An optimized histological proceeding to study the female gametophyte development in grapevine

BACKGROUND: Reproductive success in seed plants depends on a healthy fruit and seed set. Normal seed development in the angiosperms requires the production of functional female gametophytes. This is particularly evident in seedless cultivars where defects during megagametophyte's developmental processes have been observed through cytohistological analysis. Several protocols for embryo sac histological analyses in grapevine are reported in literature, mainly based on resin- or paraffin-embedding approaches. However their description is not always fully exhaustive and sometimes they consist of long and laborious steps. The use of different stains is also documented, some of them, such as hematoxylin, requiring long oxidation periods of the dye-solution before using it (from 2 to 6 months) and/or with a differentiation step not easy to handle. Paraffin-embedding associated to examination with light microscope is the simplest methodology, and with less requirements in terms of expertise and costs, achieving a satisfactory resolution for basic histological observations. Safranin O and fast green FCF is an easy staining combination that has been applied in embryological studies of several plant species. RESULTS: Here we describe in detail a paraffin-embedding method for the examination of grapevine ovules at different phenological stages. The histological sample preparation process takes 1 day and a half. Sections of 5 µm thickness can be obtained and good contrast is achieved with the safranin O and fast green FCF staining combination. The method allows the observation of megasporogenesis and megagametogenesis events in the different phenological stages examined. CONCLUSIONS: The histological sample preparation process proposed here can be used as a routine procedure to obtain embedded ovaries or microscope slides that would require further steps for examination. We suggest the tested staining combination as a simple and viable technique for basic screenings about the presence in grapevine of a normally and fully developed ovule with embryo sac cells, which is therefore potentially functional.