Vitrification in relation to stomatal deformation and malfunction in carnation leaves in vitro

Abstract

Morphological and physiological disorders in leaves are often observed under culture conditions intended to promote rapid bud proliferation. High water potential in the medium and relative humidity in the culture vessel, elevated levels of sucrose and growth regulators and the presence of certain ions in unfavorable ratios, as well as low light intensity induce anomalous leaf anatomy and morphology termed ‘vitrification’. Abnormal shoot morphogenesis has been termed hyperhydration, glassiness, succulence and translucence, depending on the severity of the phenomenon. These descriptions are based on visual characterizations and depend on the external morphology of the studied species [12, 24, 33]. In vitro plants having abnormal leaves function poorly ex vitro due to inadequate photosynthetic activity and high rates of water loss which lead eventualy to plant desiccation [3, 7, 9, 32, 34, 37]. The major causes for poor plant survival are underdeveloped mesophyll, poor cuticular wax formation and malfunctioning guard cells [9,27, 34, 35]. Stomata from vitreous leaves remained open and did not respond to darkness, abscisic acid (ABA), high Ca²⁺ and hypertonic solutions — stimuli which usually cause stomatal closure [3, 22, 29, 34, 35]. Ziv et al. [37] reported that the defect in stomatal function resided in the guard cell walls which, unlike the protoplasts, did not contract in hypertonic solution. The mechanical properties of the guard cell walls in the stomatal apparatus are determined by the cell wall structure and composition [22]. Since both hyperhydration and guard cell malfunction result from changes in the cell walls, this research investigated the relationship between leaf hyperhydration and stomatal deformation and malfunction with the purpose of using stomatal function as a simple, more accurate parameter to evaluate vitrification.