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.     

Distribution of indole-3-acetic acid in Petunia hybrida shoot tip cuttings and relationship between auxin transport, carbohydrate metabolism and adventitious root formation

To determine the contribution of polar auxin transport (PAT) to auxin accumulation and to adventitious root (AR) formation in the stem base of Petunia hybrida shoot tip cuttings, the level of indole-3-acetic acid (IAA) was monitored in non-treated cuttings and cuttings treated with the auxin transport blocker naphthylphthalamic acid (NPA) and was complemented with precise anatomical studies. The temporal course of carbohydrates, amino acids and activities of controlling enzymes was also investigated. Analysis of initial spatial IAA distribution in the cuttings revealed that approximately 40 and 10 % of the total IAA pool was present in the leaves and the stem base as rooting zone, respectively. A negative correlation existed between leaf size and IAA concentration. After excision of cuttings, IAA showed an early increase in the stem base with two peaks at 2 and 24 h post excision and, thereafter, a decline to low levels. This was mirrored by the expression pattern of the auxin-responsive GH3 gene. NPA treatment completely suppressed the 24-h peak of IAA and severely inhibited root formation. It also reduced activities of cell wall and vacuolar invertases in the early phase of AR formation and inhibited the rise of activities of glucose-6-phosphate dehydrogenase and phosphofructokinase during later stages. We propose a model in which spontaneous AR formation in Petunia cuttings is dependent on PAT and on the resulting 24-h peak of IAA in the rooting zone, where it induces early cellular events and also stimulates sink establishment. Subsequent root development stimulates glycolysis and the pentose phosphate pathway.     

Polar transport and accumulation of indole-3-acetic acid during root regeneration by Pinus lambertiana embryos

Summary The relation of indoleacetic acid (IAA) transport to accumulation of auxin at the base of cuttings and to polar root formation was investigated with small cuttings from germinating embryos of Pinus lambertiana.The transport of endogenous auxin participates in regeneration of roots. This is shown by the facts that (1) more than 40% of the cuttings rooted without addition of exogenous indoleacetic acid; (2) the first regeneration always occurred at the basal tip of a slanting cut; and (3) 2,3,5-triiodobenzoic acid (TIBA), a specific inhibitor of auxin transport, totally inhibited rooting. Addition of IAA to the medium increased the number of roots formed per rooting hypocotyl.Sections of hypocotyls excised from dormant embryos and tested immediately after 2 h hydration were capable of polar transport of IAA. This polarity increased during the first 3 days of culture because of a marked increase in basipetal transport. Culturing the cuttings in 1 M IAA for 3–5 days doubled both the basipetal transport of 1-¹⁴C-IAA by hypocotyl segments and the accumulation of radioactivity at the base of cuttings.The extent of the accumulation at the base of cuttings was similar at early (2 days, first mitoses) and late stages (5 days, organized meristem) of regeneration and was not affected by removal of the regenerating region immediately prior to uptake and transport of ¹⁴C-IAA. The accumulation was inhibited by TIBA. In terms of increase in wet and dry weight and mitotic activity, the cotyledons rather than the regenerating root meristems were the most actively growing region of the cuttings. The upper part of the hypocotyl elongated more than the region of the slanting cut where regeneration was occurring.These results provide no support for the idea that the regenerating root controls the direction of polar transport by acting as a sink. The results are consistent with the view that polar auxin transport delivers auxin to the base of the cutting and raises the local concentration to levels sufficient to promote root formation.     

Levels of endogenous indole-3-acetic acid and indole-3-acetyl-aspartic acid during adventitious rooting in avocado microcuttings

Quantification of endogenous IAA and lAAsp was carried out duringadventitious root formation in avocado microcuttings. Both auxinand conjugate were monitored in control cuttings (rooted inthe absence of auxin) as well as in cuttings treated with arooting promotor (IBA) or an auxin transport inhibitor (TIBA).Additionally, a histological study to follow root differentiationwas carried out. In control cuttings IAA levels remained constantthroughout the rooting process, however, in IB A-treated cuttingsIAA levels increased 2-fold during the first 6 d. Addition of200 µM TIBA induced a slight decrease of IAA levels andinhibited root formation. As for IAAsp levels, both control and IBA-treated cuttings showeda big increase before root differentiation occurred and as theprocess went on, a progressive decrease took place. However,in TIBA-treated cuttings IAAsp levels not only did not increasebut diminished progressively during the process. The role ofauxin conjugates during the rooting process of avocado is discussed. Key words: Avocado, IAA, IAAsp, rooting     

A strigolactone signal is required for adventitious root formation in rice

Background and Aims Strigolactones (SLs) and their derivatives are plant hormones that have recently been identified as regulating root development. This study examines whether SLs play a role in mediating production of adventious roots (ARs) in rice (Oryza sativa), and also investigates possible interactions between SLs and auxin.Methods Wild-type (WT), SL-deficient (d10) and SL-insensitive (d3) rice mutants were used to investigate AR development in an auxin-distribution experiment that considered DR5::GUS activity, [³H] indole-3-acetic acid (IAA) transport, and associated expression of auxin transporter genes. The effects of exogenous application of GR24 (a synthetic SL analogue), NAA (α-naphthylacetic acid, exogenous auxin) and NPA (N-1-naphthylphalamic acid, a polar auxin transport inhibitor) on rice AR development in seedlings were investigated.Key Results The rice d mutants with impaired SL biosynthesis and signalling exhibited reduced AR production compared with the WT. Application of GR24 increased the number of ARs and average AR number per tiller in d10, but not in d3. These results indicate that rice AR production is positively regulated by SLs. Higher endogenous IAA concentration, stronger expression of DR5::GUS and higher [³H] IAA activity were found in the d mutants. Exogenous GR24 application decreased the expression of DR5::GUS, probably indicating that SLs modulate AR formation by inhibiting polar auxin transport. The WT and the d10 and d3 mutants had similar expression of DR5::GUS regardless of exogenous application of NAA or NPA; however, AR number was greater in the WT than in the d mutants.Conclusions The results suggest that AR formation is positively regulated by SLs via the D3 response pathway. The positive effect of NAA application and the opposite effect of NPA application on AR number of WT plants also suggests the importance of auxin for AR formation, but the interaction between auxin and SLs is complex.     

Patterns of adventitious root formation in English ivy

Adventitious root formation by debladed petiole cuttings of English ivy (Hedera helix L.) proceeds via a direct rooting pattern for the easy-to-root juvenile phase, while the difficult-to-root mature phase roots through an indirect rooting pattern. Juvenile petiole cuttings treated with α-naphthaleneacetic acid (NAA, 100 μM) plus the polyamine biosynthesis inhibitor, difluoromethylarginine (DFMA, 1 mM), formed an increased number of roots per cutting initiated by the indirect rooting pattern. The increased root formation and change in rooting pattern were reversed by the addition of putrescine (1 mM). Delaying auxin application to petiole cuttings for 15 days also induced juvenile petioles to root by the indirect pattern. This could be reversed by rewounding the base of the cutting prior to auxin application after day 15. The data support the use of the terms “competent root-forming cells” and “induced competent root-forming cells” to describe the target cells for the initial events of root formation for the direct and indirect rooting patterns, respectively.     

Gibberellins inhibit adventitious rooting in hybrid aspen and Arabidopsis by affecting auxin transport

Knowledge of processes involved in adventitious rooting is important to improve both fundamental understanding of plant physiology and the propagation of numerous plants. Hybrid aspen (Populus tremula × tremuloïdes) plants overexpressing a key gibberellin (GA) biosynthesis gene (AtGA20ox1) grow rapidly but have poor rooting efficiency, which restricts their clonal propagation. Therefore, we investigated the molecular basis of adventitious rooting in Populus and the model plant Arabidopsis. The production of adventitious roots (ARs) in tree cuttings is initiated from the basal stem region, and involves the interplay of several endogenous and exogenous factors. The roles of several hormones in this process have been characterized, but the effects of GAs have not been fully investigated. Here, we show that a GA treatment negatively affects the numbers of ARs produced by wild‐type hybrid aspen cuttings. Furthermore, both hybrid aspen plants and intact Arabidopsis seedlings overexpressing AtGA20ox1, PttGID1.1 or PttGID1.3 genes (with a 35S promoter) produce few ARs, although ARs develop from the basal stem region of hybrid aspen and the hypocotyl of Arabidopsis. In Arabidopsis, auxin and strigolactones are known to affect AR formation. Our data show that the inhibitory effect of GA treatment on adventitious rooting is not mediated by perturbation of the auxin signalling pathway, or of the strigolactone biosynthetic and signalling pathways. Instead, GAs appear to act by perturbing polar auxin transport, in particular auxin efflux in hybrid aspen, and both efflux and influx in Arabidopsis.     

Effect of changing the endogenous concentration of auxins and cytokinins and the production of ethylene in pea stem cuttings on adventitious root formation

Decapitation or treatment with naphthylphthalamic acid (NPA) ndash; an inhibitor of IAA transport ndash; or a synthetic cytokinin N-(2-chloro-4-pyridyl)-N-phenylurea (CPPU) caused a decrease in rooting of pea cuttings. However the mode of action of the above treatments was different. Decapitation resulted in a decrease of indole-3-acetic acid (IAA) and a simultaneous increase in cytokinin content at the base of the cuttings. NPA decreased IAA even more, but did not influence cytokinins. CPPU alone or in combination with amino-ethoxy-vinylglycine (AVG) ndash; an ethylene biosynthesis inhibitor ndash; increased IAA at the rooting zone, but CPPU was transported from apex to the base of the cuttings where it inhibited rooting. NAA, applied alone after decapitation, stimulated rooting, probably partly by being an auxin, and partly by inhibiting the accumulation of cytokinins at the base of the cuttings. Treatment with AVG neither influenced rooting nor auxin or cytokinin content. Therefore, ethylene production does not seem to be one of the main factors involved in the reduced rooting after the various treatments.     

Glutathione and glutathione disulfide affect adventitious root formation and growth in tomato seedling cuttings

To study the relationship between glutathione and rooting, tomato seedling cuttings, grown on basal- or on auxin-supplemented media, were treated with the reduced (GSH) or oxidized (GSSG) form of this antioxidant. In turn, the consequences of the depletion of GSH pool on rooting were tested using l-buthionine sulfoximine (BSO), a specific inhibitor of GSH biosynthesis. Effects of the aforementioned treatments on rooting response were assessed. GSH treatment promoted root formation on cuttings grown on both basal- and auxin-supplemented media. Whereas GSSG did not affect the number of roots formed by cuttings grown on basal medium, it strongly enhanced the rooting stimulatory effect of auxin treatment. GSH depletion resulting from BSO application did not change the number of roots formed. All the tested compounds, namely GSH, GSSG, BSO and auxin, had a strong inhibitory effect on the elongation of regenerated roots. Supplementing the rooting medium with glutathione efficiently increased the GSH level in the rooting zones, while addition of BSO led to a strong decrease in endogenous GSH level. Neither of the treatments affected the level of GSSG. Exogenous auxin affect neither GSH nor GSSG levels in rooting zones; however, in the regenerated roots, GSH level was significantly higher when the organs were formed on auxin-supplemented medium. Patterns of GSH distribution in the roots regenerated on basal- and auxin-enriched media were studied using the GSH-specific dye monochlorobimane and confocal laser scanning microscopy. GSH was found in the root apical meristem and in the elongation zone. Auxin did not change the GSH distribution; however, the number of fluorescent cells was higher when roots were regenerated on auxin-supplemented medium.     

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.     

The Effects of Ascorbate on Root Regeneration in Seedling Cuttings of Tomato

The changes in ascorbate (ASC) and dehydroascorbate (DHA) levels and the activities of ascorbate metabolising enzymes were examined during adventitious root formation in cuttings of tomato (Lycopersicon esculentum Mill. cv. Paw) seedlings. The effects of ASC, DHA and the immediate ascorbate precursor – galactono-γ-lactone (GalL) supplemented to the culture medium on the rooting response, ascorbate content and the activities of the ASC-metabolising enzymes were also investigated. The cuttings treated with abovementioned compounds formed more roots then control plants. However, in contrast to the number of regenerated organs, the elongation of newly formed roots was markedly inhibited. Treatment with auxin (IAA) resulted in a similar phenotype. The inhibitor of auxin polar transport-TIBA (2,3,5-triiodobenzoic acid) effectively blocked rooting. The inhibitory effect of TIBA was reversed by auxin and ASC treatments, while DHA and GalL were ineffective. Both auxin and ASC stimulated cell divisions in an area of pericycle layer of TIBA-treated rooting zones, that enabled cuttings to form roots in the presence of the inhibitor of auxin polar transport. It has been found that the first stages of rooting, preceding the emergence of roots, are accompanied by an increase in endogenous content of ASC with a peak in the 3rd day of rooting. Subsequent stages, when elongation of newly formed roots occurs, are characterised by low level of ASC. The activities of the ascorbate peroxidase (APX), ascorbate oxidase (AOX), ascorbate free radical reductase (AFRR) and dehydroascorbate reductase (DHR) increased in the first 3 days of root formation. The initial period of rooting was also accompanied by the increase of the hydrogen peroxide content and the activities of catalase (CAT) and guaiacol peroxidase (GPX) in the rooting zones. IAA, ASC, DHA as well as Gal stimulated the APX activity, however the rise of the enzyme's activity induced by ASC, DHA and Gal was reversed by TIBA, which was found to inhibit APX. Only exogenous IAA was able to maintain the high level of APX activity in the TIBA-treated cuttings. AOX was strongly affected by ASC and GalL – treatments, its activity increased in the cuttings grown on the media containing ASC in the absence as well as in the presence of TIBA. On the other hand, GalL-dependent stimulation of its activity was suppressed if TIBA was present in a rooting medium.     

Factors affecting the inhibitory effect of indolylacetic acid on root formation in pea cuttings

Indol-3yl-acetic acid (IAA) present in the rooting solution for 1–4 days appreciably decreased the number of roots subsequently formed in pea stem cuttings. Removal of the lowest 10 mm of the cutting base after IAA treatment abolished the inhibitory effect of IAA almost completely. The number of roots formed from the remaining part of the base internode after excision of the basal centimeter of the internode 4 days after the cuttings were taken was similar to that of the control. It is concluded that the first root primordia, which develop near the cut surface, have an inhibitory influence on development of further roots. The inhibitory effect of IAA may be explained as a strengthening of this dominance phenomenon.
Addition of charcoal to the rooting solution delayed the appearance of roots but increased the number of roots developed during a standard rooting period. Adsorption of stimulatory and inhibitory compounds at the surface of the cutting base is believed to be the reason for these results as no evidence of accumulation of inhibitory compounds in the solution was obtained. Charcoal reversed the inhibitory effect of IAA if added after the IAA treatment. This reversal was almost complete if the IAA treatment lasted for one day only but decreased drastically if the IAA treatment was extended over 4 days.     

Effects of exogenous cytokinins on root formation in pea cuttings

Benzylaminopurine (BAP) or zeatin continuously supplied through the rooting solution to cuttings of pea ( Pisum sativum L. cv. Weibull's Marma), inhibited root formation down to a concentration of 3.10⁻⁹ M . The inhibitory effect of BAP in the concentration range 10⁻⁸–10⁻⁷ M was readily reversible if the cuttings were transferred to solutions without cytokinin after treatment for 1–4 days. A slight increase in the number of roots formed was obtained after treatment with low cytokinin concentrations for 1–2 days. Evidence from microscopic studies of primordia formation indicates that BAP inhibits differentiation of primordia at an early stage in their development. Growth of already formed primordia, or root elongation, was considerably less sensitive to the inhibitory effect of BAP. The results indirectly support the hypothesis that endogenous cytokinins prevent root formation in stems of intact plants and may be of importance for the regulation of rooting in cuttings.     

Adventitious rooting in hypocotyls of sunflower (Helianthus annuus) seedlings. IV. The role of changes in endogenous free and conjugated indole‐3‐acetic acid

We have studied the role of endogenous auxin on adventitious rooting in hypocotyls of derooted sunflower (Helianthus annuus L. var. Dahlgren 131) seedlings. Endogenous free and conjugated indole-3-acetic acid (IAA) were measured in three segments of hypocotyls of equal length (apical, middle, basal) by using gas chromatography-mass spectrometry with [¹³C₆]-IAA as an internal standard. At the time original roots were excised (0 h), the free IAA level in the hypocotyls showed an acropetally decreasing gradient, but conjugated IAA level increased acropetally; i.e. free to total IAA ratio was highest in the basal portion of hypocotyls. The basal portion is the region where most of root primordia were found. Some primordia were seen in this region within 24 h after the roots were excised. The quantity of free IAA in the middle portion of the hypocotyl increased up to 15 h after excision and then decreased. In this middle region there were fewer root primordia, and they could not be seen until 72 h. In the apical portion the amount of free IAA steadily increased and no root primordia were seen by 72 h. Surgical removal of various parts of the hypocotyl tissues caused adventitious root formation in the hypocotyl regions where basipetally transported IAA could accumulate. Reduction in the basipetal flow of auxin by N-1-naphthylphthalamic acid and 2,3,5-tri-iodobenzoic acid resulted in fewer adventitious roots. The fewest root primordia were seen if the major sources of endogenous auxin were removed by decapitation of the cotyledons and apical bud. Exogenous auxins promoted rooting and were able to completely overcome the inhibitory effect of 2,3,5-tri-iodobenzoic acid. Exogenous auxins were only partially able to overcome the inhibitory effect of decapitation. We conclude that in sunflower hypocotyls endogenously produced auxin is necessary for adventitious root formation. The higher concentrations of auxin in the basal portion may be partially responsible for that portion of the hypocotyl producing the greatest number of primordia. In addition to auxins, other factors such as wound ethylene and lowered cytokinin levels caused by excision of the original root system cuttings must also be important.     

生长素、乙烯和一氧化氮对拟南芥下胚轴插条形成不定根的调节

研究生长素、乙烯和一氧化氮（NO）对拟南芥下胚轴插条形成不定根的调节,以及生长素和乙烯信号转导成员在IAA促进不定根形成中的作用的结果表明：拟南芥切条以IAA和硝普钠（N0供体）单独处理7d后的不定根形成均受到促进,其中以50μmol·L^-1 IAAμmol·L^-1 SNP的促进作用为最强,乙烯的促进作用不明显;生长素运输和信号转导以及乙烯信号转导相关突变体对IAA促进生根作用的敏感性比野生型有所下降,特别是IAA14功能获得型的突变体。IAA和NO在促进不定根形成中有协同效应。     

Auxin and Ethylene-stimulated Adventitious Rooting in Relation to Tissue 9

We have previously shown that both endogenous auxin and ethylenepromote adventitious root formation in the hypocotyls of derootedsunflower (Helianthus annuus) seedlings. Experiments here showedthat promotive effects on rooting of the ethylene precursor,1-aminocyclopropane-l-carboxylic acid (ACC) and the ethylene-releasingcompound, ethephon (2-chloro-ethylphosphonic acid), dependedon the existence of cotyledons and apical bud (major sourcesof auxin) or the presence of exogenously applied indole-3-aceticacid (IAA). Ethephon, ACC, aminoethoxyvinylglycine (an inhibitorof ethylene biosynthesis), and silver thiosulphate (STS, aninhibitor of ethylene action), applied for a length of timethat significantly influenced adventitious rooting, showed noinhibitory effect on the basipetal transport of [³H]IAA. Theseregulators also had no effect on the metabolism of [³H]IAA andendogenous IAA levels measured by gas chromatography-mass spectrometry.ACC enhanced the rooting response of hypocotyls to exogenousIAA and decreased the inhibition of rooting by IAA transportinhibitor, N-1-naphthylphthalamic acid (NPA). STS reduced therooting response of hypocotyls to exogenous IAA and increasedthe inhibition of rooting by NPA. Exogenous auxins promotedethylene production in the rooting zone of the hypocotyls. Decapitationof the cuttings or application of NPA to the hypocotyl belowthe cotyledons did not alter ethylene production in the rootingzone, but greatly reduced the number of root primordia. We concludethat auxin is a primary controller of adventitious root formationin sunflower hypocotyls, while the effect of ethylene is mediatedby auxin. Key words: Auxin, ethylene, adventitious rooting, sunflower     

Patterns of adventitious root formation in English ivy

Adventitious root formation by debladed petiole cuttings of English ivy (Hedera helix L.) proceeds via a direct rooting pattern for the easy-to-root juvenile phase, while the difficult-to-root mature phase roots through an indirect rooting pattern. Juvenile petiole cuttings treated with -naphthaleneacetic acid (NAA, 100 M) plus the polyamine biosynthesis inhibitor, difluoromethylarginine (DFMA, 1 mM), formed an increased number of roots per cutting initiated by the indirect rooting pattern. The increased root formation and change in rooting pattern were reversed by the addition of putrescine (1 mM). Delaying auxin application to petiole cuttings for 15 days also induced juvenile petioles to root by the indirect pattern. This could be reversed by rewounding the base of the cutting prior to auxin application after day 15. The data support the use of the terms competent root-forming cells and induced competent root-forming cells to describe the target cells for the initial events of root formation for the direct and indirect rooting patterns, respectively.Kentucky Experiment Station publication 90-10-122.