Modification of Gene Expression, DNA Methylation and Small RNAs Expression in Rice Plants under In Vitro Culture.

Tissue culture is an important experimental technique widely used for plant transformation and can induce somaclonal variation that is shown to be associated with genetic and epigenetic changes. However, the molecular basis of somaclonal variation and plant cell response to tissue culture has yet to be fully understood. In this study, we investigated gene expression, DNA methylation, and small RNA changes in regenerated lines (RL) compared with the wild-type progenitor plants (WT) of rice cv. Hitomebore. Using microarray, we identified many genes that were differentially expressed in the shoot-tip tissue and showed that TEs were generally activated in RL. Methylation Sensitive Amplification Polymorphism (MSAP) analysis of 5′CCGG sites combined with bisulfite sequencing detected a generally reduced DNA methylation in the RL lines. Small RNA sequencing analysis detected widespread changes in small RNA accumulation between RL and WT. In particular, repeat and TE-associated 24-nt size class of small RNAs, the inducer of RNA-directed DNA methylation, was in general down-regulated in RL, consistent with reduced CHG and CHH methylation at some of the differentially methylated TE loci. A large number of differentially expressed miRNAs were identified in RL and WT lines, including known and novel miRNAs. The expression of some of these miRNAs exhibited inverse correlation with the predicted target genes, suggesting a regulatory function. The RL plants looked similar to WT plants under normal conditions but showed significant phenotypic alterations under abiotic stress conditions. The widespread changes in DNA methylation, small RNA accumulation and gene expression in regenerated plants supports the role of epigenetic changes in tissue culture-induced somaclonal variation. [ABSTRACT FROM AUTHOR]