Preliminary Study On The Mechanism Of Somatic Embryogenesis Of Ponkan(Citrus Reticulata Blanco)

Somatic embryogenesis technique in plant rapid propagation, germplasm conservation, genetic transformation, genetic engineering and other fields have broad prospect of application and economic value. It has been indicated that the occurrence of somatic embryogenesis is closely related to the oxidative stress, DNA methylation status and so on. So far, the molecular and physiological mechanism of citrus somatic embryogenesis is not clear. Based on this, the induction of somatic embryos in the Citrus Embryogenic callus as the starting material, Analyzed the process of somatic embryogenesis ofthe soluble protein content, the three kinds of antioxidant enzyme activity and the dynamic changes of the level of reactive oxygen species, in order to understand the role of antioxidant system in somatic embryogenesis; Using the data from the published orange genome identified the members of the family of NADPH oxidase gene and analyzed their expression in the process of ponkan induction of somatic embryogenesis, in order to select and somatic embryogenesis of specific NADPH oxidase gene; Using the MSAP technology analyzed ponkan DNA methylation level during induction of somatic embryo and pattern change, in order to reveal preliminarily its apparent change rule. The main results were as follows:1.Ponkan selected four stages during induction of somatic embryo culture, namely the embryonic callus were just inoculated (EI0), embryonic callus turn to yellow (Ell), the embryos can be seen with the naked eye (EI2) and More than 50% of the embryos(EI3),that H2O2 content and antioxidant enzyme (POD, CAT and SOD) activity has been determined. The results showed that the content of soluble protein increased firstly and then decreased, and the content of hydrogen peroxide rised gradually first of all after decreased; the three enzyme activity variation from EIO to EI2 shows first increased and then decreased, but in the EI2 to the EI3 process, POD and CAT activity decreased, increased SOD activity.2. Using bioinformatics methods in citrus genome database to screen 13 citrus NADPH oxidase gene, and analyze these genes in ponkan expression in the process of somatic embryo induction. Results showed that the expression of 8 genes were detected and the trend of the expression has obvious differences in the stages (CsRboh1、 CsRboh2、CsRboh5、CsRboh6、CsRboh7、CsRboh10、CsRbohl1、CsRbohl 2);no detectable expression of the gene is CsRboh3、CsRboh4、CsRboh8、CsRboh9 and CsRboh11. In CE and CEF stages expression of CsRboh7 gene is likely involved in the generation of reactive oxygen species in embryo-like.3. Using MSAP technology study on Citrus somatic embryogenesis process DNA methylation levels and patterns of change.Results show that the all DNS methylatiorr rise,the methylation in EI1 and EI3 these embryoids appeared two peaks, the Methylation as the main methylation modification pattern, the lateral half cytosine methylation modify mode is relatively in the process of citrus of small Somatic embryogenesis.