Microculture effects on leaf epidermis and root structure in Sclerocarya birrea subsp. caffra

Micropropagation of woody plants is beset by a number of challenges such as cell and tissue abnormality, which ultimately affect survival of plantlets ex vitro. The aim of the study was to evaluate the effects of in vitro microenvironments, in particular photoperiod, on the structure of the leaf epidermis and adventitious roots under photomixotrophic conditions. Control plants were grown under photoautotrophic conditions in a growth chamber. Stomatal density (number of stomata per mm2) on the abaxial leaf surfaces was highest for the control (221.9 ± 13.99) and the 16-h photoperiod treatment (206.6 ± 15.28). Stomata of plants grown under a 16-h photoperiod had fully expanded kidney-shaped guard cells that closely resembled stomata in the control plants. Formation of epicuticular wax was least for in vitro plants grown under a 24-h photoperiod. One of the main structural differences was the relative thickness of the inner and outer periclinal cell walls of the root epidermis between the control plants and in vitro plantlets. Control plants developed a thicker epidermis compared to that of in vitro plantlets. The root endodermis in the control plants was also comparatively thicker and more regular than that of in vitro plantlets. Control plants had a greater proportion of vascular tissue relative to the total area of the root cross-section compared to in vitro plants due to the development of xylem and phloem tissues. A micropropagation system that facilitates the modification of the in vitro microclimate may reduce the structural abnormalities of conventional microcultures.

► Stomatal density highest for the control and 16-h photoperiod in vitro plants.
► Stomata length and width were greatest for 24-h photoperiod in vitro plants.
► There is an inverse relationship between density and size of stomata.
► Epidermal layer thickness: Control plants (1.03 μm); in vitro plants (0.57 μm).