Pyramiding of triple Clubroot resistance loci conferred superior resistance without negative effects on agronomic traits in Brassica napus.

Clubroot disease caused by Plasmodiophora brassicae is becoming a serious threat to rapeseed (Brassica napus) production worldwide. Breeding resistant varieties using CR (clubroot resistance) loci is the most promising solution. Using marker-assisted selection and speed-breeding technologies, we generated Brassica napus materials in homozygous or heterozygous states using CRA3.7, CRA08.1, and CRA3.2 loci in the elite parental line of the Zhongshuang11 background. We developed three elite lines with two CR loci in different combinations and one line with three CR loci at the homozygous state. In our study, we used six different clubroot strains (Xinmin, Lincang, Yuxi, Chengdu, Chongqing, and Jixi) which are categorized into three groups based on our screening results. The newly pyramided lines with two or more CR loci displayed better disease resistance than the parental lines carrying single CR loci. There is an obvious gene dosage effect between CR loci and disease resistance levels. For example, pyramided lines with triple CR loci in the homozygous state showed superior resistance for all pathogens tested. Moreover, CR loci in the homozygous state are better on disease resistance than the heterozygous state. More importantly, no negative effect was observed on agronomic traits for the presence of multiple CR loci in the same background. Overall, these data suggest that the pyramiding of triple clubroot resistance loci conferred superior resistance with no negative effects on agronomic traits in Brassica napus.

Effects of Exogenous Ergothioneine on Brassica rapa Clubroot Development Revealed by Transcriptomic Analysis.

Clubroot disease is a soil-borne disease caused by Plasmodiophora brassicae that leads to a serious yield reduction in cruciferous plants. In this study, ergothioneine (EGT) was used to culture P. brassicae resting spores, the germination of which was significantly inhibited. Further exogenous application of EGT and P. brassicae inoculation in Chinese cabbage showed that EGT promoted root growth and significantly reduced the incidence rate and disease index. To further explore the mechanism by which EGT improves the resistance of Chinese cabbage to clubroot, a Chinese cabbage inbred line BJN3-2 susceptible to clubroot treated with EGT was inoculated, and a transcriptome analysis was conducted. The transcriptome sequencing analysis showed that the differentially expressed genes induced by EGT were significantly enriched in the phenylpropanoid biosynthetic pathway, and the genes encoding related enzymes involved in lignin synthesis were upregulated. qRT-PCR, peroxidase activity, lignin and flavonoid content determination showed that EGT promoted the lignin and flavonoid synthesis of Chinese cabbage and improved its resistance to clubroot. This study provides a new insight for the comprehensive prevention and control of cruciferous clubroot and for further study of the effects of EGT on clubroot disease.

Genome-Wide Identification and Characterization of the Trehalose-6-Phosphate Synthetase Gene Family in Chinese Cabbage (Brassica rapa) and Plasmodiophora brassicae during Their Interaction.

Trehalose is a nonreducing disaccharide that is widely distributed in various organisms. Trehalose-6-phosphate synthase (TPS) is a critical enzyme responsible for the biosynthesis of trehalose, which serves important functions in growth and development, defense, and stress resistance. Although previous studies have found that the clubroot pathogen Plasmodiophora brassicae can lead to the accumulation of trehalose in infected Arabidopsis organs, it has been proposed that much of the accumulated trehalose is derived from the pathogen. At present, there is very little evidence to verify this view. In this study, a comprehensive analysis of the TPS gene family was conducted in Brassica rapa and Plasmodiophora brassicae. A total of 14 Brassica rapa TPS genes (BrTPSs) and 3 P. brassicae TPS genes (PbTPSs) were identified, and the evolutionary characteristics, functional classification, and expression patterns were analyzed. Fourteen BrTPS genes were classified into two distinct classes according to phylogeny and gene structure. Three PbTPSs showed no significant differences in gene structure and protein conserved motifs. However, evolutionary analysis showed that the PbTPS2 gene failed to cluster with PbTPS1 and PbTPS3. Furthermore, cis-acting elements related to growth and development, defense and stress responsiveness, and hormone responsiveness were predicted in the promoter region of the BrTPS genes. Expression analysis of most BrTPS genes at five stages after P. brassicae interaction found no significant induction. Instead, the expression of the PbTPS genes of P. brassicae was upregulated, which was consistent with the period of trehalose accumulation. This study deepens our understanding of the function and evolution of BrTPSs and PbTPSs. Simultaneously, clarifying the biosynthesis of trehalose in the interaction between Brassica rapa and P. brassicae is also of great significance.

R gene triplication confers European fodder turnip with improved clubroot resistance.

Clubroot is one of the most important diseases for many important cruciferous vegetables and oilseed crops worldwide. Different clubroot resistance (CR) loci have been identified from only limited species in Brassica, making it difficult to compare and utilize these loci. European fodder turnip ECD04 is considered one of the most valuable resources for CR breeding. To explore the genetic and evolutionary basis of CR in ECD04, we sequenced the genome of ECD04 using de novo assembly and identified 978 candidate R genes. Subsequently, the 28 published CR loci were physically mapped to 15 loci in the ECD04 genome, including 62 candidate CR genes. Among them, two CR genes, CRA3.7.1 and CRA8.2.4, were functionally validated. Phylogenetic analysis revealed that CRA3.7.1 and CRA8.2.4 originated from a common ancestor before the whole-genome triplication (WGT) event. In clubroot susceptible Brassica species, CR-gene homologues were affected by transposable element (TE) insertion, resulting in the loss of CR function. It can be concluded that the current functional CR genes in Brassica rapa and non-functional CR genes in other Brassica species were derived from a common ancestral gene before WGT. Finally, a hypothesis for CR gene evolution is proposed for further discussion.

Fine mapping and candidate gene analysis of CRA3.7 conferring clubroot resistance in Brassica rapa.

KEY MESSAGE: In this study, we fine-mapped a clubroot resistance gene CRA3.7 in Chinese cabbage and developed its closely linked marker syau-InDel3008 for marker-assisted selection in CR cultivars breeding. Chinese cabbage is an important leafy vegetable rich in many nutrients widely grown in China. Clubroot disease caused by an obligate biotrophic pathogen Plasmodiophora brassicae was rapidly spread and challenged to Chinese cabbage production. A clubroot resistance (CR) gene, CRA3.7, was mapped on chromosome A03 of Brassica rapa. A Chinese cabbage line 'CR510', which harbor homozygous resistance locus CRA3.7 was selected from a BC4F3 family. 'CR510' was crossed with a clubroot susceptible Chinese cabbage inbred line '59-1'. Total 51 recombinant plants were identified from an F2 population including 3000 individuals. These recombinants were selfed and the clubroot resistance of F2/3 families was evaluated. Finally, a clubroot resistance gene CRA3.7 was fine-mapped to an interval of approximately 386 kb between marker syau-InDel3024 and syau-InDel3008. According to the reference genome, total 54 genes including five encoding the TIR-NBS-LRR proteins was annotated in the fine-mapped region. Further, nine candidate's gene expression in parental lines at 7, 14 and 21 days after inoculation of P. brassicae were evaluated. Bra019376, Bra019401, Bra019403 and Bra019410 are highly expressed in 'CR510' than '59-1'. Gene sequence of Bra019410 from 'CR510' was cloned and identified different from CRa. Therefore, Bra019376, Bra019401, Bra019403 and Bra019410 are the most likely candidates for CRA3.7. Our research provides a valuable germplasm resource against P. brassicae Pb3 and CRA3.7 closely linked marker for marker-assisted selection in CR cultivars breeding.

Transferring of clubroot-resistant locus CRd from Chinese cabbage (Brassica rapa) to canola (Brassica napus) through interspecific hybridization.

Clubroot, caused by Plasmodiophora brassicae is one of the most severe threats to brassica species in China and worldwide. Breeding for clubroot resistant varieties is one of the best ways to overcome this disease. In this study, we introduced clubroot resistance (CR) gene CRd from Chinese cabbage (85-74) into elite Brassica napus inbred line Zhongshuang 11 through interspecific hybridization and subsequent backcrossing with whole-genome molecular marker-assisted selection (MAS). The resistant test of CRd to P. brassicae isolates was evaluated in the greenhouse as well as in field conditions. Close linkage markers and the whole-chromosome background marker selection approach improved the recovery rate from 78.3% in BC1 to 100% in BC3F1. The improved clubroot-resistant variety, Zhongshuang11R, was successfully selected in the BC3F2 generation. The greenhouse and field resistant tests revealed that Zhongshuang11R was resistant to P. brassicae pathotypes. The agronomic characteristics of Zhongshuang11R were similar to those of its recurrent parental line, including oil content, composition of fatty acid, plant height, primary effective branches, grain yield per plant and thousand-seed weight. In addition, the oil quality could satisfy the quality requirements for commercial rapeseed oil. Our results will enrich the resistant resources of canola and will certainly accelerate clubroot resistance breeding programs in B. napus.

A Loop-Mediated Isothermal DNA Amplification (LAMP) Assay for Detection of the Clubroot Pathogen Plasmodiophora brassicae.

Clubroot caused by Plasmodiophora brassicae is a serious threat to cruciferous crops around the world. The resting spores of P. brassicae are a primary source of infection and can survive in soil for many years. Detection of resting spores in soil is essential for forecasting clubroot prevalence. Detection of P. brassicae has been relying on plant bioassays or PCR-based methods. The loop-mediated isothermal DNA amplification (LAMP) is a promising approach for microorganism detection with the advantage of high sensitivity, accuracy, and convenience in viewing. In this study, we developed a LAMP assay for detection of P. brassicae in soil, roots, and seeds. This method can detect P. brassicae at a minimal amount of 1 fg of plasmid DNA or 10 resting spores in the soil. Compared with conventional PCR, the LAMP was more sensitive in detection of P. brassicae at the lower levels in soil samples. In conclusion, we elaborated a sensitive, accurate, and easy-to-use LAMP assay to detect P. brassicae, which will facilitate sustainable clubroot management and planning.

Sugar Transporters in Plasmodiophora brassicae: Genome-Wide Identification and Functional Verification.

Plasmodiophora brassicae, an obligate intracellular pathogen, can hijack the host's carbohydrates for survival. When the host plant is infected by P. brassicae, a large amount of soluble sugar accumulates in the roots, especially glucose, which probably facilitates the development of this pathogen. Although a complete glycolytic and tricarboxylic acid cycle (TCA) cycle existed in P. brassicae, very little information about the hexose transport system has been reported. In this study, we screened 17 putative sugar transporters based on information about their typical domains. The structure of these transporters showed a lot of variation compared with that of other organisms, especially the number of transmembrane helices (TMHs). Phylogenetic analysis indicated that these sugar transporters were far from the evolutionary relationship of other organisms and were unique in P. brassicae. The hexose transport activity assay indicated that eight transporters transported glucose or fructose and could restore the growth of yeast strain EBY.VW4000, which was deficient in hexose transport. The expression level of these glucose transporters was significantly upregulated at the late inoculation time when resting spores and galls were developing and a large amount of energy was needed. Our study provides new insights into the mechanism of P. brassicae survival in host cells by hijacking and utilizing the carbohydrates of the host.

Identification and Characterization of Circular RNAs in Brassica rapa in Response to Plasmodiophora brassicae.

Plasmodiophora brassicae is a soil-borne pathogen that attacks the roots of cruciferous plants and causes clubroot disease. CircRNAs are noncoding RNAs, widely existing in plant and animal species. Although knowledge of circRNAs has been updated continuously and rapidly, information about circRNAs in the regulation of clubroot disease resistance is extremely limited in Brassica rapa. Here, Chinese cabbage (BJN 222) containing clubroot resistance genes (CRa) against P. brassicae Pb4 was susceptible to PbE. To investigate the mechanism of cicRNAs responsible for clubroot disease resistance in B. rapa, circRNA-seq was performed with roots of 'BJN 222' at 0, 8, and 23 days post-inoculated (dpi) with Pb4 and PbE. A total of 231 differentially expressed circRNAs were identified between the groups. Based on the differentially expressed circRNAs, the circRNA-miRNA-mRNA network was constructed using the target genes directly or indirectly related to plant resistance. Upregulated novel_circ_000495 suppressed the expression of miR5656-y, leading to the upregulation of Bra026508, which might cause plant resistance. Our results provide new insights into clubroot resistance mechanisms and lay a foundation for further studies exploring complex gene regulation networks in B. rapa.

Progress on plant orphan genes.

Orphan genes are located in a special evolutionary branch and have no significant sequence similarity with any other identified genes. Orphan genes are prevalent in every species, comparative genomics analyses found that all sequenced species contained a portion of orphan genes, and the number of orphan genes obtained by distinct screening conditions is different. Orphan genes are often associated with various stress responses, species-specific evolution and substance metabolism regulation. However, most of the orphan genes have not been well annotated or even have no recognizable functional domains, which brings some difficulties to the functional characterization of orphan genes. Compared with conserved genes, there is less research on orphan genes, which leads to the possibility that the importance of orphan genes may be "unrewarded". In this review, we summarize the origin and evolution of orphan genes, plant orphan gene screening and functions, and analyse the existing challenges and future research priorities and solutions, which provide theoretical basis for the study of orphan gene function and action mechanisms.

Spatiotemporal Quantification of Plasmodiophora brassicae Inoculum in Relation to Clubroot Development Under Inoculated and Naturally Infested Field Conditions.

Clubroot caused by Plasmodiophora brassicae is a destructive disease of cruciferous plants worldwide. A quantitative PCR (qPCR) system specific to P. brassicae was developed. Analysis of the qPCR sensitivity indicated that the lower limit of detection was 1 × 101 resting spores/ml, 1 × 102 spores/g of soil, and 1 × 103 spores/g of roots and seeds. The regression curves generated from the qPCR data of different samples had a parallel relationship. The difference between the theoretical and actual concentrations was lowest at 1 × 105 spores/g of sample, compared with other concentrations. The P. brassicae biomass in soil and plant root tissues after inoculated with different spore concentrations was correlated. A correlation analysis confirmed that the clubroot incidence and disease index at 6 weeks after inoculation increased as the spore concentration increased. Under field conditions, the natural inoculum density of the P. brassicae population decreased at the early stage and then increased, with P. brassicae mainly being detected at a soil depth of 0 to 50 cm. The horizontal distribution of P. brassicae varied in the field with occurrences of hot spots. This study established a qPCR-based method for quantitative detection of clubroot. The developed assay is useful for monitoring the spatiotemporal dynamics of P. brassicae in the field. It may also be applicable for clubroot forecasting as a part of proactive disease management.

Mining of Brassica-Specific Genes (BSGs) and Their Induction in Different Developmental Stages and under Plasmodiophora brassicae Stress in Brassica rapa.

Orphan genes, also called lineage-specific genes (LSGs), are important for responses to biotic and abiotic stresses, and are associated with lineage-specific structures and biological functions. To date, there have been no studies investigating gene number, gene features, or gene expression patterns of orphan genes in Brassica rapa. In this study, 1540 Brassica-specific genes (BSGs) and 1824 Cruciferae-specific genes (CSGs) were identified based on the genome of Brassica rapa. The genic features analysis indicated that BSGs and CSGs possessed a lower percentage of multi-exon genes, higher GC content, and shorter gene length than evolutionary-conserved genes (ECGs). In addition, five types of BSGs were obtained and 145 out of 529 real A subgenome-specific BSGs were verified by PCR in 51 species. In silico and semi-qPCR, gene expression analysis of BSGs suggested that BSGs are expressed in various tissue and can be induced by Plasmodiophora brassicae. Moreover, an A/C subgenome-specific BSG, BSGs1, was specifically expressed during the heading stage, indicating that the gene might be associated with leafy head formation. Our results provide valuable biological information for studying the molecular function of BSGs for Brassica-specific phenotypes and biotic stress in B. rapa.

Integrated analysis of leaf morphological and color traits in different populations of Chinese cabbage (Brassica rapa ssp. pekinensis).

KEY MESSAGE: QTLs and candidate gene markers associated with leaf morphological and color traits were identified in two immortalized populations of Brassica rapa, which will provide genetic information for marker-assisted breeding. Brassica rapa is an important leafy vegetable consumed worldwide and morphology is a key character for its breeding. To enhance genetic control, quantitative trait loci (QTLs) for leaf color and plant architecture were identified using two immortalized populations with replications of 2 and 4 years. Overall, 158 and 80 QTLs associated with 23 and 14 traits were detected in the DH and RIL populations, respectively. Among them, 23 common robust-QTLs belonging to 12 traits were detected in common loci over the replications. Through comparative analysis, five crucifer genetic blocks corresponding to morphology trait (R, J&U, F and E) and color trait (F, E) were identified in three major linkage groups (A2, A3 and A7). These might be key conserved genomic regions involved with the respective traits. Through synteny analysis with Arabidopsis, 64 candidate genes involved in chlorophyll biosynthesis, cell proliferation and elongation were co-localized within QTL intervals. Among them, SCO3, ABI3, FLU, HCF153, HEMB1, CAB3 were mapped within QTLs for leaf color; and CYCD3;1, CYCB2;4, AN3, ULT1 and ANT were co-localized in QTL regions for leaf size. These robust QTLs and their candidate genes provide useful information for further research into leaf architecture with crop breeding.

Identification and analysis of anthocyanin components in fruit color variation in Schisandra chinensis.

BACKGROUND: Fruit color is an important index and parameter for measuring fruit quality. As an important pigment, anthocyanin is a determinant which appears in all sorts of colors of fruits in nature. RESULTS: Color parameters were measured using a spectrometer and used as a basis to divide the materials into three groups: reddish-orange, orange and yellow. A validated high-performance liquid chromatographic-electrospray ionization-mass spectrometric method was used for the analysis of anthocyanin in Schisandra chinensis and for determining major anthocyanin components in S. chinensis fruits, i.e. cyanidin xylosyl-glucoside (CyXylGlu), cyanidin glucosyl-rutinoside (CyGluRutin), cyanidin rutinoside (CyRutin) and cyanidin xylosyl-rutinoside (CyXylRutin). The anthocyanin contents vary obviously in different colored fruits in S. chinensis. The impact of anthocyanin on coloration of fruits was investigated by multiple regression analysis between color parameters and anthocyanin components, which indicated that CyRutin is the primary cause of fruit color variation in S. chinensis. CONCLUSION: The content and type of anthocyanin determine fruit coloration in S. chinensis, laying the early foundations for systematically interpreting the mechanism of fruit coloration in S. chinensis. © 2015 Society of Chemical Industry.

[Comparative Analysis of Different Inonotus obliquus].

OBJECTIVE: To screen optimal Inonotus obliquus strains that is suitable to cultivation, extracting effective components on industrialized production, genetics and breeding. METHODS: Inonotus obliquus mycelia from different countries were used. High quality strain was screened by comparing the antagonism between Inonotus obliquus and penicillium. The contents of crude fat, crude ash, crude protein and crude fiber were analyzed. RESULTS: It was showed that antagonism ability of Finnish birch was the strongest, its mycelia diameter was up to 53. 27 mm. It was also showed that nutritional ingredient was abundant in artificial cultured Inonotus obliquus strains. The contents of crude fat and crude protein in Finnish birch were higher, which was 1. 93% and 20. 23%, respectively. The contents of crude fat and crude protein in NBRC8681 strains were lower, which was 1. 55% and 19. 85%, respectively. The content of crude ash of JL04 strains was higher(8. 76%). The content of crude ash of NBRC8681 strains was lower(6. 8%). Crude fiber content was higher in HLJ01 strains, its content reached to 45. 09%. The least content appeared in MAFF420101 strains, whose crude fiber content was only 38. 27%. CONCLUSION: The strains of Finnish birch, HLJ01 and JL04 are suitable to exploitation.

[Comparison of component from different species of Inonotus obliquus].

In this paper, water content, mineral element and active ingredient concentration on fifteen Inonotus obliquus which comes from all over the world were studied. In the aspect of water content, the free water content of Finnish birch was the highest, reaching 77.21%, significantly better than that of other strains. The free water content of JL04 is the lowest, only 54.6%. The bound water content of HLJ01 which from Heilongjiang is the highest, reaching 10. 74% , significant differences among other strains. The bound water content of Birch Russia was the lowest. In the aspect of mineral element, the calcium content of NBRC9788 was the high- est (3.49 mg · g(-1)), significantly better than other strains. The second was Finnish birch. The lowest was CX02. The phosphorus content of NBRC9788 was the highest (210.12 µg · g(-1)), significantly superior to other strains. The lowest was JL04. In the aspect of active ingredient concentration, the triterpenoids content of HLJ01 was highest (23.7 mg · g(-1)), significantly better than other strains. It was good strains for biological products research and development and then was Finnish birch. The betulin content of MAFF420165 and MAFF420308 was low, they were not suitable for production. The polysaccharide content of Finnish birch was the highest (9.7%), significantly better than the other strains, it is one of the most ideal and good strains to develop polysaccharide. The polysaccharide content from MAFF420308 and MAFF420256 was 1.2%, lower than other strains. We suggest that avoid using these strains in the study of polysaccharide product development.

Functional analysis of the key BrSWEET genes for sugar transport involved in the Brassica rapa-Plasmodiophora brassicae interaction.

Plasmodiophora brassicae, the causative agent of clubroot disease, establishes a long-lasting parasitic relationship with its host by inducing the expression of sugar transporters. Previous studies have indicated that most BrSWEET genes in Chinese cabbage are up-regulated upon infection with P. brassicae. However, the key BrSWEET genes responsive to P. brassicae have not been definitively identified. In this study, we selected five BrSWEET genes and conducted a functional analysis of them. These five BrSWEET genes showed a notable up-regulation in roots after P. brassicae inoculation. Furthermore, these BrSWEET proteins were localized to the plasma membrane. Yeast functional complementation assays confirmed transport activity for glucose, fructose, or sucrose in four BrSWEETs, with the exception of BrSWEET2a. Mutants and silenced plants of BrSWEET1a, -11a, and -12a showed lower clubroot disease severity compared to wild-type plants, while gain-of-function Arabidopsis thaliana plants overexpressing these three BrSWEET genes exhibited significantly higher disease incidence and severity. Our findings suggested that BrSWEET1a, BrSWEET11a, and BrSWEET12a play pivotal roles in P. brassicae-induced gall formation, shedding light on the role of sugar transporters in host-pathogen interactions.

A Ca2+ sensor BraCBL1.2 involves in BraCRa-mediated clubroot resistance in Chinese cabbage.

Clubroot disease caused by Plasmodiophora brassicae (P. brassicae) severely threatens the cultivation of Cruciferous plants, especially Chinese cabbage. Recently, resistance genes in plants have been reported to encode for a Ca2+-permeable channel in the plasma membrane, which can mediate the cytosolic Ca2+ increase in plant cells upon pathogen attack. However, the downstream Ca2+ sensor and decoder are still unknown. In this study, we identified the virulent and avirulent P. brassicae isolates (Pbs) of two near isogenic lines, CR 3-2 and CS 3-2, with CR 3-2 harboring clubroot resistant gene BraCRa. The transcriptomic analysis was then conducted with CR 3-2 after inoculating with virulent isolate PbE and avirulent isolate Pb4. From the differentially expressed genes of transcriptomic data, we identified a Ca2+-sensor encoding gene, BraCBL1.2, that was highly induced in CR 3-2 during infection by Pb4 but not by PbE. Moreover, GUS histochemical staining and subcellular localization analysis revealed that BraCBL1.2 was specifically expressed in the root hair cells of Arabidopsis and encoded a putative Ca2+ sensor localized in the plasma membrane. We also developed an assay to investigate the BraCRa-mediated hypersensitive response (HR) in tobacco leaves. The results suggest that BraCBL1.2 is involved in the BraCRa-mediated plant ETI immune response against P. brassicae. In addition, we verified that overexpression of BraCBL1.2 enhanced clubroot resistance in Arabidopsis. Collectively, our data identified the involvement of a Ca2+ sensor in BraCRa-mediated clubroot resistance in Chinese cabbage, providing a theoretical basis for further research on the resistance of Chinese cabbage to P. brassicae.

[Effects of nitrogen, phosphorus and potassium fertilizers on the yield, quality and nutrient uptake of Pulsatilla cernua].

OBJECTIVE: To study the effects of nitrogen (N), phosphorus (P2O5) and potassium (K2O) fertilizers on the growth, yield total saponins content and nutrient absorption of Pulsatilla cernua and provide a theoretical basis for good agriculture practice. METHODS: Field plot experiments was conducted, based on the D-saturation optimal design with three factors of nitrogen, phosphorus and potassium. Samples collected periodically were used for determination the contents of nutrient and total saponins, and for measurement of yield and agronomic characters. RESULTS: Nutrient contents in Pulsatilla cernua varied with growth stage and part under the same growth stage. Nutrient contents in aerial part were higher than that in root, while the proportion of nutritional absorption from seedling stage to the middle growth stage was larger than that at the late growth stage. Yield and total saponins content of Pulsatilla cernua were significantly influenced by the N1P2O5 and K2O applications, among three factors, N had the greatest effects, the next was K2O and P5O2. CONCLUSION: Pulsatilla cerntua under field cultivation should be fertilized properly, top-dressing with these fertilizers during the early growth stage and increasing the proportion of potassium. According to total saponin production of Pulsatilla cernua, the optimum fertilization model for high yield and good quality is 180 kg/hm2 of N, 79.74 kg/hm2 of P2O5, and 225 kg/hm2 of K20, with a N : P2O : K2O ratio of approximately 2.3 : 1 : 2.8.

[Study on plant regeneration from somatic embryos of vulnerable medicinal plant Glehnia littoralis].

OBJECTIVE: To study the cause of the seeds dormancy of Glehnia littoralis in vitro and to establish plant regeneration methods via somatic embryos. METHOD: The effects of endosperm and exogenous hormone on the seed dormancy breaking of G. littoralis and the effect of hormone concentration on embryonic callus induction and plant regeneration via somatic embryos were observed, RESULTS: The germination rate of the seeds with 1/3 endosperm was the highest which achieved 31%. TDZ, 6-BA and GA3 treatment could not break seed dormancy but easily lead to abnormal seedlings. Embryogenic callus induction rates was up to 57% on MS supplemented with 1.0 mg x L(-1) 2,4-D. After 20 days culture, embryogenic calli were transferred to MS medium and cotyledonary embryos were formed in 40 days. The regenerated plants were obtained in 20 days. CONCLUSION: An effective system of plant regeneration of G. littoralis was established in this study.