野生坚龙胆及其组培苗中龙胆苦苷的含量分析

利用含有α-萘乙酸(NAA)、6-苄氨基嘌呤(6-BA)和激动素(KT)等激素的MS固体培养基,对野生坚龙胆进行组培苗诱导培养。同时,应用高效液相色谱技术对野生坚龙胆和其组培苗的根、茎和叶中的龙胆苦苷进行含量分析和比较研究。结果发现,野生坚龙胆中的龙胆苦苷主要储存于根部,而组培坚龙胆中龙胆苦苷在根中的含量甚微,主要集中在茎、叶部位,且在出根初期组培坚龙胆叶中的龙胆苦苷含量近似于或略大于野生坚龙胆根中的含量。研究结果提示,坚龙胆中的绿色组织是龙胆苦苷合成的部位,这些部位同时具有龙胆苦苷的储藏功能。     

栽培条叶龙胆根中龙胆苦苷含量差异的研究

采用高效液相色谱法,测定栽培条叶龙胆根中龙胆苦苷的含量,结果表明:花末期(9月3日)龙胆苦苷含量高于果期(9月18日)和枯萎期(10月10日);同一根系中二龄根的含量显著高于一龄根;有性后代个体之间龙胆苦苷含量差异显著,对条叶龙胆进行高含量育种研究是可行的。     

条叶龙胆离体根培养条件的初步研究

本试验对条叶龙胆(Gentiana manshurica)离体根生长的培养基种类、光、通气进行了试验,并在此基础上用正交试验法对影响离体根生长的7个主要成分进行了试验。找出了适合条叶龙胆离体根生长和鲜重增加的培养基是white+CH 50 mg/l,IBA 0.1 mg/l,基本培养中相应的VB_1改为0.5 mg/l,蔗糖30 g/l,pH4。     

滇龙胆不同部位裂环烯醚萜类物质含量比较分析

采用高效液相色谱法,对云南临沧云县滇龙胆不同部位龙胆苦苷、獐牙菜苦苷、当药苷含量进行分析.结果显示:(1)龙胆苦苷、獐牙菜苦苷、当药苷在滇龙胆根、茎、叶、花中均有分布;龙胆苦苷、獐牙菜苦苷在根部含量较高,分别为(35.88±5.89)mg/g和(1.35±0.33)mg/g;当药苷在花部含量最高,为(0.97±0.24)mg/g.(2)不同部位有效成分间具有显著(P<0.05)或极显著(P<0.01)的相关性.(3)相同海拔条件下,不同栽培方式下滇龙胆根部龙胆苦苷含量差异不显著(P>0.05),但茎部、叶部獐牙菜苦苷含量差异显著(P<0.05).     

滇龙胆中萜类物质积累的动态变化

滇龙胆（Gentiana rigescens）为传统中药材。采用单因素方差分析比较不同生长季节滇龙胆的根部及茎叶部位龙胆苦苷、獐牙菜苦苷及当药苷含量的动态变化。结果表明,在不同生长季节,滇龙胆的根部与茎叶部位3种有效成分的含量具明显差异。龙胆苦苷主要积累于根部;獐牙菜苦苷、当药苷主要积累于茎叶部位。相关性分析表明,龙胆苦苷含量受气候因子的影响,月平均温度与茎叶部位龙胆苦苷含量呈极显著负相关（R=-0.57,P〈0.01）,月降水量与根部、茎叶部位龙胆苦苷含量呈显著（R=-0.48,P〈0.05）或极显著（R=-0.74,P〈0.01）负相关;根中当药苷含量与獐牙菜苦苷含量呈极显著正相关（R=0.62,P〈0.01）,茎叶部位獐牙菜苦苷含量与当药苷含量呈显著正相关（R=0.38,P〈0.05）。研究结果表明,滇龙胆中龙胆苦苷含量受降水量和温度的影响;龙胆苦苷、獐牙菜苦苷及当药苷的含量变动具相关性;9-11月较适宜滇龙胆药材的采收。     

条叶龙胆离体根培养条件的初步研究

本试验对条叶龙胆(Gentiana manshurica)离体根生长的培养基种类、光、通气进行了试验,并在此基础上用正交试验法对影响离体根生长的7个主要成分进行了试验。找出了适合条叶龙胆离体根生长和鲜重增加的培养基是white+CH 50 mg/L, IBA 0.1 mg/L, 基本培养中相应的VB1改为0.5 mg／L,蔗糖30 g／L,pH4。     

苦龙胆酯苷的研究进展

苦龙胆酯苷是一种裂环烯醚萜苷类化合物,又称为苦龙苷或龙胆苦酯苷。其分子式为:C27H28O14,分子量为576.52,是已知最苦的裂环烯醚萜苷类化合物。目前已证明可从川东獐牙菜、印度獐牙菜、龙胆草以及辐花肋柱花中提取。辐花肋柱花是最新证明的可提取苦龙胆酯苷的植物。苦龙胆酯苷具有助消化,保肝,抗皮肤肿瘤,抗黑热病等药理活性。在古代印度传统医药以及中药藏药中,苦龙胆酯苷的来源植物是治疗消化系统相关疾病的一味常见的草药,如保肝、抗糖尿病等。在体内药代动力学研究中,兔静脉注射苦龙胆酯苷,在血中有较快的清除率(2.62±0.41 L/h/kg)和广泛的体内组织分布(1.08±0.44 L/kg);采用游离、脂质体和囊泡体形式给仓鼠用药具有明显保护肝肾功能且未发现明显不良反应。     

药用植物组织培养的研究——Ⅲ、三分三愈伤组织的分化对莨菪碱、东莨菪碱含量的影响

从三分三种子萌发的根、茎、叶和花药、种皮诱导的愈伤组织,均含有莨菪碱、东莨菪碱。其含量和生长速度均以茎、叶愈伤组织为最高。根、茎、叶愈伤组织,在培养基中分别加入各种植物激素,培养时表明:BA(6-苄基嘌呤)促进芽的分化,NAA促进根的分化,2,4-D则抑制根的分化,GA_3(赤霉酸)影响很小。获得了三种愈伤组织只分化根不分化芽(加NAA 1毫克/升)和只分化芽不分化根(每升中加BA0.2毫克和2,4-D 0.2毫克)的良好结果。分化的这些芽或根的诱导频率,以茎、叶愈伤组织为高。分化的芽或根中均含有莨菪碱及东莨菪碱,但其含量相应地比它们的未分化的愈伤组织约低1～4倍。薄层层析结果表明,愈伤组织中有6种生物碱,茎、叶愈伤组织中,还有一种在紫外光下显蓝色荧光的物质,而分化的根或芽及花药愈伤组织中只有4种生物碱。看来,在离体培养下的三分三愈伤组织是有莨菪碱和东莨菪碱合成的全能性。这两种生物碱似乎并非最终的代谢产物,它们可能参与了器官建成中的代谢过程。文中还讨论了莨菪碱和东莨菪碱的合成部位问题。     

龙胆叶肉原生质体再生愈伤组织的研究

从龙胆试管苗的叶肉细胞分离得原生质体。培养4—5天后能见到第一次分裂,一个月后形成肉眼可见的小愈伤组织。试管苗的低温预处理是龙胆叶肉原生质体能否分裂的重要条件;似乎只有体积小的原生质体能保持持续分裂。获得的愈伤组织正在诱导器官分化。     

龙胆酵素的发酵工艺优化及其生化功效研究

以龙胆草为发酵底物,龙胆苦苷为评价指标,采用正交实验优化龙胆酵素的发酵工艺,用高效液相色谱(HPLC)分析龙胆酵素中龙胆苦苷含量,并评估其生化功效。结果表明,最优龙胆酵素发酵工艺为菌株组合酿酒酵母加植物乳杆菌1∶1、接种量10%、发酵温度30℃、发酵时间72 h。在此优化工艺条件下,发酵液龙胆苦苷含量达到(1 823±21)μg/mL,其透明质酸酶抑制率、DPPH、·OH和O2-·自由基的清除能力分别达到89.2%、77.5%、70.2%和74.1%,表明该工艺的发酵液具有很好的抗敏和自由基清除功效。     

Stimulation of root and somatic embryo production in Euonymus europaeus L. by an inhibitor of polyamine biosynthesis

In vitro formation of roots and somatic embryos is obtained from cotyledon explants of a Spindle tree (Euonymus europaeus L.) cultured on two different media: a medium inducing callus formation and the production of roots, and a medium inducing callus formation, root and somatic embryo production. We studied the effects of -difluoromethylornithine (DFMO), a specific, irreversible inhibitor of ornithine decarboxylase (ODC) on root and somatic embryo production, growth and titers of putrescine in Euonymus explants and explant-derived calli. Early changes in putrescine levels were detected in both cultures before the visible emergence of roots or somatic embryos. DFMO rapidly inhibited putrescine accumulation and growth in non-embryogenic calli and highly stimulated rooting activity. DFMO partially inhibited putrescine accumulation in embryogenic calli. This inhibition had no effects on callus growth but significantly reduced the time of emergence of roots and highly stimulated somatic embryo production. The relationship among putrescine, putrescine metabolism, growth, root and somatic embryo formation is discussed.     

猕猴桃愈伤组织的生理差异与原生质体生长和分化的关系

美味猕猴桃和中华猕猴眺子叶愈伤组织系A_(16)N_1,A_(11)B_2和A_(14)N_7,A_(14)B_2的生理分析表明:愈伤组织系A_(16)N_1和A_(14)N_7的原生质体再生细胞能持续分裂。而愈伤组织系A_(11)B_2和A_(14)B_2的原生质体再生细胞不能持续分裂。前两个愈伤组织系的多胺含量高,酚酸含量低,超氧物歧化酶活性高。过氧化物酶活性低,可溶性蛋白质和氨基酸含量高,说明用于取得并分离原生质体的材料的生理状态对原生质体生长、分化有影响。     

Determination of gentiopicroside contents during somatic embryogenesis in Gentiana straminea Maxim

A protocol for regeneration of Gentiana straminea Maxim recently established in our laboratory, somatic embryogenesis was obtained from its leaf derived calli. The gentiopicroside contents of embryogenic calli, globular-, heart-, torpedo-, and cotyledon-shaped embryoids as well as regenerated plantlets were determined by high-performance liquid chromatography. Gentiopicroside was detectable in all materials tested. Embryogenic calli showed the lowest gentiopicroside content. The changes of gentiopicroside contents were not significant (P < 0.05) with the development of somatic embryos. The highest gentiopicroside content (30.7 mg g^?1 dry weight) was achieved in regenerated plantlets. The contents of gentiopicroside were not significant (P < 0.05) differences between control plants and embryogenic calli, different stages of somatic embryos and regenerated plantlets. This protocol could be employed for producing gentiopicroside or other medicinal compounds.     

Propagation of <Emphasis Type="Italic">Gentiana macrophylla</Emphasis> (Pall) from hairy root explants via indirect somatic embryogenesis and gentiopicroside content in obtained plants

An effective protocol for plant regeneration from hairy root (HR) via indirect somatic embryogenesis was established in medicinal plant Gentiana macrophylla, a perennial herb in Gentianaceae. On the MS medium containing 0.5–2.5 mg l^?1 2,4-dichlorophenoxyacetic acid (2,4-D) or 2,4-D plus benzylaminopurine (BAP), all the HR explants produced embryogenic calli (ECs). After transfer to plant growth regulator (PGR)-free MS medium, up to 94% of the ECs produced somatic embryos (SEs) of various stages, including cotyledonary SEs. When the calli with cotyledonary SEs were transferred to PGR-free MS medium, the cotyledonary SEs on the calli developed into plantlets (1–12 ones per callus). The cotyledonary SEs showed two types: solitary and fasciculate. The former developed into single plantlets and the latter into fasciculate ones. After transplantation into soil, a half of the plantlets survived, and one of the survivors flowered without fruiting. Morphologically, about 30% plantlets appeared similar to the wild type (WT)-plants, and 70% of them displayed wrinkled dark green leaves with relatively small and dense stomata, long and thick main root with dense lateral roots. The biomass of roots and leaves of the plantlets increased by five- and one-fold, respectively, and the content of gentiopicroside of their roots raised by 72.4%, in comparison with WT-plants. Polymerase chain reaction revealed that the rolC gene integrated into HR genome still existed in the regenerated plants. This study offers us an effective method and material for producing gentiopicroside or other medicinal compounds.     

The Influence of Different Factors on Cell Growth and α-tocopherol Content of Culture from Carthamus tinctorius

Calli were induced from root, hypocotyl, cotyledon and flower bud of Carthamtus tinctorius. All calli had the capability to synthesize α-tocopherol. Among these calli, the hvpocotylcallus was better than others in cell growth rate and α-toeopherol content. Culture conditions could intensively influence the growth rate and α-tocopherol production of callus from Carthamus tinctorius. Sucrose (30g/L) was good for callus growth and glucose (30g/L) was good for α-tocopherol accumulation. High concentration (0.55%) of inositol could obviously stimulate both growth rate and α-tocopherol synthesis of callus. The inoculum quantity for best callus growth was 0.035 to 0.067g dry wt/flask (50ml volume). In addition, α-tocopherol content was effectively increased by culture callus in high CO2 concentration. Studies on optimum cuhure conditions of callus culture showed that the callus growth rate, α-tocopherol content and yield were 1.88, 2.03 and 3.30 times respectively as high as of the control by administration of 0.45%–0.55% inositol, 10% coconut milk, 0.1–0.5% casamino acid, 30g/L sucrose and 10g/L glucose.     

Genotype and explant-type dependent morphogenesis and silicon response of common reed (Phragmites australis) tissue cultures

The effects of silicon on the growth and development of Phragmites australis (Cav.) Trin. Ex Steud. (common reed) stem nodal and root embryogenic calli were investigated. Silicon is considered to be a beneficial or quasi-essential nutrient for several Gramineaceous plants, including reed. Seven callus lines of four geographical locations (genotypes 1-4) within Hungary were investigated. Callus lines 1A, 2A and 3A were produced from stem nodal explants, while lines 1B, 2B, 3B and 4 were produced from roots. For the assay of silicon-dependent growth of callus lines of identical genotype but originating from different explants, we measured the increase of fresh weight of lines 1A and 1B. The studied developmental parameters were the increase of the number of somatic embryos (for callus lines 1A and 1B) and plant or root production from somatic embryos (for all genotypes/callus lines). Silicon was added to the culture medium as sodium silicate. In control cultures, plant or root regeneration from embryogenic calli was strongly genotype- and explant type-dependent. Stem nodal explants developed plants on regeneration medium in case of callus lines 2A and 3A, while line 1A produced roots only. All root derived calli developed roots on regeneration medium. Silicon stimulated the growth of both stem nodal and root calli (callus lines 1A, B) however, the concentration optima were different. Somatic embryogenesis of root calli, but not of stem nodal calli, was stimulated by silicate at low concentrations. However, for both of these callus lines, root development was stimulated by silicon. It had genotype-dependent influences on plant regeneration: while stimulation was observed in case of callus line 2A, inhibition occurred for line 3A. Root morphogenesis on calli was significantly influenced by silicon and depended on the callus line studied. Root production was stimulated on callus lines 1A, B and 2B, while in case of callus line 3B, it was significantly inhibited. The morphogenetic effects of Si were similar for different explants of the same geographical origin, i.e. plant or root production was similarly stimulated or inhibited by this element. We can conclude that the effects of Si on plant or root development depend on reed genotype used for callus induction. Its effect on growth and somatic embryogenesis depends on the explant type used for callus production. This is the first detailed report on the role of silicon in plant vegetative development and morphogenesis of a Gramineaceous plant.     

Development of NaCl-tolerant line in Chrysanthemum morifolium Ramat. through shoot organogenesis of selected callus line

Plants were regenerated successfully through shoot organogenesis of a NaCl-selected callus line of Chrysanthemum morifolium Ramat. cv. Maghi Yellow (a salt sensitive cultivar), developed through stepwise increase in NaCl concentration (0-100mM) in the MS medium. The stepwise increase in NaCl concentration from a relatively low level to cytotoxic level was found to be a better way to isolate NaCl-tolerant callus line, since direct transfer of callus to high saline medium was detrimental to callus survival and growth. The selected callus line exhibited significant increase in superoxide dismutase (EC 1.15.1.1), ascorbate peroxidase (EC 1.11.1.11) and glutathione reductase (EC 1.6.4.2) activities compared to control callus (grown in medium devoid of NaCl). Stability of salt tolerance character of the selected callus line was checked by growing the calli in NaCl-free medium for 3 consecutive months followed by re-exposure to higher salinity stress (120mM NaCl). Among different growth regulator treatments, a combination of 5mgl(-1) TDZ (Thidiazuron) along with 0.25mgl(-1) NAA and 0.5mgl(-1) GA(3) was found to be the most effective for shoot organogenesis in selected callus line. The regeneration potential of the NaCl-tolerant callus ranged from 20.8% to 0% against 62.4% to 0% in control callus line. Under elevated stress condition (medium supplemented with 250mM NaCl), selected calli derived regenerants (S1 plants) exhibited significantly higher SOD and APX activities over both PC (positive control: control callus derived plants grown on MS medium devoid of NaCl) and NC (negative control: control callus derived plants subjected to 250mM NaCl stress) plants. In addition, the NC plants showed stunted growth, delayed root initiation, and had lesser number of roots as compared to S1 plants. Based on growth performance and antioxidant capacity, the S1 plants could be considered as NaCl-tolerant line showing all positive adaptive features towards the salinity stress. Further study on agronomic performance of these S1 plants under saline soil condition need to be undertaken to check the genetic stability of the induced salt-tolerance.     

<Emphasis Type="Italic">In vitro</Emphasis> organogenesis and antioxidant enzymes activity in <Emphasis Type="Italic">Acanthophyllum sordidum</Emphasis>

The effect of various hormonal combinations on callus formation and regeneration of shoot and root from leaf derived callus of Acanthophyllum sordidum Bunge ex Boiss. has been studied. Proteins and activity of antioxidant enzymes were also evaluated during shoot and root organogenesis from callus. Calli were induced from leaf explants excised from 30-d-old seedlings grown on Murashige and Skoog medium containing 4.52 μM 2,4-dichlorophenoxyacetic acid + 4.65 μM kinetin. Maximum growth of calli and the most efficient regeneration of shoots and roots occurred with 2.69 μM 1-naphthalene acetic acid (NAA), 2.69 μM NAA + 4.54 μM thidiazuron and 2.46 μM indole-3-butyric acid. Protein content decreased in calli and increased significantly during regeneration of shoots from callus. Superoxide dismutase activity decreased in calli comparing to that of seedlings, then increased in regenerated shoots and roots. High catalase activity was detected in seedlings and regenerated shoots, whereas high peroxidase activity was observed in calli and regenerated roots.     

Somatic Embryogenesis and Plant Regeneration from Papaya Suspension Cultures

Compact embryogenetic calli were obtained from explants on P3 medium after 4 weeks of culture and high-frequency somatic embryogenesis occurred after these calli were transferred into suspension culture. Experimental data showed that low level (0.2%W/V) of activated charcoal had beneficial effects on somatic embryogenesis. Abundant calli on P4 medium however, showed no embryogenesis. On the other hand, callus induction and somatic embryogenesis varied with different rarities of exptants. The efficiency of somatic embryogenesis was much higher, if roots were used as explants, whereas stems were more suitable for callus formation Mature somatic embryos with cotyledons were cultured on MS medium containing different plant hormones. The optimum medium for germination and growth of entire plantlet was Mso medium. The somatic embryos on MS2, MS and MS3 media germinated rapidly, but formed excessive callus from the surface of germinating embryos.     

Stimulation of root and somatic embryo production in Euonymus europaeus L. by an inhibitor of polyamine biosynthesis

In vitro formation of roots and somatic embryos is obtained from cotyledon explants of a Spindle tree (Euonymus europaeus L.) cultured on two different media: a medium inducing callus formation and the production of roots, and a medium inducing callus formation, root and somatic embryo production. We studied the effects of α-difluoromethylornithine (DFMO), a specific, irreversible inhibitor of ornithine decarboxylase (ODC) on root and somatic embryo production, growth and titers of putrescine in Euonymus explants and explant-derived calli. Early changes in putrescine levels were detected in both cultures before the visible emergence of roots or somatic embryos. DFMO rapidly inhibited putrescine accumulation and growth in non-embryogenic calli and highly stimulated rooting activity. DFMO partially inhibited putrescine accumulation in embryogenic calli. This inhibition had no effects on callus growth but significantly reduced the time of emergence of roots and highly stimulated somatic embryo production. The relationship among putrescine, putrescine metabolism, growth, root and somatic embryo formation is discussed.