Morphogenesis of potato plants in vitro. I. Effect of light quality and hormones

Stem cuttings of potato plants (Solanum tuberosum L., cv. Miranda) were cultured in vitro on MS medium with sucrose either without or with addition of indole-3-acetic acid (IAA) or kinetin (K) under red light (R) or blue light (B). Plants on medium without hormones under R were thin, long, with very small leaves, and produced no or only a few microtubers (after longer-lasting cultivations). In B, plants remained short, thick, with large, wellde-veloped leaves and produced a significant amount of microtubers. Darkening of both roots and shoots strongly promoted tuber formation; the tubers were formed on the darkened part of the plant. IAA had no pronounced effect on plant development in B except for slight lengthening of the stem, and, in longer cultivations, slightly enhanced tuber formation as well. In R, IAA brought about several significant effects: stem reduction and induction of tuber formation being the most significant. Kinetin in R increased tuber formation slightly. In B, kinetin not only strongly stimulated tuber formation, but also increased the total fresh weight and root (+ stolons)/shoot ratio. Results are discussed with regard to the possible role of auxins and/or cytokinins in mediating the morphogenetic effects of light.     

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.     

Changes in the levels of free IAA and cytokinins in potato tubers during dormancy and sprouting

Changes in the levels of free indol-3-ylacetic acid (IAA) and free cytokinins were determined in the course of dormancy and sprouting period in potato tubers(Solanum tuberosum L., cv. Nevskii) stored at 4 °C. The same analyses were performed in potato tubers after Ethrel application, which prolongs dormancy. No significant changes were found in free IAA level during dormancy followed by a rapid decrease during sprouting. After Ethrel application a significant lower IAA level was found 3 weeks after application, but further on no changes in free IAA level between treated and non-treated tubers were detected. Cytokinin level was relatively low and constant till sprouting and increases then by about 55 %, mainly due to an increase in the level of zeatin riboside and isopentenyladenosine. Ethrel application decreased cytokinin level during dormancy slightly, but postpones the increase coupled with sprouting by about 1 month. Thus, IAA does not seem to have a significant effect on tuber dormancy, while cytokinins are probably necessary for sprouting initiation.     

Effects of NO3−, NH4+ and urea nutrition on endogenous levels of IAA and four cytokinins in two epiphytic bromeliads

The long-term effects of different nitrogen sources on the endogenous IAA and cytokinin levels in two bromeliad species were investigated. In nature, Vriesea philippocoburgii is a tank-forming epiphytic bromeliad which uses the tank water reservoir as a substitute for soil, whereas Tillandsia pohliana is a tankless atmospheric epiphytic species. A culture was established from seeds germinated in aseptic condictions, and the plantlets were grown for 6 months in a modified Knudson medium to which was added 8 mol m⁻³ of nitrogen in the form of NO₃⁻, NH₄⁺ or urea. The hormonal contents of the bromeliad shoots were determined by means of high-performance liquid chromatography (HPLC), coupled to an enzyme-linked immunosorbent assay (ELISA) for indole-3-acetic acid (IAA), isopentenyladenine (iP), isopentenyladenosine ([9R]iP), zeatin (Z) and zeatin riboside ([9R]Z). Nitrogen supplied in the form of urea gave the highest values of fresh and dry weights for both species, and this was positively correlated to IAA levels. The cytokinin patterns showed that isopentenyladenosine was the predominant form for both species in all samples. However, urea induced the highest level of this riboside form and also the highest level of total cytokinins for V. philippocoburgii, while NH₄⁺ had the same effect on the atmospheric species. These results are discussed in terms of the different growth habits of these two species in nature. It is suggested that urea may be an important source of nitrogen often found inside the tank of V. philippocoburgii. NO₃⁻ treatment increased the IAA/Cks balance, mainly for V. philippocoburgii, while urea and NH₄⁺ shifted this ratio in favour of cytokinins, thus apparently inhibiting root development in both species.     

Phytohormones,Rhizobium mutants,and nodulation in legumes. VII. Identification and quantification of cytokinins in effective and ineffective pea root nodules using radioimmunoassay

Radioimmunoassays (RIA), employing antisera raised in rabbits against bovine serum albumin conjugates of zeatin riboside, dihydrozeatin riboside, and isopentenyladenosine, were used to estimate levels of these cytokinins and their corresponding bases in samples of effective (nitrogen-fixing, Fix⁺), ineffective (nonnitrogen-fixing, Fix^–) pea root nodules and uninoculated roots. Assays were done on extracts of nodule tissue, 1–2 g fresh weight, or approximately 10 g fresh weight of root tissue, and high specific activity [³H]zeatin riboside was added during preparation of the extract for use as a recovery marker. Two different purification procedures were employed, each involving several purification steps. High performance liquid chromatography (HPLC) was the final step in both procedures. Fractions from HPLC were analyzed by RIA using the appropriate antiserum. The cytokinins, zeatin, zeatin riboside, dihydrozeatin riboside, isopentenyl adenine, and isopentenyladenosine were detected and quantified in nodule tissue, and similarly, in root tissue (with the exception of zeatin, which we were unable to quantify in root tissue). Cytokinin levels in nodule tissue were higher than those in root tissue. The major cytokinins detected in nodule tissue were zeatin, followed by zeatin riboside and then dihydrozeatin riboside. The levels of zeatin and zeatin riboside estimated in nodules in the present study by RIA were of the same order of magnitude, though tending to be a little higher, than values obtained previously by bioassay. Dihydrozeatin riboside was identified with confidence for the first time in nodule tissue. There was a general decline with age in cytokinin levels in nodules, but no major qualitative change in nodule cytokinins with age. For theRhizobium strains examined, the data did not indicate a clear correlation between nodule cytokinin levels and the effectiveness of nodules in nitrogen fixation.     

Changes in Gene Expression from Diploid to Autotetraploid Status of Lycopersicon esculentum

The cytokinin activity of the root exudate, the leaves, and the apices of vegetative and flowering white lupin plants (Lupinus albus L.) was investigated. The level of cytokinin activity in the root exudate decreased over the 11-week experimental period. Four peaks of cytokinin activity were recorded in the root exudate of 8-week-old plants after fractionation on Sephadex LH-20. Two of these peaks co-eluted with zeatin and zeatin riboside. It is suggested that the remaining peaks represent nucleotide and glucoside cytokinins. The cytokinin levels in extracts of the mature leaves fluctuated slightly over the experimental period. Three peaks of activity co-eluting with zeatin, zeatin riboside and the glucoside cytokinins were recorded in extracts of mature leaves of 8-week-old plants. In the apices cytokinin activity could only be detected in the inflorescences of flowering plants. It would appear that cytokinins produced by the roots accumulate in the fully expanded mature leaves, but are utilized in the rapidly growing apical region and in young expanding leaves.     

Effects of Root Cutting on Cytokinin Content in the Shoot Apex Cells of Arabidopsis Plants

The dependence of cytokinin accumulation in the shoot apexes of Arabidopsis plants on the delivery of these hormones from the roots was studied. For the estimation of cytokinin content in the cells, the immunohistochemical localization method using antibodies against zeatin riboside was used. Differential conjugation of free cytokinin bases and their ribosides was used to prevent washing of cytokinins during the dehydration process. Root cutting decreased the immunostaining of zeatin in the cells of the shoot apical meristem, thereby supporting the hypothesis about dependence of cytokinin accumulation in these cells on the hormone delivery from the roots. The level of cytokinins in the cells of the shoot apex decreased under the effect of protonophore, indicating the important role of the secondary-active transmembrane transport process of cytokinins in the maintenance of their level in the cells of the shoot apex.     

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     

Changes in the Content of Indole-3-Acetic Acid and Cytokinins in Spruce, Fir and Oak Trees after Herbicide Treatment

Treatment of spruce, fir and oak trees with herbicides, which may be one of the forest damage inducing agents, caused pronounced changes in the contents and distribution of indole-3-acetic acid (IAA) and cytokinins (CKs) one year after treatment, i.e. at the time of the first microscopically visible damage in treated trees. In Picea pungens IAA content increased in the terminal buds by about 105 % and in the apical buds of the first order branches by 220 %. The same was true for young sprouts of Abies nordmanniana, while in leaves of oak trees IAA content was decreased by 15 % after glyphosate treatment and by 30 % after 2,4-dichlorophenoxyacetic acid (2,4-D) treatment. Another striking feature was a significantly decreased content of IAA in the lower parts of roots in Picea pungens (50 % of the control), which is accompanied by an increase in IAA content in the middle part of the roots (130 %). On the other hand, the IAA content of both sprouts and roots of A. nordmanniana was significantly increased after herbicide treatment.In P. pungens, the content of free cytokinins (sum of zeatin, zeatin riboside, isopentenyladenine and isopentenyladenosine) decreased due to herbicide treatment. The strongest decrease was seen in roots, especially in their upper and middle parts (the average reduction of cytokinin content in roots was 63 %). In the above-ground organs the reduction was seen namely for isopentenyladenine and isopentenyladenosine, while the abundance of zeatin riboside was, on the other hand, higher in treated plants. In Quercus robur leaves, the total content of cytokinins also decreased, namely after glyphosate treatment. In consequence of these changes, CK/IAA ratio decreased pronouncedly in all organs of herbicide-treated trees, with the exception of oak leaves treated by 2,4-D.     

Comparison of effects of bacterial strains differing in their ability to synthesize cytokinins on growth and cytokinin content in wheat plants

The content of cytokinins and pigments together with the morphological parameters and fresh weight were estimated in durum wheat (Triticum durum Desf.) plants 2–4 days after introduction into their rhizosphere of an aliquot of Bacillus suspension using the strains that differed in their ability of producing cytokinins. The experiments were performed under laboratory conditions at the optimum light intensity and mineral nutrition. Inoculation with microorganisms incapable to synthesize cytokinins did not affect the total cytokinin content in the wheat plants, whereas the presence of cytokinin-producing microorganisms in the rhizosphere was accompanied by a considerable increase in the total cytokinin content and the accumulation of individual hormones. On the second day after inoculation, a dramatic increase in zeatin riboside and zeatin O-glucoside contents was observed in the roots, and at the next day the accumulation of zeatin riboside and zeatin was registered in the shoots of treated plants. The increase in cytokinin content promoted plant growth (the increased leaf length and width and a faster accumulation of plant fresh and dry weight). Plant treatment with a substance obtained from microorganisms incapable to synthesize hormones resulted in the insignificant growth stimulation. Plant treatment with a substance obtained from cytokinin-producing microorganisms increased leaf chlorophyll content; in this case, the level of chlorophylls was comparable to that observed in the plants treated with a synthetic cytokinin benzyladenine. The role of cytokinins of microbial origin as a factor providing for growth-stimulating effect of bacteria on plants is discussed.     

Effect of applied and internal hormones on vitrification and apical necrosis of different plants cultured in vitro

Development of vitrification and apical necrosis was followed in Camellia sinensis, Gerbera jamesonii, Malus domestica and hybrid Populus tremula x P. alba shoots cultured in vitro on Murashige & Skoog (MS) medium with different concentrations of growth regulators. High humidity in the culture vessels and excess of BA in the medium were found to be the major factors influencing vitrification. Lack of exogenous cytokinin in the medium during successive subcultures induced apical necrosis in poor-rooting species (Malus domestica, Camellia sinensis). The level of internal phytohormones (ABA, IAA, IPA, 2iP, Z, ZR) was determined in the apple shoots by means of ELISA. The content of internal cytokinins in the vitrified apple shoots was several times greater than in normal ones, which supports the hypothesis that excess of cytokinins, inducing rapid divisions of cells in meristems in the atmosphere with high humidity, is responsible for vitrification. Apical necrosis of the plantlets that appeared after cultivation on cytokinin-free medium is the result of deficiency in endogenous hormones in apple shoots and this being confirmed by analysis of endogenous hormones in apple shoots.Abbreviations BA benzyladenine - BHT butylated hydroxy-toluene - ABA abscisic acid - IAA indole-3-acetic acid - ELISA enzyme-linked immunosorbent assay - IPA isopentenyladenosine - 2iP isopentenyladenine - NAA naphthyl-3-acetic acid - TBS trishydroxymethylaminomethane buffered saline - TLC thin layer chromatography - Z zeatin - ZR zeatin riboside     

The control of bud dormancy in potato tubers. Measurement of the seasonal pattern of changing concentrations of zeatin-cytokinins

A radioimmunoassay, combined with high-performance liquid chromatography, has been used to analyse the zeatin-type cytokinins of potato (Solanum tuberosum L. cv. Majestic) tubers and tuber buds throughout growth and storage. During tuber growth, zeatin riboside was the predominant cytokinin detected in all tissues. Immediately after harvest, the total cytokinin concentration fell dramatically in the storage tissue, largely as a consequence of the disappearance of zeatin riboside. During storage, levels of cytokinins in the storage tissue remained relatively constant, but increased in the tuber buds. In the buds of tubers stored at 2°C there was a 20-to 50-fold increase in total cytokinin over six weeks, coinciding with the natural break of innate dormancy. At 10°C the rise in the level of bud cytokinins was slower, correlating with the longer duration of innate dormancy. Injecting unlabelled cytokinins into tubers in amounts known to induce sprouting gave rise to increases in cytokinin concentrations in the buds of the same order as the increase associated with the natural break of dormancy. Metabolism of injected cytokinins was greater in non-dormant than in dormant tubers. The roles of cytokinin concentration and the sensitivity of the buds to cytokinin in the control of dormancy are discussed.Abbreviations CK cytokinin - FW fresh weight - HPLC high-performance liquid chromatography - RIA radioimmunoassay - tio⁶ade 6-(4-hydroxy-3-methylbut-trans-2-enylamino)-purine=zeatin - tio⁶adeglc⁹ 6-(4-hydroxy-3-methylbut-trans-2-enylamino)-9--D-glucopyranosyl purine=zeatin-9-glucoside - tio⁶ado 6-(4-hydroxy-3-methylbut-trans-2-enylamino)-9--D-ribofuranosyl purine=zeatin riboside - tio⁶ado-[³H]-diol a radioactive derivative of zeatin riboside, synthesised by periodate-oxidation followed by [³H]NaBH₄-reduction - tio⁶AMP 6-(4-hydroxy-3-methylbut-trans-2-enylamino)-9--D-5-phosphoribofuranosyl purine=zeatin riboside 5-monophosphate - t(ioglc⁴)⁶ade 6-(4-O--D-glucopyranosyl-3-methylbut-trans-2-enylamino)-purine=zeatin-O-glucoside     

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.     

Auxin and cytokinin relationships in 24 microalgal strains1

Endogenous auxins and cytokinins were quantitated in 24 axenic microalgal strains from the Chlorophyceae, Trebouxiophyceae, Ulvophyceae, and Charophyceae. These strains were in an exponential growth phase, being harvested on day 4. Acutodesmus acuminatus Mosonmagyaróvár Algal Culture Collection‐41 (MACC) produced the highest biomass and Chlorococcum ellipsoideum MACC‐712 the lowest biomass. The auxins, indole‐3‐acetic acid (IAA) and indole‐3‐acetamide (IAM) were present in all microalgal strains. No other auxin conjugates were detected. IAA and IAM concentrations varied greatly, ranging from 0.50 to 71.49 nmol IAA · g^?1 DW and 0.18 to 99.83 nmol IAM · g^?1 DW, respectively. In 19 strains, IAA occurred in higher concentrations than IAM. Nineteen cytokinins were identified in the microalgal strains. Total cytokinin concentrations varied, ranging from 0.29 nmol · g^?1 DW in Klebsormidium flaccidum MACC‐692 to 21.40 nmol · g^?1 DW in Stigeoclonium nanum MACC‐790. The general trend was that cis‐zeatin types were the predominant cytokinins; isopentenyladenine‐type cytokinins were present in moderate concentrations, while low levels of trans‐zeatin‐type and very low levels of dihydrozeatin‐type cytokinins were detected. Ribotides were generally the main cytokinin conjugate forms present with the cytokinin free bases and ribosides present in similar but moderate levels. The levels of O‐glucosides were low. Only one N‐glucoside was detected, being present in nine strains in very low concentrations. In 15 strains, the auxin content was 2‐ to 4‐fold higher than the cytokinin content.     

Loading and translocation of various cytokinins in phloem and xylem of the seedlings of Ricinus communis L.

The phloem sap of Ricinus seedlings was analyzed for cytokinins and the concentration was compared with that in cotyledons and xylem sap. The dominant cytokinin in the phloem sap was isopentenyladenine (70 nM) when the endosperm was attached to the cotyledons; zeatin, dihydrozeatin and cytokinin-ribosides were present at relatively low concentrations (1–2 nM). Removal of the endosperm and incubation of the cotyledons in buffer led to a sharp decrease in the level of isopentenyladenine in the phloem sap, down to the value for zeatin, namely 1–2 nM. Similar low cytokinin concentrations were found in the xylem sap, too, whereas in the cotyledons the cytokinin content was at least 10-fold higher. Incubation of the cotyledons with various cytokinins (isopentenyladenine, zeatin and their ribosides) led to an increase of each of the applied cytokinins in the phloem sap, including also the metabolically closely related cytokinins. Zeatin was especially well loaded. It is concluded that the phloem translocates most free bases and ribosides of the various cytokinin species, if they are offered to the phloem. The data also show that the cytokinin levels in the phloem, which may be far higher than in the xylem, are subject to strong fluctuations depending on the physiological situation.This work was supported by the Deutsche Forschungsgemeinschaft (SFB 137). The experimental assistance by P. Geigenberger and the help in cytokinin analysis by Dr. A. Fußeder, Dr. B. Wagner, W. Peters (all Bayreuth) and by Prof. E. Weiler (Bochum) is gratefully acknowledged. Also the constructive discussions with Profs. E. Weiler (Bochum) and E. Beck (Bayreuth) are much appreciated.     

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.     

Transgenic tobacco plants co-expressing Agrobacterium iaa and ipt genes have wild-type hormone levels but display both auxin- and cytokinin-overproducing phenotypes

Transgenic tobacco lines simultaneously expressing the Agrobacterium iaaM, iaaH and ipt genes, obtained by crossing lines expressing ipt with lines expressing iaaM and iaaH, were used to study in planta interactions between auxin and cytokinins. All phenotypic traits of the respective parental lines characteristic of cytokinin and auxin overproduction were present in the cross. Indole-3-acetic acid (IAA) and combined zeatin riboside (ZR) and zeatin riboside-5'-monophosphate (ZRMP) contents were analysed by mass spectrometry in young, developing leaves from the cross, the parental lines and the wild type. Unexpectedly, hormone levels in the cross were very similar to wild-type levels. Thus IAA levels in the cross were much lower throughout vegetative development than in the parental IAA overproducing line, although expression of the bacterial IAA biosynthesis genes was not reduced. The results suggest that effects on apical dominance, adventitious root formation, leaf morphology and other traits commonly +/- associated with IAA and cytokinin overproduction, and observed in the iaa E ipt cross, cannot be explained solely by analysis of auxin and cytokinin contents in individual organs. As traits associated with both hormones are expressed in close spatial and temporal proximity, it is likely that cellular resolution of hormone contents is essential to explain physiological responses to auxins and cytokinins.     

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.     

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)     

Mass-spectrometric quantitation of cytokinins in tobacco crown-gall tumours induced by mutated octopine Ti plasmids of Agrobacterium tumefaciens

The levels of the major cytokinins, zeatin, zeatin riboside, zeatin riboside-5-monophosphate and zeatin-7-glucoside were measured in tobacco (Nicotiana tabacum L.) crown-gall tissues carrying insertion and deletion mutations in the T-DNA. Measurements were made by combined gas chromatography-mass spectrometry using selected ion monitoring with ¹⁵N- and ²H-labelled internal standards. The results demonstrate that, relative to wild-type tumour tissue, cytokinin levels are considerably elevated in tissues lacking functional T-DNA auxin-biosynthetic genes. From a detailed analysis of the major cytokinin metabolites it is concluded that a reduction in the extent of cytokinin degradation via N⁶-side-chain cleavage is an important factor leading to increased cytokinin levels in these tissues.Abbreviations IAA indole-3-acetic acid - SIM selected ion monitoring - Z zeatin - [7G]Z zeatin-7-glucoside - [9R]Z zeatin-9-riboside - [9R-5P]Z zeatin riboside-5-monophosphate