Scutellaria polysaccharide mediates the immunity and antioxidant capacity of giant freshwater prawn (Macrobrachium rosenbergii).

The giant freshwater prawn (Macrobrachium rosenbergii) is a commercially valuable freshwater crustacean species that frequently appears a death affected by various diseases, resulting in substantial economic losses. Improving the survival rate of M. rosenbergii is a hot and essential issue for feeding the prawns. Scutellaria polysaccharide (SPS) extracted from Scutellaria baicalensis (a Chinese medicinal herb) is conducive to the survival rate of organisms by enhancing immunity and antioxidant ability. In this study, M. rosenbergii was fed 50, 100, and 150 mg/kg of SPS. The immunity and antioxidant capacity of M. rosenbergii were tested by mRNA levels and enzyme activities of related genes. The mRNA expressions of NF-κB, Toll-R, and proPO (participating in the immune response) in the heart, muscle, and hepatopancreas were decreased after four weeks of SPS feeding (P < 0.05). This indicated that long-term feeding of SPS could regulate the immune responses of M. rosenbergii tissues. The activity levels of antioxidant biomarkers, alkaline phosphatase (AKP), and acid phosphatase (ACP) had significant increases in hemocytes (P < 0.05). Moreover, catalase (CAT) activities in the muscle and hepatopancreas, as well as superoxide dismutase (SOD) activities in all tissues, significantly decreased after four weeks of culture (P < 0.05). The results demonstrated that long-term feeding of SPS could improve the antioxidant capacity of M. rosenbergii. In summary, SPS was conducive to regulating the immune capacity and enhancing the antioxidant capacity of M. rosenbergii. These results provide a theoretical basis for supporting SPS addition to the feed of M. rosenbergii.

The genomes of medicinal skullcaps reveal the polyphyletic origins of clerodane diterpene biosynthesis in the family Lamiaceae.

The presence of anticancer clerodane diterpenoids is a chemotaxonomic marker for the traditional Chinese medicinal plant Scutellaria barbata, although the molecular mechanisms behind clerodane biosynthesis are unknown. Here, we report a high-quality assembly of the 414.98 Mb genome of S. barbata into 13 pseudochromosomes. Using phylogenomic and biochemical data, we mapped the plastidial metabolism of kaurene (gibberellins), abietane, and clerodane diterpenes in three species of the family Lamiaceae (Scutellaria barbata, Scutellaria baicalensis, and Salvia splendens), facilitating the identification of genes involved in the biosynthesis of the clerodanes, kolavenol, and isokolavenol. We show that clerodane biosynthesis evolved through recruitment and neofunctionalization of genes from gibberellin and abietane metabolism. Despite the assumed monophyletic origin of clerodane biosynthesis, which is widespread in species of the Lamiaceae, our data show distinct evolutionary lineages and suggest polyphyletic origins of clerodane biosynthesis in the family Lamiaceae. Our study not only provides significant insights into the evolution of clerodane biosynthetic pathways in the mint family, Lamiaceae, but also will facilitate the production of anticancer clerodanes through future metabolic engineering efforts.

De Novo Assembly and Species-Specific Marker Development as a Useful Tool for the Identification of Scutellaria L. Species.

Scutellaria L. (family Lamiaceae) includes approximately 470 species found in most parts of the world and is commonly known as skullcaps. Scutellaria L. is a medicinal herb used as a folk remedy in Korea and East Asia, but it is difficult to identify and classify various subspecies by morphological methods. Since Scutellaria L. has not been studied genetically, to expand the knowledge of species in the genus Scutellaria L., de novo whole-genome assembly was performed in Scutellaria indica var. tsusimensis (H. Hara) Ohwi using the Illumina sequencing platform. We aimed to develop a molecular method that could be used to classify S.indica var. tsusimensis (H. Hara) Ohwi, S. indica L. and three other Scutellaria L. species. The assembly results for S.indica var. tsusimensis (H. Hara) Ohwi revealed a genome size of 318,741,328 bp and a scaffold N50 of 78,430. The assembly contained 92.08% of the conserved BUSCO core gene set and was estimated to cover 94.65% of the genome. The obtained genes were compared with previously registered Scutellaria nucleotide sequences and similar regions using the NCBI BLAST service, and a total of 279 similar nucleotide sequences were detected. By selecting the 279 similar nucleotide sequences and nine chloroplast DNA barcode genes, primers were prepared so that the size of the PCR product was 100 to 1000 bp. As a result, a species-specific primer set capable of distinguishing five species of Scutellaria L. was developed.

Comparative Genome Analysis of Scutellaria baicalensis and Scutellaria barbata Reveals the Evolution of Active Flavonoid Biosynthesis.

Scutellaria baicalensis (S. baicalensis) and Scutellaria barbata (S. barbata) are common medicinal plants of the Lamiaceae family. Both produce specific flavonoid compounds, including baicalein, scutellarein, norwogonin, and wogonin, as well as their glycosides, which exhibit antioxidant and antitumor activities. Here, we report chromosome-level genome assemblies of S. baicalensis and S. barbata with quantitative chromosomal variation (2n = 18 and 2n = 26, respectively). The divergence of S. baicalensis and S. barbata occurred far earlier than previously reported, and a whole-genome duplication (WGD) event was identified. The insertion of long terminal repeat elements after speciation might be responsible for the observed chromosomal expansion and rearrangement. Comparative genome analysis of the congeneric species revealed the species-specific evolution of chrysin and apigenin biosynthetic genes, such as the S. baicalensis-specific tandem duplication of genes encoding phenylalanine ammonia lyase and chalcone synthase, and the S. barbata-specific duplication of genes encoding 4-CoA ligase. In addition, the paralogous duplication, colinearity, and expression diversity of CYP82D subfamily members revealed the functional divergence of genes encoding flavone hydroxylase between S. baicalensis and S. barbata. Analyzing these Scutellaria genomes reveals the common and species-specific evolution of flavone biosynthetic genes. Thus, these findings would facilitate the development of molecular breeding and studies of biosynthesis and regulation of bioactive compounds.

Enhanced flavonoid production in hairy root cultures of Scutellaria bornmuelleri by elicitor induced over-expression of MYB7 and FNSП2 genes.

For the purpose of the current study, hairy root induction in S. bornmuelleri, which is an important medicinal plant, was examined using a particular protocol. Accordingly, some factors such as four strain types of Agrobacterium rhizogenes (A4, A13, MSU440 and ATCC15834), three different explants, namely stem, petiole and leaf, two co-cultivation media, i.e. full and half-MS were studied. Besides, two inoculation methods including injection and immersion as well as three inoculation times (5, 7 and 10 min) were closely taken into account. Utilizing injection method by MSU440 strain, hairy root induction took place in stem explants, and a remarkable increase in transformation frequency (100%) was observed in half-strength MS medium. Methyl jasmonate (MeJA, 100 µM), methyl-b-cyclodextrin (b-CD, 0.7, 7 and 14 mM) and Chitosan (Chi, 50, 100 and 200 mg/l) were used either individually or in a combined way to elicitation. Based on the HPLC results, production of chrysin, wogonin and baicalein increased 9.15, 10.56 and 13.25 times after elicitation of hairy roots by MeJA + Chi. In addition, transcripts of FNSП2 and MYB7, two important genes involved in the flavonoid biosynthesis pathway, were studies. By applying Chi and MeJA + Chi elicitor, the expression of both genes increased noticeably. It can be concluded that the mentioned hairy root culture system of S. bornmuelleri can be an alternative to flavonoids production. Moreover, there is a direct and positive relationship between the expression of FNSП2 and MYB7 genes as well as the level of three flavonoids.

Transcriptomic Insight into Terpenoid Biosynthesis and Functional Characterization of Three Diterpene Synthases in Scutellaria barbata.

Scutellaria barbata (Lamiaceae) is an important medicinal herb widely used in China, Korea, India, and other Asian countries. Neo-clerodane diterpenoids are the largest known group of Scutellaria diterpenoids and show promising cytotoxic activity against several cancer cell lines. Here, Illumina-based deep transcriptome analysis of flowers, the aerial parts (leaf and stem), and roots of S. barbata was used to explore terpenoid-related genes. In total, 121,958,564 clean RNA-sequence reads were assembled into 88,980 transcripts, with an average length of 1370 nt and N50 length of 2144 nt, indicating high assembly quality. We identified nearly all known terpenoid-related genes (33 genes) involved in biosynthesis of the terpenoid backbone and 14 terpene synthase genes which generate skeletons for different terpenoids. Three full length diterpene synthase genes were functionally identified using an in vitro assay. SbTPS8 and SbTPS9 were identified as normal-CPP and ent-CPP synthase, respectively. SbTPS12 reacts with SbTPS8 to produce miltiradiene. Furthermore, SbTPS12 was proven to be a less promiscuous class I diterpene synthase. These results give a comprehensive understanding of the terpenoid biosynthesis in S. barbata and provide useful information for enhancing the production of bioactive neo-clerodane diterpenoids through genetic engineering.

An Untapped Resource in the Spotlight of Medicinal Biotechnology: The Genus Scutellaria.

BACKGROUND: This review is intended to draw the attention of pharmaceutical and biotechnological communities to the untapped potential of the Scutellaria genus. Skullcaps, as they are more widely known, are found in one of the oldest materia medica in the world, that of ancient Chinese pharmacology, and their numerous wide range of medicinal bioactivities have been studied both in vivo and in vitro. For thousands of years, chemical compounds from the Scutellaria species have been safely used as antitumor, antibacterial, antiviral, anti-inflammatory, antioxidant or hepatoprotective factors. OBJECTIVE: As these effects are well known, reflected in the presence of Scutellaria plants in national pharmacopoeias, it is clear that the plant has yet enormous unexploited potential. The European pharmacological market has turned to the resources of Scutellaria only in the last two decades, and although the construction and clinical processing of a new drug is a long process, the general impression is that very few medical products in pharmacies have been inspired by the phytochemistry of skullcaps. CONCLUSION: This paper presents the current state of knowledge on the wealth of Scutellaria chemical compounds with treatment applications, its tissue culture and biotechnological achievements, especially in the context of the production of secondary metabolites.

Continuation of the genetic divergence of ecological speciation by spatial environmental heterogeneity in island endemic plants.

Divergent selection plays a critical role not only as a speciation driver but also in maintaining post-speciation divergence. In the absence of direct evidence, ancestral interspecific gene flow between incipient species can reflect ancient selective pressure for ecological speciation. In the present study, two late-Pleistocene diverged species endemic to Taiwan, Scutellaria playfairii and S. tashiroi, were spatially and ecologically partitioned with partial overlap. Multilocus genome-scan analyses and in silico evaluation revealed ancestral interspecific gene flow but distinct genetic compositions, implying that adaptive divergence contributed to their speciation. Ecological niche modeling and principal component analysis suggested incomplete divergent niches between the two species; the species distribution is therefore consistent with Hutchinson's metaphor of multidimensional hypervolume niches rather than attributable to a single factor. Constraint ordination analysis supported this inference of a combination of variables explaining the genetic structure. The rare occurrence of hybrids in the sympatric population suggested hybrid breakdown, providing further evidence of divergent selection blocking gene flow. The correlation of environmental variables with integrated genetic components demonstrated that environmental heterogeneity maintains the species and population differentiation. This study highlights the importance of environmental heterogeneity and divergent selection for the rapid speciation and recent diversification of island plants.

Imbalanced positive selection maintains the functional divergence of duplicated DIHYDROKAEMPFEROL 4-REDUCTASE genes.

Gene duplication could be beneficial by functional division but might increase the risk of genetic load. The dynamics of duplicated paralogs number could involve recombination, positive selection, and functional divergence. Duplication of DIHYDROFLAVONOL 4-REDUCTASE (DFR) has been reported in several organisms and may have been retained by escape from adaptive conflict (EAC). In this study, we screened the angiosperm DFR gene focusing on a diversified genus Scutellaria to investigate how these duplicated genes are retained. We deduced that gene duplication involved multiple independent events in angiosperms, but the duplication of DFR was before the divergence of Scutellaria. Asymmetric positive selective pressures resulted in different evolutionary rates between the duplicates. Different numbers of regulatory elements, differential codon usages, radical amino acid changes, and differential gene expressions provide evidences of functional divergence between the two DFR duplicates in Scutellaria, implying adaptive subfunctionalization between duplicates. The discovery of pseudogenes accompanying a reduced replacement rate in one DFR paralogous gene suggested possibly leading to "loss of function" due to dosage imbalance after the transient adaptive subfunctionalization in the early stage of duplication. Notwithstanding, episodic gene duplication and functional divergence may be relevant to the diversification of ecological function of DFR gene in Scutellaria.

Diversifying selection of the anthocyanin biosynthetic downstream gene UFGT accelerates floral diversity of island Scutellaria species.

BACKGROUND: Adaptive divergence, which usually explains rapid diversification within island species, might involve the positive selection of genes. Anthocyanin biosynthetic pathway (ABP) genes are important for floral diversity, and are related to stress resistance and pollination, which could be responsible for species diversification. Previous studies have shown that upstream genes of ABP are subject to selective constraints and have a slow evolutionary rate, while the constraints on downstream genes are lower. RESULTS: In this study, we confirmed these earlier observations of heterogeneous evolutionary rate in upstream gene CHS and the downstream gene UFGT, both of which were expressed in Scutellaria sp. inflorescence buds. We found a higher evolutionary rate and positive selection for UFGT. The codons under positive selection corresponded to the diversified regions, and the presence or absence of an α-helix might produce conformation changes in the Rossmann-like fold structure. The significantly high evolutionary rates for UFGT genes in Taiwanese lineages suggested rapid accumulation of amino acid mutations in island species. The results showed positive selection in closely related species and explained the high diversification of floral patterns in these recently diverged species. In contrast, non-synonymous mutation rate decreases in long-term divergent species could reduce mutational load and prevent the accumulation of deleterious mutations. CONCLUSIONS: Together with the positive selection of transcription factors for ABP genes described in a previous study, these results confirmed that positive selection takes place at a translational level and suggested that the high divergence of ABP genes is related to the floral diversity in island Scutellaria species.

[Karyotype and evolutionary trend analysis of Scutellaria species in Jinyun mountain, Chongqing].

Traditional squash method was used to analyze chromosome number and karyotypes of four Scutellaria species in Chongqing Jinyun Mountain Natural Reserve： Scutellaria tsinyunensis, S.yunnanensis, S.franchetiana and S.indica.The result showed that the chromosome numbers were 26 except for S.franchetiana, which had 24 chromosomes.These species were all diploid with metacentric and submetacentric chromosomes.Their karyotypes were symmetrical and primitive.The karyotype formula of S.tsinyunensis is 2n=2x=26=24m+2sm, 1B type, As.k=55.28%; the karyotype formula of S.yunnanensis var.salicifolia is 2n=2x=26=26m, 1B type, As.k=56.11%; the karyotype formula of S.franchetiana is 2n=2x=24=20m+4sm, 2B type, As.k=58.50%; the karyotype formula of S.indica is 2n=2x=24=20m+4sm, 2B type, As.k=58.41%.The results were compared with the reported data of S.baicalensis and S.alaschanica.S.alaschanica is expected to be the most advanced one whereas S.tsinyunensis, and S.yunnanensis var. salicifolia primitive.These results are expected to provide some references to the origin and differentiation of genus Scutellaria.

Positive selection and functional divergence of R2R3-MYB paralogous genes expressed in inflorescence buds of Scutellaria species (Labiatae).

Anthocyanin is the main pigment forming floral diversity. Several transcription factors that regulate the expression of anthocyanin biosynthetic genes belong to the R2R3-MYB family. Here we examined the transcriptomes of inflorescence buds of Scutellaria species (skullcaps), identified the expression R2R3-MYBs, and detected the genetic signatures of positive selection for adaptive divergence across the rapidly evolving skullcaps. In the inflorescence buds, seven R2R3-MYBs were identified. MYB11 and MYB16 were detected to be positively selected. The signature of positive selection on MYB genes indicated that species diversification could be affected by transcriptional regulation, rather than at the translational level. When comparing among the background lineages of Arabidopsis, tomato, rice, and Amborella, heterogeneous evolutionary rates were detected among MYB paralogs, especially between MYB13 and MYB19. Significantly different evolutionary rates were also evidenced by type-I functional divergence between MYB13 and MYB19, and the accelerated evolutionary rates in MYB19, implied the acquisition of novel functions. Another paralogous pair, MYB2/7 and MYB11, revealed significant radical amino acid changes, indicating divergence in the regulation of different anthocyanin-biosynthetic enzymes. Our findings not only showed that Scutellaria R2R3-MYBs are functionally divergent and positively selected, but also indicated the adaptive relevance of regulatory genes in floral diversification.

Floral development in Scutellaria pinnatifida (Lamiaceae): the ontogenetic basis for sepal reduction.

The Scutellaria is a Labiatae genus (subfamily Scutellarioideae) with a highly specialised floral structure. The genus is characterised by a peculiar two-lobed calyx with a projecting appendage, named the scutellum. Here, we present a detailed analysis of floral development, using epi-illumination light microscopy, to clarify open questions about its floral organisation. Floral whorls appeared in an acropetal sequence, with a marked temporal overlap of petal and stamen appearance. Organ appearance in each whorl proceeded unidirectionally from the abaxial to the adaxial side. Significant developmental features included the formation of the scutellum, reduction of sepal lobes and formation of a three-lobed nectary disc. Our study revealed that both loss of organ initiation and fusion of primordia are responsible for the reduction in sepal members in Scutellaria. The nectary structure was markedly different from most other studied Lamiaceae.

Diversification, biogeographic pattern, and demographic history of Taiwanese Scutellaria species inferred from nuclear and chloroplast DNA.

The ragged topography created by orogenesis generates diversified habitats for plants in Taiwan. In addition to colonization from nearby mainland China, high species diversity and endemism of plants is also present in Taiwan. Five of the seven Scutellaria species (Lamiaceae) in Taiwan, for example, are endemic to the island. Hypotheses of multiple sources or in situ radiation have arisen to explain the high endemism of Taiwanese species. In this study, phylogenetic analyses using both nuclear and chloroplast markers revealed the multiple sources of Taiwanese Scutellaria species and confirmed the rapid and recent speciation of endemic species, especially those of the "indica group" composed of S. indica, S. austrotaiwanensis, S. tashiroi, and S. playfairii. The common ancestors of the indica group colonized first in northern Taiwan and dispersed regionally southward and eastward. Climate changes during glacial/interglacial cycles led to gradual colonization and variance events in the ancestors of these species, resulting in the present distribution and genetic differentiation of extant populations. Population decline was also detected in S. indica, which might reflect a bottleneck effect from the glacials. In contrast, the recently speciated endemic members of the indica group have not had enough time to accumulate much genetic variation and are thus genetically insensitive to demographic fluctuations, but the extant lineages were spatially expanded in the coalescent process. This study integrated phylogenetic and population genetic analyses to illustrate the evolutionary history of Taiwanese Scutellaria of high endemism and may be indicative of the diversification mechanism of plants on continental islands.

Enhanced accumulation of secondary metabolites in hairy root cultures of Scutellaria lateriflora following elicitation.

Hairy root cultures of Scutellaria lateriflora were established using Agrobacterium rhizogenes A4 and produced acteoside (18.5 mg g(-1) dry wt), baicalin (14.5 mg g(-1) dry wt) and wogonoside (12 mg g(-1) dry wt). Yeast extract (50 µg ml(-1)) increased acteoside production 1.4-fold and flavone production 1.7-fold after 7 and 14 days of elicitation. Addition of Pectobacterium carotovorum lysate in the stationary phase of the hairy root culture stimulated only the accumulation of wogonin to 30 mg g(-1) dry wt. The production of wogonin in hairy roots could be associated with its role as a phytolaexin.

Characterization of 24 transferable microsatellite loci in four skullcaps (Scutellaria, Labiatae).

PREMISE OF THE STUDY: Transferable polymorphic microsatellite loci for four skullcaps, Scutellaria indica, S. taiwanensis, S. austrotaiwanensis, and S. playfairii, were developed for future studies of the mating system and population structure of these species. Interspecific amplification was also tested in various Scutellaria species. METHODS AND RESULTS: Twelve novel polymorphic microsatellite loci were isolated from four S. taiwanensis specimens, and seven are interspecifically transferable. Microsatellite loci developed from S. austrotaiwanensis in a previous study were also analyzed in the other three species, and 12 loci were found to be transferable. Allele numbers of the total 24 loci for S. indica, S. taiwanensis, S. playfairii, and S. austrotaiwanensis are two to four, two, two to five, and two to three, respectively, with an expected heterozygosity ranging from 0.114-0.661, 0.062-0.499, 0.280-0.730, and 0.268-0.662, respectively. The interspecies transferability of these 24 loci was further tested in another 10 Scutellaria species, including three species native to Taiwan. Seventeen loci were found to be interspecifically amplifiable, especially among the Taiwan native species. CONCLUSIONS: These highly polymorphic and transferable loci will be useful for future studies of the mating system of closely related Scutellaria species.

Isolation and characterization of the anthocyanidin genes PAL, F3H and DFR of Scutellaria viscidula (Lamiaceae).

Anthocyanidin is a group of flavonoid compounds used as a vegetable pigment and plays an important role in flower coloration and environmental adaptations of the Chinese ornamental plant Scutellaria viscidula. We determined the cDNA sequences of phenylalanine ammonia-lyase (SvPAL), flavanone 3-hydroxylase (SvF3H) and dihydroflavonol 4-reductase (SvDFR) genes in S. viscidula. Comparative analysis showed that the protein products of these three genes did not have a transit peptide at their N-terminal portion, which indicated that these enzymes were directly involved in the substrate conversion in the cytoplasmic matrix. Bioinformatic analysis further revealed that Svpal, Svf3h and Svdfr were the members of flavonoid biosynthetic genes with highly conserved motifs. Based on phylogenetic tree analysis, it appears that PAL, F3H or DFR from different plants might have originated from the same ancestor. This study can help to map and regulate the important stages involved in anthocyanidin biosynthesis by genetic engineering to diversify flower color and improve the ornamental value of S. viscidula.

[Oryzalin-induced chromosome doubling of Scutellaria barbata].

OBJECTIVE: To obtain polyploidy Scutellaria barbata. METHODS: Explants of stem segment of Scutellaria barbata were treated with oryzalin at various concentration and time duration in vitro. RESULTS: Comparing with the characteristics of diploid, mutant plants had significant changes in plant morphology inducing compact plant, darker leaf color, smaller leaf index, thicken stem, significantly larger stomata, guard cells and chloroplasts in stomata guard cells and significantly lower stomata density. Mutant plants were plant chimera of diploid and tetraploid assessed by chromosome counting and flow cytometry. CONCLUSION: The technique could be used for chromosome doubling in Scutellaria barbata.

[Cloning of Scutellaria viscidula CHS gene and construction of its sense and antisense plant expression vectors].

OBJECTIVE: To get Scutellaria viscidula CHS gene sense and antisense plant expression vectors,and explore the mechanism of CHS's effect on the flavonoid biosynthetic pathway through the transformation of Scutellaria viscidula. METHODS: CHS gene fragment was amplified by Reverse Transcription Polymerase Chain Reaction (RT-PCR) from total RNA of Scutellaria viscidula and cloned into PMD18-T vector, sequenced and bioinformaticly analyzed. RESULTS: The DNA sequence was correct and we simultaneously guessed the amino acid sequence and the three-dimensional structure of CHS. Cloned fragments and pCAMBIA1304+ vector were digested using Blg II and BstE II at the same time, then they were connected with CHS gene fragment and pCAMBIAI304+ big skeleton fragment to get CHS sense and antisense plant expression vectors. CONCLUSION: The successful construction of Scutellaria viscidula CHS gene sense and antisense plant expression vectors, and the transformation into C58C1 and LBA4404 through the freeze-thaw method, lay the foundation for Agrobacterium-mediated transformation of Scutellaria viscidula CHS gene in plant heredity.

[Induction and culture of hairy roots in Scutellaria viscidula and its baicalin production].

OBJECTIVE: To establish a culturing system of hairy roots of Scutellaria viscidula, and study the hairy roots growth and biosynthesis of flavonoid in the culturing system. METHOD: Hairy roots of S. viscidula were obtained from infected stem explants after infection with the disarmed Agrobacterium tumefaciens strain C58C1, elite strains were screened and growth curves were determined. The transformation of Ri T-DNA was examined through PCR and baicalin content was examined through HPLC. RESULT: Hairy roots appeared in vitro 8 days after inoculation of S. viscidula with disarmed A. tumefaciens strain C58C1. After 24 days the frequency of stems explants was up to 81%. Transformation was confirmed by the amplification of rolC genes from the hairy roots of S. viscidula. The results demonstrated that rolC genes could be expressed in hairy roots of S. viscidula. Under the 36 days suspension culture of S. viscidula hairy roots in 1/2MS medium, dry weight of hairy root increased 17.42 times, the content and baicalin increased 21.60 and 25.56 times. CONCLUSION: The establishment of the culturing system of hairy root of S. viscidula provided a foundation for further industrial production of active drug component.