Identification of kinetin and kinetin riboside in coconut (Cocos nucifera L.) water using a combined approach of liquid chromatography-tandem mass spectrometry, high performance liquid chromatography and capillary electrophoresis

Kinetin (free base and riboside), which was assumed by many scientists to be a synthetic cytokinin plant growth hormone, has been detected for the first time in the endosperm liquid of fresh young coconut fruits ("coconut water"). To facilitate the study, we developed a sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the identification and quantification of kinetin and kinetin riboside in purified coconut water extract sample. Following a solid-phase extraction of cytokinins in coconut water using C18 columns, the samples were further purified by Oasis MCX columns and analyzed by LC-MS/MS for kinetin and kinetin riboside. Detection by mass spectrometry was carried out using selected reaction monitoring (SRM) mode, by identifying the putative kinetin and kinetin riboside based on their characteristic fragments. Based on a signal-to-noise ratio of 3, the limits of detection in SRM mode were 0.02 microM and 0.005 microM for kinetin and kinetin riboside, respectively. Furthermore, optimal conditions for a baseline chromatographic separation of 18 cytokinin standards by high performance liquid chromatography (HPLC) were developed. The HPLC method had been employed for the confirmation and further fractionation of kinetin in coconut water extracts. The confirmation and fractionation of kinetin riboside was carried out using a further modified HPLC program due to the presence of other interfering material(s) in the sample matrix. Finally, fractions of putative kinetin and kinetin riboside collected from HPLC eluate of coconut water sample were further authenticated by independent capillary zone electrophoresis (CZE) experiment.     

Judicious use of kinetin to improve growth and yield of rice in nickel contaminated soil

The present study was conducted to evaluate the effect of kinetin on growth and yield of rice in the presence and absence of nickel contamination. Rice seedlings were dipped in kinetin solution (10^?3, 10^?4 and 10 M^?5) for 2 hours and transplanted in pots having soil contaminated with nickel sulfate @ 130 mg kg^?1. Experiment was laid out according to completely randomized design with four replications. Results revealed that kinetin significantly improved growth and yield of rice grown in nickel contamination. Kinetin @ 10^?4 M showed maximum improvement in plant height, paddy yield, 1000 grain weight, number of tillers and panicles up to 9.76, 15.72, 11.77, 11.87, and 10.90%, respectively, as compared to plants grown in contaminated soil without kinetin. Kinetin also improved the uptake of nutrients (NPK) in straw and grain of plants grown in Ni contaminated soil. Plants treated with kinetin had more concentration of Ni in shoot but less in grain compared to plants grown in Ni contaminated soil without application of kinetin. The application of kinetin can reduce stress effect on plants through improvement in the biomass of plant. This strategy could be used to increase the phytoextraction of Ni from the contaminated soil.     

Regulation of Growth in Avena Stem Segments by Gibberellic Acid and Kinetin

Kinetin at physiological concentrations causes significant reduction of GA₃-promoted growth in excised Avena stem segments. Kinetin is therefore considered to be a gibberellin-antagonist in this system. A Lineweaver-Burke plot reveals that kinetin acts non-competitively with GA₃. The kinetin inhibition of GA₃-promoted growth can be seen within 6 hours. It was found that soluble protein is markedly increased by kinetin in the tissue during the first 3 hours, thus preceding the inhibition of GA₃-promoted growth by several hours. At the cellular level, kinetin negated the blocking effect of GA₃ on cell division in the intercalary meristem portions of these segments. In fact, kinetin promotes both lateral and longitudinal cell divisions in intercalary meristem cells.     

Salinity and hormone interactions in affecting growth,transpiration and ionic relations ofPhaseolus vulgaris

Addition of either abscisic acid (ABA) or kinetin at 10⁻⁶ M to salinized media (20–120mM NaCl) induced remarkable effects on growth ofPhaseolus vulgaris plants. Whereas ABA inhibited the plant growth and the rate of transpiration, kinetin induced stimulation of both parameters. Moreover, ABA increased proline and phosphorus concentrations in the salinized plants whilst kinetin decreased them. ABA induced stimulation of the transport of K, Ca and Cl from root to shoot, accumulation of K, Na and Cl in root cells and inhibits the transport of Na and accumulation of Ca. Kinetin appeared to inhibit the transport and accumulation of Na and Cl, transport of K, and stimulates the accumulation of K and Ca as well as the transport of Ca. The highest influence of both ABA and kinetin was mostly observed when these hormones were used in combination with the highest concentration of NaCl (120 mM) in the medium.     

Paclobutrazol enhances minituber production in Norland potatoes

The effect of two plant growth regulators, paclobutrazol and kinetin, on minituber yield in greenhouse-grown Norland potatoes was investigated. Plants were treated with paclobutrazol at 450 mg/L, kinetin at 10 mg/L, or a combination of paclobutrazol at 450 mg/L + kinetin at 10 mg/L as single foliar applications at early stolon initiation. A set of plants sprayed with water served as the control. The experiment was conducted twice. In both cases, paclobutrazol nearly doubled the number of usable tubers/plant without affecting total tuber yield. Kinetin had no effect either on tuber number or tuber weight. Kinetin applied as a combination with paclobutrazol decreased the effectiveness of paclobutrazol on tuber number by 13–20%. Paclobutrazol treatments prolonged tuber dormancy by approximately 3 weeks. The results suggest that paclobutrazol treatment would be effective in enhancing potato minituber production under greenhouse conditions.Abbreviations PTZ paclobutrazol - KIN kinetin     

Influence of arbuscular mycorrhizal fungi and kinetin on the response of mungbean plants to irrigation with seawater

Increasing use of saline water in irrigation can markedly change the physical and chemical properties of soil. An experiment was carried out to investigate the interaction between the mycorrhizal fungus Glomus clarum, isolated from a saline soil, and kinetin on the growth and physiology of mungbean plants irrigated with different dilutions of seawater (0, 10, 20, and 30%). The growth, chlorophyll concentration and sugar content of mycorrhizal plants was greater than that of non-mycorrhizal plants under all conditions (with or without seawater). The dry weight of both mycorrhizal and non-mycorrhizal mungbean plants irrigated with 10% seawater was significantly increased by treatment with kinetin. The mycorrhizal symbiosis increased root:shoot dry weight ratio, concentrations of N, P, K, Ca and Mg, plant height, protein content, nitrogen or phosphorus-use efficiencies, and root nitrogenase, acid or alkaline phosphatase activities of seawater-irrigated mungbean plants, with little or no effect of kinetin. Kinetin treatment generally decreased chlorophyll concentration and sugar content in mycorrhizal plants as well as Na/N, Na/P Na/K, Na/Ca and Na/Mg ratios. Root colonization by G. clarum was increased by irrigation with seawater, and kinetin had no consistent effect on fungal development in roots. This study provides evidence that arbuscular mycorrhiza can be much more effective than kinetin applications in protecting mungbean plants against the detrimental effects of salt water.     

Effect of kinetin concentration and methods of application on seed germination, yield components, yield and fiber properties of the Egyptian cotton (Gossypium barbadense)

Two field experiments in 1993 and 1994 as well as a laboratory germination experiment were conducted on the Egyptian cotton cultivar Giza 75 (Gossypium barbadense) to determine the effect of six concentrations of kinetin (6-furfurylaminopurine) ranging from 0 to 10.0 mg l(-1) and three different methods of application: (A) seeds were soaked for 24 h before germination (laboratory experiment) or sowing (field experiment) in solutions of different kinetin concentration, (B) cotton plants were sprayed twice with different kinetin concentrations at 60 and 75 days after sowing (DAS) during the square initiation and the beginning of bolling stages, at volume solution of 480 l ha(-1). (C) Seeds were soaked in kinetin solutions before sowing as method A. In addition cotton plants were sprayed twice as method B with the same kinetin concentrations. Kinetin application improved seed viability and seedling vigour as shown by lengths of the hypocotyl, radicle and the entire seedling, as well as seedling fresh weight. Moreover, significant increases were recorded in the number of open bolls/plant, boll weight, lint and seed indices, seed cotton yield/plant, and seed cotton and lint yields/plot. The highest means were obtained at 5 mg kinetin l(-1) concentration and under method C of application. Treatments generally, had no significant effects on lint percentage, yield earliness and fiber properties. These results show that, the use of kinetin at 5 mg l(-1) for pre-soaking seeds before planting and spraying cotton plants at 60 and 75 DAS with the same concentration could improve cotton germination, seed cotton and lint yields.     

Development of Asparagus plumosus Shoot Tips Grown in vitro

Lateral shoot tips from young Asparagus setaceus (Kunth) Jessop (syn. A. plumosus Baker) shoots were grown on a modified Murashige and Skoog medium. Tips from 5 to 20 mm lateral shoots had significantly better growth and development than tips from lateral shoots (2 mm) still covered by leaf-scale. The optimum temperature for growth and development of the explants was 17 to 24°C. The initial growth was fast at 24°C but stopped after about 4 weeks. At 17°C the growth was slow but in return the cultures continued to grow. Kinetin was necessary for growth. Without any kinetin all cultures died. Optimum growth was found with 2 mg/l kinetin. There was no growth at all with IAA alone. A low IAA concentration had no effect, but at high concentrations IAA inhibited the kinetin induced growth.     

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.     

The Effect of Abscisic Acid on the Metabolism of Kinetin in Detached Leaves of Rumex pulcher

The effect of abscisic acid (ABA) and of gibberellic acid (GA₂)on the metabolism of kinetin in detached leaves was investigated.Kinetin labelled in either the purine ring or the N-6 substitutedside chain was fed into leaves of Rumex pulcher. The leaveswere extracted 4 or 20 h later and the extract chromatographedon paper which was subsequently scanned for radioactivity. Fourmajor radioactive peaks were revealed. Two peaks were tentativelyidentified, one kinetin and its riboside, and the other adenine.ABA effected a significant decrease in the transformation ofkinetin to adenine while GA₃ had no consistent effect. The possiblemeaning of this finding is discussed.     

Effects of Polyamines and Kinetin on In Vitro Frond Senescence in Lemna aequinoctialis 6746

Both polyamines and kinetin could retard the loss of chlorophyll during dark-induced senescence in excised frond of Lernna aequinoctialis 6746. The effect of polyamines on retarding the chlorophyll loss was stronger than that of kinetin. Kinetin remarkably inhibited the loss of soluble proteins and the increase of protease activity, while no similar effects were observed from polyamines. An inhibitor of polyamine biosynthesis, methylglyoxal bis- (guanyl- hydrazone) (MGBG), slightly increased the loss of chlorophyll and soluble proteins. During senescience, both the increase of putrescine (Put) content and the decrease of spermidine (Spd) content were inhibited by kinetin at the concentration of 0.05 mmol/L, but the spermine (Spm) level was not affected by kinetin. The arginine decarboxylase (ADC) activity was dominant in frond of Lemna aequinoctialis 6746. Kinetin slightly increased ADC activity, while it had no marked effect on ornithine decarboxylase (ODC) and s-adenosylmethionine decarboxylase (SAMDC). The possible relationship between polyamines and cytokinins in retarding senescence was also discussed.     

多胺与激动素对稀脉浮萍离体叶状体衰老的影响

多胺与ＫＴ 都可抑制暗诱导衰老的稀脉浮萍（Ｌｅｍ ｎａ ａｅｑｕｉｎｏｃｔｉａｌｉｓ）离体叶状体的叶绿素损失,且多胺的作用大于ＫＴ。ＫＴ 还显著抑制蛋白质的损失与蛋白酶活性的上升,而多胺对此却无大的影响。０．０５ ｍ ｍ ｏｌ／Ｌ的甲基乙二醛二脒基－腙（ＭＧＢＧ）轻微促进叶绿素和蛋白质的损失。０．０５ ｍ ｍ ｏｌ／Ｌ的ＫＴ 可抑制衰老过程中腐胺（Ｐｕｔ）的上升和亚精胺（Ｓｐｄ）的下降,而对精胺（Ｓｐｍ ）无明显影响。在稀脉浮萍中,精氨酸脱羧酶（ＡＤＣ）活性占优势。ＫＴ 可轻微促进ＡＤＣ 活性,而对鸟氨酸脱羧酶（ＯＤＣ）和Ｓ－腺苷甲硫氨酸脱羧酶（ＳＡＭＤＣ）活性无显著影响。讨论了多胺与细胞分裂素在抑制植物叶片衰老过程中作用途径的可能关系     

The Action of Kinetin in Improving the Water Balance and Delaying Senescence Processes of Cut Rose Flowers

Kinetin delays the fading of cut rose (Rosa hyb. cv. Golden Wave) flower shoots exposed to water stress conditions. Using leafless flower shoots (i.e., a plant system devoid of stomates), we demonstrated that the main initial effect of kinetin was on increasing water uptake and petal growth. Later, kinetin slowed down processes associated with both senescence and stress (RNase activity and dry weight reduction), and maintained petal turgidity for an extended period.     

EFFECTS OF PLANT GROWTH SUBSTANCES ON IN VITRO FIBER DEVELOPMENT FROM UNFERTILIZED COTTON OVULES

Fertilization of cotton ovules was prevented by removal of styles and stamens on the morning of anthesis. Forty-eight hr later ovaries were harvested and ovules were aseptically transferred to liquid culture medium supplemented with various plant growth substances. In the absence of phytohormones, ovules browned and failed to increase in size or produce fibers. Indoleacetic acid and gibberellic acid provided for ovule growth and fiber development. Kinetin provided for ovule growth only. The ovule's capacity for indoleacetic acid- or gibberellic acid-stimulation of fiber development was reduced by high concentrations of kinetin or abscisic acid. Low concentrations of kinetin partially reversed the inhibitory effect of abscisic acid.     

Auxin-gibberellin relationships in their effects on hypocotyl elongation of light-grown cucumber seedlings VI. Promotive and inhibitory effects of kinetin

The interaction of kinetin with IAA and GA₃ on the elongationof hypocotyl sections of Cucumis sativus L. cv. National Picklingwas studied. Kinetin in the concentration range of 10^–7M to 10^–4 M markedly inhibited IAA-induced elongation,while in a lower range from 10^–10 M to 10^–8 M, itsynergistically enhanced IAA-induced elongation. Kinetin alonein this range had no effect. A 5-to 15-min pulse treatment seemsenough to induce the maximum effect for both inhibition andpromotion. Since the magnitude of the maximum inhibition dependedon the concentration and not on the duration of treatment, thereaction in the cell caused by kinetin seemed to be completedwithin a short period. Washing of the sections with distilledwater after kinetin treatment (30 min) did not significantlyeliminate the kinetin effect. This probably indicates that thebinding of kinetin molecules to a supposed acceptor is not reversible.Interaction of kinetin with GA₃ in their pretreatment effectson IAA-induced elongation shows that in the inhibitory concentrationrange, the kinetin effect was partly overcome by GA₃, and thatin the promotive range, the magnitude of the enhancement wasdetermined by kinetin regardless of the presence of GA₃. Theeffect of kinetin seems to dominate over that of GA₃ indicatingthat the modes of their pretreatment effects differ from oneanother. (Received June 24, 1977; )     

Auxin and Kinetin Induced Changes of Callus Cell Wall Composition of Abutilon avicennae Gaertn

In a previous report we have shown that the arrangement of callus cell wail fibrils of Abutilon avicennae could be induced to change under IAA (2 ppm) and kinetin (10 ppm) treatments. Kinetin at this concentration was shown to be able to induce callus cell differentiation and form tracheary elements by changing the orientation of the wall fibrils. It was thus assumed that the hormonal induction of cellular differentiation and structual change of the cell wall may possibly be accompanied by the simultaneous changes of chemical composition of the wall. Attempt was therefore made to investigate if such changes do occur in vitro under the influence of phytohormones. Suspension cell-culture of Abutilon avicennae was used in this experiment to study the hormonal effect on the incorporation of H3-glucose into the cell wall polysaccharides. Analysis of neutral sugars of the cell wall following IAA (2ppm) and kinetin (10ppm) treatments was carried out with a gas chromatography. The results obtained in this experiment are shown in tables 1-2 and figures 1, It was found that the auxin was capable of promoting the synthesis of all neutral sugars, among which the glucose and the maunose in particular, increased tremendously. When H3-glucose was added to the culture medium, IAA was found to enhance the incorporation of the isotopes into the matrix polysaccharides (hemiceUulose and pectin). The result demonstrates clearly that the primary function of IAA is to stimulate the synthesis of hemicellulose composition and, as a consequence, the cell wall retained at the primary growth stage. Kinetin, on the other hand, showed an inhibitory effect on most of the neutral sugars except glucose and mannose. It appeared to have a striking inhibitory action on the synthesis of arabinose and rhanmose (a special composition of pectic substance). It also limited the incorporation of H3-glucose into the pectic substance. It is, therefore, suggested that the action of kinetin may mainly be inhibitory on the synthesis of pectic composition. The decreased rate of pectin synthesis would implicate that the cell wall has been advan ced into the phase of secondary growth. The results presented here agree fairly well with our connotation that there is a parallel relationship between cellular morphology and biochemical characteristics during cell wall differentiation and growth.     

]Effect of kinetin on the formation of chromosome aberrations during irradiation of Crepis capillaris L. seeds and its radioprotective effect

The object of this investigation was the mutagenic effect of kinetin (6-phurphuryl-aminopurine) on chromosomes of Crepis capillaris L. Dry seeds were soaked in the 0.05% kinetin solution for 4, 6 and 8 hours before the irradiation with X-rays. It is observed that soaking of dry seeds in kinetin solution stimulates the appearance of chromatid aberrations for almost two hours earlier, than in the control (soaking seeds in water). A regular increase of the chromatid-type rearrangements with increasing the duration of soaking in the kinetin solution was observed (3.3%, 16-20% and almost 50% at 4 hours; 6 hours' and 8 hours' soaking respectively). This increase is indicative for the effect of kinetin on the processes taking place in the cell before the irradiation. Apparently kinetin stimulates the onset of the stage S for a certain proportion of cells, and thus at the moment of irradiation the cell population is at the asynchronous state. A considerable radioprotective effect of kinetin was observed in all the variants of the experiment, which is not associated with the capacity of stimulating the initiation of chromatid aberrations inherent in kinetin. Kinetin alone (without irradiation) exerts no mutagenic effect on the cells of Crepis capillaris.     

Specific Effects of Kinetin on the Uptake of Monovalent Cations by Sunflower Cotyledons

Kinetin promoted the uptake of K⁺ and Rb⁺ into detached sunflower cotyledons. This action was concomitant with an acceleration of growth. A slighter promotion of Li⁺ uptake was also noted, but there was no consistent influence on that of Na⁺. A small inhibitory effect on NH₄⁺₄ uptake was apparent when the latter was computed per average weight of sample during the course of incubation. Light also promoted the growth of the cotyledons, but depressed their capacity to absorb potassium. The action of kinetin on cotyledons removed from 5–7 day old seedlings was weaker than on those removed from 2–4 day old seedlings with regard to growth but stronger with regards to K⁺ uptake. When K⁺ uptake by cotyledons taken from 7-day old seedlings was followed with time the kinetin effect was already detectable within a few hours, but it became more pronounced after 10 hours' incubation. Kinetin did not accelerate growth or K⁺ uptake in hypo-cotyl segments. IAA, which was previously shown to promote these processes in hypocotyl segments, inhibited them in cotyledons. A working hypothesis is suggested according to which endogenous auxins and cytokinins regulate the absorption of K⁺ in shoot cells of the intact plant in a manner similar to that in which they act in excised tissues and in this way affect the distribution and redistribution of K⁺ in the shoot; and that they are among the factors which determine the selectivity of ion uptake in the intact plant.     

THE INFLUENCE OF GROWTH SUBSTANCES ON THE INDUCTION OF APOGAMY IN PTERIDIUM GAMETOPHYTES

Experiments employing sequences of three media demonstrated the effect of growth substances on the induction of apogamy. The most effective sequence was 4% sucrose 1–14 days, 4% sucrose plus growth substance 15–28 days, and 0.1% sucrose 29–56 days. In this sequence concentrations of NAA, IAA, and GA promoted apogamous shoot formation. A higher NAA concentration than optimal for shoot formation stimulated apogamous root formation in all medium sequences. Kinetin was without effect or inhibitory to apogamy. Combination of kinetin/GA or GA/NAA concentrations did not increase the apogamous response. One combination of the kinetin/NAA concentrations had a synergistic effect on the apogamous shoot formation. Additions of GA to the synergistic kinetin/NAA combination had an antagonistic effect on the apogamous response.     

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.