Using cryomethods around SEM technologies to characterize waxes on the petals surface of Rosa damascena

Abstract

The rosaceae is one of the major family of flowering plants with 3200 species in 115 genera. The genus Rosa encompasses approximately 190 species and more than 18 000 cultivar forms of the plant have been identified [1]. The species Rosa damascena is well known for its intensely fragrant flowers.In the cosmetic field, essential oils and waxes are two major components which are extracted from therose flower. Rose essential oils are very often used in fine fragrances whereas waxes can be used as active components to play a major role in the skin physiology. Rose petals' surfaces have already been studied by conventional SEM technologies. The rose scent compounds are emitted by both epidermal layers [2]. Residues of secretory substances have already been observed on both surfaces [2,3]. The wax components are localized in the cuticle on the surface of the epidermal cells. The cuticle of floral organs presents a micro‐relief defined as cuticular patterns [4]. Although the shape of epidermal cells is variable between both the petal rose faces, the cuticle presents some common patterns which results in parallel folds. Circular wax patterns were also observed on the abaxial surface [2,3,5]. The aim of our study was principally to characterize more precisely the wax aspect at thesurface of petals of the Rosa damascena . Wax microstructures are fragile and to avoid sample preparation artifacts we decided to work on fresh hydrated specimens and innovate by mixing cryomethods with different observation modes in SEM. In a first approach we manually plunged the petal sample into liquid nitrogen and transferred it into the SEM chamber equipped with Peltier freezing stage. The surface analysis using SE and BSE detectors in variable pressure mode allowed us to distinguish two shapes of epidermal cells on the abaxial face. Both are elliptical but one of them shows globular structures (Figure 1). This latter shape would appear to be associated with circular wax models. In a second time, the cryofixation was carried out with a semi‐automated plunge freezing device. The specimen was etched, coated and then transferredfrom the cryo preparation chamber onto a SEM‐FEG cryo‐stage. High resolution imaging with an in‐lens SE detector at low voltage permitted us to analyse the extreme surface of petals and observe the structure of the rose petal waxes. We confirmed by this study the interest of mixing cryomethods to preserve the rose petal wax patterns as close as their native state. In addition using numerous and complementary detectors in different vacuum modes allowed us to collect new information on the wax aspect at thesurface of petals of the Rosa damascena . The optimizing protocols of cryomethods can also be an opportunity to have a better understanding of the scent molecules release from the epidermal cells though the cuticle. Our first observations in this direction are encouraging.