Showing papers on "Zeatin published in 1989"

Alterations of Endogenous Cytokinins in Transgenic Plants Using a Chimeric Isopentenyl Transferase Gene.

Abstract: Cytokinins, a class of phytohormones, appear to play an important role in the processes of plant development. We genetically engineered the Agrobacterium tumefaciens isopentenyl transferase gene, placing it under control of a heat-inducible promoter (maize hsp70). The chimeric hsp70 isopentenyl transferase gene was transferred to tobacco and Arabidopsis plants. Heat induction of transgenic plants caused the isopentenyl transferase mRNA to accumulate and increased the level of zeatin 52-fold, zeatin riboside 23-fold, and zeatin riboside 5[prime]-monophosphate twofold. At the control temperature zeatin riboside and zeatin riboside 5[prime]-monophosphate in transgenic plants accumulated to levels 3 and 7 times, respectively, over levels in wild-type plants. This uninduced cytokinin increase affected various aspects of development. In tobacco, these effects included release of axillary buds, reduced stem and leaf area, and an underdeveloped root system. In Arabidopsis, reduction of root growth was also found. However, neither tobacco nor Arabidopsis transgenic plants showed any differences relative to wild-type plants in time of flowering. Unexpectedly, heat induction of cytokinins in transgenic plants produced no changes beyond those seen in the uninduced state. The lack of effect from heat-induced increases could be a result of the transient increases in cytokinin levels, direct or indirect induction of negating factor(s), or lack of a corresponding level of competent cellular factors. Overall, the effects of the increased levels of endogenous cytokinins in non-heat-shocked transgenic plants seemed to be confined to aspects of growth rather than differentiation. Since no alterations in the programmed differentiation pattern were found with increased cytokinin levels, this process may be controlled by components other than absolute cytokinin levels.

Effect of Zinc Nutritional Status on Growth, Protein Metabolism and Levels of Indole-3-acetic Acid and other Phytohormones in Bean (Phaseolus vulgaris L.)

Abstract: Bean (Phaseolus vulgaris L. var. Prelude) plants were grown for 17 d under controlled environmental conditions with varied Zn supply in the nutrient solution. The concentrations of amino acids; indole-3-acetic acid, IAA; abscisic acid, ABA; isopentenyl adenine, I-Ade; isopentenyl adenosine, I-Ado; zeatin, Z; and zeatin riboside, ZR were determined in various shoot fractions. The growth of plants, especially shoot growth, was severely depressed under conditions of Zn deficiency. Simultaneously, concentrations of soluble protein and chlorophyll decreased, whereas amino acid concentrations increased several-fold. In the Zn deficient plants, the level of IAA in the shoot tips and young leaves decreased to about 50% of that in Zn-suflicient plants. A similar decrease occurred in the ABA levels of shoot tips. In contrast, Zn deficiency was without effect on cytokinin levels in the leaves. Re-supply of Zn to the deficient plants for up to 96 h significantly increased shoot growth, soluble protein, and IAA levels up to the values of Zn-sufficient plants. Simultaneously, the concentration of amino acids dropped to low levels. The effect of Zn nutritional status on the tryptophan level was parallel to that of most of the other amino acids. The results confirm the role of Zn in protein synthesis and demonstrate that the decrease in IAA level in Zn-deficient plants is not brought about by impaired synthesis of tryptophan. It is also unlikely that in Zn-deficient plants the conversion of tryptophan to IAA is specifically inhibited.

Somatic Embryogenesis and Plant Regeneration in Suspension Cultures of Dessert (AA and AAA) and Cooking (ABB) Bananas ( Musa spp.)

Abstract: Proembryogenic calli were initiated from basal leaf sheaths and rhizome tissue on modified Schenk and Hildebrandt (SH) medium with 30 μM 3,6–dichloro–2–meth–oxybenzoic acid (Dicamba). Cell suspensions were maintained in half–strength Murashige and Skoog (MS) medium supplemented with 20 μM Dicamba. The development of somatic embryos was promoted in cell suspensions 3–4 weeks after subculture in liquid modified MS medium with 5 μM zeatin. Characteristic stages of embryonic development were recapitulated and histological examination confirmed bipolar organization of somatic embryos. Conversion into plantlets took place in double layer media system composed of solid half strength MS medium with 5 μM zeatin and 1 g/l charcoal and liquid, hormone–free, half strength MS medium. In four Musa genotypes several hundred plantlets were regenerated and transferred into soil where they continued to grow. Somatic embryogenesis represents a significant step in developing a new breeding strategy for apomictic banana and plantain species.

Cytokinin Production by Bradyrhizobium japonicum

Abstract: Although there is considerable circumstantial evidence for the involvement of cytokinins in legume nodulation, the cytokinins produced by rhizobia have not been well characterized. Bradyrhizobium japonicum 61A68, a bacterium which nodulates soybean (Glycine max [L.] Merr.), was grown in defined medium. Cytokinins were purified from the culture medium by Amberlite XAD-2 chromatography and fractionated by column chromatography on Sephadex LH-20 in 35% ethanol. Pooled fractions from the Sephadex column were analyzed for cytokinin activity with the tobacco callus bioassay. Cytokinin activity was observed in fractions corresponding to the elution volumes of zeatin, ribosylzeatin, and methylthiozeatin. No activity corresponding to the elution volumes of isopentenyladenine or its riboside was found. Total cytokinin activity in the B. japonicum culture filtrate was equivalent to approximately 1 microgram of kinetin per liter. Transfer RNA was isolated from B. japonicum cells by phenol extraction, followed by potassium acetate extraction, cetyltrimethylammonium bromide precipitation, and DEAE cellulose chromatography. Transfer RNA was enzymically hydrolyzed to nucleosides. High performance liquid chromatographic analysis of cytokinin nucleosides showed peaks corresponding to the retention times of trans-ribosylzeatin, methylthioribosylzeatin, isopentenyladenosine, and methylthioisopentenyladenosine. Analysis of the tRNA hydrolysate by Sephadex LH-20 chromatography and tobacco bioassay showed cytokinin activity in fractions corresponding to ribosylzeatin, methylthioribosylzeatin, and isopentenyladenosine. The presence of the trans isomer of ribosylzeatin was also determined by enzyme immunoassay.

Biosynthesis of cytokinins by Azotobacter chroococcum

Abstract: Cytokinins were detected in culture nitrates of Azotobacter chroococcum, A. beijerinckii, A. vinelandii, Pseudomonas fluorescens and P. putida . The most prolific producer of cytokinins was A. chroococcum . Several purine ring constituents [adenine (ADE), adenosine, adenosine-5′-monophosphate, hypoxanthine, inosine and inosine-5′-monophosphate] and isoprenoid compounds [isopentyl alcohol (IA), 3-methyl-3-buten-1-ol, 3-methyl-2-buten-1-ol and isoprene] were tested for their suitability as physiological precursors to cytokinin biosynthesis. The isolation and quantification of zeatin riboside, dihydrozeatin riboside, t - and c -zeatin, isopentyl adenine and its riboside were monitored by high performance liquid chromatography (HPLC), u.v. spectrometry and bioassay. The stimulation of A. chroococcum to produce cytokinins by the addition of ADE and IA was monitored during the growth of the bacterium and tested under various environmental conditions in liquid media.

An enzyme mediating the conversion of zeatin to dihydrozeatin in phaseolus embryos.

Abstract: A reductase catalyzing the conversion of zeatin to dihydrozeatin was detected in soluble fractions of immature Phaseolus vulgaris embryos. The enzyme was partially purified by ammonium sulfate fractionation and affinity, gel filtration, and anion exchange chromatography. NADPH was the only cofactor required for enzyme activity, and the pH optimum was 7.5 to 8.0. The enzyme did not recognize compounds closely related to zeatin, such as ribosylzeatin, cls-zeatin, O-xylosylzeatin, N6-(Δ2-isopentenyl)adenine, or N6-(Δ2-isopentenyl)adenosine. No conversion of dihydrozeatin to zeatin by the enzyme was observed. Two forms of the reductase could be separated by either gel filtration or anion exchange high performance liquid chromatography. The high molecular weight isozyme (Mr 55,000 ± 5,000) eluted as the second peak from the anion exchange column, while the low molecular weight isozyme (Mr 25,000± 5000) was less negatively charged. The results suggest that side chain reduction occurs at the free base level. In addition, Phaseolus embryos are useful for the detection of zeatin-specific metabolic enzymes.

Zeatin Glycosylation Enzymes in Phaseolus Isolation of O-Glucosyltransferase from P. lunatus and Comparison to O-Xylosyltransferase from P. vulgaris

Abstract: An enzyme catalyzing the formation of O-glucosylzeatin in immature embryos of Phaseolus lunatus was purified 2500-fold using ammonium sulfate precipitation followed by affinity and anion exchange chromatography. The enzyme uses trans-zeatin as substrate (K(m) 28 micromolar) but not cis-zeatin, ribosylzeatin, or dihydrozeatin. Both UDP-glucose and UDP-xylose can serve as glycosyl donors, with K(m)s of 0.2 and 2.7 millimolar, respectively, for the formation of O-glucosylzeatin and O-xylosylzeatin. In comparison, the UDPxylose-zeatin:O-xylosyltransferase (JE Turner, DWS Mok, MC Mok, G Shaw [1987] Proc Natl Acad Sci USA 84: 3714-3717) isolated by the same procedures from P. vulgaris embryos uses only UDP-xylose as donor substrate and the K(m)s for both zeatin and UDP-xylose are much lower (2 and 3 micromolar, respectively). The chromatographic behavior on affinity columns and molecular weights (approximate M(r) 44,000 daltons) of the two enzymes are similar. Results from substrate competition experiments and enzyme separation by anion exchange HPLC indicate a single, distinct, zeatin O-glycosylation enzyme occurs in embryos of each of these Phaseolus species.

In vitro shoot regeneration of Populus deltoides: effect of cytokinin and genotype.

Abstract: Treatment differences were observed in the in vitro adventitious shoot regeneration response from internodal explants of three genotypes of Populus deltoides cultured on media supplemented with five concentrations each of the cytokinins 6-benzyladenine, 2-isopentyladenine, and zeatin. For each of the three genotypes, the greatest number of shoots were consistently regenerated on media containing the cytokinin zeatin. Tissue necrosis resulted when explants from any of the three genotypes were cultured on media supplemented with 6-benzyladenine. A zeatin concentration by genotype interaction was also observed. Genotypic differences in shoot regeneration were observed for 16 genotypes of Populus deltoides when cultured on medium supplemented with 0.5 mgL−1 zeatin. Six genotypes were highly recalcitrant and failed to regenerate shoots. The percent of explants regenerating was greater than 50% for four genotypes.

The effect of sucrose on the levels of abscisic acid, indoleacetic acid and zeatin/zeatin riboside in wheat ears growing in liquid culture

Abstract: The influence of a varied sucrose supply on grain size and hormonal contents of detached wheat ears (Triticwn aestivum L. cv. Schirokko) was investigated throughout grain development. In ears led limited amounts, or no sucrose, grain weights in both proximal and distal grain positions of the ear were reduced. Radioimmunoassay for abscisic acid, indoleacetic acid and zeatin/zeatin riboside showed that the changes in the levels of these hormones in grains and bracts were comparable to intact ears when detached ears were well supplied with sucrose. Under conditions of limited sucrose supply, higher abscisic acid levels in the distal and proximal grains of detached ears were found compared to ears supplied with adequate sucrose. Limiting sucrose supply to the ear did not alter the levels of indoleacetic acid or zeatin/zeatin riboside in either the grains or bracts of detached ears.

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

Abstract: 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.

Hormones and Pod Development in Oilseed Rape (Brassica napus).

Abstract: The endogenous levels of several plant growth substances (indole acetic acid, IAA; abscisic acid, ABA; zeatin, Z; zeatin riboside, [9R]Z; isopentenyladenine, iP; and isopentenyladenosine, [9R]iP were measured during pod development of field grown oilseed Rape (Brassica napus L. var oleifera cv Bienvenu) with high performance liquid chromatography and immunoenzymic (enzyme-linked immunosorbent assay, ELISA) techniques. Results show that pod development is characterized by high levels of Z and [9R]Z in 3 day old fruits and of IAA on the fourth day. During pod maturation, initially a significant increase of IAA and cytokinins was observed, followed by a progressive rise of ABA levels and a concomitant decline of IAA and cytokinin (except iP) levels. The relationship between hormone levels and development, especially pod number, seed number per pod, and seed weight determination, will be discussed.

Protoplast regeneration and organogenesis from pea protoplasts

Abstract: Protoplasts of young Pisum Sativum L. seedlings from 7 different genotypes were isolated and regenerated to the callus stage. Germinating embryos were cultivated with cotyledons removed, thus avoiding intracellular starch accumulation in donor tissue. The first lateral shoots provided a source of homogenous meristematic cells which gave rise to sustained protoplast division and resulted in callus formation within 4, weeks. Root formation occurred on hormone-free medium and shoots developed on medium containing kinetin, 2iP or zeatin in the third subculture, when subcultured in monthly intervals.

The in ovulo environment and its relevance to cloning wheat via somatic embryogenesis

Abstract: Induction of somatic embryogenesis refers to processes that initiate embryo-forming cellular activity. Competence of somatic tissues for embryo formation is measured by the number of somatic embryos produced following induction. Recent findings indicate that competence of wheat explants can be enhanced by treatments that alter hormone levels during differentiation of explantsin vivo. Wheat spikes have been detached at anthesis and cultured with culms in detached-spike-culture medium. The addition of abscisic acid, indole-3-acetic acid or zeatin to the medium depresses embryogenic competence of immature-embryo explants (excised and cultured 11–13 days after anthesis) while deleting hormones from the medium enhances competence. Differentiation of somatic embryos is improved by exposing embryogenic cultures to conditions that simulate nutritional, hormonal and gas-phase conditions of ovules. In particular the lowering of oxygen availability to callus cultures promotes growth of embryogenic callus and suppresses growth of nonembryogenic callus. Finally procedures that simulate seed desiccation significantly increase germination percentages of somatic embryos of wheat.

Callus induction and plant regeneration from excised zygotic embryos of the seed-propagated yams Dioscorea composita Hemsl. and D. cayenensis Lam.

Abstract: A tissue culture method for regeneration of plantlets from calluses of Dioscorea composita Hemsl. and Dioscorea cayenensis L. is described. Zygotic embryos were used as initial explants. Calluses were obtained on Murashige & Skoog basal medium supplemented with 18 μM 2,4-D and plantlets were regenerated on media containing 0.1 μM zeatin and 3.3 mM glutamine according to previously described protocols [3]. Inclusion of 0.3% (w/v) activated charcoal in media did not increase callusing. Regeneration of plantlets from D. cayenensis calluses occurred only at low levels of 2,4-D (2.25 μM) contained in the media tested. The results indicated that there were genotype-dependent differences between the yam species in their ability to regenerate plantlets in vitro.

Shoot Tip Culture of Japanese Persimmon (Diospyros kaki Thunb.)

Abstract: Aseptic shoot cultures were initiated from shoot tips (1mm) of Diospyros kaki Thunb. cv. ‘Nishimurawase’. Shoot tips were excised from dormant axillary buds and cultured in vitro. The best shoot growth was obtained on MS medium containing half strength nitrogen supplemented with 6-(4-Hydroxy-3-methyl-trans-2-butenylamino) purine (zeatin) at 10-5M; 6-benzylaminopurine (BAP) and 6-(γ, γ-dimethylallylamino) purine (2iP) was ineffective. A five-fold proliferation rate was obtained by single node cutting of elongated shoot.

High frequency regeneration and heritable somaclonal variation in Brassica juncea

Abstract: Multiple shoot formation in cotyledonary callus of Indian mustard (Brassica juncea cv. Prakash) was induced on modified MS media supplemented with high cytokinin (kinetin or zeatin) and low IAA concentrations. Complete plants were obtained on prolonged incubation of shoots on the same medium. 6-Benzyladenine alone or in combination with IAA or NAA did not support plantlet regeneration. A total of 71 plants were transferred to greenhouse. The seed, however, could be collected from 37 plants only. The seed was sown in the field to evaluate the material for somaclonal variation in R1 generation. Data were recorded for yield, plant height, number of primary branches, siliqua number, 1,000 seed weight and oil content. Somaclonal lines showed tremendous amount of variation for all the characters studied. A number of plants in this generation showed significantly higher yield and/or other improved agriculturally important characteristics as compared to the control. A line with dwarf plant type was also identified. A number of plants were selected from this generation and carried forward to R2 generation. Most of these lines bred true in R2 generation. The material seems to be very promising for future breeding programmes.

Propagation of Camellia japonica cv. Alba Plena by tissue culture

Abstract: SummaryShoot cultures of Camellia japonica cv. Alba Plena have been established and multiplied in vitro using original material from a mature tree 50 years old. Three kinds of stage II expiants were distinguished for the purposes of shoot multiplication: ST1, the shoot tips of harvested shoots longer than 13–14 mm; n, nodal segments constituting the lower parts of the shoots from which the ST1 were taken; and ST2, whole harvested shoots 5–10 mm long. The ST1 explants were less responsive than the others. Of the various macro-nutrient formulae tried in shoot multiplication cultures the most satisfactory was Woody Plant Medium. The most suitable growth regulator combination for achieving shoot multiplication was: 2 mg l−1 of BA, 2 mg l−1 of zeatin, 2 mg l−1 of 2iP and 0.01 mg l−1 of IBA, used during four weeks, then the cultures were transferred to the same medium for another four weeks the cytokinin concentration being reduced to 1 mg l−1 (transfer medium). The addition of a liquid medium of the same compo...

Quantitative changes of free-base, riboside, ribotide and glucoside cytokinins in developing rice grains

Abstract: Rice grains at various growth stages were analysed for endogenous free-base, riboside, ribotide and glucoside cytokinins on the basis of GC/MS and GC/SIM using deuterium-labeled internal standards. Cytokinins identified were trans- and cis-zeatins, trans- and cis-ribosylzeatins, isopentenyladenosine, isopentenyladenosine monophosphate, trans- and cis-ribosylzeatin monophosphates, trans- and cis-zeatin-O-glucosides, trans- and cis-ribosylzeatin-O-glucosides and zeatin-9-glucoside (trans/cis geometry was not determined). The highest amounts of cytokinins were recorded at the early growth stage, namely either heading, anthesis or milk stage, suggesting that cytokinins may play important roles in the development of the grain. Cis isomers of zeatin derivatives were always present and more abundant than trans isomers. It seemed unlikely that cis isomers were released from t-RNAs during the extraction procedure.

Plant regeneration from immature and mature embryo derived calli of Hordeum marinum

Abstract: Callus cultures were obtained from immature and mature embryos of Hordeum marnium on MS media containing 0.5 mg l-1 parachlorophenoxyacetic acid or 2 mg l-1 2,4-dichlorophenoxyacetic acid. Regeneration occurred after transferring calli to MS either devoid of hormones or supplemented with 1 mg l-1 indole-3-acetic acid and 1 mg l-1 zeatin. The regeneration capacity of the immature embryo derived calli (94%) was about 5 times higher than that of mature embryo derived calli (17%). A total of 30 and 964 plantlets were obtained from 21 mature and 59 immature embryo derived calli, respectively. Low frequency (less than 1%) of albino plantlets was obtained from both explants after 3–9 months in culture. Plants expressing transient chlorophyll deficiency were produced from immature embryo derived cultures at a frequency of 10%. However, when transferred to soil, these plantlets became green.

Plant regeneration from cell suspension cultures ofArabidopsis thaliana heynh.

Abstract: A technique is described for the establishment, maintenance, and regeneration of plants from cell suspension cultures ofArabidopsis thaliana (L) Heynh. Friable, rapidly growing cell suspension cultures were initiated from leaf or hypocotyl callus cultures and these have been maintained in liquid culture for 24 months. The cells grown in liquid culture were used to study the effects of growth regulators, medium salts composition, culture temperature, sucrose concentration and medium solidifying agents on morphogenesis. The most important parameters for plant regeneration were culture temperatures lower than 25°C, the medium solidification agent gelrite at 0.2% (w/v) and zeatin or thidiazuron as the choice of cytokinin. These cell suspensions continue to regenerate fertile plants with a total of over 200 plants having been rooted to date and they also serve as convenient sources of cells for protoplast isolation, biochemical, and molecular assays.

Cytokinin effects on pollen embryogenesis in cultured anthers of Hyoscyamus niger

Abstract: Addition of the cytokinins, benzylaminopurine, N6-Δ-(2-isopentenyl)-adenine, kinetin, zeatin, and zeatin riboside to a basal medium containing mineral salts and sucrose induced characteristic changes in pollen embryogenesis in cultured anthers and anther segments of Hyoscyamus niger. In anthers cultured in media containing 0.01–10.0 mg/L of each cytokinin, there was a progressive delay in the appearance of plantlets outside the anther locule and in the morphology of plantlets formed. Among the cytokinins tested, only zeatin riboside promoted anther efficiency; however, all compounds reduced pollen efficiency by as much as 40–60% of the control even in the lowest concentration tested. The effects of cytokinins were particularly noticeable in the failure of pollen grains to form embryoids by the division of the generative cell and in the decrease in the number of embryoids formed by the division of both generative and vegetative cells. At the morphogenetic level, embryoids formed in media containing cytokin...

Identification of cytokinins in primary crown gall tumours of tomato

Abstract: . Identification of the cytokinin complex of primary crown gall tumours of tomato (Lycopersicon esculentum L.) by combined gas chromatography-mass spectrometry has been described. Several cytokinins have been identified which included zeatin, dihydrozeatin, isopentenyladenine, and their respective riboside and nucleotide derivatives. In addition, 6-benzylaminopurine, its riboside and the corresponding nucleotide have also been identified as major endogenous compounds in this tissue. This would appear to be the first report on the identification of cytokinins from a primary crown gall tumour tissue using unequivocal methods.