Structural and chemical study of callus formation from leaves of Rubia tinctorum

Abstract

Dedifferentiation was monitored in Rubia tinctorum L. leaves over a 14-d period after callus induction using transmission electron microscope (TEM), high performance liquid chromatography (HPLC), spectroscopy and thin layer chromatography (TLC). Photosynthetic pigment loss of leaves took 3-5 d coinciding with the first period of anthraquinone accumulation. Callus cells were discernible in the region of the vascular bundles and wounded edges of leaves after 10-14 d. Characteristic ultrastructural alterations were manifested in vacuolization, appearance of mitochondria, amount of smooth endoplasmatic reticulum and cytoplasm, caryolympha density of nuclei and cytoplasm content of cells. There were special events in the transfer cells: unequal divisions of dedifferentiated plastids and lytic activity in the cell wall. Our results show that mesophyll cells seem to be stopped at a particular level of dedifferentiation, while transfer cells embodied in veins of leaves pass through further alterations and lead to callus formation. Findings suggest that a sort of dedifferentiation drift manifests in the various cells of R. tinctorum leaves during callus induction and depending on their specialized status they achieve different levels of dedifferentiation. Approximately 4 weeks after callus induction, root growth has started from the young calli. © 2007 Institute of Experimental Botany, ASCR.