DNA Methylation Analysis During Proliferation And Differentiation Of Pinus Massoniana Lamb Embryogenic Cultures

Although the Pinus massoniana lamb somatic embryogenesis system had been established, but there were still some difficult to maintain of embryogenic cultures and improve differentiation rate. Difficult to maintain of embryogenic cultures and improve differentiation rate. Embryogenic cultures during the proliferation had significantly declined. The development of somatic embryogenesis associated with its epigenetic gene expression. DNA methylation as an important way to epigenetic regulation may participate in the different stages of somatic embryogenesis. In this study, we restored the DNA methylation lever and DNA methylation pattern during the proliferation and differentiation of pinus massoniana Lamb embryogenic cultures.Embryogenic cultures from five resurrection regulation stages were investigated to assess the extent and pattern of cytosine methylation by methylation sensitive amplified polymorphism (MSAP) analysis. The results show that there were changes both in DNA methylation lever and DNA methylation pattern during the proliferation and differentiation of pinus massoniana Lamb embryogenic cultures.The results could lead to the somatic embryogenesis molecular mechanism of Pinus massoniana Lamb, and were summarized as the following:1. There were changes in DNA methylation pattern during the5resurrection regulation stages. MSAP analysis showed that the resurrection regulation phase had the most total methylation ratios and full methylation ratios and the stage of early and bloom proliferation had the most hemi-methylations. DNA hyper-methylation was most from decline stage to resurrection regulation stage and the most de-methylation was from resurrection regulation stage to stable proliferation stage. There were difference between the two treatment both in hyper-methylation and de-methylation.2. Different stages of differentiation phase had a significant correlation with its-In. Function of-In and development stages during3d-50d was y=-3.8E-6x4+0.0004x3-0.0143x2+0.1901x+0.8321and R2was almost one. Which indicated that embryogenesis events and their-In had significant correlation.3. Changes of DNA methylation patterns and levels were detected by MSAP at11 durations after ABA induction. The results showed that the50d had the most total methylation ratios and hemi-methylation ratios; the least total methylation ratios and hemi-methylation ratios was2d after ABA induction; The most full methylation ratios were at3days and15days had the least full methylation. DNA hyper-methylation was the highest from35d-50d and was the lowest from0.5h-6h.DNA de-methylation was most from3d-5d and was least from Oh-0.5h.