Selection and evaluation of reference genes for qRT-PCR of Scutellaria baicalensis Georgi under different experimental conditions.

Scutellaria baicalensis Georgi is a famous medicinal plant with its dried roots having been used as a traditional Chinese medicinal for more than 2000 years. Although its genome sequence has previously been published and molecular biology methods have been used to study this species, no suitable internal reference genes have been investigated for standardization of gene expression via quantitative real-time polymerase chain reaction (qRT-PCR). Here, the stabilities of 10 candidate reference genes, ACT11, ACT7, α-TUB, ß-TUB, GAPDH, UBC, RPL, SAM, HSP70, and PP2A, were analyzed by four different procedures of GeNorm, NormFinder, BestKeeper, and RefFinder. Their expression stabilities were evaluated under various conditions, including different tissue types (root, stem, leaf, and flower), hormone stimuli treatments (methyl jasmonate, salicylic acid, and abscisic acid), and abiotic stresses (heavy metal, salt, drought, cold, and wounding). The results indicated that ß-TUB was the most stable gene for all tested samples, while ACT11 was the most unstable. The most stable reference gene was not consistent under different conditions. ß-TUB exhibited the highest stability for different tissue types and abiotic stresses, while for hormone stimuli treatments, ACT7 showed the highest stability. To confirm the applicability of suitable reference genes, we selected to SbF6H and SbF8H as target genes to analyze their expression levels in different tissues. This study helps to the accurate quantification of the relative expression levels of interest genes in S. baicalensis via qRT-PCR analysis.

Separate and Combined Response to UV-B Radiation and Jasmonic Acid on Photosynthesis and Growth Characteristics of Scutellaria baicalensis.

The negative effects of enhanced ultraviolet-B (UV-B) on plant growth and development have been reported with many species. Considering the ability of jasmonic acid (JA) to improve plant stress tolerance, the hypothesis that JA pretreatment could alleviate the adverse effects of UV-B on S. baicalensis was tested in this study with photosynthesis and growth characteristics. The results showed that UV-B or JA alone both induced photosynthesis inhibition and decreased biomass in stems and leaves. However, the photosynthetic reduction caused by increased UV-B was mainly related to the effect of nonstomatal-limitation, while that of JA was a stomatal-limitation effect. JA pretreatment prior to UV-B could remit the photosynthetic inhibition via the recovery of chlorophyll content, stomatal conductance; and intercellular CO2 concentration (especially the maximum electron transport rate increase). Furthermore, the coaction of JA and enhanced UV-B alleviated some disadvantageous effects on the leaf and did not aggravate the growth damage induced by their separate actions.

Organ-Specific Metabolic Shifts of Flavonoids in Scutellaria baicalensis at Different Growth and Development Stages.

Scutellaria baicalensis Georgi is a traditional Chinese herbal medicine mainly containing flavonoids that contribute to its bioactivities. In this study, the distributions and dynamic changes of flavonoid levels in various organs of S. baicalensis at different development stages were investigated by UHPLC-QTOF-MS/MS and HPLC-DAD methods. The results indicated that the metabolic profiles of S. baicalensis changed with growth and development. During the initial germination stage, the seeds mainly contained flavonols. With growth, the main kinds of flavonoids in S. baicalensis changed from flavonols to flavanones and flavones. The results also revealed that the accumulation of flavonoids in S. baicalensis is organ-specific. The flavones without 4'-OH groups mainly accumulate in the root and the flavanones mainly accumulate in aerial organs. Dynamic accumulation analysis showed that the main flavonoids in the root of S. baicalensis accumulated rapidly before the full-bloom stage, then changed to a small extent. The results suggested the proper harvest time for the aerial parts was at the initial stage of reproductive growth and the flower buds should be collected before flowering. This study deepening the knowledge of S. baicalensis should provide valuable information for guiding the scientific cultivation of this plant and the development and utilization of S. baicalensis.

[Influence of cutting seedling on growth, quality and yield of both aerial and underground part by cutting seedling in Scutellaria baicalensis].

By measuring the growth data of Scutellaria baicalensis in different cutting-seedling and determined active ingredient contents by HPLC and ultraviolet spectrophotometric determination. such as flavonoids. baicalin. wogonoside. baicalein. wogonin. oroxylin A. scutellarin. luteolin. and apigenin in the whole plant. Under circumstances of guaranteeing the quality and yield of medicinal materials. the yield of medicinal materials. and stems and leaves reached 193.60,63.21 kg/mu after twice cutting seedling. Not only yield but also active ingredient contents have been improved to some extent. the contents of flavonoids. baicalin. wogonoside. baicalein. wogonin. oroxylin A reached 18.52%. 15.13%. 4.03%. 1.04%. 1.04%. 0.12%. respectively in roots. Luteolin was not detected in young stems and leaves of S. baicalensis,the contents of other active ingredients such as scutellarin. luteolin and apigenin reached 7.00%. 0.96%. 0.04% respectively under twice cutting seedling. Therefore. regular cutting seedling could be regard as a new cultivation technique for wider range of promotion. And gaining high quality and yield of medicinal materials and tea with the purpose of rational utilization of natural resources and promoting the development of integration of herbal combination.

[Influence of different germination stage on plant biomass and active compounds of Scutellaria baicalensis germchit].

This paper is aimed to study plant biomass and active compounds of Scutellaria baicalensis germchit in different five stages (from germination to transplant). The length of shoot and root, the diameter and the weight of root were determined. HPLC method was used to determine the content of active compounds (baicalin, scutellarin, wogonoside, baicalein, wogonin). According to the results, various biological indicators increased with the germination of seedling. However, the drying rate of the root declined to 27.96% from 32.90%. The contents of scutellarin and baicalein increased firstly, and then decreased. The maximums of them were 3.22,3.89 mg•g⁻¹ while the data of shoot/root was 0.35. The maximums of the contents of baicalin and wogonoside were 107.39,16.11 mg•g⁻¹ while the data of shoot/root was 0.23 and 0.06. The contents of wogonin gradually increased to the maximum of 0.88 mg•g⁻¹ while the data of shoot/root was 0.50. In conclusion, the contents of baicalin, scutellarin, wogonoside, baicalein and wogonin reached or approached the maximum at germination stage while the data of shoot/root was 0.35. The rate of shoot and root can be used as a judging index of active compounds for S. baicalensis germchit.

[Variation and correlation analysis on phenotypic traits of Scutellaria baicalensis from different strains].

To research the differences and correlation between Scutellaria baicalensis about phenotypic traits of different strains, 10 aboveground traits and 6 root traits of S. baicalensis in two-year-transplanted plants from 14 different strains were compared respectively, and the SPSS 17.0 statistical software was used for data analysis. It showed that phenotypic traits variation of different S. baicalensis strains was rich and the F value ranged from 3.169 to 71.58. The difference was significant between each other and germplasm 15 performs the most outstanding characters. Correlation analysis showed that there existed a significant correlation between the characters except for lateral root number, root diameter and length. The correlation coefficient between the fresh weight of root and the reed head diameter was up to 0.877. Principal component analysis showed that the average of overall yield per plant and root diameter could be used as the comprehensive reference index for germplasm evaluation. The differences and correlations in phenotypic traits of different S. baicalensis strains, provide theoretical basis for distinguishing germplasm and breeding good varieties of S. baicalensis.

Autotetraploidy induced in nianmaohuangqin (Radix Scutellariae Viscidulae) with colchicine in vitro.

OBJECTIVE: To establish and optimize the propagation of Nianmaohuangqin (Radix Scutellariae Viscidulae) and induce and characterize polyploidy of Nianmaohuangqin (Radix Scutellariae Viscidulae). METHODS: Buds from germinating seed-derived explants were induced by tissue culture. With an orthogonal test, different concentrations of 6-benzyl-aminopurine (BAP), indole-3-acetic acid (IAA) and kinetin (KT) were used to determine the optimal concentrations for the propagation of Nianmaohuangqin (Radix Scutellariae Viscidulae). The different concentrations of IAA and rooting powder (ABT) were used to induce rooting. A 0.3% w/v colchicine solution was used to induce polyploidy and the induced buds was identified by root-tip chromosome determination and stomatal apparatus observation. RESULTS: A large number of buds could be induced directly from epicotyl and hypocotyl explants on Murashige and Skoog (MS) (Murashige and Skoog 1962) medium supplemented with 1.1-1.3 mg/L BAP and 0.2 mg/L IAA. Root induction and development could be observed within 20 days of inoculation on 1/2 MS medium supplemented with 0.2 mg/L IAA and 0.1 mg/L ABT. Furthermore, 27 lines of autotetraploid individuals were obtained with a plantlet chromosome number of 2n = 4x = 36. CONCLUSION: Autotetraploid lines could be obtained through induction with colchicine in vitro, proving that this method might be used for plant selection and breeding.

[Effect of environmental factors on photosynthetic physiology and flavonoid constituent of Scutellaria baicalensis].

To discuss the effect of environmental factors and photosynthesis on the growing of plant and the content of active components in Scutellaria baicalensis, the photosynthetic physiology index and diurnal changes of flavonoid constituent of S. Baicalensis were observed and tested in flowering and fruiting stages, and in the meantime environmental parameters were recorded. The obtained data were analyzed data by using path analysis and gray correlation analysis. The results showed that PAR and SWC were important environmental factors impacting on photosynthesis of S. baicalensis. SWC, RH and Ca were important environmental factors impacting on baicalin content. PAR, Po and Ta were important environmental factors impacting on baicalein content.

[Study on rapid propagation of Scutellaria baicalensis variation].

OBJECTIVE: To establish and optimize the technology of taking root and promoting seedlings of white flower Scutellaria baicalensis test tube plantlet, and provide the theory and technology base for efficient factorization production system of white flower Scutellaria baicalensis. METHODS: Stem segments with axillary bud were cultured onto the different basic medium with different kinds and concentration of cytokinin and auxins to take root and produce strong seedling. RESULTS: The suitable culture medium for taking root of white flower Scutellaria baicalensis was 1/2 MS (all substance reduced half) + IBA 0.02 mg/L + sucrose 2%, the induction rate of root was 100%; The best medium for promoting seedling was 1/2 MS (all substance reduced half) + PP333 0.2 mg/L + IBA 0.02 mg/L + sucrose 2%, the seedling was green, the internode was normal, and its growth was vigorous and healthy. CONCLUSION: 1/2 MS (all substance reduced half) culture medium and relatively low concentration of sucrose is beneficial to inducing roots; Media adding appropriate concentration of IBA can significantly increase the root induction rate, the seedling has many stout roots; PP333 has dwarfing effect on seedlings and suitable concentration of PP333 can significantly improve the quality of the plantlets. A good technology of taking root and producing strong test tube plantlets is established.

Integrated metabolomics and proteomics analysis to study the changes in Scutellaria baicalensis at different growth stages.

Scutellaria baicalensis is a functional food that has the potential to treat various diseases. Scutellaria baicalensis can be divided into two types: Ziqin (strip types) and (rotten xylem). Ziqin is used to clear lower energizer large intestine heat syndrome, while Kuqin is used for the treatment of upper energizer lung heat syndrome. At present, the substance basis of the differences between Ziqin and Kuqin is not clear. The changes in metabolite accumulation and protein expression between them were analyzed by the non-targeted metabolomic technique in combination with the label-free proteomics approach. The results showed that the differentially accumulated metabolites and abundant proteins were mainly enriched in the pathways of phenylalanine, tyrosine and tryptophan biosynthesis, phenylpropanoid biosynthesis, flavonoid biosynthesis, flavone and flavonol biosynthesis, isoflavonoid biosynthesis, and anthocyanin biosynthesis. Collectively, these results reveal the changes of Scutellaria baicalensis in different growth years and provide a reference for selecting the appropriate harvest period.

[Effects of plant growth regulator mepiquat chloride on content of active compounds in Scutellaria baicalensis].

OBJECTIVE: To study the effect of plant growth regulator mepiquat chloride (DPC) on the growing development of plant and the content of active compounds in Scutellaria baicalensis. METHOD: After spraying DPC during the seedling period, the length of taproot, fresh weight of root, diameter of taproot and the length of stem were measured. The contents of baicalin, baicalein and wogonin were determined by HPLC, respectively. Total flavonoids and scavenging DPPH were determined with ultraviolet spectrophotometry. RESULT: After spraying DPC, fresh weight of S. baicalensis root was significant increased, and root diameter was also increased. The contents of baicalin and total flavonoids were significantly increased and baicalein and wogonin were decreased remarkably. Compared with controls, scavenging activity of ethanol extracts on DPPH free radical was no significant changed after spraying DPC. CONCLUSION: Plant growth regulator DPC could regulate the growth on the ground and underground effectively, and could enhance the content of flavonoids compounds of S. baicalensis.

[Effect of different developmental stage on plant growth and active compounds in Scutellaria baicalensis].

OBJECTIVE: To study the developmental phase on the growth and active compounds in Scutellaria baicalensis. METHOD: Seeds of wild plants were collected from Laiwu and sowed in Fangshan (Beijing) and Laiwu (Shandong). Samples of aerial and underground parts were collected in five growth periods of sprouts, seedlings, flowering, seed drop and withered periods respectively. The length of taproot, fresh weight of root, diameter of taproot and the length of stem were determined. The content of active compounds and total flavonoids were determined by HPLC and ultraviolet spectrophotometry respectively. The transcripted level of PAL1, PAL2, PAL3, C4H, 4CL, CHS, GUS and UBGAT were analyzed with RT-PCR. RESULT: The results showed that the aerial part of S. baicalensis grew quickly before flowering stage, and the underground part grew mostly between the periods of flowering and withered. In the whole growing developmental periods, the content of total flavonoids was not changed significantly, the content of baicalin was increased gradually and the content of baicalein was decreased gradually. Expression level of PAL and 4CL was the highest in withered period, CHS was increased between flowering and seed drop and decreased in withered period. CONCLUSION: Seedlings and withered periods may be the key phase affecting the growth and active compounds in S. baicalensis.

[Effects of AM fungi on growth and microelement of Scuteliaria baiealensis under different applied phosphorous levels].

The effects of AM fungi on the growth, nutrient uptake and microelement of Scutellaria baicalensis under different applied phosphorous levels in the pot culture were studied. The results showed that applied phosphorous levels had remarkable effect on AM fungal inoculation. Inoculation effect of AM fungi was the best under 0.12-0.18 g x kg(-1) soil. At those levels, the effect of AM fungi on the growth of S. baicalensis was the best, but either lower or higher phosphorous level showed suppressed infecting of AM fungi to S. baicalensis. After inoculation, the contents of biomass, total P, baicalin, total N of root were increased as well as the microelements of K, Ca, Mg, Fe, Zn, but the contents of total N of shoot, N/P were decreased as well as the microelements of Mn, Cu. In conclusion, the growth of host plant and using rate of phosphorus fertilizer can be promoted by inoculation of AM fungi.

[Pollen germination in vitro and pollen tube growth of Scutellaria baicalensis].

OBJECTIVE: To study the pollen germination in vitro and pollen tube growth of Scutellaria baicalensis. METHOD: Two years-old S. baicalensis that in the experiment field of Shangluo university were used as the materials. The effects of culture medium, sucrose, PEG, pH value, culture time and temperature on the pollen germination and tube growth of S. baicalensis were investigated using the method of liquid culture. RESULT AND CONCLUSION: The results showed that 100 g x L(-1) sucrose was helpful for the germination and growth of pollens. However, higher concentration of sucrose would inhibit strongly pollen germination and the growth of pollen tubes. PEG could accelerate pollen germination and tube growth, and the optimal concentration was 150 g x L(-1). The optimum culture medium was BK and BK + 10% sucrose + 150 g x L(-1) PEG (pH 5.8) , and the latter was more benefit for pollen germination and tube growth. The pollen of germination was better at 25-35 degrees C, and it was the best at 30 degrees C for 2-3 hours, while pollen tube growth was slower at 25 degrees C.

[Methods on construction of core germplasm collection of Scutellaria baicalensis by ISSR marker].

OBJECTIVE: To build up primary core germplasm of Scutellaria baicalensis. METHODS: The genetic diversity of 40 germplasm resources of Scutellaria baicalensis in different province were analyzed by ISSR, and the primary core germplasm were constructed with progressive sampling method of smallest genetic distance. RESULTS: 15 primers, which showed good repetitive, special bands and distinct polymorphism, were selected from 51 random ISSR primers. Then the total 248 loci were amplified by these selected 15 primers, with a 97.17% polymorphic loci. The average of Shannon information index (I), Nei's genetic diversity (H), number of alleles and effective number of alleles (NE) by POPGENE 32 analysis were 0.4353, 0.2819, 1.9640 and 1.4617, respectively. It showed there was highly genetic diversity in the 40 germplasm resources. The result of analysis by NTSYS-PC software shows the genetic similarity (Gs) were among 0.64 and 0.80, and there was upper coherence between the clustering result and source core germplasm collection except individual germplasms. The result showed the percentage of polymorphic loci was obviously reduced and the Shannon's information index and Nei's genetic diversity were increased a little, but the index change of germplasm genetic diversity was less than that before sampling. The core germplasms from No. 3 sampling were most representative, whose sampling number was about 30% of the initial sampling, and the percentage of polymorphic loci was that of before sampling 96.8%. CONCLUSIONS: It was practicable that the methods would be used to construct core germplasm collection of Scutellaria baicalensis by ISSR marker.

[Effects of light on flavonoids accumulation and related gene expression in suspension cultures of Scutellaria baicalensis].

OBJECTIVE: To investigate the effects of light on baicalin,baicalein accumulation and related genes expression in suspension cultures of Scutellaria baicalensis. METHOD: The content of baicalin, baicalein in suspension cultures of S. baicalensis was determined by HPLC. The related genes expression was analyzed by semi-quantitative PCR. RESULT: Active ingredients in suspension cultures of S. baicalensis were promoted significantly by light, PAL gene transcription under the conditions of light was higher than that under the dark conditions, the UBGAT gene expression levels was significantly related to the baicalein (r = -0.995). CONCLUSION: Active ingredients and related genes (PAL, UBGAT) in suspension cultures of S. baicalensis were promoted significantly by light.

[Determination of six mineral elements in roots, stems, leaves, flowers and seeds of Scutellaria baicalensis by FAAS].

A study was carried out on the contents of six trace elements, Ca, Cu, Fe, Mn, Zn and K, in roots, stems, leaves, flowers and seeds of planted Scutellaria baicalensis, by flame atomic absorption spectrophotometry (FAAS). The results indicated that Scutellaria baicalensis was rich in trace elements, meaning that it has a relatively high nutritive value. In stems of Scutellaria baicalensis, the content sequence of the six trace elements was found to be Fe > Mn > Zn = Cu > K > Ca. In leaves, the content sequence of the six trace elements was Fe> Mn > Zn > Cu > K > Ca. In flowers and seeds it was Ca > Fe > Mn > Zn > Cu > K, and Ca > Fe > Zn > Mn > Cu >K, separately, and in roots it was Ca > Fe > Cu > Mn > Zn > K. The stems, leaves, flowers and seeds are rich in Fe, whose content is higher than that in pork liver, Mn and Zn, but lower in Ca. The flowers, seeds and roots are especially rich in Ca, whose content is higher than that in bone, indicating that different parts of Scutellaria baicalensis may accumulate different mineral element. This study, for the first time, researched into Ca, Cu, Fe, Mn, Zn and K contents in different parts of Scutellaria baicalensis, which helps explain the multifunction of Scutellaria baicalensis and provides theoretical basis for further developing its medical and edible value.

[Effects of N P K on yield and baicalin contents of Scutellaria baicalensis].

OBJECTIVE: To find out the effects of different levels and combinations of nitrogen, phosphorus and potassium on the growth and secondary metabolites of Scutellaria baicalensis. METHODS: The quantities of growth, physiological indicators and baicalin contents in different fertilization treatment were calculated and compared. RESULTS: The growth physiological indicators and baicalin contents of S. baicalensis in complex fertilization treatment were better than those in single element fertilization treatment, and nutrition balance types better than maladjustment type. The reasonebly fertilitzing in complex fertilization treatment, nitrogen could improve the fertilizer efficiency of phosphorus. CONCLUSION: According to the fertilization mathematic model, the optimum fertilization dosage should be primarily defined as N: 22.6-25.0 g/m2, P: 30.6-44.6 g/m2, K: 5.4-21.4 g/m2, that is 2N:3P:1K.

Influence of Different Carbohydrates on Flavonoid Accumulation in Hairy Root Cultures of Scutellaria baicalensis.

Carbohydrate sources play important roles in energy and growth of plants. Therefore, in this study, we investigated the optimal carbohydrate source in hairy root cultures (HRCs) of Scutellaria baicalensis infected with Agrobacterium rhizogenes strain R1000. The hairy roots were cultured in half-strength B5 liquid medium supplemented with seven different carbohydrates sources (sucrose, fructose, glucose, galactose, sorbitol, mannitol and maltose), each at a concentration of 100 mM, in order to identify the best carbon sources for the production of major flavones, such as wogonin, baicalin and baicalein. Sucrose, galactose and fructose markedly influenced the production of major flavones and were therefore chosen for subsequent experiments. HRC growth and flavone accumulation were examined following culture with 30, 100 and 150 mM sucrose, galactose and fructose, respectively. From these data, 150 mM sucrose was found to be the optimal carbon source for the enhancement of baicalein production and growth of S. baicalensis HRCs. Fructose caused the greatest increase in baicalin accumulation. Additionally, galactose was the optimal carbon source for wogonin production. These results provide important insights into the optimal growth conditions, particularly the appropriate carbohydrate source, for S. baicalensis.

[Study on identification of wild and cultirated Radix Scutellariae in different growing years].

OBJECTIVE: To identify Radix Scutellariae (Huangqin) of different growth years, to distinguish whether it's wild or cultivated and to provide useful information for the quality control of Huangqin crude drug. METHOD: By using morphological and histological methods, we studied 87 individuals of 45 specimens from 12 habitats of 5 provinces of China, which grew wild or were cultivated in different growing years. Moreover, 22 commercial samples of Huangqin from 7 provinces were also investigated. RESULT: The identification was performed base on morphological and histological characteristics, such as, the shape, color, cork, remaining stems, decayed central xylem, and vessels arrangement, xylem cork ring, growth rings, etc. CONCLUSION: We established an identification method for distinguishing Huangqin wild or cultivated in different growing years. Furthermore, the structure of annual rings in the transection of Radix Scutellariae has been discovered for the first time.