The Molecular Mechanims Of Jasmonic Acid Promoting Secondary Somatic Embryos Generation From Globular Embryo Epidermal Cells

Cotton is an important economic crop. For a long time, the conventional cotton breeding based on selection of target traits is time-consuming, it can’t satisfy the market demand. The deep research of cotton cell engineering and gene engineering has injected fresh blood into the conventional breeding technology and made up for the deficiency of the conventional breeding. The cotton transgenic breeding technology developed rapidly. The cotton somatic embryogenesis is the foundation of the research of cotton transgenic technology. We found that in the early stage of cotton somatic embryo development, especially in the globular embryo stage, adding jasmine acid in the culture medium can change the developmental direction of the globular embryo epidermal cells, a large number of secondary somatic embryos can be produced. A systematic study on JA mediated somatic embryo development was performed. We will use the globular embryos as explants to provide practical possibility for Agrobacterium mediated transformation.1. At an appropriate jasmonic acid concentration in the medium,the somatic embryos was generated from globular embryo epidermal cells. After jasmine acid treatment, the globular embryos were observed through microscope、scanning electron microscope and paraffin section. The secondary somatic embryos was developed from the globular embryo, which mainly occurred in the hypocotyledonary axis of the embryo. Compared with the original globular embryos,there was no significant difference in the histological observation and subsequent development of the secondary embryos and the original globular embryos.and they all can develop into regenerated plants.2. Analysis of the expression of the limiting enzyme AOS in the JA synthesis pathway was performed. JA plays an important role in regulating the proliferation of cotton somatic embryos, and AOS is the key enzyme in the pathway of JA biosynthesis in plants. We identified the AOS gene expressed in the embryonic development process, based on the results of quantitative Real-Time PCR, the expression pattern of these genes can be divided into 3 categories: the relative expression levels of GhAOS1 and GhAOS6 are high during the development of cotton somatic embryo; the relative expression levels of GhAOS2 and GhAOS3 in cotton callus and embryogenic callus are high; the relative expression levels of GhAOS4 and GhAOS5 in stem and leaf are high. It is suggested that the expression of these genes has spatial and temporal specificity, and GhAOS1 and GhAOS6 may play an important role in the development of cotton somatic embryo. Subcellular localization analysis showed that most of the genes were located in the peroxysome,and which may be involved in light respiration, somatic embryo maturation and germination. The promoter element analysis showed that they may be regulated by hormones and stress in somatic embryogenesis. It laid a foundation for further study on the function of AOS gene family in cotton somatic embryo development.3. Through the analysis of the chromosome location, subcellular location, promoter and expression pattern of AOS gene, we screened 3 AOS genes, Gh AOS1, GhAOS2 and GhAOS6.the constructed over expression vector and VIGS interference vectorwere transformed into cotton. We found that whether the over-expression or the interference of AOS gene, there was no significant difference in the amount of secondary somatic embryos developed from globular embryos,therefore we believed that the expression of AOS gene was not closely related to the development of secondary somatic embryos from globular embryo epidermal cells.4.After the JA treatment of globular embryo, the RNA sequencing analysis showed that the most of the differentially expressed genes were found in 0.5h, and the number of up-regulated genes was greater than that of down-regulated genes. The genes related to stress were more prominent, especially related to the hormones. Under stress stimulation, the differentially expressed genes were closely related to auxin, ABA and ethylene. We mainly analyzed the genes related to hormone response, stress response and somatic embryo development related genes. It is speculated that the JA mediated somatic embryos may be through interaction with other hormones.