Wild-type ovule development in Arabidopsis thaliana: a light microscope study of cleared whole-mount tissue

A detailed morphological description of wild-type ovule development in Arabidopsis thaliana is presented. The entire process from the formation of the ovule protrusion until the eight-nuclear endosperm stage is described. The study is based on a light-microscopical analysis of stained and subsequently optically cleared whole-mount ovules. It is supplemented by confocal laser scanning microscopy of propidium iodide-stained whole-mount ovules. It has been shown that the combination of both techniques eliminates the need for sections to a large extent, and hence allows the rapid morphological inspection of a large number of ovules in Arabidopsis. The ovule constitutes a relatively simple organ. During development, three discrete major pattern elements are laid down along the proximal-distal axis: the nucellus at the distal end (harbors the megaspore/gametophyte lineage), the chalaza (flanked by the integuments) and the funiculus (includes the vascular strand) at the proximal end. These three pattern elements already appear at a very early stage, when the initially formed protrusion, consisting of files of uniform cells, is transformed into a patterned primordium. Subsequent morphogenesis results in the manifestation of the morphological characters of each pattern element. It was possible to dissect this developmental process into distinct, morphologically discernible steps at a high resolution. A classification scheme of ovule developmental stages is proposed, which is based on ovule-specific, discrete, and easy-to-score markers.