| Abstract | 第8-10页 |
| List of Abbreviations | 第11-13页 |
| 1. Introduction | 第13-21页 |
| 2. Review of Literature | 第21-41页 |
| 2.1 Genetic improvement of citrus | 第21-22页 |
| 2.2 Application of biotechnological techniques in citrus genetic improvement | 第22-23页 |
| 2.3 Citrus in vitro regeneration | 第23-25页 |
| 2.4 Citrus genetic transformation | 第25-27页 |
| 2.5 Citrus genetic transformation methods | 第27-35页 |
| 2.5.1. Protoplast Transformation | 第28页 |
| 2.5.2. Biolistic Transformation | 第28-29页 |
| 2.5.3. Agrobacterium-mediated transformation | 第29-35页 |
| 2.5.3.1 The process of gene transfer | 第30-32页 |
| 2.5.3.2 Factors affecting Agrobacterium mediated transformation | 第32-35页 |
| 2.6 Use of GFP as a reporter gene | 第35-36页 |
| 2.7 Genetics of cold tolerance in crop species | 第36-38页 |
| 2.8 Genetic modification of plants growth | 第38-41页 |
| 3. Regeneration and characterization of plants derived from leaf in vitro culture of two sweet orange (Citrus sinensis (L.) Osbeck) cultivars | 第41-56页 |
| 3.1 Introduction | 第41-43页 |
| 3.2 Materials and methods | 第43-45页 |
| 3.2.1 Plant material | 第43页 |
| 3.2.2 Induction of shoots from leaf discs | 第43-44页 |
| 3.2.3 Shoot elongation and rooting | 第44页 |
| 3.2.4 Random amplification polymorphic DNA (RAPD) analysis of the regenerated plants | 第44-45页 |
| 3.2.5 Statistical analysis | 第45页 |
| 3.3 Results | 第45-50页 |
| 3.3.1 Shoot regeneration from leaf explants | 第45-47页 |
| 3.3.2 Effect of medium composition on shoot induction | 第47页 |
| 3.3.3 Effect of cultivar on shoot induction | 第47-48页 |
| 3.3.4 Effect of leaf age on shoot regeneration | 第48-49页 |
| 3.3.5 Effect of lighting conditions on shoot induction | 第49-50页 |
| 3.3.6 Rooting of elongated shoots and RAPD analysis of regenerated plants | 第50页 |
| 3.4 Discussion | 第50-56页 |
| 4. Agrobacterium-mediated genetic transformation and regeneration of Valencia' Sweet orange (Citrus sinensis L. Osbeck) plants from leaf eplants via direct organogenesis | 第56-88页 |
| 4.1 Introduction | 第56-60页 |
| 4.2 Materials and methods | 第60-67页 |
| 4.2.1 Plant material and preparation of explants | 第60-61页 |
| 4.2.2 Preparation of Agrobacterium tumefaciens for transformation | 第61-62页 |
| 4.2.3 Sensitivity of explants to Kanamycin concentrations | 第62-63页 |
| 4.2.4 Sensitivity of explants to disinfectant antibiotic (Cefatoxime) | 第63页 |
| 4.2.5 Evaluation of factors influencing regeneration and transformation efficiency | 第63-64页 |
| 4.2.6 Transformation, selection and regeneration | 第64页 |
| 4.2.7 Elongation and in vitro rooting of regenerated shoots | 第64-65页 |
| 4.2.8 Detection of GFP fluorescence by stereomicroscopy | 第65页 |
| 4.2.9 DNA isolation and PCR analysis | 第65-66页 |
| 4.2.10 Flow cytometry | 第66页 |
| 4.2.11 Statistical analysis | 第66-67页 |
| 4.3 Results and discussion | 第67-86页 |
| 4.3.1 Kanamycin concentration affecting survival efficiency of leaf discs | 第67-69页 |
| 4.3.2 Cefatoxime concentrations affecting survival efficiency of leaf discs | 第69-70页 |
| 4.3.3 Optimization of factors affecting transformation efficiency | 第70-79页 |
| 4.3.3.1 Effect of Agrobacterium tumefaciens concentration (OD_(600)) | 第71-72页 |
| 4.3.3.2 Effect of Immersion time | 第72-74页 |
| 4.3.3.3 Effect of age of leaf discs | 第74-75页 |
| 4.3.3.4 Effect of co-cultivation period and temperature | 第75-78页 |
| 4.3.3.5 Effect of Acetosyringone concentration | 第78-79页 |
| 4.3.4 Production of transgenic plants and transgene expression | 第79-84页 |
| 4.3.5 Molecular analysis of GFP expressing Valencia sweet orange plants | 第84-86页 |
| 4.3.6 Ploidy level of regenerated plants | 第86页 |
| 4.4 Conclusion | 第86-88页 |
| 5. Agrobacterium tumefaciens mediated transformation of agronomically important genes in citrus | 第88-129页 |
| 5.1 Agrobacterium mediated genetic transformation and over-expression of PtrICE gene in cell suspension cultures of citrus cultivar 'GWZ' (Gailiangcheng orange x Weizhang Satsuma mandarin) | 第89-116页 |
| 5.1.1 Introduction | 第89-93页 |
| 5.1.2 Materials and methods | 第93-99页 |
| 5.1.2.1 Plant material | 第93页 |
| 5.1.2.2 Explant preparation and transformation | 第93-95页 |
| 5.1.2.3 PCR analysis for confirmation of PtrICE gene integration | 第95-96页 |
| 5.1.2.4 Isolation of RNA and RT-PCR | 第96页 |
| 5.1.2.5 Cold treatments | 第96-99页 |
| 5.1.3 Statistical analysis | 第99-100页 |
| 5.1.4 Results | 第100-112页 |
| 5.1.4.1 Selection of transformed cells and production of transgenic plantlets | 第100-102页 |
| 5.1.4.2 Overexpresssion and PCR analysis of PtrICE gene in transgenic callus lines of GWZ | 第102-103页 |
| 5.1.4.3 Changes in relative electrolyte leakage (EL) and Malondialdehyde (MDA) contents under cold stress | 第103-105页 |
| 5.1.4.4 Effects of PtrICE gene over-expression on antioxidant enzyme activities | 第105-107页 |
| 5.1.4.5 Cell viability measured as TTC reduction assay | 第107-108页 |
| 5.1.4.6 Cell viability assay measured by Evans Blue staining method | 第108-110页 |
| 5.1.4.7 Low temperature effect on H_2O_2 content | 第110-111页 |
| 5.1.4.8 Changes in fresh cells growth rate under cold stress treatments | 第111-112页 |
| 5.1.5 Discussion | 第112-115页 |
| 5.1.6 Conclusion | 第115-116页 |
| 5.2 Transformation and characterization of TfGA2-ox2 gene in cell suspension cultures of 'Bingtangcheng' Sweet orange (Citrus sinensis L. Osbeck) | 第116-129页 |
| 5.2.1 Introduction | 第116-118页 |
| 5.2.2 Materials and methods | 第118-121页 |
| 5.2.2.1 Plant material | 第118页 |
| 5.2.2.2 Binary vector and transformation | 第118-120页 |
| 5.2.2.3 Evaluation of factors influencing transformation | 第120页 |
| 5.2.2.4 DNA extraction and PCR analysis | 第120-121页 |
| 5.2.2.5 Statistical analysis | 第121页 |
| 5.2.3 Results and discussion | 第121-127页 |
| 5.2.3.1 Effect of pre-culture period on transformation | 第121-123页 |
| 5.2.3.2 Effect of Agrobacterium infection time on transformation | 第123页 |
| 5.2.3.3 Effect of co-cultivation period on transformation | 第123-124页 |
| 5.2.3.4 PCR analysis of the resistant calluses | 第124-125页 |
| 5.2.3.5 Callus transformation and recovery of the transformed callus lines | 第125-127页 |
| 5.2.4 Conclusion | 第127-129页 |
| REFERENCES | 第129-159页 |
| PROTOCOLS | 第159-169页 |
| ACKNOWLEDGEMENTS | 第169-170页 |
