Study of physiological and biochemical events leading to vitrification of Arbutus unedo L. cultured in vitro

Abstract

Vitrified shoots of strawberry tree propagated in vitro showed poor lignification when analysed by FTIR-ATR and by the acetyl bromide method, as well as marked disturbances on leaf tissues organization. Arbutus unedo (Ericaceae), commonly known as strawberry tree, is a bush or small tree grown mainly for the production of edible fruits that can be eaten fresh, processed or used to make liquors. In vitro propagation of A. unedo in liquid media is an advantageous procedure that reduces the time for multiplication. However, tissue culture in liquid media often leads to excessive accumulation of water in the apoplast that results in a physiological mechanism characterized by low lignification, impaired stomatal function and reduced mechanical strength of tissue culture-generated plants. Fourier-transform infrared spectroscopy in attenuated total reflectance mode (FTIR-ATR) was employed to assess the chemical-derived differences between vitrified and non-vitrified cell walls. FTIR-based spectral assignments suggested higher contents of aromatic compounds in leaves than in stems. Lignin measurements by the acetyl bromide procedure showed that there are no significant differences in the percentage of lignin by dry weight between the different types of leaves. Anatomical studies using light microscopy and scanning electron microscopy showed changes on epidermis organization of vitrified leaves as well as the presence of abnormal stomata in the abaxial surface. These observations may explain the difficulty of vitrified plants to balance absorption and evapotranspiration, and cope with hydric stress during the acclimatization phase. Moreover, cross-sectioned leaves revealed broad lacunar spaces and cross-sectioned stems had spaced-hypertrophic cortical parenchyma cells, where the water vapor may accumulate. Finally, the characterization of vitrified tissues was considered an important contribution to uncover the mechanisms that cause plant vitrification, not just by improving the methods developed for the micropropagation of this species, but also contributing for its agroeconomic valorization.