Embryogenic cell lines initiated from immature zygotic embryos of Pinus nigra Arn. ssp. Austriaca were characterized in terms of macromorphological traits (colour, bipolar structures formation, germination ability) and their embryogenic potential was defined as high, medium or null. Quantification of global genomic DNA methylation revealed the existence of specific DNA methylation levels for the determinated embryogenic potentials. The line considered as effectively embryogenic, i.e., with the ability of develop the whole embryogenic program and producing plants, showed the lowest methylation levels. There was also proved the existence of an inverse relationship between total contents of free PAs and embryogenic potential, being the highest contents of free putrescine and spermidine in the non-embryogenic line and the lowest in the effectively embryogenic one. Relationships among DNA methylation levels, profiles of free individual polyamine contents and embryogenic potentials based on the ability to produce well-formed somatic embryos with effective plant conversion are discussed.

The developmental transition from a fertilized ovule to a dispersed diaspore (seed or fruit) involves complex differentiation processes of the ovule's integuments leading to the diversity in mature seed coat structures in angiosperms. In this study, comparative imaging and transcriptome analysis were combined to investigate the morph-specific developmental differences during outer seed coat differentiation and mucilage production in Aethionema arabicum, the Brassicaceae model for diaspore dimorphism. One of the intriguing adaptations of this species is the production and dispersal of morphologically distinct, mucilaginous and non-mucilaginous diaspores from the same plant (dimorphism). The dehiscent fruit morph programme producing multiple mucilaginous seed diaspores was used as the default trait combination, similar to Arabidopsis thaliana, and was compared with the indehiscent fruit morph programme leading to non-mucilaginous diaspores. Synchrotron-based radiation X-ray tomographic microscopy revealed a co-ordinated framework of morph-specific early changes in internal anatomy of developing A. arabicum gynoecia including seed abortion in the indehiscent programme and mucilage production by the mucilaginous seed coat. The associated comparative analysis of the gene expression patterns revealed that the unique seed coat dimorphism of Ae. arabicum provides an excellent model system for comparative study of the control of epidermal cell differentiation and mucilage biosynthesis by the mucilage transcription factor cascade and their downstream cell wall and mucilage remodelling genes. Elucidating the underlying molecular framework of the dimorphic diaspore syndrome is key to understanding differential regulation of bet-hedging survival strategies in challenging environments, timely in the face of global climatic change.

How the biophysical properties of overlaying tissues control growth, such as the embryonic root (radicle) during seed germination, is a fundamental question. In eudicot seeds the endosperm surrounding the radicle confers coat dormancy and controls germination responses through modulation of its cell wall mechanical properties. Far less is known for grass caryopses that differ in tissue morphology. Here we report that the coleorhiza, a sheath-like organ that surrounds the radicle in grass embryos, performs the same role in the grass weed Avena fatua (common wild oat). We combined innovative biomechanical techniques, tissue ablation, microscopy, tissuespecific gene and enzyme activity expression with the analysis of hormones and oligosaccharides. The combined experimental work demonstrates that in grass caryopses the coleorhiza indeed controls germination for which we provide direct biomechanical evidence. We show that the coleorhiza becomes reinforced during dormancy maintenance and weakened during germination. Xyloglucan endotransglycosylases/hydrolases may have a role in coleorhiza reinforcement through cell wall remodelling to confer coat dormancy. The control of germination by coleorhiza-enforced dormancy in grasses is an example of the convergent evolution of mechanical restraint by overlaying tissues.

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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.