Preliminary Studies On The Mechanism Of Somatic Embryogenesis In Eleutherococcus Senticosus Maxim

Plant somatic embryogenesis means the process of morphogenesis of somatic cell developing into embryoid without sexual cell fusion under given conditions. It has broad application prospect and huge economic values in rapid propagation, artificial seeds, germplasm conservation, plant genetic engineering and other fields. Somatic embryogenesis is not only a concrete manifestation of totipotence of cells, but also a reappearance process of the zygotic embryo morphogenesis, so the study on somatic embryogenesis and developmental mechanisms has a very important significance for revealing the molecular biology mechanisms of cell differentiation, cell development and morphogenesis.In this study, the somatic embryogenesis of Eleutherococcus senticosus was studied on the basis of the morphological observation, physiological and biochemical characteristics test. The relationship between dynamic changes of callose and somatic embryogenesis was analyzed through plasmolyzing the zygotic embryos of Eleutherococcus senticosus to induce somatic embryogenesis; meanwhile, through Solexa high-throughput sequencing technology, transcriptome of somatic embryo, which are in three periods of early development of Eleutherococcus senticosus, were analyzed. Further more, the digital gene expression profiles of those materials were analyzed, including function analysis and classification of differentially expressed genes, as well as screening genes related to early somatic embryogenesis of Eleutherococcus senticosus. The purpose is to explore mechanism of early somatic embryogenesis, which will provide molecular resources for further study on somatic embryogenesis of Eleutherococcus senticosus. The main results as follows:1. The somatic embryogenesis of Eleutherococcus senticosus includes five phases: globular, heart-shaped, torpedo, cotyledon embryo and finally somatic embryo seedling. Soluble sugars, starch, soluble protein content, superoxide dismutase, peroxidase activity in embryonic calli, globular, torpedo, cotyledon embryos, and seedlings were measured. The content of soluble sugar was the highest in embryonic calli and gradually reduced with developing of somatic embryos; there are two peaks of starch content during the whole development:in globular and cotyledon embryo period. Soluble protein content increased along the development of somatic embryos, with higher speed when somatic embryos developing from embryonic calli to globular embryos; SOD activity increased gradually from embryonic calli to somatic embryos seedlings; The POD activity was the highest in embryonic calli, which showed tend of down first and up later. The changes of these substances might be related with the differentiation and development of somatic embryos.2. There was positive correlation between somatic embryogenesis and callose produced after plasmolysis treatment.①The best reagent is1.0M mannitol and the optimum treatment time was12hours for zygotic embryos of Eleutherococcus senticosus treated by plasmolysis.②The callose content changed a lot during plasmolysis treatment and somatic embryogenesis, with the trends of decrease first and then increase with somatic embryogenesis. Callose content was higher and the number of somatic embryos was the biggest after12hours treatment of plasmolysis.③Confocal laser scanning microscope was used to observe callose deposition, which showed great difference in different tissues during somatic embryogenesis after being treated by plasmolysis. There were a large number of callose deposition in the epidermis and vascular tissue mainly around the cytomembrane, while very little in other tissues.④A number of callose deposition can be observed in embryonic calli, but not in non-embryonic calli.⑤Adding of2-DDG decreased the induction of somatic embryos and the numbers of somatic embryos produced by each explants, while incidence rate of somatic embryo was only11.5%.3.58,327,688sequencing reads were obtained by transcriptome sequencing of Eleutherococcus senticosus, which were eventually assembled into75,803unigenes. In order to understand these gene functions, they were annotated with Clusters of Orthologous Groups (COG), gene orthology (GO), and the Kyoto Encyclopedia of Genes and Genomes (KEGG). Digital gene expression (DGE) profile showed differences of gene expression at different development stages (EC, YEC, and GE). The differentially expressed genes were analyzed by GO and Pathway. More than5.6million tags per sample were obtained and a large number of differentially expressed genes at different stages of somatic embryogenesis were identified, which included some genes related with early somatic embryogenesis.