Enhanced salidroside production in liquid-cultivated compact callus aggregates of Rhodiola sachalinensis: manipulation of plant growth regulators and sucrose

Liquid-cultivated compact callus aggregates (CCA) of Rhodiola sachalinensis were initiated from the explant of stems. Microscopic analysis showed that CCA displayed some level of cellular/tissue differentiation, which was perhaps responsible for the increased salidroside production. A higher ratio of cytokinin/auxin was found to be beneficial both for CCA growth and salidroside accumulation. Sucrose (> 100 g l-1) had a positive effect on salidroside synthesis, which was not due to an osmotic effect of the medium. The medium containing 40 g l-1 sucrose, 1.5 μM naphthaleneacetic acid and 15 μM 6-benzylaminopurine yielded ca. 120 mg l-1 salidroside after 24 days of culture. This revised version was published online in June 2006 with corrections to the Cover Date.     

Establishment of <i>Rhodiola quadrifida</i> Hairy Roots and Callus Culture to Produce Bioactive Compounds

Rhodiola quadrifida is a rare mountain medicinal plant whose root extracts are used in traditional Chinese medicine as a hemostatic, antitussive, and tonic in the treatment of gynecological diseases. The aim of the study was to obtain R. quadrifida cultures at different degrees of differentiation in vitro and compare their growth characteristics and the content of salidroside and rosavin. Hairy roots were obtained by incubating cotyledons and hypocotyls in a suspension of Agrobacterium rhizogenes strain A4. The presence of the rolB and rolC genes was proven by polymerase chain reaction. The obtained roots were cultivated in Murashige-Skoog medium (MS). Calluses were obtained from the hairy roots in MS medium with the addition of hormones: 3 mg/L 2,4 D and 0.5 mg/L BAP. The presence of the main secondary metabolites of R. quadrifida, salidroside and rosavin, in calluses and salidroside in hairy roots by HPLC/MS was confirmed. The content of salidroside in callus culture was significantly higher than in hairy roots, 0.158 and 0.047%, respectively. The content of rosavin in callus culture was 0.07%. The content of rosavin and salidroside in callus culture was close to the level of these substances in the rhizomes of R. quadrifida plants growing in vivo, making this culture promising for its possible biotechnological use.     

Production of salidroside and polysaccharides in Rhodiola sachalinensis using airlift bioreactor systems

Rhodiola sachalinensis is widely used in traditional Chinese medicine, and salidroside and polysaccharides are its important bioactive compounds. This study used airlift bioreactor systems to produce mass bioactive compounds through callus culture. Several factors affecting callus biomass and bioactive compound accumulation were investigated. Callus growth was vigorous in a bioreactor system, and the growth ratio was 2.8-fold higher in bioreactor culture than in agitated-flask culture. Callus biomass and polysaccharide content were favorable at 0.1 air volume per culture volume per min (vvm), whereas favorable salidroside content was observed at a high air volume (0.2 vvm). The maximum yields of salidroside (7.90 mg l^?1) and polysaccharide (2.87 g l^?1) were obtained at 0.1 vvm. Inoculum density greatly affected callus biomass and bioactive compound accumulation, and the highest biomass and contents or yields of salidroside and polysaccharide were determined at a high inoculum density of 12.5 g l^?1. The level of hydrogen ion concentration (pH) at 5.8 improved callus biomass accumulation. Acidic medium (pH 4.8) stimulated salidroside synthesis but higher pH level (7.8) promoted polysaccharide accumulation. The highest yields of both bioactive compounds were obtained at pH 5.8. Methyl jasmonate (MeJA) participated in synthesis promotion of bioactive compounds, and the contents and yields of salidroside [4.75 mg g^?1 dry weight (DW), 58.43 mg l^?1] and polysaccharides (392.41 mg g^?1 DW, 4.79 g l^?1) were at maximum at 125 and 150 μmol of MeJA. Therefore, bioreactor systems can be used to produce R. sachalinensis bioactive compounds, and callus culture in a bioreactor can be as an alternative method for supplying materials for commercial drug production.     

Strategies for the improvement of salidroside production in cell suspension cultures of Rhodiola sachalinensis

Strategies of elicitation and precursor feeding were applied to improve salidroside production in cell suspension cultures of Rhodiola sachalinensis. Of the seven elicitors examined, that extracted from Aspergillus niger was the most effective, increasing the salidroside content by five-fold when added on the day of inoculation 40 mg carbohydrate is medium. Three possible precursors for salidroside synthesis, l-phenylalanine, l-tyrosol and l-tyrosine were added to the cultures. A high content of salidroside (1.440%) was attained with an initial l-tyrosol concentration of 0.5 mm in the medium. Combined application of the two strategies resulted in a significantly high salidroside content of 1.734%, corresponding to a salidroside yield of 200 mg/l. Received: 5 November 1996 / Revision received: 17 February 1997 / Accepted 11 April 1997     

Asiaticoside production from centella (Centella asiatica L. Urban) cell culture

Petiole explants of centella plants (Centella asiatica L. Urban) were cultured on Murashige and Skoog (MS) solid medium containing 20 g/L sucrose, supplemented with 1.0 mg/L benzylaminopurine and 1.0 mg/L naphthaleneacetic acid for callus production. To establish a cell suspension culture, 2 g of fresh callus was cultured in 50 mL of the same medium but without solid agent at a 100 rpm agitation speed. Every 2 g of culture was subcultured in fresh MS liquid medium for maintenance. After 24 days of culture at a 120 rpm agitation speed, the centella cell biomass reached a maximum of 9.03 g/50 mL on the same MS medium with 30 g/L sucrose and a 3 g inoculum size. A high performance liquid chromatography analysis showed that asiaticoside content in 24-day old suspension cultured cells (45.35 mg/g dry weight) was significantly higher (4.5 fold) than that of in planta leaves (10.55 mg/g dry weight).     

The Effects of Agitated Liquid Medium on in vitro Cultures of Hevea brasiliensis

The initiation and prolonged growth of callus, from stem explants of young plants of Hevea brasilienies on solid medium yielded a heterogeneous callus, with areas which are the result of compact growth interspersed with brown necrotic tissue and soft white tissue formations. Subculturing this callus (O callus) to agitated liquid medium and returning it to solid medium resulted in the production of a homogeneous friable and rapidly growing callus (Rl callus) The two established lines O and Rl have remained stable over one year in culture and differ in gross morphology, anatomy, growth and auxin content. Both were maintained on Murashige and Skoog's medium, with 2 mg/1 2,4-D and 0.5 mg/I kinetin. R 1 but not O showed enhanced growth at the lower 2,4-D level of 0.2 mg/l: both lines failed to continue growing when 2,4-D was omitted. It is suggested that the changes resulting from subculture in agitated liquid medium are related to those undergone by callus cultures which become habituated. Thus the Rl callus line is regarded as partially habituated. Subculture in agitated liquid medium also resulted in the production of large numberr of polyploid cells but these did not persist over the long periods of subsequent growth on agar medium, Enhanced auxin production by the establihed Rl callus line was thus observed in the absence of a detectable level of polyploidy.     

Somatic embryogenesis in Vigna radiata (L.) Wilczek.

Somatic embryogenesis was achieved from immature cotyledon derived callus of mungbean, V.radiata (L.) Wilczek in MS liquid medium. Embryogenic callus was induced on MS medium with NAA (5 mg/L). Differentiation of somatic embryos was observed when embryogenic callus was transferred to MS liquid medium containing 2,4-D (1.5 mg/L) and L-proline (50 mg/L). The torpedo shaped embryos were transferred to MS liquid medium with BAP and ABA (1 mg/L each) for maturation and germination. Fifty per cent of torpedo shaped embryos were converted into tiny plants (8-9 plants out of 17) after one week of culture. The germinated embryos were isolated and transferred to MS half strength basal (solid) medium for further development.     

高山红景天细胞悬浮培养中pH值对红景天甙胞外释放及细胞活性的影响

高山红景天(Rhodiola sachalinensis A.Bor.)细胞悬浮培养中,通过降低培养基pH值能有效地诱导培养细胞中红景天甙的胞外释放。红景天甙的跨膜运输是一个与H~ 对运的动态过程,培养基pH值决定了红景天甙在胞内外含量的分布。细胞组织在pH值大于3的培养基中处理3h以内,对细胞活性的影响不大。将诱导释放处理过的细胞组织转入到新鲜的生产培养基中,细胞仍具有合成红景天甙的能力。     

Production of salidroside and tyrosol in cell suspension cultures of Rhodiola crenulata

Salidroside and its aglycone tyrosol are important compounds found in Rhodiola plants. In this study, callus derived from Rhodiola crenulata was induced and grown when explants were incubated on a Murashige and Skoog (MS) medium containing various concentrations of 6-benzyaldenine (BA), naphthalene acetic acid (NAA) and thidiazuron (TDZ). Callus was easily initiated from juvenile leaves in half strength MS medium supplemented with 0.5 mg/L BA and 3.0 mg/L NAA, while full strength MS containing 0.5 mg/L TDZ and 0.5 mg/L NAA was the best for callus subculture and subsequent cell suspension culture. The activities of l-phenylalanine ammonia lyase (PAL) and β-d-glucosidase, two key enzymes in salidroside synthesis, increased at first and subsequently decreased in cell suspension cultures. The salidroside and tyrosol levels in the cell suspension cultures were determined using high-performance liquid chromatography. High levels of salidroside and tyrosol were detected in cell suspension cultures of R. crenulata extracted with 75 % methanol, demonstrating that the biotechnological production of these compounds using plant cell suspension cultures derived from R. crenulata may be an attractive alternative to harvest-based production.     

Rosmarinic acid synthesis in transformed callus culture of Coleus blumei benth

Agrobacteria mediated Coleus blumei tumour tissues were cultured in vitro on MS medium. Sixteen diversified transformed callus cultures were maintained for several years in the absence of plant growth regulators and antibiotics without affecting the growth rate. Rosmarinic acid was detected spectrophotometrically in all tissue lines but in different quantities. The highest rosmarinic acid accumulation detected was 11% of dry tissue mass. The relation between culture growth and rosmarinic acid production was investigated in three callus lines. The lines showed different rosmarinic acid accumulation in relation to their growth rate; it was either parallel or inversely related to the tissue growth. The effects of certain medium constituents on the callus growth and rosmarinic acid accumulation were examined in four tumour cell lines. Addition of 4% or 5% sucrose stimulated rosmarinic acid synthesis and decreased callus growth. Nitrogen reduction to one half or one quarter of initial concentration did not affect rosmarinic acid synthesis and decreased callus growth in three lines, while it increased rosmarinic acid accumulation and callus growth in one line. Addition of 0.1 mg/l Phe stimulated rosmarinic acid production in two lines but had little effect on the rosmarinic acid level in others. Rosmarinic acid production was significantly improved on modified macronutrients, where the Ac2 line produced 16.5 mg of rosmarinic acid per tube (0.2 g of dry wt) after being in culture for 35 days.     

遮荫和红膜处理对高山红景天根生物量及红景天甙含量季节变化的影响

采用纱布遮荫(覆以透明膜)和红色滤光膜处理人工种植生长3年和4年的高山红景天,观察了根生物量和红景天甙含量的季节变化结果表明,纱布遮荫处理(相对光强为全光照的51．8％)下根的生物量显著下降;红景天甙含量略有增加,红景天甙产量(红景天甙含量与根生物量乘积)则有所降低,但差异均不显著,红色滤光膜处理(相对光强为全光照的51．8％)与纱布遮荫处理相比,根生物量减少但不显著,红景天甙的含量和产量则显著增加,生长季末前者的红景天甙含量为后者的1．63倍(生长3年)和1．55倍(生长4年),红景天甙产量为后者的1．44倍(生长3年)和1．45倍(生长4年)。     

Enhancement of cyclosporin production in a Tolypocladium inflatum strain after epichlorohydrin treatment.

Following treatment of conidia of the cyclosporin producer fungus, Tolypocladium inflatum, with 0.15 M epichlorohydrin, strain M6 was isolated. The new strain exhibited a similar growth rate to the parent organism but more extensive conidiation and several-fold higher overall cyclosporin production. Strain M6 reached titres of 318 mg l-1 cyclosporin A in agar cultures, whereas in liquid medium it produced 140 mg l-1 cyclosporin A and 68 mg l-1 cyclosporin C. It also maintained a steady volumetric productivity of 0.48 mg l-1 h-1 cyclosporin A over 2 weeks of submerged cultivation in maltose-based semisynthetic medium. The new strain holds potential for improved cyclosporin production due to the superior titres and demonstrated capacity to sustain elevated production of cyclosporin for periods greater than the wild type.     

箭叶秋葵愈伤组织诱导及增殖培养的激素组合筛选

箭叶秋葵[ Abelmoschus sagittifolius(Kurz.) Merr.]又名红花马宁、铜皮、五指山参、小红芙蓉、岩酸,系锦葵科(Malvaceae)秋葵属(Abelmoschus Medic.)多年生亚灌木状草本;其根既可食用又可入药,具有滋养强壮之功效,可用于治疗神经衰弱、头晕、腰腿痛、胃痛及腹泻等[1].     

红景天甙生物合成途径：酪醇合成的起始反应及其糖基化

红景天甙(Salidroside)生源途径分子机制的解析是利用基因工程、代谢工程技术合成目标化合物的基础。糖基化是红景天甙生物合成的最后一步反应。在前期工作中,本课题组率先报道了与红景天甙生物合成相关的3个尿苷二磷酸葡萄糖基转移酶(UGTs)基因,在体外酶学性质研究的基础上,利用根癌农杆菌和发根农杆菌介导分别建立了相关转基因体系,鉴别了红景天甙生物合成最适UGT及植物和毛状根生物反应器系统合成红景天甙的效率差异;酪醇(Tyrosol)是红景天甙糖基化反应的甙元底物分子,其具体的代谢通路及其调控机制仍不明确。针对酪醇生物合成来源主要存在两种观点:一是酪醇可能来自于苯丙烷代谢途径产生的4-香豆酸,该途径起源于苯丙氨酸;二是生物碱代谢途径的中间产物酪胺可能是酪醇生物合成的前体,该途径则起源于酪氨酸。在后续工作中,否定了酪醇来源于苯丙烷代谢途径的可能性,进一步的工作证实酪氨酸脱羧酶(TyrDC)在酪醇生物合成的起始反应中担负着重要功能,酪醇作为一种苯乙烷类化合物衍生物,其生物合成来源于生物碱代谢途径。     

不同来源高山红景天材料中有效成分的HPLC分析

本文用HPLC测定、比较了5种不同来源的高山红景天材料中有效成分—红景天甙及其甙无酪醇的含量,并建立了高山红景天有效成分分析的样品制备方法,结果表明:固体培养、液体培养及反应器培养的高山红景天组织中均含有上述两种有效成分.其中固体培养的材料中有效成分的含量最高.三种提取方法比较表明,用索氏提取器连续回流提取明显优于冷浸提取和批式回流提取.     

Medium composition and methyl jasmonate influence the amount and spectrum of secondary metabolites in callus cultures of Zanthoxylum stenophyllum Hemsl.

Callus cultures of Zanthoxylum stenophyllum were initiated in vitro and the effect of growth regulators and elicitors was tested both upon callus growth and secondary metabolite production. On a medium containing naphthaleneacetic acid, kinetin, and 2,4-dichlorophenoxyacetic acid, a yellowish and friable callus was obtained from 90% of cotyledon explants. Callus growth and secondary metabolite accumulation was followed after sub-culturing the established callus culture on different media containing various hormonal combinations. Results indicate that medium containing naphthaleneacetic acid and a higher concentration of 2,4-dichlorophenoxyacetic acid gave the highest stimulation of growth. Addition of an organic nitrogen source also had a positive effect on growth. Rapid HPLC screening of methanol extractable secondary metabolites from calluses showed that phytohormones and nutrients were able to modify the chromatographic pattern of compounds. Calluses grown on the medium giving the highest stimulation of growth show a reduced accumulation of some secondary products, but not all. In response to elicitation by methyl jasmonate, metabolite production was different for the different classes of compounds, and hormonal composition of the culture medium influenced the response. Thus, results confirm the importance of the reciprocal interactions between hormones, nutrients, and elicitors when attempts are made to enhance secondary metabolite accumulation in in vitro cultures.     

Chemical potential of Aphelandra sp. cell cultures

Six different callus lines and three different suspension culture lines were established from plants of two Aphelandra species (Acanthaceae). All established lines were analyzed for secondary metabolite accumulation. A discrepancy between secondary metabolites accumulated in the plants and in the cell cultures could be observed. All established Aphelandrasp. cell cultures produced verbascoside (acteoside) as the major extractable metabolite. Time course experiments were carried out to investigate the relationship between cell growth and verbascoside production. In the present study it was shown that verbascoside accumulation was growth dependent and positively related to the presence of 2,4-D in the medium. The conditions in which verbascoside represents ca. 18% of cell culture weight have been defined. Free polyamines were detected in the cell culture lines cultivated in MS liquid medium (cysteine 10 mg l^-1, thiamine 1 mg l^-1, 2,4-D 1 mg l^-1, kinetin 0.2 mg l^-1 and sucrose 30 g l^-1). Putrescine and spermidine accumulated within 8 days to a maximum of 8.4 μmol g^-1 of dry wt and 2.6 μmol g^-1 of dry wt respectively and thereafter their concentration decreased rapidly. There was no evidence for the presence of spermine or any other type of free or conjugated polyamines in the tested cell culture lines. This revised version was published online in June 2006 with corrections to the Cover Date.     

Production of solasodine by hairy root,callus, and cell suspension cultures of Solanum aviculare Forst.

The production of the steroidal alkaloid solasodine, an alternative to diosgenin as a precursor for the commercial production of steroid drugs, was studied in hairy root, callus, and cell suspension cultures of Solanum aviculare Forst. through manipulation of culture medium. The individual and combined effects of medium components on the growth index and the production of solasodine were analyzed using factorial analysis of variance. Solasodine content was optimized to 6.2 mg g⁻¹in the hairy root, 1.4 mg g⁻¹callus, and 0.7 mg g⁻¹in cell suspension cultures (dry weight). An improved isocratic reversed phase high performance liquid chromatographic method provided selective determination of the solasodine content of these samples. Analysis of growth and solasodine content of hairy root cultures and callus cultures demonstrated that the production of solasodine was shown to be growth-dependent in hairy root cultures but not in callus cultures. This revised version was published online in June 2006 with corrections to the Cover Date.