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.     

Endogenous plant growth regulators in carnation tissue cultures under different conditions of ventilation

Endogenous indole-3-acetic acid (IAA), abscisic acid (ABA) and cytokinins (zeatin, zeatin riboside, dihydrozeatin, (diH)Z, dihydrozeatin riboside, (diH)[9R]Z, N⁶-isopentenyl adenine and N⁶-isopentenil adenine riboside) levels were evaluated in normal (N) and hyperhydric (H) microplants of Dianthus caryophyllus cultured under different aeration conditions in hormone-free liquid medium. The morphological differences between N and H explants grown under ventilated conditions were correlated with differences in their endogenous hormonal levels: after 15 and 30 days of culture, H explants showed lower IAA and ABA contents than N explants, as well as higher cytokinin levels, mainly of (diH)Z and (diH)[9R]Z. This was associated with less tissue differentiation and with an inability of H microplants to survive under ex vitro conditions. However, these relationships could not be observed between H and N explants grown under non-ventilated conditions probably due to the difficulty in discerning the plant status (N or H) and therefore, an underestimation of H microplants. This assumption is supported by the low ability for acclimatization to ex vitro of N plants grown without ventilation.     

Effect of ambiol and 2-chloroethylphosphonic acid on the content of phytohormones in potato leaves and tubers

The effects of the antioxidant Ambiol and 2-chlorethylphosphonic acid (2-CEPA) on individual concentrations and concentration ratios of phytohormones, photosynthesis and photophosphorylation rates, sucrose and starch content in tubers, and plant productivity were studied in potato (Solanum tuberosum L). Ambiol increased the ratio of indoleacetic acid (IAA) to abscisic acid (ABA), IAA/ABA, and that of zeatin (Z) and zeatin riboside (ZR) to ABA, (Z + ZR)/ABA. These effects were underlain by an increase in the content of auxins and cytokinins and a decrease in ABA. Unlike Ambiol, 2-CEPA increased the level of ABA, the effect being the most pronounced in the tubers. Ambiol increased the rates of photosynthesis and noncyclic photophosphorylation in chloroplasts isolated from potato leaves. The relation of this phenomenon to auxin and cytokinin accumulation, Ambiol- and 2-CEPA-induced changes in the hormonal balance of potato tubers, carbon metabolism, and plant productivity is discussed.     

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     

菊苣薄层培养花芽,营养芽分化中内源激素的动态变化

菊苣（Ｃｉｃｈｏｒｉｕｍ ｉｎｔｙｂｕｓＬ．）花梗薄层细胞培养于ＭＳ附加ＮＡＡ 和ＢＡ 或ＩＡＡ 和ＢＡ 的ＭＳ培养基上有花芽或营养芽分化． 花芽分化中内源ＩＡＡ、ＤＨＺ＋ ＤＨＺＲ、ｉＰＡ 含量明显增加,而Ｚ＋ ＺＲ变化不明显．营养芽分化中内源细胞分裂素含量增加明显,而ＩＡＡ 在培养前７ ｄ 含量下降,随后有所增加,在原基形成时含量达原初水平的２／３． 可见,花芽分化比营养芽分化所需内源ＩＡＡ／ＣＴＫ 比值要高     

Effects of Ambiol and 2-Chlorethylphosphonic Acid on the Content of Phytohormones in Potato Leaves and Tubers

The effects of the antioxidant Ambiol and 2-chlorethylphosphonic acid (2-CEPA) on individual concentrations and concentration ratios of phytohormones, photosynthesis and photophosphorylation rates, sucrose and starch content in tubers, and plant productivity were studied in potato (Solanum tuberosum L). Ambiol increased the ratio of indoleacetic acid (IAA) to abscisic acid (ABA), IAA/ABA, and that of zeatin (Z) and zeatin riboside (ZR) to ABA, (Z + ZR)/ABA. These effects were underlain by an increase in the content of auxins and cytokinins and a decrease in ABA. Unlike Ambiol, 2-CEPA increased the level of ABA, the effect being the most pronounced in the tubers. Ambiol increased the rates of photosynthesis and noncyclic photophosphorylation in chloroplasts isolated from potato leaves. The relation of this phenomenon to auxin and cytokinin accumulation, as well as Ambiol- and 2-CEPA-induced changes in the hormonal balance of potato tubers, carbon metabolism, and plant productivity, is discussed.     

Effect of apex excision and replacement by 1-naphthylacetic acid on cytokinin concentration and apical dominance in pea plants

As known from literature lateral buds from pea ( Pisum sativum ) plants are released from apical dominance when repeatedly treated with exogenous cytokinins. Little is known, however, about the endogenous role of cytokinins in this process and whether they interact with basipolar transported IAA, generally regarded as the main signal controlling apical dominance. This paper presents evidence that such an interaction exists.
The excision of the apex of pea plants resulted in the release of inhibited lateral buds from apical dominance (AD). This could be entirely prevented by applying 1-naphthylacetic acid (NAA) to the cut end of the shoot. Removal of the apex also resulted in a rapid and rather large increase in the endogenous concentrations of zeatin riboside (ZR), isopentenyladenosine (iAdo) and an as yet unidentified polar zeatin derivative in the node and internode below the point of decapitation. This accumulation of ZR and iAdo, was strongly reduced by the application of NAA. The observed increase in cytokinin concentration preceded the elongation of the lateral buds, suggesting that endogenous cytokinins play a significant role in the release of lateral buds from AD. However, the effect of NAA on the concentration of cytokinins clearly demonstrated the dominant role of the polar basipetally transported auxin in AD. The results suggest a mutual interaction between the basipolar IAA transport system and cytokinins obviously produced in the roots and transported via the xylem into the stem of the pea plants.     

Changes of Endogenous Hormone Contents During Floral Bud and Vegetative Bud Differentiation in Thin Cell Layer Culture of Cichorium intybus L. Explant

The sectioned thin cell layers (TCL) of flower stalk of Cichorium intybus L. were cultured in MS medium supplemented with NAA and BA or IAA and BA where floral and vegetative buds were developed from the explant. Endogenous IAA, DHZ+DHZR, iPA increased significantly during the floral bud formation, while Z+ZR remained changed. The levels of cytokinins, DHZ +DHZR, iPA, and Z-f-ZR all increased significantly during the vegetative bud formation, however IAA level was reduced during the first 7 days of culture and increased to two-thirds of initial values on the day when the bud primordia were formed. The results suggested that the initiation of floral buds was associated with a high IAA/CTK ratio, whereas the induction of vegetative bud differentiation was related to a low IAA/CTK ratio.     

Hormone Levels and Apical Dominance in the Aquatic Fern Marsilea drummondii A. Br

Terminal buds and successively subjacent lateral buds of the water fern, Marsilea drummondii, were examined to determine the pattern of hormone distribution in relation to apical dominance. Quantitative levels of indole-3-acetic acid (IAA), abscisic acid (ABA), zeatin and zeatin riboside (Z and ZR), and isopentenyladenosine (iPA) were determined by a solid-phase immunoassay using polycional antihormone antibodies. Enzyme-linked immunosorbent assay was used following a one-step HPLC purification procedure to obtain the free hormones. Active shoot apices contained the most IAA and Z-type cytokinins and inhibited buds the least. No significant differences in ABA levels were found leading to the conclusion that ABA did not play any role in apical dominance. The normal precedence of the most rapid outgrowth of the youngest inhibited bud as observed previously in decapitated plants was well correlated with its very high level of iPA observed in this study. The same phenomenon was observed in the median buds but with a weaker amplitude. The presence of this storage form could indicate that a bud at its entry into quiescence eventually looses the ability to hydroxylate iPA to Z-type cytokinins when it is fully inhibited. IAA and Z + ZR are concluded to be essential for lateral bud growth.     

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     

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.     

The Influence of Different Cytokinins and Auxins on Flower Neoformation in Thin Cell Layers of Nicotiana tabacum L.

Neoformation of flower buds was studied in thin cell layersderived from floral branches of Nicotiana tabacum L. Organogenesiswas found to be expressed in different ways according to thekind of auxin and cytokinin present in the culture medium. Inthe presence of synthetic auxins (NAA, CTA), more flower budswere generated than with the natural auxins (IAA, IBA), if theculture medium was also supplemented with a cytokinin. Theseconditions also stimulated the development of the buds to flowersor inflorescences. The most active cytokinin was BA. Naturalcytokinins (zeatin, IPA) appeared much less active but led toremarkable development of roots together with flower buds. Anorganogenetic effect of 2,4-D was observed in this experimentalsystem, leading to the conclusion that organogenetic activityof cytokinins and auxins in thin cell layer cultures stronglydepends on the molecular structure of the growth regulators. (Received September 30, 1987; Accepted March 23, 1988)     

Cytokinins in photoperiodic induction of flowering in Chenopodium species

Changes in cytokinin (zeatin – Z, zeatin riboside – ZR, isopentenyladenine – iP, isopentenyladenosine – iPA) levels were determined under light regimes inductive and non-inductive for flowering in leaves, stems, roots and apical parts of short-day Chenopodium rubrum and long-day Chenopodium murale. In leaves. stems and roots of both plant species the level of cytokinins (in C. rubrum of Z and ZR, in C. murale of Z. ZR, iP and iPA) decreased by about 50% during the dark period and increased again during the subsequent light period, No significant changes in cytokinin levels were observed in continuous light. In apical parts of C. rubrum cytokinin level (Z, ZR, iP) was dramatically increased (by 400–500%) at the end of the dark period and decreased to about the original value during the following light period, while no changes were observed in continuous light. In apical parts of C. murale the level of cytokinins doubled during floral induction consisting of 10 days of continuous light. A red (R) break (15 min at the 6th h of darkness), which prevents flowering in C. rubrum , has no significant effect on cytokinin levels in leaves at the end of darkness. Cytokinin levels increased 1 h after R and decreased again rapidly. On the other hand, the increase of cytokinin level in the apical parts of C. rubrum was largely prevented by the R break. These effects of R on cytokinin levels were not reverted by far-red (FR), while the effect on flowering was reverted. It may be concluded that there is no correlation between changes in cytokinin levels in leaves. Stems and roots and photoperiodic flower induction, as both species, representing different photoperiodic types, showed similar changes under the same light regime. The increase of cytokinin levels in apical parts of both photoperiodic species during floral induction suggests a role (increased cell division and branching) for cytokinins in apex evocation.     

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     

Thidiazuron influences the endogenous levels of cytokinins and IAA during the flowering of isolated shoots of Dendrobium

This study reports the effects of thidiazuron (TDZ) on the endogenous levels of indoleacetic acid (IAA), zeatin, zeatin riboside ([9R]Z), isopentenyladenine and isopentenyladenosine ([9R]iP) as well as structural changes in the apical meristem of Dendrobium Second Love shoots during flower induction and initial development in vitro. The results revealed that the presence of 1.8microM TDZ had a profound effect on the endogenous cytokinins (CKs) and IAA levels of the explants, when compared to those grown on a TDZ-free medium. A significant increase in CKs (especially [9R]iP and [9R]Z) and IAA in the first samples (taken at day 5) grown on TDZ-enriched medium was associated with flower induction, while a second increase in the level of these hormones after 25d of culture was related to flower development. The histological changes detected in the shoot apical meristem of explants grown in the presence of 1.8microM TDZ during 30d of culture are also described. Based on these findings, it is suggested that both auxin and CKs seem to be involved with the floral transition of Dendrobium Second Love in vitro. However, a possible direct effect of TDZ on flower formation is not discarded.     

Cytokinins in Rosa hybrida in relation to bud break

To assess the role of endogenous cytokinins in growth and development of Rosa hybrida , their concentrations in bleeding sap and in roots, stem, leaves, axillary shoots and bottom breaks in three stages of development were quantified. Cytokinins were purified by means of immunoaffinity chromatography and HPLC, and identified by retention time, UV spectrum and GC-MS. The major translocation form in the xylem was zeatin riboside (ZR). In all mature tissues, cytokinins of the zeatin-type were predominant, amounting to 80–90% of the total cytokinin concentration. The stems contained high concentrations of cytokinins, probably caused by lateral movement of ZR from the xylem to adjacent stem tissue and the ability of the stem to metabolize cytokinins. In young leaves the contribution of isopentenyl adenine (iP)-type cytokinins to the total cytokinin pool was about 50%, indicating that these leaves might be capable of de novo synthesis of cytokinins. In older leaves, the concentration of an unidentified cytokinin-like compound increased to more than 50% of total cytokinins. This compound, which was also found in the roots, might be a storage form of cytokinins. In young axillary shoots, about 50% of the cytokinins are iP-compounds, suggesting either import of iP-type cytokinins via the phloem or de novo synthesis of cytokinins. In buds forming bottom breaks, ZR and zeatin riboside monophosphate (ZRMP) are the main cytokinins, indicating that these buds receive their cytokinins from the roots.     

Identification and quantification of the major endogenous cytokinins in pistachio seedlings

The major endogenous cytokinins, Z, ZR, DHZ, DHZR, iP and iPR in pistachio seedlings (Pistacia vera L. cv. Ohadi) were purified by HPLC and their identities confirmed using GC-MS. The aerial parts of two-year old pistachio seedlings including mature leaves, young leaves, lateral buds, debarked stems and bark were subjected to analysis. All of the above mentioned cytokinins were identified in the aerial parts except DHZ which was only present in mature leaves. Z-type cytokinins contributed almost 43% of the total cytokinins. ZR and DHZR were identified as the major ribosides and iP as the main base. The greatest concentration of ZR was detected in the bark, amounting to about 48%. DHZR and ZR constituted the major portion of the total cytokinins detected in both young and mature leaves while Z was detected as a minor cytokinin in leaves. The sharp increase of iP concentration during leaf maturation indicates that mature leaves are probably capable of de novo biosynthesis of cytokinins. The absence of DHZ (except in mature leaves) and the presence of considerable concentrations of DHZR in pistachio stems suggest that these tissues are able to metabolize DHZ to DHZR. The large amount of ZR in pistachio leaves suggests that root-derived ZR is transported into the leaves after loading into the xylem. The presence of high amounts of iP in pistachio lateral buds indicates that iP has been accumulated in these parts. The occurrence of a totally different cytokinin distribution pattern in buds, as compared with the other aerial parts, possibly results from their different metabolism.     

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.     

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.     

Endogenous cytokinins in Cocos nucifera L. in vitro cultures obtained from plumular explants

Auxin induces in vitro somatic embryogenesis in coconut plumular explants through callus formation. Embryogenic calli and non-embryogenic calli can be formed from the initial calli. Analysis of endogenous cytokinins showed the occurrence of cytokinins with aromatic and aliphatic side chains. Fourteen aliphatic cytokinins and four aromatic cytokinins were analysed in the three types of calli and all the cytokinins were found in each type, although some in larger proportions than others. The most abundant cytokinins in each type of callus were isopentenyladenine-9-glucoside, zeatin-9-glucoside, zeatin riboside, isopentenyladenine riboside, dihydrozeatin and dihydrozeatin riboside in decreasing order. Total cytokinin content was compared between the three types of calli, and it was found to be lower in embryogenic calli compared to non-embryogenic calli or initial calli. The same pattern was observed for individual cytokinins. When explants were cultured in media containing exogenously added cytokinins, the formation of embryogenic calli in the explants was reduced. When 8-azaadenine (an anticytokinin) was added the formation of embryogenic calli and somatic embryos was increased. These results suggest that the difference in somatic embryo formation capacity observed between embryogenic calli and non-embryogenic calli is related to their endogenous cytokinin contents.