Abstract
Phase change-related epigenetic and physiological changes in the micropropagation process of Betula platyphylla. To evaluate the epigenetic regulation and the cell redox state during micropropagation, Betula platyphylla was used to regenerate plants from callus. Axillary bud, callus (20 days after induction), aged callus (40 days after induction), budding callus, regenerating shoots and regenerated root were estimated for the cell redox state using 18 biochemical parameters, such as the steady-state levels of reactive oxygen, content of peroxidation products and enzymatic or non-enzymatic protective systems. These biochemical parameters at different developmental phases were found to be significantly different. DNA methylation and corresponding enzyme activities were also investigated at different phases. The average percentage of methylated CCGG sites in the three birch lines varied from 11.92 to 17.03 % at different phases. The level of DNA methylation in the axillary bud and young callus was 13.84 and 11.92 %, respectively. It increased to 12.36 % at budding callus phase and to 15.24 % in the regenerated bud. At rooting phase, DNA methylation was observed to be the highest (17.03 %). When compared between different phases, younger callus was found to have a lower level of DNA methylation (11.92 %) than that of the older callus (14.50 %), indicating that DNA methylation took place as the callus aged. The results also showed that CCGG site methylation patterns (hemi-methylation of external C sites and full methylation of internal C sites) were different at various differentiation phases. The results of principal component analysis clearly demonstrated that there were phase change-related epigenetic and physiological changes in the micropropagation process. These data lead to the identification of biochemical parameters during the process of micropropagation and demonstrate a dynamic connection between plant micropropagation in vitro, cell redox state and DNA methylation.
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