Plant Growth Regulation 23: 159–166, 1997. 159 c 1997 Kluwer Academic Publishers. Printed in the Netherlands. Metabolism of zeatin riboside in a hormone autonomous genetic tumour line of tobacco Shyamal K. Nandi & Lok Man S. Palni Plant Cell Biology Group, Research School of Biological Sciences, The Australian National University, G.P.O. Box 475, Canberra City, ACT 2601, Australia; present address: Department of Environmental Physiology & Biotechnology, G.B. Pant Institute of Himalayan Environment & Development, Kosi-Katarmal, Almora-263 643, U.P., India ( author for correspondence) Received 6 February 1997; accepted in revised form 27 August 1997 Key words: Cytokinin metabolism, genetic tumour, hormone, autonomous Abstract The metabolic fate of externally applied [ 3 H]-zeatin riboside ([9R]Z) was studied in a cultured genetic tumour line of Nicotiana glauca (Grah.) N. langsdorffii (Weinm.), which grows on auxin and cytokinin free medium. Metabolism by 3.5-week-old tissues showed enhanced stability of supplied [9R]Z; unmetabolized [9R]Z accounted for 48.7 and 37.5% of extracted radioactivity following 8 and 24 h incubation, respectively; tissues of different ages (1–10 weeks following subculture) also indicated high cytokinin stability following 8 h incubation (unmetabolized [9R]Z accounted for 32.5–53.0% of extracted radioactivity). All analyses were performed by thin layer chromatography (TLC) and the results subsequently confirmed by high performance liquid chromatography (HPLC). Side-chain cleavage and modification of the purine ring were the major forms of metabolism; metabolites with an intact cytokinin moiety included zeatin (Z), [9R]Z nucleotides and glucosyl derivatives. Detailed analysis of metabolites carried out in the experiments using 3.5-week-old tissues indicated that both dihydro-derivatives as well as cis isomers of Z and [9R]Z were not formed. Adenine, adenosine and its nucleotide(s) were the main degradative metabolites; in 3.5-week-old tissues these metabolites accounted for about 5.9 and 7.8% (of 3 H extracted) following 8 and 24 h incubation, respectively. In tissues of different ages (1–10 weeks following subculture), these metabolites accounted for about 7.6–22.9% of the extracted 3 H. Some metabolites (zeatin, adenine and adenosine) were also detected in the staled incubation media. The observed high [9R]Z stability in this tissue may reflect low levels of cytokinin oxidase activity and/or some form of compartmentation. Abbreviations: Ade = adenine; Ado = adenosine; (diH)Z = dihydrozeatin; (diH)[9R]Z = dihydrozeatin riboside; D1–5 = dyes 1–5; HPLC = high performance liquid chromatography; iP = isopentenyladenine; [9R]iP = isopen- tenyladenosine; TLC = thin layer chromatography; Z = zeatin; [9R]Z = zeatin riboside; [7G]Z = zeatin-7-glucoside; [9G]Z = zeatin-9-glucoside Introduction Genetic tumours arise spontaneously, without any apparent external cause, in certain interspecific hybrids, particularly in Nicotiana spp., and can be maintained indefinitely without the addition of auxin and cytokinin to the culture medium. Although phyto- hormone imbalance has been implicated in the induc- tion and maintenance of these tumours [1], recent evi- dence suggests that cytokinins are possibly involved in genetic tumour formation in Nicotiana hybrids [9, 10, 15] and tumourous tissues have been shown to contain relatively higher cytokinin levels in comparison to nor- mal plants [6]. The cytokinins in 3-week-old genetic tumour lines of Nicotiana glauca N. langsdorffii and N. suaveolens N. langsdorffii have been iden- tified and quantified by radioimmunoassay [15] and the dynamics of endogenous cytokinins determined