Clonal and physiological variation in the cytokinin content of tobacco-cell lines differing in cytokinin requirement and capacity for neoplastic growth

Abstract

A new enzyme-linked immunoassay for isopentenyladenine (IP)-type cytokinins is described. We measured the content of IP, isopentenyladenosine (IPA), zeatin (Z) and zeatin riboside (ZR) in cloned, axenically cultured lines of tobacco cells that differ in cytokinin requirement and capacity for neoplastic growth. IP or Z were not detected in any of the tissues. Sister clones isolated from the same cloned line of crown-gall cells obtained by transformation with the pTiT37 plasmid showed an up to eight-fold difference in their ZR and IPA content (30–230 and 21–77 pmol/g fresh weight, respectively). This variation could not be accounted for by differences in the time course of cytokinin accumulation. Fully habituated tissues, which are not transformed by the tumour-inducing (Ti) plasmid, contain high levels of ZR and IPA, indicating that the production of these cytokinins is not a unique property of crown-gall cells. Although high levels of cytokinin were correlated with tumour autonomy, they were not correlated with the capacity for cytokinin-autotrophic growth. No cytokinin (< 1 pmol/g fresh weight) was detected in sister clones of non-transformed cytokinin-requiring and cytokinin-autotrophic tissues. This suggests either that the cytokinin level needed for autotrophic growth is below the detection limit of our assay or that substances other than the cytokinins assayed are important for growth. The incubation of tissues on auxin-containing medium dramatically inhibited ZR and IPA accumulation in some of the cell lines, suggesting that auxins may be important in regulating cytokinin metabolism.