Scanning electron microscopy (Cryo-SEM)

Visualization of the three dimensional surface wax structures was carried out with a scanning-electron microscope (Philips XL30 ESEM) equipped with a cryo preparation unit (Alto 2500, Gatan, UK). For this purpose, three intact clusters inserted between the second and third nodes of the shoot were collected early in the morning from the experimental plots. Immediately after sampling, three berries of the same size were removed from the inside of each cluster, from which a slice of the berry skin of approx. 3 mm x 3 mm was excised with a scalpel. The slices were mounted with a low temperature glue on a specimen holder by carefully avoiding touching the waxy surface. Cryofixation was performed with nitrogen slush (< -185°C). The frozen samples were sputtered with 20 nm Au in a high vacuum cryo preparation chamber and examined with a SE detector operating with acceleration voltage of 5–10 kV at high vacuum and -150°C. In this way, the specimens were cryo-fixed, sputtered and ready for analysis within 90 min after sampling in the field. A total of nine samples from each variety were analyzed, the surface structure of each specimen was documented from at least three positions at 8.000x magnification.