THE COMPETENCE OF CELLS FOR CELL-DIVISION AND REGENERATION IN TOBACCO EXPLANTS DEPENDS ON CELLULAR LOCATION, CELL-CYCLE PHASE AND PLOIDY LEVEL

This work concerns application of flow cytometry (FCM) and confocal laser scanning microscopy (CLSM) to investigate the competence of cells for cell division and regeneration. FCM analysis of freshly-cut thin cell layer (TCL) explants of Nicotiana tabacum, excised from upper internodes of vegetative plants, revealed that one-quarter of the cells had the 2C nuclear DNA content, whereas of the other cells most nuclei had the 4C and some had the 8C DNA content. Cytometric examination using CLSM showed that the 2C nuclei were mainly located in the epidermis and subepidermis, and the 4C nuclei predominantly in the cortical tissue. During culture of the explants, part of the cortical cells went through mitosis from the first day onwards, and formed callus from which predominantly diploid and some tetraploid roots regenerated at low frequency. Most cortical cells were thus in the G(2) phase of the diploid cell cycle. FCM analysis showed that another fraction of the 4C cortical cells was induced to endoreduplicate to 8C cells. These cells thus had previously switched to the G(1) phase of the tetraploid cell cycle. CLSM analysis revealed that subepidermal and epidermal cells, respectively, underwent cell division from the second and third day onwards. Shoot primordia developed from cells of both cell layers together. Most shoot regenerants were normal diploids, some were mixoploids or tetraploids. The combination of FCM and CLSM allowed identification of the cell cycle phase, the ploidy level, the position of the cell, and the cellular development. The results give insight into the involvement of these parameters in the competence for cell division and regeneration at the level of the individual explant cells, and are therefore relevant for cellular and molecular approaches to plant transformation.