The Impact of Light Wavelength and Darkness on Metabolite Profiling of Korean Ginseng: Evaluating Its Anti-Cancer Potential against MCF-7 and BV-2 Cell Lines.

Korean ginseng is a source of functional foods and medicines; however, its productivity is hindered by abiotic stress factors, such as light. This study investigated the impacts of darkness and different light wavelengths on the metabolomics and anti-cancer activity of ginseng extracts. Hydroponically-grown Korean ginseng was shifted to a light-emitting diodes (LEDs) chamber for blue-LED and darkness treatments, while white fluorescent (FL) light treatment was the control. MCF-7 breast cancer and lipopolysaccharide (LPS)-induced BV-2 microglial cells were used to determine chemo-preventive and neuroprotective potential. Overall, 53 significant primary metabolites were detected in the treated samples. The levels of ginsenosides Rb1, Rb2, Rc, Rd, and Re, as well as organic and amino acids, were significantly higher in the dark treatment, followed by blue-LED treatment and the FL control. The dark-treated ginseng extract significantly induced apoptotic signaling in MCF-7 cells and dose-dependently inhibited the NF-κB and MAP kinase pathways in LPS-induced BV-2 cells. Short-term dark treatment increased the content of Rd, Rc, Rb1, Rb2, and Re ginsenosides in ginseng extracts, which promoted apoptosis of MCF-7 cells and inhibition of the MAP kinase pathway in BV-2 microglial cells. These results indicate that the dark treatment might be effective in improving the pharmacological potential of ginseng.

Isolation and Characterization of the Genes Involved in the Berberine Synthesis Pathway in Asian Blue Cohosh, Caulophyllum robustum.

Caulophyllum robustum, commonly named Asian blue cohosh, is a perennial herb in the family Berberidaceae. It has traditionally been used for folk medicine in China. We isolated berberine from the leaves, stem, roots, and fruits of C. robustum, and this is the first report on berberine in this species. Transcriptome analysis was conducted for the characterization of berberine biosynthesis genes in C. robustum, in which, all the genes for berberine biosynthesis were identified. From 40,094 transcripts, using gene ontology (GO) analysis, 26,750 transcripts were assigned their functions in the categories of biological process, molecular function, and cellular component. In the analysis of genes expressed in different tissues, the numbers of genes in the categories of intrinsic component of membrane and transferase activity were up-regulated in leaves versus stem. The berberine synthesis genes in C. robustum were characterized by phylogenetic analysis with corresponding genes from other berberine-producing species. The co-existence of genes from different plant families in the deepest branch subclade implies that the differentiation of berberine synthesis genes occurred early in the evolution of berberine-producing plants. Furthermore, the copy number increment of the berberine synthesis genes was detected at the species level.

Effect of Light and Dark on the Phenolic Compound Accumulation in Tartary Buckwheat Hairy Roots Overexpressing ZmLC.

Fagopyrum tataricum 'Hokkai T10' is a buckwheat cultivar capable of producing large amounts of phenolic compounds, including flavonoids (anthocyanins), phenolic acids, and catechin, which have antioxidant, anticancer, and anti-inflammatory properties. In the present study, we revealed that the maize transcription factor Lc increased the accumulation of phenolic compounds, including sinapic acid, 4-hydroxybenzonate, t-cinnamic acid, and rutin, in Hokkai T10 hairy roots cultured under long-photoperiod (16 h light and 8 h dark) conditions. The transcription factor upregulated phenylpropanoid and flavonoid biosynthesis pathway genes, yielding total phenolic contents reaching 27.0 ± 3.30 mg g-1 dry weight, 163% greater than the total flavonoid content produced by a GUS-overexpressing line (control). In contrast, when cultured under continuous darkness, the phenolic accumulation was not significantly different between the ZmLC-overexpressing hairy roots and the control. These findings suggest that the transcription factor (ZmLC) activity may be light-responsive in the ZmLC-overexpressing hairy roots of F. tataricum, triggering activation of the phenylpropanoid and flavonoid biosynthesis pathways. Further studies are required on the optimization of light intensity in ZmLC-overexpressing hairy roots of F. tataricum to enhance the production of phenolic compounds.

De novo assembly and characterization of transcriptome in the medicinal plant Euphorbia jolkini.

BACKGROUND: Euphorbia jolkini, a medicinal herb that grows on the warm beaches in Japan and South Korea, is known to be used for traditional medicines to treat a variety of ailments, including bruises, stiffness, indigestion, toothache, and diabetes. OBJECTIVE: It is to analyze the whole transcriptome and identify the genes related to the phenylpropanoid biosynthesis in the medicinally important herb E jolkini. METHODS: Paired-end Illumina HiSeq™ 2500 sequencing technology was employed for cDNA library construction and Illumina sequencing. Public databases like TAIR (The Arabidopsis Information Resource), Swissprot and KEGG (Kyoto Encyclopedia of Genes and Genomes) were used for annotations of unigenes obtained. RESULTS: The transcriptome of E. jolkini generated 139,215 assembled transcripts with an average length of 868 bp and an N50 value of 1460 bp that were further clustered using CD-HIT into 93,801 unigenes with an average length of 847 bp (N50-1410 bp). Sixty-three percent of the coding sequences (CDS) were annotated from the longest open reading frame (ORF). A remarkable percentage of unigenes were annotated against various databases. The differentially expressed gene analysis revealed that the expression of genes related to the terpenoid backbone biosynthesis pathway was higher in the flowers, whereas that of genes related to the phenylpropanoid biosynthesis pathway was both up- and downregulated in flowers and leaves. A search of against the transcription factor domain found 1023 transcription factors (TFs) that were from 54 TF families. CONCLUSION: Assembled sequences of the E. jolkini transcriptome are made available for the first time in this study E. jolkini and lay a foundation for the investigation of secondary metabolite biosynthesis.

Genome-wide transcriptome profiling of the medicinal plant Zanthoxylum planispinum using a single-molecule direct RNA sequencing approach.

High-throughput RNA sequencing has revolutionized transcriptome-based studies of candidate genes, key pathways and gene regulation in non-model organisms. We analyzed full-length cDNA sequences in Zanthoxylum planispinum (Z. planispinum), a medicinal herb in major parts of East Asia. The full-length mRNA derived from tissues of leaf, early fruit and maturing fruit stage were sequenced using PacBio RSII platform to identify isoform transcriptome. We obtained 51,402 unigenes, with average 1781 bp per gene in 82.473 Mb gene lengths. Among 51,402, 3963 unigenes showed variety of isoform. By selection of one representative gene among each of the various isoforms, we finalized 46,306 unique gene set for this herb. We identified 76 cytochrome P450 (CYP450) and related isoforms that are of the wide diversity in the molecular function and biological process. These transcriptome data of Z. planispinum will provide a good resource to study metabolic engineering for the production of valuable medicinal drugs and phytochemicals.

RNA-Seq De Novo Assembly and Differential Transcriptome Analysis of Korean Medicinal Herb Cirsium japonicum var. spinossimum.

Cirsium japonicum belongs to the Asteraceae or Compositae family and is a medicinal plant in Asia that has a variety of effects, including tumour inhibition, improved immunity with flavones, and antidiabetic and hepatoprotective effects. Silymarin is synthesized by 4-coumaroyl-CoA via both the flavonoid and phenylpropanoid pathways to produce the immediate precursors taxifolin and coniferyl alcohol. Then, the oxidative radicalization of taxifolin and coniferyl alcohol produces silymarin. We identified the expression of genes related to the synthesis of silymarin in C. japonicum in three different tissues, namely, flowers, leaves and roots, through RNA sequencing. We obtained 51,133 unigenes from transcriptome sequencing by de novo assembly using Trinity v2.1.1, TransDecoder v2.0.1, and CD-HIT v4.6 software. The differentially expressed gene analysis revealed that the expression of genes related to the flavonoid pathway was higher in the flowers, whereas the phenylpropanoid pathway was more highly expressed in the roots. In this study, we established a global transcriptome dataset for C. japonicum. The data shall not only be useful to focus more deeply on the genes related to product medicinal metabolite including flavolignan but also to study the functional genomics for genetic engineering of C. japonicum.

Transcriptome and metabolome analysis in shoot and root of Valeriana fauriei.

BACKGROUND: Valeriana fauriei is commonly used in the treatment of cardiovascular diseases in many countries. Several constituents with various pharmacological properties are present in the roots of Valeriana species. Although many researches on V. fauriei have been done since a long time, further studies in the discipline make a limit due to inadequate genomic information. Hence, Illumina HiSeq 2500 system was conducted to obtain the transcriptome data from shoot and root of V. fauriei. RESULTS: A total of 97,595 unigenes were noticed from 346,771,454 raw reads after preprocessing and assembly. Of these, 47,760 unigens were annotated with Uniprot BLAST hits and mapped to COG, GO and KEGG pathway. Also, 70,013 and 88,827 transcripts were expressed in root and shoot of V. fauriei, respectively. Among the secondary metabolite biosynthesis, terpenoid backbone and phenylpropanoid biosynthesis were large groups, where transcripts was involved. To characterize the molecular basis of terpenoid, carotenoid, and phenylpropanoid biosynthesis, the levels of transcription were determined by qRT-PCR. Also, secondary metabolites content were measured using GC/MS and HPLC analysis for that gene expression correlated with its accumulation respectively between shoot and root of V. fauriei. CONCLUSIONS: We have identified the transcriptome using Illumina HiSeq system in shoot and root of V. fauriei. Also, we have demonstrated gene expressions associated with secondary metabolism such as terpenoid, carotenoid, and phenylpropanoid.

Effect of Auxins on Anthocyanin Accumulation in Hairy Root Cultures of Tartary Buckwheat Cultivar Hokkai T1O.

This study investigated the influence of auxins on the growth of hairy roots and accumulation of anthocyanins, including cyanidin 3-O-glucoside (C3gl) and cyanidin 3-0-rutinoside (C3r), in the hairy root culture of tartary buckwheat cultivar Hokkai TIO. C3gl and C3r contents were evaluated using high- performance liquid chromatograph (HPLC). Four auxins, 2,4-dichlorophenoxyacetic acid (2,4-D), indoleacetic acid (IAA), indolebutyric acid (IBA) and naphthaleneacetic acid (NAA), were added to the medium of the hairy root cultures at diverse concentrations. IAA, IBA and 2,4-D promoted the growth of hairy roots since the dry weight of the roots was slightly higher than or comparable with that of the control. However, NAA at all concentrations suppressed the growth of hairy roots. Generally, auxin treatments resulted in higher accumulation of C3gl and C3r than that of the control except for 2.85 µM IAA and 2.69 µM NAA. The amount of C3gl and C3r after treatment with 4.92 µM IBA was the highest among all treatments and was 3.24 times more than that of the control. Our results suggested that auxins at appropriate concentrations might facilitate hairy root growth of tartary buckwheat and enhance the production of C3gl and C3r.

Accumulation of Rutin and Betulinic Acid and Expression of Phenylpropanoid and Triterpenoid Biosynthetic Genes in Mulberry (Morus alba L.).

Mulberry (Morus alba L.) is used in traditional Chinese medicine and is the sole food source of the silkworm. Here, 21 cDNAs encoding phenylpropanoid biosynthetic genes and 21 cDNAs encoding triterpene biosynthetic genes were isolated from mulberry. The expression levels of genes involved in these biosynthetic pathways and the accumulation of rutin, betulin, and betulinic acid, important secondary metabolites, were investigated in different plant organs. Most phenylpropanoid and triterpene biosynthetic genes were highly expressed in leaves and/or fruit, and most genes were downregulated during fruit ripening. The accumulation of rutin was more than fivefold higher in leaves than in other organs, and higher levels of betulin and betulinic acid were found in roots and leaves than in fruit. By comparing the contents of these compounds with gene expression levels, we speculate that MaUGT78D1 and MaLUS play important regulatory roles in the rutin and betulin biosynthetic pathways.

Accumulation of phenylpropanoids and correlated gene expression in hairy roots of tartary buckwheat under light and dark conditions.

Differential expression patterns of flavonoid biosynthetic pathway genes in the hairy roots of tartary buckwheat cultivars "Hokkai T8" and "Hokkai T10" were studied over a time course of the light-dark cycle. The Agrobacterium rhizogenes-mediated transformation system was applied for inducing hairy roots. Further, a total of six phenolic compounds and two anthocyanins were analyzed in the hairy roots which were exposed to both light and dark conditions, and their amounts were estimated by HPLC. The gene expression levels peaked on day 5 of culture during the time course of both dark and light conditions. Notably, FtPAL, Ft4CL, FtC4H, FtCHI, FtF3H, FtF3'H-1, and FtFLS-1 were more highly expressed in Hokkai T10 than in Hokkai T8 under dark conditions, among which FtPAL and FtCHI were found to be significantly upregulated, except on day 20 of culture. Significantly higher levels of phenolic compound, rutin, along with two anthocyanins were detected in the hairy roots of Hokkai T10 under both conditions. Furthermore, among all the phenolic compounds detected, the amount of rutin in Hokkai T10 hairy roots was found to be ∼5-fold (59,01 mg/g dry weight) higher than that in the control (12.45 mg/g dry weight) at the respective time periods under light and dark conditions.

EST sequencing and gene expression profiling in Scutellaria baicalensis.

Scutellaria baicalensis is an important medicinal plant, but few genomic resources are available for this species, as well as for other non-model plants. One of the major new directions in genome research is to discover the full spectrum of genes transcribed from the whole genome. Here, we report extensive transcriptome data of the early growth stage of S. baicalensis. This transcriptome consensus sequence was constructed by de novo assembly of shotgun sequencing data, obtained using multiple next-generation DNA sequencing (NGS) platforms (Roche/454 GS_FLX+ and Illumina/Solexa HiSeq2000). We show that this new approach to obtain extensive mRNA is an efficient strategy for genome-wide transcriptome analysis. We obtained 1,226,938 and 161,417,646 reads using the GS_FLX and the Illumina/Solexa HiSeq2000, respectively. De novo assembly of the high-quality GS_FLX and Illumina reads (95 % and 75 %) resulted in more than 82 Mb of mRNA consensus sequence, which we assembled into 51,188 contigs, with at least 500 bp per contig. Of these contigs, 39,581 contained known genes, as determined by BLASTX searches against non-redundant NCBI database. Of these, 20,498 different genes were expressed during the early growth stage of S. baicalensis. We have made the expressed sequences available on a public database. Our results demonstrate the utility of combining NGS technologies as a basis for the development of genomic tools in non-model, medicinal plant species. Knowledge of all described genes and quantitation of the expressed genes, including the transcription factors involved, will be useful in studies of the biology of S. baicalensis gene regulation.

Metabolomic analysis and phenylpropanoid biosynthesis in hairy root culture of tartary buckwheat cultivars.

Buckwheat, Fagopyrum tataricum Gaertn., is an important medicinal plant, which contains several phenolic compounds, including one of the highest content of rutin, a phenolic compound with anti-inflammatory properties. An experiment was conducted to investigate the level of expression of various genes in the phenylpropanoid biosynthetic pathway to analyze in vitro production of anthocyanin and phenolic compounds from hairy root cultures derived from 2 cultivars of tartary buckwheat (Hokkai T8 and T10). A total of 47 metabolites were identified by gas chromatography-time-of-flight mass spectrometry (GC-TOFMS) and subjected to principal component analysis (PCA) in order to fully distinguish between Hokkai T8 and T10 hairy roots. The expression levels of phenylpropanoid biosynthetic pathway genes, through qRT-PCR, showed higher expression for almost all the genes in T10 than T8 hairy root except for FtF3'H-2 and FtFLS-2. Rutin, quercetin, gallic acid, caffeic acid, ferulic acid, 4-hydroxybenzoic acid, and 2 anthocyanin compounds were identified in Hokkai T8 and T10 hairy roots. The concentration of rutin and anthocyanin in Hokkai T10 hairy roots of tartary buckwheat was several-fold higher compared with that obtained from Hokkai T8 hairy root. This study provides useful information on the molecular and physiological dynamic processes that are correlated with phenylpropanoid biosynthetic gene expression and phenolic compound content in F. tataricum species.

Metabolic profiling of glucosinolates, anthocyanins, carotenoids, and other secondary metabolites in kohlrabi (Brassica oleracea var. gongylodes).

We profiled and quantified glucosinolates, anthocyanins, carotenoids, and other secondary metabolites in the skin and flesh of pale green and purple kohlrabis. Analysis of these distinct kohlrabis revealed the presence of 8 glucosinolates, 12 anthocyanins, 2 carotenoids, and 7 phenylpropanoids. Glucosinolate contents varied among the different parts and types of kohlrabi. Glucoerucin contents were 4-fold higher in the flesh of purple kohlrabi than those in the skin. Among the 12 anthocyanins, cyanidin 3-(feruloyl)(sinapoyl) diglucoside-5-glucoside levels were the highest. Carotenoid levels were much higher in the skins than the flesh of both types of kohlrabi. The levels of most phenylpropanoids were higher in purple kohlrabi than in pale green ones. trans-Cinnamic acid content was 12.7-fold higher in the flesh of purple kohlrabi than that in the pale green ones. Thus, the amounts of glucosinolates, anthocyanins, carotenoids, and phenylpropanoids varied widely, and the variations in these compounds between the two types of kohlrabi were significant.

Accumulation of tilianin and rosmarinic acid and expression of phenylpropanoid biosynthetic genes in Agastache rugosa.

Korean mint (Agastache rugosa), a perennial, medicinal plant of the Labiatae family, has many useful constituents, including monoterpenes and phenylpropanoids. Among these, tilianin and rosmarinic acid, 2 well-known natural products, have many pharmacologically useful properties. Chalcone synthase (CHS) and chalcone isomerase (CHI) catalyze the first and second committed steps in the phenylpropanoid pathway of plants, leading to the production of tilianin. In this study, cDNAs encoding CHS (ArCHS) and CHI (ArCHI) were isolated from A. rugosa using rapid amplification of cDNA ends (RACE)-PCR. Amino acid sequence alignments showed that ArCHS and ArCHI shared high sequence identity and active sites with their respective orthologous genes. Quantitative real-time PCR analysis was used to determine the expression levels of genes involved in tilianin and rosmarinic acid biosyntheses in the flowers, leaves, stems, and roots of A. rugosa. High-performance liquid chromatography (HPLC) revealed that the accumulation pattern of tilianin matched the expression patterns of ArCHS and ArCHI in different organs of A. rugosa. Moreover, acacetin, the precursor of tilianin, also demonstrated an accumulation pattern congruent with the expression of these 2 genes. The transcription levels of ArPAL, ArC4H, and Ar4CL were the highest in the leaves or flowers of the plant, which also contained a relatively high amount of rosmarinic acid. However, the roots showed a significant content of rosmarinic acid, although the transcription of ArPAL, ArC4H, and Ar4CL were low. The findings of our study support the medicinal usefulness of A. rugosa and indicate targets for increasing tilianin and rosmarinic acid production in this plant.

Accumulation of phenylpropanoids and correlated gene expression during the development of tartary buckwheat sprouts.

Buckwheat sprouts are considered an excellent dietary source of phenolic compounds. The time duration and amount of light for sprouting strongly affect the nutritional quality of sprouts. In this study, these two factors were investigated in two cultivars of tartary buckwheat sprouts: Hokkai T8 and T10. The transcriptional levels of flavonoid biosynthetic genes were investigated in light/dark- and dark-treated sprouts. Among the main flavonoid biosynthesis structural genes, FtPAL, Ft4CL, FtF3H, FtDFR, and FtANS exhibited higher transcriptional levels than others as compared to that of a housekeeping gene (histone H3) during sprouting; FtF3'H1, FtF3'H2, FtFLS2, and FtANS were substantially upregulated at 2, 4, and 6 days in light/dark-treated T10 sprouts than in dark-treated ones. However, FtDFR was downregulated in 8 and 10 day old light/dark-treated sprouts of both cultivars. High-performance liquid chromatography (HPLC) analysis revealed that increasing the culture time did not affect the accumulation of flavonoids or anthocyanins. However, light contributed the production of anthocyanins in Hokkai T10 sprouts. The anthocyanins included cyanidin 3-O-glucoside, cyanidin 3-O-rutinoside, and delphinidin-3-O-coumarylglucoside, which were identified by HPLC and electrospray ionization-tandem mass spectrometry. Instead of anthocyanins, Hokkai T8 sprouts produced large amounts of 4 flavonoid C-glycosylflavone compounds in both light/dark and dark conditions: orientin, isoorientin, vitexin, and isovitexin. These results indicate that these two types of tartary buckwheat sprouts have different mechanisms for flavonoid and anthocyanin biosynthesis that also vary in light/dark and dark conditions.

Overexpression of cinnamate 4-hydroxylase gene enhances biosynthesis of decursinol angelate in Angelica gigas hairy roots.

Angelica gigas is a medicinal plant that produces pyranocoumarins, including decursin (D) and decursinol angelate (DA), which have neuroprotective, anticancer, and antiandrogenic effects. In this study, the coumarin biosynthetic pathway was engineered to increase the production of DA. Specifically, a vector was constructed which contained the A. gigas phenylalanine ammonia-lyase (AgPAL) and cinnamate 4-hydroxylase (AgC4H) genes that were driven by the cauliflower mosaic virus (CaMV) 35S promoter. Transgenic hairy roots that overexpressed AgPAL or AgC4H genes were obtained by using an Agrobacterium rhizogenes-mediated transformation system. Among them, only AgC4H-transgenic hairy root lines produced more DA than control transgenic hairy root lines. The enhanced gene expression corresponded to elevated C4H activities. This study showed the importance of C4H in the production of DA in A. gigas hairy root culture.

Enhancement of rutin in Fagopyrum esculentum hairy root cultures by the Arabidopsis transcription factor AtMYB12.

Common buckwheat (Fagopyrum esculentum Moench) is rich in phenolic compounds and may be useful for the treatment of metabolic syndrome in humans. To improve the production of rutin in buckwheat, we overexpressed the flavonol-specific transcription factor, AtMYB12 using Agrobacterium rhizogenes into hairy root culture systems. This induced the expression of flavonoid biosynthetic genes encoding phenylalanine ammonia lyase, cinnamate 4-hydroxylase, 4-coumarate:CoA ligase, chalcone synthase, chalcone isomerase, flavone 3-hydroxylase, flavonoid 3'-hydroxylase, and flavonol synthase. This led to the accumulation of rutin in buckwheat hairy roots up to 0.9 mg/g dry wt. PAP1 expression, however, did not correlate with the production of rutin.

Carotenoid accumulation and characterization of cDNAs encoding phytoene synthase and phytoene desaturase in garlic (Allium sativum).

Phytoene synthase (PSY) and phytoene desaturase (PDS), which catalyze the first and second steps of the carotenoid biosynthetic pathway, respectively, are key enzymes for the accumulation of carotenoids in many plants. We isolated 2 partial cDNAs encoding PSY (AsPSY-1 and AsPSY-2) and a partial cDNA encoding PDS (AsPDS) from Allium sativum. They shared high sequence identity and conserved motifs with other orthologous genes. Quantitative real-time PCR analysis was used to determine the expression levels of AsPSY1, AsPSY2, and AsPDS in the bulbils, scapes, leaves, stems, bulbs, and roots of garlic. High-performance liquid chromatography demonstrated that carotenoids were not biosynthesized in the underground organs (roots and bulbs), but were very abundant in the photosynthetic organs (leaves) of A. sativum. A significantly higher amount of ß-carotene (73.44 µg·g(-1)) was detected in the leaves of A. sativum than in the other organs.

Differential expression of anthocyanin biosynthetic genes and anthocyanin accumulation in tartary buckwheat cultivars 'Hokkai t8' and 'Hokkai t10'.

Six genes involved in anthocyanin biosynthesis in tartary buckwheat have been cloned, namely, FtC4H, Ft4CL, FtCHI, FtF3H, FtF3'H, and FtANS, which encode cinnamate 4-hydroxylase (C4H), 4-coumarate:CoA ligase (4CL), chalcone isomerase (CHI), flavones 3-hydroxylase (F3H), flavonoid 3'-hydroxylase (F3'H), and anthocyanidin synthase (ANS), respectively. Then, these cDNAs were used, along with previously isolated clones for phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS), to compare gene expression in different organs, flowering stages, and maturing seeds of tartary buckwheat cultivars 'Hokkai T8' and 'Hokkai T10'. Quantitative real-time polymerase chain reaction analysis showed that these anthocyanin biosynthetic genes were most highly expressed in the stems and roots of Hokkai T10. The FtANS gene was more highly expressed than other genes during flowering and maturing seeds. In addition, the anthocyanin concentration was higher in 'Hokkai T10' than in 'Hokkai T8'; however, naringenin chalcone, a flavonoid, was absent from 'Hokkai T10' seedlings based on fluorescence microscopy.

An efficient protocol for genetic transformation of Platycodon grandiflorum with Agrobacterium rhizogenes.

The balloon flower (Platycodon grandiflorum) is a popular traditional medicinal plant used in Korea to treat conditions such as bronchitis, asthma, tuberculosis, diabetes, and inflammatory diseases. Recently, immunopharmacological research identified triterpenoid and saponin as important active compounds in P. grandiflorum. To study and extract these compounds and other metabolites from P. grandiflorum, a technique was developed for producing hairy root cultures, which are a reliable source of plant compounds. To achieve this, the activity of Agrobacterium rhizogenes was exploited, which can transfer DNA segments into plant genomes after infecting them. In this study, the A. rhizogenes strain R1000 was determined that had the highest infection frequency (87.5%) and induced the most hairy roots per plant, and the concentration of antibiotics (75 mg/l kanamycin) was elucidated for selection after transformation. Wild-type and transgenic hairy roots contained various phenolic compounds, although both of them had similar concentrations of phenolic compounds. In the future, the protocols described here should be useful for studying and extracting valuable metabolites such as phenolic compounds from P. grandiflorum hairy root cultures.