Cytokinins and Flower Bud Formation in Vitro in Tobacco

Abstract

Explants from flower stalks of Nicotiana tabacum L. were cultured on different cytokinins to induce flower bud formation. All cytokinins tested except zeatin and zeatin-riboside induced the same maximal number of flower buds. Benzyladenine, benzyladenosine, and dihydrozeatin were the most active compounds whereas isopentenyladenosine and isopentenyladenine acted at a 20-fold higher concentration. These data suggest that the active cytokinins bind to the same receptor with different affinities. The presence of benzyladenine in the medium was necessary only during the first 2 days of culture (initiation period). The equilibrium between benzyladenine and its conjugates (the riboside, glucoside, and nucleotides) after a 4-day pulse was independent of the benzyladenine concentration whether it was inductive or noninductive for bud formation. The level of all derivatives was proportional to the benzyladenine concentration in the medium. Isopentenyladenine was used as a competitive inhibitor of benzyladenine conjugation. Isopentenyladenine concentrations that were too low for bud formation led to a synergistic increase in bud number when applied together with benzyladenine. Isopentenyladenine decreased benzyladenine uptake and conjugation. In spite of the lower uptake, the concentration of free benzyladenine inside the explants was higher in the presence of isopentenyladenine than in its absence whereas the concentration of the 7-glucoside of benzyladenine was lower. It was concluded that the free cytokinin base is the main active compound.The regeneration of organs in small explants from plant tissues offers the possibility to study differentiation on tissues spatially isolated from the whole plant. In this way the qualitative and quantitative response to auxins and cytokinins involved in the regulation of developmental processes can be studied. In tobacco, both the effects of the concentrations and of the ratios of these plant growth regulators on the induction and frequency of roots and/or shoots ( 16) or flower buds (20) have been described. In attempting to understand the mechanism of action of these plant growth regulators, it is important to relate the number of regenerated organs to the actual endogenous auxin and cytokinin concentrations. These concentrations cannot be determined from the amount of growth ' Present address: Department of Botany, University of Guelph, Guelph, Ontario N1G 2W1, Canada. 565regulator taken up because of the conversion of the biologically active molecules into derivatives of unknown activity or no activity at all (1,6,8,9,12,23,24). The cytokinin conjugates formed are glucosides, ribosides, and ribotides. Generally, the glucosides are considered stable and inactive. In contrast, the free base and perhaps also the ribosides and ribotides are active (1 1, 13).Little research has been performed on cytokinin metabolism in relation to developmental processes in plants. Van der Krieken et al. (22) (18), is very well suited to study cytokinin metabolism in relation to flower b...

Comparison of Benzyl Adenine Metabolism in Two Petunia hybrida Lines Differing in Shoot Organogenesis

“…Explant transfer experiments are often used to determine the critical time in which exogenous hormones mediate the commitment of explants to produce organs (7,22,24). The model of Christianson and Warnick (6,8) defined the induction phase as the time during which the hormone concentration in the medium controls the developmental commitment to organ initiation.…”

“…The developmental response of explants to exogenous hormones is the result of a variety of biochemical processes including hormone uptake, transport, and metabolism (10). Thus, although explant growth is typically described in relation to the hormone concentration in the medium, it is important to realize that the hormone concentration does not necessarily reflect the level of active hormone within the explant (3,24). Unfortunately, these biochemical processes are rarely investigated with respect to their role in controlling plant development.…”

“…For petunia leaf explants, the exogenous cytokinin BA2 can control the commitment of leaf explants to produce shoots in tissue culture. Understanding the action of cytokinin requires that the actual concentration of active molecules within the tissue be characterized throughout the developmental process (9,24). The major conjugates of BA and other cytokinins have been characterized in a few plant tissues, and their activities differ significantly (12)(13)(14)(15).…”

In Vitro Flower Bud Formation in Tobacco: Interaction of Hormones

“…The most abundant metabolites are compounds not yet identified, but the hydrolysis results suggest that they are conjugates. Furthermore, we show that IAA metabolism is not affected by the presence of BA and vice versa (22). Conjugation of IAA with amino acids is regarded as an important mechanism for detoxification, defense against decarboxylation, and a source of free IAA, which is considered to be the biologically active compound (1, 4, 5, 10).…”

“…It was determined that this period was 4 d for NAA (16) and 3 d for BA (22), both at a concentration of 1 AM.…”