The Effects Of DNA Methylation On Callus Growth And Shoot Regeneration Of Leaves Of Fragaria Vesca

Based on the theory of plant cell pluripotency,plant tissue culture has been gradually mature,and it has been widely used in agriculture and horticulture fields.The tissue culture of Strawberry can provide a large number of virus-free seedlings.Fragaria vesca(Fragaria vesca)is an ideal model plant for studying the function of strawberry genes.The research of Fragaria vesca on tissue culture can provide theoretical reference and support for the study on strawberry breeding and transgenic strawberry.The regulatory role of epigenetics in plant development has been paid extensive attention.Epigenetic modifications,such as DNA methylation,play an important role in regulating gene expression,but few studies were done to investigate their effects on strawberry.On the one hand,different concentrations of DNA methylation inhibitors were applied to Fragaria vesca leaves in this study.On the other hand,The RNA interference strains of DNA methylation pathway key genes DECREASE IN DNA METHYLA TION1(FveDDM1)and RNA interference strains of DNA demethylase DEMETER-LIKE 1(FveDML1)were used as materials to study the regulation role of DNA methylation pathway interference on the callus growth and shoot regeneration process of Fragaria vesca leaves.The differentially expressed genes were identified,and we analyzed key genes that regulate and participate in the pluripotency of strawberry leaves and the pathway of bud regeneration in vitro.Our study provides a theoretical basis for the improvement of strawberry researches by genetic engineering and the application of seedlings of strawberry tissue culture in industry.The main findings are as follows:1.Different concentrations of the DNA methylation inhibitor 5-azacitidine(5-AzaC)and the histone deacetylase inhibitor TSA(Trichostatin A)were applied in the induction medium of the leaves of Fragaria vesca.Leaves were treated with 0 μM 5-AzaC(Control),5 μM 5-AzaC,10 μM 5-AzaC,20 μM 5-AzaC and 3 μM TSA respectively.The callus and shoot regeneration of the leaves were observed after incubation for a period.It was found that 5-AzaC and TSA inhibited the callus and shoot regeneration of the leaves of Fragaria vesca.It is speculated that DNA methylation and histone deacetylation process are involved in the pluripotency and in vitro shoot regeneration of Fragaria vesca leaves.2.The callus and shoot regeneration of FveDDM1-RNAi explants and FveDML1-RNAi explants during tissue culture were observed and recorded.By statistical analysis,we found that the silencing of FveDDM1 gene promoted the callus and shoot regeneration of Fragaria vesca.But there was no significant difference between the growth of callus and shoot regeneration of FveDML1-RNAi explants and Fragaria vesca.3.By analyzing the differentially expressed genes of FveDDM1-RNAi explants and FveDML1-RN Ai explants,we found that the significant difference genes of FveDDM1-RNAi explants mainly functioned in protein-binding and ATP-binding pathway.However,significant differences in FveDML1-RNAi explants mainly functioned in oxidation-reduction reactions,protein-binding,and ATP-binding pathways.4.By cluster analysis of differentially expressed genes,we found that compared with the control group,the expression level of some genes in FveDDM1-RNAi explants was significantly up-regulated,including CUC2(CUP SHAPED COTELYDON2),transcription factor MYB11 and ammonium-based transporters.The expression of CUC2 genes increased by 5.63 times than control group.It is speculated that DNA methylation may directly or indirectly affect these key genes,thereby participating in the callus formation and shoot regeneration pathways of Fragaria vesca.