In vitro morphogenetic response and distribution of endogenous plant hormones in hypocotyl segments of snapdragon (Antirrhinum majus L.)

Snapdragon seedlings, 20 mm in length, were cut into 5 segments from the cotyledon to the root, which were cultured in vitro on hormone-free MS medium. Adventitious shoot formation was highest in the basal hypocotyl segments with stimulation by the addition of BA. Endogenous cytokinins were higher in the basal hypocotyl segments than in the two upper hypocotyl segments, whereas auxin content was higher in the two upper than in the basal hypocotyl segments. Ratios of cytokinins to auxin were also the highest in the basal hypocotyl segment. A general principle in in vitro culture that a high concentration of cytokinin and a low concentration of auxin promotes the induction of shoot morphogenesis was confirmed from measurements of endogenous growth regulator concentrations.     

Changes in endogenous levels of three cytokinins during development of excised watermelon cotyledons

We have determined by an immunological method the endogenous levels of three cytokinins: dihydrozeatin riboside (DHZR), transzeatin riboside (tZR) and isope-ntenyladenosine (IPA) in watermelon (Citrullus vulgaris Schrad., cv. Fairfax) cotyledons that were either attached to the seedling or excised from the seed after imbibition and then grown on water. Both seedlings and cotyledons were grown either for 5 days in continuous light or for 3 days in the dark and 2 days in light. Our aim was to verify whether endogenous cytokinin levels are lower in excised than in attached cotyledons as could be expected since excised cotyledons are much more sensitive to exogenous cytokinin application. The levels of the three cytokinins were very low immediately after imbibition, but gradually increased during the following days. They were higher in excised cotyledons after 5 days of culture in the dark than in cotyledons of the same age that had developed on the seedling. Dihydrozeatin riboside was by far the most abundant of the three cytokinins in cotyledons as well as in the hypocotyl and the root.
Irradiation reduced the level of DHZR, negating the concept that light promotes cotyledon development by increasing endogenous cytokinins. Transzeatin riboside when supplied exogenously, stimulated cotyledon development at a lower concentration than the other two cytokinins. Exogenous supply of ben-zyladenine (BA) induced a strong increase in endogenous tZR already after 24 h.     

Effects of the embryonic axis and exogenous growth regulators on sunflower cotyledon storage protein mobilization

Cotyledons of sunflower seedlings ( Helianthus annuus L. cv. Giant gray stripe) expand and their protein content first rises then begins to decrease during the first three days of growth. Storage protein structures, which are visible with scanning electron microscopy, undergo modification that leads to storage protein disappearance by day 4 post-imbibition. Expansion of cotyledons detached from seeds prior to imbibition is greatly reduced, total protein levels remain high, and storage protein structures remain visible in cells of these cotyledons. Incubation of excised cotyledons in 1.0 μM benzyladenine or kinetin increases the rates of cotyledon expansion and storage protein loss to levels higher than in intact seedling cotyledons, Incubation in 10 μM indole-3-acetic acid inhibits cotyledon expansion and protein mobilization. More rapid hydrolysis of storage proteins in cotyledons of intact seedlings or detached cotyledons treated with cytokinin is further indicated in day 2 specimens by SDS-polyacrylamide gel electrophoresis. These results suggest a possible mechanism for regulation of cotyledon development by interactions of the promotive effects of cytokinin and inhibitory effects of auxin.     

Comparative Study on the Bioassay between N6, O2'-Dibutyryl Cyclic Adenosine 3,5-Monophosphate and Cytokinin

The physiological activity of diBcAMP by various classical bioassay methods of cytokinin was studied. It exhibits the cytokinin-like activity in many ways of biological response, such as: the unfolding of etiolated barley leaf, the expansion of radish cotyledon, the expansion and greening of cucumber cotyledon and the inhibition of the elongation of mung-bean hypocotyl, etc. The activity of 1 mM diBcAMP was approximately the same as the activity of 0.044 mM benzyladenine in the most of the tests. However, the activity of 1 mM diBcAMP was found to be higher than the optimal concentration (0.044 mM) of benzyledenine in the radish cotyledon under the expansion test. There was no toxicity of diBcAMP in all of the bioassay methods used, even with higher concentration.     

Light-stimulated cotyledon expansion in the blu3 and hy4 mutants of Arabidopsis thaliana.

Cotyledon expansion in response to blue light was compared for wild-type Arabidopsis thaliana (L.) Heynh. and the mutants blu3 and hy4, which show reduced inhibition of hypocotyl growth in blue light. White, blue, and red light stimulated cotyledon expansion in both intact and excised cotyledons of wild-type seedlings (ecotypes No-0, WS, Co-0, La-er). Cotyledons on intact blu3 and hy4 seedlings did not grow as well as those on the wild type in response to blue light, but pretreatment of blu3 seedlings with low fluence rates of red light increased their responsiveness to blue light. Excision of cotyledons alleviated the mutant phenotype so that both mutant and wild-type cotyledons grew equally well in blue light. The loss of the mutant cotyledon phenotype upon excision indicates that the blu3 and hy4 lesions affect cotyledon expansion indirectly via a whole-plant response to light. Furthermore, the ability of excised, mutant cotyledons to grow normally in blue light shows that this growth response to blue light is mediated by a photosystem other than the ones impaired by the blu3 and hy4 lesions.     

Control by cytokinins of the cellular behavior in the plate meristem of zucchini cotyledons

The temporal and spatial effects of exogenous cytokinins on both cell expansion and division activity in the plate meristem of cultured zucchini cotyledons were studied. N ⁶-benzylaminopurine (1–100 μM) and N-(2-chloro-4pyridyl)-N′-phenylurea (4PU-30) (0.1–100 μM) greatly stimulated the cell growth and division. They provoked multiple cell cycles, formation of larger clusters of daughter cells and an increase of the final number of cells. Both cytokinins led to earlier achievement of final cotyledon size and shortened the cell doubling time. By contrast to the purine cytokinin, phenylurea cytokinin 4PU-30 enlarged the cotyledon predominantly in length. Zeatin and kinetin were less effective, particularly in stimulating cell expansion. In low concentrations, all cytokinins were more effective in stimulating division activity rather than expansion. The cells in the cotyledon margins displayed a higher division activity, especially when treated with exogenous cytokinins. The final cotyledon and cluster areas were not of the strict proportional dependence upon the number of their cells. These results provide a novel example where stimulated cell division fails to evoke a respective increase in the final organ size.     

Differential effects of methyl jasmonate on growth and division of etiolated zucchini cotyledons

The jasmonates are well studied in the context of plant defence but increasingly are also recognised as playing roles in development. In many systems, jasmonates antagonise the effects of cytokinins. The aim of the present work was to elucidate interactions between methyl jasmonate and cytokinin (benzyladenine) in regulating growth of zucchini (Cucurbita pepo L., cv. Cocozelle, var. Tripolis) cotyledons, taking advantage of the ability to simultaneously quantify cell enlargement and division from paradermal sections of the first palisade layer. Growth regulators were applied to cotyledons, excised from dry seeds and grown in darkness. Cytokinin stimulated expansion and division whereas, surprisingly, jasmonate stimulated expansion but inhibited division. Jasmonate antagonised the stimulating effect of cytokinin on division but worked cooperatively with cytokinin in increasing expansion. However, expansion with jasmonate was more isotropic than with cytokinin. Jasmonate also stimulated the loss of cellular inclusions and soluble protein. Soluble proteins revealed a partial antagonism between jasmonate and cytokinin. These results illustrate the complex interplay between jasmonates and cytokinin in the regulatory network of cotyledon development following germination.     

Design and synthesis of photolabile caged cytokinin

Cytokinins are phytohormones that regulate diverse developmental processes throughout the life of a plant. trans-Zeatin, kinetin, benzyladenine and dihydrozeatin are adenine-type cytokinins that are perceived by the AHK cytokinin receptors. Endogenous cytokinin levels are critical for regulating plant development. To manipulate intracellular cytokinin levels, caged cytokinins were designed on the basis of the crystal structure of the AHK4 cytokinin receptor. The caged cytokinin was photolyzed to release the cytokinin molecule inside the cells and induce cytokinin-responsive gene expression. The uncaging of intracellular caged cytokinins demonstrated that cytokinin-induced root growth inhibition can be manipulated with photo-irradiation. This caged cytokinin system could be a powerful tool for cytokinin biology.     

Changes in Endogenous Cytokinins During Germination and Seedling Establishment of <Emphasis Type="Italic">Tagetes minuta</Emphasis> L.

Endogenous cytokinins were quantified and identified in germinating achenes and developing seedlings of Tagetes minuta L. incubated at 25 °C over a 144 h period. The process of germination (radicle emergence) was completed 38 h after commencement of imbibition. Subsequent growth was considered to cover seedling establishment. Eighteen isoprenoid cytokinins, belonging to the zeatin (9), dihyrozeatin (5) and isopentenyladenine (4) groups and one aromatic cytokinin, benzyladenine, were identified. The total isoprenoid cytokinin concentration increased upon imbibition, reached a peak by 48 h and subsequently decreased with seedling development. The individual cytokinin groups and the respective derivatives within each group did, however, not follow such a consistent trend. During the course of the experiment, the ribotides and ribosides were present in the highest concentrations, reaching a peak at 48 h and decreasing thereafter. The free bases and O-glucoside remained at low levels throughout the experiment. Isopentenyladenine-9-glucoside increased dramatically in the developing seedlings and after 144 h was the predominate cytokinin. Benzyladenine was the only aromatic cytokinin detected throughout the experiment. It was present in high concentrations in the dry achenes and declined rapidly upon imbibition.     

The effect of cytokinins on in vitro cultured inbred lines of Raphanus sativus var. radicula Pers. with genetically determined tumorigenesis

The higher plant tumors are convenient models for studying the genetic control mechanism of plant cell division. There are two types of tumors: induced by the pathogenic factor and genetically determined. The development of both tumor types was related to the changes in cytokinin metabolism and/or signal transduction. In this work, the effect of synthetic cytokinins on the in vitro morphogenesis of cotyledon explants and isolated apices of radish seedlings was studied in several inbred radish lines (Raphanus sativus var. radicula Pers.) that differed in their in vivo tumorigenic properties. It was noted that root formation was stronger affected by kinetin while the treatment with thidiazuron tended to induce active callus formation in cotyledon explants of all inbred lines, except IIa. Growing with benzyladenine produced an intermediate effect as regards all morphogenetic responses. Cytokinin treatment of tumorigenic lines enhanced necrotic development in cotyledon explants. Culturing isolated apices of regenerated plants produced tumors anatomically and morphologically similar to those developing in vivo. Some of the lines nontumorigenic in vivo with enhanced formation of calli on cotyledon explants also developed tumors on apical explants in vitro when treated with cytokinins. These data suggest that different mechanisms for tumor formation operate in various radish lines. The radish lines are classified into three types: (1) necrotic lines with tumor formation putatively related to endogenous cytokinin level, (2) callus-forming lines with cell division enhanced in response to cytokinins, and (3) necrosis-and callus-forming lines with both mechanisms of tumor formation involved.     

Cytokinin action is coupled to ethylene in its effects on the inhibition of root and hypocotyl elongation in Arabidopsis thaliana seedlings.

Cytokinins have profound effects on seedling development in Arabidopsis thaliana. Benzyladenine (BA) inhibits root elongation in light- or dark-grown seedlings, and in dark-grown seedlings BA inhibits hypocotyl elongation and exaggerates the curvature of apical hooks. The latter are characteristic ethylene responses and, therefore, the possible involvement of ethylene in BA responses was examined in seedlings. It was found that the inhibitory effects of BA on root and hypocotyl elongation were partially blocked by the action of ethylene inhibitors or ethylene-resistant mutations (ein1-1 and ein2-1). Ethylene production was stimulated by submicromolar concentrations of BA and could account, in part, for the inhibition of root and hypocotyl elongation. It was demonstrated further that BA did not affect the sensitivity of seedlings to ethylene. Thus, the effect of cytokinin on root and hypocotyl elongation in Arabidopsis appears to be mediated largely by the production of ethylene. The coupling between cytokinin and ethylene responses is further supported by the discovery that the cytokinin-resistant mutant ckr1 is resistant to ethylene and is allelic to the ethylene-resistant mutant ein2.     

Cytokinin concentration in relation to mineral nutrition and benzyladenine treatment in Plantago major ssp. pleiosperma

Cytokinins in shoot and root tissue were studied in plants of Plantago major L. ssp. pleiosperma (Pilger) grown on a concentrated and on a dilute nutrient solution. Cytokinins of plants transferred from the concentrated to the dilute solution were compared with plants treated similarly but with 10⁻⁸ M benzyladenine (BA) added to the dilute solution. Cytokinin concentrations were also measured in plants that had been grown throughout on one of the two nutrient solutions. A restricted supply of minerals was correlated with low cytokinin concentrations. Transfer from a concentrated nutrient solution to a dilute solution depressed the cytokinin concentration by 50% within two days of transfer. This decline did not appear if similar plants were supplied with 10⁻⁸ M BA in the dilute solution. The glucosides were the only major cytokinins enhanced by mineral shortage. This effect of low mineral supply was retarded but not entirely prevented by exogenous BA. The increased synthesis of glucosides in the BA-treated plants was accompanied by lowered concentrations of free bases and their ribosides and of nucleotides. The sum of cytokinins in BA-treated plants was thus similar to that in plants grown at the high mineral level. The results are discussed in relation to a possible role of cytokinins in regulating growth responses to changes in mineral nutrition.     

Tissue culture and plant regeneration of okra (Abelmoshus esculentus)

Explants (hypocotyl, cotyledon, cotyledonary node and leaf segment) were excised from aseptically grown okra (Abelmoschus esculentus) seedlings. The explants were cultured on a Murashige and Skoog basal nutrient medium supplemented with auxins, cytokinins and auxin-cytokinin combinations. Callus formation and root differentiation occurred in a medium containing naphthaleneacetic acid (NAA) or indoleacetic acid. There was a greater proliferation of roots on medium supplemented with NAA. The addition of 2,4-dichlorophenoxyacetic acid (2,4-D) to the growth medium suppressed root formation. No shoot bud or shoot development was observed at any of the auxin levels tested. Both kinetin (KN) and zeatin (Z) also proved ineffective in inducing shoot buds or shoots. Shoots were produced on cotyledon and cotyledonary node explants cultured in a medium supplemented with benzyladenine and NAA. These shoots developed roots on the same medium. The plantlets, on transfer to soil, grew normally.     

Arabidopsis thaliana wild type, pho1, and pho2 mutant plants show different responses to exogenous cytokinins.

Despite the involvement of cytokinins in phosphate (Pi) signaling being highlighted, the physiological processes involved remain unclear. In this study, we have evaluated the effect of cytokinins on different physiological responses using wild type (wt) and two Arabidopsis mutants with altered shoot Pi content (pho1 and pho2). Physiological studies were related with those previously described as cytokinin-regulated: including hypocotyl elongation, root growth, anthocyanin accumulation, senescence and relative gene expression. Generally, pho1 mutants showed decreased sensitivity to cytokinin, whereas pho2 mutants showed increased sensitivity to the hormone. This observation applies to inhibition of hypocotyls and root growth and anthocyanin accumulation. However, this effect was not shown during senescence or in the expression of ARR6 (Arabidopsis response regulator, ARR). Interestingly, Pi content in shoot of pho1 mutants increased to wt levels after treatment with cytokinins. These results suggest that the interaction between phosphate signaling and cytokinin signaling may be bidirectional while the differential behavior in response to cytokinin is discussed further.     

Cytokinin-Regulated Mesophyll Cell Division and Expansion during Development of Cucurbita pepo Leaves

The role of cytokinins in the development of mesophyll structure was studied in developing pumpkin Cucurbita pepo L. leaves. Leaves were treated with cytokinins at different stages of growth: when they reached 25 or 50% of their final size (S _max), immediately after leaf growth ceased, and during senescence. At the early stages of leaf development, treatment with exogenous benzyladenine accelerated division of mesophyll cells. At the later stages of development, BA treatment activated expansion of growing cells and those, which have just accomplished their growth. The exogenous cytokinin did not affect the senescent leaf cells. The content of endogenous cytokinins changed during mesophyll development. The juvenile leaves (25% of S _max) were characterized by low level of these phytohormones. In the expanding leaves (50% of S _max), the content of phytohormones increased and decreased when leaf growth ceased. In the senescent leaves, the cytokinin content decreased markedly. It was concluded that the response of mesophyll cells to cytokinin depended on the cell growth phase at the moment of hormone action. Furthermore, in the young leaves, lower cytokinin concentrations were required for division of mesophyll cells in vivo than for cell expansion at the final stage of leaf development.     

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     

hoc: An Arabidopsis mutant overproducing cytokinins and expressing high in vitro organogenic capacity

A novel Arabidopsis thaliana mutant, named hoc, was found to have an high organogenic capacity for shoot regeneration. The HOC locus may be involved in cytokinin metabolism leading to cytokinin-overproduction. In vitro, hoc root explants develop many shoots in the absence of exogenous growth regulators. The mutant displays a bushy phenotype with supernumerary rosettes and with normal phyllotaxy, resulting from precocious axillary meristem development. Genetic and molecular analyses show that the high shoot regeneration and the bushy phenotype are controlled by a recessive single gene, located on chromosome I, next to the GAPB CAPS marker. The mapping data and allelism tests reveal that the hoc mutant is not allelic to other reported Arabidopsis growth-regulator mutants. In darkness the hoc mutant is de-etiolated, with a short hypocotyl, opened cotyledons and true leaves. Growth regulator assays reveal that the mutant accumulates cytokinins at about two- and sevenfold the cytokinin level of wild-type plants in its aerial parts and roots, respectively. Consequently, the elevated amounts of endogenous cytokinins in hoc plants are associated with high organogenic capacity and hence bushy phenotype. Thus hoc is the first cytokinin-overproducing Arabidopsis mutant capable of auto-regenerating shoots without exogenous growth regulators.     

Analysis of Variability in the Amaranthus Bioassay for Cytokinins: Effects of Water Stress on Benzyladenine- and Fusicoccin-dependent Responses

The use of only the upper part of the hypocotyl and the cotyledons in the Amaranthus tricolor bioassay for cytokinins, instead of the whole seedling, was found to reduce the endogenous response and give a higher benzyladenine-dependent response. There is no marked difference in the uptake or metabolism of benzyladenine in whole seedlings compared with that in excised cotyledons.