Biochemical characterization of embryogenic calli of Vanilla planifolia in response to two years of thidiazuron treatment.

Vanilla planifolia embryogenic calli were cultured for two years on a medium containing thidiazuron (TDZ). Due to the presence of TDZ, these calli were under permanent chemical treatment and the differentiation of adventitious shoots from protocorm-like-bodies (PLBs) was blocked. When embryogenic calli were transferred onto a medium without TDZ, shoot organogenesis and plantlet regeneration occurred. To gain better knowledge about the biochemical and molecular processes involved in the morphoregulatory role of TDZ, hormonal and metabolomic analyses were performed. Our results indicate that in the presence of TDZ, embryogenic calli contained a high amount of abscisic acid (ABA) essentially metabolized into abscisic acid glucosyl ester (ABAGE) and phaseic acid (PA), which was the most abundant. When transferred onto a medium without TDZ, shoot regeneration and development take place in four stages that include: embryogenic calli growth, differentiation of PLBs from meristmatic cells zones (MCZ), shoot organogenesis from PLBs and the elongation of well-formed shoots. From a hormonal perspective, the significant reduction in ABA metabolism and its readjustment in the ABAGE pathway triggered PLBs formation. However, this first morphogenesis was stimulated by a strong reduction in IAA metabolism. The organogenesis of PLBs into shoots is associated with an increase in ABA catabolism and a gradual shift in cellular metabolism towards shoot differentiation. Thus, the initiation of the elongation process in shoots is correlated with an alteration in metabolite composition, including an increase in energy reserves (sucrose/starch) and a rapid decrease in alanine content. Our data highlighted the relationship between endogenous hormone signalling, carbohydrate metabolism and shoot organogenesis in Orchid plants.