Distinct modes of adventitious rooting in Arabidopsis thaliana

The literature describes different rooting protocols for Arabidopsis thaliana as models to study adventitious rooting, and results are generally perceived as comparable. However, there is a lack of investigations focusing on the distinct features, advantages and limitations of each method in the study of adventitious rooting with both wild-type (WT) ecotypes and their respective mutants. This investigation was undertaken to evaluate the adventitious rooting process in three different experimental systems, all using A. thaliana, analysing the same rooting parameters after transient exposure to auxin (indole-3-acetic acid) and control conditions: excised leaves, de-rooted plants and etiolated seedlings. The founding tissues and sites of origin of roots differed depending on the system used, whereas all rooting patterns were of the direct type (i.e., without callus formation). None of the systems had an absolute requirement for exogenous auxin, although rooting was enhanced by this phytohormone, with the exception of de-rooted plants, which had adventitious rooting strongly inhibited by exogenous auxin. Root elongation was much favoured in isolated leaves. Auxin-overproducing mutants could not be used in the detached leaf system due to precocious senescence; in the de-rooted plant system, these mutants had a WT-like rooting response, whereas the expression of the 'rooty' phenotype was only evident in the etiolated seedling system. Adventitious rooting of etiolated WT seedlings in the presence of exogenous auxin was inhibited by exogenous flavonoids, which act as auxin transport inhibitors; surprisingly, the flavonoid-deficient mutant chs had a lower rooting response compared to WT. Although Arabidopsis is an excellent model system to study adventitious rooting, physiological and developmental responses differed significantly, underlining the importance of avoiding data generalisation on rooting responses derived from different experimental systems with this species.     

Involvement of free and conjugated polyamines and free amino acids in the adventitious rooting of micropropagated cork oak and grapevine shoots

The recent advances in biotechnology have boosted interest in the differentiation processes, including adventitious rooting. Differentiation processes depend on endogenous factors, among which auxins and polyamines are believed to play a major role. A positive correlation between polyamine accumulation and the induction of adventitious rooting by auxin has been observed in numerous woody species, suggesting that polyamines could be used as markers of the rooting process. The aim of the present work is to investigate whether primary and secondary nitrogen metabolism is involved in adventitious root induction by auxin treatments in cork oak (Quercus suber L.) and grapevine (Vitis vinifera L.) shoots cultured in vitro. For this purpose, we followed the profile of free and conjugated polyamines, free amino acid pools and ¹⁵N-labelling profiles during root induction and expression. We have also observed the effects of cyclohexylamine (CHA), an inhibitor of spermidine synthase. Taking the results together, it is possible to conclude that: (a) glutamine is the most abundant free amino acid in grapevine, while in cork oak, asparagine and arginine are the major amino acids; (b) in grapevine, auxin did not significantly affect the glutamine levels, but changed the ¹⁵N-enrichment and labelling pattern of arginine; (c) auxin affected asparagine levels and ¹⁵N-labelling pattern of glutamine in cork oak; (d) in both cork oak and grapevine, free putrescine (Put) can be considered as a marker of in vitro root induction; (e) in both species, the presence of CHA resulted in the accumulation of free Put; (f) no Put catabolism was detected in the form of ¹⁵N-NMR products, namely ¹⁵N-γ-aminobutyric acid; (g) the CHA-induced accumulation of Put only increased grapevine rooting rate.     

Stimulation of adventitious rooting of Taxus species by thiamine

Summary Results obtained from using root inducing compounds on Taxus species cuttings suggested that rooting could be significantly enhanced by the presence of thiamine. This observation was verified using a root inducing solution containing a set concentration of IBA (0.2%), NAA (0.1%), and supplemented with various concentrations of thiamine. The best rooting response for Taxus cuspidata stem cuttings was found using this solution supplemented with 0.08% thiamine. Rooted cuttings were easily established and developed into vigorous plants. In addition, Taxus brevifolia shoots obtained from tissue cultures via in vitro organogenesis also responded favorably to this 0.08% thiamine supplemented rooting solution.     

Strigolactones suppress adventitious rooting in Arabidopsis and pea

Adventitious root formation is essential for the propagation of many commercially important plant species and involves the formation of roots from nonroot tissues such as stems or leaves. Here, we demonstrate that the plant hormone strigolactone suppresses adventitious root formation in Arabidopsis (Arabidopsis thaliana) and pea (Pisum sativum). Strigolactone-deficient and response mutants of both species have enhanced adventitious rooting. CYCLIN B1 expression, an early marker for the initiation of adventitious root primordia in Arabidopsis, is enhanced in more axillary growth2 (max2), a strigolactone response mutant, suggesting that strigolactones restrain the number of adventitious roots by inhibiting the very first formative divisions of the founder cells. Strigolactones and cytokinins appear to act independently to suppress adventitious rooting, as cytokinin mutants are strigolactone responsive and strigolactone mutants are cytokinin responsive. In contrast, the interaction between the strigolactone and auxin signaling pathways in regulating adventitious rooting appears to be more complex. Strigolactone can at least partially revert the stimulatory effect of auxin on adventitious rooting, and auxin can further increase the number of adventitious roots in max mutants. We present a model depicting the interaction of strigolactones, cytokinins, and auxin in regulating adventitious root formation.     

Nitric oxide modulates specific steps of auxin-induced adventitious rooting in sunflower

Present work on indole-3-acetic acid (IAA)-induced adventitious rooting in sunflower hypocotyl highlights a clear demarcation of nitric oxide (NO)-dependent and NO-independent roles of auxin in this developmental process. Of the three phases of adventitious rooting, induction is strictly auxin-dependent though initiation and extension are regulated by an interaction of IAA with NO. A vital role of auxin-efflux transporters (PIN) is also evident from 1-napthylphthalamic acid (NPA)-triggered suppression of adventitious roots (AR). Use of actin depolymerizing agent, latrunculin B (Lat B), has demonstrated the necessity of functional actin filaments in auxin-induced AR response, possibly through its effect on actin-mediated recycling of auxin transporter proteins. Thus, evidence for a linkage between IAA, NO and actin during AR formation has been established.Key words: adventitious roots, auxin, sunflowerAdventitious roots (AR) are post-embryonic roots known to originate from stem, leaf petiole and non-pericycle tissue of old roots. In young stem, AR commonly arise from the interfascicular parenchyma while they appear from vascular rays near the cambium in older stem. Formation of AR begins with re-differentiation of predetermined cells which switch from their morphogenetic path to act as mother cells for initiation of root primordia.¹ The process of AR formation consists of three physiologically interdependent phases: induction, initiation and extension.¹ Induction phase comprises of various molecular and biochemical events but no morphologically visible changes appear during this phase. Formation of multilayered cells and conception of root primordia occurs during initiation phase. During expression phase, root primordia exhibit intra-stem growth and their emergence through epidermis. Various environmental and endogenous factors, such as temperature, light, hormones (particularly auxin), sugars and mineral salts, act as cues for promoting redifferentiation of predetermined cells resulting in root induction.The three phases of AR formation are known to be regulated by alterations in the endogenous level of auxin.² A transient increase in auxin concentration has been reported during induction phase, which is followed by a decrease and again an increase during expression phase.³ Auxin transport to and from the responding region is essential for root organogenesis. Acropetal transport of auxin occurs through the vascular cylinder and the basipetal transport takes place through the epidermal and subtending cortical cells.⁴ Polar transport of auxin from the shoot apical meristem to the rooting region is primarily facilitated by auxin-influx (AUX1) and -efflux (PIN) transporters. Asymmetric localization of these transporter proteins in the vascular cambium cells is responsible for differential distribution of auxin in a particular zone of cells.⁵ Polar auxin transport is known to be inhibited by 1-napthylphthalamic acid (NPA, a phytotropin). This inhibition is mediated through a binding of NPA molecule to putative NPA-binding protein (NBP), which is functionally associated with PIN proteins.⁶ Efflux transporters exhibit rapid turnover in plasma membrane.⁷ High affinity of NBP for actin filaments,^8,9 suggests its involvement in the cycling and polar distribution of PIN proteins.¹⁰ Organization of actin filaments is known to be rapidly, reversibly and specifically disrupted by Latrunculin B (Lat B), a macrolide toxin isolated from Latrunculia magnificia, a red sea sponge.¹¹ Lat B associates with actin monomers in 1:1 ratio, thereby preventing their repolymerization into filaments, resulting in a complete shift from F-actin to G-actin.¹² Owing to its well-understood and simple mode of action and low effective dosage, Lat B has supplanted the classic actin-depolymerizing drug cytochalasin D¹³ in pharmacological investigations. In the past few years, significant work has been done on NO as a signaling molecule in a variety of plant developmental processes.¹⁴ Nitric oxide is known to play a crucial role in root development.¹⁵Using sunflower as a model system, present work has been undertaken to investigate the possible role of NO during IAA-induced adventitious rooting in hypocotyl explants. Since auxin action is principally based on PIN-regulated polar transport of IAA molecules, and PIN proteins are known to exhibit actin-asssisted rapid recycling in the target cells, attempts have been made in the present work to find a correlation between auxin transport, actin and NO, using specific pharmacological agents. Additionally, effect of Cyclosporin A (CsA), an inhibitor of nitric oxide synthase (NOS) in animal systems,¹⁶ has also been investigated. These specific agents have been used to monitor root initiation at the target sites in our attempts to decipher a signaling cascade for AR.Seeds of Sunflower (Helianthus annuus L. cv. Morden) were germinated on moist germination sheets at 25 ± 2°C under continuous illumination of 4.3 Wm⁻². Hypocotyls from 4 d old seedlings and with similar growth rate, were excised up to 6 cm below the cotyledonary node. Hypocotyl explants with apical meristem intact but cotyledons excised were selected for the present work with a view to provide a continuity of the endogenous auxin source. Similar explants were also recently used by Huang et al.¹⁷ to investigate indole-3-butyric acid (IBA)-induced AR formation in mung bean (Phaseolus radiatus L.). Using IAA instead of IBA for such investigations was preferred for the present work keeping in view that the two auxins seem to employ different transport proteins for their polar transport.¹⁸ Freshly harvested explants were put upright in glass vials with their proximal cut ends dipped in 1 ml of different concentrations (1, 5, 10 and 15 µM) of IAA, thus bathing the hypocotyls up to 5 mm of their lower ends. Explants were maintained in dark during the course of experiments. The number of AR visible on hypocotyl surface was recorded daily up to 4 days of incubation. Concentration of IAA thus observed optimal for rooting (10 µM) was used for all further experiments. Similarly, various concentrations of other test solutions, namely NPA (auxin efflux blocker; 1 and 10 µM), Lat B (an inducer of actin depolymerization; 25, 50 and 100 nM), CsA (an inhibitor of cyclophilins; 1, 5 and 10 µM), sodium nitroprusside (SNP; NO donor; 1, 5, 10 and 100 µM) and 2-phenyl-4,4,5,5-tetramethyllimidazoline-1-oxyl-3-oxide (PTIO; NO scavenger; 1 and 1.5 mM), were initially used to select their respective optimal concentrations. Based on these preliminary experiments NPA, Lat B, CsA, SNP and PTIO were used at 10 µM, 100 nM, 10 µM, 100 µM and 1.5 mM, respectively, for all subsequent experiments. Some other treatment combinations, namely NPA (10 µM) + IAA (10 µM); LatB (100 nM) + IAA (10 µM); CsA (10 µM) + IAA (10 µM); SNP (100 µM) + NPA (10 µM) and PTIO (1.5 mM) + IAA (10 µM) were also used to investigate their effects on adventitious rooting. Hypocotyl explants incubated in distilled water served as control. Morphological observations of rooting response were imaged after 7 days of incubation, using Nikon digital camera fitted on a stereomicroscope (Stemi 2000, Zeiss, Germany). Detailed evaluation of root initiation was observed after clearing by immersing the explants in a 3:1 solution of ethanol: acetic acid overnight. They were then transferred to 2 N NaOH solution, left overnight, washed once with distilled water and stained with safranin solution for 2–3 min. Excess stain was removed by repeated washing in distilled water. The lower 2 cm region of hypocotyl explants was then cut and mounted on a glass slide to examine endogenous root initials, using a stereomicroscope (Stemi 2000, Zeiss, Germany) fitted with a Nikon camera.Root initiation and extension in the basal region of hypocotyl explants maintained in distilled water indicates the expected basipetal transport of the inducing factor (endogenous IAA) from the intact meristem, as also reported earlier.¹⁹ Treatment with IAA (10 µM) elicited two effects on hypocotyl explants in comparison to those subjected to distilled water treatment: (1) Formation of greater number of root initials, (2) Greater extension of the initiated roots (Figs. 1 and ​and22). A response similar to that evoked by IAA is also evident in hypocotyl explants treated with 100 µM of SNP (Figs. 1 and ​and22). Recently SNP (NO donor) has been reported to evoke dose-dependent response on AR formation in marigold.²⁰ In the present work, treatment with variable concentrations of SNP, ranging from 1–100 µM, lead to a gradual increase in the number and extension growth of AR till 100 µM. Pagnussat et al.²¹ and Liao et al.²⁰ have used 10 µM and 50 µM as effective SNP concentration in cucumber and marigold, respectively. Thus, optimal concentration of SNP for AR formation is species-dependent. In presence of PTIO (1.5 mM; a specific NO scavenger), complete suppression of AR was evident in sunflower, as also reported earlier in mung bean.¹⁷ Combination of PTIO with IAA lead to root initiation only (no extension growth). NPA (10 µM) blocked AR initiation by endogenous (distilled water treatment) and exogenous IAA (Fig. 1). Application of NPA inhibits polar auxin transport, thus reducing the optimal concentration of IAA required for AR formation at the hypocotyl base (zone of AR formation). Thus, no evidence of root initials was evident in presence of NPA, which is also reported in cucumber²² and loblolly pine,²³ respectively. Though NO is expected to act downstream of IAA²⁴ but a treatment of SNP in combination with NPA (present work) lead to complete suppression of AR formation. Our unpublished observations have indicated the expression of NO in the interfascicular cells after induction phase (i.e., during AR iniation and extension). Thus, it can be proposed that IAA is involved in induction phase of adventitious rooting independent of NO, while initiation and extension phases appear to involve IAA-NO interaction. CsA-cyclophilin complex is known to inhibit calcineurin (a protein phosphatase) and NOS activity in animal systems.²⁵ Treatment of hypocotyl explants with CsA (10 µM) lead to formation of fewer number of roots which exhibited extension growth. Oh et al.²⁶ reported a significant reduction in the number of roots in the presence of CsA in hypocotyl explants from tomato. Subjecting hypocotyl explants with a combination of CsA and IAA lead to formation of fewer number of root initials, reaffirming the involvement of NO in auxin action in the developmental process under investigation (AR). However, further investigations on the role of cyclophilins and NOS in auxin-modulated AR formation are required to pinpoint their specific sites of action in this developmental process. Treatment with Lat B (+ and − IAA) lead to complete AR suppression in sunflower hypocotyl explants. Actin-mediated polar localization of PIN proteins is responsible for polar auxin transport and disruption of microfilaments by Lat-B would thus, directly affect IAA transport leading to the observed AR suppression. These observations indicate a convergence of the effect of IAA with that of NO and a role of a well organized actin in the responding cells.Open in a separate windowFigure 1Effect of IAA and various other pharmacological agents on adventitious rooting in hypocotyl explants. Morphological observations of rooting response (A and C). Evaluation of endogenous root initiation and elongation observed in cleared explants stained with safranin (B and D). Scale bar represents 3 mm.Open in a separate windowFigure 2Quantitative analysis of AR initiation in presence of distilled water, IAA and various pharmacological agents in hypocotyl explants of sunflower. Each datum presents a mean and standard error from at least three observations.To sum up, present investigations provide evidence for a linkage between auxin-induced AR response in seedling hypocotyls and NO (Fig. 3). Both endogenous and exogenous IAA-mediated AR induction seem to depend on actin. Significance of actin in this developmental response has become evident via its role in the cycling of auxin efflux proteins (PIN). The three phases of AR formation can be differentiated from each other in terms of their sensitivities to IAA and NO. AR induction phase seems to be governed by auxin alone, independent of NO. NO seems to become operative in this auxin-modulated response (AR) during initiation and extension phase only. Investigations are being undertaken in the author''s laboratory to visualize and quantitate the NO signal in the IAA-responding hypocotyl explants so that the phasing of the role of NO during AR formation can be precisely predicted.Open in a separate windowFigure 3Schematic presentation of the probable events associated with NO-mediated adventitious rooting.     

The lose of juvenility elicits adventitious rooting recalcitrance in apple rootstocks

For perennial woody plants, softwood cutting is an efficient technique for larger scale propagation and adventitious rooting of cuttings is one of the most crucial steps. To evaluate the significance of juvenility on adventitious rooting, rooting rates was compared between softwood cuttings collected from apomictic seedlings (juvenile), in vitro cultured plants (rejuvenated), suckers (juvenile like) and canopy shoots (adult) of reproductively mature trees in Malus xiaojinensis. After pre-treatment with indole-3-butytric acid (IBA) (3,000 mg L^?1) + H₂O₂ (50 mM), rooting rates in cutting from juvenile, juvenile like and rejuvenated donor plants were significantly higher (>90 %) than that from adult trees. The effects of IBA on adventitious rooting were enhanced significantly by exogenous H₂O₂. After 15 passages of in vitro subculture, the micro-shoots from adult phase explants were rejuvenated successfully, marked by the elevated expression of miR156 in the leaflets of the micro-shoots. But the rooting ability of rejuvenated micro-shoots was recovered delayed at the 18th or 21st passage of subculture. During the process of rejuvenation, the leaf indole-3-acetic acid contents and the expressions of rooting related genes CKI1, ARRO-1, ARF7 and ARF19 increased significantly. In contrary, the leaf abscisic acid contents decreased. A lack of juvenility is the most important limiting factor governing adventitious rooting of softwood cuttings in apple rootstocks.     

Dynamic thermal time model of cold hardiness for dormant grapevine buds

Background and Aims

Grapevine (Vitis spp.) cold hardiness varies dynamically throughout the dormant season, primarily in response to changes in temperature. The development and possible uses of a discrete-dynamic model of bud cold hardiness for three Vitis genotypes are described.

Methods

Iterative methods were used to optimize and evaluate model parameters by minimizing the root mean square error between observed and predicted bud hardiness, using up to 22 years of low-temperature exotherm data. Three grape cultivars were studied: Cabernet Sauvignon, Chardonnay (both V. vinifera) and Concord (V. labruscana). The model uses time steps of 1 d along with the measured daily mean air temperature to calculate the change in bud hardiness, which is then added to the hardiness from the previous day. Cultivar-dependent thermal time thresholds determine whether buds acclimate (gain hardiness) or deacclimate (lose hardiness).

Key Results

The parameterized model predicted bud hardiness for Cabernet Sauvignon and Chardonnay with an r² = 0·89 and for Concord with an r² = 0·82. Thermal time thresholds and (de-)acclimation rates changed between the early and late dormant season and were cultivar dependent but independent of each other. The timing of these changes was also unique for each cultivar. Concord achieved the greatest mid-winter hardiness but had the highest deacclimation rate, which resulted in rapid loss of hardiness in spring. Cabernet Sauvignon was least hardy, yet maintained its hardiness latest as a result of late transition to eco-dormancy, a high threshold temperature required to induce deacclimation and a low deacclimation rate.

Conclusions

A robust model of grapevine bud cold hardiness was developed that will aid in the anticipation of and response to potential injury from fluctuations in winter temperature and from extreme cold events. The model parameters that produce the best fit also permit insight into dynamic differences in hardiness among genotypes.     

莼菜休眠芽解除休眠及生根的研究

莼菜为世界著名珍稀水生蔬菜,具有很高的食用和药用价值。已有研究显示,可通过冬芽实现莼菜复壮,找出休眠解除的最佳方法是复壮的基本前提。本实验以莼菜冬季型休眠芽（冬芽）为实验材料,通过光照、温度等处理探求莼菜冬芽的休眠解除方法。莼菜冬芽生根率随着光照强度的增加,其生根率由全黑暗0%显著增长至（16.0±1.3）%;温度和处理时间对莼菜冬芽生根率、根长、生根根数和含水量有显著性影响。其中,35 °C水浴处理6 h后各项指标均到达较大值,上述指标分别为（96.1±2.8）%、（7.2±0.2） cm、74.5±6.0和（83.6±0.3）%;40 °C（6 h）、45 °C（1、3和6 h）和50 °C（10、20和30 min）水浴处理后莼菜冬芽绝大部分失绿变黄;随着培养时间的延长,其冬芽全部腐烂。光照能够促进莼菜冬芽生根;土培生根效果较为显著;适度水浴加热处理能有效解除莼菜冬芽的休眠,35 °C水浴处理6 h为最佳处理方案。     

Auxin and light control of adventitious rooting in Arabidopsis require ARGONAUTE1

Adventitious rooting is a quantitative genetic trait regulated by both environmental and endogenous factors. To better understand the physiological and molecular basis of adventitious rooting, we took advantage of two classes of Arabidopsis thaliana mutants altered in adventitious root formation: the superroot mutants, which spontaneously make adventitious roots, and the argonaute1 (ago1) mutants, which unlike superroot are barely able to form adventitious roots. The defect in adventitious rooting observed in ago1 correlated with light hypersensitivity and the deregulation of auxin homeostasis specifically in the apical part of the seedlings. In particular, a clear reduction in endogenous levels of free indoleacetic acid (IAA) and IAA conjugates was shown. This was correlated with a downregulation of the expression of several auxin-inducible GH3 genes in the hypocotyl of the ago1-3 mutant. We also found that the Auxin Response Factor17 (ARF17) gene, a potential repressor of auxin-inducible genes, was overexpressed in ago1-3 hypocotyls. The characterization of an ARF17-overexpressing line showed that it produced fewer adventitious roots than the wild type and retained a lower expression of GH3 genes. Thus, we suggest that ARF17 negatively regulates adventitious root formation in ago1 mutants by repressing GH3 genes and therefore perturbing auxin homeostasis in a light-dependent manner. These results suggest that ARF17 could be a major regulator of adventitious rooting in Arabidopsis.     

Endogenous auxin levels in terminal stem cuttings of Chrysanthemum morifolium during adventitious rooting

Free and ester-bound IAA were determined in Chrysanthemum morifolium Ramat cv. 'Yellow Galaxy' by means of a solid phase enzyme immunoassay. In shoots, free auxin decreases basipetally whereas ester IAA reaches a maximum in the middle part. After making the cuttings, a strong increase in both free and ester IAA (or total IAA, respectively) is found up to the time when the first adventitious roots become visible. Only prolonged irradiance of stock plants at high light intensities (40 W m⁻²) delays this increase in the cuttings, concomitantly with a lower number of roots compared to the controls (4.5 W m⁻²), although root growth as determined by measuring root length or fresh weight is not affected. A distinct relation is found between IAA content of stock plants at the time when cuttings are taken and the number of adventitious roots formed by the cuttings 20 days later.     

Induction,growth and direct rooting of adventitious shoots of Begonia x hiemalis

The efficiency of commercial micropropagation programs for Begonia x hiemalis depends on the production of large adventitious shoots for easy handling and on effective rooting and acclimatization procedures. Maximum induction of adventitious buds on petiole segments occurred in response to NAA (0.1 mg, l^-1) and BA (0.5 mg l^-1), but continued shoot growth was limited. With a lower concentration of BA (0.1 mg l^-1) fewer shoots were produced but shoot growth was enhanced. With a combined agar/liquid culture program the low BA (0.1 mg l^-1) medium produced 50 percent more shoots larger than 1 cm than did the high BA (0.5 mg l^-1) medium. In vitro rooted explants developed weak root systems and acclimatization losses occurred during adaptation to greenhouse conditions. Adventitious shoots treated with commercial rooting powder and placed directly in mist frames produced much stronger root systems and could be adapted to greenhouse conditions without loss. The elimination of the in vitro rooting stage also simplifies the micropropagation program.Contribution No. 743     

The role of strigolactones in photomorphogenesis of pea is limited to adventitious rooting

The recently discovered group of plant hormones, the strigolactones, have been implicated in regulating photomorphogenesis. We examined this extensively in our strigolactone synthesis and response mutants and could find no evidence to support a major role for strigolactone signaling in classic seedling photomorphogenesis (e.g. elongation and leaf expansion) in pea (Pisum sativum), consistent with two recent independent reports in Arabidopsis. However, we did find a novel effect of strigolactones on adventitious rooting in darkness. Strigolactone‐deficient mutants, Psccd8 and Psccd7, produced significantly fewer adventitious roots than comparable wild‐type seedlings when grown in the dark, but not when grown in the light. This observation in dark‐grown plants did not appear to be due to indirect effects of other factors (e.g. humidity) as the constitutively de‐etiolated mutant, lip1, also displayed reduced rooting in the dark. This role for strigolactones did not involve the MAX2 F‐Box strigolactone response pathway as Psmax2 f‐box mutants did not show a reduction in adventitious rooting in the dark compared with wild‐type plants. The auxin‐deficient mutant bushy also reduced adventitious rooting in the dark, as did decapitation of wild‐type plants. Rooting was restored by the application of indole‐3‐acetic acid (IAA) to decapitated plants, suggesting a role for auxin in the rooting response. However, auxin measurements showed no accumulation of IAA in the epicotyls of wild‐type plants compared with the strigolactone synthesis mutant Psccd8, suggesting that changes in the gross auxin level in the epicotyl are not mediating this response to strigolactone deficiency.     

Phytotoxic effects of Heterothalamus psiadioides (Asteraceae) essential oil on adventitious rooting

Essential oils have many functions in plant development; among them, they take part in plant–plant interactions, such as allelopathy. The phytotoxicity of the essential oil of Heterothalamus psiadioides was evaluated on Arabidopsis thaliana adventitious rooting. This development process has demonstrated great sensitivity to phytotoxic essential oils and has been primarily studied by our research group. The essential oil of H. psiadioides showed highly negative effects on A. thaliana wild type (WT) adventitious rooting in very small amounts. As the hormone auxin has an important role on adventitious rooting, A. thaliana mutant superroot (sur1) that overproduces indole-3-acetic acid was tested to verify if it could better respond to the oil than WT. The mutant had a WT-like rooting response and the expression of its phenotype was not evident in treatments that had oil application. The essential oil also induced the generation of H₂O₂, a reactive oxygen species in high levels and trying to recover these effects with the aid of the antioxidant Trolox^® was unsuccessful. Increasing levels of H₂O₂ may affect leaf pigmentation of WT microcuttings and auxin levels of both WT and sur1 microcuttings. Since the expression of auxin-responsive genes is decreased by H₂O₂ treatment via mitogen-activated protein kinase activation, microcuttings growth and rooting are impaired causing altered development pattern.     

Histological analysis of adventitious rooting in Arabidopsis thaliana (L.) Heynh seedlings

ABSTRACT

Adventititous rooting is essential for the post-embryonic growth of the root apparatus in various species. In Arabidopsis thaliana, adventitious rooting has been reported in some mutants, and auxin seems to be the inducer of the process. The objective of the study was to identify the tissues involved in adventitious rooting in the most commonly used ecotypes for molecular and genetic studies (i.e. Columbia, Wassilewskija and Landsberg erecta) both in the presence and absence of exogenous auxin. Seedlings of the three ecotypes were grown under various conditions. When grown under 16 hours light/day for 11 days, all seedlings showed adventitious roots, both with and without auxin, however, both adventitious and lateral rooting were enhanced by exogenous auxin (2 µM naphthaleneacetic acid). Independently of the presence of auxin and of the ecotype, the hypocotyl pericycle produced adventitious roots directly (i.e., according to the same pattern of lateral root formation by the pericycle cells in the primary root). However, in the presence of auxin, roots of indirect origin also, and mainly, formed and their formation was preceded by the exfoliation of the tissues external to the stele. Exfoliation was caused by cell hypertrophy, separation, and disintegration, which mainly involved the endodermis. At the exfoliation site, the pericycle, with a minor contribution of a few endodermal cells, produced the callus from which indirect roots arose. The finding that adventitious rooting occurs in the absence of auxin (all ecotypes) indicates that this process is part of the normal root apparatus in Arabidopsis, with the hypocotyl pericycle as the target tissue of the process. Exogenous auxin alters adventitious rhizogenesis mainly affecting the endodermis response.     

Weakly cytokinin-active diphenylurea derivatives influence adventitious rooting in M26 Malus pumila microcuttings

The cytokinin activity of 8 previously unstudied diphenylurea (DPU)derivatives, differing in either type or position of the substituents of thephenyl rings, was investigated. Cytokinin activity was assessed using thebetacyanin (so-called amaranthin) accumulation test and the tomato regenerationtest. We also assayed their capacity to enhance adventitious root formation inmicrocuttings of apple rootstock M26 and in a tomato cotyledon rooting test,while their possible auxin-like activity was tested using the pea stemelongation test. All the compounds showed weak cytokinin activity, and thismight be responsible for the enhanced adventitious rooting which is induced bysome of the compounds. None of the DPU derivatives showed auxin-like activity.     

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.