A Comparative Auxin and Cytokinin Study in Normal and to-2 Mutant Tomato Plants

Torosa-2 (to-2), a tomato mutant with strong apical dominance,was studied in order to determine the mechanism of shoot outgrowthcontrol. In decapitated or defoliated to-2 plants only a fewshoots grew and IAA or morphactin application had little oronly short term effects. No differences were found in auxinand cytokinin activities between normal and mutant plants upto 20 days after sowing. In the period from 40 to 90 days theIAA content increased equally in both genotypes. During thesame period, however, cytokinin increased only in normal plants.The results obtained with the to-2 mutant indicate that budsdo not shoot, probably because bud differentiation does notoccur. The ratio of auxin to cytokinin clearly was affectedby the low level of cytokinin in all tissues of the to-2 plants.This led us to the conclusion that insufficient quantities ofcytokinin for lateral bud differentiation is the cause of thestrong apical dominance in to-2. (Received January 20, 1982; Accepted April 26, 1982)     

Apical Dominanace in Xanthium strumarium: A DISCUSSION IN RELATION TO CURRENT HYPOTHESES OF CORRELATIVE INHIBITION

The influence of the spectral distribution of illumination onthe gibberellin, cytokinin, auxin, and abscisic acid levelsand the correlation with the degree of branching in Xanthiumstrumarium is presented and discussed. Gibberellins do not appearto play a major role in apical dominance but may be importantfor bud extension following the initial release from dominance.The cytokinin level was much higher in inhibited buds than inreleased buds. It is suggested that the cytokinins present wereprobably not able to participate in bud growth because of anauxin-induced accumulation of abscisic acid in the buds themselves.The concentration of abscisic acid as measured by bioassay andgas-liquid chromatography was between 50 and 250 times thatoccurring in all other plants parts examined. This level felldramatically following release from apical dominance by decapitation.The results are discussed in relation to current hypothesesof apical dominance.     

The Effect of Applications of a Synthetic Cytokinin on Shoot Dominance and Grain Yield in Spring Barley

Spring barley cv Ark Royal was grown in pots in the open, andsoil drenches containing 6-benzyl-aminopurine were repeatedlyapplied during the pre-heading phase starting either ‘early’when the main shoot apex had reached the glume primordium stage,during tillering, or ‘later’ at the anther primordiumstage, at the start of stem elongation Although grain yield was unaffected by the ‘late’cytokinin treatments, it was increased by up to 57 per centby the ‘early’ treatments. The increase was dueto greater grain yields of the smaller shoots of each plantthere was no effect on the yields of the two largest shootsor on the total number of fertile shoots The increased grain yield of these smaller shoots was attributableto increased weights of their individual kernels, especiallyin the basal and distal parts of the ears such that a greateruniformity of kernel size within the ear was achieved Thus the cytokinin treatment increased uniformity both betweenshoots and between spikelets within the ears Hordeum vulgare L, spring barley, cytokinin, 6-benzylaminopunne, shoot dominance, harvest index, grain yield     

Cytokinin/Auxin Control of Apical Dominance in Ipomoea nil

Although the concept of apical dominance control by the ratioof cytokinin to auxin is not new, recent experimentation withtransgenic plants has given this concept renewed attention.In the present study, it has been demonstrated that cytokinintreatments can partially reverse the inhibitory effect of auxinon lateral bud outgrowth in intact shoots of Ipomoea nil. Althoughless conclusive, this also appeared to occur in buds of isolatednodes. Auxin inhibited lateral bud outgrowth when applied eitherto the top of the stump of the decapitated shoot or directlyto the bud itself. However, the fact that cytokinin promotiveeffects on bud outgrowth are known to occur when cytokinin isapplied directly to the bud suggests different transport tissuesand/or sites of action for the two hormones. Cytokinin antagonistswere shown in some experiments to have a synergistic effectwith benzyladenine on the promotion of bud outgrowth. If theratio of cytokinin to auxin does control apical dominance, thenthe next critical question is how do these hormones interactin this correlative process? The hypothesis that shoot-derivedauxin inhibits lateral bud outgrowth indirectly by depletingcytokinin content in the shoots via inhibition of its productionin the roots was not supported in the present study which demonstratedthat the repressibility of lateral bud outgrowth by auxin treatmentsat various positions on the shoot was not correlated with proximityto the roots but rather with proximity to the buds. Resultsalso suggested that auxin in subtending mature leaves as wellas that in the shoot apex and adjacent small leaves may contributeto the apical dominance of a shoot. (Received September 24, 1996; Accepted March 16, 1997)     

Types, Formation, and Metabolism of Cytokinins in Leaves of Alnus Glutinosa (L.) Gaertn.

The nature of the cytokinins present in mature, expanded leavesof alder (Alnus glutinosa (L.) Gaertn.) was investigated usinga chromatographic system capable of partially resolving zeatinfrom (?)-dihydrozeatin. A zeatin-like cytokinin, present asboth a ‘free’ form and as a polar conjugate, postulatedto the the O-ß-D-glucoside of zeatin, accounted forthe bulk of the cytokinin activity (detected by the soybeancallus assay). Studies on the fate of [8-¹⁴C]zeatin supplied to detached alderleaves indicated this cytokinin to undergo extensive metabolism.An appreciable proportion of the radioactivity was incorporatedinto 80% methanol-insoluble and soluble acidic/neutral fractions,while adenosine- and adenine-like peaks were prominent metabolitesin the basic n-butanol-soluble fraction. Small amounts of glucosides,with the properties of both zeatin-O-ß-D-glucosideand dihydrozeatin-O-ß-D-glucoside were formed, thelatter becoming the most prominent with time. The ability of alder leaves to form glucoside directly from(?)-dihydrozeatin was assessed using the soybean callus assay.(?)-Dihydrozeatin was subject to a relatively rapid, continuousand substantial conversion to its putative O-ß-D-glucosideand cytokinin activity was consequently conserved while, incontrast, leaves supplied with zeatin exhibited a progressiveloss of cytokinin activity and, in agreement with the radioactivityexperiments, produced only a small amount of activity attributableto the putative O-glucosides. The significance of the observed cytokinin metabolism is discussedin relation to the endogenous cytokinin status of the leaf.     

Effect of Zinc Sulfate and Boric Acid on the Hormonal Status of Potato Plants in Relation to Tuberization

Potato (Solanum tuberosum L.) plants were grown in a greenhouse using zinc- and boron-deficient soil. The effects of seed-tuber treatment with 3 mM zinc sulfate and 8 mM boric acid on the content and ratio of phytohormones in the leaves and mature tubers, the indices of photosynthetic activity, the rate and NaF-sensitivity of respiration, and the tuber growth were studied. Zinc-sulfate treatment shifted the hormonal balance toward a substantial increase in the cytokinin content and the cytokinin/ABA ratio, as well as a decrease in the IAA/cytokinin ratio. Boric-acid treatment resulted in an increase in the IAA content and IAA/cytokinin ratio. Zinc-sulfate treatment abolished the apical dominance and increased the tuber weight due to their increased number and the number of phellem (cork) cell layers. Boric-acid treatment increased cell diameter in the tuber perimedullary zone; an increase in tuber weight per plant was related to tuber growth. A relationship between changes in the plant hormonal status induced by zinc-sulfate and boric-acid treatments and the activity of physiological processes is discussed.     

Role of Cytokinin in Vessel Regeneration in Wounded Coleus Internodes

Cytokinin was found to be a controlling or limiting factor invessel regeneration around a wound in internodes of Coleus blumeiBenth. in which the endogenous cytokinin level was minimized.The cytokinin was applied in aqueous solution to the base ofexcised, mature internodes that had an active vascular cambium.Each internode also received IAA in lanolin at its apical end.Under low (0.1 %, w/w) or high (10%, w/w) auxin concentrations,the control internodes (without exogenous cytokinin) exhibitedsmall amounts of vessel regeneration. At appropriate concentrationszeatin, kinetin and 6-benzylamino-purine (BAP) induced a significantincrease in vessel regeneration around the wound. The threecytokinins also induced novel patterns of supplementary regenerationfurther from the wound surface. Kinetin and BAP showed the strongestpromoting effect at 5 and 10 µg ml^–1, while zeatinwas most effective at 20 µg ml^–1. At a low (0.1%) auxin level zeatin was the most effective cytokinin, whereaskinetin was the most effective one at high (1 %) auxin. An inhibitoryeffect on vessel regeneration was observed at the highest kinetinconcentration tested (50 µg ml^–1). The regenerationof vessels induced by cytokinin was very polar. Many more regeneratedvessel members differentiated below the wound than above it,and the regeneration process proceeded acropetally from thebase of the internode to its upper parts. Our results implya basipetal polar increase in cambium responsiveness along thestem axis from internode 5 to 7. The possible significance ofsuch a basipetal increase in cambium sensitivity in wood formationin trees is discussed. Auxin, Coleus blumei, cytokinin, vascular differentiation, vessel regeneration, wound xylem     

The Pattern of Reproductive Development in Chenopodium rubrum L.

Individual plants of Chenopodium rubrum were given differentnumbers of inductive cycles in a 12 h photoperiod and the patternof reproductive development was analysed after 40 d of growth.At least 2 inductive cycles are required to form any determinatereproductive organs and at least 12 cycles are required fornormal reproductive development. Individuals given a singleinductive cycle display a loss of apical dominance at thosenodes formed immediately after the treatment without the subsequentformation of any floral structures. Plants given between 2 and12 mductive cycles display both determinate reproductive organsand indeter minate vegetative shoots. The pattern of reproductivedevelopment on such plants depends upon the number of cyclesrelative to the developmental age of newly initiated primordia.It is suggested that the early events of floral induction mayinvolve a radical decrease in the ratio of auxin to cytokinin.     

Effects of Storage Temperature and Wounding on Cytokinin Levels in Potato Tubers

The level of butanol-soluble cytokinin in potato tubers (Solanumtuberosum L.) decreased greatly after harvest of the tubers,remained low during rest-period and then began to increase priorto the end of the period. In warm-stored tubers (25?C) the levelrose faster than in cold-stored ones (4?C). On the other hand,the level of water-soluble cytokinin, which was high duringthe rest-period, decreased after the rest was broken. Wounding the tubers caused an increase in butanol-soluble cytokinin.The increase in cytokinin within the first 24 hr after woundingwas smaller in warm-stored tubers than in cold-stored ones andwas decreased with increasing storage time of the tubers. Thelevel of water-soluble cytokinin decreased by wounding. Theseresults suggest that water-soluble cytokinin is a storage formand that the level of butanol-soluble cytokinin is regulatedby an interconversion between the two. (Received February 6, 1982; Accepted May 13, 1982)     

Exposure of Petunia Seedlings to Ethylene Decreased Apical Dominance by Reducing the Ratio of Auxin to Cytokinin

Seedlings of Petunia x hybrida ‘Orchid’ treated with the ethylene-releasing compound ethephon at 0.9, 1.7, and 3.5 mM evolved ethylene at a higher rate as the concentration of ethephon increased. Regardless of the concentration of ethephon applied, ethylene evolution peaked 6 to 8 h following application. Evidence that ethephon application decreased apical dominance included an increase in the number of new nodes on the main stem and a sustained increase in the length of new and existing lateral shoots compared to the control (no ethephon). Plants treated with 3.5 mM ethephon developed mild chlorosis, whereas a concentration of 1.7 mM ethephon decreased apical dominance without phytotoxic effects. The auxin/cytokinin ratio decreased in the apical shoot section as early as 1 h after ethephon treatment. In contrast, a decrease in the ratio in the subapical shoot section was not detected until 24 h after ethephon application. Reduction in auxin/cytokinin ratio was a result of a decrease in indole-3-acetic acid (IAA) and an increase of zeatin riboside (ZR), but not isopentenyladenosine (iPA). These results suggest that exposing ‘Orchid’ petunia seedlings to ethylene via ethephon lowers the auxin/cytokinin ratio, thereby promoting the outgrowth of lateral shoots.     

Involvement of Calcium in Adventitious Bud Initiation in Torenia Stem Segments

In Torenia stem segments cultured in vitro, active meristematicdivisions are induced in the epidermis by treatment with cytokinin,resulting in the formation of adventitious buds. Applicationof the calcium ionophore A23187 [GenBank] was found to induce meristematicdivisions in the absence of cytokinin. The induction by A23187 [GenBank] was inhibited by simultaneous addition of auxin, but not byanti-cytokinin. A two hour pre-treatment with A23187 [GenBank] was alsoeffective, but only when it was applied to the explants justafter their excision from mother plants. The A23187 [GenBank] -inducedmeristematic zones developed into dome-shaped structures, butnot into complete adventitious buds. Complete elimination ofcalcium from the culture medium caused 50% inhibition of A23187 [GenBank] -and/or cytokinin-induced initiation of meristematic divisions.When the explants were preincubated with EGTA and then culturedon a Ca-free medium containing EGTA, cytokinin failed to inducebud initiation. Similar inhibition was also obtained by lanthanum,a calcium antagonist, by verapamil, a calcium channel inhibitor,and by trifluoperazine and chlorpromazine, calmodulin inhibitors.These results support the idea that adventitious bud initiationinduced by cytokinin in Torenia stem segments may be mediated,at least partially, by an increase in the level of intracellularCa²⁺. ¹Bioscience Research Center, Mitsui Petrochemical IndustriesLtd., Waki-cho, Kuga-gun, Yamaguchi 740, Japan. (Received May 9, 1985; Accepted October 5, 1985)     

The Effect of Cytokinins on Vitrification in Melon and Carnation

The influence of exogenous cytokinin on vitrification of melonand carnation buds grown in vitro was assessed. In melon, theanatomical traits of vitrification were found to be similarto those previously described for carnation grown on a highconcentration of auxin. In carnation, the plantlets treatedwith high cytokinin concentrations became bushy but the anatomicalstructure remained normal. The relation of cytokinin and vitrificationis discussed. Cucumis melo L., Dianthus caryophyllus L., vitrified plantlets, habituation, cytokinin     

Endogenous Cytokinins in Flower Bud Forming Explants of Tobacco

The accumulation of endogenous cytokinins was studied in pedicelexplants of tobacco (Nicotiana tabacumL.) during regenerationof flower buds in vitro. Maximal bud formation was induced onmedia containing 1.0 mmol m^–3 of benzyladenine or dihydrozeatin.No buds were formed in the absence of cytokinin. The levelsof dihydrozeatin, zeatin, and the corresponding ribosides weredetermined in explants cultured in the presence or absence ofcytokinin by means of a competitive ELISA technique. In explantsincubated without a cytokinin, only the dihydrozeatin concentrationincreased significantly during the first day of incubation anddecreased during the second day. No increase was observed inexplants incubated in the presence of benzyladenine. The concentrationof dihydrozeatin in these bud-forming explants was only 10 to15% of the concentration built up in explants cultured on dihydrozeatininstead of benzyladenine. This suggests that the endogenouscytokinins only play a minor role in the regeneration of flowerbuds in vitro. Key words: cytokinin, flower bud development, tissue culture, tobacco     

Involvement of tRNA Bound Cytokinin on the Gall Formation in Zizania

To understand the role of the conjugated forms of cytokininin gall formation, tRNA was extracted and purified from vegetativeand flowering shoots of Zizania, then hydrolysed by RNase T,or acid to release the conjugated forms of cytokinin. The resultsindicated that the level of cw-zeatin riboside fromZizania galltissue was 3 to 5 times higher than that of flowering Zizania,and the cytokinin activity in terms of kinetin equivalence was1.6 times higher in Zizania gall tissue than that of floweringZizania. It was found that higher amounts of cytokinin alsopromote protein synthesis. Due to the difference in levels ofthe conjugated forms of cytokinin between vegetative and floweringZizaniashoots, it is implied that the conjugated form of cytokininmay play an important role during the process of gall formationin Zizania latifolia. Key words: Gall formation, cytokinin, Zizania latifolia, RNA, smut fungus, water oat     

Response of cytokinin concentration in the xylem exudate of bean (Phaseolus vulgaris L.) plants to decapitation and auxin treatment,and relationship to apical dominance

When xylem exudate of previously untreated Phaseolus vulgaris plants was analysed for cytokinins by radioimmunoassay, a low concentration (about 5 ng · ml^–1) was found. However, when the plants were decapitated about 16 h before the xylem exudate was collected, an almost 25-fold increase in cytokinin concentration was observed. Twenty-four hours after decapitation this increase even reached 4000 compared to control plants. Applying naphthaleneacetic acid (NAA) to the shoot of decapitated plants almost eliminated the effect of shoot tip removal on cytokinin concentration, suggesting that cytokinins in the xylem exudate of intact plants are under the control of the polar auxin transport system. Other xylem constituents, such as potassium or free amino acids did not show this strong increase after decapitation and did not respond to NAA application. It is concluded that the observed auxin/cytokinin interaction has an important regulatory role to play, not only in apical dominance but in many other correlative events as well.Abbreviations AD apical dominance - CKs cytokinin(s) - iAde/iAdo isopentenyladenine/iospentenyladenosine - NAA naphthaleneacetic acid - Z/ZR zeatin/zeatin riboside     

Effects of Wounding on Adventitious Bud Formation in Torenia Stem Segments Cultured In Vitro

In in vitro cultured stem segments of Torenia fournieri Lind.,the formation of adventitious buds can be induced when the culturemedium contains cytokinin. When long stem segments (2.0 cm ormore) were cultured with cytokinin, a large number of buds wereformed in the marginal regions, namely, within the limits of0.5 cm from the cut ends of explants, while only a few budswere initiated in the middle part of the explants. If a slightinjury was made transversely with a scalpel in the central partof an explant, a significant increase in the number of budswas noted within the limits of 0.5 cm from the wound site. Whena wounding treatment was given lengthwise to an explant, a largenumber of adventitious buds were formed over the entire surfaceof the explant compared to the control. Excision itself of explantsfrom mother plants and the additional wounding given to theexplants seemed to trigger the induction of adventitious buddifferentiation in Torenia stem segments. These wounding treatmentsdid not affect the uptake into explants and/or the distributionpattern of radioactive benzyladenine applied to the culturemedium. Key words: Torenia fournieri, Adventitious bud formation, Cytokinin, Wounding     

Relationship between the Growth Rate of a Filament and the Ability to Differentiate a Bud in the Moss, Physcomitrium sphaericum

Protonemata filaments of the moss Physcomitrium sphaericum displaygrowth rates that change remarkably before they differentiatebuds under normal conditions, that is, without cytokinin treatment.Before bud differentiation occurred, the growth rate of thefilament first decreased to a minimum and then increased toa maximum and decreased again. Study of whether the change inthe growth rate is related to bud differentiation showed thatbud differentiation was closely related to the maximum growthrate and the fluctuation range, that is, the difference betweenthe minimum and the succeeding maximum growth rate. The frequencyof bud-differentiated filaments increased as both the maximumgrowth rate and the fluctuation range of growth rate increased.Examination of the relationship between the time of cytokininapplication and the frequency of bud-differentiated filamentsshowed that the cytokinin treatment given at the time when thegrowth rate increased was most effective to induce buds. Therelationships between cytokinin, growth rate of filaments andthe ability to differentiate buds are discussed. (Received September 17, 1985; Accepted March 13, 1986)     

Influence of mycorrhizae and Rhizobium on cytokinin content in drought-stressed alfalfa

The objective of this research was to study the growth responseto drought of arbuscular mycorrhizal and non-mycorrhizal alfalfa(Medicago sativacv. Aragn) in relation to leaf cytokinin levels.In the experiment, four treatments were used: (a) plants inoculatedwith Clomus fasciculatum (Taxter sensu Gerd.) Gerdemann andTrappe and Rhizobium meliloti 102 F51 strain, (MR); (b) plantsinoculated with only Rhizobium (RP); (c) plants inoculated withonly mycorrhizae (MN); and (d) plants non-inoculated (NP). Non-mycorrhizalplants were supplemented with phosphorus and nonnodulated oneswith nitrogen to achieve similar size in all treatments. Plantswere subjected to drought by withholding irrigation in a cyclicway. The effects of drought on growth, number of stems, degreeof senescence, and leaf cytokinin levels were measured. Results of identification of cytokinins showed that dihydrozeatinriboside (dHZR) and ortno-topolin riboside (oTR) were predominantin alfalfa leaves. Nonsymbiotic plants (NP) showed higher totalcytokinin concentrations (dHZR and oTR). Under drought, NP plantsshowed the largest percentage drop in cytokinins and lower numberof stems as well as increased degree of senescent leaf tissuerelative to control values. By contrast, stressed symbioticplants (RP, MN and MR) showed higher green leaf weight thannonsymbiotic ones (NP) due to delay of leaf senescence and maintenance(RP) or increase (MN, MR) of stem leaf cytokinin levels duringdrought. The relationships between growth and the different cytokininsare discussed, suggesting an important role of mycorrhizal symbiosisin maintaining cytokinin levels under drought. Key words: Alfalfa, arbuscular mycorrhizae, cytokinins, drought, leaf senescence     

Endogenous Cytokinins in Developing Fruits of Seeded and Seedless Citrus Cultivars

This report describes the isolation and identification of endogenouscytokinins from Citrus ovaries. Cytokinin active fractions wereobtained by extraction with 80% (v/v) ethanol, followed by purificationwith hexane, n-butanol and a polyvinylpyrrolidone and SephadexLH-20 column chromatography. Five fractions with cytokinin activity were found in the organicphase, using the tobacco callus assay. The main active compoundsin these fractions were separated by HPLC, bioassayed and identifiedby GC—MS as ribosyl zeatin, zeatin and isopentenyl adenosine.Hydrolysis of the first fraction with B-glucosidase gave cytokininactive compounds that in paper chromatography had R_F's similarto those of zeatin and ribosyl zeatin. Treating the aqueousphase with alkaline phosphatase produced a cytokinin activecompound that in paper chromatography had the same R_F as isopentenyladenosine indicating that their ribotide was probably the majorphosphorylated cytokinin present in Citrus ovaries. Key words: Citrus, cytokinin fruit set and development     

Controlled Cytokinin Production in Transgenic Tobacco Using a Copper-Inducible Promoter

The cytokinin group of plant hormones regulates aspects of plant growth and development, including the release of lateral buds from apical dominance and the delay of senescence. In this work the native promoter of a cytokinin synthase gene (ipt) was removed and replaced with a Cu-controllable promoter. Tobacco (Nicotiana tabacum L. cv tabacum) transformed with this Cu-inducible ipt gene (Cu-ipt) was morphologically identical to controls under noninductive conditions in almost all lines produced. However, three lines grew in an altered state, which is indicative of cytokinin overproduction and was confirmed by a full cytokinin analysis of one of these lines. The in vitro treatment of morphologically normal Cu-ipt transformants with Cu²⁺ resulted in delayed leaf senescence and an increase in cytokinin concentration in the one line analyzed. In vivo, inductive conditions resulted in a significant release of lateral buds from apical dominance. The morphological changes seen during these experiments may reflect the spatial aspect of control exerted by this gene expression system, namely expression from the root tissue only. These results confirmed that endogenous cytokinin concentrations in tobacco transformants can be temporally and spatially controlled by the induction of ipt gene expression through the Cu-controllable gene-expression system.