Presence Of Indole-3-butyric Acid Research Articles

An Efficient Micropropagation Protocol for the Endangered European Shrub February Daphne (Daphne mezereum L.) and Identification of Bacteria in Culture

Daphne mezereum of the Thymelaeaceae family is a medicinal shrub occurring naturally in Europe and under legal protection in Poland. In the present study, a protocol developed for mass propagation of February daphne from nodal explants is presented. Micropropagation is one of the in vitro techniques that allow the preservation of rare and valuable plants by developing efficient methods for their propagation. In the proliferation stage, explants were cultured in the Woody Plant Medium (WPM) with different cytokinins, and in the rooting stage on the semi-solid WPM medium with perlite, indole-3-butyric acid (IBA) in various concentrations was used. The maximum proliferation rate (five shoots per explant) was observed on the medium containing 4.14 μM of meta-Topolin (mT). When the regenerated shoots were rooted in vitro in the presence of IBA in various concentrations, that of 19.68 μM induced the highest number of roots per shoot (6.63) and the maximal root length (2.15 cm). It is also worth remembering that plants are often colonized by different groups of microorganisms, which also affect the diversity of the ecosystem. The endophytic bacteria inhabiting the D. mezereum shoots are Mycobacterium.

Characteristics of composition and content of auxins in tissues of Siberian apple tree <i>Malus baccata</i> L. Borkh.

It is known that the highest content of auxins is found primarily in the areas of active mitosis, including young leaves, apical meristems, cambium, embryos and endosperm, as well as developing fruits, which determines, among other things, plant habitus and fruit size. In agriculture and biotechnology, auxins (indole-3-butyric acid (IBA) and indole-3-acetic acid (IAA) are used to stimulate lateral development, including in apple tree rootstocks. Despite the crucial role of endogenous IBA in rooting, its presence in apple tree tissues remains unconfirmed, although its content in planta was shown for many other plants. Therefore, in this work, the composition and content of auxins in 2 tissue types of Siberian apple tree of lowand high-growing forms, along with the presence of IBA in them, was compared. Tissues of ovary and bark of Siberian apple tree (Malus baccata L. Borkh.) of 2 forms (low and tall) growing on an experimental plot of Siberian Institute of Plant Physiology and Biochemistry SB RAS were used in the study. Bark samples were collected from the 1st year shoots at the stage of intensive sap flow. The ovary samples were collected in July at the stage of active growth. Auxins purified by solid-phase extraction were analysed by GC-MS. In addition to IAA-3, the following auxins were found in fast-growing tissues of the Siberian apple tree both in the cambium layer of annual gain during intensive sap flow and set fruit during the active growth stage: IBA, indole-1-acetic acid, indole-3-carboxylic acid and indole-3-propionic acid. Similar to other plants, the content of these auxins was significantly lower than that of endogenous IAA.

Overexpression of UGT74E2, an Arabidopsis IBA Glycosyltransferase, Enhances Seed Germination and Modulates Stress Tolerance via ABA Signaling in Rice.

UDP-glycosyltransferases (UGTs) play key roles in modulating plant development and responses to environmental challenges. Previous research reported that the Arabidopsis UDP-glucosyltransferase 74E2 (AtUGT74E2), which transfers glucose to indole-3-butyric acid (IBA), is involved in regulating plant architecture and stress responses. Here, we show novel and distinct roles of UGT74E2 in rice. We found that overexpression of AtUGT74E2 in rice could enhance seed germination. This effect was also observed in the presence of IBA and abscisic acid (ABA), as well as salt and drought stresses. Further investigation indicated that the overexpression lines had lower levels of free IBA and ABA compared to wild-type plants. Auxin signaling pathway gene expression such as for OsARF and OsGH3 genes, as well as ABA signaling pathway genes OsABI3 and OsABI5, was substantially downregulated in germinating seeds of UGT74E2 overexpression lines. Consistently, due to reduced IBA and ABA levels, the established seedlings were less tolerant to drought and salt stresses. The regulation of rice seed germination and stress tolerance could be attributed to IBA and ABA level alterations, as well as modulation of the auxin/ABA signaling pathways by UGT74E2. The distinct roles of UGT74E2 in rice implied that complex and different molecular regulation networks exist between Arabidopsis and rice.

Morphophysiology, Phenotypic and Molecular Diversity of Auxin-induced Passiflora mucronata Lam. (Passifloraceae).

Genetic diversity allows identification of potential intraspecific genotypes in the genus Passiflora. The objective of this study was to examine the morphological and genetic diversity of auxin-induced Passiflora mucronata. The experiments were arranged in a complete randomized block design, with a 9 x 2 factorial arrangement (nine genotypes x presence and absence of auxin, indole-3-butyric acid (IBA)), with four replicates of 16 cuttings. The rooting and vegetative growth responses were variable. Genotype 5 was more responsive in the absence of IBA and genotypes 3, 8 and 9 were more responsive in the presence of IBA. Auxin increased rooting rate and percentage, reducing the average time of root protrusion in eight days. IBA also contributed to increase photosynthesis and dry root and shoot mass in 55.55 and 44.44% of the genotypes, respectively. The highest relative contribution to phenotypic diversity in the absence of auxin was rate (38.75%) and percentage (20.27%) of rooting, whereas in the presence of auxin was stomatal conductance (23.19%) and root dry mass (20.91%). Similarity was found for phenotypic and molecular divergence in the presence of IBA, in which genotypes 1 and 6; genotypes 5, 8 and 9; and genotype 3 were clustered in distinct groups.

<i>In Vitro</i> Propagation and Recovery of Eight Apple and Two Pear Cultivars Held in a Germplasm Bank

Eight apple (Malus x domestica) and two pear (Pyrus communis) traditional cultivars of commercial interest were selected from the germplasm bank CIAM (Centro de Investigaciones Agrarias de Mabegondo, NW of Spain) one of the most important collections of The Iberian Peninsula. In order to define the establishment protocol, apical buds 1.0 - 1.5 millimetre length of the selected cultivars were established in solid mineral medium Murashige and Skoog (MS) with modified vitamins (thiamine 10X) and supplemented with 1 mg·L-1 6-benzyladenine (BA), 0.2 mg·L-1 gibberellic acid (GA3) and 0.3 mg·L-1 of indole-3-butyric acid (IBA). The highest shoot initiation rates were achieved with apple cultivar “Principe Grande” (55%) and “Barburina” pear cultivar (50%); the lowest rate was obtained with apple cultivar “Camoesa” (11%). Initiation rates for shoots culture varied between 22% and 37% for the other cultivars. The cytokinins BA, zeatin, 2-isopentyladenine (2iP) and thidiazuron (TDZ) were tested at concentrations of 0, 0.25, 0.5 and 1 mg·L-1 in the absence or presence of IBA (0.1 mg·L-1) for the multiplication step. For all cultivars, the highest multiplication rates were obtained with medium supplemented with TDZ and also in media containing BA. The in vitro rooting success of shoots in medium with 0.1 mg·L-1 IBA without cytokinins, and the survival rate of plantlets acclimatized under greenhouse conditions depended on the genotype of the different cultivars. In vitro propagation of these ten traditional apple and pear cultivars in NW Spain has not previously been reported.

Plant regeneration of ‘Amethyst’ purple raspberry (Rubus occidentalis×R. idaeus‘Amethyst’) from in vitro leaf tissues

Genotype-specific limitations in plant regeneration can greatly inhibit use of plant tissue culture and biotechnology in plant improvement. An experiment was conducted to discover the optimal plant growth regulator combinations for plant regeneration of a cold-hardy purple raspberry cultivar (Rubus occidentalis × R. idaeus 'Amethyst'). Regeneration of adventitious shoots was analyzed when in vitro leaf tissues were cultured on woody plant medium (WPM) with varying thidiazuron (TDZ) and indole-3-butyric acid (IBA) concentrations. The maximum regeneration rate occurred on WPM medium containing 1.0 μM TDZ and 0 μM IBA with an average of 93.3% of the explants forming shoots. The number of shoots produced from each leaf explant varied with the TDZ concentrations. An average of 3.9 shoots per explant was produced on 0.5 TDZ and 2.3 shoots on 1 µM TDZ medium. Higher TDZ or presence of IBA in the medium decreased the shoot number. Regenerated shoots were transferred to Murashige and Skoog (MS) medium with 1 to 10 μM IBA with or without polyvinylpyrrolidone (PVP) for in vitro rooting. More roots were induced in 10 μM IBA medium than lower amounts. Addition of PVP significantly enhanced rooting. Rooted explants were transferred to potting medium and grown in the greenhouse. This research will assist future raspberry improvement programs that use biotechnology techniques such as in vitro mutation and genetic transformation.

Effect of indole-3-butyric acid on root formation in Alnus glutinosa microcuttings

A study of the in vitro rooting process in mature alder (Alnus glutinosa (L.) Gaertn.) shoots is described. Microcuttings from shoots cultured in vitro were transferred to a half-strength Woody Plant Medium containing 0 or 0.1 mg l indole-3-butyric acid (IBA) for 0 to 7 days. The presence of IBA in the medium increased the rooting percentage, number of roots, percentage of lateral roots, and length of the shoots. Histological studies were carried out with shoots treated with 0 or 0.1 mg l IBA for 7 days. According to these criteria, treatment with IBA for 2–3 days proved to be the most successful. In both treatments, substancial reactivation of cell division was observed at the base of the shoots after 1 day. Some cambial zone and adjacent phloem cells became dense cytoplasm, having nuclei with prominent nucleoli. The first cell divisions were also observed at this time. In the treatment with IBA (0.1 mg l for 7 days), meristemoids became individualized, consisting of densely staining cells, by day 3. Identifiable conical shaped root primordia with several cell layers were visible after 4–5 days. Roots with an organized tissue system emerged from the stem after 6 days in the IBA-treated shoots. Meristemoid formation was delayed until the fourth day and root emergence until the eight day in the control treatment (no IBA).–1–1–1

IN VITRO REGENERATION FROM PUMMELO (CITRUS GRANDIS OSBECK.) COTYLEDON-DERIVED CALLUS

Pummelo, a citrus fruit, is an underutilized fruit crop with a potential for commercialization in warm humid climates. Improvement of crops through bio-technological approaches mainly involves availability of efficient in vitro regeneration protocols. A study was conducted to develop a regeneration protocol for a local cultivar of pummelo under cultivation in Devanahalli, Bangalore, India, using cotyledon explants obtained from six-week-old in vitro grown seedlings. Callus was induced on MS (Murashige and Skoog) media by use of α-naphthaleneacetic acid (NAA) at 1, 3 and 5 mg/L, and 2,4-dichlorophenoxyacetic acid (2,4-D) at 0.5, 1 and 2 mg/L, each one individually and also in combination with 6-benzyl adenine (BA) at concentrations of 0.5 and 1 mg/L. The best callusing was obtained in media containing 5 mg/L NAA plus 0.5 mg/L BA (100% callusing with 1.2 g callus fresh weight per explant). In order to induce shoots in the calli, they were transferred in MS media containing each of BA (1 to 8 mg/L) and Adenine Sulphate (AdS) (1 to 40 mg/L), with or without 0.25 mg/L indole-3-butyric acid (IBA). 2,4-D-induced calli showed ability of regenerating shoots better than NAA-induced calli which failed almost totally. Shoot regeneration was best (90% with average 1.9 shoots/explant) when BA was used at 5 mg/L alone. AdS was less effective than BA in shoot induction in the calli though its best result, 70% shoot induction with average of 2 shoots/explant at 20 mg/L, was also considerable. Presence of IBA in the media with each of the two cytokinins used, generally, affected shoot regeneration negatively. The regenerated microshoots were rooted in half strength MS medium supplemented with 4 mg/L IBA. The produced protocol in this study can be useful for improvement of pummelo through biotechnological techniques which involve callus tissue.

Adventitious rooting performance in micropropagated Cornus mas

Axillary buds sampled from a mature 27-year-old Cornus mas cv. Macrocarpa were grown in vitro on modified woody plant medium (WPM). Adventitious rooting performance of microshoots was assayed on half-strength WPM supplemented with 1-naphthaleneacetic acid (NAA) or indole-3-butyric acid (IBA) under various pH. NAA induced significantly higher rooting frequencies than IBA. The pH of 6.8 inhibited rooting, and differentiated roots were extremely thick and fragile. The highest rooting frequency was recorded on half-strength WPM supplemented with 5.37 µM NAA at the pH value adjusted to 6.2 (73 % of rooted shoots). In the presence of IBA, the formation of adventitious roots was observed only in the basal part of the microshoot dipped into rooting medium. In the case of NAA, however, adventitious roots arose also from the parts of microshoots that were not in contact with medium. The growth of aerial roots was always positively gravitropic. The nuclear microsatellite Cf-G17 gave a monomorphic fingerprinting pattern across the mother shrub and micropropagated plantlets. Acclimatized plants did not show any visually detectable morphological variation and the aerial adventitious root formation was no longer observed.

ELIMINATION OF GRAPEVINE LEAFROLL ASSOCIATED VIRUS 1 AND GRAPEVINE RUPESTRIS STEM PITTING ASSOCIATED VIRUS FROM GRAPEVINE cv. AGIORGITIKO, AND A MICROPROPAGATION PROTOCOL FOR MASS PRODUCTION OF VIRUS-FREE PLANTLETS

Grapevine leafroll-associated virus 1 (GLRaV-1) and Grapevine rupestris stem pitting-associated virus (GRSPaV) were eradicated from Vitis vinifera L. cv. Agiorgitiko by combining in vitro thermotherapy and tissue culture. GRSPaV is known to be quite recalcitrant to elimination, whereas GLRaV-1 is more easily knocked out. In this study, the effectiveness of two different virus elimination techniques, including meristem- and shoot-tip culture, was evaluated. Results showed that meristem-tip culture combined with thermotherapy was the most effective for eliminating both viruses as confirmed by nested RT-PCR assays. Success rate for GLRaV-1 (91.2%) was higher than for GRSPaV-1 (67.6%). The ratio of virus elimination to survival was higher for meristem-tip culture than for shoot tips (1.108 and 0.469 respectively). The effect of six basal media on in vitro shoot proliferation of virus-free explants of cv. Agiorgitiko was also studied and woody plant medium (WPM) proved to be the most effective. The presence of cytokinin 6-benzyladenine (BA) alone resulted in chlorotic plantlets, while supplementation with the auxin naphthaleneacetic acid (NAA) enhanced proliferation rate. Root induction at two temperature regimes (22±2°C and 26±2oC) revealed that higher temperature was more effective in the presence of IBA (indole-3-butyric acid) rather than NAA.

Mössbauer spectroscopic evidence for iron(III) complexation and reduction in acidic aqueous solutions of indole-3-butyric acid

Mossbauer spectroscopic studies were carried out in acidic (pH 2.3) 57FeIII nitrate containing aqueous solutions of indole-3-butyric acid (IBA), rapidly frozen in liquid nitrogen at various periods of time after mixing the reagents. The data obtained show that in solution in the presence of IBA, iron(III) forms a complex with a dimeric structure characterised by a quadrupole doublet, whereas without IBA under similar conditions iron(III) exhibits a broad spectral feature due to a slow paramagnetic spin relaxation which, at liquid nitrogen temperature, results in a large anomalous line broadening (or, at T = 4.2 K, in a hyperfine magnetic splitting). The spectra of 57FeIII+IBA solutions, kept at ambient temperature under aerobic conditions for increasing periods of time before freezing, contained a gradually increasing contribution of a component with a higher quadrupole splitting. The Mossbauer parameters for that component are typical for iron(II) aquo complexes, thus showing that under these conditions gradual reduction of iron(III) occurs, so that the majority (85%) of dissolved iron(III) is reduced within 2 days. The Mossbauer parameters for the iron(III)-IBA complex in aqueous solution and in the solid state (separated from the solution by filtration) were found to be similar, which may indicate that the dissolved and solid complexes have the same composition and/or iron(III) coordination environment. For the solid complex, the data of elemental analysis suggest the following composition of the dimer: [L2Fe-(OH)2-FeL2] (where L is indole-3-butyrate). This structure is also in agreement with the data of infrared spectroscopic study of the complex reported earlier, with the side-chain carboxylic group in indole-3-butyrate as a bidentate ligand. The Mossbauer parameters for the solid 57FeIII-IBA complex at T = 80 K and its acetone solution rapidly frozen in liquid nitrogen were virtually identical, which indicates that the complex retains its structure upon dissolution in acetone.

Agrobacterium rhizogenes vs auxinic induction for in vitro rhizogenesis of Prosopis chilensis and Nothofagus alpina

The induction and improvement of in vitro rhizogenesis of microshoots of Prosopis chilensis (Mol.) Stuntz and Nothofagus alpina (Poep. et Endl. Oerst.) were compared using Agrobacterium rhizogenes (Ar) versus indole-3-butyric acid (IBA) in the culture media. Microshoots of P. chilensis (1-2 cm length), coming from in vitro grown seedlings, were cultivated in a modified Broadleaved Tree Medium (BTMm) containing half salt concentration of macronutrients and 0.05 mg x L(-1) benzilaminopurine (BAP). After 30 days, microshoots with 2-4 leaves were selected and cultured in BTMm-agar in presence or abscense of Ar and in combination with IBA. For N. alpina, the apical shoots with the first 2 true leaves, from 5 weeks old seedlings, were cultured in the abovementioned medium, but with 0.15 mg x L(-1) of BAP. After 2 months, microshoots with 2-3 leaves were selected and cultured in BTMm-agar, supplemented with 5 mg x L(-1) IBA or in liquid BTMm on perlite and, in the presence or absence of A. rhizogenes (Ar) and in combination with 3 mg x L(-1) IBA. Rooting in P. chilensis reached 100.0% when Ar infection was produced in the presence of IBA, increasing both, the number and dry weight of roots. In N. alpina, 90.0% of rooting efficiency was obtained when Ar infection was produced in liquid culture and in the absence of auxin.

Manipulation of the morphogenetic pathways of Lilium longiflorum transverse thin cell layer explants by auxin and cytokinin

Excised tissues from transverse young stem sections of Lilium longiflorum were cultured on Murashige and Skoog medium supplemented with growth regulators at various concentrations. After 45 d in culture, the presence of α-naphthaleneacetic acid (NAA) in the culture medium at 5.4 μM resulted in bulblet formation while 2,4-dichlorophenoxyacetic acid (2,4-D) at 2.2 μM resulted in root formation. The presence of IBA (indole-3-butyric acid) in the culture medium at 1.0 μM resulted in shoot formation while plantlet formation occurred when IBA was added at a concentration of 2.0 μM. When 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea (TDZ) was added to the culture medium at 1.1 μM, protocorm-like bodies (PLBs) formed, while 2.2 μM resulted in shoot formation (on abaxial and adaxial surfaces). The presence of NAA and TDZ in the culture medium at 5.4 μM and 0.4, 1.1 or 2.2 μM, respectively, resulted in somatic embryo formation while NAA- and 6-benzylaminopurine-(BA) containing culture medium formed callus or bulblets. The establishment of different regeneration systems when explants are exposed to various growth regulators demonstrates that the choice of growth regulator combinations and concentrations are of significance in determining the morphogenetic response and plant regeneration capacity.

Indole-3-acetic acid and indole-3-butyric acid in tissues of carrot inoculated withAgrobacterium rhizogenes

The role of auxins in induction of roots byAgrobacterium rhizogenes was studied in carrot root disks. Transformed roots were produced on root disks by inoculation withA. rhizogenes, A4. Measurement of indole-3-acetic acid (IAA) by gas chromatography-mass spectrometry (GC-MS) indicated that there was a significant increase in the concentration of IAA in transformed callus and induced roots compared with initial IAA concentrations in carrot disks. Indole-3-butyric acid (IBA) was found to occur naturally in carrot roots. The presence of IBA, a potent root inducer, must be taken into account when assessing the role of auxin during transformation and induction of roots byA. rhizogenes.