inducing-hairy-roots-agrobacterium-rhizogenes-mediated-transformation

Inducing Hairy Roots by <em>Agrobacterium rhizogenes</em>-Mediated Transformation in Tartary Buckwheat (<em>Fagopyrum tataricum</em>)

Tartary buckwheat (TB) [Fagopyrum tataricum (L.) Gaertn] possesses various biological and pharmacological activities because it contains abundant ...

China Academy of Chinese Medical Sciences

China Academy Of Chinese Medical Sciences

Indirubin, one of the key components of medicinal plants including Isatis tinctoria, Polygonum tinctorium, and Strobilanthes cusia, possesses great medicinal efficacy in the treatment of chronic myelocytic leukemia (CML). Due to misidentification and similar name, materials containing indirubin and their close relatives frequently fall prey to adulteration. In this study, we selected an internal transcribed spacer 2 (ITS2) for distinguishing these indirubin-containing species from five of their usual adulterants, after assessing identification efficiency of matK, rbcL, psbA-trnH, and ITS2 among these species. The results of genetic distances and neighbor-joining (NJ) phylogenetic tree indicated that ITS2 region is a powerful DNA barcode to accurately identify these indirubin-containing species and discriminate them from their adulterants. Additionally, high performance liquid chromatography (HPLC) was used to verify indirubin in different organs of the above species. The results showed that indirubin had been detected in the leaves of Is. tinctoria, P. tinctorium, S. cusia, and Indigo Naturalis (made from their mixture), but not in their roots, or in the leaves of their adulterants. Therefore, this study provides a novel and rapid method to identify and verify indirubin-containing medicinal plants for effective natural treatment of CML. 

Rapid Identification and Verification of Indirubin-Containing Medicinal Plants.

Saffron is one of the most expensive species of Chinese herbs and has been subjected to various types of adulteration because of its high price and limited production. The present study introduces a loop-mediated isothermal amplification (LAMP) technique for the differentiation of saffron from its adulterants. This novel technique is sensitive, efficient and simple. Six specific LAMP primers were designed on the basis of the nucleotide sequence of the internal transcribed spacer 2 (ITS2) nuclear ribosomal DNA of Crocus sativus. All LAMP amplifications were performed successfully, and visual detection occurred within 60 min at isothermal conditions of 65 °C. The results indicated that the LAMP primers are accurate and highly specific for the discrimination of saffron from its adulterants. In particular, 10 fg of genomic DNA was determined to be the limit for template accuracy of LAMP in saffron. Thus, the proposed novel, simple, and sensitive LAMP assay is well suited for immediate on-site discrimination of herbal materials. Based on the study, a practical standard operating procedure (SOP) for utilizing the LAMP protocol for herbal authentication is provided.

Rapid authentication of the precious herb saffron by loop-mediated isothermal amplification (LAMP) based on internal transcribed spacer 2 (ITS2) sequence.

Mu-tong (Akebiae Caulis) is a traditional Chinese medicine commonly used as a diuretic and antiphlogistic. A common adulterant of Mu-tong is Guan-mu-tong (Aristolochiae Manshuriensis Caulis), which is derived from the stem of Aristolochia manshuriensis Komarov, and contains carcinogenic aristolochic acids. We used an alternative technique, loop-mediated isothermal amplification (LAMP), to differentiate Mu-tong from Guan-mu-tong because LAMP is quick, highly sensitive, and specific. We designed a set of four common primers (G-F3, G-B3, G-FIP, and G-BIP) and a loop primer (G-LB) for LAMP based on the internal transcribed spacer 2 sequence of Ar. manshuriensis. We successfully amplified the LAMP assays and visual detection occurred within 60 min at isothermal conditions of 65°C. The LAMP reaction exhibited a tenfold increase in detection (4.22 pg/μl DNA) over conventional polymerase chain reaction demonstrating that LAMP is a useful technique to detect Guan-mu-tong. We conclude that the LAMP technique is a potentially valuable safety control method for simple and efficient discrimination of Mu-tong from its adulterant Guan-mu-tong. 

Rapid Identification of Officinal Akebiae Caulis and Its Toxic Adulterant Aristolochiae Manshuriensis Caulis (Aristolochia manshuriensis) by Loop-Mediated Isothermal Amplification.

Pulsatillae radix is a conventional traditional Chinese medicine (TCM) with common name Baitouweng, and has notable effects on inflammation and dysentery. Pulsatilla chinensis (Bge.) Regel is the only source plant of Baitouweng recorded in Chinese Pharmacopoeia, but its adulteration often occurs in the market that possibly affects medicinal efficacy and safety. We have established an internal transcribed spacer 2 (ITS2) barcode library based on 105 plant samples from 12 Pulsatilla species and 10 common adulterants. Results indicate that ITS2 barcoding can accurately distinguish Pulsatilla species from their adulterants. Pulsatilla chinensis can be discriminated from 11 congeneric species by two stable single nucleotide polymorphisms (SNPs) in the ITS2 region. Additionally, a quick specific PCR-RFLP identification assay based on the ITS2 barcode was developed. Using specific primers ITS2/PR1 combined with restriction enzyme Bgl I, Pu. chinensis can rapidly be differentiated from other species via simple and low-cost test procedures. Furthermore, 30 commercial Baitouweng products were tested and only two products were derived from authentic Pu. chinensis. Thus, these two molecular approaches provide practical tools for quick identification of commercial Baitouweng products and can help ensure the safe use of this TCM product.

Rapidly discriminate commercial medicinal Pulsatilla chinensis (Bge.) Regel from its adulterants using ITS2 barcoding and specific PCR-RFLP assay.

There is an unmet need for herbal medicine identification using a fast, sensitive, and easy-to-use method that does not require complex infrastructure and well-trained technicians. For instance, the detection of adulterants in  herbal product has been challenging, since current detection technologies are not effective due to their own limits. High Resolution Melting (HRM) has emerged as a powerful new technology for clinical diagnosis, research in the food industry and in plant molecular biology, and this method has already highlighted the complexity of species identification. In this study, we developed a method of species specific detection of  using HRM analysis combined with internal transcribed spacer 2. We then applied this method to commercial products purporting to contain . . Our results demonstrated that HRM can differentiate  from six common adulterants. HRM was proven to be a fast and accurate technique for testing the authenticity of  in herbal products. Based on these results, a HRM approach for herbal authentication is provided.

Study of Commercially Available  Products Using Bar-HRM Technology.

Herbal medicine is a major component of complementary and alternative medicine, contributing significantly to the health of many people and communities. Quality control of herbal medicine is crucial to ensure that it is safe and sound for use. Here, we investigated a comprehensive quality evaluation system for a classic herbal medicine, Danggui Buxue Formula, by applying genetic-based and analytical chemistry approaches to authenticate and evaluate the quality of its samples. For authenticity, we successfully applied two novel technologies, third-generation sequencing and PCR-DGGE (denaturing gradient gel electrophoresis), to analyze the ingredient composition of the tested samples. For quality evaluation, we used high performance liquid chromatography assays to determine the content of chemical markers to help estimate the dosage relationship between its two raw materials, plant roots of Huangqi and Danggui. A series of surveys were then conducted against several exogenous contaminations, aiming to further access the efficacy and safety of the samples. In conclusion, the quality evaluation system demonstrated here can potentially address the authenticity, quality, and safety of herbal medicines, thus providing novel insight for enhancing their overall quality control. : We established a comprehensive quality evaluation system for herbal medicine, by combining two genetic-based approaches third-generation sequencing and DGGE (denaturing gradient gel electrophoresis) with analytical chemistry approaches to achieve the authentication and quality connotation of the samples.

A Comprehensive Quality Evaluation System for Complex Herbal Medicine Using PacBio Sequencing, PCR-Denaturing Gradient Gel Electrophoresis, and Several Chemical Approaches.

Artemisinin, which has been isolated from  L., is the most effective antimalarial drug and has saved millions of lives. In addition, artemisinin and its derivatives have anti-tumor, anti-parasitic, anti-fibrosis, and anti-arrhythmic properties, which enhances the demand for these compounds. Improving the content of artemisinin in  is therefore becoming an increasing research interest, as the chemical synthesis of this metabolite is not viable. Ultraviolet B and C irradiation have been reported to improve the artemisinin content in , but they are harmful to plant growth and development. Therefore, we screened other light sources to examine if they could promote artemisinin content without affecting plant growth and development. We found that red and blue light could enhance artemisinin accumulation by promoting the expression of the genes that were involved in artemisinin biosynthesis, such as  (ADS) and  (CYP71AV1) genes. Thus, in addition to being the main light sources for photosynthesis, red and blue light play a key role in plant secondary metabolism, and optimizing the combination of these light might allow for the productionof artemisinin-rich .

Red and Blue Light Promote the Accumulation of Artemisinin in Artemisia Annua L.

Seed is not only the main reproductive organ of most of herbal plants but also an important part of Traditional Chinese Medicine (TCM). Seed TCMs possess important medicinal properties and have been widely used as components of pharmaceutical products. In parallel with the increasing popularity and accessibility of seeds as medicinal products in recent years, numerous substitutes and adulterants have also appeared on the market. Due to the small volume and similar appearances of many seed TCMs, they are very difficult to accurately identify the constituent plant species through organoleptic methods. Usage of the wrong herb may be ineffective or may worsen the condition and even cause death. Correct identification of seed herbal medicines is therefore essential for their safe use. Here, we acquired 177 ITS2 sequences and 15  sequences from 51 kinds of seed TCMs belonging to 64 species that have been described in the Chinese Pharmacopoeia. Tree-building analysis showed that the ITS2 sequences of 48 seed TCMs can be differentiated from each other, and they formed distinct, non-overlapping groups in the maximum-likelihood tree. Furthermore, all of the sequences acquired in this study have been submitted to the public DNA barcoding system for herbal medicine, and this integrated database was used to identify 400 seed TCM samples purchased from medicinal markets, drug stores, and the Internet, enabling the identification of 7.5% of the samples as containing non-declared species. This study provides a brief operating procedure for the identification of seed TCMs found in herbal medicine. In the future, researchers and traditional herbal medicine enterprises can use this system to test their herbal materials.

Identifying the Species of Seeds in Traditional Chinese Medicine Using DNA Barcoding.

The genus , which contains about 30 species around the world and seven species in China, is the source of the medicinal plant , which is commonly used as a hemostatic agent as well as to treat burns and scalds. Here we report the complete chloroplast (cp) genome sequences of four  species (, , , and  var. ). These four  cp genomes exhibit typical quadripartite and circular structures, and are 154,282 to 155,479 bp in length, consisting of large single-copy regions (LSC; 84,405⁻85,557 bp), small single-copy regions (SSC; 18,550⁻18,768 bp), and a pair of inverted repeats (IRs; 25,576⁻25,615 bp). The average GC content was ~37.24%. The four  cp genomes harbored 112 different genes arranged in the same order; these identical sections include 78 protein-coding genes, 30 tRNA genes, and four rRNA genes, if duplicated genes in IR regions are counted only once. A total of 39⁻53 long repeats and 79⁻91 simple sequence repeats (SSRs) were identified in the four  cp genomes, which provides opportunities for future studies of the population genetics of  medicinal plants. A phylogenetic analysis using the maximum parsimony (MP) method strongly supports a close relationship between  and  var. , followed by , and finally . The availability of these cp genomes provides valuable genetic information for future studies of  identification and provides insights into the evolution of the genus .

Complete Chloroplast Genomes from : Identity and Variation Among Four Species.

Tartary buckwheat (Fagopyrum tataricum) not only provides a supplement to primary grain crops in China but also has high medicinal value, by virtue of its rich content of flavonoids possessing antioxidant, anti-inflammatory, and anticancer properties. Light is an important environmental factor that can regulate the synthesis of plant secondary metabolites. In this study, we treated tartary buckwheat seedlings with different wavelengths of light and found that red and blue light could increase the content of flavonoids and the expression of genes involved in flavonoid synthesis pathways. Through coexpression analysis, we identified a new MYB transcription factor (FtMYB116) that can be induced by red and blue light. Yeast one-hybrid assays and an electrophoretic mobility shift assay showed that FtMYB116 binds directly to the promoter region of flavonoid-3'-hydroxylase (F3'H), and a transient luciferase activity assay indicated that FtMYB116 can induce F3'H expression. After transforming FtMYB116 into the hairy roots of tartary buckwheat, we observed significant increases in the content of rutin and quercetin. Collectively, our results indicate that red and blue light promote an increase in flavonoid content in tartary buckwheat seedlings; we also identified a new MYB transcription factor, FtMYB116, that promotes the accumulation of rutin via direct activation of F3'H expression.

The light-induced transcription factor FtMYB116 promotes accumulation of rutin in Fagopyrum tataricum.

DNA barcoding has been used for decades, although it has mostly been applied to somesingle-species. Traditional Chinese medicine (TCM), which is mainly used in the form ofcombination-one type of the multi-species, identification is crucial for clinical usage.Next-generation Sequencing (NGS) has been used to address this authentication issue for the pastfew years, but conventional NGS technology is hampered in application due to its short sequencingreads and systematic errors. Here, a novel method, Full-length multi-barcoding (FLMB) vialong-read sequencing, is employed for the identification of biological compositions in herbalcompound formulas in adequate and well controlled studies. By directly sequencing the full-lengthamplicons of ITS2 and  through single-molecule real-time (SMRT) technology, thebiological composition of a classical prescription Sheng-Mai-San (SMS) was analyzed. At the sametime, clone-dependent Sanger sequencing was carried out as a parallel control. Further, anotherformula- (SJS)-was analyzed with genes of ITS2 and CO1. All the ingredients inthe samples of SMS and SJS were successfully authenticated at the species level, and 11 exogenousspecies were also checked, some of which were considered as common contaminations in theseproducts. Methodology analysis demonstrated that this method was sensitive, accurate andreliable. FLMB, a superior but feasible approach for the identification of biological complexmixture, was established and elucidated, which shows perfect interpretation for DNA barcodingthat could lead its application in multi-species mixtures.

Full-Length Multi-Barcoding: DNA Barcoding from Single Ingredient to Complex Mixtures.

Investigation of aged Chinese herbal materials will help us to understand their use and sources in ancient time and broaden the historical perspective of Chinese material medica. To reach this aim, the basic understanding of aged herbal materials, including physical and chemical characters, is of great importance. Delayed luminescence (DL) technique was developed as a rapid, direct, systemic, objective and sample loss-free tool to characterize the properties of Chinese herbal materials. In this study, we measured DL values in aged Chinese herbal materials that were transported from Asia to Europe during the 20th century and stored in Naturalis Biodiversity Center and the Utrecht University museum, and compared these with modern material of the same species.

A comparative study of aged and contemporary Chinese herbal materials by using delayed luminescence technique.

With people's increasing needs for health concern, rutin and emodin in tartary buckwheat have attracted much attention for their antioxidant, anti-diabetic and reducing weight function. However, the biosynthesis of rutin and emodin in tartary buckwheat is still unclear; especially their later glycosylation contributing to make them more stable and soluble is uncovered. Based on tartary buckwheat' genome, the gene structures of 106 UGTs were analyzed; 21 candidate FtUGTs were selected to enzymatic test by comparing their transcript patterns. Among them, FtUGT73BE5 and other 4 FtUGTs were identified to glucosylate flavonol or emodin in vitro; especially rFtUGT73BE5 could catalyze the glucosylation of all tested flavonoids and emodin. Furthermore, the identical in vivo functions of FtUGT73BE5 were demonstrated in tartary buckwheat hairy roots. The transcript profile of FtUGT73BE5 was consistent with the accumulation trend of rutin in plant; this gene may relate to anti-adversity for its transcripts were up-regulated by MeJA, and repressed by ABA.

Article	Year
Rapid Identification and Verification of Indirubin-Containing Medicinal Plants. Evidence-based complementary and alternative medicine : eCAM\| PubMed ID: 26089942	2015
Rapid authentication of the precious herb saffron by loop-mediated isothermal amplification (LAMP) based on internal transcribed spacer 2 (ITS2) sequence. Scientific reports\| PubMed ID: 27146605	2016
Rapid Identification of Officinal Akebiae Caulis and Its Toxic Adulterant Aristolochiae Manshuriensis Caulis (Aristolochia manshuriensis) by Loop-Mediated Isothermal Amplification. Frontiers in plant science\| PubMed ID: 27379153	2016
Rapidly discriminate commercial medicinal Pulsatilla chinensis (Bge.) Regel from its adulterants using ITS2 barcoding and specific PCR-RFLP assay. Scientific reports\| PubMed ID: 28059130	2017
Study of Commercially Available Products Using Bar-HRM Technology. Frontiers in plant science\| PubMed ID: 28360920	2017
A Comprehensive Quality Evaluation System for Complex Herbal Medicine Using PacBio Sequencing, PCR-Denaturing Gradient Gel Electrophoresis, and Several Chemical Approaches. Frontiers in plant science\| PubMed ID: 28955365	2017
Red and Blue Light Promote the Accumulation of Artemisinin in Artemisia Annua L. Molecules (Basel, Switzerland)\| PubMed ID: 29857558	2018
Identifying the Species of Seeds in Traditional Chinese Medicine Using DNA Barcoding. Frontiers in pharmacology\| PubMed ID: 30018552	2018
Complete Chloroplast Genomes from : Identity and Variation Among Four Species. Molecules (Basel, Switzerland)\| PubMed ID: 30149578	2018
The light-induced transcription factor FtMYB116 promotes accumulation of rutin in Fagopyrum tataricum. Plant, cell & environment\| PubMed ID: 30375656	2019
Full-Length Multi-Barcoding: DNA Barcoding from Single Ingredient to Complex Mixtures. Genes\| PubMed ID: 31067783	2019
A comparative study of aged and contemporary Chinese herbal materials by using delayed luminescence technique. Chinese medicine\| PubMed ID: 31969930	2020
Genome-wide analyses reveals a glucosyltransferase involved in rutin and emodin glucoside biosynthesis in tartary buckwheat. Food chemistry\| PubMed ID: 32126466	2020

Yuhua Shi

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