Hypocotyl Cuttings Research Articles

DNA Hypomethylation Activates the RpMYB2-Centred Gene Network to Enhance Regeneration of Adventitious Roots.

Plants, being immobile, are exposed to environmental adversities such as wind, snow and animals that damage their structure, making regeneration essential for their survival. The adventitious roots (ARs) primarily emerge from a detached explant to uptake nutrients; therefore, the molecular network involved in their regeneration needs to be explored. DNA methylation, a key epigenetic mark, influences molecular pathways, and recent studies suggested its role in regeneration. In our research, the application of 5-azacytidine (5-azaC), an inhibitor of DNA methylation, caused the earlier initiation and development of root primordia and consequently enhanced the AR regeneration rate in Robinia psuedoacacia L (black locust). The whole-genome bisulfite sequencing (WGBS) revealed a decrease in global methylation and an increase in hypomethylated cytosine sites and regions across all contexts including CHH, CHG and mergedCG caused transcriptional variations in 5-azaC-treated sample. The yeast two-hybrid (Y2H) assay revealed a RpMYB2-centred network of transcriptionally activated transcription factors (TFs) including RpWRKY23, RpGATA23, RpSPL16 and other genes like RpSDP, RpSS1, RpBEN1, RpGULL05 and RpCUV with nuclear localization suggesting their potential co-localization. Additionally, yeast one-hybrid (Y1H) assay showed the interaction of RpMYB2 interactors, RpGATA23 and RpWRKY23, with promoters of RpSK6 and RpCDC48, and luciferase reporting assay (LRA) validated their binding with RpSK6. Our results revealed that hypomethylation-mediated transcriptomic modifications activated the RpMYB2-centred gene network to enhance AR regeneration in black locust hypocotyl cuttings. These findings pave the way for genetic modification to improve plant regeneration ability and increase wood production while withstanding environmental damage.

Utilization of Kandelia candel (L.) Druce and hypocotyl cutting seedling for mangrove rehabilitation in Indonesia

Kandelia candel (L.) Druce is one of the rare mangrove species in Indonesia. Additionally, mangrove rehabilitation requires effective and efficient methods. Accelerating the growth of mangrove species using vegetative propagation, specifically hypocotyl cutting, is necessary. The objectives of this study were to investigate the scientific literature on the use of K. candel for mangrove rehabilitation in Indonesia and to analyze the growth of K. candel seedling from hypocotyl cuttings. Scientific literature was collected from 2012 to 2022 in some search engines and library databases. An experimental study was carried out with different treatments (hypocotyl cuttings, planting media, plant growth regulator) and three replications. We found 576 articles on mangrove rehabilitation in Indonesia and only an article uses K. candel (0,002%) as a species in mangrove rehabilitation. There was no study on hypocotyl cutting of K. candel. The best combination of intact hypocotyl treatment was intact hypocotyl, mud+sand growing medium, and growth regulator 15.000 ppm. In contrast, the best combination of cut hypocotyl treatment was bottom hypocotyl, mud+sand planting medium, and growth regulator 15.000 ppm.

Effects of ectomycorrhizal fungal inoculation on growth and rooting of carob tree (Ceratonia siliqua L.)

The present work investigated the ability of the ectomycorrhizal (ECM) fungi, Pisolithus tinctorius (Pers.) Coker and Couch to improve the growth and root formation in carob seedlings (Ceratonia siliqua L.). Root initiation of hypocotyl cuttings in vitro was enhanced by treatments with different concentrations of either fungal culture filtrate or indole-3-butyric acid (IBA) or in combination. The ectomycorrhization protocol for carob seedlings comprised of three different treatments: in vivo or in vitro inoculation and non-inoculated seedlings. Results obtained after six months of acclimatization showed that inoculation had a positive effect on the growth and rooting of carob plants. Moreover, plants mycorrhized in vitro or in vivo outperformed non-mycorrhized ones in plant height, number of internodes, number of leaves, leaf length, number of roots, maximal root length, and fresh and dry weights of both stems and roots. Indeed, the optimum response was encountered in the seedlings inoculated in vitro. The application of culture filtrate on hypocotyl cuttings revealed significant differences between experimental treatments for the number and length of roots. The best response was recorded in medium containing fungal culture filtrate with 40%. Root formation of hypocotyl cuttings of carob plants was also enhanced by the use of IBA, where the best rooting was manifested in treatment with IBA at a concentration of 2 mg/l. The application of P. tinctorius culture filtrate to the culture medium of hypocotyl cuttings after pretreatment by immersing in IBA was even more effective in improving root formation than the treatment of hypocotyls base with IBA alone. The present study demonstrated the possibility of developing procedures to improve the rooting growth of carob plants by establishing a successful artificial symbiosis with P. tinctorius. Highlights from this work could play a crucial role in arid ecosystem rehabilitation and controlled mycorrhization programs.

High-Efficiency Agrobacterium rhizogenes-Mediated Transgenic Hairy Root Induction of Lens culinaris

Previous efforts to transform lentil have been considerably hampered by the crop’s recalcitrant nature, giving rise to particularly low transformation and regeneration frequencies. This study aimed at optimizing an Agrobacterium rhizogenes-mediated transformation protocol for the generation of composite lentil plantlets, comprised of transgenic hairy roots and wild-type shoots. Transformation was performed by inoculating the cut hypocotyl of young lentil seedlings, while optimization involved the use of different bacterial strains, namely R1000, K599 and Arqua, and protocols differing in media composition with respect to the presence of acetosyringone and MES. Composite plantlets had a transgenic hairy root system characterized by an increased number of hairy roots at the hypocotyl proximal region, occasionally showing plagiotropic growth. Overall findings underline that transformation frequencies are subject to the bacterial strain, media composition as well as their combined effect. Among strains tested, R1000 proved to be the most capable of hairy root formation, while the presence of both acetosyringone and MES in inoculation and culture media yielded considerably higher transformation rates. The transgenic nature of hairy roots was demonstrated by the Ri T-DNA-mediated transfer of the rolB2 gene and the simultaneous absence of the virCD sequence of A. rhizogenes. Our findings provide strong evidence that A. rhizogenes-mediated transformation may be employed as a suitable approach for generating composite seedlings in lentil, a species whose recalcitrance severely hampers all efforts addressed to transformation and whole plant regeneration procedures. To the best of our knowledge, this is the first report on the development of a non-laborious and time-efficient protocol for the generation of transgenic hairy roots in lentil, thus providing an amenable platform for root biology and gene expression studies in the context of improving traits related to biotic and abiotic stress tolerance.

Effect of polar auxin transport and gibberellins on xylem formation in pine cuttings under adventitious rooting conditions

Maturation-related decline of adventitious root formation is one of the major factors affecting adventitious rooting in forest tree species. We demonstrate that inhibition of polar auxin transport promoted cambium and xylem differentiation in rooting-competent hypocotyl cuttings from Pinus radiata under conditions of adventitious root formation. Treatments with bioactive gibberellins inhibited rooting while at the same time inducing both the differentiation of a continuous ring of cambium and xylem formation. Treatments with inhibitors of gibberellin biosynthesis did not affect the rooting response. The results demonstrate that xylem parenchyma and procambial cells at the xylem poles of rooting-competent hypocotyl cuttings after excision and under conditions of adventitious root induction become adventitious root meristems or xylem, depending on the directional auxin flow. Gibberellin may interact with this pathway, inducing xylem differentiation and inhibiting rooting. We conclude that modifications of auxin flow at the rooting sites, and the priming of cambial cells to differentiate into xylem during tree ageing, may be associated with the maturation-related decline of adventitious root formation.

Reactive oxygen species regulate auxin levels to mediate adventitious root induction in Arabidopsis hypocotyl cuttings.

Adventitious root (AR) formation from leafy stem cuttings is critical for breeding of many forest and horticultural species. In addition to the plant hormone auxin, wound-induced signaling caused by the cutting excision is also essential for AR initiation. Here we found that reactive oxygen species (ROS) are rapidly generated at the excision site as a wound-induced signal and propagated throughout the hypocotyl cutting after excision of the Arabidopsis (Arabidopsis thaliana) primary root. ROS propagation was not observed in the presence of an NADPH oxidase inhibitor (diphenylene iodonium chloride) or in a knockout mutant of the NADPH oxidase gene respiratory burst oxidase homolog protein D (RBOHD). Respiratory burst oxidase homolog protein D was specifically upregulated in hypocotyl cuttings at 0.5 h post excision (hpe). Together, these data suggest that RBOHD mediates ROS propagation in hypocotyl cuttings. We also found that auxin levels increased significantly in the shoot apex at 5 hpe and at the base of the cutting at 6 hpe; these effects were blocked by treatment with ROS scavengers. Consistent with this, transcript levels of auxin biosynthesis and polar-transport genes generally increased between 1 to 6 hpe. Collectively, our results suggest that wound-induced ROS participate in AR induction through regulation of auxin biosynthesis and transport.

Nitric oxide donor improves adventitious rooting in mung bean hypocotyl cuttings exposed to cadmium and osmotic stresses

Exogenous application of a nitric oxide donor, sodium nitroprusside (SNP), improved the formation and growth of adventitious roots in mung bean hypocotyl cuttings. The combined applications of SNP and CdCl2 or mannitol partially overcame the negative effects of Cd and mannitol on adventitious rooting compared with Cd or mannitol alone. SNP significantly increased the endogenous NO level and the glutathione (GSH), ascorbic acid (ASA), polyphenol, and proline (Pro) levels during the root induction and/or initiation stages. However, the malondialdehyde (MDA) levels were significantly reduced by the SNP treatment during the entire time course. To unveil the role of NO in mediating the plant response to heavy metal and osmotic stresses during adventitious rooting, SNP together with CdCl2 or mannitol was applied in the experiments. The results obtained showed that the Cd- or mannitol-induced changes in the levels of GSH, ASA, polyphenol, Pro, and MDA and the activities of ascorbate peroxidase (APX), peroxidase (POD), catalase (CAT), superoxide dismutase (SOD), and indole-3-acetic acid oxidase (IAAO) were completely or partially reversed by SNP supplementation in the SNP + Cd/mannitol treatments, suggesting that NO mediated the redox state via the regulation of antioxidative systems to prevent membrane lipid peroxidation in response to the stresses during adventitious rooting. Furthermore, the protective effects of NO on the cellular antioxidative systems under the stresses were similar to those of IBA, implying that auxin and NO shared common pathways to respond to the stresses and leading to adventitious rooting in mung bean hypocotyl cuttings under heavy metal and osmotic stresses. NO mediates the redox state and prevents membrane lipid peroxidation during adventitious rooting in plants exposed to abiotic stresses via the regulation of antioxidant levels and antioxidative enzyme activities. NO shares common signaling pathways with auxin or acts as a downstream mediator of auxin in response to abiotic stresses during adventitious rooting.

Genome-wide Identification and Expression of ARF Gene Family during Adventitious Root Development in Hot Pepper (Capsicum annuum)

Auxin response factors (ARFs) are transcription factors that activate or repress the expression of primary/early auxin response genes by binding to auxin-responsive elements (AuxREs) in their promoter regions. The ARFs play important roles in diverse developmental processes. To explore the ARF gene family in hot pepper (Capsicum annuum L.), we performed a genome-wide identification and expression analysis. In this study, 19 pepper ARF genes (CaARFs) clustered into three phylogenetic groups (I, II, and III) were comprehensively analyzed. Conserved domain analysis showed that all CaARFs contained a B3 DNA-binding domain and a middle domain, but two members lacked the carboxy-terminal dimerization (CTD) domain. The number of introns in CaARF genes ranged from 1 to 13 and the gene structure was similar among genes in the same phylogenetic group. Additionally, prediction of CaARFs promoter elements and putative targets for microRNAs suggested that the regulation of CaARFs may occur at both transcriptional and posttranscriptional levels. Most CaARFs were expressed in more than one tested tissue, and most CaARFs were identified as being responsive to exogenous auxin. Moreover, time-course transcription profiles of CaARFs revealed their roles in adventitious rooting of hypocotyl cuttings from pepper seedlings. Therefore, our results will provide a foundation for better understanding the regulatory mechanisms and molecular functions of CaARFs in hot pepper.

Transcriptomic profiling provides molecular insights into hydrogen peroxide-induced adventitious rooting in mung bean seedlings

BackgroundHydrogen peroxide (H2O2) has been known to function as a signalling molecule involved in the modulation of various physiological processes in plants. H2O2 has been shown to act as a promoter during adventitious root formation in hypocotyl cuttings. In this study, RNA-Seq was performed to reveal the molecular mechanisms underlying H2O2-induced adventitious rooting.ResultsRNA-Seq data revealed that H2O2 treatment greatly increased the numbers of clean reads and expressed genes and abundance of gene expression relative to the water treatment. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses indicated that a profound change in gene function occurred in the 6-h H2O2 treatment and that H2O2 mainly enhanced gene expression levels at the 6-h time point but reduced gene expression levels at the 24-h time point compared with the water treatment. In total, 4579 differentially expressed (2-fold change > 2) unigenes (DEGs), of which 78.3% were up-regulated and 21.7% were down-regulated; 3525 DEGs, of which 64.0% were up-regulated and 36.0% were down-regulated; and 7383 DEGs, of which 40.8% were up-regulated and 59.2% were down-regulated were selected in the 6-h, 24-h, and from 6- to 24-h treatments, respectively. The number of DEGs in the 6-h treatment was 29.9% higher than that in the 24-h treatment. The functions of the most highly regulated genes were associated with stress response, cell redox homeostasis and oxidative stress response, cell wall loosening and modification, metabolic processes, and transcription factors (TFs), as well as plant hormone signalling, including auxin, ethylene, cytokinin, gibberellin, and abscisic acid pathways. Notably, a large number of genes encoding for heat shock proteins (HSPs) and heat shock transcription factors (HSFs) were significantly up-regulated during H2O2 treatments. Furthermore, real-time quantitative PCR (qRT-PCR) results showed that, during H2O2 treatments, the expression levels of ARFs, IAAs, AUXs, NACs, RD22, AHKs, MYBs, PIN1, AUX15A, LBD29, LBD41, ADH1b, and QORL were significantly up-regulated at the 6- and/or 24-h time points. In contrast, PER1 and PER2 were significantly down-regulated by H2O2 treatment. These qRT-PCR results strongly correlated with the RNA-Seq data.ConclusionsUsing RNA-Seq and qRT-PCR techniques, we analysed the global changes in gene expression and functional profiling during H2O2-induced adventitious rooting in mung bean seedlings. These results strengthen the current understanding of H2O2-induced adventitious rooting and the molecular traits of H2O2 priming in plants.

Dynamics and control of phloem loading of indole-3-acetic acid in seedling cotyledons ofRicinus communis

During seed germination, sugars and auxin are produced from stored precursors or conjugates respectively, and transported to the seedling axis. To elucidate the mode of travel of indole-3-acetic acid (IAA) into the phloem, a solution of [(3)H]IAA, together with [(14)C]sucrose, was injected into the endosperm cavity harboring the cotyledons of germinating seedlings of Ricinus communis Phloem exudate from the cut hypocotyl was collected and the radioactivity recorded. Sucrose loading into the phloem was inhibited at higher IAA levels, and the rate of filling of the transient pool(s) was reduced by IAA. IAA was detected within 10min, with the concentration increasing over 30min and reaching a steady-state by 60min. The kinetics indicated that phloem loading of IAA involving both an active, carrier-based, and a passive, diffusion-based component, with IAA traveling along a pathway containing an intermediary pool, possibly the protoplasts of mesophyll cells. Phloem loading of IAA was altered by sucrose, K(+), and a range of non-specific and IAA-specific analogs and inhibitors in a manner that showed that IAA moves into the phloem from the extra cotyledonary solution by multiple pathways, with a carrier-mediated pathway playing a principal role.

1,3-di(benzo[d]oxazol-5-yl)urea acts as either adventitious rooting adjuvant or xylogenesis enhancer in carob and pine microcuttings depending on the presence/absence of exogenous indole-3-butyric acid

Asexual propagation in Ceratonia siliqua L. (carob), species of economic value, is difficult because of adventitious rooting recalcitrance. In Pinus radiata adventitious rooting of hypocotyl cuttings is enhanced by two urea-derivatives, 1,3-di(benzo[d]oxazol-5-yl)urea (5-BDPU) and 1,3-di(benzo[d]oxazol-6-yl)urea (6-BDPU), combined with exogenous indole-3-butyric acid (IBA). The research was aimed to define the role of these urea-derivatives in adventitious root (AR) formation of carob, and to identify morphogenic roles induced in carob, but also in pine, a distantly-related forest species. In carob, 5-BDPU (10 μM) highly promoted AR formation in combination with IBA (1 μM) when applied for 3 days, followed by a transfer onto hormone free medium (HF) up to culture end (4 weeks). IBA alone (1 μM) was more effective than IBA + kinetin (Kin, 10 nM), whereas Kin alone and 5-BDPU alone were not AR-inductive. The histological analysis showed that the cambial cells initiated the ARs, and similar numbers of AR-primordia were visible at day 12, independently of the AR-inductive treatment (i.e., IBA, IBA + 5-BDPU, IBA + Kin). No cutting treated with Kin alone, and rare HF (±5-BDPU)-treated ones, showed AR-primordia at day 12. The number of AR-forming explants increased under IBA + 5-BDPU. By contrast, the cambial cells were stimulated to initiate deuteroxylem instead of ARs under 5-BDPU alone. The histological analysis in pine microcuttings treated with IBA and/or 5-BDPU at the same concentrations confirmed that 5-BDPU applied alone enhanced xylogenesis, highlighting that this urea-derivative exhibits a dual morphogenic role being involved in the switching between adventitious rooting and xylogenesis depending on the presence of exogenous auxin in both species.

Activity of seaweed extracts and polysaccharide-enriched extracts from Ulva lactuca and Padina gymnospora as growth promoters of tomato and mung bean plants

Although marine seaweeds have been used as biostimulants since the beginning of modern agriculture, studies have only recently focused on the ability of seaweed extracts and their polysaccharides to enhance growth of plants. In this work, two bioassays were carried out to study the growth-promoting activity of seaweed extracts and polysaccharide-enriched extracts from Ulva lactuca and Padina gymnospora, obtained in neutral and alkaline conditions. Initially, the effect of seaweed extracts and polysaccharide-enriched extracts on seed germination and growth-promoting activity on tomato (Solanum lycopersicum cv. Rio Grande) plants under in vitro conditions was studied. Half-strength Murashige-Skoog (MS) medium with or without sucrose was supplemented with different concentrations of seaweed extracts (2, 4, and 10 mg mL−1) or polysaccharide-enriched extracts (0.2, 0.4, and 1.0 mg mL−1). The parameters evaluated were germination percentage, radicle and shoot length, and dry weight. In a second experiment, polysaccharide-enriched extracts at 1.0 mg mL−1 and indole-3-butyric acid as the control were studied for root inducer activity in mung bean (Vigna radiata). The majority of seaweed extracts had an inhibitory effect on seed germination. However, a significant effect (P ≤ 0.05) on tomato seedling growth (except for dry weight) was shown with seaweed extracts at 2 mg mL−1 included in half-strength MS medium with sucrose (30 g L−1). Moreover, 10 mg mL−1 neutral and alkaline seaweed extracts had an inhibitory effect on the parameters evaluated. In contrast, polysaccharide-enriched extracts obtained from U. lactuca and P. gymnospora promoted germination and stimulated growth of tomato plants compared to the controls. Additionally, treatment of mung bean hypocotyl cuttings with polysaccharide-enriched extracts of U. lactuca and P. gymnospora induced rooting more rapidly and in greater number compared to the controls. These results provide evidence that polysaccharide-enriched extracts act as an effective growth-promoting treatment.

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

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

Identification of systemic responses in soybean nodulation by xylem sap feeding and complete transcriptome sequencing reveal a novel component of the autoregulation pathway

Establishment of the nitrogen-fixing nodulation symbiosis between legumes and rhizobia requires plant-wide reprogramming to allow infection and development of nodules. Nodulation is regulated principally via a mechanism called autoregulation of nodulation (AON). AON is dependent on shoot and root factors and is maintained by the nodulation autoregulation receptor kinase (NARK) in soybean. We developed a bioassay to detect root-derived signalling molecules in xylem sap of soybean plants which may function in AON. The bioassay involves feeding of xylem extracts via the cut hypocotyl of soybean seedlings and monitoring of molecular markers of AON in the leaf. Transcript abundance changes occurring in the leaf in response to feeding were used to determine the biological activity of the extracts. To identify transcript abundance changes that occur during AON, which may also be used in the bioassay, we used an RNA-seq-based transcriptomics approach. We identified changes in the leaves of bioassay plants fed with xylem extracts derived from either Bradyrhizobium japonicum-inoculated or uninoculated plants. Differential expression responses were detected for genes involved in jasmonic acid metabolism, pathogenesis and receptor kinase signalling. We identified an inoculation- and NARK-dependent candidate gene (GmUFD1a) that responds in both the bioassay and intact, inoculated plants. GmUFD1a is a component of the ubiquitin-dependent protein degradation pathway and provides new insight into the molecular responses occurring during AON. It may now also be used in our feeding bioassay as a molecular marker to assist in identifying the factors contributing to the systemic regulation of nodulation.

Allelopathic Effects of Ginsenosides from Panax ginseng on the Root Development of Four Cultivar Plants in Their Early Growth Stages

Some allelochemicals, including ginsenosides secreted from the roots of ginseng and American ginseng, have been considered to play a regulatory role in population regeneration. This study was conducted to investigate the allelopathic effects of ginsenosides on the root development of several cultivar plants at the early growth stage. The different inhibitions in wheat, cabbage, and cucumber roots resulting from ginsenosides treatment at different concentrations were observed. The root vigor of the three receptor plants constantly decreased compared with the control when the treatment concentration increased; the root length and root fresh weight exhibited similar trends. However, ginsenoside treatment showed negligible effect on the hypocotyl adventitious roots of cucumber and mung bean. The root number, root length, root fresh weight, and root vigor showed no significant changes; however, the antioxidant enzyme activities were slightly enhanced compared with that of the control. These results indicate that the ginsenosides inhibited the root growth of all indicator plants, and these inhibitory effects were stronger on wheat and cucumber than on cabbage. However, no evidence indicates that ginsenosides inhibited the growth of hypocotyl cuttings of cucumber and mung bean.

Distribution of hydrogen peroxide during adventitious roots initiation and development in mung bean hypocotyls cuttings

Previous studies have shown that hydrogen peroxide (H2O2) may mediate the auxin response during the formation of adventitious roots (AR). However, the mechanism and distribution of H2O2 during AR formation remains unclear. In this study, we investigate the spatiotemporal changes and role of H2O2 in AR initiation and development. Application of 5–100 mM H2O2 to Mung bean (Phaseolus radiatus L.) hypocotyl cuttings induced AR formation in a dose-dependent manner. The effect was blocked by ascorbic acid (AA), an important reducing substrate for H2O2 reduction. Depletion of endogenous H2O2 by AA resulted in the significant reduction of AR emergence, suggesting a physiological role for H2O2 in the regulation of AR formation. Determination of H2O2 content showed that the level of H2O2 increased gradually and reached the highest value 60 h after induction of AR. Further detection of endogenous H2O2 by the specific fluorescent probe dichlorofluorescein diacetate (H2DCF-DA) and 3,3′-diaminobenzidine (DAB) staining in transverse sections of the basal region of cuttings revealed that obvious H2O2 signals were observed in the pericycle cells between the vascular bundles 24 h after the primary roots were removed. With the development of root primordia, H2O2 signals increased gradually and were mainly distributed in the root meristem. AA significant inhibited the H2O2-dependent fluorescence and the formation of AR, suggesting an essential role of H2O2 generation during AR initiation and development. Furthermore, the involvement of Ca2+ during H2O2-mediated AR formation was evaluated. Ca2+ channel inhibitors LaCl3 and ruthenium red (RR) and Ca2+ chelator ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) prevent H2O2-induced AR formation, which indicate that the hypocotyl cuttings response to H2O2 depends on the availability of both intracellular and extracellular Ca2+ pools, and Ca2+ is a downstream messenger in the signaling pathway triggered by H2O2 to promote adventitious root formation.

USING BIOTEST TO ESTIMATE TAXOL PRODUCTION FROM CELL SUSPENSION CULTURE OF TAXUS WALLICHIANA ZUCC. IN DIFFERENT CONDITIONS OF CULTURE

Calli and cell suspension cultures were initiated from Lam Dong Yew (Taxus wallichiana Zucc.) young stem explants, on B5 medium supplemented with 2,4-D and kinetin at appropriate concentrations. Taxol content was estimated by using a root primordium biotest with hypocotyl cuttings of Phaseolus aureus Roxb. (green bean). Results showed that taxol production was influenced by different conditions of culture, such as culture time, temperature, medium osmotic pressure or methyl jasmonate... The growth of calli were inhibited if they were lighted or maintained for a long time on medium culture but taxol synthesis was stimulated. Phenylalanine (as organic precusor), methyl jasmonate (as biotic elicitor) as well as a high osmotic pressure induced taxol accumulation.

Induction of Periploca sepium in vitro plantlet and studies on dynamic accumulation of periplocin

To analyze the content of periplocin in different part of the Periploca sepium in vitro plantlet and study its dynamic variation during the process of differentiation. The seeds were generated seedling under aseptic condition, and the cut hypocotyl was induced to form the callus and adventitious buds on the MS culture medium with the hormone of IBA 0.1 mg x L(-1) + BA 1 mg x L(-1). The seedling was cut down when the buds grew up to 3 cm and then the root was cultured in the 1/2 MS culture medium with the hormone of IBA 0.5 mg x L(-1) to form intact plantlet. Different parts of it were collected and the content of periplocin was measured during the process of differentiation. The contents of periplocin varied widely in different parts during the process of differentiation, with the highest in the roots and then callus, stem and leaf of intact plantlet, stem and leaf of plantlet without root from high to low. The periplocin of the secondary metabolite is more likely to be produced and accumulated in root and callus. Periplocin in stem and leaf is probably transported by conducting tissue.

Hydrogen peroxide generated by copper amine oxidase involved in adventitious root formation in mung bean hypocotyl cuttings

The role of hydrogen peroxide (H2O2) generated by copper-containing diamine oxidases (CuAO) in adventitious rooting in mung bean hypocotyl was studied. The physiological data show that aminoguanidine (AG), an irreversible inhibitor of CuAO, inhibited adventitious rooting significantly. Among major catalysed and metabolised products or substrates of CuAO, only H2O2 obviously induced adventitious rooting and notably reversed the inhibitory effect of AG on adventitious rooting. The spatio-temporal change of CuAO activity in the rooting region of hypocotyls was histochemically measured using a peroxidase-coupled assay with the artificial substrate 3,3-diaminobenzidine tetrahydrochloride (DAB) as chromogen. DAB staining was detected in the parenchyma tissue between the vascular bundles, where roots originated before the emergence of root primordium, and then in the forming root primordium. With the origination of adventitious roots, the density and area of DAB staining increased rapidly. Most cells in root primodium had an evidently DAB staining, and strong staining mostly distributed in root meristem. H2O2, which was detected with the H2O2-sensitive probe 2,7-dichlorofluorescein diacetate, had a similar change to CuAO activity in this process. AG largely suppressed DAB staining and H2O2 fluorescence, and prevented root primordium formation. Taken together, these results clearly prove that, CuAO is responsible for H2O2 production during adventitious rooting, and H2O2 generated by CuAO is required for adventitious rooting in mung bean hypocotyls.

Characterization and expression of a Pinus radiata putative ortholog to the Arabidopsis SHORT-ROOT gene

We characterized a Pinus radiata D. Don putative ortholog to the Arabidopsis thaliana (L.) Heynh. SHORT--ROOT gene (AtSHR) and analyzed its expression in different organs during vegetative development and in response to exogenous auxin during adventitious rooting. The predicted protein sequence contained domains characteristic of the GRAS protein family and showed a strong similarity to the SHORT--ROOT (SHR) proteins. Quantitative reverse transcriptase--polymerase chain reaction (qRT-PCR) and in situ hybridization showed that the gene is predominantly expressed in roots, root primordia and in the cambial region of hypocotyl cuttings. Increased mRNA levels were observed, independently of the presence or absence of exogenous auxin, in the cambial region and rooting competent cells of hypocotyl cuttings within the first 24 h of adventitious rooting, before the activation of cell divisions and the organization of the adventitious root meristem. The expression pattern in organs and during adventitious rooting was similar to that of a Pinus radiata SCARECROW-LIKE (PrSCL1) gene, except that PrSCL1 is induced in response to exogenous auxin. Results suggest that the Pinus radiata SHORT-ROOT (PrSHR) gene has a role in root meristem formation and maintenance and in the cambial region of hypocotyl cuttings.