Allometric relationships between leaf and bulb traits of Fritillaria przewalskii Maxim. grown at different altitudes.

Plants adapt to high altitudes by adjusting the characteristics of their above and underground organs. Identifying the species-specific plant traits changed in response to altitude is essential for understanding ecophysiological processes at the ecosystem level. Multiple studies analyzed the effects of altitude on above and underground organ traits in different species. Yet, little is known about those responses in the alpine Fritillaria przewalskii Maxim. (Liliaceae). F. przewalskii is a perennial medicinal plant with meager annual growth and vanishing wild populations. We analyzed leaf and bulb functional traits, and their allometric relationships in F. przewalskii plants grown at three altitudes: 3000, 2700, and 2400 m. Leaf thickness, leaf biomass, leaf biomass allocation, and the aboveground:underground ratio increased significantly with increasing altitude. Conversely, bulb allocation decreased at higher altitudes. The altitude influenced the allometric growth trajectories of specific leaf and bulb traits: higher altitudes led to thicker and broader leaves and changed the shape of the bulbs from more circular, which is ideal (at 2700 m), to more elongated (at 3000 m). Those variations had remarkable ecological significance. Hence, bulb biomass is the largest at 2700 m of altitude for which their vertical and longitudinal ratio is unaffected. which is economically favorable. Our findings show that F. przewalskii has a notable potential of growth and morphological plasticity along the altitude gradient and that 2700 m might be ideal for developing its artificial cultivation.

[Breeding and variety characteristics of a new variety of Fritillaria thunbergii "Zhebei 3"].

A new variety "Zhebei 3(Zhejiao Pharmaceutical 2018002)" was selected and bred from multi seeded Fritillaria thunbergii mutants by systematic breeding method. From 2012 to 2016, the traits assessment, disease resistance appraisal, plot ratios and regional trials of the variety were continuously carried out. The results showed that "Zhebei 3" emerged early and had late seedlings. The average growth period was about 100 days, which was 6 days and 12 days higher than the "Zhebei 1" and "Zhebei 2". The average yield was 5 095.5 kg·hm~(-2), which was 14.42% and 17.71% higher than of the control respectively. The average proliferation rate of bulbs was 261.2%, which was 37.46% and 31.58% higher than that of the control, respectively. The propagation coefficient of bulbs was about 1∶2.6, and the total amount of peimine and peiminine was 0.172 2%, which was 4.49% and 29.47% higher than the control, respectively. The identification of disease resistance showed that it was resistance to bulb stem(soft) rot, better than the control. "Zhebei 3" has stable characters, high yield, good quality, strong disease resistance, and moderate propagation coefficient which is suitable for planting in Zhejiang province.

Corolla retention after pollination facilitates the development of fertilized ovules in Fritillaria delavayi (Liliaceae).

Corollas (or perianths), considered to contribute to pollinator attraction during anthesis, persist after anthesis in many plants. However, their post-floral function has been little investigated within a cost-benefit framework. We explored the adaptive significance of corolla retention after anthesis for reproduction in Fritillaria delavayi, a perennial herb endemic to the alpine areas of the Hengduan Mountains, southwestern China. We examined whether the persistent corollas enhance reproductive success during seed development. Persistent corollas increased fruit temperature on sunny days, and greatly decreased the intensity of ultraviolet-B/C (UV-B/C) radiation reaching fruits. When corollas were removed immediately after pollination, fecundity and progeny quality were adversely affected. Measurements of flower mass and size showed no further corolla growth during fruiting, and respiration and transpiration tests demonstrated that both respiration rate and transpiration rate of corollas were much lower during fruiting than during flowering, indicating a slight additional resource investment in corolla retention after anthesis. Thus, seed production by F. delavayi may be facilitated by corolla retention during seed development at only a small physiological cost. We conclude that corolla retention may be an adaptive strategy that enhances female reproductive success by having a protective role for ripening seeds in the harsh conditions at high elevation.

Identification of genes involved in steroid alkaloid biosynthesis in Fritillaria imperialis via de novo transcriptomics.

Crown imperial (CI) has been used in traditional medicine. Today it is known that such beneficial effects are due to its richness in steroidal alkaloids (SA). Using de novo transcriptomics, orthologues/paralogues finder, phylogenetic analysis and tissue- and developmental stage-specific expression analysis, we identified ten genes and several TFs involved in the biosynthesis of SA in CI. The comparative analysis of ten genes expression profiles revealed the possibility of their co-regulation, which may imply the possibility of their organization in metabolic gene clusters. Having in mind convergent evolution of steroidal biosynthetic pathways in flowering plants and records of convergent evolution of specific proteins, observed expression patterns open a reasonable interest to investigate the possibility of the existence of genes cluster organization in SA pathway in the family Liliaceae or at least in some species of genus Fritillaria. Obtained results support transcriptomics as useful approach in elucidating genes underlying complex biochemical pathways.

Metabolomics Study of Cultivated Bulbus Fritillariae Cirrhosae at Different Growth Stages using UHPLC-QTOF-MS Coupled with Multivariate Data Analysis.

INTRODUCTION: Bulbus fritillariae cirrhosae (known as Chuan bèi mǔ in China, BFC) contain fritillaria steroidal alkaloids as the bioactive ingredients and are widely used as traditional Chinese medicine for the treatment of cough and phlegm. Due to limited wild resources, the cultivated species are becoming predominantly used in Chinese traditional medicine markets. OBJECTIVE: To assess the impact of different growth stages on the alkaloids of cultivated BFC and establish a reference for quality control and guidance for appropriate harvesting practices. METHODS: The ultra-high performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) metabolomic strategy was applied to determine potential chemical markers for the discrimination and quality control of cultivated BFC in different growth stages. The molecular feature extraction and multivariate statistical analysis were applied to alkaloid extraction and full metabolomic profiling of cultivated BFC for classification and marker compound characterisation. RESULT: This approach allowed the establishment of a fast and efficient comparative multivariate analysis of the metabolite composition of 42 samples covering growth of cultivated BFC ranging in age from one to seven years old. Four alkaloid compounds were identified in cultivated BFC based on accurate mass, retention time, and MS/MS fragments. These compounds may be used as potential chemical markers for the classification and discrimination of cultivated BFC samples indifferent growth stages. CONCLUSIONS: The proposed analytical method in combination with multivariate statistical analysis comprised a useful and powerful strategy to explore the chemical ingredients and transforming mechanisms of cultivated BFC and for quality evaluation and control. Copyright © 2018 John Wiley & Sons, Ltd.

[Functional production regionalization for Fritillariae Cirrhosae Bulbus based on growth and quality suitability assessment].

The major contributing factors for growth of endangered medicinal plants of Fritillariae Cirrhosae Bulbus were screened on the GIS platform by using the MaxEnt model, and spatial distribution data of the medicine quality suitability were generated by geostatistics interpolation based on reported measured data of ecology and quality suitability assessment. On this basis, a functional production cultivation regionalization with high feasibility and operability were formatted for protection, wild monitoring, and cultivation of this plant by fuzzy superposition of spatial suitability data of ecology and quality, as well as integrated with land use and cover data. Therefore, a novel assessment and regionalization method were presented for ecology, growth and quality suitability of the Chinese traditional medicinal plants. This method is expected to overcome shortage of traditional regionalization methods difficult to distinguish the contribution of ecological factors and quality factors, which provide an innovative theory and methodology for regionalization, and is helpful to practical application of wild resource protection, monitoring, and commercialization cultivation for traditional Chinese medicinal plants.

[In vitro Regeneration from Bulbous Scales of Fritillaria sonnikovae, an Endemic Species].

Features of in vitro regeneration of Fritillaria sonnikovae from bulb scales were studied. The initiation of shoot formation was obtained on a nutrient medium BDS, supplemented with 5 µM 6-benzylaminopurine and 2 µM α-naphthaleneacetic acid. Optimization of propagation stage was carried out using obtained microbulbs as explants. High regenerative response of explants and shoot multiplication rate were observed on both media supplemented with growth regulators (up to 47% and 4.2 ± 0.6 pcs./explant, respectively) and hormone-free medium (48% and 4.1 ± 0.2 shoots per explant, respectively). It has been established that the ad- dition of growth regulators on the stage of cultivation does not cause increased morphogenic response but contributes to the accelerated initiation and development of microbulbs. Morphological and histological analysis revealed the dynamics of the formation of shoots de novo. In vitro development of F. sonnikovae follows the path of direct organogenesis from the epidermal tissue of the explant.

[Study on accumulation law of peimine in different Fritillaria thunbergii cultivar].

In order to study the accumulation of Fritillaria thunbergii cultivar, peimine content in Xiaye, Kuanye, Duozi and Xiaosanzi bulbs of different sizes and parts was determined by HPLC-ELSE. The results indicated that the peimine content varied significantly with the cultivar type, the size and part of bulb. The distribution laws of peimine were as follow: Xiaosanzi > Duozi > Xiaye > Kuanye, small-size bulb > big-size bulb, core bud > scale. The peimine yield per plant in Duozi was the highest.

[Different ratio of hormone effects on Fritillaria cirrhosa bulb induction].

OBJECTIVE: To study the effects of different hormone induced bulb of Fritillaria cirrhosa seedling leaves, in order to provide an effective way to expant Fritillaria cirrhosa source. METHODS: Fritillaria cirrhosa seedling leaves were used as explants, orthogonal test was carried out to study the medium consisting of 6-BA, NAA, 2, 4-D and KT, and proliferation induced by direct bulb. Induction rate and average number of bulb producing was treated as the assessment indices, and the content of alkaloid of bulb was determined. RESULTS: The best medium formula for Fritillaria cirrhosa tissue culture seedling was MS+6-BA 2.0 mg/L +NAA 1.0mg/L +KT 1.0mg/L, the bulb induction rate was 83.37% and the average number of bulb was 17.27. The alkaloid content of induced bulb was 0.389%, which was 1.64 times that of wild Fritillaria cirrhosa bulb. CONCLUSION: This study provides a simple and rapid mehod for the production of tissue culture of Fritillaria cirrhosa bulb. It is useful for reasonable development and utilization of Fritillaria cirrhosa resources, as well as improvement of the quality of medicinal materials.

Neither protogynous nor obligatory out-crossed: pollination biology and breeding system of the European Red List Fritillaria meleagris L. (Liliaceae).

For 4 years we studied pollination biology and breeding system of the critically endangered, Red List plant Fritillaria meleagris L. (Liliaceae), in the larger of the two remaining populations of the plant in SE Poland. Our observations indicated that, contrary to literature data, the species is not dichogamous nor is it obligatorily out-crossing. Selfing, although rare in natural populations, results in fully developed seeds. Flowers are visited by several insect species, mostly social and solitary bees. In spite of extremely low visitation rates to this early spring-flowering plant, the species is not pollen limited. Although the largest pollen loads are transferred by solitary bees, the key pollinators are bumblebees (mostly the most common species, Bombus terrestris and B. lapidarius) due to their seasonal and floral constancy, and tolerance of bad weather conditions. The current decline of the studied population seems not to be related to the species' pollination or breeding systems but to plant habitat loss. It is suggested, however, that in smaller populations, the species' dependence on generally rare pollinators and largely out-crossed breeding system may accelerate local extinction.

[Quantitative determination of peimisin and total alkaloids in Fritillaria taipaiensis of different growing stage].

OBJECTIVE: To study the content of peimisine and total alkaloids in Fritllaria taipaiensis of different growing stage. METHODS: High performance liquid chromatography coupled with evaporative light scattering detector was employed, and peimisine was separated and quantified upon an Agilent Extend C18 (250 mm x 4.0 mm, 5 microm), using acetonitrile-water-diethylamine as the mobile phase at the flow rate of 1.0 mL min(-1); UV spectrophotometry was used for the determination of total alkaloids. RESULTS: During from two to three years period, the content of peimisine in F. taipaiensis increased with growing stage, from four to six years, it decreased with growing stage; From two to four years, the content of total alkaloids in F. taipaiensis increased with growing stage, from five to six years, it decreased with growing stage. CONCLUSION: It is suitable to harvest F. taipaienssi of four years growing stage.

[Study on the production of alkaloid by cell mass suspension culture of Fritillaria cirrhosa].

OBJECTIVE: Set up Fritillaria cirrhosa cell mass suspension culture system to rapidly screen the best culture conditions for cell mass proliferation and hormone combination. METHODS: Using MS medium as the basic medium, the impact of inoculum size, hormone combination, growth regulators for Fritillaria cirrhosa cell mass suspension culture were compared, and also the growth of cell mass at different culture conditions was compared, and the total alkaloids content in proliferative cell mass was measured. RESULTS AND CONCLUSION: Fritillaria cirrhosa grow significantly faster in cell mass suspension culture than in the solid culture. The total alkaloid content in cell mass is higher than commercial and wild bulb of Fritillaria cirrhosa. The optimal inoculum size for cell mass suspension culture is 30 g/L and the optimal culture media is MS +6-BA 2.0 mg/L + NAA 0.2 mg/L.

Differences in soil properties and bacterial communities between the rhizosphere and bulk soil and among different production areas of the medicinal plant Fritillaria thunbergii.

To explore rhizosphere effects, geographical differences and their effects on the bacterial community associated with the geoherb Fritillaria thunbergii, some physicochemical properties of soil samples (3 sampling sites × 2 habitats (rhizosphere and bulk soil)) were measured and the soil bacterial community detected by PCR-denaturing gradient gel electrophoresis (DGGE). Among the three regions, soil pH varied between 4.48 and 7.73 indicating that F. thunbergii could grow both in acid and slightly alkaline soil. As the authentic Dao-di producing area, Ningbo showed the highest soil quality with the highest content of organic matter (OM) (2.46%), phosphatase (268 mg kg(-1) 24 h(-1)) and urease activity (1481 mg kg(-1) 24 h(-1)). In comparison with the bulk soil, pH, organic carbon content, and phosphatase and urease activities were all lower in the rhizosphere, suggesting that the roots may secrete some unique metabolites in root exudates. Statistical analyses showed that soil properties of Ningbo and Panan in Zhejiang province were more similar to each other than those in Nantong in Jiangsu province. In addition, PCR-DGGE analysis showed that main bacterial population identified in F. thunbergii was proteobacteria (18 bands, 55%), acidobacteria (4, 12%), actinobacteria (4, 12%) and bacterioidetes (6, 18%). Overall, soil properties and microbial communities varied not only between the rhizosphere and bulk soil but also among the three regions. We suggest that the plant, together with the soil properties, cooperatively shape the structure of the rhizosphere bacteria, and that the soil properties have a close relationship with the geoherbalism of F. thunbergii.

Flight metabolic rate has contrasting effects on dispersal in the two sexes of the Glanville fritillary butterfly.

Evolution of dispersal is affected by context-specific costs and benefits. One example is sex-biased dispersal in mammals and birds. While many such patterns have been described, the underlying mechanisms are poorly understood. Here, we study genetic and phenotypic traits that affect butterfly flight capacity and examine how these traits are related to dispersal in male and female Glanville fritillary butterflies (Melitaea cinxia). We performed two mark-recapture experiments to examine the associations of individuals' peak flight metabolic rate (MR(peak)) and Pgi genotype with their dispersal in the field. In a third experiment, we studied tethered flight in the laboratory. MR(peak) was negatively correlated with dispersal distance in males but the trend was positive in females, and the interaction between MR(peak) and sex was significant for long-distance dispersal. A similar but nonsignificant trend was found in relation to molecular variation at Pgi, which encodes a glycolytic enzyme: the genotype associated with high MR(peak) tended to be less dispersive in males but more dispersive in females. The same pattern was repeated in the tethered flight experiment: the relationship between MR(peak) and flight duration was positive in females but negative in males. These results suggest that females with high flight capacity are superior in among-population dispersal, which facilitates the spatial spreading of their reproductive effort. In contrast, males with high flight capacity may express territorial behaviour, and thereby increase the number of matings, whereas inferior males may be forced to disperse. Thus, flight capacity has opposite associations with dispersal rate in the two sexes.

[The effects of in vitro culture conditions on regeneration of Fritillaria cirrhosa].

OBJECTIVE: The optimal in vitro culture conditions for the regeneration of Fritillaria cirrhosa were screened. METHODS: Through the in vitro culture of the flowering stage Fritillaria cirrhosa, the effect of illumination, culture temperature, hormone combination and the amount of ribavirin on the regeneration of Fritillaria cirrhosa were studied. RESULTS AND CONCLUSION: The explant browning level could be apparently reduced if the bulb were cultured under the ray after exposure to proper low-temperature. The optimal temperature for the regeneration of bulb is (20 +/- 2) degrees C. The hormone has obvious promotional effect on the regeneration of bulb, and the best combination is 6-BA 2.0 mg/L plus NAA 0.2 mg/L Concentration of 10 mg/L of ribavirin is best for the regeneration of bulb.

[Study on the Fritillaria cirrhosa seed germination and polyploid induction].

OBJECTIVE: In order to improving the existent breeds of Fritillaria cirrhosa, increasing its medicinal ingredients and enriching the resources of heredity breeding. METHODS: By using of low-temperature stratification treatment, the seed of Fritillaria cirrhosa completed its after-ripening of physiology and morphology. The induction treatment by different concentrations of colchicine solution and different treatment times for Fritillaria cirrhosa seeds were compared. RESULT: Detected the plant morphology and chromosome number, it is shown that the induced material obviously possessed the characteristics of polyploid. CONCLUSION: With the treatment of 30 mg/L GA3 for 32 h and stratification for 70 d, the seed germination rate of Fritillaria cirrhosa reached 67.0%. After treated with 0.1% colchicine solution for 48 h, the stratificationed mature seed showed polyploid inductivity of 85.7%.

[Preliminary study of fertilizer effect on yield and quality of Fritillaria ussuriensis].

OBJECTIVE: To preliminarily study the effect of fertilizer methods on the yield and quality of Fritillaria ussuriensis. METHOD: Through the investigation of the available nitrogen, phosphorus, potassium in soil fertility of F. ussuriensis planting field as well as the measurement of total nitrogen, phosphorus, potassium contents in bulbus of F. ussuriensis, the balanced fertilization plan for cultivation of F. ussuriensis was made. RESULT: The optimal fertilizing amount was 13.0 kg x m(-2) pig dung, or 5.5 kg x m(-2) deer dung and 6.5 kg x m(-2) pig dung, or 11.0 kg x m(-2) deer dung. CONCLUSION: Effective fertilizing may increase significantly the yield and quality of F. ussuriensis.

[Effect of temperature on physiologic indexes, bulb yielding and total alkaloids contents of Fritillaria cirrhosa].

OBJECTIVE: To study the physiologic indexes, yield and the contents of alkaloids of Fritillaria cirrhosa D. Don under different temperature. METHOD: The growth temperatures (15, 20, 25, 30 degrees C) of F. cirrhosa were controlled by using artificial climate, the growth was observed, the contents of chlorophyll a and b, soluble sugar, MAD, proline of the leaves of F. cirrhosa were tested, and the yield and the alkaloids content of the bulbs were analyzed. RESULT: The growth period of F. cirrhosa under 15, 20 degrees C were appropriately extended. The difference of the content of leaves chlorophyll b under four temperatures and the contents of total chlorophyll and chlorophyll a under 15, 20, 30 degrees C were not significant. The contents of soluble sugar, MAD and proline of leaves and the growth ratio, dry weight and content of alkaloids of bulb increased with the temperature decrease. CONCLUSION: Higher temperature is not suitable for the growth of F. cirrhosa. Under the relatively lower temperature, the growth period of F. cirrhosa extended, the bulb can grow properly, and the content of alkaloid increased. F. cirrhosa can improve its cold tolerance by increasing the content of proline and soluble sugar, and it also can maintain the normal content of chlorophyll under the lower temperature.

[Research progress on resources in Bulbus Fritillariae Cirrhosae].

The paper reviews the recent developments in germplasm, identification, ecological factors and cultivation of bulbus fritillariae cirrhosae. During past years, new methods for identification such as mass spectrometry (MS), polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and DNA chips have been invented and identification works became easier. The relationship between ecological factors such as climate, soil, community types and the quality of bulbus fritillariae cirrhosae has been revealed, thus a new cultivation model "semi-wild cultivation" have been developed and it is beneficial to material conservation. The present problems and the future research direction on bulbus fritillariae cirrhosae were also discussed.

[Study on bulblet induction of Fritillaria anhuiensis in vitro].

OBJECTIVE: To study the optimal condition for bulb induction of Fritillaria anhuiensis in vitro. METHOD: Effects of sucrose, salicylic acid, active carbon and 5 degrees C pre-incubation on bulb formation in vitro were investigated by adopting the number of bulb and fresh weight as indexes. RESULT: The number of bulb and fresh weight in medium added with 50 g x L(-1) sucrose were significantly higher than those with other treatments. The medium with different concentrations of salicylic acid showed no significant promotion on callus differentiation. However, 5 g x L(-1) active carbon (AC) treatment was better than other treatments. Meanwhile, a large number of plantlets formed after 5 degrees C pre-incubation for 30-40 days was suitable for bulb formation and growth. CONCLUSION: Bulblet formation was optimal in MS + KT 2 mg x L(-1) + NAA 2 mg x L(-1) + 50 g x L(-1) sucrose +5 g x L(-1) AC after callus pre-incucation at 5 degrees 1 for 30-40 days.