Involvement of xylem sap zeatin-O-glucoside in cucumber shoot greening

A fraction of xylem sap collected from squash (Cucurbita maxima) root and separated with gel filtration showed strong promoting activity in a greening bioassay using etiolated cucumber cotyledons. This activity decreased with waterlogging of the root. The promoting factor was purified with sequential gel-permeation, and reverse-phase and normal-phase column chromatographies. Zeatin-O-glucoside (ZOG) was identified as the promoting factor by monitoring liquid chromatography tandem mass spectrometry. Although the glycosyl conjugate of zeatin was thought to be inactive, ZOG had a promoting activity that was 100 times higher than those of zeatin and zeatin riboside between concentrations of 10^–9 to 10^–4 M in the greening bioassay. These results suggest that in some developmental stages, the conjugation of cytokinins with a sugar moiety, such as glucose, might be a key factor in the control of shoot greening by roots.     

Comparative Effects of Abscisic Acid and Methyl Jasmonate in Greening Cucumber Cotyledons and Its Application to a Bioassay for Abscisic Acid

In the cucumber cotyledon greening system, abscisic acid (ABA)is more potent inhibitor of growth and chlorophyll productionand/or destruction than methyl jasmonate (MJ). The inhibitoryeffect of ABA is apparent within 5 h of exposure to light whereasMJ is ineffective at all concentrations tested (10^–6 to10^–3 M). With longer exposure of 24 h to light and inthe presence of 40 mM KC1, the inhibition of growth and chlorophyllproduction by ABA is more pronounced whereas MJ does not inhibitgrowth and inhibits chlorophyll levels only at the higher concentrations.Both benzyladenine and KC1 stimulate chlorophyll productionand increase the fresh weights of the cucumber cotyledons andeither one of these compounds reverse the inhibitory effectsof ABA. Inhibition of chlorophyll production by ABA is valuableas a simple and rapid bioassay for abscisic acid. Under similarconditions cytokinins increase chlorophyll production and hencethe cucumber cotyledon greening system is ideal for detectingboth ABA which inhibits and cytokinins which stimulate chlorophyllproduction. (Received December 6, 1982; Accepted June 9, 1983)     

An improved disk bioassay for determining activities of plant growth regulators

An improved filter paper disk bioassay for determining activities of plant growth regulators was developed and evaluated. Indole-3-acetic acid (IAA), (±)-abscisic acid (ABA), and 6-furfurylamino-purine (kinetin) were dissolved in 95% ethanol at fixed dilutions. Specific concentrations of each growth regulator were then evenly dropped onto individual 6-cm paper disks and the solvent evaporated. The activities of the above three water-insoluble plant growth regulators in both solution and the disk assay were compared using the excised cucumber (Cucumis sativus L.) cotyledon root formation bioassay (IAA), wheat (Triticum aestivum L.) coleoptile straight growth bioassay (ABA), and cucumber cotyledon expansion bioassay (kinetin), and similar results were obtained at each concentration. The possible principle of this method has been studied using the cucumber cotyledon expansion bioassay. The results suggested that the disks, as carriers, had highly dispersed kinetin molecules on them and greatly accelerated the dissolution and diffusion of kinetin from disks to water.     

An improvement of cucumber cotyledon greening bioassay for cytokinins

The cucumber cotyledon greening bioassay for cytokinins was modified by using 95% acetone-ethanol instead of 80% acetone as extraction solvent. The cotyledons were extracted directly with a 2:1 (v/v) acetone-ethanol solution in dark for 24 hours, omitting the homogenization and centrifugation operations of the previous bioassay. The modified bioassay is more convenient and especially useful in screening cytokinin-active substance from a large number of samples.     

The Effect of Potassium on Cotyledon Expansion Induced by Cytokinins

Potassium has been found to enhance greatly the expansion response of cucumber cotyledons to cytokinins. A reduction of the response to kinetin is obtained with increasing age of the cotyledons. The lesser response is associated with lower levels of potassium remaining in the cotyledon. A high level of KCI in the incubation medium offsets the lower potassium content of the tissue and enables a much larger response to the cytokinins. At 40 mM KCI the response to kinetin is 4.2 times greater than in the absence of KCI. Calcium increases the effect of potassium on the response to kinetin. When incubated in 40 mM KCI and 10 mM CaCI₂ with 10 mg/I 6-benzylamino-purine, the final weight of the cotyledons is 6.8 times the initial weight after just 4 days. This KCI-CaCI₂ combination is also found to promote chlorophyll synthesis in the usual cucumber cotyledon bioassay.     

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.     

An in vitro technique for the production de novo of multiple shoots in cotyledon explants of cucumber (Cucumis sativus L.)

A technique is described for the production de novo of cucumber (Cucumis sativus L.) shoots in the presence of cytokinin using cotyledon explants. The shoots, which arose from adventitious buds and not from enhanced axillary branching, are confined to a specific region at the base of the cotyledon. Concentrations (4 mgl^–1 or less) of the cytokinins 6-benzylaminopurine, kinetin and N⁶-(2-isopentenyl)adenine, are all effective in producing adventitious buds. It is possible to achieve a yield of 23 shoots per cotyledon by removal of the axillary bud. The yield is increased to 50 shoots per cotyledon by cutting the basal region of the cotyledon into small pieces prior to culturing. These techniques may be useful for transformation studies in cucumber.     

The Uptake of Cytokinins by Protoplasts and Root Sections of Brassica campestris

The effect of cytokinins was studied on the incorporation of ¹⁴C-labelled precursors into the nucleic acid fraction of protoplasts isolated from callus or roots of Brassica campestris. Protoplasts from callus and roots took up ¹⁴C-uridine from the incubation medium and incorporated this precursor into the ribonucleic acid fraction during the experimental period of 16 h. Low concentrations of kinetin (10^?8-5 × 10^?6M) did not stimulate the incorporation, and kinetin inhibited this process at higher concentrations (5 × 10^?5M). This result led to an investigation on the uptake of cytokinins by protoplasts of roots. In contrast to a rapid uptake of radio-actively labelled adenine and uridine. protoplasts from roots took up only small amounts of labelled kinetin. zeatin, zeatin riboside and zeatin nucleotides from the incubation medium. Root sections took up far more adenine and kinetin than protoplasts from roots. The ratio between the amount of kinetin taken up and applied was much higher for the sections than for protoplasts, indicating that intact root cells took up kinetin far more rapidly than protoplasts. It is suggested that the plasmalemma and cell wall play an essential role in the uptake of cytokinins or that the differences in the uptake rates are related to differences between the rates of metabolism of cytokinins in root sections and in protoplasts.     

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     

A simple bioassay for abscisic acid using cucumber hypocotyls

A simple bioassay based on the inhibition by abscisic acid (ABA) of cucumber (Cucumis sativus L., cv. National Pickling) hypocotyl elongation was developed. Sections of 3-day-old dark-grown cucumber hypocotyl taken from 0–5 mm immediately below the cotyledon were used for the assay. A dark incubation period of 20 h was followed by an exposure to light for 24 h. Under these conditions, the inhibition of hypocotyl elongation is proportional to the abscisic acid applied. The minimum detectable level of abscisic acid was 10^–9 M, and the range of linear response to abscisic acid was between 10^–7 and 10^–3 M. This assay is 10 times more sensitive than the cucumber cotyledon greening bioassay for abscisic acid.     

Effects of gibberellic acid and zeatin on the growth response of cucumber cotyledons

In contrast to cytokinin, gibberellic acid has no effect on the growth of the isolated cucumber cotyledon in darkness. Like cytokinins in light, gibberellic acid causes increases in fresh weight and area of the cotyledon at concentrations from 10^–7 to 10^–3 M. Radiant energies in the blue, red, and far-red regions of the spectrum all induce the growth responses to gibberellic acid. The effect of the far red is greater than that of the red, which is greater than that of the blue. Gibberellic acid is ineffective in the promotion of chlorophyll development, whereas cytokinins are very effective. Although zeatin and gibberellic acid both cause an increase in fresh weight and area of the cotyledons in light, they appear to have entirely separate actions in the growth responses.     

A comparative study of different cytokinins on the formation of Rhododendron forrestii Balf. f. ex diels. axillary shoots in vitro

The effect of different concentrations and activities of cytokinins on the morphogenesis of regenerated Rhododendron forrestii Balf. f. ex Diels. shoots taken from nodal segments were tested. We evaluated zeatin, zeatin riboside, izopentyladenine, izopentyladenine riboside, kinetin, kinetin riboside, benzylaminopurine, benzylaminopurine riboside. The experimental results were evaluated by mathematical methods and regression analysis describing the effect of isoprenic and aromatic type of cytokinins. On the basis of this modelling, maximum axillary shoot production was attained with medium supplemented with 2.0 mg·l⁻¹ izopentyladenine riboside, 2.0 mg·l⁻¹ benzylaminopurine and 20 g·l⁻¹ sucrose. Minimal axillary shoots were produced with kinetin and kinetin riboside.     

Cytokinin stimulation of abscission in lemon pistil explants

The stylar abscission bioassay was used to identify five stimulators of lemon (Citrus limon cv. Lisbon) abscission in pistil explants. The stimulators (N-phenyl-N′-1,2,3-thiadiazol-5-ylurea, N6-benzyladenine, kinetin, zeatin, and N6-isopentenyladenine), which are all cytokinins, accelerated the timing of expiant abscission when they were added as supplements (100 μM) to the test medium. To study possible relationships between cytokinins, ethylene, and abscission, we measured accumulating ethylene concentrations in sealed cultures and endogenous 1 -aminocyclopropane-1-carboxylic aicd (ACC) in explants incubated on test medium plus or minus 100 μM N-phenyl-N′-1,2,3-thiadiazol-5-ylurea (thidiazuron), 100 μM N6-benzyladenine (bzl⁶Ade), or 2 μM picloram, an inhibitor of stylar abscission. Although ethylene accumulated to similar levels in all treatments, the concentrations obtained with picloram and thidiazuron were, respectively, higher and lower than those obtained in control cultures. The accumulation of ethylene in cultures with bzl⁶Ade, on the other hand, was not significantly different from controls. ACC concentrations in explants remained fairly constant in all treatments during the incubations, except in explants on thidiazuron, in which case the ACC concentration declined slightly. We conclude that cytokinins can stimulateCitrus abscissionin vitro and that this stimulation is not accompanied by marked effects on either measurable ethylene or ACC concentrations. Our finding that 100 μM aminoethoxyvinylglycine, an ethylene biosynthesis inhibitor, counteracts the stimulation of abscission by bzl⁶Ade suggests that a minimum level of ethylene production is required for the cytokinin effect. The possibility that cytokinins affect other aspects related to ethylene, such as biosynthetic rates, metabolism, or tissue retention, is not excluded by our results.     

Genotypic and media effects on plant regeneration from cotyledon explant cultures of someBrassica species

Conditions were established for efficient plant regeneration from cotyledon explant calli in different cultivars ofBrassica juncea, B. campestris andB. carinata on Murashige & Skoog's (MS) medium supplemented with various combinations of cytokinins and auxins. Regeneration frequency, however, varied with genotype and the different growth hormone combinations in media. Almost in all species, MS medium with zeatin (1.0 mg 1^-1) and IAA (0.1 mg l^-1) was found to be best for shoot organogenesis followed by the ones containing high kinetin (2.0 mg l^-1) and low IAA (0.02 or 0.2 mg l^-1) concentrations. On these media, the cotyledonary explants invariably underwent callusing followed by multiple shoot formation, which could be separated and subcultured for further propagation. Number of shoots per cotyledon explant cultured varied from 0 to as many as 50. InB. juncea andB. campestris, the regeneration frequency declined sharply in the absence of auxin in medium. BAP in combination with NAA yielded no or a reduced number of shoots. Shoot organogenesis also declined with the reduction in photoperiod from continuous light to 16 hours. Shoots were easily rooted during prolonged incubation on the same medium and whole plants were transferred to pots in the greenhouse and grown to maturity.Abbreviations BAP 6-benzylaminopurine - KIN kinetin - IAA indole-3-acetic acid - MS medium after Murashige & Skoog [8] - NAA -napthaleneacetic acid - ZEA Zeatin     

Effects of cytokinins on the respiration of soybean callus tissue

A technique which incorporates a brief blending step to disperse callus tissue into small clumps of cells was developed, and the effects of cytokinins on respiration of soybean (Glycine max [L.] Merrill var. Acme) callus tissue prepared in this way were studied. Adenine alone did not affect respiration, but kinetin and zeatin showed effects correlating with their reported effects on growth of this tissue; after about 3 hours both hormones promoted respiration at concentrations which promote growth, while kinetin, but not zeatin, also exhibited inhibition at higher concentrations. Studies with 2,4-dinitrophenol led to the suggestion that although the respiration of this tissue is largely under the control of ATP levels, kinetin does not exert its control on respiration through effects on ATP levels or oxidative phosphorylation during the monitoring period. Further inhibitor and substrate studies provided evidence that the promotion of respiration by kinetin results from an increase in substrate entering the tricarboxylic acid cycle, perhaps by an effect on pyruvate metabolism.     

Dimethyl sulfoxide: A solvent for cytokinins in theAmaranthus bioassay

Dimethyl sulfoxide present in the agar medium at concentration 0.2 % (v/v) and lower does not inhibit cytokinin-induced betacyanin synthesis in theAmaranthus caudatus seedlings. The activity of kinetin, N⁶-(Δ²-isopentenyl)adenine andtrans- zeatin is the same when these cytokinins are dissolved in either water or dimethyl sulfoxide and incorporated into the medium after autoclaving. A simple method is described which allows the cytokinin activity of slightly water-soluble and thermolabile compounds,e.g. aromatic urea and thiourea derivatives, to be determined in theAmaranthus bioassay.     

The effect of selected plant hormones on in vitro proliferation of hyphae of Glomus fistulosum

Effects of N⁶-benzyladenine, kinetin, zeatin, N⁶-benzyladenosine, kinetin riboside, zeatin riboside, jasmonic acid, indole-3-acetic acid, indole-3-butyric acid, indole-3-propionic acid, abscisic acid and gibberellic acid on proliferation of hyphae of arbuscular mycorrhizal (AM) fungus Glomus fistulosum were studied under axenic conditions in vitro. The growth of intraradical hyphae of G. fistulosum was fully suppressed by 30 μM indole-3-acetic acid, but a perceptible decrease in the proliferation of the hyphae was observed already at 3 μM. Because such concentration is near the concentrations common in root tissues in vivo, the effect may be biologically significant. Similar effect was also observed for Glomus mosseae. Inhibitory effects of abscisic acid and cytokinins occurred only at very high, non-physiological concentrations. Ribosylated cytokinins showed stronger inhibition effects than their non-ribosylated counterparts. No stimulation of proliferation of hyphae by any plant hormone tested was observed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Influence of Different Cytokinins on the Germination of Lettuce (Lactuca sativa) and Celery (Apium graveolens) Seeds

The naturally occurring cytokinins, zeatin, zeatin riboside and dihydrozeatin did not promote the germination of celery (Apium graveolens L.) seeds and 6-Δ²-isopentenyladenine (2iPA) and its riboside were only moderately active. Of the synthetic cytokinins, kinetin, kinetin riboside, and the disubstituted urea, N-phenyl-N′-pyridyl urea (NC5392) were moderately active, and 6-benzyl-aminopurine (BA) and its derivatives BA riboside and 6-benzyl-amino-9(tetrahydropyran-2yl)purine (SD8339) were the most active cytokinins tested. 6-(o-hydroxybenzyl)aminopurine (hyd-BA) and its naturally occurring riboside inhibited germination under normally inductive conditions. All the cytokinins examined were more active in promoting germination of lettuce (Lactuca sativa L.) than celery seeds. BA, BA riboside and SD8339 were again the most active cytokinins. In contrast to the results with celery, zeatin and zeatin riboside were highly active. The other cytokinins also showed high activity with the exception of dihydrozeatin, hyd-BA and hyd-BA riboside which were less active. Cytokinin ribosides were less active than the corresponding free bases during the early period of the lettuce seed incubation but total germination after 90 h was similar.     

Some effects of plant growth regulators on tissue cultures of the marine red alga Agardhiella subulata (Gigartinales,Rhodophyta)

We examined whether auxins and cytokinins, either singly or in combination, stimulate cell division in tissue cultures of a red seaweed. Our experimental model consisted of filamentous and callus-like growths that developed from cross-sectional discs cut from young branches of Agardhiella subulata. Plant growth regulators were added to the medium to give combinations of an auxin with a cytokinin over a range of concentrations (1 µg L^–1 –10 mg L^–1). Several mixtures of auxins and cytokinins, as well as some single auxins, cytokinins and phenolics, stimulated cell division and growth in the tissue cultures beyond that of controls. The treatments that were effective included: phenylacetic acid/zeatin; phenylacetic acid/6-benzylaminopurine; -naphthaleneacetic acid/zeatin; 2,4,5-trichlorophenoxyacetic acid/6-benzylaminopurine; and indoleacetic acid/kinetin. High concentrations of cytokinins (i.e. 10 mg L^–1) inhibited the regeneration of plants in some of the cell cultures. These results provide further evidence that growth regulators can be used for the tissue culture of seaweeds and for the study of developmental phenomena in these plants.     

The role of cytokinin in sieve tube regeneration and callose production in wounded coleus internodes

Cytokinin proved to be a controlling factor in sieve tube regeneration around wounded collateral bundles in an in vivo system in which the endogenous cytokinin level had been minimized. Both kinetin and zeatin were applied in aqueous solution to the bases of excised, mature internodes of Coleus blumei Benth. that had an active vascular cambium. Each internode also received indoleacetic acid (IAA) in lanolin at its apical end. Under either low (0.1% w/w) or high (1.0% w/w) auxin concentrations, the control internodes (without exogenous cytokinin) exhibited small amounts of sieve tube regeneration. At appropriate concentrations, both kinetin and zeatin induced a significant increase in sieve tube regeneration around the wound. However, the highest concentration of kinetin tested (50 μg/mL) completely inhibited this process. Kinetin was the most effective with high auxin (1.0% IAA), while zeatin was the most effective with low auxin level (0.1% IAA). Kinetin and zeatin showed the strongest promotive effect at 10 μg/mL and 20 μg/mL, respectively. Both cytokinins also induced supplementary phloem regeneration further from the wound surface. In addition to their effects on vascular tissue regeneration, both cytokinins promoted callose production. This was most evident on the sieve plates of the regenerated sieve tube members and on the walls of the parenchyma cells around the wound. The largest deposits of callose were found in both regenerated sieve tube members and parenchyma cells at the highest cytokinin concentration tested (50 μg/mL). The possible role of cytokinin in controlling callose accumulation in the sieve tubes during autumn is discussed.