Análisis de los cambios estructurales de células in vitro de Tagetes erecta L. sometidas a biobalística

Abstract

Tagetes erecta L callus cells ("porte bajo” material) were used as a plant model in order to delivery lcy-β gene (from carotenoids route) by means of biobalistic transformation. The aim was to evaluate the probable transformation and analyze the cellular morphological changes caused by the bombardment of callus. The pC35Sβ and pBI426 plasmids were extracted from E. coli and analyzed by restriction assays. Transient expression assays were carried out, using gold particles bombardment coated with pBI426 construction. Three pressures and three distances were assessed to determine the conditions of bombardment in low-pressure chamber. The condition evaluated 60psi/14.5 cm showed the highest values of transient expression, so these conditions were used to bombard the callus with pC35Sβ construction. The bombarded callus could not be subcultured, despite of showed positive values of cell viability (67%). This feature was evaluated by differential staining with DAF and PI fluorochromes. It has been reported that cell viability does not confirm that the cells are able to develop cell mass on subcultures. Fluorescent micrographs were analyzed with IP staining to investigate in cell nuclei changes from bombarded and not bombarded callus cells. Digital Image Analysis were carried out to analyze nuclei morphology of the bombarded cells, it was found a tendency to smaller area and perimeter sizes than non-bombarded nuclei cells. The nuclei cells showed morphological differences in the arrangement and shape of the chromatin in the course of 22 days of culture. Bombarded cells displayed repeatedly, highly condensed nuclear bodies in the nucleoplasm, but not in the non-bombarded cells. Nevertheless, transformation callus could not be assured. On the other hand, during the capture of fluorescent micrographs, tracheids were observed in both cell lines (bombarded and non-bombarded callus cells). Environmental Scaning Electronic Microscopy also revealed the presence of differentiated cells in the callus cultures of T. erecta these differentiated cells in the callus were identified as tracheids, in which it has been reported thickening of the cell wall and lignin synthesis. There are no reports of these structures on in vitro cultures of T. erecta. This model could be used to abound in the study of xylem and lignin synthesis, at molecular and cellular level. Trachea elements were observed in both bombarded and non-bombarded callus.