Genome-Wide Characterization of SPL Gene Family in Codonopsis pilosula Reveals the Functions of CpSPL2 and CpSPL10 in Promoting the Accumulation of Secondary Metabolites and Growth of C. pilosula Hairy Root.

SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors play critical roles in regulating diverse aspects of plant growth and development, including vegetative phase change, plant architecture, anthocyanin accumulation, lateral root growth, etc. In the present study, 15 SPL genes were identified based on the genome data of Codonopsis pilosula, a well-known medicinal plant. Phylogenetic analysis clustered CpSPLs into eight groups (G1-G8) along with SPLs from Arabidopsis thaliana, Solanum lycopersicum, Oryza sativa and Physcomitrella patens. CpSPLs in the same group share similar gene structure and conserved motif composition. Cis-acting elements responding to light, stress and phytohormone widely exist in their promoter regions. Our qRT-PCR results indicated that 15 CpSPLs were differentially expressed in different tissues (root, stem, leaf, flower and calyx), different developmental periods (1, 2 and 3 months after germination) and various conditions (NaCl, MeJA and ABA treatment). Compared with the control, overexpression of CpSPL2 or CpSPL10 significantly promoted not only the growth of hairy roots, but also the accumulation of total saponins and lobetyolin. Our results established a foundation for further investigation of CpSPLs and provided novel insights into their biological functions. As far as we know, this is the first experimental research on gene function in C. pilosula.

[Improvement effects of quicklime and calcium magnesium phosphate fertilizer on acidified soil cultivating Codonopsis tangshen.] / ç”ŸçŸ³ç°å’Œé’™é•ç£·è‚¥å¯¹é ¸åŒ–å·å šå‚åœŸå£¤çš„æ”¹è‰¯æ•ˆæžœ.

To solve the problem of soil acidification in the cultivation of Codonopsis tangshen, laboratory experiments were carried out to investigate C. tangshen seed germination, seedling growth and soil exchangeable acid, microbial community structure after applying quicklime (QL) and calcium magnesium phosphate fertilizer (CMP). The results showed that QL and CMP treatments significantly improved the survival rate of C. tangshen seedlings from 147.7% to 326.7% and from 270.1% to 311.2%, respectively. The maximum increase rates of the height of C. tangshen seedlings were 516.7% and 546.3%, and that of root length were 798.0% and 679.2% in the treatments of QL and CMP, respectively. 1‰-4‰ QL or CMP treatments increased the relative chlorophyll content, antioxidant enzyme activity and the content of soluble protein of C. tangshen seedlings, decreased the content of malondialdehyde and superoxide anion radical of seedlings, increased soil pH by 0.88-2.02 units and 0.23-1.19 units, and decreased the exchangeable aluminum content in soil by 53.0%-95.3% and 17.6%-81.3%, respectively. Soil bacterial and actinomycetic abundances were significantly higher in 2‰-4‰ QL or CMP treatments than that in the control. Soil fungal abundance was significantly lower in the QL treatment of 2‰ and CMP treatment of 4‰. 1‰-4‰ QL or CMP treatments significantly increased fresh weight of C. tangshen tubers by 40.5%-78.5% and 28.4%-78.8%, respectively. In conclusion, the suitable quantity of QL and CMP for acidified soil (pH=4.12, ρb=1.15 g·cm-3, tillage layer=15 cm) amendment were 1.73-3.45 t·hm-2 and 3.45-6.90 t·hm-2, and QL and CMP amendment could fit the optimum soil pH (5.5-6.5) for the growth of C. tangshen seedlings.

Supplementary Light Source Affects the Growth and Development of Codonopsis lanceolata Seedlings.

Codonopsis lanceolata is widely used in traditional medicine and diets. However, there is no optimal protocol for the commercial production of C. lanceolata seedlings. This study was carried out to find the optimum supplementary light source for the production of C. lanceolata seedlings. Seedlings were grown for four weeks in a glasshouse with an average daily light intensity of 490 µmol·m-2·s-1 photosynthetic photon flux density (PPFD) coming from the sun and a 16-h daily supplementary lighting at 120 µmol·m-2·s-1 PPFD from either high-pressure sodium (HPS), metal halide (MH), far-red (FR), white LED (LED-w), or mixed (white: red: blue = 1:2:1) LEDs (LED-mix). The results showed that the greatest total biomass, stem diameter, ratio of shoot weight to shoot length, root biomass, and ratio of root weight to shoot weight were found in seedlings grown under supplementary LED-mix. Meanwhile, the stomatal properties and soluble sugar contents were improved for seedlings in LED-mix. The contents of starch, total phenols, and flavonoids were the greatest for seedlings in LED-w and LED-mix. The expression of photosynthetic proteins and genes in seedlings was also enhanced by LED-mix. Overall, these results suggest that LED-mix is advantageous to the photosynthetic potential and the accumulation of biomass, carbohydrates and secondary metabolites in C. lanceolata.

[Influence of different cultivation measures on the content of three free carbohydrates of Codonopsis Radix].

To provide a scientific evidence for the quality control of Codonopsis Radix, a method was established for determining the content of three free carbohydrates of Codonopsis Radix. The developed method showed good linearity. The calibration curves were linear within the range of 2.312 5-18.500 0 µg for sucrose, 1.500 0-12.000 0 µg for glucose, and 2.000 0-16.000 0 µg for fructose, resgectwely. The recoveries varied between 96.31%-101.8%. The method is simple, accurate and reproducible, and can be used for determining the content of sucrose, glucose and fructose of Codonopsis Radix. The results showed that different cultivation measures had an effect on the content of three free carbohydrates of Codonopsis Radix. According to the content of sucrose, using Zhuanggenling>not using Zhuanggenling. While, not pinching, shelving>not pinching, not shelving>pinching, not shelving>pinching, shelving. According to the content of glucose and fructose, not using Zhuanggenling>using Zhuanggenling. While, pinching, shelving>not pinching, not shelving>not pinching, shelving>pinching, not shelving. In consideration of the differences of sweetness and content of the three free carbohydrates in Codonopsis Radix, we recommend that the content of free carbohydrates could be considered as the marker to evaluate the quality of Codonopsis Radix.

[Effect of different fertilization treatments on yield and secondary metabolites of Codonopsis pilosula].

The research studies the effect of different fertilization treatments on yield and accumulation of secondary metabolites of Codonopsis pilosula by using single factor randomized block design, in order to ensure reasonable harvesting time and fertilization ratio, and provide the basis for standardized cultivation of C. pilosula. According to the clustering results, the nitrogen fertilizer benefitted for the improvement of root diameter and biomass of C. pilosula. The phosphate fertilizer could promote the content of C. pilosula polysaccharide. The organic fertilizers could increase the content of lobetyolin. With the time going on, C. pilosula's yield, polysaccharide and ehanol-soluble extracts increased while the content of lobetyolin decreased. According to various factors, October is a more reasonable harvest period. Organic fertilizers are more helpful to the yield and accumulation of secondary metabolites of C. pilosula.

[Study on distribution division of Codonopsis Radix].

This paper is aimed to clarify the distribution of Codonopsis Radix in China and search the main ecological factors that affect the suitability distribution. Through literature reading, National Specimen Information Infrastructure researching, field investigation and general survey of Chinese medicine resources, the distribution information was acquired. The MaxEnt model and ArcGIS technology were applied to analyze the main environmental factors influencing the suitability of Codonopsis Radix with integrated 55 environmental factors. The results showed the precipitation and altitude were the major factors impacting the ecology suitable of Codonopsis Radix. The ecological suitable region of C. pilosula was mainly concentrated in south Gansu, Shanxi, Shanxi, Ningxia and, and the ecological suitable region of C. pilosula var. modesta was mainly concentrated in south Gansu, northwest Guizhou, northeast Sichuan, and the ecological suitable region of C. tangshen was mainly concentrated in west Hubei, east Chongqing, middle Sichuan. Combined with the investigation and cultivation of Codonopsis Radix distribution information, the results of ecological suitability of Codonopsis Radix were verified. The ecological suitability distribution result of Codonopsis Radix was consistent with each species actual distribution, which could provide scientific basis for carrying out the rational cultivation of Codonopsis Radix.

Improved Growth and Metabolite Accumulation in Codonopsis pilosula (Franch.) Nannf. by Inoculation of Bacillus amyloliquefaciens GB03.

Codonopsis pilosula (Franch.) Nannf. is a traditional Chinese herbal medicinal plant and a low-cost succedaneum for Panax ginseng and contains various bioactivity components. In this work, we first evaluated the effects of the inoculation of the plant growth-promoting rhizobacteria Bacillus amyloliquefaciens strain GB03 on growth and metabolite accumulation of C. pilosula. The results demonstrated that application of B. amyloliquefaciens GB03 significantly improved the growth of C. pilosula compared to DH5α, Luria broth medium, and water treatment, respectively. On the other hand, we observed that the content of lobetyolin, one of the most important secondary metabolites in C. pilosula, was obviously improved by inoculation of GB03 and almost reached twice that compared to the other three treatments. In addition, some amino acids of roots were elevated by GB03, although not significantly. In conclusion, B. amyloliquefaciens GB03 could induce positive effects on the growth and further stimulate accumulation of secondary metabolites in C. pilosula.

[Resource investigation of wild Codonopsis pilosula in Tanchang county of Gansu].

Tanchang county is the distribution of wild medicinal plant resource-rich region, in order to ascertain Tanchang county Codonopsis pilosula wild resources and reserves of the status quo, according to the fourth national Chinese medicine resources survey technology solutions, using sets of plots and investigating combined route survey method, the county wild C. pilosula var. modesta and C. pilosula resources were investigated by a comprehensive survey designed to reveal the distribution of the county's wildlife resources and herbs C. pilosula reserves. The results showed that in Tanchang county seven ecological zones 53 plots, wild C. pilosula distributed in there were 6 ecological zones 11 plots, accounting for 85.71% of the survey area, wild C. pilosula var. modesta was found only in an ecoregions plots, overlapping with C. pilosula region, accounting for 14.29% of the survey area. C. pilosula herbs reserves were calculated as about 461.85 t, economic capacity of 254.02 t, annual amount of acceptance 25.40 t. C. pilosula var. modesta herbs reserves were calculated as 67.75 t, economic capacity of 36.16 t, acceptance annual amount 3.62 t. The total ash C. pilosula was 3.25%, alcohol-soluble extract was 63.86%, while the C. pilosula var. modesta total ash was 3.69%, alcohol-soluble extract was 68.32%. C. pilosula is suitable for broad range, but wild resource is scarce, C. pilosula var. modesta is suitable for relatively narrow scope, and wild resource is scarce, it is recommended to strengthen the protection of wild resources and the rational development and utilization.

Chemical fingerprinting of Codonopsis pilosula and simultaneous analysis of its major components by HPLC-UV.

Although Codonopsis pilosula (C. pilosula) has long been considered as an important herbal medicine, no analytical method of marker compounds for quality assessment is registered in the Korean Pharmacopoeia. We developed a simple and robust analytical method of three marker components lobetyolin (1), lobetyol (2), and tangshenoside I (3) using HPLC-UV method. We also confirmed the three marker components using UPLC-qTOF/MS method. Various extraction conditions were optimized to achieve three marker compounds with faster extraction kinetics and higher recovery. The analytical condition was then validated by determining the linearity, accuracy, precision, limit of detection, limit of quantification, recovery, repeatability, robustness, and stability. By this method, the three markers were successfully quantified in 38 commercial samples along with three related species that are sometimes used as alternatives to C. pilosula. Finally, principal component and hierarchical clustering analyses were conducted to show the practicality of the method developed for the quality evaluation of C. pilosula.

[Endophytic bacterial diversity in Codonopsis pilosula, Ephedra sinica, and Lamiophlomis rotate: a study with LH-PCR].

To investigate the endophytic bacterial diversity in the three medicinal plant species Codonopsis pilosula, Ephedra sinica, and Lamiophlomis rotata in Ganzi of Sichuan, Southwest China, the total DNA of the three species were extracted by stringent surface sterilization, and studied with length heterogeneity-PCR (LH-PCR) method. For the same plant species, their root-, stem-, and leaf LH-PCR profiles were in a high level of similarity, with little differences in band richness. However, there existed great differences in the LH-PCR profiles among different plant species. C. pilosula had the biggest band richness, followed by E. sinica, and L. rotata. In the three plant species, the endophytic bacteria with an approximately 474 bp DNA length were dominant. The endophytic bacterial diversity of the plants was negatively correlated with rhizosphere soil available phosphorus content, but positively correlated with rhizosphere soil pH. Elevation and rhizosphere soil total nitrogen content were the important environmental factors affecting the distribution of enophytic bacteria in these plant species. The information of population diversity obtained from LH-PCR could more intuitively reflect the differences of bacterial diversity among different plant species, and thus, LH-PCR would be available to be used for analyzing the endophytic bacterial diversity in medicinal plants, providing information and guidance for the further isolation of microbial resources.

[Studies on classification criteria of Codonopsis pilosula var. modesta seedlings in southern of Gansu].

OBJECTIVE: To reveal the quality grade of Codonopsis pilosula var. modesta seedlings from Gansu and provide the scientific basis for establishment of the seedling quality classification standard. METHOD: Thirty batches of C. pilosula var. modesta seedlings collected from varied producing area of Gansu were selected as testing materials, the fresh weight of single plant, water content, length of seedling, diameter of seedling, length of bud and number of buds were determined and the grading standard were systemically analyzed by K-Means Cluster. RESULT: The C. pilosula var. modesta seedlings could be divided into 3 grades of criteria, i. e. the first-grade seedlings were at least 14.1 g of the fresh weight of seedling, 24.0 cm of length of seedling, 1.17 cm of diameter of seedling; the second-grade seedlings were 14.0-3.5 g of the fresh weight of seedling, 23.9-21.6 cm of length of seedling, 1. 16-0. 68 cm of diameter of seedling; the third-grade seedlings were 3.4-1.9 g of the fresh weight of seedling, 21.5-13.5 cm of length of seedling, 0.67-0.54 cm of diameter of seedling. CONCLUSION: Among 30 batches of C. pilosula var. modesta seedlings, 66.7% of the seedlings belonged to the first and the second grades. It suggests that the first and the second grade seedlings should be used in the standard production.

[Effect of Astragalus membranaceus var. mongholicus seed extracts on seed germination and seedling growth of different Codonopsis pilosula caltiver].

OBJECTIVE: To reveal the allelopathy effect of Astragalus membranaceus var. mongholicus seeds and provide information for the intercrop production. METHOD: The A. membranaceus. var. mongholicus seeds were soaked in distilled water for different time (12, 24, 36, 48, 60 h) , and then the seed extracts were used to study their effects on the seed germination, seedling growth and development of two Codonopsis pilosula. RESULT: The A. membranaceus var. mongholicus seeds contained some allelopathy compounds. Their soaked liquid had significantly influence on the seed germination and seedling growth of C. pilosula. The seed germination rate, germination power, germination index and vigor index of two C. pilosula calrivar were improved and then inhabited with soaking time elongation. The extract soaking for 24 h significantly improved the germination traits but the extract for 60 h appeared different degrees of inhibiting vigor. The seed extracts soaking ranging between 12 and 60 h all significantly improved the above plant growth of C. pilosula but significant inhibited their radicle growth in length. And with the soaking time elongation the facilitation effect weakened and the inhibiting effect enhanced, especially more significant in the C. pilosula caltivar (Baitiaodangshen). CONCLUSION: The A. membranaceus var. mongholicus seeds have allelopathic compounds and the endogenous inhibitor can be extracted when soaked for more than 24 h in water with intact seeds, resulting in improvement of seed germination rate. The C. pilosula could be intercropped in A. membranaceus var. mongholicus field, however, when intercroped it should notice that the intercrop proportion should vary with the caltivar.

[Study on seed germination testing standardization of Codonopsis tangshen].

OBJECTIVE: To study the influence of different factors (temperature, light and germinating beds) on seed germination of Codonopsis tangshen. METHOD: The general germination method was applied. RESULT AND CONCLUSION: The optimum seed germination conditions of C. tangshen were as follows: temperature 25 degrees C, light, germinating-beds top of paper (TP) or between of paper (BP). The first seedling--counting time was the 10th day after beginning the test; the final time was the 18th day. The test also showed that gibberellin notably increased the seed germination rate of C. tangshen.

[An overview of research on germplasm resources of Radix Codonopsis and their utilization].

Radix Codonopsis an edible Chinese herb, is resourceful and distributed widely in China. The research on its germplasm resources and utilization has been reviewed in this paper. For germplashm resources, the works of collection, arrangement, evaluation and the selected breeding need to be carried out urgently. It is necessary that unofficial Dangshen and aerial parts of all medicinal Dangshen should be utilized extensively. The effective ingredient obtained by tissue culture will provide a new resource.

[Analysis of RAPD on medicinal plants of Codonopsis pilosula].

OBJECTIVE: To study genetic diversity and genetic relationship among 9 cultivated and 4 nature populations of Codonopsis pilosula and C. pilosula var. modesta form Gansu Province at DNA level. METHODS: DNA of 217 individuals was amplified by 14 decamer primers from 200 radom primers. The polymorphic bands of RAPD were counted. And the results were analyzed by clustering analysis NTSYS-PC software. RESULTS: A total of 125 loci were detected by 14 arbitrary primers, among which 109 and 106 loci were polymorphic in C. pilosula and C. pilosula var. modesta respectively, and the percentage of polymorphic bands (PPB) were 87.20% and 84. 80%. The cluster analysis showed that 13 populations were divided into 2 groups. The one included 8 cultivated populations of C. pilosula, the other group consisted of a cultivated population and 4 nature populations of C. pilosula var. modesta. CONCLUSION: The RAPD results indicated that there was abundant genetic diversity in cultivated populations of C. pilosula and C. pilosula var. modesta. The genetic differentiation among 8 cultivated populations of C. pilosula was very small and related to their geographical distance. Only the cultivation in Wen County originated from C. pilosula var. modesta, while the other populations cultivated in Gansu Province belongs to C. pilosula.

[Study on dynamic growth of Codonopsis pilosula].

OBJECTIVE: To study on the dynamic changes of Codnopsis pilosula for the guidance of the field management. METHOD: Using the random method the influences of the cultivating density and fertilizing weight at four different levels was observed. RESULT AND CONCLUSION: The dynamic changes of the biomass, growth period andbiological changes at different growth stages were found out. The most predominant combination of the two factors, the 1.05 million roots/hm2 of the cultivating density and 240 kg x hm(-2) of the fertilizing weight reached the highest accumulation of stems & leaves; 652 mg x d(-1) the 0.6 million roots/hm2 and 240 kg x hm(-2) reached the heaviest single fresh root. The results could be used for the cultivating, fertilizing and practicing SOP of C. pilosula.

[Study on biomass dynamic changes of Codnopsis pilosula under the planting density and fertilizing amount].

Study on the biomass dynamic changes of Codnopsis pilosula (Franch.) Naff. under the different planting density and fertilizing amount of the four levels respectively via the random methods have showed out: the best combination for the stems and leaves are 1.05 million plant/hm2 and 240 kg fertilizer/hm2. Its biomass accumulation can achieve 652 mg/d; That for the root are 0.6 million plant/hm2 and 240 kg fertilizer/hm2 and the weight of every fresh root achieved 14. 88g. The results will be helpful to cultivating and fertilizing of Codnopsis pilosula.

[The effects of fertilization on yield and root diameter of Codonopsis pilosula var. modesla].

Field trials were conducted using three factors regression design of quadratic orthogonal rotation. The result indicated that under the condition of cultivation, yield and average root diameter of Codonopsis Pilosula var. modesla (Nannf) L.T. Shen showed a positive correlation with N fertilizer application on high fertilization level; The effect of major three fertilizer on yield and average diameter of root was K > P > N. In the course of selective optimization yield which is higher than 3750 kg/hm2 (fresh weight) and average diameter of root which is more than 1.5 cm, the index of fertilizer application for N, P and K pure nutrient is 155 kg/hm2, 250 kg/hm2 (P2O5) and 60 kg/hm2 (K2O) respectively. The ration of N:P:K is 1:1.6:0.4.