Cytochemical and ultrastructural analysis of pro-embryogenic masses in Tabebuia roseo-alba

Abstract

Somatic embryogenesis can be used for the propagation of woody species that have difficult propagation. Tabebuia roseo-alba is a native species with white colors, from the Brazilian savanna, propagated by seeds, but these seeds have low germination rate and short viability period. Thus, in vitro propagation studies have been carried out and aimed at the large scale production of seedlings. The objective of this study was to evaluate the cytochemical characteristics and structures of pre-embryogenic masses of Tabebuia roseo-alba cultivated in vitro. Callus were induced from cotyledon leaves on MS medium supplemented with 3% sucrose and 0.6% agar. The effect of different NAA concentrations (0.0, 2.0, 4.0, 6, 0 and 8.0 mg L-1) in the induction medium was evaluated. After 70 days, cytochemical (double staining with carmine acetic acid and Evans blue) and ultrastructure analyses (Scanning Electronic Microscopy – SEM) of callus were performed. Callus growth curve was established by quantifying fresh and dry mass. The material was randomly collected at every 3 days until curve stabilization. Cell viability was determined by the Tetrazolium test during the growth curve establishment. NAA growth regulator was effective in inducing callus in all concentrations. SEM and double staining were used to characterize the formation of pre-embryogenic masses, and in both, the concentration of 6 mg L-1 provided better features (isodiametric cells and strong reaction to carmine acetic acid dye). The growth curve was established in a period of 36 days, showing sigmoidal behavior. For the formation of embryogenic callus, the optimal concentration indicated is 6 mg L-1 NAA. Subcultures should be carried out before the onset of the stationary growth phase.