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

This work concerns application of flow cytometry (FCM) and confocal la ser scanning microscopy (CLSM) to investigate the competence of cells for cell division and regeneration. FCM analysis of freshly-cut thin c ell layer (TCL) explants of Nicotiana tabacum, excised from upper inte rnodes of vegetative plants, revealed that one-quarter of the cells ha d the 2C nuclear DNA content, whereas of the other cells most nuclei h ad the 4C and some had the 8C DNA content. Cytometric examination usin g CLSM showed that the 2C nuclei were mainly located in the epidermis and subepidermis, and the 4C nuclei predominantly in the cortical tiss ue. During culture of the explants, part of the cortical cells went th rough mitosis from the first day onwards, and formed callus from which predominantly diploid and some tetraploid roots regenerated at low fr equency. Most cortical cells were thus in the G(2) phase of the diploi d cell cycle. FCM analysis showed that another fraction of the 4C cort ical cells was induced to endoreduplicate to 8C cells. These cells thu s had previously switched to the G(1) phase of the tetraploid cell cyc le. CLSM analysis revealed that subepidermal and epidermal cells, resp ectively, underwent cell division from the second and third day onward s. Shoot primordia developed from cells of both cell layers together. Most shoot regenerants were normal diploids, some were mixoploids or t etraploids. 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 involvemen t of these parameters in the competence for cell division and regenera tion at the level of the individual explant cells, and are therefore r elevant for cellular and molecular approaches to plant transformation.