Detection of DNA methylation changes during somatic embryogenesis of Siberian ginseng ( Eleuterococcus senticosus)

Abstract

DNA methylation is known to play an important role in the regulation of gene expression in eukaryotes. In this study, we assessed the extent and pattern of cytosine methylation during somatic embryogenesis in Eleuterococcus senticosus, using two methods to evaluate DNA methylation rates: (1) direct determination of 5-methyl-deoxycytidine (5mdC) amounts in genomic DNA by HPLC separation and quantification of nucleosides and (2) methylation-sensitive amplification polymorphism (MSAP) technique. The tissue assayed include embryogenic and non-embryogenic callus. Leaf segments were cultured on modified Murashige and Skoog (MS) medium containing 2,4-dichlorophenoxyacetic acid (2,4-D). Opaque and friable embryogenic callus formed on modified MS medium with 2.26 μM 2,4-D. Initiation and development of somatic embryos occurred in the same medium containing 2.26 μM 2,4-D. Non-embryogenic callus failed to regenerate even after prolonged maintenance and subculture on to the same media. Dark treatment significantly increased the number of mature somatic embryos. HPLC analyses on genomic DNA from embryogenic and non-embryogenic callus showed that global DNA methylation rates were significantly lowered in embryogenic calli. Similarly, 16.99% of 5′-CCGG-3 sites in the genome of non-embryogenic callus were cytosine methylated, where as 11.20% were methylated in case of embryogenic callus tissue, as detected by MSAP technique. Hypermethylation of DNA in non-embryogenic callus compared with embryogenic callus reflects the marked expression of this molecular feature, which may well contribute to the developmental gene expression.