Variations in DNA methylation in Picea omorika (Panč) Purk. embryogenic tissue and the ability for embryo maturation

Developmental changes in response to phytohormone treatments are commonly accompanied by variations in DNA methylation. To improve somatic embryo maturation in Picea omorika, a long-term embryogenic tissue line was exposed, for one to four weeks, to a variety of auxin/cytokinin ratios, or to hormone-free medium, before placement onto a maturation medium containing 40 ìM ABA. The extent of cytosine methylation in thus pretreated explants was estimated with the aim to reveal possible interdependence of the global DNA methylation status and the ability for subsequent embryo maturation on ABA-containing medium. The differences in the electrophoretic pattern of RAPD bands following DNA digestion with HpaII and MspI were the criteria used for evaluating the levels of DNA methylation. The latter varied depending on the exogenous hormone supply. Reduction of the 2,4-D level or its omission from the culture medium resulted in decreased methylation. A one-week exposure to hormone-free medium supplemented with activated charcoal coincided with reduced DNA methylation and improved subsequent somatic embryo maturation on the ABA-containing medium. A similar reduction in DNA methylation was observed after one-week exposure to 5-azaC in the presence of 2,4-D and BA, but this treatment did not affect subsequent embryo development.