Transgenic Hairy Root Cultures Research Articles

Metabolic engineering of hairy root cultures in Beta vulgaris for enhanced production of vanillin, 4-hydroxybenzoic acid, and vanillyl alcohol.

The flavor of vanilla is a complex blend of compounds, with vanillin as the most prominent, along with vanillyl alcohol and 4-hydroxybenzoic acid. Natural vanillin extracted from vanilla beans is expensive, so researchers use heterologous synthesis to produce nature-identical vanillin in plant hosts. Consequently, alternative traditional farming and gathering methods are required to bridge the significant disparity between supply and demand. The current research successfully developed a method to induce hairy root formation from leaves. It integrated the Vanillin synthase (VpVAN) gene into transgenic hairy root lines of Beta vulgaris, synthesizing vanillin-related compounds. The presence of the VpVAN gene in transgenic roots was confirmed using PCR analysis. Additionally, RT-qPCR analysis demonstrated the expression of the VpVAN gene in the transgenic root lines. The transgenic hairy root clones H1, H2, and H5 showed enhanced vanillin production, vanillyl alcohol, and 4-hydroxybenzoic acid. Elicitation with methyl jasmonate (MJ) and salicylic acid (SA) further improved the production of these compounds in B. vulgaris hairy roots. The maximum hairy root biomass was observed after 60 days, with the maximum synthesis of vanillin and 4-hydroxybenzoic acid obtained from hairy root clones H5 and HR2, respectively. Vanillyl alcohol HR2 was obtained on the 45th day of cultivation. Elicitation with wound-associated hormone methyl jasmonate and salicylic acid enhanced the yield of vanillin, vanillyl alcohol, and 4-hydroxybenzoic acid, with a 215-fold increase in vanillin, a 13-fold increase in vanillyl alcohol, and a 21 fold increase in 4-hydroxybenzoic acid. The study results indicate that establishing transgenic hairy root cultures with the VpVAN gene is a promising alternative method for enhancing the production of vanilla flavor compounds such as vanillin, vanillyl alcohol, and 4-hydroxybenzoic acid. A cost-effective protocol has been developed to mass-produce phenolic compounds using a hairy root culture of B. vulgaris. This approach addresses the increasing demand for these substances while reducing the cost of natural vanillin production, making it suitable for industrial-scale applications.

Effect of silver nanoparticles on tropane alkaloid production of transgenic hairy root cultures of Hyoscyamus muticus L. and their antimicrobial activity

The utilization of nanotechnology and biotechnology for enhancing the synthesis of plant bioactive chemicals is becoming increasingly common. The hairy root culture technique can be used to increase secondary metabolites such as tropane alkaloids. Agrobacterium was used to induce hairy roots from various explants of Hyoscyamus muticus. The effect of nano-silver particles (AgNPs) at concentrations of 0, 25, 50, 100, and 200 mg/L on tropane alkaloids synthesis, particularly hyoscyamine and scopolamine, was studied in transgenic hairy root cultures. Different types of explants obtained from 10-day-old seedlings of H. muticus were inoculated with two strains of Agrobacterium rhizogenes (15,834 and A4). The antimicrobial activity of an ethanolic extract of AgNPs-induced hairy root cultures of H. muticus was tested. The frequency of hairy roots was higher in hypocotyl, root, leaf, and stem explants treated with A. rhizogenes strain A4 compared to those treated with strain 15,834. In transgenic hairy root cultures, AgNPs application at a concentration of 100 mg/L resulted in the highest total tropane alkaloid production, which exhibited broad-spectrum antimicrobial activity. The study demonstrated the potential of nano-silver as an elicitor for promoting the production of target alkaloids in Hyoscyamus muticus hairy root cultures, which exhibit high biological activity.

Impact of Light and Dark Treatment on Phenylpropanoid Pathway Genes, Primary and Secondary Metabolites in Agastache rugosa Transgenic Hairy Root Cultures by Overexpressing Arabidopsis Transcription Factor AtMYB12.

Agastache rugosa, otherwise called Korean mint, has a wide range of medicinal benefits. In addition, it is a rich source of several medicinally valuable compounds such as acacetin, tilianin, and some phenolic compounds. The present study aimed to investigate how the Tartary buckwheat transcription factor AtMYB12 increased the primary and secondary metabolites in Korean mint hairy roots cultured under light and dark conditions. A total of 50 metabolites were detected by using high-performance liquid chromatography (HPLC) and gas chromatography-time-of-flight mass spectrometry (GC-TOFMS). The result showed that the AtMYB12 transcription factor upregulated the phenylpropanoid biosynthesis pathway genes, which leads to the highest accumulation of primary and secondary metabolites in the AtMYB12-overexpressing hairy root lines (transgenic) than that of the GUS-overexpressing hairy root line (control) when grown under the light and dark conditions. However, when the transgenic hairy root lines were grown under dark conditions, the phenolic and flavone content was not significantly different from that of the control hairy root lines. Similarly, the heat map and hierarchical clustering analysis (HCA) result showed that most of the metabolites were significantly abundant in the transgenic hairy root cultures grown under light conditions. Principal component analysis (PCA) and partial least-squares discriminant analysis (PLS-DA) showed that the identified metabolites were separated far based on the primary and secondary metabolite contents present in the control and transgenic hairy root lines grown under light and dark conditions. Metabolic pathway analysis of the detected metabolites showed 54 pathways were identified, among these 30 were found to be affected. From these results, the AtMYB12 transcription factor activity might be light-responsive in the transgenic hairy root cultures, triggering the activation of the primary and secondary metabolic pathways in Korean mint.

Generation of Stable Catharanthus roseus Hairy Root Lines with Agrobacterium rhizogenes.

Agrobacterium rhizogenes is the bacterial agent that causes hairy root disease in dicots and is purposefully engineered for the development of transgenic hairy root cultures. Due to their genetic and metabolic stability, hairy root cultures offer advantages as a tissue culture system for investigating the function of transgenes and as a production platform for specialized metabolites or proteins. The process for generating hairy root cultures involves first infecting the explant with A. rhizogenes, excising and eliminating A. rhizogenes from the emerging hairy roots, selecting for transgenic hairy roots on plates containing the selective agent, confirming genomic integration of transgenes by PCR, and finally adapting the hairy roots in liquid media. Here we provide a detailed protocol for developing and maintaining transgenic hairy root cultures of our medicinal plant of interest, Catharanthus roseus.

Genetically engineered hairy root cultures of Hyoscyamus senecionis and H. muticus: ploidy as a promising parameter in the metabolic engineering of tropane alkaloids.

Tetraploidy improves overexpression of h6h and scopolamine production of H. muticus, while in H. senecionis, pmt overexpression and elicitation can be used as effective methods for increasing tropane alkaloids. The effects of metabolic engineering in a polyploid context were studied by overexpression of h6h in the tetraploid hairy root cultures of H. muticus. Flow cytometry analysis indicated genetic stability in the majority of the clones, while only a few clones showed genetic instability. Among all the diploid and tetraploid clones, the highest level of h6h transgene expression and scopolamine accumulation was interestingly observed in the tetraploid clones of H. muticus. Therefore, metabolic engineering of the tropane biosynthetic pathway in polyploids is suggested as a potential system for increasing the production of tropane alkaloids. Transgenic hairy root cultures of Hyoscyamus senecionis were also established. While overexpression of pmt in H. senecionis was correlated with a sharp increase in hyoscyamine production, the h6h-overexpressing clones were not able to accumulate higher levels of scopolamine than the leaves of intact plants. Applying methyl jasmonate was followed by a sharp increase in the expression of pmt and a drop in the expression of tropinone reductase II (trII) which consequently resulted in the higher biosynthesis of hyoscyamine and total alkaloids in H. senecionis.

Draft Genome Sequence of Agrobacterium rhizogenes Strain NCPPB2659.

This work reports the draft genome sequence of Agrobacterium rhizogenes strain NCPPB2659 (also known as strain K599). The assembled genome contains 5,277,347 bp, composed of one circular chromosome, the pRi2659 virulence plasmid, and 17 scaffolds pertaining to the linear chromosome. The wild-type strain causes hairy root disease in dicots and has been used to make transgenic hairy root cultures and composite plants (nontransgenic shoots with transgenic roots). Disarmed variants of the strain have been used to produce stable transgenic monocot and dicot plants.

Silencing the Transcriptional Repressor, ZCT1, Illustrates the Tight Regulation of Terpenoid Indole Alkaloid Biosynthesis in Catharanthus roseus Hairy Roots.

The Catharanthus roseus plant is the source of many valuable terpenoid indole alkaloids (TIAs), including the anticancer compounds vinblastine and vincristine. Transcription factors (TFs) are promising metabolic engineering targets due to their ability to regulate multiple biosynthetic pathway genes. To increase TIA biosynthesis, we elicited the TIA transcriptional activators (ORCAs and other unidentified TFs) with the plant hormone, methyl jasmonate (MJ), while simultaneously silencing the expression of the transcriptional repressor ZCT1. To silence ZCT1, we developed transgenic hairy root cultures of C. roseus that expressed an estrogen-inducible Zct1 hairpin for activating RNA interference. The presence of 17β-estradiol (5μM) effectively depleted Zct1 in hairy root cultures elicited with MJ dosages that either optimize or inhibit TIA production (250 or 1000μM). However, silencing Zct1 was not sufficient to increase TIA production or the expression of the TIA biosynthetic genes (G10h, Tdc, and Str), illustrating the tight regulation of TIA biosynthesis. The repression of the TIA biosynthetic genes at the inhibitory MJ dosage does not appear to be solely regulated by ZCT1. For instance, while Zct1 and Zct2 levels decreased through activating the Zct1 hairpin, Zct3 levels remained elevated. Since ZCT repressors have redundant yet distinct functions, silencing all three ZCTs may be necessary to relieve their repression of alkaloid biosynthesis.

Biodegradation of γ-hexachlorocyclohexane by transgenic hairy root cultures of Cucurbita moschata that accumulate recombinant bacterial LinA

γ-HCH was successfully degraded using LinA-expressed transgenic hairy root cultures of Cucurbita moschata . Fusing an endoplasmic reticulum-targeting signal peptide to LinA was essential for stable accumulation in the hairy roots. The pesticide γ-hexachlorocyclohexane (γ-HCH) is a persistent organic pollutant (POP) that raises public health and environmental pollution concerns worldwide. Although several isolates of γ-HCH-degrading bacteria are available, inoculating them directly into γ-HCH-contaminated soil is ineffective because of the bacterial survival rate. Cucurbita species incorporate significant amounts of POPs from soils compared with other plant species. Here, we describe a novel bioremediation strategy that combines the bacterial degradation of γ-HCH and the efficient uptake of γ-HCH by Cucurbita species. We produced transgenic hairy root cultures of Cucurbita moschata that expressed recombinant bacterial linA, isolated from the bacterium Sphingobium japonicum UT26. The LinA protein was accumulated stably in the hairy root cultures by fusing an endoplasmic reticulum (ER)-targeting signal peptide to LinA. Then, we demonstrated that the cultures degraded more than 90% of γ-HCH (1ppm) overnight and produced the γ-HCH metabolite 1,2,4-trichlorobenzene, indicating that LinA degraded γ-HCH. These results indicate that the gene linA has high potential for phytoremediation of environmental γ-HCH.

Induction of hairy roots by Agrobacterium rhizogenes-mediated transformation of spine gourd (Momordica dioica Roxb. ex. willd) for the assessment of phenolic compounds and biological activities

An efficient protocol for hairy root induction of spine gourd (Momordica dioica) was established using Agrobacterium rhizogenes (KCTC 2703). This study evaluates the phenolic compound production, antioxidant and antimicrobial (antibacterial, antifungal and antiviral) activities of transgenic hairy root cultures in M. dioica . Hairy roots were induced from leaves, petiole, and internodal explants. Molecular analysis of PCR and gene sequencing using specific primers of rolC and aux1 revealed T-DNA integration in the hairy root clones and RT-PCR analysis confirmed the expression of hairy root inducible genes (rolC and aux1). The greatest biomass accumulation of hairy roots on MS liquid medium supplemented with 3% sucrose was observed at 22 days. Ultra-HPLC was used to compare the individual phenolic compound contents of transgenic and non-transgenic roots. Moreover, transgenic hairy roots efficiently produced several phenolic compounds, such as flavonols, hydroxycinnamic acid and hydroxybenzoic acid derivatives. The total phenolic, flavonoid contents and biological (antioxidant, antibacterial, antifungal and antiviral) activities were higher in hairy roots compared to non-transformed roots. These results demonstrate the greater potentiality of M. dioica hairy root cultures for the production of valuable phenolic compounds and for studies of their biological activity.

Molecular cloning and transgenic characterization of the genes encoding chalcone synthase and chalcone isomerase from the Tibetan herbal plant Mirabilis himalaica.

Mirabilis himalaica is an endangered medicinal plant species in the Tibetan Plateau. The two genes respectively encoding chalcone synthase (MhCHS) and chalcone isomerase (MhCHI) were isolated and characterized from M. himalaica. The sequence analysis revealed that the two genes were similar with their corresponding homologous genes in other plants. The tissue profiles showed that both MhCHS and MhCHI had higher expression levels in roots than in stems and leaves. Transgenic hairy root cultures respectively with overexpressing MhCHS and MhCHI were established. The genomic PCR detection confirmed the authority of transgenic hairy root lines, in which either MhCHS or MhCHI expression levels were much higher than that in non-transgenic hairy root line. Finally, the HPLC detection results demonstrated that the rotenoid contents in MhCHS/MhCHI-transformed hairy root lines were enhanced. This study provided two candidate genes that could be used to genetic engineering rotenoid biosynthesis in M. himalaica and an alternative method to produce rotenoid using transgenic hairy root cultures.

Determination of terpenoid indole alkaloids in hairy roots of Rhazya stricta (Apocynaceae) by GC-MS.

Rhazya stricta Decne. (Apocynaceae) is a medicinal plant rich in terpenoid indole alkaloids (TIAs), some of which possess important pharmacological properties. The study material including transgenic hairy root cultures have been developed and their potential for alkaloid production are being investigated. In this study, a comprehensive GC-MS method for qualitative and quantitative analysis of alkaloids from Rhazya hairy roots was developed. The composition of alkaloids was determined by using GC-MS. In quantification, the ratio between alkaloid and internal standard was based on extracted ion from total ion current (TIC) analyses. The developed method was validated. An acceptable precision with RSD ≤ 8% over a linear range of 1 to 100 µg/mL was achieved. The accuracy of the method was within 94-107%. Analysis of hairy root extracts indicated the occurrence of a total of 20 TIAs. Six of them, pleiocarpamine, fluorocarpamine, vincamine, ajmalicine and two yohimbine isomers are reported here for the first time in Rhazya. Trimethylsilyl (TMS) derivatisation of the extracts resulted in the separation of two isomers for yohimbine and also for vallesiachotamine. Clearly improved chromatographic profiles of TMS-derivatives were observed for vincanine and for minor compounds vincamine and rhazine. The results show that the present GC-MS method is reliable and well applicable for studying the variation of indole alkaloids in Rhazya samples.

Evaluation of phenolic compounds, antioxidant and antimicrobial activities from transgenic hairy root cultures of gherkin (Cucumis anguria L.)

The production of secondary metabolites from hairy root cultures of gherkin (Cucumis anguria L.) along with their antioxidant and antimicrobial activities were studied. The explants from in vitro seedlings were inoculated with Agrobacterium rhizogenes strain KCTC 2703 for the induction of hairy roots. Transgenic clones of hairy roots were confirmed by polymerase chain reaction (PCR) and reverse transcription-PCR using rolC specific primers. Transgenic hairy roots developed in MS liquid medium supplemented with 3% sucrose exhibited the highest accumulation of biomass 98.55g/l fresh mass (FM) and 10.15g/l dry mass (DM) were observed at 21days. Biomass accumulation of hairy root lines was 9.3-fold higher compared to in vitro grown roots. Moreover, transgenic hairy roots effectively produced several phenolic compounds, such as flavonols, hydroxycinnamic and hydroxybenzoic acids derivatives. Due to these metabolic changes, the antioxidant and antimicrobial activities were increased with hairy roots compared to non-transformed roots. The total phenolics, flavonoids content, antioxidant and antimicrobial activity were higher in hairy roots compared to non-transformed roots. Our study described the greater potentiality of C. anguria hairy root cultures for the production of important phenolic compounds and uses of their biological activities.

Influence of Agrobacterium oncogenes on secondary metabolism of plants

The transgenic hairy root cultures have revolutionized the role of tissue culture of plants in the production of secondary metabolites. It was shown that hairy roots often exhibit about the same or higher biosynthetic capacity for secondary metabolite production comparing to their normal roots. Using this methodology, a big number of chemical compounds has been synthesized. The role of single rol genes in secondary metabolite production was studied. Stimulatory effect on the production of secondary metabolites has been shown for genes rolA, rolB, rolC. These genes are present in naturally trasgenic Linaria and Nicotiana plants that are discussed as a model for study of possible evolutionary function of rol genes in the control of secondary metabolites.

An efficient transformation method for estrogen-inducible transgene expression in Catharanthus roseus hairy roots

Efficient genetic engineering of the medicinal plant, Catharanthus roseus, is essential in improving the production of the pharmaceutically important anticancer compounds, vinblastine and vincristine. Here we optimize several steps in the Agrobacterium-mediated transformation of C. roseus, including antibiotic selection and Agrobacterium elimination parameters. With the optimized protocol, stable transgenic hairy root cultures expressing green fluorescent protein (GFP) under control of the estrogen-inducible XVE system were established with a transformation efficiency of 33 %. The estrogen-inducible system has not been previously tested in C. roseus hairy roots but offers several advantages as an inducible system. Prior to induction, low basal levels of Gfp are observed. Upon induction with 5 mM 17β-estradiol, Gfp transgene expression is highly and rapidly induced within 1 h, increasing after 24 h. Strongest GFP expression is observed in the meristematic root tips and is sustained over 5 days in the presence of estradiol. Neither the 17β-estradiol induction nor the estrogen-inducible system elicits a defense response in C. roseus hairy roots.

Robust in vitro assay system for quantitative analysis of parasitic root-knot nematode infestation using Lotus japonicus

Root-knot nematodes are sedentary endoparasites that induce permanent infestation sites inside the roots of a broad range of crop plants. The development of effective control strategies require understanding the root-knot nematode parasitic process, however, the key molecular determinants for host manipulation during infestation remain elusive. One limiting factor has been the lack of a standardized conventional method for quantitative measurement of host parasitism by root-knot nematodes, particularly one that enables efficient downstream analyses and is free from other biological sources of variability. We report here a robust, highly reproducible system for quantitative analysis of all stages of root-knot nematode infestation using the legume Lotus japonicus as the plant host. This system provides a high quality nematode inoculum that maintains consistency in juvenile age and viability even between independently prepared populations. An optimized root transformation protocol was also developed for L.japonicus to facilitate downstream molecular studies in conjunction with the quantitative assay. Hairy root transformation efficiencies up to 91% were achieved. Root-knot nematodes formed egg masses at the root surface of both intact plants and transgenic hairy root cultures within eight weeks, confirming the assay conditions support an efficient completion of the infestation cycle. The in vitro assay system described here is compatible with other plant hosts and will benefit agricultural biotechnology research as it now enables specific high-throughput screening of nematode resistance traits together with subsequent mechanistic elucidation of the causative factors.

Engineering the MEP pathway enhanced ajmalicine biosynthesis

The 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway genes encoding DXR and MECS from Taxus species and STR from Catharanthus roseus were used to genetically modify the ajmalicine biosynthetic pathway in hairy root cultures of C. roseus. As expected, the STR-overexpressed root cultures showed twofold higher accumulation of ajmalicine than the control. It was important to discover that overexpression of the single DXR or MECS gene from the MEP pathway also remarkably enhanced ajmalicine biosynthesis in transgenic hairy root cultures, and this suggested that engineering the MEP pathway by overexpression of DXR or MECS promoted the metabolic flux into ajmalicine biosynthesis. The transgenic hairy root cultures with co-overexpression of DXR and STR or MECS and STR had higher levels of ajmalicine than those with overexpression of a single gene alone such as DXR, MECS, and STR. It could be concluded that transgenic hairy root cultures harboring both DXR/MECS and STR possessed an increased flux in the terpenoid indole alkaloid biosynthetic pathway that enhanced ajmalicine yield, which was more efficient than cultures harboring only one of the three genes.

The influence of Agrobacterium rhizogenes on induction of hairy roots and ß-carboline alkaloids production in Tribulus terrestris L.

We have developed an efficient transformation system for Tribulus terrestris L., an important medicinal plant, using Agrobacterium rhizogenes strains AR15834 and GMI9534 to generate hairy roots. Hairy roots were formed directly from the cut edges of leaf explants 10-14days after inoculation with the Agrobacterium with highest frequency transformation being 49%, which was achieved using Agrobacterium rhizogenes AR15834 on hormone-free MS medium after 28days inoculation. PCR analysis showed that rolB genes of Ri plasmid of A. rhizogenes were integrated and expressed into the genome of transformed hairy roots. Isolated transgenic hairy roots grew rapidly on MS medium supplemented with indole-3-butyric acid. They showed characteristics of transformed roots such as fast growth and high lateral branching in comparison with untransformed roots. Isolated control and transgenic hairy roots grown in liquid medium containing IBA were analyzed to detect ß-carboline alkaloids by High Performance Thin Layer Chromatograghy (HPTLC). Harmine content was estimated to be 1.7μgg(-1) of the dried weight of transgenic hairy root cultures at the end of 50days of culturing. The transformed roots induced by AR15834 strain, spontaneously, dedifferentiated as callus on MS medium without hormone. Optimum callus induction and shoot regeneration of transformed roots in vitro was achieved on MS medium containing 0.4mgL(-1) naphthaleneacetic acid and 2mgL(-1) 6-benzylaminopurine (BAP) after 50days. The main objective of this investigation was to establish hairy roots in this plant by using A. rhizogenes to synthesize secondary products at levels comparable to the wild-type roots.

Overexpression of a Catharanthus tryptophan decarboxylase (tdc) gene leads to enhanced terpenoid indole alkaloid (TIA) production in transgenic hairy root lines of Rauwolfia serpentina

To enhance the production of terpenoid indole alkaloids in Rauwolfia serpentina, Catharanthus tryptophan decarboxylase (Crtdc) gene was over-expressed in transgenic hairy root cultures using Agrobacterium rhizogenes-mediated transformation. Among six transgenic hairy root lines, line RT4 accumulated the highest alkaloid content, with 0.1202 % dry weight (DW) reserpine and 0.0064 % DW ajmalicine, after 10 weeks of culture. Whereas, wild-type roots accumulated 0.0596 ± 0.003 % DW reserpine and 0.0011 ± 0.001 % DW ajmalicine. Transgenic hairy root line RT7 produced the lowest alkaloid content (reserpine: 0.0896 ± 0.002 % DW; ajmalicine: 0.002 ± 0.0 % DW). On the basis of alkaloid content the six hairy root lines were grouped as RT4/RT2 > RT3/RT5 > RT7/RT8. Analysis of gene expression profile indicated that Crtdc was expressed at a higher level in transgenic lines, which could be correlated with enhanced metabolite accumulation in roots. This study confirms that over-expression of Crtdc is a superlative method to improve the biosynthetic potential of Rauwolfia hairy root cultures. Enhanced reserpine and ajmalicine production can serve as an alternative choice to provide resources for relative pharmaceutical industries.

Molecular Breeding of Solasodine Glycoside for Solanum khasianum using Anti-Solamargine Single-Chain Fv Gene

A recombinant single chain fragment variable (scFv) antibody was constructed from hybridoma cell lines expressing anti-solamargine (As) monoclonal antibody (MAb). The characteristics of the As-scFv protein, expressed in both recombinant Escherichia coli and hairy root, were almost same with those of the original MAb. Up to 220 ng recombinant As-scFv was produced per milligram of soluble protein in transgenic hairy root cultures of S. khasianum. The solasodine glycosides concentration was 2.3 fold higher in the transgenic, than in the wild-type hairy root, reflecting from the soluble As-scFv level. The recombinant S. khasianum plant regenerated from recombinant hairy roots contained 2.3 fold concentration of solasodine glycoside, compared to the regenerated plant from wild-type hairy root. These results suggested that the scFv antibody expressed in transgenic plant controlled the antigen level. This methodology is the first success for molecular breeding of secondary metabolites, without understanding of biosynthetic enzyme.

Overexpression of phenylalanine ammonia-lyase improves flavones production in transgenic hairy root cultures of Scutellaria baicalensis

Transgenic hairy root lines from Scutellaria baicalensis overexpressing phenylalanine ammonia-lyase (PAL) (SbPAL1, SbPAL2, and SbAPL3) were established using an Agrobacterium rhizogenes-mediated transformation system. Stable genetic transformation with the SbPAL genes was confirmed by real-time PCR. These transgenic hairy root lines produced higher quantities flavones (i.e., baicalin, baicalein, and wogonin) than the control hairy root line. In particular, the wogonin content was increased by 4–11 times in the SbPAL1-, SbPAL2-, and SbPAL3-overexpressing hairy roots than the S. baicalensis wild-type roots. This research showed the importance of PAL in flavone biosynthesis and demonstrated the efficiency of metabolic engineering in S. baicalensis hairy roots.