Leaf Morpho-Anatomical Evaluation of Echeveria Cultivars using Different Fixation Methods

Echeveria succulent plants are among the most diverse ornamental plants belonging to the Crassulaceae family, showing increased demand in horticultural markets owing to their rosette formation and high adaptability to water-stress environments. Owing to this level of demand and its desirable qualities, several studies have attempted to define environmental adaptation for this plant as well as its phenotypes and taxonomy, usually through leaf morpho-anatomical research. Leaf anatomical analysis has been widely employed to study these relationships; however, in the case of Echeveria, there are several barriers owing to their unique leaf structure, meaning that relatively few studies have been conducted. Several techniques use chemical fixation to investigate leaf anatomy; however, there are no universal methods due to equipment constraints and a lack of comparative studies. In this study, different fixation methods using formaldehyde, ethanol, and methanol were used to visualize the leaf morpho-anatomical structures of 15 Echeveria cultivars under light and scanning electron microscopy (LM and SEM), whereas freehand sectioning was used to obtain transverse sections. Each fixative had a different effect on each cultivar despite belonging to the same genus. Among the three fixation methods, methanol provided better preservation of the cell morphology as observed using LM and SEM. In addition, freehand sectioning is a cost-effective method that requires skill and patience, limited and affordable materials, and can be generally used without any sophisticated equipment. The results of this study provide a benchmark for future research employing simple and effective methods for related species that possess succulent leaf characteristics.