Androgenesis in Citrus aurantifolia (Christm.) swingle

By means of gas chromatography-selected ion monitoring-mass spectrometry using an isotope-dilution assay with 4,5,6,7-tetradeutero-indole-3-acetic acid as the internal standard, indole-3-acetic acid has been estimated to be present in aseptically cultured gametophytes of wild-type Physcomitrella patens (Hedw.) B.S.G. at a level of 0.075 g g^–1 dry weight or 2.1 ng g^–1 fresh weight.Abbreviations IAA indole-3-acetic acid - d₄IAA 4,5,6,7-tetra-deutero-indole-3-acetic acid - [¹⁴C]IAA indole-3-[2-¹⁴C]-acetic acid - GC-SIM-MS gas chromatography-selected ion monitoring-mass spectrometry     

Morphogenetic potential of foliar explants in Duboisia myoporoides R.Br. (Solanaceae)

The effects of serial combinations of either indole-3-acetic acid, indole-3-butyric acid or -naphthaleneacetic acid (0.5–10.0 mg/l) with either kinetin, 6-benzyl-amino-purine, zeatin or 6-methylaminopurine (0.5–5.0 mg/l) have been investigated to assess the morphogenetic potential of foliar explants of Duboisia myoporoides. Shoot buds developed either directly or via a callus interphase. Combinations involving indole-3-acetic acid with any of the cytokinins were more effective in inducing shoot bud formation compared to those containing indole-3-butyric acid or -napthalenacetic acid as an auxin. Among cytokinins, zeatin, kinetin and 6-benzylamino-purine were equally effective for shoot formation. However, optimum response with zeatin could be achieved at low concentrations (0.5–2.0 mg/l), while kinetin and 6-benzylamino-purine exhibited comparable efficacy at higher levels (3.0–5.0 mg/l). 6-Methylaminopurine proved least effective in all concentrations and combinations tested. Rooting of the differentiated shoots was readily achieved with -naphthaleneacetic acid alone (0.5 mg/l) after changing the physical form of the medium from gel to static liquid. Regenerated plantlets were transferred to pots and grown to maturity in the field with a high rate of survival (80–90%).Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MAP 6-methylaminopurine - MS Murashige and Skoog (1962) medium - NAA -naphthaleneacetic acid - PVP polyvinyl pyrrolidone     

The Pseudomonas savastanoi tryptophan-2-mono-oxygenase is biologically active in Nicotiana tabacum

It has been proposed that the eukaryotic T-DNA-encoded indole-3-acetic acid (IAA) biosynthesis genes of Agrobacterium tumefaciens and their prokaryotic counterpart in Pseudomonas savastanoi originated from common ancestor genes. This paper provides additional evidence for the functional similarity between the gene products. We have demonstrated that a chimeric gene consisting of the coding sequence of the P. savastanoi tryptophan-2-mono-oxygenase (iaaM gene) and a plant promoter encodes an active enzyme in Nicotiana tabacum. Transformants obtained with this chimeric gene grew as a callus on hormone-free media. No stably transformed plantlets could be isolated. The callus tissues contained extremely high levels of indole-3-acetamide and slightly elevated levels of IAA. Either indole-3-acetamide by itself has a low auxin activity or, alternatively, it is converted aspecifically and at low rates into IAA. The P. savastanoi tryptophan-2-mono-oxygenase activity in plants is also able to detoxify the amino-acid analogue 5-methyltryptophan. This property can be used for positive selection of transformed calli.Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - IAM indole-3-acetamide - NAA naphthalene-1-acetic acid - NPT-II neomycin phosphotransferase II - T-DNA transferred DNA     

Radioimmunoassay for pmol-quantities of indole-3-acetic acid for use with highly stable [125I]- and [3H]IAA derivatives as radiotracers

A radioimmunoassay for the detection of as little as 0.5–1 pmol indole-3-acetic acid (IAA) in unpurified or partially purified plant extracts is described. The assay makes use of either IAA[¹²⁵I]tyrosine methyl ester or [³H]IAA methyl ester as radioactive antigens and IAA methyl ester as the assay standard (measuring range: 1–200 pmol). Levels of extractable IAA in a number of biological samples have been estimated.Abbreviations BSA bovine serum albumin - 2,4-D 2,4-dichlorophenoxy acetic acid - DMF dimethyl formamide - GC-MS gas chromatography-mass spectroscopy - IAA indole-3-acetic acid - RIA radioimmunoassay - SICM selected ion current monitoring - TLC thin layer chromatography - TME tyrosine methyl ester Part 18 in the series: Use of immunoassay in plant science     

Effects of ascorbic acid on axillary shoot induction in Tylophora indica (Burm. f.) Merrill.

A procedure for rapid in vitro multiplication of Tylophora indica (Burm. f.) Merrill., an important indigenous medicinal plant, has been developed. Addition of ascorbic acid was essential to induce sprouting of axillary buds. Optimum multiplication was observed on MS medium containing 6-benzylamino purine (5.0 mg l^–1), -naphathalene-acetic acid (0.5 mg l^–1) and ascorbic acid (100 mg l^–1). Rooting of in vitro produced shoots was readily achieved with indole-3-acetic acid alone (1.0 mg l^–1) in MS. The plantlets thus obtained were successfully transferred to pots in large numbers which grew normally.Abbreviations BAP 6-benzylamino purine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - 2ip 2-isopentenyladenine - Kn kinetin - MS Murashige & Skoog media - NAA -naphthalene acetic acid     

Levels of indole-3-acetic acid in intact and decapitated coleoptiles as determined by a specific and highly sensitive solid-phase enzyme immunoassay

A specific solid-phase enzyme immunoassay for the detection of as little as 3–4 pg of indole-3-acetic acid (IAA) is described. The assay involves minimal procedural efforts and requires only standard laboratory equipment. Up to 50 samples in triplicate, processed simultaneously, can be assayed and evaluated in 2.5 h. As little as 1 mg oat coleoptile tissue is sufficient for a quantitative IAA analysis and little or no extract purification is necessary. Using this assay, levels of IAA have been determined in coleoptiles of maize and oat. The distribution of IAA within single coleoptiles was quantitated and the production of IAA during the regeneration of the physiological tip in Avena coleoptiles was investigated. The changes in levels of IAA and other major phytohormones were quantitated during the growth of oat coleoptiles.Abbreviations ABA abscisic acid - BHT butylated hydroxytoluene - BSA bovine serum albumin - IAA indole-3-acetic acid - TBS Trishydroxymethylaminomethane buffered saline Part 21 in the series Use of Immunoassay in Plant Science     

l-Tryptophan metabolism in wound-activated and Agrobacterium tumefaciens-transformed potato tuber cells

The in vivo metabolism of L-tryptophan in wound-activated and Agrobacterium tumefaciens , strain C 58, transformed tissues of white potato tubers ( Solanum tuberosum L. cv. Saskia) was investigated. The following metabolites of L-tryptophan were identified in both tissues by co-chromatography with authentic standards in several thinlayer chromotography (TLC) and high pressure liquid chromatographic (HPLC) systems: indole-3-acetic acid (IAA), indole-3-acetaldehyde, indole-3-ethanol, indole-3-acetamide and tryptamine. Labelled indole-3-acetaldoxime was only found in transformed tissue. Crown gall tissue generally incorporated [¹⁴C]-L-tryptophan into precursors of IAA at a distinctly higher rate than did wound tissue. Tryptamine and indole-3-ethanol accumulated about ten-fold more label in crown gall cells than in cells from wounded tissue. The incorporation of radioactivity into indole-3-acetamide as determined by 2 consecutive TLC systems followed by HPLC analysis was rather low, though consistently observed in both tissues. An indole-3-acetamide hydrolyzing enzyme, the putative product of gene 2 on the T-DNA, could be extracted from the transformed tissue only. The indole-3-ethanol level was 4.3 nmol (g dry weight)⁻¹ and 41 nmol (g dry weight)⁻¹ for wounded tissue and primary crown gall tissue, respectively, as determined by HPLC with a [¹⁴C]-labelled internal standard. The experiments are critically discussed in relation to recent reports on a T-DNA encoded enzyme of IAA biosynthesis in crown gall tumors.     

Indole-3-butyric acid in Arabidopsis thaliana III. In vivo biosynthesis

Indole-3-butyric acid (IBA) was identified by HPLC and GC-MS as one of the reaction products after incubation of sterile cultures of Arabidopsis thaliana seedlings with labeled indole-3-acetic acid (IAA). This is the first demonstration of IBA biosynthesis in a dicotyledonous plant. After 1 h of incubation most of the IBA was found in the free form, while after longer periods of incubation most of it was detected in conjugated forms. Formation of IBA conjugates was inhibited by the addition of unlabeled IBA. The biosynthesis of IBA and its conjugates was followed throughout the development of the seedlings and at different pH values. All parts of the plant (isolated roots, leaves, shoots and flowers) were able to convert IAA to IBA to the same extent.IAA was more readily transported than IBA in mature Arabidopsis plants. Feeding of labeled phenylacetic acid (PAA) and -naphthylacetic acid (NAA) to Arabidopsis seedlings resulted in a new small peak which was hydrolyzed by 7N NaOH, but the formation of compounds with longer side chains (analogous to IBA) could not be detected.Abbreviations IAA indole-3-acetic acid - IBA indole-3-butyric acid - NAA -naphthylacetic acid - PAA phenylacetic acid     

Plant regeneration from cotyledon protoplasts of Xinjiang muskmelon

Cotyledon protoplasts were isolated from seedlings of Xinjiang muskelon (Cucumis melo var.saccharinus) grown under sterile conditions and cultured in modified Miller medium. High frequency division of the protoplast-derived cells was observed. Agarose bead culture with B₆S₃ tobacco crown gall nurse cells was found most suitable for the cotyledon protoplasts when compared with other culture methods. Intact plants were regenerated from the protocalli by a two-step culture procedure with liquid and then solid media.Abbreviations BAP 6-benzylaminopurine - B₆S₃ crown gall tumor cells of tobacco - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - MES 2(Nmorpholino) ethanesulfonic acid - MS Murashige and Skoog medium(1962) - NAA -naphthaleneacetic acid - N6 Zhu et al. medium (1975)     

Adventitious bud regeneration from leaf expiants of the shrubby ornamental honeysuckle,Lonicera nitida Wils. cv. ‘Maigrün’: effects of thidiazuron and 2,3,5-triiodobenzoic acid

Different combinations of auxins and cytokinins were employed to assess the regeneration capacity from in vitro leaf explants of Lonicera nitida Wils. cv Maïgrün. A high frequency of rhizogenesis was noticed, with 2.3 M thidiazuron plus 2.9 M indole-3-acetic acid as the only hormonal combination to support caulogenic responses. Increasing thidiazuron concentration and/or suppressing auxin did not improve caulogenesis. Combining thidiazuron with 2,3,5-triiodobenzoic acid produced a dramatic increase in the percentage of caulogenic explants. A maximum of 74% of adventitious bud forming explants was obtained with 2.3 M thidiazuron plus 20 M 2,3,5-triiodobenzoic acid. Buds were often in a rosette form and were vitreous, so that shoot elongation was difficult to obtain. The effect of the duration of the 2,3,5-triiodobenzoic acid treatment on shoot elongation was investigated.Abbreviations BAP benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA3 gibberellic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - 2 IP 2-isopentenyladenine - MS Murashige and Skoog - NAA -naphthylacetic acid - TDZ N-phenyl-N-1,2,3-thidiazol-5-ylurea (thidiazuron) - TIBA 2,3,5-triiodobenzoic acid - Z zeatin     

35S-β-glucuronidase gene blocks biological effects of cotransferred iaa genes

The iaaM and iaaH genes of Agrobacterium tumefaciens and Agrobacterium rhizogenes play an important role in crown gall and hairy root disease. The iaaM gene codes for tryptophan monooxygenase which converts tryptophan into indole-3-acetamide (IAM). IAM is converted into the auxin indole-3-acetic acid (IAA) by indoleacetamide hydrolase, encoded by the iaaH gene. In functional studies on the activity of the iaa genes of the TB region of the A. tumefaciens biotype III strain Tm4, the frequently used 35S--glucuronidase (35S-UidA or GUS) marker gene was found to inhibit IAA synthesis and root induction encoded by the TB iaa genes. To exert this inhibition, the 35S-UidA gene must be cotransferred with the iaaH gene. The 35S promoter alone is sufficient to cause the inhibitory effect.     

Changes in cell ultrastructure and endogenous abscisic acid and indole-3-actic acid concentrations in Fatsia japonica leaves under polyethylene glycol-induced water stress

Ultrastructural alterations in epidermal and mesophyll cells as well as variations in bulk leaf endogenous ABA and IAA concentrations were studied in PEG-treated plants of Fatsia japonica Decne & Plank. Under stress induced by PEG vesicles containing fibrous material and electron-dense bodies associated with plasma membranes were observed. Cytochemical examination indicated that electron-dense bodies corresponded to lipids and the fibrous material of the vesicles were polysaccharides. Chloroplasts, mitochondria, nuclei and Golgi apparatus also showed modifications. A strong relationship was found between increasing PEG-induced water stress, increasing endogenous ABA and ultrastructural changes. In relation with leaf ontogeny and ABA concentration a higher ABA level was observed in younger than in older leaves. The differences in the endogenous concentrations of indole-3-acetic acid are unclear, except after 7 days of PEG-treatment. The increase in the endogenous abscisic acid concentration could be related with the ultrastructural changes.Abbreviations ABA = abscisic acid - IAA = indole-3-acetic acid - PEG = polyethylene glycol - = leaf water potential - TEM = transmission electron microscope     

Changes in indole-3-acetic acid levels during tomato (Lycopersicon esculentum Mill.) seed development

Summary The changes in the level of indole-3-acetic acid (IAA) were investigated in seeds and fruit tissues-placenta and mesocarp-during tomato (Lycopersicon esculentum Mill.) zygotic embryogenesis, which was characterized through eight morphological embryo stages [from globular (stage 1) to mature embryo (stage 8)]. In whole seeds, IAA levels increased mainly at stage 3 (young torpedo) and at stage 5 (late torpedo stage). As the seed matured and dehydrated, IAA levels decreased and showed a new distribution pattern within seed structures, preferentially in endosperm tissue. IAA contents in fruit tissues were lower but followed the same pattern as those of seeds. These data support the hypothesis of IAA biosynthesis in seeds with a transient role of the endosperm at the end of embryo development and suggest a role of IAA in fruit and seed growth. Moreover a comparison of IAA and ABA changes suggests that IAA could be especially necessary for the beginning of embryo growth, whereas ABA could act mainly at the end of the growth phase.Abbreviations ABA abscisic acid - ABTS 2,2-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) - BHT butylhydroxytoluene - DW dry weight - ELISA enzyme linked immunosorbent assay - HPLC high performance liquid chromatography - IAA indole-3-acetic acid. PGRs: plant growth regulators     

Plants regenerated from auxin-auxotrophic variants are inviable

Summary Shoots regenerated from auxin-auxotrophic variants of Nicotiana plumbaginifolia were inviable when cultured in vitro in the absence of auxin. Variant shoots survived longer when grafted to wild-type stocks but eventually died after a characteristic pattern of degeneration. The auxin auxotrophs were isolated after mutagen treatment by a total isolation method as infrequent variants amongst haploid protoplast-derived cell colonies. The variants responded to several active auxins but, unlike the wild type, not to cytokinin. Plant regeneration from variant cultures ceased at early stages of shoot formation after complete withdrawal of auxin from the medium.Abbreviations BAP 6-benzylaminopurine - BUdR 5-bromo-2-deoxyuridine - 2,4-D 2,4-dichlorophenoxyacetic acid - DMSO dimethyl sulfoxide - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MNNG N-methyl-N-nitro-N-nitrosoguanidine - NAA 1-naphthaleneacetic acid     

Formation in vitro of ripe tomato fruits from thin layer explants of flower pedicels

Thin longitudinal sections cut from pedicels of fifteen cultivars of tomato (Lycopersicon esculentum) were grown in vitro on Murashige-Skoog medium supplemented with various concentrations of different auxins and cytokinins. Isatin (an auxin precursor slowly converted to an active auxin) was the most effective source of auxin for the formation of buds without prior root formation, while zeatin was the most effective cytokinin for growth and development of the buds. Flower buds and ripe fruits developed consistently from explants of the cultivar Pixie Hybrid II treated with 10 M isatin plus 3 M zeatin as the cytokinin. Fruits developed parthenocarpically, grew to a diameter of about 15 mm, ripened promptly, and possessed normal color and flavor.Abbreviations BAP benzylaminopurine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - IPA isopentyladenosine - NAA -napthaleneacetic acid     

The biosynthesis and conjugation of indole-3-acetic acid in germinating seed and seedlings ofDalbergia dolichopetala

Germinating seed ofDalbergia dolichopetala converted both [²H₅]l-tryptophan and [²H₅]indole-3-ethanol to [²H₅]indole-3-acetic acid (IAA). Metabolism of [2-¹⁴C]IAA resulted in the production of indole-3-acetylaspartic acid (IAAsp), as well as several unidentified components, referred to as metabolites I, II, IV and V. Re-application of [¹⁴C]IAAsp to the germinating seed led to the accumulation of the polar, water-soluble compound, metabolite V, as the major metabolite, together with a small amount of IAA. Metabolites I, II and IV were not detected, nor were these compounds associated with the metabolism of [2-¹⁴C]IAA by shoots and excised cotyledons and roots from 26-d-oldD. dolichopetala seedlings. Both shoots and cotyledons converted IAA to IAAsp and metabolite V, while IAAsp was the only metabolite detected in extracts from excised roots. The available evidence indicates that inDalbergia, and other species, IAAsp may not act as a storage product that can be hydrolysed to provide the plant with a ready supply of IAA.Abbreviations HPLC-RC high-performance liquid chromatography-radiocounting - IAA indole-3-acetic acid - IAAsp indole-3-acetylaspartic acid - IAlnos 2-O-indole-3-acetyl-myo-inositol - IEt indole-3-ethanol     

Tip-localised H+-fluxes and the applicability of the acid-growth hypothesis to tip-growing cells: Control of chloronemal extension in Funaria hygrometrica by auxin and light

The role of tip-localised H⁺ secretion in regulating chloronemal tip growth in the moss Funaria hygrometrica Hedw. was investigated. pH was monitored with pH microelectrodes placed close to the cell surface while the rate of extension growth was manipulated by illumination and by the application of indole-3-acetic acid. Growth stimulations were accompanied by acidification of the external solution; this acidification was most pronounced at the growing tip. The timing and extent of acification external to the tip correlated well with the magnitude and time course of growth stimulations. The maintenance of both growth and H⁺ efflux under CO₂-free conditions indicated that neither photosynthetic nor respiratory CO₂ metabolism were involved. Artificially acidifying the nutrient solution rapidly but transiently stimulated elongation in both white light and darkness. Furthermore, the stimulation of elongation caused by white light was inhibited if the nutrient solution was buffered strongly near neutrality. We conclude that the acid growth hypothesis is applicable to tip growth in Funaria and that light and exogenous indole-3-acetic acid act at least in part by stimulating localised H⁺-ion efflux.Abbreviations D darkness - IAA indole-3-acetic acid - WL white light     

Growth of and quassin accumulation by cultures of Quassia amara

Callus and suspension cultures were established from Quassia amara, a member of the Simaroubaceae. Analysis of the tissue culture showed that quassin was present in both callus and suspension cultures. The effect of variation in auxin and cytokinins on both callus growth and the presence of quassin was examined. The suspension culture was grown in a 7 liter bioreactor when good yields of quassin were achieved.Abbreviations IAA indole-3-acetic acid - IBA indolebutyric acid - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - 6BA 6-benzyladenine - IpA N⁶ (-isopentenyl) adenine - IpAR N⁶ ( isopentenyl) adenine riboside - td doubling time     

Effects of water stress on cellular ultrastructure and on concentrations of endogenous abscisic acid and indole-3-acetic acid in Fatsia japonica leaves

Ultrastructural alterations in mesophyll cells as well as variations in bulk leaf endogenous ABA and IAA concentrations were studied in water-stressed field-grown plants of Fatsia japonica. Under water deficit cellular membranes were modified and an increase in vesicles was observed. The main damage to the chloroplasts included thylakoid swelling and disruption of the chloroplast envelope. Concomitant variations in abscisic acid and indole-3-acetic acid were observed. Despite the expected increased in endogenous ABA concentration in relation to water stress, after the highest concentration of ABA, observed at predawn in severely stressed plants (29-1), there was a sharp decline from 2768 pmol g fw^–1 to 145 pmol g fw^–1; thus in severely stressed plants ABA levels were not related to changes in bulk leaf ABA contents. Water stress did not influence the concentrations of indole-3-acetic acid, although the increase in the endogenous abscisic acid concentration could be related with the ultrastructural changes.Abbreviations ABA abscisic acid - IAA indole-3-acetic acid - leaf water potential     

Primary Action of Indole-3-acetic Acid in Crown Gall Tumors: Increase of Solute Uptake

Exogenously added indole-3-acetic acid at a concentration of 100 micromolars stimulates d-glucose uptake (or 3-O-methyl-d-glucose uptake) by 25% in crown gall tumors induced on potato tuber tissue by Agrobacterium tumefaciens strain C 58. The titration of the endogenous IAA with the auxin antagonist 2-naphthaleneacetic acid at 100 micromolars reduces d-glucose uptake by about 80%. The apparent inhibition constant K_i is 21 micromolars. Other auxin antagonists like 1-naphthoxyacetic acid and 2-(p-chlorophenoxy)-2-methylpropionic acid show similar effects. The uptake of the amino acids leucine, methionine, tryptophan, lysine, and aspartic acid is also inhibited by 2-naphthaleneacetic acid to similar degrees. The auxins 1-naphthaleneacetic acid and 2-naphthoxyacetic acid at concentrations between 10 and 100 micromolars inhibit solute uptake only slightly (inhibition less than 20%). The impact of the results on the postulated role of indole-3-acetic acid as a modifier of the electrochemical proton gradient across the plasmalemma in crown gall tumor tissue is discussed.