| Citrus is an important fruit crop for agricultural economy in the world, and the production is inhibited by citrus canker which severely occurres worldwide. Agronomic practice and bactericide can only solve the problem in some context, and some bactericide can cause heavy environmental pollution. These make the disease resistance breeding a best selection for fighting against the bacterial pathogens. Conventional disease resistance breeding is difficult because of polyembryony, apomixis, long juvenility, high heterozygosity and parthenogenesis; as well as limitation for reproduction barrier among different species. Resistant genes cloning and transgenic approach offer a potentially bactericide-free and environment-friendly solution for canker pathogen control. In this study, the complete dominant gene Xa21, which has a wide spectrum of resistance to Xoo, was introduced into citrus with different explants by Agrobacterium-mediated transformation, meanwhile transformation system was optimized by GFP gene. Histochemical GUS assay, PCR and Southern blotting analysis were used to confirm the integration of transgenes. RT-PCR and Real-time RT-PCR were used to analyze the expression of Xa21. Disease resistance test indicated that resistance was improved on some transgenic lines. And the resistance mechanism of Xa21 in citrus was discussed. The main results are as follows:1. Optimization of regeneration system in vitro for citrus. 1) A statistical analysis of the effect on the regeneration of somatic embryogenesis in different culture medium revealed that most appendages could improve the embryogenic capacity of citrus calli which had competence for somatic embryogenesis, and the most appropriate media for somatic embryogenesis of Anliucheng from calli was MT basal medium contain 2% glycerin. But different culture medium had little influence on citrus calli which lost competence for somatic embryogenesis. 2) Adventitious shoots of Anliucheng regeneration efficiency increased with enhanced BA concentration, and peaked at 4.0 mg/L. Regeneration efficiency of different explants was inversely proportional to its damage degree. The dark treatment reduced the damage healing of explants and increased the numbers of adventitious shoot, and had no effect on regeneration of trimmed etiolated shoot/root region. 3) Shoot-tip graft in vitro in greenhouse could improve the efficiency and shorten the time for transgenic plant regeneration.2. Agrobacterium-mediated transformation with explants, such as somatic embryo, epicotyl segment, the trimmed etiolated shoot/root region seedling, cotyledon, seed, root, makes the explant source rich in citrus transformation. The main results are as follows: Using GFP as a reporter gene, transformation system that using explant somatic embryo of Valencia was established. 12 transgenic lines were obtained by GFP detecting and molecular analysis. Then Xa21 gene was introduced into citrus genetypes, Anliucheng (6 lines) and Valencia (5 lines), with selective marker hygromycin. Stable integration of transgenes was confirmed by GUS assay and molecular analysis. It could be concluded that transgenic plants regenerated via somatic embryogenesis could reduce the efficiency of chimera occurred, and decreased transformation steps compared with calli.Transformation system that using explant trimmed etiolated shoot/root region of Anliucheng seedling was established by using GFP as a reporter gene. The transgenes integration was proved by GFP detecting and molecular analysis. Agrobacterium mediated transformation of this explant produced ten independently transgenic plants, three of which had a single copy transgene. And using the explant, transgenic plants could be obtained within 80 d. Compared with epicotyl, high regeneration and transformation efficiency was obtained. But GFP dependence limited its practice. The results also revealed that cells competent for transformation are located in the newly formed tissure, such as cambial cells of cut surface of epicotyls. And chimera is formed from several transgenic or non-transgenic cells. The line of chimera is clear in the early stage of chimera formation and indistinct with transgene growing, and disappeared in the end.3. Transformation system establishment with hygromycin selective system through Agrobacterium-mediated calli transformation and resistance analysis to citrus canker for transgenic lines. In this study, only 2 genotypes, Anliucheng (95 transgenic lines) and Valencia (32 transgenic lines), were regenerated into transgenic plants from 13 transgeic calli of different genotypes. PCR analysis revealed that the ratio of positive Anliucheng and Valencia transgenic plants were 90.2% and 85.6% respectively. And in calli transformation, correlation analysis suggested that transformation efficiency is in direct ratio to the proliferation and differentiation capacity of calli.Integration of the transgene into citrus genome was confirmed by histochemical GUS staining, Southern blot and Real-time RT-PCR. GUS staining indicated that the ratio of positive plants was 98.0%, and few chimeras occurred in regeneration of transgenic plants. This also suggested regeneration of most somatic embryoids could be riginated from single cells. PCR and Southern blot revealed that the three genes integrated into citrus genome with 1 -2 copies, and no gene lost and rearranged during transgenes regeneration. Real-time RT-PCR analysis indicated that Xa21 expression levels were low. By pathogen inoculation in vitro, transgenic plants displayed varied resistance levels with about 63.7% transgenic resistance increase. Hence, Xa21 gene has a potential in canker resistance breeding. The results also suggested that position effects and backgroud were more obvious than dosage effects and copy number on the citrus canker resistance level of the transgenic plants.Aiming at the problems that the transgenes leaves could not extend fully, plant height and leaf index had no significant difference with that of control by statistic analysis, and no ploidy variation occurred between transgenes and non-transgenes. It may be concluded that the gene insertion result in transgenic variation. The variation in codon usage of LRR gene family of citrus was analysis by the program-Codontree. The results indicated that LRR genes of citrus had a specific codon usage bias differed from NBS-LRR of rice and genome of citrus. We predict that transgenic plant morphology and transgenes expression were probably due to the codon usage bias of species.The selection of gene receptor in plant transformation, aspects for enhancing the transformation efficiency, mechanism of transformation occurrence and formation, the impact of codon usage bias of gene family on exogenous gene expression, and the potential usage of resistant genes in citrus breeding were also discussed in the present study.
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