DNA methylation during sexual embryogenesis and implications on the induction of somatic embryogenesis in Castanea sativa Miller

Abstract

From anthesis to mature seed formation, burrs from cross-pollinated adult Castanea sativa Miller trees were characterized and seven developmental stages defined based on macro and micromorphological traits. In order to get an insight into the involvement of epigenetic mechanisms in sexual embryogenesis and to define somatic embryogenesis induction capability, global DNA methylation and the somatic embryogenic competence were quantified. On cross-pollinated trees once fertilization takes place, at least one ovule per ovary becomes dominant, and transient DNA demethylation occurs coinciding with the start of the sexual embryogenic programme. Unfertilized ovules from the same cluster, which maintain their prior size, increase their methylation level and undergo degeneration. These results were validated using non-cross-pollinated trees and the asynchrony of flower receptivity. When testing in vitro somatic embryogenesis response of isolated dominant ovules and axes from zygotic embryos under cross-pollinated conditions, the highest competence was found for reaching seed maturity. Thus, a "developmental window" of somatic embryogenesis in chestnut has been characterized. It includes from fertilization to embryo maturity, and a transient decrease in methylation is necessary after fertilization for the development of the somatic embryogenesis response.

New Insights into Somatic Embryogenesis: LEAFY COTYLEDON1, BABY BOOM1 and WUSCHEL-RELATED HOMEOBOX4 Are Epigenetically Regulated in Coffea canephora

“…For instance, during the phase of dedifferentiation or embryogenic calli generation in E. senticosus and Rosa hybrida, DNA demethylation events are frequent [34], [51]. Recent studies in C. sativa and Acca sellowiana have also showed the importance of demethylation events during SE induction or prior to the start of embryo differentiation [20], [52]. We observed two increases in DNA methylation during the SE of C. canephora (Figure 4A).…”

“…This strongly suggests a rapid cellular response to in vitro conditions [47], [48] accompanied by a drastic chromatin remodeling [49], [50]. Previous reports have shown that embryogenic cell formation is related to the increase of DNA methylation [20], [32], [33]. However, an increase in DNA methylation is not always the condition that has been reported to be important for SE; a decrease in DNA methylation levels seems to be fundamental for embryo development in some species.…”

“…However, Charkrabarty et al [34] reported that in order to obtain an embryogenic calli from Eleuterococus senticosus, low levels of DNA methylation are needed. More recently, Viejo et al [20] showed in Castanea sativa that DNA demethylation is required for SE induction and further development of somatic embryos in this species. On the other hand, it was recently reported that both DNA methylation and H3K9me2 modulate WUSCHEL expression in vitro during shoot regeneration in Arabidopsis [35].…”

Dynamic DNA cytosine methylation in the Populus trichocarpa genome: tissue-level variation and relationship to gene expression

“…In micropropagated Acacia , shoots with juvenile leaves exhibited higher DNA methylation levels than shoots with mature leaves [37]. Transient DNA methylation of ovules accompanied embryogenesis in chestnut [38]. As noted above, in poplar drought stress induced changes in total cellular DNA methylation [30] and was associated with transcriptome changes within separately propagated clones [31].…”

Cryopreservation of embryogenic tissues from mature holm oak trees

“…Several authors described changes at gene transcription level such as DNA methylation or histone modification linked to the embryogenic response [60], somatic embryo maturation [55] and complete somatic embryo development and conversion [37]. Such changes were also observed in the different steps of the cryopreservation procedure [17].…”