Adventitious Root Formation Research Articles

Integrating histology and phytohormone/metabolite profiling to understand rooting in yellow camellia cuttings

Vegetative propagation through cutting is a widely used clonal approach for maintaining desired genotypes. However, some woody species have difficulty forming adventitious roots (ARs) with this approach, including yellow camellia (YC) C. nitidissima. Yellow camellias, prized for their ornamental value and potential health benefits in tea, remain difficult to propagate clonally due to this rooting recalcitrance. As part of the efforts to understand YC cuttings’ recalcitrance, we conducted a detailed investigation into AR formation in yellow camellia cuttings via histology and endogenous phytohormone dynamics during this process. We also compared YC endogenous phytohormone and metabolite phytohormone profiles with those of easy-to-root poplar and willow cuttings. Our results indicate that the induction of ARs in YC cuttings is achievable through auxin treatment, and YC ARs are initiated from cambial derivatives and develop a vascular system connected with that of the stem. During AR induction, endogenous hormones showed a dynamic profile, with IAA continuing to increase starting 9 days after auxin induction. JA, JA-Ile, and OPDA showed a similar trend as IAA but decreased by the 45th day. Cytokinin first decreased to its lowest level by the 18th day and then increased. SA largely exhibited an increasing trend with a drop on the 36th day, while ABA first increased to its peak level by the 18th day and then decreased. Compared to poplar, YC cuttings had a low level of IAA, IAA-Asp, and OPDA, and a high level of cytokinin and SA. Metabolite profiling highlighted significant down-accumulation of compounds associated with AR formation in yellow camellias, such as citric and ascorbic acid, fructose, sucrose, flavonoids, and phenolic acid derivatives. Our study reveals the unfavorable endogenous hormone and metabolite profiles underlying the rooting recalcitrance of YC cuttings, providing valuable knowledge for addressing this challenge in clonal propagation.

Physiological and biochemical aspects of successful stenting in Rosa hybrida L.: Role of rootstock

Stenting is a commercial propagation method for roses that combines grafting and cutting in one action and the resulting plantlet is named ‘stentling’. Several physiological and biochemical factors can significantly influence graft union healing and adventitious root formation. The effect of rootstock and its cutting-prepared region have not been well studied for successful stenting. We provide a comprehensive overview of the physiological and biochemical aspects of the stenting process, utilizing rootstock positions as the source of variation among treatments. Morphophysiological and biochemical assays were performed to evaluate the effect of three different stem parts (bottom, middle, and top) of ‘Natal Briar’ as rootstock on graft union healing and adventitious root formation of the rose stentlings. When the rootstock originating from the bottom and middle positions improved the physiological, biochemical, and morphological responses of the stentling, significantly. In this regard, the improved biochemical properties, including total soluble sugars, total phenolic and flavonoid contents, phenylalanine ammonia-lyase, peroxidase and lignin content seem to play a special role in the success of grafting and subsequently the adventitious root formation. Assessing morphophysiological and biochemical parameters paves the way for a better understanding of the mechanism of successful stenting.

CDPK5 and CDPK13 play key roles in acclimation to low oxygen through the control of RBOH-mediated ROS production in rice

Abstract CALCIUM-DEPENDENT PROTEIN KINASE (CDPK) stimulates reactive oxygen species (ROS)-dependent signaling by activating RESPIRATORY BURST OXIDASE HOMOLOG (RBOH). The lysigenous aerenchyma is a gas space created by cortical cell death that facilitates oxygen diffusion from the shoot to the root tips. Previously, we showed that RBOHH is indispensable for the induction of aerenchyma formation in rice (Oryza sativa) roots under low-oxygen conditions. Here, we showed that CDPK5 and CDPK13 localize to the plasma membrane where RBOHH functions. Mutation analysis of the serine at residues 92 and 107 of RBOHH revealed that these residues are required for CDPK5- and CDPK13-mediated activation of ROS production. The requirement of Ca2+ for CDPK5 and CDPK13 function was confirmed using in vitro kinase assays. CRISPR/Cas9-based mutagenesis of CDPK5 and/or CDPK13 revealed that the double knockout almost completely suppressed inducible aerenchyma formation, whereas the effects were limited in the single knockout of either CDPK5 or CDPK13. Interestingly, the double knockout almost suppressed the induction of adventitious root formation, which is widely conserved in vascular plants, under low-oxygen conditions. Our results suggest that CDPKs are essential for the acclimation of rice to low-oxygen conditions and also for many other plant species conserving CDPK-targeted phosphorylation sites in RBOH homologs.

Peculiarities of adventitious root formation in stem cuttings of Black Chokeberry (Aronia melanocarpa (Michx.) Elliott)) varieties depending on the influence of biologically active substances

Peculiarities of adventitious root formation in stem cuttings of Black Chokeberry (Aronia melanocarpa (Michx.) Elliott)) varieties depending on the influence of biologically active substances

PagMYB180 regulates adventitious rooting via a ROS/PCD-dependent pathway in poplar

The formation of adventitious roots (AR) is an essential step in the vegetative propagation of economically woody species. Reactive oxygen species (ROS) function as signaling molecules in regulating root growth and development. Here, we identified an R2R3-MYB transcription factor PagMYB180 as a regulator of AR formation in hybrid poplar (Populus alba × Populus glandulosa). PagMYB180 was specifically expressed in the vascular tissues of poplar roots, stems and leaves, and its protein was localized in the nucleus and acted as a transcriptional repressor. Both dominant repression and overexpression of PagMYB180 resulted in a significant reduction of AR quantity, a substantial increase of AR length, and an elevation of both the quantity and length of lateral roots (LR) compared to the wild type (WT) plants. Furthermore, PagMYB180 regulates programmed cell death (PCD) in root cortex cells, which is associated with elevated levels of ROS. Transcriptome and reverse transcription-quantitative PCR (RT-qPCR) analyses revealed that a series of differentially expressed genes are related to ROS, PCD and ethylene synthesis. Taken together, these results suggest that PagMYB180 may regulate AR development via a ROS/PCD-dependent pathway in poplar.

Application of commercial seaweed extract-based biostimulants to enhance adventitious root formation in ornamental cutting propagation protocols: a review

Despite significant advancements in stem-cutting propagation, insufficient rooting efficiency remains an economic burden for the ornamental nursery industry. IBA and NAA play a critical role in generating adventitious roots (AR) when applied exogenously. In sustainable agriculture, the substitution of chemical inputs, with alternative natural eco-friendly products presents a key challenge. Biostimulants can form part of a solution to mitigate such risks deriving from the use of agrochemicals, they are generally considered to be non-toxic, non-polluting, biodegradable, and non-hazardous. The current knowledge of the use of commercial seaweed extract (SE) products applied to ornamental cutting propagation has not been summarized until now. Therefore, we conducted a systematic review, and we hypothesized that SE-based biostimulant application to ornamental stem cuttings improves AR formation in terms of rooting percentage, root number, and architecture. Moreover, they increase the overall quality of a rooted cutting as dry biomass and organic compound content. The authors chose SE-based biostimulants because they have been proven to have an extremely low carbon footprint; moreover, they are expected to account for more than 33% of the global market for biostimulants and reached a value of 894 million Euros by 2022. This review focuses on (i) SE-based biostimulants, in particular, brown algae; (ii) technical information on five commercial products: Goteo®, Kelpak®, AlgaminoPlant, Bio Rhizotonic, Actiwawe and others, less known, also used as phytoregulators substitutes; (iii) applied protocols, describing dose, application method, number of treatments, cutting type; (iv) effects of applied protocols on rooting rate, root architecture and overall rooted cutting quality. Outcomes show that findings vary based on crops, cuttings, location, raw materials, composition, dose, application number and procedures, and growth environment.

Comparative transcriptome profiling reveals that light coordinates auxin to inhibit adventitious root formation in grapevine

Grapevine (Vitis sp.) is one of the most important economic fruit crops all over the world, and the formation of adventitious roots (ARs) is crucial for the vegetative reproduction of grapes. However, studies on the regulatory mechanisms of this process are currently lacking. In this study, we applied an efficient and convenient leave-petiole (LP) system for studying ARs, revealing a significant inhibition of root primordia formation under continuous-light treatment. The results showed that isolated ARs of grapevine were induced and originated from ray cells near the vascular cambium, with the process categorized into induction, initiation, and extension stages. LP samples under light and dark conditions were used for transcriptome sequencing and endogenous hormone measurements at three critical time points of AR formation. A total of 37 155 transcripts were obtained, and 7041 genes showed significantly different expression levels in the petiole. An integrated analysis, including Gene Ontology (GO) enrichment analysis, weighted gene co-expression network analysis (WGCNA), and hormonal content determination, showed that several genes (ARF4, LAX1, PIN1, SUS2, APX1, TPXL1, CHS3, etc.) associated with hormone signals, sugar synthesis and transport, reactive oxygen species (ROS) scavenging, cell wall biogenesis, flavonoid biosynthesis, microtubule remodeling, and some transcription factors (HY5, COP1, ERF2, MYB15, etc) played vital roles in light-induced AR formation. A hypothetical model was initially constructed, which illustrated the centrality of auxin in HY5-dependent AR formation and the complex crosstalk among various factors. The results of this study provided abundant genetic resources and a novel perspective for understanding the molecular mechanisms of AR formation in grapevine.

Unraveling Key Factors for Hypoxia Tolerance in Contrasting Varieties of Cotton Rose by Comparative Morpho-physiological and Transcriptome Analysis.

The cotton rose (Hibiscus mutabilis) is a plant species commonly found in tropical and subtropical regions. It is remarkably resilient to waterlogging stress; however, the underlying mechanism behind this trait is yet unknown. This study used hypoxia-tolerant "Danbanhong" (DBH) and more hypoxia-sensitive "Yurui" (YR) genotypes and compared their morpho-physiological and transcriptional responses to hypoxic conditions. Notably, DBH had a higher number of adventitious roots (20.3) compared to YR (10.0), with longer adventitious roots in DBH (18.3 cm) than in YR (11.2 cm). Furthermore, the formation of aerenchyma was 3-fold greater in DBH compared to YR. Transcriptomic analysis revealed that DBH had more rapid transcriptional responses to hypoxia than YR. Identification of a greater number of differentially expressed genes (DEGs) for aerenchyma, adventitious root formation and development, and energy metabolism in DBH supported that DBH had better morphological and transcriptional adaptation than YR. DEG functional enrichment analysis indicated the involvement of variety-specific biological processes in adaption to hypoxia. Plant hormone signaling transduction, MAPK signaling pathway and carbon metabolism played more pronounced roles in DBH, whereas the ribosome genes were specifically induced in YR. These results show that effective multilevel coordination of adventitious root development and aerenchyma, in conjunction with plant hormone signaling and carbon metabolism, is required for increased hypoxia tolerance. This study provides new insights into the characterization of morpho-physiological and transcriptional responses to hypoxia in H. mutabilis, shedding light on the molecular mechanisms of its adaptation to hypoxic environments.

PagWOX11/12a from hybrid poplar enhances drought tolerance by modulating reactive oxygen species

WOX11/12 is a homeobox gene of WOX11 and WOX12 in Arabidopsis that plays important roles in crown root development and growth. It has been reported that WOX11/12 participates in adventitious root (AR) formation and different abiotic stress responses, but the downstream regulatory network of WOX11/12 in poplar remains to be further investigated. In this study, we found that PagWOX11/12a is strongly induced by PEG-simulated drought stress. PagWOX11/12a-overexpressing poplar plantlets showed lower oxidative damage levels, greater antioxidant enzyme activities and reactive oxygen species (ROS) scavenging capacity than non-transgenic poplar plants, whereas PagWOX11/12a dominant repression weakened root biomass accumulation and drought tolerance in poplar. RNA-seq analysis revealed that several differentially expressed genes (DEGs) regulated by PagWOX11/12a are involved in redox metabolism and drought stress response. We used RT-qPCR and yeast one-hybrid (Y1H) assays to validate the downstream target genes of PagWOX11/12a. These results provide new insights into the biological function and molecular regulatory mechanism of WOX11/12 in the abiotic resistance processes of poplar.

Anatomical limitations in adventitious root formation revealed by magnetic resonance imaging, infrared spectroscopy, and histology of rose genotypes with contrasting rooting phenotypes.

Adventitious root (AR) formation is one of the most important developmental processes in vegetative propagation. Although genotypic differences in rose rooting ability are well known, the causal factors are not well understood. The rooting of two contrasting genotypes, 'Herzogin Friederike' and 'Mariatheresia', was compared following a multiscale approach. Using magnetic resonance imaging, we non-invasively monitored the inner structure of stem cuttings during initiation and progression of AR formation for the first time. Spatially resolved Fourier-transform infrared spectroscopy characterized the chemical composition of the tissues involved in AR formation. The results were validated through light microscopy and complemented by immunolabelling. The outcome demonstrated similarity of both genotypes in root primordia formation, which did not result in root protrusion through the shoot cortex in the difficult-to-root genotype 'Mariatheresia'. The biochemical composition of the contrasting genotypes highlighted main differences in cell wall-associated components. Further spectroscopic analysis of 15 contrasting rose genotypes confirmed the biochemical differences between easy- and difficult-to-root groups. Collectively, our data indicate that it is not the lack of root primordia limiting AR formation in these rose genotypes, but the firmness of the outer stem tissue and/or cell wall modifications that pose a mechanical barrier and prevent root extension and protrusion.

VEGETATIVE PROPAGATION FROM SHOOT CUTTINGS OF MATURE ROSEWOOD TREES (ANIBA ROSAEODORA DUCKE)

The production of seedlings by cuttings is an alternative to reconcile exploration and conservation of Rosewood (Aniba rosaeodora Ducke). In this study, our objective was to evaluate the rooting of cuttings from epicormic shoots of adult rosewood strains, through substrate tests, auxin concentrations, and cutting types, as well as to describe the anatomical process of the formation of adventitious roots. We carried out two experiments: E1 - substrate test (sand [A], vermiculite [V], and A+V) and concentrations of indole butyric acid (IBA) via talc (0.0; 1.5; 3.0; 6 .0 g kg-1); E2 – evaluation of the type of cutting: apical, intermediate, basal and regrowth; in addition to anatomical analyzes on samples of rooted cuttings. In the experiments, the variables survival (%), rooting (%), number of roots, and length of the largest root (cm) were evaluated. In E2, shoots (%), number of shoots, length of the largest shoot (cm), and callosity (%) were added. The rooting at 3.0 g kg-1 IBA was better than the control and vermiculite as a substrate was superior to sand. Even without interaction, auxin showed a tendency for better rooting when used with one of the vermiculite substrates. Among the types of cuttings, the regrowth was superior for most variables, differing from the others in terms of sprouting and callosity, while the basal was inferior in the concentration of total carbohydrates and starch.

Adventitious root formation confers waterlogging tolerance in cowpea (Vigna unguiculata (L.) Walp.)

Crop adaptation to waterlogging stress necessitates alterations in their morpho-physiological and biochemical characteristics. Cowpeas, which serve as a dual-purpose legume crop (food and fodder), are sensitive to waterlogging stress, especially when exposed to extended periods of water stagnation during the early growth stage. In this study, we subjected five distinct and superior cowpea varieties to 10 days of waterlogging stress at the early seedling stage (V2, 15 days post emergence for 10 days) under controlled conditions. The aim was to comprehend the response of these varieties and identify the ideal trait for screening a large collection of cowpea genetic resources for waterlogging tolerance. We measured and analyzed changes in morpho-physiological and root parameters to gain a deeper understanding of the mechanism underlying waterlogging tolerance. The treatment (waterlogging and control), genotype, and their interactions had a significant impact on the most studied traits (p < 0.05). The results indicated a significant reduction in morpho-physiological parameters such as plant height, leaf area, leaf number, Normalized Difference Vegetation Index (NDVI), chlorophyll content, and chlorophyll fluorescence parameters under stress treatment than control conditions. However, root parameters like the number of adventitious roots (AR) and their length (ARL) significantly increased under waterlogging stress in tolerant cowpea varieties like DC15 and PL4. Correlation and PCA analyses further revealed a positive and significant association between cowpeas’ waterlogging tolerance and AR formation and its AR length. Therefore, the current study reveals that swift development of AR and ARL may serve as potential traits conferring waterlogging tolerance in cowpeas. Using suitable mapping populations, these traits could reveal genomic regions associated with waterlogging tolerance in cowpeas. The tolerant varieties and key traits identified in this study could be beneficial in breeding programs aimed at enhancing waterlogging tolerance in cowpeas.

Cинтетические укрытия вегетационных сооружений с интегрированным фотолюминофором в укоренении черенков туи западной

We have explored the effectiveness of using light-correcting covering materials for greenhouse structures when rooting the cuttings of various ornamental forms and varieties of the northern white-cedar (Thuja occidentalis L.). Two types of shelters have been tested: the modified spunbond at a density of 30 g/m2 with a photoluminescent phosphor integrated into its structure at a concentration of 0.8 % and the unmodified spunbond at the same density. Europeum-doped yttrium oxysulfide (Y2O2SEu) has been chosen as a photoluminescent phosphor. We have analyzed 11 varieties and forms of Thuja occidentalis L.: Rheingold, Mr. Bowling Ball, Woodwardii, Tiny Tim, Golden Smaragd, Mirjam, Golden Globe, Danica, Sunkist, Smaragd and Brabant. The response of the indicators of callusogenesis and rhizogenesis to the influence of a transformed light flux has been tested. A positive effect of the use of photoluminescent phosphors in rooting the stem cuttings of the northern white-cedar has been established. An increase in the indicators of regenerative ability and post-regenerative development of the root systems and the aboveground parts of the cuttings has been recorded, such as: the activity of callusogenesis, the number of adventitious roots formed, the length of the leading root, the total length of the root system, the height of the aboveground part and the diameter of the root collar. Callusogenesis has increased from 67.59 ± 2.05 % in the check to 76.81 ± 1.61 % when having integrated the photoluminescent phosphor. The number of adventitious roots formed has increased from 9.64 ± 0.43 to 11.44 ± 0.34 pcs., correspondingly. The total length of the root systems has increased from 64.56 ± 3.70 to 75.52 ± 2.77 cm, correspondingly. One-way analysis of variance confirmed the significance of the differences between the tested forms and varieties in each of the shelter options for most of the tested indicators. Two-way analysis of variance has revealed a significant effect of the positive impact of photoluminescent phosphors on callusogenesis, adventitious root formation, post-regenerative development of the root systems and the aboveground parts of the cuttings.

Nitric oxide, energy, and redox-dependent responses to hypoxia.

Hypoxia occurs when oxygen levels fall below the levels required for mitochondria to support respiration. Regulated hypoxia is associated with quiescence, particularly in storage organs (seeds) and stem cell niches. In contrast, environmentally induced hypoxia poses significant challenges for metabolically active cells that are adapted to aerobic respiration. The perception of oxygen availability through cysteine oxidases, which function as oxygen-sensing enzymes in plants that control the N-degron pathway, and the regulation of hypoxia-responsive genes and processes is essential to survival. Functioning together with reactive oxygen species (ROS), particularly hydrogen peroxide (H2O2) and reactive nitrogen species (RNS), such as nitric oxide (·NO), nitrogen dioxide (·NO2), S-nitrosothiols (SNOs), and peroxynitrite (ONOO-), hypoxia signaling pathways trigger anatomical adaptations such as formation of aerenchyma, mobilization of sugar reserves for anaerobic germination, formation of aerial adventitious roots, and the hyponastic response. NO and H2O2 participate in local and systemic signaling pathways that facilitate acclimation to changing energetic requirements, controlling glycolytic fermentation, the γ-aminobutyric acid (GABA) shunt, and amino acid synthesis. NO enhances antioxidant capacity and contributes to the recycling of redox equivalents in energy metabolism through the phytoglobin (Pgb)-NO cycle. Here, we summarize current knowledge of the central role of NO and redox regulation in adaptive responses that prevent hypoxia-induced death in challenging conditions such as flooding.

Genetic diversity and candidate genes for transient waterlogging tolerance in mungbean at the germination and seedling stages.

Mungbean [Vigna radiata var. radiata (L.) Wilczek] production in Asia is detrimentally affected by transient soil waterlogging caused by unseasonal and increasingly frequent extreme precipitation events. While mungbean exhibits sensitivity to waterlogging, there has been insufficient exploration of germplasm for waterlogging tolerance, as well as limited investigation into the genetic basis for tolerance to identify valuable loci. This research investigated the diversity of transient waterlogging tolerance in a mini-core germplasm collection of mungbean and identified candidate genes for adaptive traits of interest using genome-wide association studies (GWAS) at two critical stages of growth: germination and seedling stage (i.e., once the first trifoliate leaf had fully-expanded). In a temperature-controlled glasshouse, 292 genotypes were screened for tolerance after (i) 4 days of waterlogging followed by 7 days of recovery at the germination stage and (ii) 8 days of waterlogging followed by 7 days of recovery at the seedling stage. Tolerance was measured against drained controls. GWAS was conducted using 3,522 high-quality DArTseq-derived SNPs, revealing five significant associations with five phenotypic traits indicating improved tolerance. Waterlogging tolerance was positively correlated with the formation of adventitious roots and higher dry masses. FGGY carbohydrate kinase domain-containing protein was identified as a candidate gene for adventitious rooting and mRNA-uncharacterized LOC111241851, Caffeoyl-CoA O-methyltransferase At4g26220 and MORC family CW-type zinc finger protein 3 and zinc finger protein 2B genes for shoot, root, and total dry matter production. Moderate to high broad-sense heritability was exhibited for all phenotypic traits, including seed emergence (81%), adventitious rooting (56%), shoot dry mass (81%), root dry mass (79%) and SPAD chlorophyll content (70%). The heritability estimates, marker-trait associations, and identification of sources of waterlogging tolerant germplasm from this study demonstrate high potential for marker-assisted selection of tolerance traits to accelerate breeding of climate-resilient mungbean varieties.

Engineered poplar for bioproduction of the triterpene squalene.

Building sustainable platforms to produce biofuels and specialty chemicals has become an increasingly important strategy to supplement and replace fossil fuels and petrochemical-derived products. Terpenoids are the most diverse class of natural products that have many commercial roles as specialty chemicals. Poplar is a fast growing, biomassdense bioenergy crop with many species known to produce large amounts of the hemiterpene isoprene, suggesting an inherent capacity to produce significant quantities of other terpenes. Here we aimed to engineer poplar with optimized pathways to produce squalene, a triterpene commonly used in cosmetic oils, a potential biofuel candidate, and the precursor to the further diversified classes of triterpenoids and sterols. The squalene production pathways were either re-targeted from the cytosol to plastids or co-produced with lipid droplets in the cytosol. Squalene and lipid droplet co-production appeared to be toxic, which we hypothesize to be due to disruption of adventitious root formation, suggesting a need for tissue specific production. Plastidial squalene production enabled up to 0.63 mg/g fresh weight in leaf tissue, which also resulted in reductions in isoprene emission and photosynthesis. These results were also studied through a technoeconomic analysis, providing further insight into developing poplar as a production host.

Does the Physiological Age of Stock Plant Material Affect the Uptake of Indole-3-Butyric Acid (IBA) in Leafy Cuttings of Prunus subhirtella ‘Autumnalis’?

The aim of this study was to investigate the uptake of exogenously added indole-3-butyric acid (IBA) as an adventitious root (AR) inducer. The concentration of indole-3-butyric acid (IBA) in leafy cuttings of Prunus subhirtella ‘Autumnalis’ from stock material of different physiological ages was analyzed at three evaluation time points (1, 4, 24 h). We examined three stock plants: a physiologically mature stock plant (approximately 60 years old); physiologically rejuvenated plants (16 years old) that were previously propagated by cuttings from semi-mature stock material; and in vitro juvenile stock material. For IBA to effectively induce AR formation, it must first be converted into the active hormone indole-3-acetic acid (IAA). The total concentration of IAA in the first 24 h after cutting was evaluated according to the physiological age of the stock material and the IAA concentration in the cuttings according to the evaluation time points was assessed. We also examined rooting success and the quality of the adventitious root system depending on the physiological age of the stock material. High performance liquid chromatography coupled with mass spectrometry (HPLC-MS/MS) was used to identify and quantify the phytohormones. The difference in rooting and quality of the developed AR was observed between semi-mature stock and mature stock material. Cuttings from semi-mature plants rooted in 95.00 ± 5.00% of cases, while cuttings from mature stock plants only rooted in 68.33 ± 4.09%. Successfully rooted cuttings from mature stock material were accompanied by callus formation, which was significantly lower in cuttings from semi-mature stock material. Our results show that the interaction between physiological age and time after severance had no effect on IBA concentration in the cuttings (p = 0.907). Duration of time elapsed since severing (evaluation time points) had a significant effect on the uptake of IBA (p = 0.002 **) and IAA (p = 0.0009 ***) in the cuttings. Physiological age had a significant effect on IAA concentration in the early stages of AR formation in the cuttings (p = 0.038 *). Our results suggest that the dynamics of other endogenous phytohormones and the physiological state of the stock plant are also important for AR formation, as the proportion of successfully rooted cuttings from semi-mature stock material was significantly higher than the proportion of successfully rooted cuttings from mature stock material, irrespective of IBA uptake in stock material of different ages.

Ethylene Action Inhibition Improves Adventitious Root Induction in Adult Chestnut Tissues.

Phase change refers to the process of maturation and transition from the juvenile to the adult stage. In response to this shift, certain species like chestnut lose the ability to form adventitious roots, thereby hindering the successful micropropagation of adult plants. While auxin is the main hormone involved in adventitious root formation, other hormones, such as ethylene, are also thought to play a role in its induction and development. In this study, experiments were carried out to determine the effects of ethylene on the induction and growth of adventitious roots. The analysis was performed in two types of chestnut microshoots derived from the same tree, a juvenile-like line with a high rooting ability derived from basal shoots (P2BS) and a line derived from crown branches (P2CR) with low rooting responses. By means of the application of compounds to modify ethylene content or inhibit its signalling, the potential involvement of this hormone in the induction of adventitious roots was analysed. Our results show that ethylene can modify the rooting competence of mature shoots, while the response in juvenile material was barely affected. To further characterise the molecular reasons underlying this maturation-derived shift in behaviour, specific gene expression analyses were developed. The findings suggest that several mechanisms, including ethylene signalling, auxin transport and epigenetic modifications, relate to the modulation of the rooting ability of mature chestnut microshoots and their recalcitrant behaviour.

NnARF17 and NnARF18 from lotus promote root formation and modulate stress tolerance in transgenic Arabidopsis thaliana

Auxin response factors (ARFs) play a crucial role in regulating gene expression within the auxin signal transduction pathway, particularly during adventitious root (AR) formation. In this investigation, we identified full-length sequences for ARF17 and ARF18, encompassing 1,800 and 2,055 bp, encoding 599 and 684 amino acid residues, respectively. Despite exhibiting low sequence homology, the ARF17- and ARF18-encoded proteins displayed significant structural similarity and shared identical motifs. Phylogenetic analysis revealed close relationships between NnARF17 and VvARF17, as well as NnARF18 and BvARF18. Both ARF17 and ARF18 demonstrated responsiveness to exogenous indole-3-acetic acid (IAA), ethephon, and sucrose, exhibiting organ-specific expression patterns. Beyond their role in promoting root development, these ARFs enhanced stem growth and conferred drought tolerance while mitigating waterlogging stress in transgenic Arabidopsis plants. RNA sequencing data indicated upregulation of 51 and 75 genes in ARF17 and ARF18 transgenic plants, respectively, including five and three genes associated with hormone metabolism and responses. Further analysis of transgenic plants revealed a significant decrease in IAA content, accompanied by a marked increase in abscisic acid content under normal growth conditions. Additionally, lotus seedlings treated with IAA exhibited elevated levels of polyphenol oxidase, IAA oxidase, and peroxidase. The consistent modulation of IAA content in both lotus and transgenic plants highlights the pivotal role of IAA in AR formation in lotus seedlings.

Transcriptome profiling of indole-3-butyric acid–induced adventitious root formation in softwood cuttings of walnut

Inducing adventitious root (AR) formation in mature walnut species (Juglans L.) is challenging. However, the AR formation of mature trees can be improved by rejuvenation. In rejuvenated cuttings, exogenous indole-3-butyric acid (IBA) is essential for AR formation, and the underlying mechanism is still not well understood. Therefore, we utilized transcriptome sequencing to investigate the mechanism of IBA-induced AR formation. Our results revealed that, in comparison to the control group, IBA treatment (9 mmol · L-1) significantly increased the endogenous indole-3-acetic acid (IAA) content, leading to an enhanced rooting rate. We performed RNA sequencing to identify differentially expressed genes (DEGs) between the IBA-treated and control (CK) groups at 1, 2, 3, and 5 days after cutting (DAC). The results showed that, compared to the control cuttings, there were 1 539, 889, 785, and 984 up-regulated genes and 2 791, 2 936, 3 017, and 1 752 down-regulated genes, at 1, 2, 3, and 5 DAC, respectively. Analysis of RNA-seq data revealed that G-type ATP-binding cassette 36/37 (ABCG36/37) and ATP-binding cassette subfamily D 1 (ABCD1), associated with IBA transport, were down-regulated in the rejuvenation cuttings. In contrast, PIN-FORMED (PIN) and PINOID (PID), associated with auxin efflux, were up-regulated. We identified 49 auxin/indole-3-acetic acid (AUX/IAA)-encoding genes, including IAA1, IAA3, IAA5, IAA6, IAA8, IAA11, IAA12, IAA19, and IAA20, which were up-regulated at 1–5 DAC in the rejuvenated cuttings. This study highlights that the overexpression of JrWOX5/11 in poplar significantly enhance AR growth, as evidenced by increased root length, surface area, volume, and quantity. Moreover, the co-expression network analysis involving JrWOX11 and JrWOX5 in walnut cuttings elucidates complex genetic interactions, underscoring their pivotal role in the formation of AR. Our data supported the following molecular mechanism of IBA-induced adventitious root formation. Firstly, IBA is converted to free IAA in peroxisomes. Then, the highly concentrated IAA in the procambium and parenchyma cells induces WUSCHEL-related homeobox 11 (WOX11) expression at two days. Finally, WOX11 acts redundantly to up-regulate WOX5, initiating the development of root primordia cells.