The physiology and control of vitrification in plants propagated in vitro using petunia as a model system

Abstract

Vitrification is a physiological and developmental disorder that affects plants propagated in vitro. The shoot apex is the source of vitreous development. Widening of the meristematic region as well as increased cell area of the tunica and corpus were characteristic of the vitrified petunia shoot apex. Longitudinal sections showed normal stems to have cells of an organized and uniform rectangular shape while cells of vitreous stems had irregular shapes. Epidermal, cortical and pith cells from transverse sections of vitrified stems were significantly larger than their morphologically normal counterparts indicating hypertrophy of the cells. Vitreous plants also had a reduction in the lignification of xylem and lack of palisade mesophyll in leaves. High concentrations of Gelrite decreased vitrification while increased sucrose levels promoted vitreous development of normal petunia nodal segments. Leaves of vitreous plants had higher levels of reducing sugars and sucrose but lower or undetectable levels of inositol as compared to normal plants. Normal plants on medium void of inositol have the ability to synthesize inositol and maintain levels equal to that found in normal plants from inositol containing media. Basal segments from normal explants, formed a higher percentage of vitreous shoots than did the upper nodes. A method was developed for including polyethylene glycol with Gelrite to obtain gelled media of varying water potentials. Media water potential from -0.31 to -1.2 MPa had no effect on occurrence of vitrification. Gelrite promoted vitrification but Gibco agar, alone or in combination with Gelrite, reduced its occurrence. Lowering media NH4 content reduced vitrification, while sealing culture vessels with parafilm increased vitreous growth. Normal growth of petunia could be controlled by the gelling agent used and whether or not the vessel was sealed. A synchronized system was developed for reverting vitreous plant growth to normal by limiting the supply of medium. As the supply of water and nutrients became limiting, the shoot apex was tranformed from a vitreous state to normal...