Cellular and morphological changes during leaf explant dedifferentiation and plant regeneration of Tagetes erecta

Plant tissue culture allows the maintenance of species of interest under controlled conditions. There have been few studies related to the morphological changes that take place during callus formation and redifferentiation processes. The aim of this work was to analyze the morphological changes during dedifferentiation and redifferentiation processes in leaf explants of marigold (Tagetes erecta L.) using transmission electron microscopy, digital image analysis and concepts of fractal dimension. Leaf explants were sown in MS medium supplemented with 2.0 mg L⁻¹ of benzyladenine and 2.0 mg L⁻¹ of 2,4-dichlorophenoxyacetic acid to induce dedifferentiation (callus formation). The redifferentiation process (bud formation) was obtained with explants sown in MS medium supplemented with 3.0 mg L⁻¹ indoleacetic acid and 0.5 mg L⁻¹ benzyladenine. During the first 8 days, the dedifferentiation process showed gradual changes in terms of the area and perimeter of the explants. The fractal dimension of the perimeter corresponding to the projected image of the calli changed from smooth to rough at day 20. These features indicate variations in tissue morphometry as a result of the callus formation process and further formation of cellular aggregates. Structural observations during the dedifferentiation process showed changes in shape and cellular organization as callus formation became evident. Round-shaped cells were observed in the callus, with few organelles and a low degree of differentiation. Indirect organogenesis was achieved over a period of 10 days; in addition, plantlets developed from the obtained buds in 20 days in growth regulator-free MS medium.