The biosynthetic relationship between littorine and hyoscyamine in transformed roots of Datura stramonium

The metabolic relationship between littorine and hyoscyamine has been monitored in transformed roots of Datura stramonium. Quantification by GC of unlabelled littorine and by GCMS of ¹³C-labelled littorine demonstrated that exogenously added littorine (0.1 mm) was significantly metabolised (35%) to hyoscyamine. In contrast, exogenously added hyoscyamine was not metabolised to littorine, indicating that this conversion is irreversible. The conversion of littorine to hyoscyamine was suppressed by P-450 oxidase inhibitors (particularly clotrimazole), implicating the involvement, at least in part of a cytochrome P-450 activity operating hyoscyamine biosynthesis. Received: 15 September 1997 / Revision received: 14 February 1998 / Accepted: 10 March 1998     

不同培育方式对橡胶树实生苗生理指标、养分及长势的影响

橡胶树(Heveabrasiliensis)种子催芽生长一般使用沙床培育,沙子是不可再生资源,为了选择一种适合橡胶树种子培育方式来替代对沙子的依赖,该研究通过水培、悬空培育和传统的沙培比较橡胶树实生苗第1蓬叶稳定时,苗木的生长势、生理指标及养分含量。结果表明,水培实生苗地上部株高、茎粗、叶面积的长势最佳,壮苗指数和生物量的含量最高,但其根太长,根相对较细。水培的叶、茎、根的可溶性糖、丙二醛、游离脯氨酸、超氧化物歧化酶的含量均较低;水培和悬空培育的叶片和茎的叶绿素、类胡萝卜素及根系活力的含量没有显著性差异,均高于沙培。水培的叶、茎、根中的氮和磷含量最低,沙培的最高;而水培实生苗根和茎中钾的含量较高,叶片中含量与悬空培育、沙培均没有显著性差异;悬空培育在叶、茎、根中钾的含量最低。水培促进了苗木的生长,降低干旱胁迫,提高养分利用率,但后续还需调控根系,建设良好根团。悬空培育的苗木长势较弱,还需进一步完善方法。     

Production of tropane alkaloids by transformed root cultures of Atropa belladonna in stirred bioreactors with a stainless steel net

A scale up of transformed root cultures of Atropa belladonna from a 300-ml flask to a 30-l tank was accomplished without any reduction in alkaloid productivity. Cutting treatment of seed cultures showed no distinct effect on root growth, morphology, and alkaloid content in conical flasks during 1 month of culture. Randomly cut roots thus grown were further cultivated in 3-l and 30-l modified stirred bioreactors for a scale-up culture. After 1 month of culture, 1490 mg of tropane alkaloids was produced by a 30-l culture of A. belladonna transformed roots. These roots contained the same level of atropine (5.4 mg/ g dw) as the roots of this plant grown in the field for 12 months and still contained a considerable amount of other alkaloids including 1.6 mg/g dw of 6-β-hydroxyhyoscyamine, 0.9 mg/g dw of scopolamine, and 2.0 mg/g dw of littorine. Received: 12 June 1998 / Revision received: 31 August 1998 / Accepted: 27 October 1998     

The Effects of Certain Environmental Factors on Alkaloid Contents of Rauvolfia

The effects of light intensity, mineral nutrients and seasonal climatic changes on the alkaloid contents of Rauvolfia yunnanensis Tsiang were studied and the experimental results were summarized as follows: The alkaloid contents of Rauvolfia roots increased with the light intensity decreased, and a maximum was reached at about 28% of the normal day light. The supply of the mineral nutrients of P, K especially N would raise the root production and alkaloid contents respectively. Determinations were carried out about the variations of alkaloid contents of the roots, stems and leaves on the samples collected every month of the year from both the wild and the cultivated Rauvolfia. The alkaloid contents in the roots of both wild and cultivated Rauvolfia appeared with only one high peak-August to September (end of the raining season), and a period with low contents appeared from March to May (dry season). The alkaloid contents in stems appeared with two high peaks, one was in coincidence with that of the roots, the other appeared between March and May (dry season) just as the period with low alkaloid contents in root. The alkaloid contents of leaves of wild Rauvolfia appeared with two high peaks, both were the same as those of the stems, but in the cultivated Rauvolfia, only one peak with high contents from July to August (raining season) was observed.     

Simultaneous determination of scopolamine, hyoscyamine and littorine in plants and different hairy root clones of Hyoscyamus muticus by micellar electrokinetic chromatography

Hyoscyamus muticus hairy root clones were established following infection with Agrobacterium rhizogenes strains A4, LBA-9402 and 15834 and with A. tumefaciens strain C58C1pRTGus104. The accumulation of tropane alkaloids hyoscyamine, littorine and scopolamine was evaluated by micellar electrokinetic capillary electrophoresis. Littorine was reported for the first time in these clones as well as in the roots of the intact plant and confirmed by collision induced dissociation-mass spectrometry. Tropane alkaloid content in hairy roots was compared with leaves and roots of normal plants at two vegetative stages. Significant differences appeared between the alkaloid contents of the different clones. In particular, all the hairy root clones and the roots of the intact plant produced 1.5-3 and 4.5-9 times more littorine than scopolamine, respectively. The only exception was clone KB7, carrying the h6h gene, which overproduced scopolamine. The aerial parts of H. muticus plants did not contain any littorine, thus indicating different transportation or translocation mechanisms of the various tropane alkaloids.     

Cytokinins and Morphological Aspects of French-bean Roots in the Presence of Rhizobium

Typical abnormalities were observed on French-bean roots when the plants were cultivated in the presence of Rhizobium phaseoli, under hydroponic conditions. Substantial quantities of cytokinins were detected in the plant culture medium and their concentration remained high until the 9th day after inoculation, when the first nodules appeared. The presence of both partners, plant and bacteria, was required for this synthesis. Exogenous cytokinin production was responsible for morphological alterations of the roots observed before nodulation.     

不同繁殖模式下甘草药用成分含量积累速度差异研究

目的:系统比较野生甘草无性繁殖的甘草根和人工甘草种子有性繁殖的甘草根中药用成分的积累速度差异情况,为进一步研究影响甘草药用成分积累的基因机制奠定理论基础。方法:本研究基于HPLC和UV等分析方法,比较野生甘草和人工甘草在甘草酸、甘草苷和甘草总黄酮以及其他类成分的含量差异。基于HPLC指纹图谱技术,全面分析不同繁殖模式下甘草根的化学信息和内在质量的差异情况。结果:除甘草素和异甘草素外,野生甘草不定根中其他三种药用成分和总黄酮的含量均高于人工甘草实生根,经SPSS分析,除异甘草素外,其他成分在两组之间均存在显著差异;根据HPLC指纹图谱,两种甘草药用成分之间差异显著,相似度为0.647。结论:野生无性繁殖的甘草根和人工有性繁殖的甘草根中主要药用成分含量之间存在显著差异,野生甘草具有快速积累药用成分的功能。本研究结果对于今后人工调控生产优质甘草药材,实现甘草资源的可持续利用具有重要的理论和实践意义。     

激素对转基因雪莲毛状根植株再生及类黄酮产生的影响

为了研究外源激素GA3和IAA对3个转基因新疆雪莲类黄酮高产毛状根系C17、C27、C46的植株再生及其总黄酮含量的影响,在培养基中添加不同浓度的GA3和IAA,结果发现,GA3浓度高于1.0 mg/L时,可诱导毛状根系产生不定芽,其中以GA3浓度为2.0 mg/L时,转基因毛状根系C17的不定芽再生率最高,可达82％。高压液相色谱以及紫外分光光度法测定结果表明,与未用激素处理的毛状根和它的再生植株相比,外源激素GA3和IAA能显著提高毛状根培养物中芹菜素和总黄酮的含量。毛状根系的组织干重与类黄酮的含量没有相关性,但毛状根系的再生率与类黄酮的含量几乎呈反相关性。     

Inhibition of flower induction on in vitro chicory root explants by hydroponic forcing pretreatment of roots

Fragments of vernalized chicory roots (Cichorium intybus L.) cultured in vitro under continuous light flower almost 100%. When chicory roots were placed in hydroponic forcing before in vitro culture, flowering percentage was reduced by half. The build-up of inhibition during 3 weeks of hydroponic forcing was studied in detail. The third week, in which growth of the chicory head is the strongest, was especially important in the inhibition process. When the root apex was eliminated during hydroponic forcing, flowering inhibition in vitro was weaker. The same observation was made when adventitious roots, developed during hydroponic forcing, were removed. The photoperiodic conditions during hydroponic forcing had no influence on the build-up of inhibition. It is suggested that activity of the apex and, possibly, of the adventitious roots during hydroponic forcing cause the flowering inhibition on chicory root fragments in vitro.     

不同品种吊兰根初生木质部原型的观察与比较

以3个品种吊兰(金边吊兰、银心吊兰、银边吊兰)为材料,采用徒手切片的方法研究了吊兰根初生木质部原型与品种、生境及根直径的t关系。结果表明,不同品种的吊兰根初生木质部原型存在着差异;在水培条件下,吊兰根初生木质部原型与土培条件下的不同,水培条件下的根初生木质部束远少于土培条件;在水培条件下,吊兰根初生木质部原型与根的直径存在正相关关系,但是在土培条件下银心吊兰和银边吊兰根初生木质部原型则和根的直径没有关系。     

Biosynthesis of C6-C3 acids in Datura innoxia

Datura innoxia plants were fed via the roots with cinnamic acid-[2¹⁴C], (±)-phenyllactic (2-hydroxy-3-phenylpropanoic) acid-[2¹⁴C] and phenylalanine-[2-¹⁴C]. In each case apohyoscine, hyoscine, hyoscyamine and littorine were isolated from the aerial parts, and hyoscine, hyoscyamine and littorine from the roots. Cinnamic acid was not incorporated into the acid moieties of the alkaloids. Phenyllactic acid served as a better precursor than phenylalanine for tropic acid (hyoscine and hyoscyamine) and atropic acid (apohyoscine). Phenylalanine served as an effective precursor for the phenyllactic acid moiety of Littorine.     

REGENERATION RESPONSES IN PEA ROOTS AFTER TIP EXCISION AT DIFFERENT LEVELS

Root tips of Pisum sativum cv Alaska were excised at seven excision distances ranging from 250 to 5,000 μm from the root cap/body junction. Three response types resulted: 1) Type A— One to three regenerated apices grew in the same plane as the root axis; 2) Type B—One to three regenerates emerged either longitudinally from a swollen wound site or at angles >90“° from the root longitudinal axis; 3) Type C—One to three lateral roots emerged at 90° from the root axis. The response type was dependent on the excision distance: Type A response resulted after excisions of 250 and 500 μm, Type B after excisions of 1,000 and 1,500 μm, and Type C after excisions of > 1,500 μm. The vascular patterns of the regenerated roots varied with excision distance. Most regenerates resulting after being excised at 250 μm exhibited variations in xylem strand number. Most of those resulting after being excised at 500 μm were ”polystelic“ and few of these showed variation in xylem strand number. With increasing excision distance the number of regenerates with variation in xylem stand number or with a ”polystelic“ condition decreased. Both the type of regeneration response and the vascular patterns within the regenerates correlated with the state of differentiation of cells at each excision distance.     

The growth of isolated barley embryos cultivated under different conditions

Isolated barley embryos were cultivated aseptically in three different complete media. The growth of the primary root of each embryo was measured during four days of cultivation. Embryos were cultivated on three different consistencies of media: on agar plates, on cellulose tissue moistened with the medium and in liquid shaken cultures. The last way of cultivation yielded the highest degree of the growth of roots. Optimum combination of conditions in shaken liquid medium was selected on the basis of systematic study. The growth of roots of isolated embryos under appropriate conditions approaches that of roots of seedlings cultived on moistened blotting paper.     

北柴胡不定根培养及茉莉酸甲酯处理对柴胡皂苷含量的影响

目的:建立北柴胡不定根培养体系,明确茉莉酸甲酯(MeJA)处理对北柴胡不定根中柴胡皂苷含量积累的影响。方法:利用固体和液体相结合的组织培养技术培养北柴胡不定根;分别以不同浓度的MeJA处理不定根不同时间,利用HPLC测定处理后不定根中柴胡皂苷含量的积累变化。结果:培养了北柴胡不定根;MeJA处理对北柴胡不定根中柴胡皂苷含量的积累有明显促进作用,当MeJA浓度为200μmol/L时,柴胡皂苷含量最高,为0.45%;以200μmol/L MeJA处理北柴胡不定根26 d时,柴胡皂苷含量最高,为0.51%。结论:北柴胡不定根培养结合MeJA诱导,可做为柴胡皂苷次生代谢合成途径及其积累规律研究的有效技术体系。     

The influence of salinity on cell ultrastructures and photosynthetic apparatus of barley genotypes differing in salt stress tolerance

A hydroponic experiment was conducted to elucidate the difference in growth and cell ultrastructure between Tibetan wild and cultivated barley genotypes under moderate (150 mM NaCl) and high (300 mM NaCl) salt stress. The growth of three barley genotypes was reduced significantly under salt stress, but the wild barley XZ16 (tolerant) was less affected relative to cultivated barley Yerong (moderate tolerant) and Gairdner (sensitive). Meanwhile, XZ16 had lower Na⁺ and higher K⁺ concentrations in leaves than other two genotypes. In terms of photosynthetic and chlorophyll fluorescence parameters, salt stress reduced maximal photochemical efficiency (F _v/F _m), net photosynthetic rate (Pn), stomatal conductance (Gs), and intracellular CO₂ concentration (Ci). XZ16 showed relatively smaller reduction in comparison with the two cultivated barley genotypes. The observation of transmission electron microscopy found that fundamental cell ultrastructure changes happened in both leaves and roots of all barley genotypes under salt NaCl stress, with chloroplasts being most changed. Moreover, obvious difference could be detected among the three genotypes in the damage of cell ultrastructure under salt stress, with XZ16 and Gairdner being least and most affected, respectively. It may be concluded that high salt tolerance in XZ16 is attributed to less Na⁺ accumulation and K⁺ reduction in leaves, more slight damage in cell ultrastructure, which in turn caused less influence on chloroplast function and photosynthesis.     

栽培与野生化血丹不同部位中两种化学成分含量分析

为探讨并分析栽培与野生化血丹植株中不同部位中两种化学成分的含量差异,该研究采用超声法提取、高效液相色谱法(HPLC)测定栽培与野生化血丹根、茎、叶、花、混合样等部位中桃叶珊瑚苷和梓醇的含量,并进行比较。结果表明:(1)桃叶珊瑚苷在栽培与野生化血丹植株内均有分布,含量均以根中最高,其在栽培与野生化血丹植株内的含量表现分别为根叶混合样茎花、根混合样茎花叶,栽培化血丹不同部位中桃叶珊瑚苷的含量均高于野生化血丹。(2)梓醇在栽培化血丹的茎中未检出,在栽培与野生化血丹其他部位均有分布,含量均以叶中最高,其在栽培与野生化血丹植株内的含量分别表现为叶花混合样根、叶混合样茎花根,野生化血丹不同部位中梓醇的含量均高于栽培化血丹。(3)桃叶珊瑚苷和梓醇在栽培和野生化血丹植株不同部位中的含量均存在显著差异(P0.05),栽培与野生同一部位间总体上无显著差异,为该濒危药用植物资源药用部位选择和合理开发利用提供实验参考。     

Lead Accumulation Ability of Selected Plants of Noccaea spp.

Several species of the Noccaea genus are known for their hyperaccumulation ability especially in the case of Cd, Ni, and Zn. However, ambiguous observations were previously published concerning their accumulation properties for Pb. The Pb accumulation properties of Noccaea rotundifolia, Noccaea montana, and Noccaea jankae hungarica plants were tested in field and pot experiments in soils differing in the mobile pool of Pb, as well as in soilless hydroponic culture. The Pb content in the dry biomass of plant shoots reached up to 54 mg/kg in field conditions and 84 mg/kg in pots regardless of the bioavailable pool of Pb in the pots. The hydroponic experiment showed a stepwise increase in Pb content in plant biomass with increasing Pb concentration in the solution, but the predominant proportion of plant Pb was retained in the roots. Although the hyperaccumulation ability of some of the Noccaea species is widely discussed in the literature, our results are in agreement with those suggesting no Pb hyperaccumulation potential in these plants.     

Effects of NaCl-salinity on amino acid and carbohydrate contents of Phragmites australis

The effects of NaCl-salinity on growth, free amino acid and sugar content and composition were assayed in roots, rhizomes and leaves of Phragmites australis (Cav.) Trin. ex Steud. Juvenile plants produced from freshwater clones, were cultured under greenhouse hydroponic conditions for 21 days. Relative growth rates were highest at a salinity level of 0 and 1.5‰, respectively, but decreased significantly at 10‰. All plants cultured at 35‰ salinity died. The osmolality in rhizomes and leaves increased with salinity. The total contents of free amino acids were highest in rhizomes>leaves>roots. In rhizomes, the amino acid content increased significantly up to four-fold from 0 to 10‰ salinity. This increase was caused by up to 200-fold increase of proline and 11-fold increase of glutamine at 10‰, whilst the share of asparagine and glutamate decreased. Leaves showed a similar response to salinity with increasing amino acid contents, and shares of proline and glutamine whereas roots did not react significantly. The contents of sucrose, glucose and fructose were highest in leaves>rhizomes>roots. In rhizomes of all three clones, the sugar contents increased up to 3.5-fold from 1.5 to 10‰ salinity level, but were lower at 1.5‰ versus the control (0‰). Sugar contents were lowest (roots) and highest (leaves) at 1.5‰ salinity. The sugar composition did not vary significantly except for leaves where the fraction of sucrose decreased with increasing salinity level at all three clones from 89.1 to 61.7% of total dissolved sugar (pooled data). The importance of free amino acids and sugars as osmolytes was similar in rhizomes and leaves (13–15% of total osmolality at 10‰). In rhizomes, free amino acids were more important as osmolyte than sugars, while the opposite was true for leaves. Proline contributed up to 2.7% to total osmolality. It is hypothesised that a strong proline accumulation indicates the exceeding of a critical salinity level.     

Effects of experimental thinning on the growth and maturation of the brown alga Undaria pinnatifida (Laminariales; Phaeophyta) cultivated in Matsushima Bay,northern Japan

In northern Japan, massive production of high-quality specimens of the kelp Undaria pinnatifida prior to the traditional harvest season is strongly needed to meet commercial demand. To address this need, we tested the effects of controlled thinning by cutting small plants on the growth and maturation of sporophytes cultivated in Matsushima Bay, northern Japan. In early December 2009, the stocking densities of cultivated kelps were thinned to 10 and 5 plants per 4 cm thread section in two experimental groups. In contrast, no thinning was conducted during cultivation until March 2010 in the control group (about 16–20 plants per 4 cm thread section). Morphological features, photosynthesis, and carbon and nitrogen contents were compared among the three groups. Compared to kelps of control group, total length, stipe width, the length and width of sporophyll, and dry weights of blade and sporophyll showed significantly higher values in kelps of the two experimental groups. However, stipe length of kelps of control group was significantly greater than those of the two experimental groups. The photosynthetic rates and nitrogen contents of kelps of the two experimental groups were significantly greater than those of the control from January to March. No significant differences occurred in all these parameters between kelps of the two experimental groups. Based on these results, the growth and maturation of U. pinnatifida sporophytes can be promoted greatly by experimental thinning, and this will improve production of high-quality specimens of kelps and increase economic returns before the traditional harvest season.     

Gibberellic acid improved shoot multiplication in Cephaelis ipecacuanha

The internodes of Cephaelis ipecacuanha elongated when cultured on Gamborg B5 solid medium supplemented with 0.5 or 1 mg/L gibberellic acid (GA₃). The size of the elongated internode doubled in length compared to the untreated shoots, and the adventitious shoots formed on the elongated internodes. The shoots grew easily into plantlets without the use of auxin for rooting. The ex vitro regenerates cultivated in the greenhouse showed normal characteristics. Emetic alkaloids were detected in the leaves of in vitro shoots and the roots of regenerates cultivated in the greenhouse. This method using GA₃ propagated numerous plants at a rate of more than 100 times compared to the method without GA₃.