An Efficient Plant Regeneration System for Different Explants of Rare and Endangered Plants in Mussaenda anomala

To establish an efficient regeneration method for the rare and endangered plant Mussaenda anomala to address problems regarding its reproductive obstacles and scarce populations.In this study,the terminal buds,axillary buds,stem segments with two axillary buds,stem segments with two axillary buds and one terminal bud,and leaves of M.anomala were used as explants.The effects of different explants and disinfection methods,plant growth regulators and substrates on plant regeneration were explored.The following results were obtained:(1)The terminal bud was a suitable explant for M.anomala tissue culture,and the disinfection method utilized was treatment with 0.2%HgCl2 for 8 min.(2)Initiate medium:MS basic medium supplemented with 0.5 mg/L 6-BA and 0.2 mg/L IBA for the high germination rate(100%)and the maximum bud height(1.70 cm)of the terminal bud.(3)Proliferation medium:MS basic medium supplemented with 3.0 mg/L 6-BA and 0.2 mg/L IBA for a high proliferation rate(96%)and proliferation time(6.0)of terminal buds.(4)Proliferation medium supplemented with 0.7 mg/L GA3 significantly increased the bud heights of multiple buds.(5)Rooting medium:MS basic medium supplemented with 0.5 mg/L IBA and 0.5 mg/L IAA for a high rooting rate(88%),root number(12.0)and root length(5.07 cm).(6)The optimal substrate for seedling acclimation and transplanting was perlite:vermiculite(1:1),which resulted in the highest survival rate(97%)and plant height(5.89 cm),as well as better growth potential for seedlings.The surfaces of M.anomala explants are densely covered with trichome,which increased the difficulty of disinfection;the plant growth regulators directly affected the growth and development in the regeneration process of M.anomala,and the substrate significantly affected the survival rate and height growth for seedling acclimation.

玉叶金花(Mussaenda pubescens)SSR引物的快速开发

利用链亲和素磁珠吸附法捕捉能与生物素标记的探针(AC)15退火结合的玉叶金花(Mussaenda pu-bescens)微卫星序列的单链限制性酶切片段.获得的单链目的片段,经PCR扩增形成双链,然后克隆到pGEMT载体上,转化至DH5α中,得到一个微卫星序列文库.经测序统计,引物得率为12.5%;根据序列设计的SSR两侧引物PL和PR,共得到5对有多态性的SSR引物.玉叶金花微卫星引物的筛选将为下一步进行玉叶金花基因组结构的分析、分子进化和系统发育研究提供重要的微卫星标记.

Determination of Chlorogenic Acid in Stems of Mussaenda pubescens Ait.f.by High Performance Liquid Chromatography

[Objectives] This study was conducted to establish a method for determination of chlorogenic acid in Mussaenda pubescens Ait.f.[Methods]HPLC used SHISEIDO C_(18) MG Ⅱ column( 5 μm,4.6 mml.D.× 250 mm) as chromatographic column and acetonitrile-0.4% phosphoric acid solution( 9∶91) as mobile phase.The separation was performed at a flow rate of 1.0 ml/min and a column temperature of 30 ℃,and the detection wavelength was set at 327 nm.[Results]Chlorogenic acid had a good linear relation in the range of 0.254 7-2.547 5 μg( R2= 0.999 9).The recovery rate was 97.8%,RSD = 1.82%( n = 9).[Conclusions]The content of chlorogenic acid in M.pubescens was determined by ultrasonic extraction and HPLC.The method was simple,stable and reliable and could be used for the quality control of M.pubescens.

A New Bis-iridoid Glucoside from Mussaenda incana

A new bis-iridoid glucoside, incaside 1 was isolated along with known iridoid, 6-0- veratroylcatalposide from the stem-bark of Mussaenda incana. The structure of the new iridoid was established mainly on the basis of the spectroscopic （including 2D NMR） and chemical studies of its octaacetate 1a.

NEW TRITERPENOIDAL SAPONINS FROM MUSSAENDA PUBESECUS

A cyclolanostene-type triterpene and two saponins derived thereform, namely MP-C,D and F,were isolated from the acid-hydrolyzate of the mixture of saponins of Mussaenda pubesecus.Their structures were elucidated on the basis of chemical and UV,IR,~1H,^(13)C NMR and MS spectral evidence.The two new saponins MP-D and F were named Mussaendoside A and C,respectively.

NEW SAPONIN MUSSAENDOSIDE M FROM MUSSAENDA PUBESECUS

The structure of a new saponin mussaendoside M obtained from Mussaenda pubesecus was identified by FAB-MS and 2D-NMR spectroscopy.

Mussaenda lancipetala X.F.Deng&D.X.Zhang,a new species of Rubiaceae from China

报道了在云南发现的玉叶金花属Mussaenda一新种——狭瓣玉叶金花M. lancipetala X. F. Deng & D. X. Zhang。狭瓣玉叶金花的性系统为柱头缩入式雌雄异位; 花冠裂片卵状披针形且具有细长的长尖头, 花冠管外被有粉末状的毛被, 可以很容易与玉叶金花属其他种类相区别。

Pre-and post-zygotic reproductive isolation between co-occurring Mussaenda pubescens var.alba and M. shikokiana(Rubiaceae)

Reproductive isolation is a fundamental require- ment for speciation and includes several sequential stages. Few studies have determined the relative contributions of pre- and post-zygotic reproductive isolation in plants, especially between relative species with clear differentiation in flower form. To investigate the mechanisms responsible for repro- ductive isolation in sympatric A4ussaenda pubescens var. alba and Mussaenda shikokiana （Rubiaceae） in Guangxi Province, China, we made observations of flowering phenology, patterns of insect visitation, and conducted pollination experiments, including artificial hybridization. The two species had over- lapping flowering times and were pollinated by overlapping pollinators; however, their relative importance differed significantly with M. pubescens visited more commonly by bees and M. shikokiana more frequently by butterflies. Using vegetative and floral characters and molecular evidence based on nuclear ribosomal internal and external transcribed spacer regions we detected seven naturally occurring hybrids among a sample of approximately 125 individuals. Hybrids were characterized by morphologies that most closely resembled their maternal parents based on chloroplast evidence. Studies of artificially synthesized and natural hybrids demonstrated that hybrid seed had very low germination rates and naturally occurring hybrids exhibited pollen sterility. Post-zygotic reproductive isolating mechanisms play a primary role in limiting gene exchange between co-occurring species and maintaining species integrity in areas of sympatry.

Reproductive isolation between sympatric sister species, Mussaenda kwangtungensis and M. pubescens var. alba

Reproductive isolation defines the biological species concept and plays a key role in the formation and maintenance of species. The relative contributions of different isolating stages has been suggested to be closely associated with phylogenetic relatedness. Few studies have focused on the relative contributions of pre- versus post-zygotic mechanisms, and even fewer have been conducted under strict phylogenetic frameworks. Pre-and post-zygotic reproductive isolation stages have been investigated in the sister species Mussaenda kwangtungensis and M. pubescens var. alba. The two species have partly overlapping distribu-tion ranges and flowering times, while the principal pollinators differed strikingly for them, demonstrating strong pre-zygotic isolations. Natural hybrids were detected by simple sequence repeat markers and their maternal parents were identified based on chloroplast gene sequen-ces. Five out of 81 individuals were suggested to be hybrids that fall into the categories F2, BC1, and BC2 by the NewHybrids analysis. Interspecific crossings resulted in significantly reduced fruit set and seed germination rates. Phylogenetic analysis revealed short Kimura-2-parameter distance between M. kwangtungensis and M. pubescens var. alba. These findings strongly supported the hypothesis that for species with a closer phylogenetic relationship, pre-zygotic isolation plays an important part in limiting gene exchange in sympatric areas.

不同产地玉叶金花质量比对研究

目的:对湖南桃源、广西玉林、广东茂名三地的玉叶金花中药材进行质量比对研究。方法:根据各地玉叶金花中药材质量标准,通过状性鉴别、薄层鉴别、显微鉴别、理化鉴别、含量测定等方法,鉴定各地玉叶金花药材质量。结果:三地的玉叶金花在薄层鉴别、理化鉴别、显微鉴别方面差别不大,外观性状方面叶面有稍微差别。湖南桃源玉叶金花中的新绿原酸、绿原酸、隐绿原酸、异绿原酸A、异绿原酸B、异绿原酸C的总量最低,为0.598%,而广西玉林和广东茂名玉叶金花中这些成分总含量较高,分别为0.986%和1.098%。结论:通过对三地玉叶金花中药材质量进行比对,可为玉叶金花中药材的鉴别、种植、质量评价提供参考。

基于UPLC-Q-TOF-MS/MS和分子网络技术快速分析玉叶解毒颗粒醇提物的化学成分

目的采用超高效液相色谱-四极杆-飞行时间串联质谱(UPLC-Q-TOF-MS/MS)整合分子网络(Molecular Networking)策略对玉叶解毒颗粒的化学成分进行快速分析与鉴定。方法根据正负离子模式下质谱扫描获取化合物二级质谱数据文件,计算MS/MS碎片模式的相似性并创建全球天然产物社会分子网络(GNPS),根据分子网络中相似结构的分子簇对玉叶解毒颗粒中的各大类成分进行快速鉴定。对于在分子网络中未体现的化合物则采用Agilent Masshunter Qualitative Analysis工作站中的分子特征提取(MFE)功能和中药成分数据库(TCM-DATA),根据一级、二级质谱数据和相关参考文献进行人工解析鉴定。结果通过液质分析从玉叶解毒颗粒中共鉴定出89个化学成分,其中酚酸类化学成分22个,黄酮及其苷类化学成分21个,环烯醚萜苷类化学成分6个,三萜皂苷类化学成分28个,其他类化学成分12个。结论采用UPLC-Q-TOF-MS/MS整合分子网络技术可对玉叶解毒颗粒进行快速和系统的化学成分识别,为其质量控制和临床应用提供了理论依据,所建立的分子网络能为中药复方成分的快速定性分析提供借鉴。

玉叶金花属及其近缘属植物的叶脉序特征研究

玉叶金花属(Mussaenda L.)种间变异式样复杂,属内杂交现象突出,其分类一直备受争议。为探索玉叶金花属及其近缘属裂果金花属(Schizomussaenda Li)、假玉叶金花属(Psudomussaenda Wernham)叶脉序特征在属间及种间分类学意义,从而为其作为药用以及园林植物资源的鉴定、开发利用提供依据,该研究采用清净法制作叶脉标本,观察了22种该类群的叶脉序特征,基于叶脉序特征进行了聚类分析,并编制了分种检索表。结果表明:(1)玉叶金花属及其近缘属叶脉序特征具有一致性,一级脉都为羽状脉,间二级脉频度均为每个脉间区少于一条,粗二级脉与中脉夹角均为锐角,三级脉多为贯穿型,脉间区排列均不规则。(2)属间或属内种间有分类学价值的叶脉序特征为粗二级脉脉序类型、粗二级脉与中脉夹角、粗二级脉间距、粗二级脉与中脉连接方式、间二级脉及近缘二级脉的存在与否、三级脉贯穿类型、四级脉类型、游离端小脉分支情况、脉间区发育情况。(3)聚类分析显示该类群22种植物聚为12支,聚类分析中,裂果金花属及假玉叶金花属嵌入玉叶金花属内,显示了较近的亲缘关系。综上可知,玉叶金花属及其近缘类群的叶脉序特征可以为其分类提供新的研究资料。

玉叶金花丛枝症病害生理学初探

为探究玉叶金花(Mussaenda Pubescens Ait.f.)丛枝症病原物的致病机理,同时也为该病的控制提供生理生化依据,对健康和患有丛枝症的玉叶金花植株叶片的叶绿素、可溶性糖、可溶性蛋白质、保护酶活性、次生物质等10项生理生化指标进行了测定。结果表明,感病的玉叶金花植株的代谢发生了明显变化,与对照相比叶绿素含量和可溶性糖含量分别下降33.62%、37.80%,差异达极显著水平;可溶性蛋白质含量下降6.68%;过氧化氢酶(CAT)、过氧化物酶(POD)活性分别升高27.26%、109.85%;总酚和单宁含量分别升高81.93%、33.24%。

玉叶金花总三萜类成分含量测定方法建立

目的：建立玉叶金花总三萜类成分含量的测定方法。方法：以熊果酸作对照品,采用L9（34）正交设计优选显色条件,并测定玉叶金花药材中总三萜类成分的含量。结果：最佳显色条件为：5%香草醛-冰醋酸溶液0.6 m L,高氯酸0.6 m L,70℃,反应15 min;该方法在0.021-0.16 mg含量范围内线性关系良好（R=0.9995）,当显色反应完全后对照品和供试品液在70 min内稳定性良好,精密度高（RSD=2.00%,n=5）,平均回收率为100.08%（RSD=1.18%,n=6）。结论：香草醛比色法适合玉叶金花总三萜类成分的含量测定,可用于其药材的质量控制。

3种常见茜草科药用植物化感作用的生物测定

以南方常见的3种茜草科药用植物鸡屎藤〔Paederia scandens(Lour.)Merr.〕、剑叶耳草(Hedyotis caudatifolia Merr.etMetcalf)、玉叶金花(Mussaenda pubescens L.)为材料,采用常规水浸提方法制备不同浓度的水提液,用生物测定法测定比较其对萝卜(Raphanus sativus L.)的化感作用,并通过测定对受体根系活力和叶片游离脯氨酸、叶绿素含量的影响,初步探讨其化感作用机理。结果表明:水提液浓度不同,其化感效应不同;供试植物不同,化感效应差别也较大,浓度较低时,化感作用较小;具体表现为,3种供试植物化感作用顺序为:鸡屎藤>剑叶耳草>玉叶金花;鸡屎藤水提液处理显著降低了受体萝卜发芽率、发芽速率指数和叶绿素含量,显著提高了受体萝卜幼苗叶片脯氨酸含量。

玉叶金花的生药鉴定

对玉叶金花进行药材性状、显微特征、薄层色谱及紫外光谱鉴别。为该药材的鉴别和开发利用提供依据。

3种茜草科药用植物乙醇浸提液对萝卜种子萌发及幼苗生长的化感作用

以3种茜草科药用植物鸡屎藤〔Paederia scandens（Lour.）Merr.〕、剑叶耳草（Hedyotis caudatifolia Merr.et Metcalf）、玉叶金花（Mussaenda pubescens L.）为试材,采用乙醇提取的方法制备不同浓度的乙醇提液,通过测定其对受体萝卜（Raphanus sativus L.）发芽率、根系活力、叶片游离脯氨酸和叶绿素含量的影响,比较化感作用强弱。结果表明：不同供试植物乙醇提液其化感效应不同,且浓度高时,化感作用强;供试植物乙醇提液均显著降低了萝卜发芽率、发芽速率指数,且剑叶耳草在这两个指标的抑制作用显著高于另二者;供试植物乙醇提液均极显著抑制萝卜胚根和胚芽的生长,其中鸡屎藤乙醇提液抑制胚芽的作用最强,玉叶金花提液抑制胚根的作用最强;鸡屎藤比玉叶金花更显著地抑制萝卜幼苗根系活力,然而剑叶耳草却显著地促进;供试植物乙醇提液显著降低萝卜幼苗叶绿素含量,显著提高叶片脯氨酸含量;鸡屎藤乙醇提液使叶绿素含量减少69.47%~87.00%,且叶绿素a/叶绿素b随处理浓度的增加而降低。

玉叶金花水提物不同萃取部位的抗炎活性研究

目的从玉叶金花水提物不同萃取部位筛选出活性部位。方法采用二甲苯致小鼠耳郭肿胀实验模型及角叉菜胶致大鼠足跖肿胀实验模型对玉叶金花水提物不同萃取部位进行抗炎活性的考察。结果玉叶金花水提物乙酸乙酯萃取部位、正丁醇萃取部位及水溶性部位可显著减轻二甲苯致炎小鼠的耳肿胀度(与空白对照组比较,P<0.05)及由角叉菜胶引起的大鼠1,2,3 h的足肿胀度(P<0.05),石油醚萃取部位则作用不显著。结论乙酸乙酯萃取部位、正丁醇萃取部位及水溶性部位有抑制急性炎症的作用,为玉叶金花水提物抗炎活性部位。

中国玉叶金花属一新记录种——长瓣玉叶金花

报道中国茜草科(Rubiaceae)玉叶金花属一新记录种——长瓣玉叶金花(Mussaenda longipetala H.L.Li)。该种在形态上与尾裂玉叶金花(M.caudatiloba D.Fang)和狭瓣玉叶金花(M.lancipetal X.F.Deng&D.X.Zhang)相似,但区别在于叶片长圆状卵形或椭圆状卵形,托叶早落,正常的萼裂片长6～8 mm,花冠管长约3 cm,裂片披针形,长约12 mm。目前仅知分布于越南北部的广宁省和中国的广西北部湾沿海地区。凭证标本存放于广西植物标本馆(IBK)。

珍稀濒危植物异形玉叶金花愈伤组织的诱导

为珍稀濒危植物异形玉叶金花种质资源的繁殖、保存与利用提供依据,以幼嫩叶片为外植体,采用L16（45）正交试验设计研究不同浓度2,4-D、6-BA、暗培养时间及叶片划痕数等因子对异形玉叶金花叶片诱导愈伤组织的影响。结果表明：各因素对愈伤组织诱导率的影响依次为2,4-D〉暗培养时间〉6-BA〉划痕数;异形玉叶金花叶片诱导愈伤组织各因素最佳组合为1.5mg/L 2,4-D＋0.3mg/L 6-BA,划痕数5~7条,暗培养20d。该条件下愈伤组织的诱导率达91.6%。