Monoclonal antibodies against the plant cytokinin isopentenyl adenosine

Sixteen monoclonal antibodies against isopentenyl adenosine (iPA) were developed and characterized for reactivity towards this cytokinin and structurally related molecules by use of a competition fluorescence enzyme immunoassay. Antibodies with suitable affinity and specificity were used in an immunoassay to detect and quantify isopentenyl adenosine. Logit/log plots of fluorescence vs pmol of the cytokinin indicated that the assay had a measurement range of 0.03–256 pmol (10–8500 pg) with high linearity (r =–0.98). This competition fluorescence enzyme immunoassay showed that three antibodies cross-reacted with N-6-benzyladenine and its riboside: cross-reactivity with dihydrozeatin and its riboside, cis -zeatin, trans -zeatin riboside, adenine and adenosine was minor. When an immunoaffinity-HPLC technique was used to measure the cross-reactivity of two of the antibodies in the presence of known quantities of multiple cytokinins, iPA was bound in preference to the other cytokinins. One of the antibodies was used to quantify this cytokinin in developing wheat ( Triticum aestivum L. cv. Stephens) seeds and in wheat seeds spiked with known amounts of certain other cytokinins. The use of these antibodies in immunoassay in combination with HPCL for quantification of iPA in plant extracts is discussed.     

Cytokinin production by fluorescent Pseudomonas in the presence of rice root exudates

Free-living bacteria may trigger the plant growth through production of phytohormones viz. gibberellins, auxins and cytokinins. A total 50 isolates of fluorescent Pseudomonas were screened for their ability to produce cytokinins such as isopentenyladenosine (IPA), dihydroxyzeatin riboside (DHZR) and zeatin riboside (ZR) as plant growth-promoting activity. Pseudomonas fluorescens AK1 and Pseudomonas aeruginosa AK2 were found higher phytohormones producing strains. Of the three cytokinins, IPA was the major cytokinin produced by both isolates in pure culture (5.5 and 2.9 pmol/ml, respectively) and with rice root exudates (5.9 and 3.4 pmol/ml, respectively). Production of ZR and DHZR for both organisms was found after 48 and 72 h. Amount of ZR and DHZR increased with time for both isolates in pure culture conditions. In presence of rice, production of ZR was increased 0.8 and 0.6 pmol/ml for P. fluorescence AK1 and P. aeruginosa AK2, respectively, in comparison with controls. There was no significant difference in the production of DHZR with rice exudates.     

Quantification of the export of cytokinins from roots to shoots of Rosa hybrida and their degradation rate in the shoot

Cytokinins from the roots may be involved in regulating rose ( Rosa hybrida ) shoot growth and development. The objective of this study was to estimate the export of cytokinins from the roots and their degradation rate in the shoot, which were expected to be correlated with plant development. Hence, the total cytokinin content of the shoot, the concentration of zeatin riboside (ZR) in bleeding sap, and the transpiration rates in three stages of development were determined. The estimations performed are based on the assumption that the cytokinin concentration in bleeding sap is representative for the cytokinin concentration in xylem sap in situ. This was verified by comparing the ZR concentration in bleeding sap and in sap obtaíned after pressurizing the root system to a level equivalent to the leaf water potential; no significant differences could be found. The import of cytokinins could not be correlated with plant development, as it increased linearly with time. The estimated relative degradation rate of cytokinins in the shoot decreased as the plants matured. The half-life of cytokinins in the shoot was found to be approximately 1 day, indicating that cytokinins are rapidly metabolized in the shoot.     

Hybrid Weakness in Phaseolus vulgaris L. I. Disruption of Development and Hormonal Allocation

A reduced concentration of cytokinins may cause the abnormal growth and development found in F1 hybrids between Andean and Mesoamerican races of Phaseolus vulgaris L. In this study, concentrations of the transportable cytokinin zeatin riboside (ZR) were measured by ELISA for ZR (cross reactivities dihydrozeatin, 14%, zeatin 7.6%) in roots, stems, and leaves of a Phaseolus Mesoamerican landrace (P. vulgaris L. cv. Redkloud), an Andean landrace (P. vulgaris L. cv. Batt), and their F1 hybrids. Concentrations of ZR in roots and leaves of F1 hybrids were significantly less than that found in roots and leaves of parental cultivars. Approximately 90% of the ZR found in F1 hybrids was found sequestered in the stems, whereas cytokinins of the parental cultivars were distributed throughout the plant (roots: Batt 37%, Redkloud, 44%; stems: Batt 35%, Redkloud 42%; leaves: Batt 28%, Redkloud 14%). These results suggest that abnormal growth and development of F1 hybrids may involve interruption of the regulation of cytokinin allocation, thereby disrupting the root-shoot feedback loop between root-sourced cytokinins and putative shoot-produced factors. Received October 15, 1998; accepted May 12, 1999     

Involvement of cis-Zeatin, Dihydrozeatin, and Aromatic Cytokinins in Germination and Seedling Establishment of Maize, Oats, and Lucerne

The aims of this study were to monitor endogenous cytokinin levels during germination and early seedling establishment in oats, maize, and lucerne to determine which cytokinin forms are involved in these processes; to quantify the transfer ribonucleic acid (tRNA)-bound cytokinins; and to measure cytokinin oxidase/dehydrogenase (CKX) activity. Cytokinins were identified using UPLC-MS/MS. The predominant free cytokinins present in the dry seeds were dihydrozeatin-type (DHZ) in lucerne and maize and cZ-type (cis-zeatin) in oats. Upon imbibition, there was a large increase in cZ-type cytokinins in lucerne although the cZ-type cytokinins remained at high levels in oats. In maize, the high concentrations of DHZ-type cytokinins decreased prior to radicle emergence. Four tRNA-bound cytokinins [cis-zeatin riboside (cZR)>N ⁶-(2-isopentenyl)adenosine (iPR), dihydrozeatin riboside (DHZR), trans-zeatin riboside (tZR)] were detected in low concentrations in all three species investigated. CKX activity was measured using an in vitro radioisotope assay. The order of substrate preference was N ⁶-(2-isopentenyl)adenine (iP)>trans-zeatin (tZ)>cZ in all three species, with activity fluctuating as germination proceeded. There was a negative correlation between CKX activity and iP concentrations and a positive correlation between CKX activity and O-glucoside levels. As O-glucosides are less resistant to CKX degradation, they may provide a readily available source of cytokinins that can be converted to physiologically active cytokinins required during germination. Aromatic cytokinins made a very small contribution to the total cytokinin pool and increased only slightly during seedling establishment, suggesting that they do not play a major role in germination.     

Relations between cytokinin level, bud development and apical control in Norway spruce, Picea abies

In conifers such as Norway spruce, the extent of shoot growth is predetermined by the size and number of embryonal organs of the buds laid down the previous year. As it is known that cytokinins have a key role in bud development a possible hypothesis is that the level of cytokinin in the buds during their formation determines their size and complexity. As a first step to test this hypothesis we compared cytokinin levels in buds of different size of annual shoots from 15- to 20-year-old trees of Picea abies (L.) Karst. Apical buds from the leaders, and from branches in lower parts of the trees, were collected in April, July and August. The difference in size of the buds and the shoots growing from them was considerable in these three positions. Extracts were purified by immunoaffinity columns, and the retained compounds were separated by high-performance liquid chromatography (HPLC). Quantification was made by enzyme-linked immunosorbent assay (ELISA), and the accuracy of this method was checked by measurements with liquid chromatography-mass spectrometry (LC-MS) and UV absorption. Zeatin riboside (ZR) was the most abundant cytokinin, but isopentenyladenosine (iPA) was also present in all samples. The large apical bud of the leader contained much higher cytokinin concentrations than the considerably smaller buds from lower positions, and during the period of secondary growth in July, similar relationships were found for annual stem tissue from different positions. The possible role of ZR as a controlling factor in bud development and apical control is discussed. Our conclusion is that the level of zeatin-type cytokinins appears to play an important role in the establishment of differences in bud size and, thereby, the architecture of the tree crown.     

Growth retardation induced by nutritional deficiency or abscisic acid in Lemna gibba: The relationship between growth rate and endogenous cytokinin content

The relationship between endogenous cytokinin content and relative growth rate (RGR) was studied in cultures of Lemna gibba L. G3 supplied with daily doses of mineral nutrients that were increased exponentially over time. At the optimal level of nutrient supply the RGR was 30–35% day^-1. The RGR was regulated by adjusting the rate of nitrogen supply, or it was restricted by addition of 0.5 M abscisic acid (ABA). Another approach used to investigate the specific roles of nitrogen (N) and phosphorus (P), was to transfer optimally growing plants to media without N or P but otherwise complete. The plants were harvested at regular intervals for determination of the RGR and levels of cytokinins of the isopentenyladenosine (iPA) and zeatinriboside (ZR) types with an enzyme-linked immunosorbent assay (ELISA). Levels of both iPA- and ZR-type cytokinins decreased when nitrogen was applied to cultures in growth limiting amounts. The cytokinin levels decreased more rapidly than the RGR when either N or P was lacking in the medium, suggesting an early influence of nutrient availability on cytokinin levels which in turn may induce adaptive response by the plant. RGR retardation induced by ABA did not affect cytokinin levels during the first 4 days of the treatment, and the later effects were small. The experiments gave no indication that ABA is involved in the adaptation response of Lemna plants to nutritional stress.Abbreviations ABA - abscisic acid - BAP - benzylaminopurine - ELISA - enzyme-linked-immunosorbent-assay - iP - isopentenyladenine - iPA - isopentenyladenosine - PBS - phosphate-buffered saline - PVP - polyvinylpyrrolidone - RGR - relative growth rate - R_N - relative nitrogen addition rate - Z - trans-zeatin - ZR - trans-zeatin riboside     

Cytokinin production by plant growth promoting rhizobacteria and selected mutants

One of the proposed mechanisms by which rhizobacteria enhance plant growth is through the production of plant growth regulators. Five plant growth promoting rhizobacterial (PGPR) strains produced the cytokinin dihydrozeatin riboside (DHZR) in pure culture. Cytokinin production by Pseudomonas fluorescens G20-18, a rifampicin-resistant mutant (RIF), and two TnphoA-derived mutants (CNT1, CNT2), with reduced capacity to synthesize cytokinins, was further characterized in pure culture using immunoassay and thin layer chromatography. G20-18 produced higher amounts of three cytokinins, isopentenyl adenosine (IPA), trans-zeatin ribose (ZR), and DHZR than the three mutants during stationary phase. IPA was the major metabolite produced, but the proportion of ZR and DHZR accumulated by CNT1 and CNT2 increased with time. No differences were observed between strain G20-18 and the mutants in the amounts of indole acetic acid synthesized, nor were gibberellins detected in supernatants of any of the strains. Addition of 10(-5) M adenine increased cytokinin production in 96- and 168-h cultures of strain G20-18 by approximately 67%. G20-18 and the mutants CNT1 and CNT2 may be useful for determination of the role of cytokinin production in plant growth promotion by PGPR.     

Induction of in vitro flowering in Dendrobium Madame Thong-In (Orchidaceae) seedlings is associated with increase in endogenous N(6)-(Delta (2)-isopentenyl)-adenine (iP) and N (6)-(Delta (2)-isopentenyl)-adenosine (iPA) levels

We analysed the endogenous cytokinin levels of Dendrobium Madame Thong-In seedlings grown in vitro during vegetative and flowering-inductive periods. HPLC was used to fractionate the extracts and radioimmunoassay (RIA) was used for assay of zeatin (Z), dihydrozeatin (DZ), N(6)-(Delta(2)-isopentenyl)-adenine (iP) and their derivatives. Coconut water used in experiments was found to contain high level (>136 pmol ml(-1)) of zeatin riboside (ZR). Protocorms and seedlings cultured in medium with coconut water were found to contain 0.5-3.9 pmol g(-1) FW of the cytokinins analysed. Seedlings (1.0-1.5 cm) cultured in flowering-inductive liquid medium containing 6-benzyladenine (BA, 4.4 muM) and coconut water (CW, 15%) contained up to 200 and 133 pmol g(-1) FW of iP and iPA, respectively. These levels were significantly higher than all other cytokinins analysed in seedlings of the same stage and were about 80- to 150-folds higher than seedlings cultured in non-inductive medium. During the transitional (vegetative to reproductive) stage, the endogenous levels of iP (178 pmol g(-1) FW) and iPA (63 pmol g(-1) FW) were also significantly higher than cytokinins in the zeatine (Z) and dihydrozeatin (DZ) families in the same seedlings. Seedlings that grew on inductive medium but remained vegetative contained lower levels of iPA. The importance of the profiles of iP and its derivatives in induction of in vitro flowering of D. Madame Thong-In is discussed.     

Postharvest changes in endogenous cytokinins and cytokinin efficacy in potato tubers in relation to bud endodormancy

Using an indirect enzyme‐linked immunosorbent assay (ELISA), the effects of postharvest storage duration and temperature on endogenous cytokinins in potato ( Solanum tuberosum L. cv. Russet Burbank) tuber apical bud tissues in relation to endodormancy status were determined. Following fractionation by HPLC, a total of eight cytokinins were detected and these were: zeatin riboside‐5'‐monophosphate (ZRMP), zeatin‐ O ‐glucoside (ZOG), zeatin (Z), zeatin riboside (ZR), isopentenyl adenosine‐5'‐monophosphate (IPMP), isopentenyl adenine‐9‐glucoside (IP‐9‐G), isopentenyl adenine (IP) and isopentenyl adenosine (IPA). Regardless of postharvest storage temperature or endodormancy status, IP‐9‐G was the most abundant cytokinin detected while ZRMP and ZOG were the least abundant ones. In tubers preincubated at a growth‐permissive temperature (20°C) prior to extraction, the loss of endodormancy was preceded by significant increases in the endogenous levels of Z, ZR, IPMP and IP‐9‐G. When stored continuously at a growth‐inhibiting temperature (3°C), significant increases in ZR, IP‐9‐G and IP + IPA were observed. The total content of cytokinins increased by over 7‐fold during postharvest storage and this increase was a result of de novo biosynthesis. Dose‐response studies using IPA and ZR demonstrated a time‐dependent increase in apparent cytokinin sensitivity during postharvest storage. With the exception of IP‐9‐G, injection of any of these endogenous cytokinins resulted in the rapid and complete termination of tuber endodormancy. The significance of these results with respect to endodormancy regulation and the possible mechanisms controlling cytokinin levels in potato tubers are discussed.     

Cytokinins in a genic male sterile line of Brassica napus

Endogenous cytokinin levels were analyzed in four different organs viz., root, stem, leaf and mature flower, of the wild type (cv. Westar) and a genic male sterile (GMS) line of Brassica napus . Various cytokinins viz., bases (Z, DZ), ribosides (ZR, DZR), glucosides (OGZ + OGZR, OGDZ + OGDZR) and nucleotides (ZNT, DZNT) were quantified using Amaranthus betacyanin bioassay and ELISA. The major cytokinin in all the organs was DZ. In general, leaves had the highest levels of cytokinins as compared to the other organs. Root, stem and mature flowers of the wild type plants had higher levels of cytokinins and their metabolites than the GMS line. However. levaves of the GMS line had greater amount of various cytokinins as compared to the wild type. The major cytokinin quantitatively different between the two lines was DZ. The results suggest the possible involvement of cytokinins in the expression of genic male sterility in Brassica napus .     

Endogenous cytokinins in rose petals and the effect of exogenously applied cytokinins on flower senescence

In extracts from rose petals cytokinin activity was detected by Amaranthus bioassay in HPLC eluates corresponding to the standards: Z, ZR, 2iP and 2iPA; subsequently, the presence of two groups of endogenous cytokinins was confirmed by ELISA.Measurements of senesence indicators (cell sap osmolarity and conductivity) and observations of flower vase-life indicated that when the above cytokinins were applied as holding solutions they delayed flower senescence by 34–56% and prolonged rose longevity.Abbreviations B.H.T. 2.6-di-t-buytl-4-methyl phenol - ELISA Enzyme linked Immunosorbent Assay - HPLC High Performance Liquid Chromatography - 2iP isopentenyladenine - 2iPA isopentenyladenosine - Z trans-zeatin - ZR trans-zeatin riboside     

Cytokinin oxidase activity and cytokinin content in roots of sunflower under water stress

Contents of trans-zeatin riboside (ZR), dihydrozeatin riboside (DZR) and N6-(delta2-isopentenyl) adenosine (iPA) was quantified by an indirect ELISA using polyclonal antibodies, in the roots, xylem sap and leaves of pot grown sunflower plants subjected to water stress (RWC of leaves approximately 65 per cent). The delivery rates of all three cytokinins decreased significantly under stress. Cytokinin levels also decreased in roots and in leaves of stressed plants. Three-fold increase in cytokinin oxidase activity was observed in stressed roots after polymin P-ammonium sulphate fractionation. Further purification using Con A agarose resulted in elution of protein with cytokinin oxidase activity and was found to be 30 kDa protein on SDS-PAGE.     

Immunodetection and Identification of N-(o-Hydroxybenzylamino)Purine as a Naturally Occurring Cytokinin in Populus x canadensis Moench cv Robusta Leaves

A highly specific and sensitive enzyme-linked immunosorbent assay (ELISA) was developed for 9-β-d-ribofuranosyl-N⁶-(o-hydroxybenzylamino)purine [(oOH)[9R]BAP] and structurally related cytokinins. As little as 3 femtomoles of the compound could be detected by this method. Cross-reactivity studies demonstrated the specificity of four polyclonal antibodies for (oOH)[9R] BAP and its free base in preference to a range of natural cytokinins and other purines. After evaluating the method by internal standardization employing [2-³H](oOH)[9R]BAP of high specific radioactivity as recovery marker by dilution analyses and by immunohistograms, it was possible to apply ELISA to quantify (oOH)[9R]BAP in plant extracts. In addition to (oOH)[9R]BAP, an unknown cytokinin reacting with the same antibody was detected in high performance liquid chromatography-fractionated extracts of mature Populus × canadensis Moench cv Robusta. The structure of the new compound was determined by gas chromatography-mass spectrometry and finally confirmed by synthesis as N⁶-(o-hydroxybenzylamino)purine.     

Ability of bacterium Bacillus subtilis to produce cytokinins and to influence the growth and endogenous hormone content of lettuce plants

Hormone production by micro-organisms selected as antagonists of pathogenic fungi and the effect of their introduction into soil on hormone content and growth of lettuce plants were studied. Hormones in bacterial cultural media and in plant extracts were immunopurified and assayed using specific antibodies to indolyl-3-acetic acid (IAA), abscisic acid (ABA), and different cytokinins (zeatin riboside (ZR), dihydrozeatinriboside (DHZR) and isopentenyladenosine (iPA)). ZR was shown to be the main cytokinin present in bacterial cultural media as a complex with a high molecular weight component. Inoculation of lettuce plants with bacteria increased the cytokinin content of both shoots and roots. Accumulation of zeatin and its riboside was greatest in roots shortly 2days after inoculation, when their content was 10 times higher than in control. Changes in the content of other hormones (ABA and IAA) were observed at the end of experiments only. Accumulation of cytokinins in inoculated lettuce plants was associated with an increase in plant shoot and root weight of approximately 30% over 8days.     

Altered morphology in transgenic tobacco plants that overproduce cytokinins in specific tissues and organs.

An auxin-inducible bidirectional promoter from the soybean SAUR gene locus was fused to a reporter gene in one direction and a cytokinin biosynthetic gene in the opposite direction and the expression of these fused genes was examined in transgenic tobacco. The Escherichia coli uidA gene, which encodes the enzyme beta-glucuronidase (GUS), was used as the reporter gene and the Agrobacterium tumefaciens ipt gene, which encodes the enzyme isopentenyl transferase, was used as the cytokinin biosynthetic gene. These constructs allowed the overproduction of cytokinins in tobacco in a tissue- and organ-specific manner. Localized overproduction of cytokinins was monitored using the GUS reporter gene and measured by an ELISA assay. The tissue- and organ-specific overproduction of cytokinins produced a number of morphological and physiological changes, including stunting, loss of apical dominance, reduction in root initiation and growth, either acceleration or prolonged delayed senescence in leaves depending on the growth conditions, adventitious shoot formation from unwounded leaf veins and petioles, altered nutrient distribution, and abnormal tissue development in stems. While some of these morphological changes result directly from the localized overproduction of cytokinins, other changes probably result from the mobilization of plant nutrients to tissues rich in cytokinins.     

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.     

Cytokinins and fruit development in the kiwifruit (Actinidia deliciosa). I. Changes during fruit development

The cytokinin content in fruit tissue of the kiwifruit ( Actinidia deliciosa [A. Chev.] C. F. Liang et A. R. Ferguson var. deliciosa cv. Hayward) was monitored during fruit development to identify which cytokinins were present and if they were linked with specific stages of fruit growth. Cytokinins were isolated and purified by column chromatography and high-performance liquid chromatography and quantified by radioimmunoassay. A novel HPLC step utilising an amine column was successfully introduced as a preparative step in the separation of the O - and 9-glucosides from the free bases and ribosides. The radioimmunoassay results were validated, and the different cytokinins identified, by gas chromatography-mass spectrometry. Cytokinins detected in fruit included the cytokinin free bases, zeatin and isopentenyladenine, their ribosides, nucleotides and both O - and 9-glucosides. Both qualitative and quantitative changes of the cytokinins occurred during fruit development. A decrease in cytokinin concentration occurred after anthesis (from 342 pmol g⁻¹ fresh weight at anthesis to 41 pmol g⁻¹ fresh weight 27 days after anthesis). A large increase in cytokinin concentration and content per fruit occurred as the fruit reached commercial maturity (to 1900 pmol g⁻¹ fresh weight). Individual cytokinins showed quite different patterns. Zeatin, in particular, showed a peak in concentration (13 pmol g⁻¹ fresh weight) 11 days after anthesis that correlated with the beginning of the cell division phase of fruit growth. The accumulation of cytokinin (mostly zeatin riboside or zeatin nucleotide) in mature fruit may be of significance for the postharvest storage of kiwifruit fruit.     

Dependence of cytokinin distribution in plants on their physical and chemical properties and transpiration rate

The effect of transpiration on cytokinin accumulation and distribution in 7-day-old wheat (Triticum durum Desf.) seedlings grown on nutrient medium supplemented with zeatin or its riboside was studied. The content of cytokinins in plants and nutrient medium was measured by the immunoenzyme analysis; cytokinin distribution between root cells was assessed immunohistochemically using antibodies against zeatin derivatives. The rate of transpiration was reduced 20-fold by plant placing in humid chamber. At normal transpiration, after 6 h of plant incubation on the solution of zeatin, the level of cytokinins in plant tissues increased stronger than after incubation on the solution of zeatin riboside (by 7.3 and 3.5 times, respectively, as compared with control), although the rates of both cytokinin uptake were equal. Most portions of cytokinins were retained in the roots, which was stronger expressed in the case of free zeatin uptake. A decrease in the rate of transpiration did not affect substantially the zeatin absorption from nutrient medium and the total level of cytokinin accumulation in plants, but these indices were sharply decreased in the case of zeatin riboside. In the zone of absorption of both control roots and roots treated with cytokinins, more intense cytokinin immunostaining was observed in the cells of the central cylinder. The interrelation between cytokinin distribution between the cells and apoplast, their inactivation, and transport over the plant and their form (zeatin or zeatin riboside) used for treatment is discussed.     

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.