| Citrus ranks the number one in the world fruit production. Creating high qualities potential citrus varieties via genetic improvement is the main solution of enhancing the competitiveness of China's citrus market. The conventional breeding is constrained by the complex biological characteristics, such as polyembryony, apomixis, long juvenility, high heterozygosity and parthenogenesis etc. In recent years, the improving transgenic technology has provided an efficient way to the improvement of citrus varieties.In this study, a procedure for efficient protoplast electroporation was established using green fluorescent protein gene (GFP) as reporter gene. Meanwhile, a number of factors affecting the efficiency of electroporation were also discussed. In addition, the transformation of GFP into different explants of Murcott Tangor by Agrobacterium was conducted. The transformants expressing GFP could be applied to further investigate the mechanism of cell fusion, apoptosis, and the related genetic transformation in citrus. The main results were as follows:1. Optimizing citrus protoplast transient expression system. Some factors influencing the transformation of mgfp5-ER gene into Guangdong tangerine were analyzed, including the pH value of electroporation buffer, The results showed that the optimal transient expression rate reached 10.3% with the direct current at 1250 V/cm, pulse time for 45 us, pulse number at 5, pulse interval for 0.5 s, alternating electric field at 50 V/cm and for 40 us. When the electroporation buffer pH value was 7, transient expression efficiency reached maximum. The transient expression efficiency was not significantly improved by ice pre-treating the protoplast for 10 min before electroporation. Moreover, compared with transformation mediated by PEG (Polyethylene glycol), the transient expression efficiency of this system had no significant difference.2. Comparison of protoplast transformation efficiencies in different citrus species. The result indicated that the transformation efficiencies of Guoqing No.1 and Guangdong tangerine were 9.7% and 10.9%, respectively. Protoplasts isolated from suspension culture callus were easier for transformation. Occasional transient expression of Valencia mesophyllic protoplast transformation could be observed. The transient expression in the callus protoplasts of Anliucheng and Murcott Tangor which were considered to be the most difficult for transformation has not been detected. 3. Transformation of mgfp5-ER genes with different Murcott Tangor explants. By making use of agrobacterium tumefaciens-mediated transformation method, Some transgenic callus and buds with stable GFP expression have been obtained by using embryonic callus, epicotyl segment, the trimmed etiolated shoot/root region seedling and cotyledon of test-tube seedlings of Murcott Tangeor as explants. The transformation efficiency was different among different explants, The results showed that higher transformation efficiencies with respectively 13.3% and 12.3% were obtained by using the callus and epicotyl segment as explants. The transformation efficiency was affected by the maturation level of seed, which were 8.0% and 12.6% by using the test-tube seedlings obtained from the immature seeds and mature seeds as explants respectively.4. The fluorescence and molecular analysis of Murcott Tangor transformants. 46 transformants were obtaind. 17 of them were still in a state of callus stage, and 29 of them were transgenic buds and bud points. The results showed that the GFP gene could be stably expressed in the resistant callus and resistant buds of Murcott Tangor by using the laser scanning Confocal microscopy. PCR detection of the fluorescence callus team of Murcott orange showed the PCR-positive rate was 100%.
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