The role of the conversion of IBA into IAA on root regeneration in apple: introduction of a test system

A test system for root regeneration was developed that consists of stem slices of apple shoots (ca. 0.5 mm thick; fresh weight ca. 1 mg). Roots regenerated synchronously without intervening callus formation and without interference with compounds originating from other parts of the plant. Supply of indolebutyric acid (IBA) or indoleacetic acid (IAA) induced maximally an average of 8 or 4.5 roots per slice, respectively. After uptake of IBA, a high degree of conjugation resulted in a recovery of 2.5% as internal free IBA (ca. 2 times the medium concentration). Due to conversion of absorbed IBA into IAA a fraction of 0.4% was recovered as (physiologically active) free IAA. After incubation on medium with IAA, 0.5% of the absorbed hormone was recovered in the free acid form. No conversion of IAA into IBA was observed. Equimolar contents of internal free IAA after incubation on IBA or IAA resulted for IBA in a higher number of roots than for IAA. This means that IBA may also act via internal free IBA or may synergistically modify the action or endogenous synthesis of IAA.Abbreviations BHT butyl hydroxy toluene - EtOH ethanol - FW fresh weight - HAc acetic acid - HPLC high performance liquid chromatography - IAA indoleacetic acid - IAA^int free (internal) IAA in the tissue derived from IBA or IAA uptake - IBA indolebutyric acid - IBA^int free (internal) IBA in the tissue derived from IBA or IAA uptake - MeOH methanol - PAR photosynthetic active radiation - SEM standard error of the mean - TLC thin layer chromatography - UV ultra violet     

Auxin Transport Inhibitors and the Rooting of Hypocotyl Cuttings from Etiolated Mung-bean Vigna radiata (L.) Wilczek Seedlings

The auxin transport inhibitors 2, 3, 5-triiodobenzoic acid (TIBA)and naphthylphthalamic acid (NPA) inhibited adventitious rootformation (ARF) induced by indol-3-butyric acid (IBA) on cuttingsfrom etiolated mung-bean seedlings floated on solutions of thegrowth regulators. The concentrations of TIBA and NPA requiredfor a 25 per cent reduction in ARF with 10 µM IBA wereestimated by linear interpolation to be 11.3 µm and 0.42µM respectively. NPA is a particularly potent inhibitorof IBA-induced ARF. The inhibitory effect of either compoundwas reversible by higher concentrations of IBA. NPA had no effectwhen applied after the auxin treatment. The inhibitory effects of TIBA or NPA could not be explainedby effects on the uptake or metabolism of [2-^14C]IAA. Consideringthis and other evidence, it is suggested that NPA and possiblyTIBA are acting as specific antagonists of auxin in the inductionof ARF. Vigna radiata (L.), mung-bean, root induction, hypocotyl cuttings, auxin inhibitors, indol-3-butyric acid, 2,3,5-triiodobenzoic acid, naphthylphthalamic acid, auxin uptake, auxin metabolism, adventitious roots     

The Effect of Indole-3-Butyric Acid and Riboflavin on the Morphogenesis of Adventitious Roots of Eucalyptus ficifolia F. Muell. Grown In Vitro

Shoot explants from seedling-derived culture of Eucalyptus ficifoliaF. Muell. cultured on a rooting medium free from indole-3-butyricacid (IBA) develop a root system (Type I) consisting of a fewcomparatively long roots and only small amounts of callus. IBAat 5.0 µM in a rooting medium free from riboflavin inducesthe development, on the shoot explants, of a compact root system(Type II) consisting of callus and many short roots. Riboflavinwhen exposed to light, is able to photo-oxidize IBA; the degreeof photo-oxidation depends on the photon fluence of the lightreceived. The rooting response of the cultures reflects thedegree of photo-oxidation of IBA: concentrations of IBA fromabout 10^–4M to 10^–6M in the rooting medium induceformation of the Type II root system whilst photo-oxidationof the auxin to concentrations of about 10^–8M or lowerinduces the formation of the Type I root system. Thus, exogenousriboflavin and exogenous IBA are linked in a distinct light-induced,riboflavin-mediated change in root morphogenesis. The anatomyof root development in the Type I and Type II root systems wasstudied and factors affecting the development were defined.Characteristics of riboflavin and IBA breakdown in various lightregimes were determined and related to root morphogenesis. Theresults and their implications are discussed. Key words: Auxin photo-oxidation, Riboflavin, Root morphogenesis, Tissue culture     

Effect of light and riboflavin on indolebutyric acid-induced root formation on apple in vitro

Maximum root formation on apple ( Malus ) shoots cultured in vitro occurred after an incubation in the dark on medium containing 3.2 or 10 μ M indolebutyric acid (IBA) plus riboflavin. Omission of riboflavin or culture in light resulted in a significant decrease in the number of roots formed. About 95% of the absorbed IBA was inactivated by conjugation, ca 4% was extracted as the free IBA acid (IBAH) and only 1% as IAAH. It was investigated whether the decrease in root formation caused by exposure to light or omission of riboflavin during culture was parallelled by a shift in the concentrations of the physiologically active auxin compounds (IBAH and IAAH) in the stem base, i.e. the location where the roots emerge. At least 90% of the absorbed ³H-IBA was located in the stem base. Omission of riboflavin, either in the dark or in the light, had no effect on the IBAH and IAAH concentrations, whereas root formation decreased significantly. Incubation in the light on medium containing 10 μ M IBA with or without riboflavin and culture in the dark on medium containing 3.2μ M IBA plus riboflavin resulted in similar IBAH and IAAH concentrations. However, the number of roots was significantly lower after culture in the light. Therefore, we conclude that the synergistic effect of riboflavin and the antagonistic effect of light on IBA-induced root formation are not solely based on changes in the concentrations of the active auxin components resulting from IBA uptake.     

Different Behaviour of Indole-3-Acetic Acid and Indole-3-Butyric Acid in Stimulating Lateral Root Development in Rice (Oryza sativa L.)

The plant hormone auxin has been shown to be involved in lateral root development and application of auxins, indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA), increases the number of lateral roots in several plants. We found that the effects of two auxins on lateral root development in the indica rice (Oryza sativa L. cv. IR8) were totally different from each other depending on the application method. When the roots were incubated with an auxin solution, IAA inhibited lateral root development, while IBA was stimulatory. In contrast, when auxin was applied to the shoot, IAA promoted lateral root formation, while IBA did not. The transport of [³H]IAA from shoot to root occurred efficiently (% transported compared to supplied) but that of [³H]IBA did not, which is consistent with the stimulatory effect of IAA on lateral root production when applied to the shoot. The auxin action of IBA has been suggested to be due to its conversion to IAA. However, in rice IAA competitively inhibited the stimulatory effect of IBA on lateral root formation when they were applied to the incubation solution, suggesting that the stimulatory effect of IBA on lateral root development is not through its conversion to IAA.     

Effect of Applied and Endogenous Auxin on Callus and Root Formation of In Vitro Shoots of the Apple: Rootstocks M26 and A2

The influence of exogenous IBA (indol-3yl-butyric acid) on rootand callus formation was studied in shoots of the apple rootstocksA2 and M26. The shoots grown in vitro were derived originallyfrom meristems of both juvenile and adult trees. Endogenousindol-3yl-acetic acid (IAA) concentrations in leaves and stemswere correlated with the responses to applied IAA. After 30 subcultures shoots from A2 and M26 rooted easily, butA2 did so more readily and even without IBA. Treatment withIBA improved percentage rooting and number of roots in bothrootstocks. Ex-adult and ex-juvenile shoots of A2 formed rootsto the same extent. However, ex-adult shoots of A2 showed ahigher IBA optimum for root number than ex-juvenile A2 and werealso less sensitive to supra-optimal IBA concentrations. Incontrast, in M26, there were no differences between ex-adultand ex-juvenile shoots. The results imply that rooting ability is associated more withdifferences between cultivars than with the origin of the explants.The best rooting occurred in ex-adult shoots of A2 which hadthe lowest endogenous IAA concentration, while callus formationwas correlated with high endogenous auxin concentration. Ex-adultA2 produced almost no callus even after exposure to high IBAconcentrations (25µM) whereas ex-adult M26 formed muchmore callus at 1/10 of the IBA concentration. Malus sylvestris (L.) Mill. var. domestica Borkh., Malus pumila Mill., apple rootstocks A2 and M26, in vitro culture, root and callus formation, HPLC analyses of IAA     

Hormonal Effects on Growth and Morphology of Normal and Hairy Roots of Hyoscyamus muticus

Treatment of normal and Agrobacterium rhizogenes-transformed root cultures of Hyoscyamus muticus with three different auxins, indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), and naphthaleneacetic acid (NAA), revealed that the response varied considerably among auxins, between transformed and normal roots, and depending on the parameter. In normal roots all three auxins provoked abundant branching, with IBA and NAA being the most effective at 2.5 and 0.5 μm, respectively, whereas IAA was most effective at low concentrations (0.05 and 0.1 μm). In transformed roots exogenously supplied auxins were generally inhibitory or, at best, without effect on growth and branching. Only 0.01 μm IAA significantly enhanced lateral root number, whereas at the higher concentrations IBA, although inhibitory, was the least effective auxin. In both root types IBA had little effect on primary root growth, but normal roots were more sensitive to IAA and NAA. These results suggest a different sensitivity to auxins of normal and transformed roots since there was no significant difference in endogenous free and conjugated IAA content nor in IAA uptake capacity. Ethylene production and biosynthesis were approximately threefold higher in hairy roots, but production could be stimulated up to tenfold that of control levels in normal roots by supplying NAA or 1-aminocyclopropane-1-carboxylic acid (ACC). Treatment with 2.5 μm NAA, but not IAA or IBA, also enhanced ethylene biosynthesis in normal roots but not in transformed ones. ACC and malonyl-1-aminocyclopropane-1-carboxylic acid accumulated to detectable levels only after treatment with an auxin (NAA). Received March 3, 1997; accepted May 28, 1997     

Total and Polar Lipid Biosynthesis during Crotalaria juncea Linn. Pollen Tube Growth: Effect of Gibberellic Acid, Indoleacetic Acid and (2-Chloroethyl)-phosphonic Acid

Gibberellic acid (GA₃) at 58 µM, indoleacetic acid (IAA)at 29 µM, and (2-chloroethyl) phosphonic acid (Ethephon)at 70 µM promoted pollen tube growth in Crotalaria junceapollen suspension cultures both in water and basal medium. GA₃stimulated [ l-¹⁴C]acetate incorporation into total lipids inboth media, whereas IAA enhanced incorporation in water culturesonly. On the contrary, Ethephon reduced the label in total lipidswhen supplemented in basal medium. Based on [l-¹⁴C lacetateincorporation into different phospho- and glycolipids, it isproposed that these growth regulators have a definite role inthe biosynthesis of lipid components of the membranes.     

Callus, Organogenesis and Plantlet Formation in Tissue Cultures of Clitoria ternatea

Decoated seeds of Clitoria ternatea L. germinated on Murashigeand Skoog (Physiologia Plantarum 1962, 15, 473–97) basalmedium (BM) and differentiated callus and bipolar embryoids(two-step method) in low frequency. Calluses developed on lateralroots [BM+KN(0.1 mg 1^–1)], on roots and hypocotyls [BM+KN(0.5mg 1^–1)], and on roots [BM+KN+IAA (0.5 mg 1^–1 ofeach)]. On basal medium with KN (0.5 mg 1^–1) and withKN+IAA (0.5 mg1^–1 of each), multiple shoot buds and embryoids(one-step method) were differentiated directly on split hypocotylsand roots. In the former, shoot buds developed even on unsplithypocotyls. Rhizogenesis on isolated shoot buds occurred efficientlyin BM+indole butyric acid (IBA 0.1 mg 1^–1) and BM+IAA(0.1 mg 1^–1 and 0.5 mg 1^–1). Profuse direct embryoidsand shoot buds developing on root systems are interesting morphogeneticphenomena rarely reported. Clitoria ternatea L., callus, embryoids, multiple shoot buds, regeneration     

Occurrence of Inducible Crassulacean Acid Metabolism in Leaves of Talimum triangulare (Portulacaceae)

In this paper we report for the first time the occurrence ofan inducible weak CAM in leaves of Talinwn triangulare (Jacq.)Willd. This plant is a terrestrial perennial deciduous herbwith woody stems and succulent leaves which grows under fullexposure and in the shade in northern Venezuela. Plants grownin a greenhouse (‘sun’ plants) and a growth cabinet(‘shade’ plants) with daily irrigation showed CO₂uptake only during the daytime (maximum rate, 4?0 µmolm^–2 s^–1) and a small acid accumulation during thenight (6?0 µmol H⁺g^–1 FW). Twenty-four hours aftercessation of irrigation, no CO₂ exchange was observed duringpart of the night. Dark fixation reached a maximum (1?0 µmolCO₂ m^–2 s^–1, 100 µmol H⁺ g^–1 FW) onday 9 of drought. By day 30 almost no gas exchange was observed,while acid accumulation was still 10 µmol H⁺ g^–1FW. Rewatering reverted the pattern of CO₂ exchange to thatof a C₃ plant within 24 h. Daytime and night-time phosphoenolpyruvatecarboxylase activity increased up to 100% (shade) and 62% (sun)of control values after 10 and 15 d of drought, respectively.Light compensation point and saturating irradiance were similarin well-watered sun and shade plants, values being characteristicof sun plants. CAM seems to be important for the tolerance ofplants of this species to moderately prolonged (up to 2 months)periods of drought in conditions of full exposure as well asshade, and also for regaining high photosynthetic rates shortlyafter irrigation. Key words: Talinum triwigulare, inducible CAM, PEP-C activity, recycling     

Effect of auxins on <Emphasis Type="Italic">Karwinskia humboldtiana</Emphasis> root cultures

Effects of auxins (IAA, IBA and NAA) on K. humboldtiana root culture cultivated in 16-h photoperiod or in dark have been observed. Light affected positively the production of biomass when cultivated on medium supplemented with NAA in 10 and 25 mol ^–1 concentrations. In the presence of IAA and IBA these values were significantly lower. The growth dynamics of root cultures depended on the auxin used. The best adventitious roots elongation and lateral roots induction on media supplemented with IBA has been ascertained. Morphological and anatomical differences in dependence on auxin used were observed. NAA supported the formation of huge callus-like mass besides mostly very short roots, especially under the light. Similarly IAA induced short roots, and IBA seems to be the most effective substance for the root elongation in this model system. NAA induced roots with larger diameter under the light compared with the other two auxins used. The reason is in the different anatomical structure of roots which was characterized by higher number of cell layers and large intercellulars in the cortex. The shape of cortical cells in the presence of IBA depended on the light conditions. Isodiametric cortical cells were present in roots cultivated in 16-h photoperiod, irregularly-shaped cells in the dark. The effect of light conditions was the smallest in the case of roots grown on IAA enriched media.     

Potassium Transport in Non-Growing Corn Root Tissue as Affected by IAA and GA3

Experiments were done to determine if the spontaneous recoveryof non-growing segments of corn root (Zea mays L.) from excisioninjury is dependent on auxin. Washing the segments with 5 runindoleacetic acid (IAA) for 2 to 4 hours gave a small but significantincrease in K⁺ (⁸⁶Rb) influx, used here as a parameter reflectingrecovery of electrogenie H⁺-efflux pumping. This promotive effectwas obtained only after an hour of washing, and was sustainedby 100 nm gibberellic acid (GA₃). Any early responses to auxinwere obscured by an adverse reaction of the root cells to externalIAA which resulted in a transitory inhibition of H⁺ pumpingand K⁺ influx. Pretreatment of excised root tips with 10 µM IAA in thegrinding medium protected a plasmalemma-enriched fraction ofthe microsomes during isolation, giving increased uncoupler-sensitiveATPase activity. Non-growing root tissue thus shows three responses to auxin:an adverse reaction at the outer surface of the plasmalemmawhich blocks H⁺ pumping; a protective or restorative effecton the H⁺-ATPase; an increased capacity for K⁺ influx duringthe developmental phase of washing, which is augmented by thepresence of GA₃. (Received March 31, 1986; Accepted September 8, 1986)     

Promotion of Plant Cell Division by an Epidermal Growth Factor

In some specified treatments, an epidermal growth factor (EGF)promoted adventitious root formation in epicotyl cuttings ofVigna angularis. The number of the roots induced in cuttingstreated with 0.1 mg liter^-1 EGF during the first 24 h and with210^-4 M IAA during the second 24 h was 15% greater than thatof the roots in cuttings treated without EGF and with IAA. Analysisof the optimum timing of EGF application was performed by dividingthe first 24 h period into three sequential 8 h periods (0–8h, 8–16 h and 16–24 h). The most effective timeperiods in terms of the root formation were 8–16 h and16–24 h. The 0–8 h period was ineffective with respectto the formation. When carrot suspension cells were culturedfor 15 days at a very low cell density (1,000 cells/3 ml Murashigeand Skoog's medium) with more than 0.1 mg liter^-1 EGF, cellnumbers were 72% higher than those cultured without EGF. Theseresults suggest that EGF promotes cell division of plants. (Received October 5, 1992; Accepted May 24, 1993)     

In Vitro Regeneration from Seedling Organs of Brassica oleracea var. italica Plenck cv. Green Comet I. Effect of Plant Growth Regulators

The calabrese cultivar Brassica oleracea var. italica cv. GreenComet was used in a study of the effects of exogenous hormoneson the growth and differentiation of seedling organs in vitro.Four types of explants were tested: hypocotyl segments, rootsegments, primary leaf discs and cotyledon discs. These explantswere incubated on media containing factorial combinations ofBAP x IBA, BAP x NAA, KN x IBA and KN x NAA (all at 0, 0.1,10 and 10.0mg l^–1). Hypocotyls were the most regenerativeexplants; shoot production was favoured by cytokinin: auxinratios greater than one and was decreased by IBA at 10 mg l^–1when callus was produced. Shoot formation from root explantsoccurred either in the absence of hormones or with low concentrations;no shoot was produced when any hormone was present at 10 mgl^–1. In contrast, shoot production from primary leaf diseswas favoured by high concentrations of both auxin and cytokininwith the combination of BAP and IBA the most effective. Shootproduction from cotyledon discs was sporadic with no consistentresponse on any auxin/cytokinin combination. After further experimentson the optimization of hormone concentration, the followingcombinations were chosen as allowing reliable regeneration:0.1 mg l^–1 BAP+0.1mg l^–1 IBA for hypocotyl segments,0.075 mg l^–1 KN +0.025 mg l^–1 IBA for root segments,and 5.0 mg l^–1 BAP+5.0 mg l^–1 IBA for leaf discs. Brassica oleracea var. italica, calabrese, tissue culture, seedling, auxin, cytokinin     

Storage Organ Development in Radish (Raphanus sativus L.). 2. Effects of Growth Promoters on Cambial Activity in Cultured Roots, Decapitated Seedlings and Intact Plants

The radish varieties Cherry Belle and Long White Icicle wereused to investigate the role of the shoot and the effects ofsynthetic growth promoters in controlling cambial activity inthe seedling axis. Development was compared in excised roots, roots with hypocotylsattached and intact seedlings cultured aseptically on a nutrientmedium. No cambial divisions were seen in isolated radicleswhich had been cultured for ten days following excision butretention of hypocotyl tissue or the entire shoot resulted incambial activity and the production of secondary vascular tissues.Enriching the culture medium by raising the sucrose conantrationto 8% and including 10^–5 M indol-3yl acetic acid (IAA)5 x 10^–6 M 6-benzylaminopurine (BA) and 5 x 10^–4Minositol enhanced root thickening, increasing stele and xylemdiameters in roots cultured both with and without attached shoottissues. The effects of shoot tissues and enrichment of themedium were additive. The effects of auxin, cytokinin and gibberellin (gibberellicacid, GA²) were also studied on daxpitated seedlings. BA wasmuch more effective in inducing cell divisions in the hypocotylthan either IAA or GA supplied separately but a mixture of IAA+GAalso produced clearly defined arcs of cambial tissue. Littlesecondary tissue had been produced after seven days' treatment,and stelar enlargement was due to the development of a cambialzone and cell expansion in the primary tissues. Only minor differencesin response were observed between the two varieties. No stimulation of storage organ development occurred when auxin,cytokinin or inositol was inwrporated into the inorganic culturesolution in which plants of Cherry Belle were grown. Rnphanus sarivus, radish, storage organ, cambial activity, growth promoters, indol-3-ylacetic acid, 6-benzylaminopurine, gibberellic acid     

Distribution of Radioactivity from Exogenously Supplied [1-14C]Indol-3-yl-acetic Acid and [3, 4-3H (N)] Gibberellin A, in Geotropically-stimulated Picea abies (L.) Karst. Roots

The distribution of exogenously-supplied radioactive labelledindol-3-yl-acetic acid (IAA) and gibberellin A₁ (GA₁) in geotropicallystimulated roots of Norway spruce (Picea abies (L.) Karst.)has been demonstrated. Seedlings were positioned with theirroot tips in 2.1 x 10^–6 M [¹⁴C]IAA or 1.3 x 10^–8m ³H-GA₁ for 4 and 20 h, respectively. After geotropic stimulationfor 90 min in the horizontal position the root tips were cutlongitudinally in 50 µm thick sections, using a freeze-microtome.The radioactivity in the ¹⁴C-IAA treated roots occurred in higherconcentration in the lower than in the upper halves (ratio 1.25:1). A similar trend was observed in the [³H]GA₁-treated rootswhere the ratio lower: upper halves was 2.04: 1. The ratio ofradioactivity in right and left halves of vertical roots wasapproximately the same in roots supplied with [¹⁴C]IAA and [³H]GA₁(1.09: 1). The supplied radioactive compounds were analysed chromatographicallyafter extraction in methanol of 6 mm apical root segments. Onlya small fraction (7–8 per cent) of the supplied [¹⁴C]IAAwas revealed unchanged in the segments. The major part of thechromatographed, labelled compound has not been identified,but on basis of its R_F value it is suggested that it may beindol-3-acetyl-aspartic acid (IAAasp). The chromatographic analysis of the [³H]GA,-treated segmentsshowed that only small fractions of this gibberellin has beenconverted to other compounds. These results have been discussed and correlated with knowledgeof plant growth regulators and their participation in root geotropism. Picea abies, spruce, geotropism, gibberellin A₁, indol-3-yl-acetic acid, growth regulators, redistribution in roots