In Vitro Propagation and Artificial Seed Production of Fritillaria cirrhosa D. Don, an Endangered Medicinal Plant

Fritillaria cirrhosa D.Don(Liliaceae)is an endangered perennial bulbous plant and its dry bulb is a valuable med-icinal material with antitussive and expectorant effects.Nevertheless,lack of resources and expensive prices make it difficult to meet clinical needs.This study presents a regeneration system aimed at overcoming the challenge of inadequate supply in F.cirrhosa,focusing on:(1)callus induction,(2)bulblets and adventitious bud induction,and(3)artificial seed production.Callus development was achieved in 84.93%on Murashige and Skoog(MS)medium fortified with 1.0 mg·L^(-1) picloram.The optimal medium for callus differentiation into regenerated bulb-lets was MS medium supplemented with 1.0 mg·L^(-1)6-benzyladenine(6-BA)and 0.2 mg·L^(-1)α-naphthaleneacetic acid(NAA).Subsequently,bulblets and adventitious buds were induced from regenerated bulblet sections cul-tured on MS medium fortified with 0.3 mg·L^(-1)6-BA+1.0 mg·L^(-1)2,4-dichlorophenoxyacetic acid(2,4-D),2.0 mg·L^(-1)6-BA+0.5 mg·L^(-1),and the induction rates were 88.17%and 84.24%,respectively.The regenerated bulblets were transplanted into a substrate of humus soil,river sand,and pearlite(1:1:1)after low-temperature treatment.The germination rate was 42.80%after culture for 30 days.Regenerated bulblets were used for encap-sulations in liquid MS medium containing 3%sucrose(w/v)+0.5 mg·L^(-1) NAA+2.0 mg·L^(-1)6-BA+3%sodium alginate(w/v)with a 10 min exposure to 2%CaCl_(2).Under non-aseptic conditions,the germination rate reached 81.67%,while the rooting rate was 20.56%after 45 days.The capsule added 1.0 g·L^(-1) carbendazim and 1.0 g·L^(-1) activated carbon was the best component of artificial seeds.This study successfully established an efficient regen-eration system for the rapid propagation of F.cirrhosa,involving in vitro bulblet regeneration and artificial seed production.This method introduces a novel approach for efficient breeding and germplasm preservation,making it suitable for large-scale industrial resource production.

Effects of Potassium-Solubilizing Bacteria on Growth, Antioxidant Activity and Expression of Related Genes in Fritillaria taipaiensis P. Y. Li

This study aimed to examine the effects of inoculating Fritillaria taipaiensis P.Y.Li leaves with different strains ofpotassium-solubilizing bacteria (KSB), or combinations thereof, focusing on aspects of photosynthesis and physiologicaland biochemical characteristics. At present, some studies have only studied the rhizosphere microbialcommunity characteristics of F. taipaiensis and have not discussed the effects of different microbial species on thegrowth promotion of F. taipaiensis. This paper will start from the perspective of potassium-solubilizing bacteria toconduct an in-depth study. Seed cultivation commenced at the base with three different KSBs in early October2022. The growth of F. taipaiensis leaves was observed after different treatments. Both single-plant and compoundinoculations were executed. A total of eight treatment groups were established, with aseptic fertilizer and sterilizedsoil functioning as the control group. The results reveal that intercellular CO_(2) concentration (Ci), stomatal conductance(Gs), and transpiration rate (Tr) were at their apex in the S7 group. Most treatment groups exhibited anincrease in leaf area, photosynthetic pigment content, soluble sugar, soluble protein, Superoxide Dismutase(SOD), Peroxidase (POD), Catalase (CAT) activities, and proline content. The expression levels of POD, SOD,and CAT genes were evaluated, following inoculation with different KSB. The highest was the S7 group. Theinoculation with various KSB, or combinations thereof, appears to bolster the growth and development of F. taipaiensis.The composite inoculation group S7, comprising Bacillus cereus, Burkholderia cepacia, and Bacillus subtilis,manifested the most favorable impact on the diverse indices of F. taipaiensis, thereby furnishing valuableinsights for the selection of bacterial fertilizer in the artificial cultivation of F. taipaiensis.

Effects of Inoculation with Phosphate Solubilizing Bacteria on the Physiology,Biochemistry,and Expression of Genes Related to the Protective Enzyme System of Fritillaria taipaiensis P.Y.Li

Fritillaria taipaiensis P.Y.Li is a widely used medicinal herb in treating pulmonary diseases.In recent years,its wild resources have become scarce,and the demand for efficient artificial cultivation has significantly increased.This article is the first to apply phosphate solubilizing bacteria isolated from the rhizosphere soil of F.taipaiensis P.Y.Li to the cultivation process of F.taipaiensis P.Y.Li.The aim is to identify suitable reference strains for the artificial cultivation and industrial development of F.taipaiensis P.Y.Li by examining the effects of various phosphate solubilizing bacteria and their combinations on photosynthesis,physiological and biochemical properties,and gene expression related to the protective enzyme system in F.taipaiensis P.Y.Li.The experiment,conducted in pots at room temperature,included a control group(CK)and groups inoculated with inorganic phosphorussolubilizing bacteria:W1(Bacillus cereus),W2(Serratia plymuthica),W12(Bacillus cereus and Serratia plymuthica),and groups inoculated with organophosphorus-solubilizing bacteria:Y1(Bacillus cereus),Y2(Bacillus cereus),Y12(Bacillus cereus and Bacillus cereus),totaling seven groups.Compared to CK,most growth indices in the bacterial addition groups showed significant differences,with W12 achieving the highest values in all indices except the leaf area index.The content of photosynthetic pigments,photosynthetic parameters,and osmoregulatory substances increased variably in each bacterial treatment group.W12 exhibited the highest content of chlorophyll a and soluble protein,while W1 had the highest free proline content.The activities of peroxidase(POD),superoxide dismutase(SOD),and catalase(CAT)in all inoculated groups were higher than in CK,with significant changes in SOD and CAT activities.The malondialdehyde(MDA)content in all inoculated groups was lower than in CK,with Y12 being the lowest,at approximately 30%of CK.Gene expression corresponding to these three enzymes also increased variably,with POD expression in Y2 being the highest at 2.73 times that of CK.SOD and CAT expression in Y12 were the highest,at 1.84 and 4.39 times that of CK,respectively.These results indicate that inoculating phosphate solubilizing bacteria can enhance the growth of F.taipaiensis P.Y.Li,with the mixed inoculation groups W12 and Y12 demonstrating superior effects.This lays a theoretical foundation for selecting bacterial fertilizers in the cultivation process of F.taipaiensis P.Y.Li.

Effects of Different Seed Stem Sizes on the Changes of Available Elements in Rhizosphere Soil of Fritillaria thunbergii Miq.

[Objectives]This study was conducted to screen suitable seed stems of Fritillaria thunbergii Miq.from different provenances and to provide a theoretical basis for the high-yielding and high-efficiency cultivation of F.thunbergii Miq.introduced to different places.[Methods]F.thunbergii Miq.from four different provenances including Zhejiang,Nantong and Chongqing were introduced and cultivated in Wanzhou of Chongqing.The contents of available Zn,Fe,Mn,Cu,Mo,N,P,K,Ca and Mg in rhizosphere soil of F.thunbergii Miq.during five growing stages were determined after selecting different stem sizes for field cultivation.[Results]Small stems of Pan an and Ningbo provenances(SSG3,121-160/kg)and middle stems of Nantong and Fengjie provenances(SSG2,81-120/kg)showed higher soil availability.[Conclusions]In the process of introduction and cultivation of F.thunbergii Miq.,high yield and high efficiency can be achieved by selecting smaller seed stems of F.thunbergii Miq.

Studies on the Chemical Constituents of Four Species of Fritillaria Plants

The bulbs of Fritillaria spp.(Liliaceae)are used in Chinese medicine as antipyretic,anti-tussive,expectorant,etc.In order to search for the bioactive constituents from this genus,thebulbs of four species of medicinal Fritillaria plants have been investigated.Thirteen compoundswere isolated from Fritillaria thunbergii Miq.,three of them were new compounds.Fourteencompounds from Fritillaria thunbergii var.chekiangensis Hsiao et K.C.Hsia collected inDongyang of Zhejiang Province,four of them are new compounds and another one was first iso-

遮荫下高原濒危药用植物川贝母(Fritillaria cirrhosa)光合作用和叶绿素荧光特征

测量了川贝母遮荫和全光下叶片光合生理参数和叶绿素荧光参数,探讨了其遮荫下生理生态学指标的变化和强光下的光适应机制,且对光合生理的测量过程给出了全面系统的介绍。川贝母日光合曲线呈双峰变化,遮荫后川贝母光合效率(Pmax)、细胞内CO2浓度(Ci)分别增加了31.1%、10%(p<0.01),有效光反应光强域值(PFD)增加了331.5[μmol/(m2.s)],最大表观量子效率(AQY)和气孔导度(COND)也有所增加,暗呼吸速率(Rd)和叶片蒸汽压亏缺(Vpdl)明显降低,此均有利用光合产物的积累;F′v/F′m(光照下反应中心能量捕获效率)、qP(光化学淬灭)、ETR(电子传递效率)、Psips2(PSⅡ的效率)分别增加了14.7%(p<0.01)、8.8%(p<0.01)、10%(p<0.01)、24.2%(p>0.05),促进了川贝母叶片对光能的利用;Fv/Fm(光化学效率)变化不明显,说明"灯笼花"阶段,全光下川贝母没有受到到明显的胁迫,光合机构未遭到破坏。当自然光强超过光饱和点时,川贝母主要通过提高非化学淬灭,耗散过多吸收的热能以防止光合机构的破坏。当出现极端气候(高温干旱)时,川贝母繁育退化现象("树儿子"或"灯笼花"阶段退回到"一匹叶"生长阶段)可能是对环境适应的主要方式。另外川贝母叶片狭小、叶片倾角较大降低了强光辐射的有效面积,在形态结构上有利于避免过高光强对叶光合器官的损伤。

Classification of Chinese Traditional Drug-"Beimu" (Bulbus Fritillariae) by Pyrolysis High Resolution Gas Chromatography-Pattern Recognition

The combination of pyrolysis high resolution gas chromatography and pat- tern recognition techniques is a powerful tool for the classification of traditional Chinese drug.A study has been completed on 55 Beimu samples of five different geographic origins: Eastern China.Central China.South-western China,North-western China and North-eastern China.Principal component analysis and SIMCA are applied to effectively classifying the samples according to the origin of the plants.The chemical information contained in the high resolution gas chromatographic data is sufficient to characterize the geographic origin of sam- pies.

Antitumor Actinity of Puqietinone,a Novel Alkaloid fromthe Bulbs of Fritillaria puqiensis

从蒲圻贝母中分得的一种新生物碱蒲贝酮碱，按一定剂量灌胃给药，显示了强的抗小鼠艾氏腹水癌（ＥＡＳ，实体型），宫颈癌（Ｕ＿（１４），实体型）及肝癌（ＨｅＰＡ，实体型）的活性。

秋水仙素诱导川贝母(Fritillaria cirrhosa D.Don)愈伤组织多倍体的研究

应用组织培养技术对川贝母 (Fritillaria cirrhosa D.Don)进行多倍体诱导。结果表明 :组织培养条件下 ,将川贝母愈伤组织在培养基中添加一定浓度的秋水仙素处理一段时间 ,或者经一定浓度的秋水仙素浸泡一段时间后再培养 ,均可诱发川贝母多倍体的产生 ,但以前者效果较好 ,在秋水仙素浓度为 10 0 0 mg/ L ,处理 5 d的条件下 ,诱导率最高达 70 %。细胞染色体鉴定结果为 :四倍体染色体数为 2 n=4x=48,而二倍体的染色体数为 2 n=2 x=2 4。

甘肃马唧山地区榆中贝母(Fritillaria yuzhongensis)的生态学特性及种子发芽规律的初步研究

榆中贝母(Fritillaria yuzhongensis)生长于马啣山的高山地带,全生育期约100 d左右,是一种生育期较短的类短命植物。在果实成熟时,种子的胚发育不全,具有后熟作用的特点,包括形态后熟和生理后热两个阶段;因此,需要一定时期的低温处理,才能完成后熟过程,以解除休眠,促使种子发芽。本试验表明:榆中贝母完成形态后熟过程的最佳温度是7℃左右,经过100d以上的低温处理才能使原胚发育成熟,继续在7℃低温下处理70d以上,才能完成生理后熟过程。榆中贝母种子休眠时间长,需要经过170d以上的低温处理,种子才能萌发。本试验结果为榆中贝母种子的萌发提出了适宜的温度范围和低温处理时间,为提高种子的出苗率和缩短栽培年限提供了科学依据。

Cytotoxic effect of methanolic extracts of Fritillaria imperialis bulbs and Eryngium caucasicum leaves on hepatoma and colon cancer cells

Objective: To evaluate antitumor activities of Fritillaria imperialis and Eryngium caucasicum methanolic extracts on human hepatoma (HepG2) and colon cancer (HCT116) cell lines in comparison to human foreskin fibroblasts as the normal cells. Methods: Methanolic extracts of Fritillaria imperialis and Eryngium caucasicum were prepared by the maceration method. The effect of the extracts at various concentrations (100, 200, 400, 600, and 800 μg/mL) on cell survival was evaluated using the MTT method. Besides, fluorescence staining was used to evaluate death patterns of the cells. Results: MTT assay showed that Fritillaria imperialis significantly decreased the viability of all cell lines after 24 and 48 hours of treatments. However, Eryngium caucasicum extract did not show any significant cytotoxicity effect on the cell lines. Fluorescence staining revealed that Fritillaria imperialis induced apoptosis of HCT116 cells at 550 μg/mL. Conclusions: Fritillaria imperialis extract has antiproliferative and cytotoxic effects on HCT116 and HepG2 cancer cells and therefore, may serve as an anticancer agent.

Steroidal alkaloids from the bulbs of Fritillaria monatha

Two new Steroidal alkaloids, named pengbeisine A and pengbeisine B, were isolated from the fresh bulbs of Fritillaria monatha Migo. along with a known alkaloid. The structures were characterized as 22,26-imino-17, 23-oxido-5α-jerv-6-oxo-2α,3β-diol and 22, 26-imino-17, 23-oxido-5α-jerv-6-oxo-2β,3α-diol on the basis of spectroscopic methods.

A new veratramine alkaloid from the bulbs of Fritillaria hupehensis

A new highly conjugated alkaloid of veratramine type, 22S,25S,5α-veratramine-7(8),12(14)-diene-3β,13β,23β-triol-6-one (1), was isolated from the bulbs of Fritillaria hupehensis Hsiao et K.C. Hsia. Its structure was determined on the basis of spectroscopic evidences.

Analysis of Hupehenine in the Total Alkaloids from Fritillaria hupehensis by HPLC-ELSD

A reverse-phase high pressure liquid chromatography （HPLC） method with evaporative light scattering detection （ELSD） has been developed for the quantitative analysis of hupehenine in the total alkaloids from Fritillaria hupehensis. Samples were analyzed on a reverse-phase column （Hypersil C-18） with a mobile phase of methanol：water：chloroform： triethylamine （85：15：1：0.6）. The ELSD was set at the drift tube temperature of 68.3℃ and gas flow rate of 1.8 L/min. Hupehenine＇s retention time was 13.7 min with an asymmetry factor of 1.2. The validity of the method has been verified with linearity, limit of detection, accuracy and precision. The logarithmic linear curve was obtained from 8.936 to 134.04 μg/mL （r=0.9993）. The detection limit （S/N〉3） of hupehenine was 1.79 μg/mL on the column. Intra-day RSD was 1.42% and inter-day RSD was 2.26% （3 days within a week）. The average recovery of hupehenine was 101.50%, and RSD was 1.62%.

药用植物平贝母(Fritillaria ussuriensis)组织培养的研究——Ⅰ.愈伤组织的诱导和器官分化及细胞学观察

本文对平贝母(Fritillaria ussuriensis Maxim)根、鳞茎、叶等器官在 MS,N_6,B_5,WS 和 H5种基本培养基上的培养进行详细观察。通过球状体及不定芽的形态发生曲线及差异显著性测定表明,不同培养基与激素及不同激素浓度与组合,对平贝母器官发生的影响,筛选出诱导高频率球状体与不定芽发生的培养基 MS+1.0 ppmIBA+1.0 ppmZT,MS+0.5 ppmIAA+2.0ppmBA,MS+2.0ppm NAA+0.5 ppmKT。培养过程中球状体与不定芽的形态发生的组织细胞学观察结果表明,球状体发生经过鳞片原基阶段,而不定芽发生不经此阶段;球状体形成后发育成不定芽,但生长情况优于直接产生的不定芽。早期球状体的染色体数目为2n=24。

A new cycloartane triterpenoid from the leaves and stems of Fritillaria hupehensis

A new triterpenoid has been isolated from the leaves and stems of Fritillaria hupehensis Hsiao et K.C. Hsia. Its structure was established as （2329-9,19-cycloart-23-ene-3α,25-diol 1 through chemical and spectroscopic studies including 2D NMR. Another known triterpenoid 9,19-cycloart-25-ene-313,24ζ-diol 2 was also isolated.

Heavy collecting induces smaller and deeper Fritillariae Cirrhosae Bulbus in the wild

Fritillariae Cirrhosae Bulbus is a well-known traditional Chine medicinal herb. However, the demand for this herb is leading to over-collection and its decline in the wild. This study aims to investigate the ecological conditions of Fritillaria cirrhosa under the influence of human disturbance, biotic species interactions and climatic conditions. We established a total of 78 plots at 14 sites of F. cirrhosa in its distribution center, the Hengduan Mountains area. At each site, we estimated the abundance of F. cirrhosa at different distances from roads. The diameter and height of F. cirrhosa fruit and bulbs were measured and compared to underground bulb depth. We then analyzed the effects of environmental conditions and human disturbance on the abundance of F. cirrhosa using variance partitioning. We found that(1)abundance of F. cirrhosa and their underground bulb depth showed a significant linear increase with the distance from the main road;(2) the diameter/height of fruits and the diameter/height of fruits/bulbs showed significantly different responses to the human disturbance;(3) the community associates,climate and spatial conditions can explain 58%, 22% and 27%, respectively, of the variance in the F. cirrhosa abundance. These results highlight the fact that human disturbance and biotic factors have a great influence on the survival of F. cirrhosa, even more than climate conditions.

贝母属(Fritillaria)植物同物异名现象与思考

贝母属(Fritillaria)植物种类丰富,分布区域广泛。在国内外植物分类研究中,很多种类的分类学地位较为混乱,出现了大量同物异名的现象,为该属植物的研究造成巨大障碍。因此以《Flora of China》中记述的贝母属植物种类为基础,整理了我国近些年来不同学者对该属植物的新发现与新归并;并结合英国皇家植物园(邱园)的Plant List数据库中记录的贝母属植物种类,对该属植物的名称进行了详细的梳理,列举了存在"同物异名"问题的贝母属物种。为研究贝母属植物的分类及该属植物的开发与利用,提供科学参考。

Cloning of GF14 Gene from the Bulbs of Fritillaria crrishoa

GF14 widely occurs in plant species functioning as a regulatory protein in cell signal transdution, including monoamine synthesis, exocytosis and cell cycle regulation. A partial sequence, 413 bp in length, is cloned from the bulbs of F. crrihosa by means of RT PCR technique. Sequence alignment with the BLAST program shows that it is highly similar to GF14 gene. A new full length cDNA, 1133 bp, is obtained by RACE techniques. It contains a major open reading frame of 262 amino acids, 5’and 3’ untranslated regions and a long Poly (A) tail. Sequence analysis of deduced GF14 protein from F. crrihosa indicates several conserved motifs of GF14 proteins are also found in sequence of GF14 protein of F. crrihosa.