<i>In Vitro</i> and <i>in Silico</i> Insights on the Biological Activities,Phenolic Compounds Composition of <i>Hypericum perforatum</i> L. Hairy Root Cultures

Three Hypericum perforatum hairy root lines (HR B, HR F and HR H) along with non-transformed roots were analyzed for phenolic compounds composition and in vitro enzyme inhibitory properties. In silico molecular modeling was performed to predict the interactions of the most representative phenolic compounds in HR clones with enzymes related to depression, neurodegeneration and diabetes. Chromatographic analyses revealed that HR clones represent an efficient source of quinic acid and hydroxybenzoic acids, epicatechin and procyanidin derivatives, quercetin and kaempferol glycosides, as well numerous xanthones. In vitro antidepressant activity of HR extracts through monoamine oxidase A (MAO-A) inhibition was attributed to the production of oxygenated and prenylated xanthones. The neuroprotective potential of HR extracts was related to the accumulation of quercetin 6-C-glucoside, epicatechin, procyanidins and γ-mangostin isomers as potential inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Vanillic acid and prenylated xanthones in HR clones as promising inhibitors of tyrosinase additionally contributed to the neuroprotective activity. Five preeminent xanthones in HR (γ-mangostin, mangiferin, garcinone C, garcinone E and 1,3,7-trihydroxy-6-metoxy-8-prenyl xanthone) along with the flavonol quercetin 6-C-glucoside effectively inhibited α-amylase and α-glucosidase indicating the antidiabetic properties of HR extracts. Transgenic roots of H. perforatum can be exploited for the preparation of novel phytoproducts with multi-biological activities.     

Cloning and Characterization of <i>EuGID1</i> in <i>Eucommia ulmoides</i> Oliver

Gibberellic acid controlled the key developmental processes of the life cycle of landing plants, and regulated the growth and development of plants. In this study, a novel gibberellin receptor gene EuGID1 was obtained from Eucommia ulmoides Oliver. The cDNA of EuGID1 was 1556 bp, and the open reading frame was 1029 bp, which encoded 343 amino acids. EuGID1 had the homology sequence with the hormone-sensitive lipase family. Amino acid sequence alignment confirmed EuGID1 protein had the highest homology with the GID1 protein of Manihot esculenta. EuGID1 was located in the nucleus and cell membrane and had expression in four plant organs. Overexpression of EuGID1 in transgenic Arabidopsis plants promoted plant elongation and increased siliques yield.     

Differential Responses of <i>NHX1</i> and <i>SOS1</i> Gene Expressions to Salinity in two <i>Miscanthus sinensis</i> Anderss. Accessions with Different Salt Tolerance

The lignocellulosic crop Miscanthus spp. has been identified as a good candidate for biomass production. The responses of Miscanthus sinensis Anderss. to salinity were studied to satisfy the needs for high yields in marginal areas and to avoid competition with food production. The results indicated that the relative advantages of the tolerant accession over the sensitive one under saline conditions were associated with restricted Na⁺ accumulation in shoots. Seedlings of two accessions (salt-tolerant ‘JM0119’ and salt-sensitive ‘JM0099’) were subjected to 0 (control), 100, 200, and 300 mM NaCl stress to better understand the salt-induced biochemical responses of genes involved in Na⁺ accumulation in M. sinensis. The adaptation responses of genes encoding for Na⁺ /H⁺ antiporters, NHX1 and SOS1 to NaCl stress were examined in JM0119 and JM0099.The cDNA sequences of genes examined were highly conserved among the relatives of M. sinensis based on the sequencing on approximate 600 bp-long cDNA fragments obtained from degenerate PCR. These salt-induced variations of gene expression investigated by quantitative real-time PCR provided evidences for insights of the molecular mechanisms of salt tolerance in M. sinensis. The expression of NHX1 was up-regulated by salt stress in JM0119 shoot and root tissues. However, it was hardly affected in JM0099 shoot tissue except for a significant increase at the 100 mM salt treatment, and it was salt-suppressed in the JM0099 root tissue. In the root tissue, the expression of SOS1 was induced by the high salt treatment in JM0119 but repressed by all salt treatments in JM0099. Thus, the remarkably higher expression of NHX1 and SOS1 were associated with the resistance to Na⁺ toxicity by regulation of the Na⁺ influx, efflux, and sequestration under different salt conditions.     

Antibacterial Activity and Mechanisms of Ethanol Extracts from <i>Scutellaria baicalensis</i> Georgi and <i>Magnolia officinalis</i> against <i>Phytophthora nicotianae</i>

Phytophthora nicotianae causes substantial economic losses in most countries where tobacco is produced. At present, the control of P. nicotianae mainly depends on chemical methods, with considerable environmental and health issues. We investigated the effects of ethanol extracts from Scutellaria baicalensis Georgi (SBG) and Magnolia officinalis (MO). On mycelial growth, sporangium formation, and zoospore release of P. nicotianae. Both extracts inhibited the growth of P. nicotianae, with mycelial growth inhibition rates of 88.92% and 93.92%, respectively, at 40 mg/mL, and EC50 values of 5.39 and 5.74 mg/mL, respectively. The underlying mechanisms were the inhibition of sporangium formation, the reduction of zoospore number, and the destruction of the mycelium structure. At an SBG extract concentration of 16.17 mg/mL, the inhibition rates for sporangia and zoospores were 98.66% and 99.39%, respectively. At an MO extract concentration of 2.87 mg/mL, the production of sporangia and zoospores was completely inhibited. The hyphae treated with the two plant extracts showed different degrees of deformation and damage. Hyphae treated with SBG extract showed adhesion and local swelling, whereas treatment with MO extract resulted in broken hyphae. Mixture of the extracts resulted in a good synergistic effect.     

Estimation of Growth and Photosynthetic Performance of Two C₄ Species (<i>Pennisetum spicatum</i> (L.) Körn. and <i>Zea mays</i> L.) under a Low Temperature Treatment

Pearl millet (Pennisetum spicatum (L.) Körn.) and maize (Zea mays L.) are C₄ grass species grown for feeding humans and animals in Almadinah Almunawwarah, which is in the western part of Saudi Arabia. During the winter, the mean temperature, which drops to 14°C, represents a major problem for the growth of these species in this region. Therefore, the objectives of this research were to investigate the growth response and the photosynthetic performance of P. spicatum and Z. mays under a low temperature stress. The treatments involved daytime and nighttime temperatures of 14/12°C (low temperature) and 24/22°C (optimum temperature). The results indicated that low temperature significantly reduced all growth and physiological parameters, including seed germination, leaf expansion, leaf area, shoot length and root length of the two species compared to those of the control. Additionally, the low temperature significantly decreased the light-saturated assimilation rate (A_sat), quantum yield (ϕ), saturated rate of carbon dioxide uptake (A_max) and efficiency of carboxylation on both species compared to those of the control. Moreover, the values of Fv/Fm and the chlorophyll contents of both species were significantly reduced by low temperature compared to those of the control. It can be concluded that both species had little tolerance to low temperatures.     

Characterization and Candidate Gene Analysis of the Yellow-Green Leaf Mutant <i>ygl16</i> in Rice (<i>Oryza sativa</i> L.)

Leaf color mutants are ideal materials for studying many plant physiological and metabolic processes such as photosynthesis, photomorphogenesis, hormone physiology and disease resistance. In this study, the genetically stable yellow-green leaf mutant ygl16 was identified from mutated “Xinong 1B”. Compared with the wild type, the pigment concentration and photosynthetic capacity of the ygl16 decreased significantly. The ultrastructural observation showed that the distribution of thylakoid lamellae was irregular in ygl16 chloroplasts, and the grana and matrix lamellae were blurred and loose in varied degrees, and the chloroplast structure was disordered, while the osmiophilic corpuscles increased. The results of the genetic analysis and mapping showed that the phenotype of ygl16 was controlled by a pair of recessive nuclear gene. The gene located in the 56Kb interval between RM25654 and R3 on the long arm of chromosome 10. The sequencing results showed that the 121st base of the first intron of the candidate gene OsPORB/FGL changed from A to T in the interval. qRT-PCR results showed that the expression of chlorophyll synthase-related genes in the mutant decreased.     

Chemical Constituents from Turnip and Their Effects on <i>α</i>-Glucosidase

Brassica rapa var. rapa (turnip) is an important crop in Qinghai-Tibet Plateau (QTP) with anti-hypoxic effect. Turnip is rich in glucosinolates, isothiocyanates and phenolic compounds with diverse biological activities, involving anti-oxidant, anti-tumor, anti-diabetic, anti-inflammatory, anti-microbial, hypolipidemic, cardioprotective, hepatoprotective, nephroprotective and analgesic properties. In this study, the ethyl acetate (EtOAc) and butanol parts of Brassica rapa were first revealed with inhibitory effects on α-glucosidase, whereas the water part was inactive. Subsequent bioassay-guided isolation on the EtOAc and butanol parts yielded 12 compounds, involving three indole derivatives, indole-3- acetonitrile (1) 4-methoxyindole-3-acetonitrile (2) and indole-3-aldehyde (3) two flavonoids, liquiritin (4) and licochalcone A (5) two phenylpropanoids, sinapic acid (6) and caffeic acid (7) two phenylethanol glycosides, 2-phenylethyl β- glucopyranoside (8) and salidroside (9) and three other compounds, syringic acid (10) adenosine (11) and (3β, 20E)-ergosta-5, 20 (22)-dien-3-ol (12) Licochalcone A (5) and caffeic acid (7) showed α-glucosidase inhibitory activity with IC₅₀ values of 62.4 ± 8.0 μM and 162.6 ± 3.2 μM, comparable to the positive control, acarbose (IC₅₀ = 142 ± 0.02 μM). Docking study suggested that licochalcone A (5) could well align in the active site of α-glucosidase (docking score = -52.88) by forming hydrogen bonds (Gln1372, Asp1420, Gln1372, Arg1510), hydrophobic effects (Tyr1251, Tyr1251, Trp1355, Phe1560, Ile1587, Trp1355, Phe1559, Phe1559) and π-π stacking interaction (Trp1355). This study provides valuable information for turnip as a new resource in searching anti-diabetic candidates.     

Genome-Wide Identification and Expression Profiling Suggest that Invertase Genes Function in Silique Development and the Response to <i>Sclerotinia sclerotiorum</i> in <i>Brassica napus</i>

Invertase (INV), a key enzyme in sucrose metabolism, irreversibly catalyzes the hydrolysis of sucrose to glucose and fructose, thus playing important roles in plant growth, development, and biotic and abiotic stress responses. In this study, we identified 27 members of the BnaINV family in Brassica napus. We constructed a phylogenetic tree of the family and predicted the gene structures, conserved motifs, cis-acting elements in promoters, physicochemical properties of encoded proteins, and chromosomal distribution of the BnaINVs. We also analyzed the expression of the BnaINVs in different tissues and developmental stages in the B. napus cultivar Zhongshuang 11 using qRT-PCR. In addition, we analyzed RNA-sequencing data to explore the expression patterns of the BnaINVs in four cultivars with different harvest indices and in plants inoculated with the pathogenic fungus Sclerotinia sclerotiorum. We used WGCNA (weighted coexpression network analysis) to uncover BnaINVregulatory networks. Finally, we explored the expression patterns of several BnaINV genes in cultivars with long (Zhongshuang 4) and short (Ningyou 12) siliques. Our results suggest that BnaINVs play important roles in the growth and development of rapeseed siliques and the defense response against pathogens. Our findings could facilitate the breeding of high-yielding B. napus cultivars with strong disease resistance.     

Effects of Different Geographical Aspects and Ontogenetic Variability on Total Hypericin Content of <i>Hypericum triquetrifolium</i> Turra. and <i>Hypericum scabrum</i> L.

The present study was conducted to determine the total hypericin contents of Hypericum triquetrifolium Turra. and Hypericum scabrum L. species which are naturally distributed in the flora of Siirt province, Turkey. Hypericin contents of Hypericum species grown in different geographical aspects (North, South, East, and West), and it was measured at different harvest times (full blooming and post blooming period). In the current study, it has been determined that total hypericin content varies considerably according to aspects, plant developmental stages (ontogenetic variance), and species. According to species x aspect interaction, the highest total hypericin content was recorded from the west aspect (3.13 mg/g) in Hypericum triquetrifolium, while, the lowest hypericin content was also obtained from the west aspect (1.22 mg/g) in Hypericum scabrum. When the highest total hypericin content was analyzed according to aspect x species x harvest time interaction, the highest total hypericin content was produced from Hypericum triquetrifolium at the harvest of west aspect with 5.28 mg/g, while the minimum amount of hypericin was obtained from the same aspect in Hypericum scabrum with 0.50 mg/g. In species x harvest time interaction, the highest total hypericin content was obtained from the full bloom (3.10 mg/g) harvest in Hypericum triquetrifolium, while the lowest hypericin was obtained from the full bloom (1.26 mg/g) harvest in Hypericum scabrum. The data suggest that the average total hypericin content was 2.26 mg/g in Hypericum triquetrifolium and 1.28 mg/g in Hypericum scabrum.     

Effects of <i>Piriformospora indica</i> on the Respiration of <i>Taxus chinensis</i> var. <i>mairei</i> under Water Stress

Seedlings of Taxus chinensis var. mairei were used as experimental materials to study the adaptation of Piriformospora indica to this plant under water stress. The materials were divided into two groups, namely, with or without inoculation with P. indica. Each group was subjected to four different levels of water stress. Vitality and physiological and biochemical indexes of the roots of T. chinensis var. mairei were regularly measured. Under water stress, T. chinensis var. mairei had significantly decreased root vitality; root vitality was higher in inoculated roots than in uninoculated roots. Under intense water stress, the inoculated roots had a higher soluble sugar content than the uninoculated roots. Under water stress, T. chinensis var. mairei experienced decreased activity of aerobic respiratory metabolic enzymes. The activity of anaerobic respiratory metabolic enzymes and alcohol dehydrogenase initially increased and then decreased, whereas that of lactate dehydrogenase increased. The inoculated roots had a higher activity of respiratory metabolic enzymes than the uninoculated roots. As water stress was further intensified, the roots had significantly decreased activity of aerobic respiratory metabolic enzymes and significantly increased activity of anaerobic respiratory metabolic enzymes. The activity of respiratory metabolic enzymes decreased faster in the uninoculated roots than in the inoculated roots. This study demonstrated that Piriformospora indica plays a positive role in enhancing the antihypoxic ability of T. chinensis var. mairei, thereby alleviating plant damage due to water stress.     

First Report of <i>Fusarium striatum</i> Causing Root Rot Disease of <i>Panax notoginseng</i> in Yunnan,China

Panax notoginseng is a traditional Chinese medicinal plant. Root rot of P. notoginseng is one of the most serious diseases affecting P. notoginseng growth and causes wilted leaves, fewer lateral roots and rotten roots. Root rot is a soil-borne disease, and mainly occurs from June to August in Yunnan Province when the temperatures are high and the air is humid. In this study, the endophytic fungal genus Fusarium isolate E-2018.1.22-#3.2 was obtained from a P. notoginseng embryo. Fusarium isolate E-2018.1.22-#3.2 was identified as Fusarium striatum based on morphological characteristics and molecular analysis. The fungus was found to have conidiophores and macroconidia, and its ITS, LSU and TEF-1α genes shared 100%, 99.2% and 99% identities with the homologous genes of Fusarium striatum, respectively. Isolate F. striatum E-2018.1.22-#3.2 can cause root rot symptoms, including black, soft roots, fewer lateral roots and leaf wilt, in 93% of the experimental P. notoginseng plants, and could be re-isolated, fulfilling Koch’s postulates. When the P. notoginseng plants were treated with the fungicide pyraclostrobin, isolate F. striatum E-2018.1.22-#3.2 was unable to cause root rot. We have therefore demonstrated that F. striatum E-2018.1.22-#3.2 is able to cause root rot disease in P. notoginseng. This is the first report of root rot disease caused by F. striatum on P. notoginseng in China.     

Participation of Auxin Transport in the Early Response of the <i>Arabidopsis</i> Root System to Inoculation with <i>Azospirillum brasilense</i>

The potential of Plant Growth Promoting Rhizobacteria (PGPR) has been demonstrated in the case of plant inoculation with bacteria of the genus Azospirillum which improves yield. A. brasilense produces a wide variety of molecules, including the natural auxin indole-3-acetic acid (IAA), as well as other phytoregulators. However, several studies have suggested that auxin induces changes in plant development during their interaction with the bacteria. The effects of A. brasilense Sp245 on the development of Arabidopsis thaliana root were investigated to help explain the molecular basis of the interaction. The results obtained showed a decrease in primary root length from the first day and remained so throughout the exposure, accompanied by a stimulation of initiation and maturation of lateral root primordia and an increase of lateral roots. An enhanced auxin response was evident in the vascular tissue and lateral root meristems of inoculated plants. However, after five days of bacterization, the response disappeared in the primary root meristems. The role of polar auxin transport (PAT) in auxins relocation involved the PGP1, AXR4-1, and BEN2 proteins, which apparently mediated A. brasilense-induced root branching of Arabidopsis seedlings.     

<i>DWARF</i> and <i>SMALL SEED1</i>, a Novel Allele of <i>OsDWARF</i>, Controls Rice Plant Architecture,Seed Size,and Chlorophyll Biosynthesis

Plant architecture is a vital agronomic trait to control yield in rice (Oryza sativa L.). A dwarf and small seed 1 (dss1) mutant were obtained from the ethyl methanesulfonate (EMS) mutagenized progeny of a Guizhou glutinous landrace cultivar, Lipingzabianhe. The dss1 mutant displayed phenotypes similar to those of brassinosteroid (BR) deficient mutants, such as dwarfing, dark green and rugose erect leaves, small seeds, and loner neck internode panicles with primary branching. In our previous study, the underlying DSS1 gene was isolated, a novel allele of OsDWARF (OsBR6ox) that encodes a cytochrome P450 protein involved in the BR biosynthetic pathway by MutMap technology. In this work, we confirmed that a Thr335Ile amino acid substitution residing in DSS1/OsDWARF was responsible for the dwarf, panicle architecture, and small seed phenotypes in the dss1 mutants by genetic transformation experiments. The overexpression of OsDWARF in the dss1 mutant background could not only recover dss1 to the normal plant height and panicle architecture but also rescued normal leaf angles, seed size, and leaf color. Thus, the specific mutation in DSS1/OsDWARF influenced plant architecture, seed size, and chlorophyll biosynthesis.     

<i>In vitro</i> Germination and Micropropagation of <i>Aconitum vilmorinianum</i>: An Important Medicinal Plant in China

Aconitum vilmorinianum, a well-known traditional Chinese herb, is recently being threatened by overexploitation and environment disturbance. This study was conducted to provide propagation methods through in vitro germination and explant cultivation. Germination was stimulated up to 66.00% on Murashige and Skoog (MS) medium containing 2.0 mg L⁻¹ 6-benzylaminopurine (BAP), 0.1 mg L⁻¹ 1-napthaleneacetic acid (NAA), and 30 g L⁻¹ sucrose. Three bacteria (Pantoea agglomerans, Erwinia persicina, and Pseudomonas tolaasii) would be responsible for consistent contamination during germination. The latter two were effectively eradicated after disinfected. The influence of explant types and hormone combinations on direct and indirect organogenesis was evaluated in the present work. The frequency of shoot induction from axillary bud explants was 100% on the MS fortified with 2.0 mg L⁻¹ BAP and 0.3 mg L⁻¹ NAA. Shoots multiplication was optimized on MS medium supplemented with 0.1 mg L⁻¹ thidiazuron (TDZ) and 0.1 mg L⁻¹ NAA. High callus induction percentage (96.67%) was obtained from stem segments on MS medium with 2.0 mg L⁻¹ 2,4-D, then successfully regenerated into shoots on MS medium in the presence of 0.1 mg L⁻¹ TDZ and 0.2 mg L⁻¹ NAA. The present work could be useful for the utilization and conservation of this valuable species.     

Evaluation of Pre-Emergence and Post-Emergence Herbicides for Weed Management in <i>Miscanthus sacchariflorus</i> and <i>Miscanthus sinensis</i>

Miscanthus, is a promising bioenergy crop, considered superior to other bioenergy crops because of its higher water and nutrient use efficiency, cold tolerance, and higher production of biomass. Broadleaf weeds and grass weeds, cause major problems in the Miscanthus field. A field experiment was conducted in 2018 and 2019, to assess the effects of pre-emergence (alachlor and napropamide) and post-emergence herbicides (nicosulfuron, dicamba, bentazon, and glufosinate ammonium) on broadleaf and grass weeds in M. sinensis and M. sacchariflorus fields. The weed control efficiency and phytotoxicity of pre- and post-emergence herbicides were evaluated at 30 days after treatment (DAT) and compared to those of the control plots. The results showed wide variations in the susceptibility of the weed species to the treated herbicides. Treatment with nicosulfuron 40 g.a.i.ha⁻¹ provided the most effective overall weed control (with 10% visual injury), without affecting the height and biomass of neither Miscanthus species in the field. Post-emergence herbicides such as glufosinate ammonium 400 g.a.i.ha⁻¹ and dicamba 482 g.a.i.ha⁻¹ were effective and inhibited the growth and density of the majority of weeds to a 100%; however, they showed significant phytotoxicity (toxicity scale of 1–10) to both species of Miscanthus. The application of glufosinate ammonium caused severe injuries to the foliar region (90% visual injury) of both Miscanthus sps. Comparatively, M. sinensis showed a slightly higher tolerance to the herbicides nicosulfuron, bentazon and napropamide with 10% visual injury at the recommended dose than M. sacchariflorus. The present study clearly showed that infestation of broadleaf and grass weeds in Miscanthus fields can cause significant damage to the growth and biomass of Miscanthus and applying pre-emergence and post-emergence herbicides effectively controls the high infestation of these weeds.     

Comparative Analysis of the Photosynthetic Characteristics and Active Compounds of <i>Semiliquidambar cathayensis</i> Chang Heteromorphic Leaves

In the present study, the variation patterns of leaf shape in different populations of individual Semiliquidambar cathayensis plants were analyzed to investigate the relationship among leaf shape variation, photosynthetic properties, and active compounds to understand the genetic characteristics of S. cathayensis and screen elite germplasms. The leaf shape of 18 offspring from three naturalS. cathayensis populations was analyzed to investigate the level of diversity and variation patterns of leaf shape. Furthermore, photosynthetic pigment content, physiological parameters of photosynthesis, and the active compounds in leaves of different shapes were determined. Statistical analysis showed that the leaf shape variation in  S. cathayensis indicated a high level of genetic diversity among and within the populations. Cluster analysis showed that the three natural populations formed two clusters, one whose offspring was dominated by entire leaves and another characterized by palmately trifoliate leaves. The differences in photosynthetic characteristics and active compounds of leaves of three different shapes were comprehensively evaluated using principal component analysis. Two principal components with a cumulative contribution rate of 92.768% were extracted, of which the highest comprehensive score was for asymmetrically lobed leaves. The leaf shape in different S. cathayensis germplasms exhibited distinct patterns, and there were some correlations between the photosynthetic properties and active compounds in leaves of different shapes. Thus, the leaf shape can be used to predict active compound content, and in turn, select varieties based on that purpose; it also provides a simple and effective method to classify S. cathayensis germplasms.     

Sexual Morph of <i>Furcasterigmium furcatum</i> (Plectosphaerellaceae) from <i>Magnolia liliifera</i> Collected in Northern Thailand

We isolated an interesting fungus from dead leaves of Magnolia liliifera collected from Chiang Mai, Thailand. The novel strain is related to Plectosphaerellaceae based on the morphology of its asexual morph and the analysis of sequence data. Phylogenetic analyses using a combined gene analysis of LSU and ITS sequence data showed that this strain is clustered in the same clade with Furcasterigmium furcatum with high statistical support. The new strains produced the asexual morph in culture which is morphologically similar to F. furcatum. Thus, we identified this strain as the sexual morph of F. furcatum. This is the first record of sexual morph for the monotypic genus Furcasterigmium and the first record of this genus on Magnolia.     

Efficient Evergreen Plant Regeneration of <i>Cinnamomum japonicum</i> Sieb. through <i>in vitro</i> Organogenesis

Cinnamomum japonicum Sieb. is an excellent roadside tree and medicinal tree species with considerable ornamental and economic value. In this study, we successfully developed a large-scale micropropagation protocol for C. japonicum for the first time. Sterilized shoots were excised and used as explants for shoot induction on several basal media, supplemented with different concentrations of plant growth regulators (PGRs), such as Thidiazuron (TDZ), N⁶ -Benzyladenine (6-benzylaminopurine) (BA), α-naphthaleneacetic acid (NAA) and Gibberellic acid (GA₃). After comparison, the most efficient medium for shoot regeneration was 1/2 Murashige and Skoog (MS) medium containing 0.5 mg L^–1 BA, 0.05 mg L^–1 NAA and 0.2 mg L^–1 GA₃, which resulted in an average number of induced shoots per explant and shoot length of 5.2 and 1.62 cm at 28 d, respectively. Then, elongated adventitious shoots were transferred to induce roots. 86.7% of shoots was able to root on 1/2 MS medium supplemented with 0.5 mg L^–1 NAA and 0.1 mg L^–1 BA. The earliest rooting time observed was after 21 d and the average root length was up to 3.3 cm after 28 d. Our study shows that C. japonicum can be successfully regenerated through de novo organogenesis, which lays a foundation for future transformation research on this tree.