A high affinity binding site for cytokinin to a particulate fraction in carrot suspension cells

Summary Carrot suspension cells contain one class of high affinity binding sites for cytokinin in an 80,000 × g particulate fraction. Binding of [8-¹⁴C]-benzylaminopurine (BA) to this fraction assayed by a sedimentation method was found to be optimal at pH 6.0 and thermolabile. Specific binding was proved in competition experiments in which labelled BA was displaced by increasing concentrations of unlabelled BA. Scatchard plots of these results displayed a dissociation constant (K_d) of 33 ± 6 nM. The number of binding sites found was 1,100 ± 120 fmol g^–1 fresh weight which is equivalent to a frequency of 23,000 binding sites per cell. The specificity of the binding sites to cytokinins and their analogues followed the sequence BA with highest affinity, kinetin, zeatin, iP and adenine. The cytokinin ribosides generally had a lower affinity than their cytokinin bases, and the affinity decreased in the order [9 R] BA, [9 R] iP, [9 R] Z, [9 R] A.     

Cytokinin inhibition of respiration by cells and mitochondria of soybean,Glycine max (L.) Merrill

Cells of a soybean tissue strain, suspended in an aerated liquid medium, caused disappearance of p-coumaric acid from the medium and oxidation of guaiacol, benzidine, pyrogallol, L-dihydroxyphenylalanine and L-epinephrine. Both the disappearance and the oxidations were inhibited by 6-benzylaminopurine (BAP) at a concentration of 0.5 mM. BAP at other concentrations either promoted or inhibited oxidation of epinephrine in precisely the pattern reported earlier for the disappearance of coumarate; therefore, the disappearance of coumarate probably involves its oxidation. The effectiveness of other cytokinins in inhibiting the oxidation was studied.At 0.5 mM, and perhaps even at 0.5 M, some of the several cytokinins tested inhibited oxygen consumption by the soybean cells. This inhibition, which did not require any of the above metabolizable compounds, was especially marked in the presence of cyanide, azide or Antimycin A, and was detectable in 10 min or less. Either Antimycin A or salicylhydroxamic acid alone promoted O₂ consumption but together they were quite inhibitory. The soybean cells apparently have an alternate respiratory pathway and cytokinins may influence its operation.Several cytokinins at 0.5 mM, and perhaps at 0.5 M, also inhibited oxygen consumption by mitochondrial preparations from the soybean cells, the inhibition being evident in about 20 s. The consumption required a substrate such as malate, succinate or NADH. Cytokinins and related compounds varied in effectiveness as follows: BAP and 6-isopentenyla-minopurine 9-tetrahydropyranyl-BAP > kinetin, ribosyl-isopentenylaminopurine, 9-methyl-BAP and 9-methoxymethyl-BAP > 6,6-dimethylaminopurine and zeatin (slight activity) > 6-methylaminopurine, nicotinamide and adenine (ineffective). To a great extent this order parallels the order of effectiveness of the compounds in causing cell division. Mitochondria, therefore, may contain a site for an important cytokinin action.Abbreviations BAP 6-benzylaminopurine - IPA 6-(²-isopentenyl)aminopurine     

Effects of Auxins and Cytokinins on Embryo Formation from Root-derived Callus of Oncidium ‘Gower Ramsey’

In an attempt to optimize somatic embryo formation in Oncidium ‘Gower Ramsey’, the effects of five auxins (2,4-D, IAA, IBA, NAA and picloram) and five cytokinins (2iP, BA, kinetin, TDZ and zeatin), used alone, was tested in vitro using root-derived callus. In general, kinetin (0.5 and 2 mg l⁻¹) and zeatin (0.5 mg l⁻¹) were found to be more effective than other auxin and cytokinin treatments to induce somatic embryogenesis from root-derived callus.     

Benzyladenine and derivatives-their significance and interconversion in plants

Recently benzyladenine has been isolated as a natural cytokinin from a number of plants. The natural occurrence of this cytokinin will change the attitude with which physiologists view this hormone. This review attempts to put into context what is known about this cytokinin and its derivatives and to compare and contrast its metabolism and the function and physiological action of its various metabolites. Nothing is known about the biosynthesis of benzyladenine. Its structure would suggest that its biosynthetic pathway may differ considerably from that of zeatin and iso-pentenyladenine.Abbreviations Ade adenine - Ado adenosine - BA benzyladenine - [9R]BA BA ribonucleoside - [9R-MP]BA BA nucleotide - [9R-DP]BA BA dinucleotide - [9R-TP]BA BA trinucleotide - [3G]BA BA 3 glucoside - [7G]BA BA 7 glucoside - [9G]BA BA 9 glucoside - [9R-G]BA BA 9-ribosylglucoside - [9Ala]BA BA alanine-conjugate - (2OH)BA BA ortho-OH - (2OH)[9R]BA BA ortho-Oh-riboside - KN kinetin - [9R]KN KN ribonucleoside - DHZ dihydrozeatin - Z trans-zeatin - [9R]Z zeatin ribonucleoside - [7G]Z zeatin-7-glucoside - [9G]Z zeatin-9-glucoside - [9Ala]Z zeatin alanine-conjugate - (OG)[9R]Z O-glucoside of zeatin ribonucleoside - [9R-MP]Z zeatin nucleotide - iP iso-pentenyladenine - [9R]iP iP ribonucleoside     

Endogenous cytokinin accumulation and cytokinin oxidase activity during shoot organogenesis of Petunia hybrida

Changes in endogenous cytokinin content and cytokinin oxidase activity were characterized in leaf explants from two Petunia hybrida Vilm. genetic lines which differed in their shoot organogenic response to exogenous N⁶-benzyladenine (BA). Endogenous cytokinin content in leaf explants of the highly shoot organogenic line, St40, increased 1.7-fold during the shoot induction phase (days 6–10) and had an additional 2.6-fold cytokinin increase correlated with the shift from induction to the shoot development phase. The cytokinins isopentenyl adenine (iP) and isopentenyl adenosine (iPAR) increased, while the cytokinins zeatin, zeatin riboside and dihydrozeatin remained at consistently low levels. In contrast, isoprenoid cytokinins did not accumulate in petunia TLV1 leaf explants which were incapable of shoot induction during 12 days of culture with BA. Cytokinin oxidase activity continuously increased in leaf explants of both petunia genotypes in response to BA, with a larger increase in St40. These results suggest that the differences in organogenic response in the two petunia genotypes may be the result of differences in BA uptake and metabolism which subsequently affects the accumulation of isoprenoid cytokinins and the activity of cytokinin oxidase in the early stages of shoot development.     

Cytokinin modification of metabolism of p-coumaric acid by a cell suspension of soybean (Glycine max (L.) Merrill)

Cells of a soybean tissue strain suspended in an aerated liquid medium caused the disappearance of p-coumaric acid from the medium. The rate of disappearance was modified by cytokinins. When the coumarate and the cytokinin were added to the medium simultaneously, disappearance was increased if the cytokinin was used in the concentration range from 0.05 to 50 M; higher concentrations inhibited the disappearance. If, however, the cytokinin was added at the beginning of the shaking period (for aeration) and the coumarate added 1 h later, the results were more complex. With this procedure, cytokinins at concentrations from 0.0005 to about 1 M inhibited, at 50 M they promoted, and at higher concentrations they inhibited the coumarate disappearance. The promotion was elicited by zeatin, ribosylzeatin, kinetin, 6-benzylaminopurine (BAP), by BAP substituted at the 9-position by methyl, methoxymethyl, cyclohexyl or tetrahydropyran-2-yl groups, by adenine with the amino group substituted by methyl, dimethyl, n-propyl, n-pentyl or n-hexyl groups, by 1,3-diphenylurea and nicotinamide, all at about 50 M. Adenine and benzimidazole were not effective. The promotion was detected in as little as 12 min. The delayed inhibitory effect required the presence of the cytokinin during the 1 h of shaking before the coumarate was added. This effect was elicited by zeatin, ribosylzeatin, kinetin, BAP, the aforementioned 9-substituted-BAP compounds, 9-glucosyl-BAP, 7-glucosyl-BAP, and 6-isopentenylaminopurine and its ribonucleoside. It was not caused by adenine, cis-ribosylzeatin, diphenylurea, benzimidazole, 6-methylaminopurine, 6,6-dimethylaminopurine or nicotinamide. The chemical specificity for this effect was much the same as that known for promotion of cell division in the soybean tissue.Abbreviations BAP 6-benzylaminopurine - NAA -naphthaleneacetic acid     

Cytokinins from terminal buds of Euphoria longana during different growth stages

The presence of endogenous cytokinins were detected in the terminal buds of longan ( Euphoria longana Lam.) after purification by ion exchange and Sephadex LH-20 chromatography, and bioassay, enzymic degradation, high-performance liquid chromatography and gas chromatography-mass spectrometry. Permethylated derivatives of two highly active cytokinin glucoside compounds from dormant buds were: 6-(4-O-β-D-glucosyl-3-methyl-but-2-enylamino) purine (zeatin-O-glucoside) and 9-β-D-ribofuranosyl-6-(4-hydroxy-3-methyl-but-2-enylamino) purine (zeatin riboside-O-glucoside). Simultaneously, four active cytokinins from buds at the stages of leaf flush and flower bud initiation were identified as 6-(4-hydroxy-3-methyl-but- trans -2-enylamino) purine (zeatin), zeatin-9-β-D-ribofuranosylpurine (zeatin riboside), 6-(3-methyl-2-butenyl) aminopurine (isopentenyladenosine, 2iPA) and N-(3-methyl-2-butenyl) adenine (isopentenyladenine, 2iP). The total cytokinin levels were low at leaf flush, with the zeatin and zeatin riboside in the buds about 70% of the total. In the transition of the terminal bud from leaf flush to dormancy, the principal cytokinins were zeatin-O-glucoside and zeatin riboside-O-glucoside. However, significant decreases in the levels of zeatin-O-glucoside and zeatin riboside-O-glucoside and increases in those of zeatin, zeatin riboside, 2iPA and 2iP were observed at flower bud initiation. It is suggested that in longan, the cytokinins that are translocated to the shoots are accumulated in the buds at the dormant stage, and that later there is a considerable increase in free cytokinins during flower bud initiation, leading to the promotion of flower bud development.     

Influence of Different Cytokinins on the Transpiration and Senescence of Excised Oat Leaves

In order to investigate the possibility that cytokinins control transpiration indirectly through affecting leaf senescence, a direct comparison was made of the effect of different cytokinins on transpiration and senescence of oat leaves (Avena sativa L. cv. Forward). Senescence was assessed by measuring chlorophyll loss. The synthetic cytokinins N⁶ benzyladenine (BA) and kinetin delayed senescence and increased transpiration of oat leaves to a greater extent than did the naturally occurring compounds zeatin, N^b-Δ² isopentenyladenine (i⁶ Ade) and 6-ø-hydroxybenzyladenosine (hyd-BA riboside). During the early stages of the transpiration experiment zeatin showed similar or greater activity than BA. This period was longest when freshly excised leaves were used, was reduced when leaves were used after incubation in distilled water in the dark for 20 h and was eliminated by incubation in cytokinin solution in the dark. After this period the activity of zeatin declined relative to BA. The effect of cytokinins in increasing transpiration occurred only in the light; no effect was observed in the dark. BA showed higher activity than zeatin in senescence tests but both cytokinins were less effective as the tests progressed, this decrease in activity being more rapid when older leaves were used. The results are discussed in relation to the mechanisms by which endogenous cytokinins might control sensecence and transpiration in oat leaves and to the value of the oat leaf senscence and transpiration bioassays as tests for cytokinin activity of plant extracts.     

Further studies on the effects of different auxins and cytokinins on flower formation in thin cell layers of Nicotiana tabacum: The effects of zeatin riboside, dihydrozeatin and dihydrozeatin riboside

Organogenesis in thin cell layers of Nicotiana tabacum L. was studied in relation to the effects of natural and synthetic auxins in combination with various cytokinins. All cytokinins tested, benzyladenine (BA), kinetin, zeatin (Z), zeatin riboside (ZR), N⁶-Δ²-isopentenyl) adenine (IPA), dihydrozeatin [(diH)Z] and dihydrozeatin riboside [(diH)ZR], seem to be active in flower bud formation. In addition to the initiation of flower buds, vegetative buds or roots were also formed on the explants in the presence of BA, Z or IPA as exogenous cytokinins. Only dihydrozeatin and its riboside stimulated the initation of flower buds alone (as is known for kinetin), especially if supplemented with indole-3-acetic acid (IAA) as exogenous auxin. A high number of explants with flower buds was also found with high cytokinin/2,4-D ratios. In these conditions the presence of (diH)Z yielded the higest number of flower buds per explant.     

Changes in Cytokinins before and during Early Flower Bud Differentiation in Lychee (Litchi chinensis Sonn.)

Lychee (Litchi chinensis) has been analyzed for cytokinins in buds before and after flower bud differentiation, using reversephase high performance liquid chromatography in combination with Amaranthus bioassay and gas chromatography-mass spectrometry-selected ion monitoring. Four cytokinins, zeatin, zeatin riboside, N⁶-(δ²-isopentenyl)adenine, and N⁶-(δ⁶-isopentenyl) adenine riboside, were detected in buds. There was an increase of cytokinin activity in the buds during flower bud differentiation. In dormant buds, the endogenous cytokinin content was low, and the buds did not respond to exogenous cytokinin application. Application of kinetin promotes flower bud differentiation significantly after bud dormancy. These results are interpreted as an indication that the increase in endogenous cytokinin levels during flower bud differentiation may be correlative rather than the cause of flower bud initiation.     

Shoot regeneration from in vitro-derived leaf and root explants of <Emphasis Type="Italic">Centaurea ultreiae</Emphasis>

In vitro culture is currently used to produce plant material for ex situ conservation of endangered species. In this study, an efficient protocol for shoot regeneration from leaves and roots was developed for Centaurea ultreiae, a critically endangered species. Organogenesis from leaf and root explants was promoted by incubating these explants on half-strength Murashige and Skoog (MS) medium in the presence of one of four different cytokinins [6-benzyladenine (BA), zeatin, kinetin or N⁶-(2-isopentenyl) adenine (2iP)], each provided at five different levels. Shoot organogenesis was induced in both explants. The best response, 90% of leaf explants producing a mean of 2.48 shoots per explants and 94.3% of root explants producing a mean of 5.60 viable shoots per explants, was observed when explants were incubated on a medium containing 0.55 μM BA. Histological studies revealed connectivity between vascular tissues of regenerated shoots and cambial cells of leaf explants. Moreover, adventitious shoots were derived from pericycle cells of root explants and parenchymatic cells of callus tissues.     

Light cytokinin interactions in shoot formation in epicotyl cuttings of Troyer citrange cultured in vitro

Ilumination did not affect the pathway of shoot regeneration at the cut edges of epicotyl explants of Troyer citrange (Moreira-Dias et al. 2000, 2001), but signigficantly affected the number of developed shoots and the response to exogenous cytokinins. Shoot regeneration at the apical end occurred through a direct organogenic pathway without callus formation. For explants incubated in the light, this regeneration did not require cytokinin addendum, but the number of shoots formed was significantly increased by benzyl adenine, but not by zeatin or kinetin. Incubation in the dark almost suppressed shoot formation at the apical end. The addition of benzyl adenine or kinetin, but not of zeatin, restored shoot formation in the dark to the value obtained in the light. At the basal end of the explants shoot regeneration occurred through an indirect organogenic pathway after the formation of a primary callus. In explants incubated in the light, callus formation and shoot growth was supported by a low (0.5–1 mg l⁻¹) benzyl adenine concentration and by zeatin. Kinetin did not support callus growth. Shoot formation was higher in the presence of benzyl adenine (0.5–1 mg l⁻¹) than of zeatin, but was inhibited by a high (5 mg l⁻¹) benzyl adenine concentration. Incubation in the dark increased callus growth and shoot formation at the basal cut as compared to explants incubated in the light. The three cytokinins tested supported callus growth and shoot formation in the dark, zeatin being the most effective and kinetin the least. In terms of number of shoots developed, the optimum cytokinin addendum depended on the pathway of organogenesis and the conditions of incubation. The maximum number of shoots developed at the apical end was obtained when the incubation was performed in the light in the presence of benzyl adenine. At the basal end, the optimal conditions were incubation in the dark in the presence of zeatin. It was not always possible to define an optimal cytokinin concentration as the curve concentration/response varied from experiment to experiment, which seemed unrelated to the endogenous cytokinin concentration in the explants.     

Growth and Endogenous Cytokinins in Tobacco Callus as Affected by N-(2-chloro-4-pyridyl)-N′-phenylurea

The effect of N-(2-chloro-4-pyridyl)-N-phenylurea (4PU-30) on the growth and content of endogenous cytokinins of adenine type in tobacco (Nicotiana tabacum L.) callus was investigated. Biomass accumulation in calli grown on Murashige and Skoog (MS) medium with 4PU-30 was higher than that on MS medium with kinetin. The obvious presence of isopentenyladenine type cytokinins and traces of zeatin type cytokinins supposes modification in the endogenous cytokinin metabolism of the tobacco callus grown on 4PU-30.     

Studies on α-Alkyl-α-amino Acids

Effects of cytokinins were studied on rotenone-sensitive NADH dehydrogenase in mitochondria from fresh potato tubers (Solarium tuberosum), in consideration of the operation of external and rotenone-insensitive internal NADH dehydrogenases that has not been fully accounted for in previous studies. In submitochondrial particles (smp), zeatin was only weakly active, and zeatin riboside (ZR) was inactive. Inhibition rates at 400 μM of isopentenyladenine (iP) and isopentenyladenosine (iPA) were 45% and 30%, respectively, and that of BA (BA) was 64%. In intact mitochondria, the inhibition by iP and BA significantly increased, I₅₀ being 50 and 250 μM, respectively, but that by zeatin and iPA decreased. A structure–activity study showed that hydrophobic and steric factors are important for the activity. Cytokinins inhibited the electron flow via natural quinone more strongly than that via synthetic quinone. These results suggest that among the cytokinins the species that can regulate the electron transport is iP rather than its riboside or zeatin.     

Cultivars of Hexaploid Wheat of Contrasting Stature and Chlorophyll Retention Differ in Cytokinin Content and Responsiveness

The work reported here compared cytokinin content and sensitivityin a selection of hexaploid wheat (Triticum aestivum L.) cultivarsusing the following measurements: leaf cytokinins at three timepoints during light-growth and at four 24 h intervals afterlight-grown plants were transferred to darkness; sensitivityof root growth to direct applications of isopentenyl adenosine([9R]iP); and, sensitivity of germination and subsequent rootand shoot growth to 18 h imbibition of seeds in benzyladenine(BA). Accumulation of zeatin riboside-type cultivars was greatestduring light-growth in Tibet Dwarf, a wheat with an extremedwarf phenotype, intermediate in Omar standard and dwarf cultivars,and lowest in the standard and dwarf versions of Itana. Cytokininlevels were otherwise not directly correlated to plant staturein these wheats. There were no cultivar-associated qualitativedifferences in the types of cytokinins detected in this study.During the 16 h light period, the content of zeatin riboside-typecytokinins increased up to tenfold and then declined to basallevels during dark growth. Chlorophyll retention during dark-growthwas correlated with leaf cytokinin content. Data collected ata restricted number of sampling points during dark-growth suggesteda cyclic accumulation of [9R]iP-type cytokinins and the apparentcycle in Tibet Dwarf was offset by 24 h. Tibet Dwarf showedthe greatest root growth inhibition after exposure of seedlingroots to [9R]iP or imbibition of seeds in BA. Neither of thesetreatments affected shoot growth in any of the cultivars. Wheat; Triticum aestivum ; cytokinin; zeatin riboside; benzyladenine; root inhibition     

Endogenous hormonal content and somatic embryogenic capacity of Corylus avellana L. cotyledons

Endogenous indole-3-acetic acid (IAA), abscisic acid (ABA) and cytokinins [zeatin (Z) zeatin riboside, dihydrozeatin, dihydrozeatin riboside, N⁶-isopentenyl adenine (iP) and N⁶-isopentenyladenine riboside] were evaluated in hazelnut (Corylus avellana L.) cotyledons of different developmental stage and genetic source for their somatic embryogenic capacity. There was an inverse correlation between the embryogenic potential of cotyledons and the degree of maturity of zygotic embryos, the first characteristic being associated with iP-type cytokinins and the second with Z-type cytokinins. Although the differences in total cytokinin, ABA and IAA contents between the cotyledons were small, the IAA/ABA and, mainly, the iP-type/Z-type cytokinin ratios were found to be two good indexes of the embryogenic competence of explants, suggesting that the endogenous hormonal balance is a very important factor defining the in vitro potential of hazelnut cotyledons. Received: 6 January 1997 / Revision received: 3 March 1997 / Accepted 1 April 1997     

Effects of Cytokinins on In Vitro Seed Germination and Early Seedling Morphogenesis in Lotus corniculatus L.

We determined the effects of zeatin (ZEA), isopentenyl adenine (2iP), kinetin (KIN), benzyladenine (BA), and thidiazuron (TDZ) on seed germination, elongation of seedling shoots and roots, frequency of regeneration, and the number of regenerants per seedling in Lotus corniculatus L. Sterilized seeds were cultured in vitro on Murashige and Skoog (1962) medium containing 3% sucrose, 0.7% agar, and various cytokinins (0, 0.08, 0.22, 0.35, 0.80, 2.20, and 3.50 μM). After 30 days, seedlings were transferred to cytokinin-free medium for another 60 days. All cytokinins stimulated the rate and percentage of seed germination at least twofold in optimum concentrations; TDZ and ZEA were the most active, followed closely by BA, whereas KIN and 2iP stimulated germination in higher concentrations only. Elongation of shoots and roots was strongly inhibited at the lowest TDZ and BA concentrations, whereas ZEA, KIN, and 2iP exerted moderate, dose-dependent inhibition. The frequency of regenerant-producing seeds was highest on ZEA and BA, whereas the greatest number of regenerants per seedling was found on TDZ. It is concluded that the culture of seeds on cytokinin-containing media, followed by transfer to cytokinin-free medium, is a suitable procedure for rapid production of a large number of uniform regenerants. The presumed role of particular cytokinins is discussed.     

Cytokinins of the Developing Mango Fruit : Isolation, Identification, and Changes in Levels during Maturation

The cytokinin activity has been isolated and identified from extracts of immature mango (Mangifera indica L.) seeds. The structures of zeatin, zeatin riboside, and N⁶-(Δ²-isopentenyl)adenine riboside were confirmed on the basis of their chromatographic behavior and mass spectra of trimethylsilyl derivatives. Both trans and cis isomers of zeatin and zeatin riboside were also identified by the retention times of high performance liquid chromatography. In addition, an unidentified compound appeared to be a cytokinin glucoside.

The concentration of cytokinins in the panicle and pulp of mango reached a maximum 5 to 10 days after full bloom and decreased rapidly thereafter. The cytokinin level in the seed remained high until the 28th day after full bloom. The quantity of cytokinins in pulp per fruit increased from the 10th day after full bloom, the maximum being attained around the 50th day after full bloom. Similarly, the amount of cytokinins per seed increased from the 10th day after full bloom, reaching a peak on the 40th day and decreasing gradually thereafter.

A high percentage of fruit set in mango was persistently maintained by supplying 6-benzylaminopurine (1.5 × 10³ micromolar) onto the panicle at the anthesis stage and by supplying gibberellic acid (7.2 × 10² micromolar) and naphthalene acetamide (3.1 × 10 micromolar) at the young fruit stage.

     

Involvement of cytokinins in the germination of chick-pea seeds

Eight cytokinins detected in germinated chick-pea (Cicer arietinum L. var. Castellana) seeds were first present in the embryonic axes but appeared in the cotyledons after 12h of germination. The cytokinins detected in the cotyledons originate in the embryonic axes, but no passage of these substances from the cotyledons to the axes was detected, except when the seeds were treated with red light.It is concluded that the role played by the embryonic axis in mobilizating the main reserves of the cotyledons is mainly effected through these cytokinins. Both natural and synthetic cytokinins exert an important regulatory role in the hydrolysis of reserve proteins and calcium could be involved as an intermediate.Abbreviations BA benzyladenine - cot. cotyledon - (diH)Z dihydrozeatin - (diH)ZR dihydrozeatin riboside - GZR glycosyl zeatin riboside - 2iP 277-1 - iPA 277-2 riboside - Kin kinetin - Z zeatin - ZG zeatin glucoside - ZR zeatin riboside     

The effect of auxin on cytokinin levels and metabolism in transgenic tobacco tissue expressing an ipt gene

The ipt gene from the T-DNA of Agrobacterium tumefaciens was transferred to tobacco (Nicotiana tabacum L.) in order to study the control which auxin appears to exert over levels of cytokinin generated by expression of this gene. The transgenic tissues contained elevated levels of cytokinins, exhibited cytokinin and auxin autonomy and grew as shooty calli on hormone-free media. Addition of 1-naphthylacetic acid to this culture medium reduced the total level of cytokinins by 84% while 6-benzylaminopurine elevated the cytokinin level when added to media containing auxin. The cytokinins in the transgenic tissue were labelled with ³H and auxin was found to promote conversion of zeatin-type cytokinins to ³H-labelled adenine derivatives. When the very rapid metabolism of exogenous [³H]zeatin riboside was suppressed by a phenylurea derivative, a noncompetitive inhibitor of cytokinin oxidase, auxin promoted metabolism to adenine-type compounds. Since these results indicated that auxin promoted cytokinin oxidase activity in the transformed tissue, this enzyme was purified from the tobacco tissue cultures. Auxin did not increase the level of the enzyme per unit tissue protein, but did enhance the activity of the enzyme in vitro and promoted the activity of both glycosylated and non-glycosylated forms. This enhancement could contribute to the decrease in cytokinin level induced by auxin. Studies of cytokinin biosynthesis in the transgenic tissues indicated that trans-hydroxylation of isopentenyladenine-type cytokinins to yield zeatin-type cytokinins occurred principally at the nucleotide level.Abbreviations Ade adenine - Ados adenosine - BA 6-benzylaminopurine - C control - Con A concanavallin A - CP cellulose phosphate - IPT isopentenyl transferase - NAA 1-naphthylacetic acid - NP normal phase - NPPU N-(3-nitrophenyl)-N-phenylurea - RIA radioimmunoassay - RP reversed phase We wish to thank Dr. J. Zwar for supplying phenylurea derivitives.