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5 International Scientific Online Conference DOI: https://doi.org/10.15414/2021.9788055224015

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STUDY OF LEAF SURFACE BY SCANNING ELECTRON MICROSCOPY
Igor Maltsov, Ivan Gurnenko, Myroslava Marynyuk

M.M. Gryshko National Botanical Garden of the National Academy of Sciences of Ukraine,
Kyiv, Ukraine; E-mail.: maltsov@yandex.ru

The micro-morphology of the leaf surface is valuable for the study of leaf development and
functions. Scanning electron microscopy is an ideal technique for the investigation of plant
surfaces including leaf surfaces. Sample preparation with dehydration is still an important
aspect of the microscope study. This study represented many-years development of the
methodology of leaf surface study.
Investigations were conducted in the Tropical and Subtropical plants Department of the
M.M. Gryshko National Botanical Garden of the NAS of Ukraine. Leaves of the middle formation
were collected and material took from the central part of the lamina. Contaminated material
processed with solution HCl (1N) during 10–25 seconds, after that was used ultrasound during
30–60 seconds. The fixation of the leaf surface was conducted in a 4 % solution of
glutaraldehyde. The dehydration of plant tissues for scanning electron microscopy poses
distinctive challenges. Many methods can be employed to overcome difficulties arising from
plant tissue characteristics, but none of them are universal. Washing of material carried out
with buffer solutions (I-III) (pH=7) three times 15 minutes (every buffer). Next, was conducted
the dehydration of material with the use of mixtures of tert-butanol with concentration
increasing: mixture I – 1 hour (20 %), mixture II – 1.5 hours (35 %), mixture III – 2 hours (60
%), mixture IV – 1 hour (75 %), mixture V – 1.5 hours (100 %), mixture VI – left tonight (100
%), mixture VII – 2 hours (100 %).
After the dehydration procedure, the material in a solution of tert-butanol 100 % was put
in aluminum bottles with 1 ml volume and frozen on a copper plate on the freezing camera at -
6 ℃ during 15–20 min. For the drying procedure, the material carried over on pre-chilled to -
5℃ ceramic. The working volume pumped out with a regime of pre-vacuum till 3.99 Pa (3*10–
2 mm Hg). Dried samples were glued on metal tables and were used carbon spraying in the
regime of the thermal evaporation and rotation of vacuum universal post VUP-5M (SELMI,
Ukraine). For obtaining quality photos we recommend spraying samples with platinum in the
regime of ion-plasma mode.
The examination and result documentation of obtained material were conducted with the
use of a scanning microscope JSM-6700F (JEOL, Tokyo, Japan).
Despite the fact that it does not exist the universal method for plant tissue processing for
scanning electron microscopy, this study can be used as a method of study of the leaf surface.
Furthermore, this algorithm was successfully used in the study of different plant species.

Keywords: leaf surface, scanning microscopy.

5 International Scientific Conference Agrobiodiversity for Improving the Nutrition, Health, Quality of Life and |98
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