced the tolerance to salt stress and dehydration stress in transgenic Arabidopsis --- p.159 / Chapter 4.6.6 --- Ectopic expression of GmDNJ1 enhanced the tolerance to salt stress and dehydration stress in transgenic rice --- p.160 / Chapter 4.6.7 --- Luciferase activity assay showed that GmDNJ 1 functioned as a DnaJ/Hsp40 in vitro --- p.161 / Chapter Chapter 5 --- Conclusion --- p.162 / References --- p.164 / Appendix I - Enzymes and major chemicals --- p.184 / Appendix II - Primers --- p.188 / Appendix III - Major commercial kits --- p.192 / Appendix IV - Major equipment and facilities --- p.193 / "Appendix V - Formulation of buffer, solution, gel, and medium" --- p.194 / Appendix VI - Plots in microarray experiments --- p.198 / Appendix VII - Clones with differential expression (>2.5-fold or >1.8-fold) between germplasms --- p.208 / Appendix VIII - Salt responsive genes revealed by quantitative trait analysis --- p.216 / Appendix IX - Supplementary data in real-time PCR analysis --- p.221 / Appendix X - Supplementary data in functional analyses --- p.233

Environmental stresses such as drought, salinity and low temperature have been major selective forces throughout plant evolution and are important factors which limit crop plant distribution and agricultural productivity. An understanding of how crops adapt to adverse conditions is not only of theoretical interest, but also has considerable practical value . Low-temperature stress subtraction libraries were constructed in a pBluescript vector with the two-step-PCR amplified cDNAs using subtractive hybridization. One insert cs18 was obtained and the sequence analysis of insert cs18 revealed that the insert cDNA had a 76% homology with the sequences of the corresponding portion of glucose dehydrogenase from Thermoplasma acidophilum and 62.0% homology with a genomic DNA of Arabidopsis. Four clones, cs18-13, cs18-14, cs18-15, and cs18-16 from low-temperature stress soybean root conventional cDNA library have been confirmed to have inserts that could hybridize to the cs18 insert. One cDNA with a Xba I and Xho I fragment of approximately 3,500 bp in length corresponded to the insert cs18 , which probably encodes for glucose dehydrogenase, was obtained. Northern blot analysis indicated that cs18 mRNA was highly expressed in soybean root but moderately expressed in leaves under low temperature. Changes in the nuclei of meristematic root cells in response to severe salinity were studied. Roots are in direct contact with the surrounding solution . Thus, they are the first to encounter the saline medium and are potentially the first site of damage or line of defence under salt stress. Nuclear deformation or degradation in the soybean root meristem with 150 mM or higher NaCI led to sequential cell degradation, cell death and cessation of plant growth . However, this study indicates that an increase in CaCI[2] concentration up to 5 mM could partially prevent salt injury to the cells. Tissue culture is an excellent tool for elucidat ing the correlation between plant organizational levels and salt tolerance because of the possibility it offers for studying the physiology of intact plantlets together with that of organs and single cells using homogenous plant material under uniform environmental conditions. One NaCI-tolerant cell line (R100) was isolated during this study. The R100 callus cell line was significantly more tolerant to salt than the salt-sensitive line (S100) during exposure to salt stress. Salt tolerance in this culture was characterized by an altered growth behaviour, reduced cell volume and relative water content, and accumulation of Na+, Cl ¯, K+, proline and sugars when grown under salt stress and with its subsequent relief. The selection of this salt tolerant cell line has potential for contributing new genetic variability to plant breeders. Sugars are not only important energy sources and structural components in plants , they are also central regulatory molecules controlling physiology, metabolism, cell cycle , development, and gene expression in plants. The concentrations of glucose and fructose increased during salt stress and after relieving salt stress, at a rate closely corresponding to the increase in relative water content. Their accumulation was the earliest response detected during the removing of salt stress indicating that glucose and fructose may play important roles during salt-stress. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 2000.

In this study we assayed the extent of genetic variation for five microsatellites in 94 accessions of wild (Glycine soja) and cultivated soybean (G. max). F₂ segregation analysis indicated that all five of the microsatellites were independently inherited and four loci were located in four independent linkage groups. The number of alleles per microsatellite locus ranged from five to 21. Overall, 43 more microsatellite alleles were detected in wild than in cultivated soybean. Allelic diversity for microsatellite loci was significantly higher in wild than in cultivated soybean. In a second study, molecular markers were used to identify and characterize quantitative trait loci (QTL) controlling seed weight in soybean, and to extend reports of orthologous seed weight genes in the genus Vigna to the genus Glycine by "comparative QTL mapping". DNA samples from 150 F₂ individuals from an interspecific soybean cross were analyzed with 91 genetic markers. Three and five markers were significantly associated with seed weight variation (P<0.01) in the F₂ and F_2:3 generations, respectively. Two-way ANOVA tests for digenic interactions identified three significant epistatic interactions in both generations. In a combined analysis, the significant marker loci and epistatic interactions explained 50 and 60% of the total variation for seed weight in the F₂ and F_2:3 generations, respectively. Comparison of our results in Glycine with those reported in Vigna indicated that both genera share orthologous seed weight genes. Moreover, a significant epistatic interaction between seed weight QTLs was conserved in both genera. The objective of the third study was to use molecular markers and interval mapping techniques to position and characterize quantitative trait loci controlling seed protein, oil, sucrose, and calcium content as well as seed weight in soybean. Two QTLs were detected for protein and calcium content, five for oil content and seed weight and six for sucrose content, respectively. Percent phenotypic variation explained by these individual QTLs ranged from 6.6 to 34.0%. The total phenotypic variation explained by all QTLs for specific traits were 42.5%, 36.7%, 49.0%, 53.1%, and 42.6% for seed weight, protein, oil, sucrose, and calcium, respectively. Of the 11 genomic intervals identified in this study, six were associated with more than one seed quality trait. These results suggest that the genetic correlations observed between seed quality traits may be due to a pleiotropic effect of a single QTL or that QTLs controlling different seed quality traits were inherited in clusters as tightly linked loci. / Ph. D.

Soybean, <i>Glycine max</i> L. Merr, is used worldwide as an important source of protein and oil for a wide spectrum of edible feed and industrial purposes. Modem cultivars are derived from relatively few plant introductions (PIs) which severely limits diversity in soybean germplasm. The United States Department of Agriculture (USDA) maintains the soybean germplasm collection. Mitochondrial DNA sequences have been used as markers of diversity at the cytoplasmic level. This project included three objectives. The first was a classification of the 208 varieties of the USDA's 'old domestic collection' of soybean varieties with two mitochondrial restriction fragment length polymorphisms (RFLP) markers. Molecular techniques were used to search for additional sources of cytoplasmic diversity available to soybean breeders. The two polymorphic markers were, a 2.3 kb <i>Hind</i>III fragment isolated from 'Williams 82' mitochondrial DNA, and a portion of the mitochondrial <i>atp</i>6 gene. These markers were used to distinguish cytoplasmic groups based on hybridization analysis of <i>Hind</i>III-digested soybean DNA Four major groups were observed with the 2.3 kb marker in the old domestic collection, and several minor subgroups were also detected. The second objective included subcloning and sequencing the 0.9 kb and 1.7 kb <i>Hind</i>III-<i>Pst</i>I clones flanking the 2.3 kb <i>Hind</i>III fragment from 'Williams 82' DNA The total 4.9 kb <i>Pst</i>I sequence from 'Williams 82' mitochondrial DNA was used to search a sequence database for any homology to known mitochondrial sequences. The third objective compared restriction maps of the four cytoplasmic types in the regions containing homology to the 4.9 kb <i>Pst</i>I fragment. DNAs from the four cytoplasmic types were digested with five enzymes and four specific clones (0.9 kb <i>Pst</i>I-<i>Hind</i>III, 0.8 kb HindIII-<i>Xba</i>I, 1.5 kb Xbal-<i>Hind</i>III, 1.7 kb <i>Hind</i>III-<i>Pst</i>I) were used as hybridization probes in Southern analysis to examine RFLP patterns and construct comparative restriction maps of the four cytoplasmic types of DNA. / Master of Science

Cheung Ming-yan. / Thesis submitted in 2000. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 169-185). / Abstracts in English and Chinese. / Thesis committee --- p.i / Statement --- p.ii / Abstract --- p.iii / Acknowledgement --- p.vi / General abbreviations --- p.viii / Abbreviations of chemicals --- p.x / List of figures --- p.xii / List of tables --- p.xv / Table of Contents --- p.xvii / Chapter Chapter 1 --- General Introduction - Using transgenic plants as bioreactor --- p.1 / Chapter 1.1 --- Plant as Bioreactor --- p.1 / Chapter 1.1.1 --- Plant transformation historical milestones --- p.1 / Chapter 1.1.2 --- Applications of transgenic plants --- p.5 / Chapter 1.1.2.1 --- Examples of in situ Application --- p.5 / Chapter 1.1.2.2 --- Examples of ex situ application of transgenic plant --- p.9 / Chapter 1.2 --- Plant Hosts for Transformation: Arabidopsis thaliana and Glycine max --- p.18 / Chapter 1.2.1 --- Essential components for plant transformation --- p.18 / Chapter 1.2.1.1 --- Marker genes --- p.18 / Chapter 1.2.1.2 --- Promoters --- p.18 / Chapter 1.2.2 --- Arabidopsis thaliana --- p.20 / Chapter 1.2.2.1 --- Agrobacterium-mediated transformation --- p.20 / Chapter 1.2.2.2 --- Transformation methods for A. thaliana --- p.21 / Chapter 1.2.3 --- Glycine max (soybean) --- p.22 / Chapter 1.2.3.1 --- Soybean cultivars for transformation --- p.23 / Chapter 1.2.3.2 --- Soybean regeneration systems --- p.24 / Chapter 1.2.3.3 --- Soybean transformation systems --- p.26 / Chapter 1.3 --- Target Pharmaceutical and Agricultural Proteins: Lymphocytic choriomeningitis virus and Goldfish Growth hormones I and II --- p.29 / Chapter 1.3.1 --- Production of pharmaceutical proteins --- p.29 / Chapter 1.3.1.1 --- Lymphocytic choriomeningitis virus --- p.30 / Chapter 1.3.1.2 --- Nucleoprotein of LCMV --- p.33 / Chapter 1.3.2 --- Agricultural protein category --- p.34 / Chapter 1.3.2.1 --- Carassius auratus --- p.34 / Chapter 1.3.2.2 --- Growth hormones I and II --- p.35 / Chapter 1.4 --- Hypothesis and Objectives --- p.37 / Chapter Chapter 2 --- Materials and Methods --- p.38 / Chapter 2.1 --- Materials --- p.38 / Chapter 2.1.1 --- "Plants, bacterial strains and vectors" --- p.38 / Chapter 2.1.2 --- Chemicals and Regents --- p.43 / Chapter 2.1.3 --- Commercial kits --- p.44 / Chapter 2.1.4 --- Primers and Adaptors --- p.45 / Chapter 2.1.5 --- Equipments and Facilities used --- p.47 / Chapter 2.1.6 --- "Buffer, solution and medium" --- p.47 / Chapter 2.2 --- Methods --- p.48 / Chapter 2.2.1 --- Molecular Techniques --- p.48 / Chapter 2.2.1.1 --- Bacterial cultures for recombinant DNA and plant transformation --- p.48 / Chapter 2.2.1.2 --- Recombinant DNA techniques --- p.48 / Chapter 2.2.1.3 --- "Preparation and transformation of DH5a, DE3 and Agrobacterium competent cells" --- p.49 / Chapter 2.2.1.4 --- Gel electrophoresis --- p.52 / Chapter 2.2.1.5 --- "DNA, RNA and protein extractions" --- p.53 / Chapter 2.2.1.6 --- Generation of cRNA probes for Southern and Northern blot analyses --- p.56 / Chapter 2.2.1.7 --- Southern blot analysis --- p.56 / Chapter 2.2.1.8 --- Northern blot analysis --- p.57 / Chapter 2.2.1.9 --- Expression of Lymphocytic choriomeningitis virus nucleoprotein (LCMV NP) in bacterial system --- p.58 / Chapter 2.2.1.10 --- Western blot analysis for LCMV NP --- p.59 / Chapter 2.2.1.11 --- Protein dot blot for detecting the presence of recombinant LCMV-NP generated from transgenic plants --- p.62 / Chapter 2.2.1.12 --- PCR techniques --- p.62 / Chapter 2.2.1.13 --- Sequencing --- p.63 / Chapter 2.2.2 --- Plant tissue culture and transformation --- p.64 / Chapter 2.2.2.1 --- Arabidopsis thaliana --- p.64 / Chapter 2.2.2.2 --- Soybean --- p.65 / Chapter 2.2.3 --- In vitro transcription and translation of target genes in rabbit reticulocyte and wheat germ systems --- p.68 / Chapter 2.2.3.1 --- In vitro transcription of target genes with with Ribomix large scale RNA production systems-T7 and SP6 (Promega) --- p.68 / Chapter 2.2.3.2 --- In vitro translation with rabbit reticulocyte lysate and wheat germ extract --- p.69 / Chapter Chapter 3 --- Results --- p.71 / Chapter 3.1 --- Expression of Lymphocytic choriomeningitis virus nucleoprotein (LCMV NP) and goldfish growth hormones I and II (GHI and GHII) in transgenic Arabidopsis thaliana --- p.71 / Chapter 3.1.1 --- Expression of LCMV-NP in transgenic Arabidopsis thaliana --- p.71 / Chapter 3.1.1.1 --- Cloning of the gene encoding LCMV NP into the binary vector system W104 --- p.71 / Chapter 3.1.1.2 --- Transformation of W104-LCMV-NP into the Agrobacterium GV3101/pMP90 --- p.78 / Chapter 3.1.1.3 --- Transformation of LCMV-NP cDNA into Arabidopsis thaliana --- p.80 / Chapter 3.1.1.4 --- Southern blot and Northern blot analyses of transgenic plant containing the LCMV-NP cDNA --- p.83 / Chapter 3.1.1.5 --- Production of recombinant LCMV-NP protein in DE3 cells --- p.90 / Chapter 3.1.1.6 --- Detection of recombinant LCMV-NP protein in transgenic A.thaliana --- p.98 / Chapter 3.1.2 --- Expression of goldfish growth hormones I and II (GHI and GHII) in transgenic Arabidopsis thaliana --- p.105 / Chapter 3.1.2.1 --- "Screening of homozygous lines of goldfish, Carassius auratus, growth hormones transgenic Arabidopsis thaliana" --- p.105 / Chapter 3.1.2.2 --- Southern blot and Northern blot analyses of transgenic plant containing the LCMV-NP cDNA --- p.109 / Chapter 3.1.2.3 --- Detection of recombinant GHI and GHII from transgenic plant --- p.112 / Chapter 3.2 --- In vitro transcription and translation of target genes in rabbit reticulocyte and wheat germ systems --- p.117 / Chapter 3.2.1 --- Subcloning of target genes in pGEM-3Zf(+) vector --- p.117 / Chapter 3.2.1.1 --- Subcloning of LCMV-NP fragment into pGEM-3Zf(+) vector --- p.117 / Chapter 3.2.1.2 --- Subcloning of goldfish GHI and GHII fragments into pGEM-3Zf(+) vector --- p.120 / Chapter 3.2.2 --- In vitro transcription of target genes with Ribomix large scale RNA production systems-T7 and SP6 --- p.125 / Chapter 3.2.3 --- In vitro translation with rabbit reticulocyte lysate and wheat germ extract systems --- p.128 / Chapter 3.3 --- Establishment of Glycine max regeneration and transformation systems --- p.130 / Chapter 3.3.1 --- The Establishment of soybean regeneration system --- p.130 / Chapter 3.3.2 --- Establishment of soybean transformation system --- p.133 / Chapter 3.3.2.1 --- Definition of transformation efficiency --- p.133 / Chapter 3.3.2.2 --- Effects of plant hosts --- p.136 / Chapter 3.3.2.3 --- Effects of Agrobacterium strains --- p.138 / Chapter 3.3.2.4 --- The application of vacuum infiltration --- p.139 / Chapter 3.3.2.5 --- Effect of kanamycin --- p.140 / Chapter 3.3.2.6 --- Effect of cocultivation duration and light/ dark treatment during germination --- p.141 / Chapter 3.3.2.7 --- Application of the detergent Silwet-77 --- p.142 / Chapter 3.3.3 --- Verification of transformation results by PCR screening --- p.143 / Chapter Chapter 4 --- Discussion --- p.147 / Chapter 4.1 --- "Expression of LCMV-NP, GHI and GHII in A. thaliana" --- p.148 / Chapter 4.2 --- Establishing a soybean transformation system --- p.157 / Chapter 4.2.1 --- Plant hosts and explants --- p.158 / Chapter 4.2.2 --- Regeneration of explants --- p.159 / Chapter 4.2.3 --- Agrobacterium strains --- p.161 / Chapter 4.2.4 --- Bacteria-plant interaction --- p.161 / Chapter 4.2.5 --- Transient versus stable transformation --- p.165 / Chapter 4.3 --- Conclusion and perspective --- p.167 / References --- p.169 / Appendix --- p.186