Differential Gene Expression in Response to Auxin Treatment in the Wild Type and rac,an Adventitious Rooting-Incompetent Mutant of Tobacco

Histological analyses of auxin-treated cuttings from the wild type and the rac mutant of tobacco (Nicotiana tabacum cv Xanthii) previously revealed that some rac phloem parenchyma or inner cortical parenchyma cells form callus in response to exogenous auxin treatment but these cells never undergo the organized divisions associated with adventitious root initiation in the wild type. Here we report the effect of the rac mutation on the temporal and spatial expression patterns of three genes previously shown to be associated with adventitious root meristems, HRGPnt3, iaa4/5, and gh3. Using histochemical staining analyses of HRGPnt3-GUS transformant cuttings, we determined that the rac mutation blocks auxin activation of the HRGPnt3 promoter. Thus, activation of the HRGPnt3 promoter occurs specifically during adventitious root initiation in tobacco cuttings. Histochemical staining analyses of iaa4/5-GUS and gh3-GUS transformant cuttings revealed that the rac mutation does not repress the auxin activation of the iaa4/5 and gh3 promoters. Based on our histochemical staining analyses, we conclude that differential gene expression occurs in response to auxin treatment during adventitious root initiation in the wild type compared with callus formation in rac cuttings. We also determined that HRGPnt3 mRNA accumulation occurs in response to components of our root-induction protocol other than auxin, indicating that HRGPnt3 expression is regulated both developmentally and environmentally.     

Influence of indole-butyric acid, boron, myo-inositol, vitamin D2 and seedling age on adventitious root development in cuttings of Phaseolus aureus

Adventitious roots develop in stem cuttings of Phaseolus aureus Roxb. seedlings when treatment with indole-butyric acid (IBA) is followed by treatment with boron. Root development varies according to the age of seedlings from which cuttings are taken. Increased root number is associated with expansion of the first leaf pair but subsequently declines, whereas root growth increases with increasing seedling age. Removal of leaves furing the first 72 h of treatment impairs root initiation whereas root growth is diminished by removal of leaves at any time during the first 120 h of treatment. IBA stimulates movement of ¹⁴C-IAA out of leaves. Vitamin D₂ and myo-inositol stimulate rooting of intact cuttings provided cuttings are subsequently supplied with boron. Hypocotyls excised from cuttings pretreated with IBA develop roots in response to myo-inositol in the absence of boron. It is proposed that endogenous auxin, arising in the leaves, and myo-inositol have roles in root initiation whilst the role of boron is suggested as one of initiating or maintaining transport from the leaves.     

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.     

Early steps of adventitious rooting: morphology,hormonal profiling and carbohydrate turnover in carnation stem cuttings

The rooting of stem cuttings is a common vegetative propagation practice in many ornamental species. A detailed analysis of the morphological changes occurring in the basal region of cultivated carnation cuttings during the early stages of adventitious rooting was carried out and the physiological modifications induced by exogenous auxin application were studied. To this end, the endogenous concentrations of five major classes of plant hormones [auxin, cytokinin (CK), abscisic acid, salicylic acid (SA) and jasmonic acid] and the ethylene precursor 1‐aminocyclopropane‐1‐carboxylic acid were analyzed at the base of stem cuttings and at different stages of adventitious root formation. We found that the stimulus triggering the initiation of adventitious root formation occurred during the first hours after their excision from the donor plant, due to the breakdown of the vascular continuum that induces auxin accumulation near the wounding. Although this stimulus was independent of exogenously applied auxin, it was observed that the auxin treatment accelerated cell division in the cambium and increased the sucrolytic activities at the base of the stem, both of which contributed to the establishment of the new root primordia at the stem base. Further, several genes involved in auxin transport were upregulated in the stem base either with or without auxin application, while endogenous CK and SA concentrations were specially affected by exogenous auxin application. Taken together our results indicate significant crosstalk between auxin levels, stress hormone homeostasis and sugar availability in the base of the stem cuttings in carnation during the initial steps of adventitious rooting.     

Root Formation in Ethylene-Insensitive Plants

Experiments with ethylene-insensitive tomato (Lycopersicon esculentum) and petunia (Petunia x hybrida) plants were conducted to determine if normal or adventitious root formation is affected by ethylene insensitivity. Ethylene-insensitive Never ripe (NR) tomato plants produced more below-ground root mass but fewer above-ground adventitious roots than wild-type Pearson plants. Applied auxin (indole-3-butyric acid) increased adventitious root formation on vegetative stem cuttings of wild-type plants but had little or no effect on rooting of NR plants. Reduced adventitious root formation was also observed in ethylene-insensitive transgenic petunia plants. Applied 1-aminocyclopropane-1-carboxylic acid increased adventitious root formation on vegetative stem cuttings from NR and wild-type plants, but NR cuttings produced fewer adventitious roots than wild-type cuttings. These data suggest that the promotive effect of auxin on adventitious rooting is influenced by ethylene responsiveness. Seedling root growth of tomato in response to mechanical impedance was also influenced by ethylene sensitivity. Ninety-six percent of wild-type seedlings germinated and grown on sand for 7 d grew normal roots into the medium, whereas 47% of NR seedlings displayed elongated tap-roots, shortened hypocotyls, and did not penetrate the medium. These data indicate that ethylene has a critical role in various responses of roots to environmental stimuli.     

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     

Influence of galactoglucomannan oligosaccharides on root culture of <Emphasis Type="Italic">Karwinskia humboldtiana</Emphasis>

Galactoglucomannan oligosaccharides (GGMOs) activity in K. humboldtiana root culture has been determined. GGMOs inhibited adventitious root growth and lateral root induction in contrast to IAA, IBA, and NAA stimulating effect in these processes. Similarly, the combination of GGMOs with natural auxins (IAA, IBA) evoked an inhibition of adventitious root growth and lateral root induction that depended on the oligosaccharides concentration and the type of auxin. The growth stimulating effect of the synthetic auxin, NAA, in adventitious roots was negatively affected by GGMOs, but they were without influence on lateral root induction. The presence of oligosaccharides triggered lateral root position on adventitious roots and the anatomy of adventitious roots (diameter, proportion of primary cortex to the central cylinder, number and size of primary cortical cells, intercellular spaces, and the number of starch grains in cells of primary cortex) in dependence on their coactions with auxin.     

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.     

Auxin effects on rooting in pea cuttings

Light-grown stem cuttingss of Pisum sativum L. cv. Weibull's Marma were rooted in a nutrient solution. The presence of 10 μ M indolylacetic acid (IAA) in the solution for 24 h or longer periods decreased the number of roots subsequently formed to about 50% of control, provided IAA was present in the solution during any of the 4 first 24 h periods. Treatment for 6 h or shorter periods caused no or small response. IAA did not appreciably change the time needed for root formation, the time course of root appearance or the pattern of root distribution along the basal internode. IAA at 100 μ M usually increased the number of roots although variable results were obtained with this IAA concentration.
The number of roots was strongly increased by treatment with indolylbutyric acid (IBA) or 2,4-dichlorophenoxyacetic acid (2,4-D). None of these or other synthetic auxins decreased the number of roots in suboptimal concentrations. Experiments with 10 μ M IBA showed that stimulation of rooting was obtained only if the auxin was present in the rooting solution for several days. Simultaneous treatment with IAA decreased the stimulating effect of IBA to some extent, whereas no such response was obtained if IAA was combined with 2,4-D.
IAA applied in lanolin to the stem of intact cuttings decreased the number of roots formed. Decapitation and debudding of the cuttings decreased the number of roots formed. If at least 2 leaves were left this decrease was efficiently counteracted by an optimal IAA dose applied to the upper part of the stem. A five times higher dose was less effective, indicating a negative effect on rooting also by IAA applied to the shoots.     

Adventitious rooting adjuvant activity of 1,3-di(benzo[d]oxazol-5-yl)urea and 1,3-di(benzo[d]oxazol-6-yl)urea: new insights and perspectives

Here we report new insights on the adventitious rooting adjuvant activity of 1,3-di(benzo[d]oxazol-5-yl)urea (5-BDPU) and 1,3-di(benzo[d]oxazol-6-yl)urea (6-BDPU), both symmetrically substituted urea derivatives that do not show either auxin- or cytokinin-like activity per se. Our data demonstrate that these synthetic molecules enhance adventitious rooting in distantly-related herbaceous and woody species, in the presence of endogenous or exogenous auxin. For the first time, we report that BDPUs enhance adventitious rooting in the presence of either indole-3-butyric acid (IBA) or 1-naphtalene acetic acid and that their optimal concentration depends on the strength of the exogenous auxin. Trying to understand the mode of action of BDPUs, we also show that their adventitious rooting adjuvant activity correlates with high mRNA levels of auxin-responsive genes related to the adventitious rooting process at the very early stages of adventitious rooting, before the activation of cell divisions in pine hypocotyls cuttings. The high mRNA levels are measured in the presence of low auxin concentrations and BDPUs. The mRNA levels quantified in these conditions are similar to those measured in the presence of high auxin concentrations but in the absence of BDPUs. In addition, the spatial distribution of endogenous auxin is localized in globular-shaped structures of cell divisions located centrifugal to the resin canals, at the positions of adventitious root formation, in the presence of urea derivatives and IBA after 6 days of the root induction process.     

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.     

Adventitious root formation in relation to irradiance and auxin supply

High irradiance during treatment of mung bean cuttings favours root formation in response to supplied auxin, whether the latter is IAA or IBA. On the other hand it is inhibitory towards root formation in the absence of supplied auxin. Light promotes the uptake of¹⁴C-IAA into cuttings and its upward movement into the leaves. When¹⁴C-IAA is applied to leaves of cuttings high irradiance favours movement of radioactivity into the epicotyl and hypocotyl. This movement is also enhanced by concomitant supply of IBA to the base of the cuttings. The irradiance under which stock plants are raised also affects the extent of root formation on cuttings. When cuttings are held in darkness without a supply of exogenous auxin they root best if prepared from seedlings raised under high irradiance. However, transport of¹⁴C-IAA out of leaves of cuttings is favoured when cuttings are prepared from seedlings grown under low irradiance. These observations are discussed in relation to auxin transport, photodestruction and, possibly, metabolism.     

Synergistic interaction between coumarin 1,2-benzopyrone and indole-3-butyric acid in stimulating adventitious root formation in Vigna radiata (L.) Wilczek cuttings: I. Endogenous free and conjugated IAA and basic isoperoxidases

Cuttings from 7-day-old Vigna radiata seedlings were treated for 24 h with various concentrations of coumarin and/or indole-3-butyric acid (IBA), applied either alone or in combination, in order to stimulate adventitious root formation (ARF). The effects of treatment on endogenous free and conjugated indole-3-acetic acid (IAA), basic peroxidase (basic PER) activity and its isoperoxidases analysis and their relation to ARF were then investigated at the potential rooting sites during the first 96 h after application. Simultaneously, combined treatments acted synergistically in inducing more adventitious roots in treated cuttings than in those treated with coumarin or IBA individually, as compared with the control. Endogenous free IAA increased transiently in treated cuttings as compared with the control and the maximum increase occurred with the combined treatment. This suggests that coumarin and IBA may act synergistically in increasing the endogenous free IAA level during the induction phase of rooting to initiate more roots. Likewise, higher level of conjugated IAA was also found in treated cuttings than in untreated ones, during the primary events of ARF, with the maximum level occurring in the combined treatment. Comparison of the dynamics of conjugated IAA and activity of basic PERs led to conclusion that the former but not the latter is responsible for downregulation of endogenous IAA levels significantly during the primary events of ARF. A sharp increases in basic PERs occurred during the secondary events of ARF, suggesting their role in root initiation and development rather than root induction.     

Response to auxin changes during maturation-related loss of adventitious rooting competence in loblolly pine (Pinus taeda) stem cuttings

Hypocotyl cuttings (from 20- and 50-day-old Pinus taeda L. seedlings) rooted readily within 30 days in response to exogenous auxin, while epicotyl cuttings (from 50-day-old seedlings) rarely formed roots within 60 days. Responses to auxin during adventitious rooting included the induction of cell reorganization and cell division, followed by the organization of the root meristem. Explants from the bases of both epicotyl and hypocotyl cuttings readily formed callus tissue in response to a variety of auxins, but did not organize root meristems. Auxin-induced cell division was observed in the cambial region within 4 days, and later spread to the outer cortex at the same rate in both tissues. Cells at locations that would normally form roots in foliated hypocotyl cuttings did not produce callus any differently than those in other parts of the cortex. Therefore, auxin-induced root meristem organization appeared to occur independently of auxin-induced cell reorganization/division. The observation that N-(1-naphthyl)phthalamic acid (NPA) promoted cellular reorganization and callus formation but delayed rooting implies the existence of an auxin signal transduction pathway that is specific to root meristem organization. Attempts to induce root formation in callus or explants without foliage were unsuccessful. Both the cotyledon and epicotyl foliage provided a light-dependent product other than auxin that promoted root meristem formation in hypocotyl cuttings.     

Adventitious root initiation in hypocotyl and epicotyl cuttings of eastern white pine (Pinus strobus) seedlings

The present paper reports results of experiments to develop a system for studying adventitious root initiation in cuttings derived from seedlings. Hypocotyl cuttings of 2-week-old eastern white pine (Pinus strobus L.) seedlings were treated for 5 min with 0, 100, 200, 300, 400, 500 or 600 mg l^?1 (0, 0.54, 1.07, 1.61, 2.15, 2.69 or 3.22 mM) 1-naphthaleneacetic acid (NAA) to determine the effect on root initiation. The number of root primordia per cutting was correlated with NAA concentration and the square of NAA concentration. Thus, the number increased from less than one per cutting in the 0 NAA treatment to approximately 40 per cutting at 300 mg l-1 NAA, above which no substantial further increase was observed. The larger number of root primordia formed in response to increasing concentrations of NAA was due to the formation of primordia over a larger proportion of the hypocotyls. Histological analysis of the timing of root primordium formation in hypocotyl cuttings revealed three discernible stages. Progression through these stages was relatively synchronous among NAA-treated hypocotyl cuttings and within a given cutting, but variation was observed in the portion of different cuttings undergoing root formation. Control-treated hypocotyl cuttings formed root primordia at lower frequencies and more slowly than NAA-treated cuttings, with fewer primordia per cutting. Epicotyl cuttings from 11-week-old seedlings also formed adventitious roots, but more slowly than hypocotyl cuttings. NAA treatment of epicotyl cuttings caused more rapid root initiation and also affected the origin of adventitious roots in comparison with nontreated cuttings. NAA-treated epicotyl cuttings formed roots in a manner analogous to that of the hypocotyl cuttings, directly from preformed vascular tissue, while control-treated epicotyl cuttings first formed a wound or callus tissue and subsequently differentiated root primordia within that tissue. This system of inducing adventitious roots in pine stem cuttings lends itself to studying the molecular and biochemical steps that occur during root initiation and development.     

Auxin metabolism and rooting in young and mature clones of Sequoia sempervirens

The capacity of young and mature Sequoia sempervirens clones to produce roots in vitro was studied after wounding and indole-3-butyric acid (IBA) treatments. Rooting was not observed in mature or in young cuttings cultivated for 30 days in medium without IBA. The presence of 25 μ M IBA in the medium resulted in the appearance of roots at the base of the cuttings. More roots appeared and grew faster on cuttings of the young than on the mature clone. This difference in rooting capacity between young and mature cuttings may be related to differences in the hormone levels at the base of the 5 mm long cuttings during the first 4 days of the root inductive period. After HPLC fractionation. IAA. IBA and related compounds, including indole-3-aspartic acid (IAAsp) and IBA-glucose ester (IBA-GE), were determined by MS and MS-MS and their levels measured by ELISA. Another immunoreactive compound was also found and determined to be N,N-dimethyltryptophan (DMT), a compound previously reported to inhibit auxin-enhanced ethylene production. Wounding of the stem without IBA treatment revealed a transient increase in IAA, IAAsp and DMT levels in young cuttings while a dramatic increase in the levels of DMT was observed in mature cuttings. Following IBA treatment. IAA levels increased in both clones, but higher levels were measured in the young than in the mature clone. IBA and IBA-GE were also found but in higher levels in the mature clone. Thus, the difficult-to-root mature clone differs from the young clone in its auxin metabolism.     

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.     

INITIATION AND DEVELOPMENT OF ROOTS IN JUVENILE AND MATURE LEAF BUD CUTTINGS OF FICUS PUMILA L.

Adventitious root formation (ARF) was studied in woody leaf bud cuttings of Ficus pumila L., creeping fig. Juvenile cuttings rooted easily, whereas only mature cuttings treated with indole-3-butyric acid (IBA) attained any rooting success. In the rooting process, both juvenile and mature material exhibited dedifferentiation of phloem ray parenchyma, root initial formation, primordia differentiation, and root elongation. The early stages of adventitious rooting were most critical since few primordia were observed in mature controls. The stages leading up to root primordia differentiation and elongation occurred more rapidly in IBA-treated juvenile vs. mature cuttings; however, time differences in both types between first observable roots and maximum rooting were comparable. Root primordia differentiated from basal callus of some cuttings, but neither these nor the few primordia in mature controls elongated into well-developed roots. Anatomical differences between the juvenile and mature material did not account for rooting disparity, nor did presence of perivascular fibers, sclereids, and laticifers retard rooting.     

Effect of Auxins on Adventitious Root Development from Single Node Cuttings of Camellia sinensis (L.) Kuntze and Associated Biochemical Changes

An attempt was made to induce rooting from single node cuttings of Camellia sinensis var. TV-20 under controlled conditions and study its biochemical changes during rooting. The nodal cuttings were pretreated with different concentrations of IAA, NAA and IBA and kept in a growth chamber (25 ±2 °C, 16 h photoperiod (55 μ mol m⁻² s⁻¹) with cool, white fluorescent lamps and 65% relative humidity) for 12 h. Among the three auxins used for pretreatment, IBA showed more positive response on rooting as compared to IAA and NAA within 2 weeks of transfer to potting medium. Among four concentrations of IBA tested, 75 ppm gave maximum percentage of rooting, number of roots and root length. Therefore, IBA was used further in experiments for biochemical investigation. The adventitious rooting was obtained in three distinct phases i.e. induction (0–12 days), initiation (12–14 days) and expression (14–18 days). IAA-oxidase activity of IBA-treated cuttings increased slightly as compared to control. The activity was found to decrease during induction and initiation phases and increase during expression phase. The peroxidase activity in IBA-treated cuttings increased up to initiation phase and declined at the expression phase. Polyphenoloxidase activity increased both in IBA-treated and control cuttings during induction and initiation phase but declined slowly during expression phase. Total phenolic content was higher in IBA-treated cuttings, particularly in initiation and expression phases and it also correlated with peroxidase activity. Phenolics might be playing key role for induction of adventitious rooting, and phenolic compounds can be used as rooting enhancer in tea plant.