Promotion of adventitious root formation by 4-chlororesorcinol: A polyphenol oxidase inhibitor

The extent of rooting in cuttings of Phaseolus vulgaris L., and Vigna radiata Wilcz. was affected by 4-chlororesorcinol, a polyphenol oxidase inhibitor. More root primordia and more roots were formed after 4-chlororesorcinol treatment both with and without 10^-5M Indole butyric acid. Promotion of rooting was observed also in cuttings of Elaeagnus pungens, Gypsophilia elegans and Kalanchoe blossfeldiana. The enhancement in bean and mung bean was accompanied by a concomitant wider spatial distribution of the primordia and the resulting adventitious roots. The formation of primordia in the treated cuttings was delayed by 12–24 hours, compared to untreated cuttings. The treatment was effective only when given during the first hours after the preparation of the cutting of bean and mung bean, suggesting involvement in the initiation stage. Hypocotyl extracts of mung bean cuttings, pretreated with 4-chlororesorcinol, exhibited reduced polyphenol oxidase activity. The inhibition was not reversed by washing of the treated extract in 50% acetone or by an overnight dialysis, suggesting tight or maybe even irreversible binding of the inhibitor to the enzyme.Abbreviations 4-CR 4-chlororesorcinol - IBA Indole butyric acid - PPO polyphenol oxidase     

Stimulation of adventitious root formation in non-woody stem cuttings by uridine

Uridine strongly stimulated adventitious root formation in stem cuttings of sunflower (Helianthus annuus L.), mung bean (Vigna radiata L.) and common bean (Phaseolus vulgaris L.). A dose response curve of uridine induced rooting showed that the optimum concentration of uridine was 0.1 µM. At all concentrations employed, uridine had no significant effect on root elongation. The rooting response of stem cuttings to the optimal concentration of indole-3-butyric acid (10 µM) in combination with 0.1 µM uridine did not significantly differ from their response to either of these compounds when applied alone. However, the rooting response of the cuttings to sub-optimal IBA (0.01 µM) was significantly stimulated by uridine. These findings suggested that uridine may have stimulated rooting by increasing the sensitivity of the rooting tissue to auxin.     

Caffeic acid affects early growth, and morphogenetic response of hypocotyl cuttings of mung bean (Phaseolus aureus)

Caffeic acid (CA) is one of the most common cinnamic acids ubiquitously present in plants and implicated in a variety of interactions including allelopathy among plants and microbes. This study investigated the possible interference of CA with root growth and the process of rhizogenesis in hypocotyl cuttings of mung bean (Phaseolus aureus=Vigna radiata). Results indicated that CA (0-1000 microM) significantly suppressed root growth of mung bean, and impaired adventitious root formation and root length in the mung bean hypocotyl cuttings. Further investigations into the role of CA in hampering root formation indicated its interference with the biochemical processes involved in rooting process at the three stages - root initiation (third day; RI), root expression (fifth day; RE), and post-expression (seventh day; PE) - of rhizogenesis. CA caused significant changes in the activities of proteases, peroxidases (PODs), and polyphenol oxidases (PPOs) during root development and decreased the content of total endogenous phenolics (TP) in the hypocotyl cuttings. The enhanced activity of PODs and PPOs, though, relates to lignification and/or phenolic metabolism during rhizogenesis; yet their protective role to CA-induced stress, especially during the PE phase, is not ruled out. At 1000 microM CA, where rooting was significantly affected, TP content was very high during the RI phase, thus indicating its non-utilization. The study concludes that CA interferes with the rooting potential of mung bean hypocotyl cuttings by altering the activities of PODs and PPOs and the endogenous TP content that play a key role in rhizogenesis.     

脱落酸对绿豆下胚轴不定根发生的影响

以10^-4 mol/L脱落酸(ABA)处理绿豆种子24 h,在幼苗下胚轴长6 cm时,切除根部作为插条,研究ABA对插条不定根发生及插条基部细胞周期时相的影响。结果表明,ABA可促进下胚轴插条不定根发生,增加生根数和生根范围;ABA提高插条基部细胞色氨酸转氨酶、吲哚丙酮酸脱羧酶和吲哚乙醛脱氢酶的比活性,增加吲哚乙酸含量,同时进入细胞周期S期的基部细胞数目增加,促进DNA合成,有利于不定根的发生。     

THE EFFECTS OF TEN PHENOLIC COMPOUNDS ON HYPOCOTYL GROWTH AND MITOCHONDRIAL METABOLISM OF MUNG BEAN

Ten phenolic compounds were examined for their effect on mung bean (Phaseolus aureus L.) hypocotyl growth and on respiration and coupling parameters of isolated mung bean hypocotyl mitochondria. Three compounds—tannic, gentisic, and p-coumaric acids—inhibited hypocotyl growth and when incubated with isolated hypocotyl mitochondria released respiratory control, inhibited respiration, and prevented substrate-supported Ca²⁺ and PO₄ transport. Vanillic acid also inhibited hypocotyl growth and reduced mitochondrial Ca²⁺ uptake but did not affect respiration or respiratory control of isolated mitochondria. This is the first compound reported to selectively inhibit Ca²⁺ uptake in plant mitochondria. Two other phenolic compounds—α, 3,5-resorcylic and protocatechuic acids—showed no significant effect on hypocotyl growth and did not affect mitochondrial oxidative phosphorylation either separately or in various combinations. Four phenolic compounds—ferulic, caffeic, p-hydroxybenzoic, and syringic acids—showed a significant reduction in mung bean hypocotyl growth but did not inhibit any of the mitochondrial processes examined. The results show that phenolic compounds which alter respiration or coupling responses in isolated mitochondria also inhibit hypocotyl growth and may reflect a mechanism of action for these natural growth inhibitors.     

Diheteroarylurea derivatives as adventitious rooting adjuvants in mung bean shoots and M26 apple rootstock

The present research investigates the biological profile of eight symmetrical diheteroarylureas and phenylheteroarylureas, testing their hypothetical cytokinin-like activity and rooting activity. Cytokinin-like activity was assayed by the betacyanin (so-called amaranthin) accumulation test and by the tomato regeneration test. The rooting activity was assessed using the mung bean rooting test, the apple stem slice test and the rooting of apple microcuttings. Three compounds, 1,3-di(pyrazin-2-yl)urea (3a), 1,3-di(benzo[d]oxazol-5-yl)urea (3b) and 1,3-di(benzo[d]oxazol-6-yl)urea (3c), enhanced adventitious root formation in apple stem slice test, but only 3b and 3c were active in the mung bean rooting test. Compound 3b, that showed the best rooting activity, was also able to enhance the adventitious root formation in apple microcuttings. None of the compounds showed cytokinin-like activity.     

Synergistic effects of plant growth retardants and IBA on the formation of adventitious roots in hypocotyl cuttings of mung bean

The synergistic effect of plant growth retardants, such as daminozide, paclobutrazol and triadimefon, and of indole-3-butyric acid (IBA) on the formation of adventitious roots in hypocotyl cuttings of mung bean was studied. The three retardants and IBA all stimulated adventitious root growth, but IBA was the most effective. However, mixtures of the retardants with IBA have proven generally more effective than IBA alone in promoting adventitious root formation. When IBA was applied to the hypocotyls one day after cutting preparation followed by the growth retardant on the second day, there were even more adventitious roots produced than if applied in the reverse order. The effectiveness of the treatments were in the order, IBA followed by growth retardant, IBA + growth retardant together, and IBA alone.Abbreviations IBA indole-3-butyric acid - GA gibberellin     

Methyl gallate and its 3-glucoside promote rooting in bean cuttings

Methyl gallate stimulated adventitious root formation in cuttings of bean (Phaseolus vulgaris L.). This polyphenol was quickly metabolized into 3-glucosyl methyl gallate to such an extent that 4 h after application no methyl gallate was detected. The isolated glucoside when supplied exogenously at 0.5 mM also enhanced rooting; the effect was 2-fold greater than that of methyl gallate. The glucoside persisted in the cuttings for 72 h after treatment. Because methyl gallate is rapidly transformed to a stable glucoside, we suggest that the root stimulation effect could be ascribed to its glucoside.     

Interaction of paclobutrazol and indole-3-butyric acid in relation to rooting of mung bean (Vigna radiata) cuttings

Paclobutrazol (PB) only slightly stimulated the rooting of mung bean cuttings but, interestingly, the number of adventitious roots formed was dramatically increased when PB was used together with indole-3-butyric acid (IBA). Application of PB in the first phase of root formation, when root initials are induced, caused the greatest enhancement of the promotive effect of IBA on rooting. Investigation of the effect of PB on uptake, transport and metabolism of [5^-3H]-IBA in mung bean cuttings revealed some changes in the rate of metabolism of IBA in comparison with control cuttings. PB was found to be involved in the partitioning of carbohydrates along the cuttings. Application of sucrose, like PB to the base of IBA-treated cuttings enhanced the effect of IBA. The patterns of the effects of PB and IBA, separately and together, on rooting were similar in defoliated and intact cuttings, however the number of roots was much lower in the defoliated cuttings, which lacked a source of assimilates. PB counteracted the effect of GA₃ in the upper regions of the cuttings and seemed to increase the sink capacity at the base of the cuttings. The results of the present study clearly demonstrated the enhancing influence of PB on IBA stimulation of the rooting of mung bean cuttings. It is suggested that PB may affect the rate of metabolism of IBA during rooting and the status of the local sink, in the base of the cuttings, thus partially contributing to the enhancement of the rooting-promotive effect of IBA.     

Changes in the Level of [C]Indole-3-Acetic Acid and [C]Indoleacetylaspartic Acid during Root Formation in Mung Bean Cuttings

Changes in the levels of [¹⁴C]indole-3-acetic acid (IAA) and [¹⁴C]indole-acetylaspartic acid (IAAsp) were examined during adventitious root formation in mung bean (Vigna radiata [L.] R. Wilcz. `Berken') stem cuttings. IAAsp was identified by GC-MS as the primary conjugate in IAA-treated cuttings. During root formation in IAA-treated cuttings, the level of [¹⁴C]IAAsp increased rapidly the first day and then declined; [¹⁴C]IAA was rapidly metabolized and not detected after 12 hours.     

Effect of abscisic acid on endogenous IAA, auxin protector levels and peroxidase activity during adventitious root initiation in Vigna radiata cuttings

Endogenous levels of free and conjugated IAA, auxin protectors (Prs) and peroxidase (PER) activity and their relation to adventitious root initiation (ARI) were investigated at the potential sites of adventitious rooting in relation to exogenous application of 250 μM ABA during the first 120 h after treatment. Cuttings from 7-day-old mung bean [Vigna radiata (L.) Wilcz.] seedlings were treated with 125, 250, and 500 μM ABA for 24 h. ABA significantly stimulated ARI but extremely inhibited epicotyl growth as compared to control. Free and conjugated IAA were measured by reversed-phase high performance liquid chromatography while Prs and PER activities were measured spectrophotometrically. The present results also indicate that endogenous free IAA levels peaked later in ABA-treated cuttings than that in control, suggesting that ABA extended the length of the induction phase of rooting process in treated cuttings and that might explain the significant delay of the appearance of roots at the treated cuttings. Higher level of IAA conjugates was found in ABA-treated cuttings than that in untreated ones. Pr level also peaked later in ABA-treated cuttings than that in control, indicating that ABA extended the period of Pr activity. An initial temporary decrease of PER activity was found in associating with high levels of free IAA and Prs during most of the primary events, while the opposite occurred during the secondary events of adventitious rooting process in both treated and untreated cuttings. Thus, ABA may stimulate ARI in mung bean Vigna radiata cuttings by regulating the concentration and /or activities of endogenous IAA, Prs, and PER activity in favor of inducing a large number of adventitious roots at their potential sites of adventitious rooting.     

Influence of Aryl Esters of Indole-3-acetic and Indole-3-butyric Acids on Adventitious Root Primordium Initiation and Development

Synthetic aryl esters of indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) greatly enhanced adventitious root primordium initiation in bean (Phaseolus vulgaris L. cv. Top Crop) and jack pine (Pinus banksiana Lamb.) cuttings, respectively. Bean cuttings produced 95 to 154% more macroscopically visible root primordia in 2 days when treated with phenyl indole-3-acetate (P-IAA), in comparison with an equal concentration of IAA. Substantial but lesser increases occurred when treatment was done with 3-hydroxyphenyl indole-3-acetate (3HP-IAA). On a molar basis, either P-IAA or 3HP-IAA were 10 or more times as efficient as IAA in inducing adventitious root primordium initiation in bean cuttings. Methyl indole-3-acetate was no more effective than IAA in these tests. Phenyl indole-3-butyrate (P-IBA) consistently enhanced the number of rooted jack pine seedling cuttings by 11 to 12% in comparison with a 27% higher concentration of IBA. The number of elongated roots (2 mm or more) after 5 days was 165 to 276% greater for P-IAA than for IAA-treated bean cuttings. Similar but lesser increases occurred as a result of 3HP-IAA treatment. P-IBA in comparison with IBA treatment did not influence either the number of roots or length of the longest root per rooted jack pine cutting. Enzymes in bean and jack pine cuttings hydrolyzed the aryl esters. However, check experiments showed that initial integrity of the esters was required for enhanced activity in inducing root primordium initiation. Treatment of bean cuttings with hydrolysates of P-IAA, or with IAA and phenol, alone or combined, did not influence root primordium initiation or development in a manner different from treatment with IAA alone.     

Plant growth regulators and adventitious root development in relation to auxin

Adventitious root formation in stem cuttings of mung bean was enhanced by ethrel, which had an additive effect when employed simultaneously with indolebutyric acid (IBA). Abscisic acid (ABA) did not influence the number of roots per cutting whereas gibberellic acid (GA₃) and kinetin were without effect on rooting at lower concentrations but were inhibitory at higher concentrations. Nevertheless, all three of these chemicals showed synergistic interactions with IBA and/or indol-3-ylacetic acid (IAA) and thereby significantly promoted root formation. A localised application of morphactin to the epicotyl of cuttings totally inhibited root production irrespective of which of the foregoing growth regulators were suppliedvia the hypocotyl. Morphactin application also prevented root formation in cuttings treated with vitamin D₂. The various growth regulators employed had differing effects on growth of roots but there was no simple relationship between their effects on root formation and subsequent root growth.     

The Weak Cytokinins N,N′-bis-(1-naphthyl)urea and N,N′-bis-(2-naphthyl)urea may Enhance Rooting in Apple and Mung Bean

The present research investigates the possibility that 2 weak urea-type cytokinins, the N,N′-bis-(1-naphthyl)urea and the N,N′-bis-(2-naphthyl)urea, enhance adventitious root formation. The rooting activity was assessed using the stem slice test, the mung bean rooting test and the rooting of apple microcuttings. The two compounds influenced the adventitious rooting process differently as regards the bioassay used. In the stem slice test, in the presence of exogenous auxin, both compounds enhanced the rooted slice percentage. In mung bean shoots, the N,N′-bis-(1-naphthyl)urea enhanced the root formation at the lowest concentration used (0.01 μM) while the N,N′-bis-(2-naphthyl)urea enhanced rooting at higher concentrations. In the rooting test of apple microcuttings the N,N′-bis-(1-naphthyl)urea and the N,N′-bis-(2-naphthyl)urea slightly enhanced only the mean root number per microcutting.     

Hydrogen Peroxide Is a Second Messenger in the Salicylic Acid-Triggered Adventitious Rooting Process in Mung Bean Seedlings

In plants, salicylic acid (SA) is a signaling molecule that regulates disease resistance responses, such as systemic acquired resistance (SAR) and hypertensive response (HR). SA has been implicated as participating in various biotic and abiotic stresses. This study was conducted to investigate the role of SA in adventitious root formation (ARF) in mung bean (Phaseolus radiatus L) hypocotyl cuttings. We observed that hypocotyl treatment with SA could significantly promote the adventitious root formation, and its effects were dose and time dependent. Explants treated with SA displayed a 130% increase in adventitious root number compared with control seedlings. The role of SA in mung bean hypocotyl ARF as well as its interaction with hydrogen peroxide (H₂O₂) were also elucidated. Pretreatment of mung bean explants with N, N’-dimethylthiourea (DMTU), a scavenger for H₂O₂, resulted in a significant reduction of SA-induced ARF. Diphenyleneiodonium (DPI), a specific inhibitor of membrane-linked NADPH oxidase, also inhibited the effect of adventitious rooting triggered by SA treatment. The determination of the endogenous H₂O₂ level indicated that the seedlings treated with SA could induce H₂O₂ accumulation compared with the control treatment. Our results revealed a distinctive role of SA in the promotion of adventitious rooting via the process of H₂O₂ accumulation. This conclusion was further supported by antioxidant enzyme activity assays. Based on these results, we conclude that the accumulation of free H₂O₂ might be a downstream event in response to SA-triggered adventitious root formation in mung bean seedlings.     

Physiological basis of the synergistic effects of IBA and triadimefon on rooting of mung bean hypocotyls

Mixtures of 1–3 × 20.32 mg L^-1 IBA and 1–3 × 289.5 µg L^-1 triadimefon (TRI) significantly increased the formation of adventitious roots in mung bean hypocotyl cuttings compared to application of either IBA or TRI alone. The physiological basis of the synergistic effects of the mixture is likely to be due to a combination of increased endogenous IAA content and peroxidase activity. It is suggested that a mixture of IBA with TRI at appropriate concentrations is an effective and simple method for promoting adventitious root formation.     

The effect of 4-chlororesorcinol on the endogenous levels of IAA,ABA and oxidative enzymes in cuttings

Treatment of bean cuttings with 4-chlororesorcinol (4-CR), known to increase the number of roots and extend their distribution, prevented the accumulation of free indol-3-yl-acetic acid (IAA) in the hypocotyls within 24 h after cutting preparation. In mung bean there was no change in the distribution (upper half vs. 1 ower half of the hypocotyl) of IAA within the hypocotyl as a result of the treatment. In bean cuttings the treatment with 4-CR prevented the accumulation of IAA in the bottom of the cutting. Oxidation of IAA as a measure of IAA oxidase activity in bean was enhanced appreciably by 4-chlororesorcinol. The level of abscisic acid in mung bean, on the other hand, remained 3–4 fold higher than in the control, yet still about 50% lower than the zero time level. In untreated mung bean cuttings the activity of peroxidase increased after cutting preparation. In contrast, the activity of peroxidase in 4-Cr-treated cuttings was consistently lower. In order to relate to the effect of exogenously applied auxin the level of peroxidase was measured also in indol-3-yl-butyric acid-treated cuttings. The overall peroxidase activity in IBA-treated cuttings was not affected. However, when assaying for the different isozymes the drop in peroxidase activity was most evident in the inducible basic isoperoxidases both in 4-CR and IBA treatments. It appears that the exposure to 4-CR exerts an effect that is similar to that of exogenously applied auxin, affecting the activity of basic peroxidases and enhancing the oxidation of endogenous IAA, thus allowing the organization of the primordia.Abbreviations ABA - abscisic acid - 4-CR - 4-chlororesorcinol - IAA - indol-3-yl-acetic acid - IBA - indol-3-yl-butyric acid     

The effects of 3,5-diiodo-4-hydroxybenzoic acid on the oxidation of IAA and auxin-induced ethylene production by cress root segments

Summary In previous research here, 3,5-diiodo-4-hydroxybenzoic acid (DIHB) was shown to promote the elongation of roots of cress (Lepidium sativum) seedlings growing in light, and to inhibit the auxin-induced production of ethylene in this tissue. Although DIHB is a cofactor for the oxidation of indole-3-acetic acid (IAA) by horse-radish peroxidase, it inhibits the decarboxylation of [1-¹⁴C]IAA by segments excised from cress roots. The inhibition by DIHB of ethylene production by this tissue does not, therefore, arise from a reduction of IAA levels. These findings are discussed in relation to the effects of DIHB on cress root growth.Abbreviations IAA indole-3-acetic acid - DIHB 3,5-diiodo-4-hydroxybenzoic acid - DCP 2,4-dichlorophenol - 2,4-D 2,4-dichlorophenoxyacetic acid This study forms part of a research project to be submitted by M.L.R. for PhD degree and supported by a grant from Consejo Nacional de Ciencia y Tecnología (México).     

N,N′-bis-(2,3-Methylenedioxyphenyl)urea and N,N′-bis-(3,4-methylenedioxyphenyl)urea enhance adventitious rooting in Pinus radiata and affect expression of genes induced during adventitious rooting in the presence of exogenous auxin

We have analyzed the effect of N,N′-bis-(2,3-methylenedioxyphenyl)urea (2,3-MDPU) and N,N′-bis-(3,4-methylenedioxyphenyl)urea (3,4-MDPU), two symmetrically substituted diphenylurea derivatives with no auxin or cytokinin-like activity, on the rooting capacity of Pinus radiata stem cuttings. Results indicate that both diphenylurea derivatives enhance adventitious rooting in the presence of exogenous auxin (indole-3-butyric acid, IBA), even at low auxin concentration, in rooting-competent cuttings, but have no effect on the adventitious rooting of low or null competent-to-root cuttings. Histological analyses show that, in the simultaneous presence of MDPUs and low concentration of exogenous auxin, adventitious root formation is induced in the cell types that retain intrinsic competence to form adventitious roots in response to auxin. The time course of cellular events leading to root formation and the time of root emergence are closely similar to that observed in cuttings treated only with higher auxin concentration. In addition, the mRNA level of a P. radiata SCARECROW-LIKE gene, which is significantly induced in the presence of the optimal concentration (10 μM) of exogenous auxin needed for cuttings to root, is increased in the presence of MDPUs and low concentration of exogenous auxin (1 μM). The expression of a P. radiata SHORT-ROOT gene in rooting-competent cuttings during adventitious rooting is also affected by the presence of MDPUs when combined with auxin. As MDPUs do not affect the expression of either gene in the absence of exogenous auxin, but only in its presence, we suggest that MDPUs could interact, directly or indirectly, with the auxin-signalling pathways in rooting-competent cuttings during adventitious rooting.     

Abscisic-acid-stimulated rooting of stem cuttings

Summary Abscisic acid (ABA) has been found to stimulate rooting of stem cuttings of mung beans and English ivy. ABA partially overcame the inhibitory effect of gibberellic acid on root formation of mung bean cuttings but at the concentrations used did not overcome the inhibitory effect of kinetin on root formation.