Enhanced indole alkaloid production in suspension compact callus clusters of Catharanthus roseus: impacts of plant growth regulators and sucrose

A special culture system, compact callus clusters, was developed from Catharanthus roseus stem explants in a modified Murashige and Skoog (MS) liquid medium containing 5.37 µM -naphthaleneacetic acid and 4.65 µM kinetin. Morphological and anatomical studies showed that the globular compact callus cluster cultures consisted of many cohesive callus aggregates displaying some level of cellular/tissue differentiation, which was also in agreement with the results from peroxidase and esterase isoenzyme pattern analysis. The compact callus cluster cultures could synthesise about 2-fold more indole alkaloids than the dispersed cell cultures, and this was postulated to be associated with their differential status. Plant growth regulators and sucrose concentration, as well as shaking speed significantly affected properties of the compact callus clusters. In detail, 2,4-dichlorophenoxyacetic acid destroyed the compact structure and reduced alkaloid production of the compact callus cluster cultures; but a high concentration of cytokinins was necessary to maintain the compact structure and high alkaloid production of the special cultures. The optimum sucrose (5–6%) gave the greatest alkaloid and biomass production, as well as the greatest degree of compaction of the compact callus clusters.     

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     

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.     

超临界CO2萃取红景天中红景天苷、苷元酪醇的研究

采用超临界CO₂ 萃取法和乙醇常温浸提法相比较, 研究从红景天中提取红景天苷、苷元酪醇的工艺条件, 结论是:采用超临界CO₂ 萃取法能萃取出红景天生药中红景天苷的1.2%, 提取率不高, 但该方法能萃取出80%的苷元酪醇, 萃取液中苷元酪醇的相对含量可达45.68%;乙醇常温浸提法能将红景天苷、苷元酪醇同时有效萃取, 且得率较高, 但是萃取液中两物质相对含量较低, 进一步分离纯化将有难度。本研究结果表明, 将超临界CO₂ 萃取法和乙醇常温浸提法有效结合, 可实现两物质的有效分离, 推进红景天有效成分的产业化进程。     

温室栽培光强和光质对高山红景天生物量和红景天甙含量的影响

为探讨光强及光质对高山红景天(Rhodiola sachalinensis)生物量和红景天甙含量的影响。于2000年4月至6月在东北林业大学温室内以移栽于大兴安岭加格达奇圃地人工种植生长3a的高山红景天为材料,通过纱布遮荫及遮以不同颜色的滤光膜分别进行了光强、光质控制实验(处理45d)。随着光强的降低,高山红景天全株生物量、根生物量、根的红景天甙含量和产量以及叶中叶绿素a、叶绿素b、总叶绿素的含量均有降低的趋势,但叶绿素含量变化很小,不同光强及对照之间的差异均未达到显著水平。相对光强为67．75％和44．71％的两种处理下的全株生物量、根生物量、根的红景天甙含量和产量差异不显著,它们的全株生物量和红景天甙含量与对照(全光照)的差异也不显著,但根生物量和红景天甙产量与对照的差异显著。当相对光强减弱至31．96％,全株生物量、根生物量、根的红景天甙含量和产量均大幅度下降,根冠比显著增加。4种滤光膜处理均使高山红景天的全株生物量及根生物量显著降低,蓝膜和绿膜处理的降低幅度大于红膜和黄膜处理的。红膜处理的红景天甙的含量和产量均高于对照,但黄膜、蓝膜和绿膜处理的红景天甙含量和产量则低于对照。通过计算去除4种滤光膜的光强因素,仅从光质的作用看。4种滤光膜处理仍是减小了全株生物量和根生物量,红膜和绿膜处理提高了红景天甙的含量和产量,而黄膜处理降低了红景天甙的含量和产量,蓝膜处理几乎没有效果。4种滤光膜处理均使叶绿素含量增加,但只有蓝膜处理的与对照差异显著。红膜处理不仅显著提高根中红景天甙的含量(为对照的3．42倍),而且对根生物量的影响较小(为对照的90．24％)。因而提高了高山红景天根的红景天甙产量,这意味着在生产上可能会有一定的实践意义。     

Microspore culture in Corchorus olitorius: effect of growth regulators, temperature and sucrose on callus formation

Culture of isolated microspores and of anthers on media containing IAA directed free microspore development to an embryogenic pathway in C. olitorius. The first division of microspores on transfer to culture media was symmetrical in contrast to the asymmetrical division seen in normal development in vivo. Initially, 10-30% microspores divided symmetrically, but only 0.2-1% of the dividing microspores continued dividing and produced multicellular microcalli. About 30% of these microcalli produced callus but only on medium with 2.0 mg/L zeatin and 0.1 mg/L IAA. Incubation in the dark at temperatures of 35 degrees C for 1 day and then 25 degrees C was found effective for induction of first embryonic division in Corchorus. The frequency of microspore callus formation was higher on medium containing either 3% or 5% sucrose. Addition of colchicine and addition of activated charcoal to the above medium did not enhance microspore division in Corchorus olitorius. On transfer to different media most calli produced roots but regeneration of shoots and embryos was not induced.     

光强和光质对野外栽培高山红景天生物量和红景天甙含量的影响

为探讨野外条件下光强及光质对高山红景天 (Rhodiola sachalinensis)生物量和红景天甙含量的影响 ,于 2 0 0 1年 5月 8日至 9月 16日在大兴安岭加格达奇的高山红景天种植圃地 ,利用纱布及红色、蓝色和绿色的滤光膜遮光处理 ,对生长 3a和 4 a的高山红景天进行了光强、光质控制实验。与温室实验类似 ,遮荫显著抑制高山红景天根的生长 ,并使红景天甙的含量略有提高。红膜处理使光强大约降低一半 ,但仅从光质的角度而言 ,红膜处理对根的生长影响不大 ,却显著增加了根中的红景天甙含量和产量 ,不过效果不如温室实验明显。绿膜处理未表现出对红景天甙积累的促进作用 ,这与温室实验结果不同。红膜处理不同天数的结果表明 ,处理时间对红景天甙含量提高的程度影响很小。这意味着在野外种植的情况下 ,可以在临近收获的最后一段时间用红膜对高山红景天进行处理 ,这样既可避免红膜处理对高山红景天根生长的抑制 (由于减弱了光照 ) ,又可显著提高根的红景天甙含量 ,从而达到大幅度提高红景天甙产量的目的。     

Effects of explants and growth regulators in garlic callus formation and plant regeneration

We intended to evaluate the effects of different explants and growth regulators on callus induction and plant regeneration in garlic (Allium sativum L.). Furthermore, we intended to differentiate among different morphological types of callus by light microscopy and to relate them with their abilities to regenerate plants in the red-garlic cultivar 069. A factorial design with BDS—basal Dunstan and Short (1977)—medium, as a control and supplemented with 0.042, 0.42 and 4.24 μM picloram or with 0.045, 0.45 and 4.5 μM 2,4-D, in both cases with and without 4.43 μM N⁶-benzylaminopurine (BAP), was used. The cultures were grown in darkness at 25 ± 2°C and they were subcultured over a 6-month period. Basal plates and meristems were highly responsive explants, while immature umbels and root-tips were less responsive ones, as indicated by percentage of induced callus, growing callus and regenerating callus. The best response was 41% regenerating callus with 0.045 μM 2,4-D and BAP from basal plates while 57, 56 and 20% regenerating callus were obtained with 0.45 μM 2,4-D from meristems, root-tips and immature umbels, respectively. Also, these treatments showed a higher percentage of nodular and embryogenic callus (type I). Thus, it can be concluded that the use of meristems and 2,4-D will enhance callus production and quality, increase plant regeneration and allows to develop a protocol suitable for further transformation experiments in garlic.     

Interactive effects of nitrate and phosphate salts, sucrose, and inoculum culture age on growth and sesquiterpene production in Artemisia annua hairy root cultures

Summary We analyzed four factors (phosphate and nitrate salts, sucrose, and culture inoculum age), simultaneously at three levels using a fractional factorial design method to determine the most suitable conditions for maximizing both root biomass and terpenoid production in transformed Artemisia annua root cultures. Optimal growth conditions were determined to be: nitrate (15 mM), phosphate (1.0 mM), sucrose content (5% wt/vol), and inoculum age (8 d-old). Determination of optimal conditions for sesquiterpene production was more complicated than for biomass production. For most experiments artemisinic acid was undetectable especially in experiments where phosphate was greater than 0.5 mM and for nearly all culture inoculum ages of 14 d. Artemisinic acid was also never detected whenever arteannuin B was present. Arteannuin B was the major artemisinic compound detected in these experiments, sometimes at levels exceeding 300 μg/g fresh weight. When the sum of artemisinin and its three precursors is analyzed, three factors (sucrose, nitrate, and inoculum age) are heavily dependent on one another, and in conjunction with possible degradation of artemisinin by peroxidase, the current analysis does not provide a clear picture regarding the most effective conditions for maximizing the production of artemisinin. Abbreviations: AA, artemisinic acid; AB, arteannuin B; AT, artemisitene; AN, artemisinin; FW, fresh weight; DW, dry weight; S, sucrose; N, potassium nitrate; P, sodium phosphate; A, inoculum culture age.     

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)。     

Variable effects on growth and defense traits for plant ecotypic differentiation and phenotypic plasticity along elevation gradients

Along ecological gradients, phenotypic differentiation can arise through natural selection on trait diversity and magnitude, and environment‐driven plastic changes. The magnitude of ecotypic differentiation versus phenotypic plasticity can vary depending on the traits under study. Using reciprocal transplant‐common gardens along steep elevation gradients, we evaluated patterns of ecotypic differentiation and phenotypic plasticity of several growth and defense‐related traits for two coexisting but unrelated plant species, Cardamine pratensis and Plantago major. For both species, we observed ecotypic differentiation accompanied by plasticity in growth‐related traits. Plants grew faster and produced more biomass when placed at low elevation. In contrast, we observed fixed ecotypic differentiation for defense and resistance traits. Generally, low‐elevation ecotypes produced higher chemical defenses regardless of the growing elevation. Yet, some plasticity was observed for specific compounds, such as indole glucosinolates. The results of this study may suggest that ecotypic differentiation in defense traits is maintained by costs of chemical defense production, while plasticity in growth traits is regulated by temperature‐driven growth response maximization.     

刺激剂(elicitor)在植物细胞培养次生代谢物生产中的应用

刺激剂（ｅｌｉｃｉｔｏｒ）在植物细胞培养中被用来作为提高次生代谢物产量的手段。文中概括介绍了微生物、寡聚糖、蛋白质、第二信使及其他物质作为刺激剂在植物细胞培养中的应用及其研究成果。     

Enhanced biotransformation of TCE using plant terpenoids in contaminated groundwater

Aims:  To examine plant terpenoids as inducers of TCE (trichloroethylene) biotransformation by an indigenous microbial community originating from a plume of TCE-contaminated groundwater.
Methods and Results:  One-litre microcosms of groundwater were spiked with 100 μmol 1⁻¹ of TCE and amended weekly for 16 weeks with 20 μl 1⁻¹ of the following plant monoterpenes: linalool, pulegone, R-(+) carvone, S-(−) carvone, farnesol, cumene. Yeast extract-amended and unamended control treatments were also prepared. The addition of R-carvone and S-carvone, linalool and cumene resulted in the biotransformation of upwards of 88% of the TCE, significantly more than the unamendment control (61%). The aforementioned group of terpenes also significantly ( P  < 0·05) allowed more TCE to be degraded than the remaining two terpenes (farnesol and pulegone), and the yeast extract treatment which biotransformed 74–75% of the TCE. The microbial community profile was monitored by denaturing gradient gel electrophoresis and demonstrated much greater similarities between the microbial communities in terpene-amended treatments than in the yeast extract or unamended controls.
Conclusions:  TCE biotransformation can be significantly enhanced through the addition of selected plant terpenoids.
Significance and Impact of the Study:  Plant terpenoid and nutrient supplementation to groundwater might provide an environmentally benign means of enhancing the rate of in situ TCE bioremediation.     

The effect of growth regulators and sucrose on anthocyanin production in Camptotheca acuminata cell cultures.

The effect of different concentrations of growth regulators and sucrose on anthocyanin production in cell suspension cultures of Camptotheca acuminata Decaisne (Nyssaceae) was described for the first time and qualitatively and quantitatively evaluated. Anthocyanin production was significantly greater in the presence of kinetin, compared to benzyladenine, with the greatest concentration observed in the presence of 2 microM kinetin. No significant differences in anthocyanin production were observed when comparing 2,4-dichlorophenoxyacetic acid to alpha-naphthaleneacetic acid, except when using 2 microM, 2,4-dichlorophenoxyacetic acid, which resulted in greater anthocyanin production. High sucrose concentration enhanced the production of anthocyanins. Based on the absence of anthocyanin production in the dark, we concluded that light was essential for stimulating anthocyanin production. The optimised medium consisted of: 2 microM kinetin, 2 microM 2,4-dichlorophenoxyacetic acid and 292 mM sucrose. HPLC/DAD and HPLC/MS analyses revealed that the main anthocyanin was Cy 3-O-galactoside and that the minor derivative was Cy 3-O-glucoside.     

杜仲愈伤组织中次生代谢产物积累动态研究

以杜仲愈伤组织为材料,研究了不同接种量及培养时间、不同来源的愈伤组织及其继代次数对愈伤组织生长和次生代谢产物含量的影响。结果表明,愈伤组织继代培养时接种量在0．35g左右比较合适。愈伤组织的生长曲线大致呈S形,在20d时达到最大值,而总黄酮和绿原酸的含量均在16d时达到最大值。在继代培养中,茎和叶愈伤组织的增长量、绿原酸和总黄酮含量均在第三代达到最大值;下胚轴诱导的愈伤组织的增长量、绿原酸和总黄酮含量均在第四代达到最大值;子叶愈伤组织的增长量和总黄酮含量在第五代达到最大值,绿原酸含量于第四代达到最大值。不同来源的愈伤组织中,叶愈伤组织中绿原酸含最最高,下胚轴愈伤组织中总黄酮含量最高。     

Effect of carbon dioxide on cell growth and saponin production in suspension cultures of Panax ginseng

The effects of carbon dioxide supply within the range of 1–5 % (along with purified air), on cell culture of Panax ginseng were investigated in a balloon type bubble bioreactor containing 4 dm³ of Murashige and Skoog (MS) medium supplemented with 7.0 mg dm⁻³ indolebutyric acid, 0.5 mg dm⁻³ kinetin and 30 g dm⁻³ sucrose. A 1 % CO₂ supply was found beneficial for the production of cell mass; however, increasing CO₂ concentration to 2.5 and 5 % decreased the biomass accumulation. CO₂ enrichment was not beneficial for saponin production and 1, 2.5, and 5 % CO₂ supply resulted in decrease in saponin accumulation up to 11.6, 19.5, and 50.6 %, respectively.     

Effect of plant growth regulators on evolution of ethylene and methane by different explants of chickpea

Shoot tips, cotyledonary nodes and hypocotyls of chickpea (Cicer arietinum L.) were grown on 3 media: plant induction medium (PIM), callus induction medium (CIM), and shoot induction medium (SIM). Maximum growth and differentiation was seen in PIM, whereas minimum was observed in CIM. Shoot tips which differentiated to multiple shoots evolved negligible amounts of ethylene. Maximum ethylene evolution was recorded by hypocotyls in PIM. Ethylene appears to have stimulatory effect on shoot bud differentiation in cotyledonary nodes. But in hypocotyls, increased ethylene inhibited growth and differentiation. Calli on media containing only auxin (PIM) evolved significantly more ethylene, whereas those on media with cytokinin (SIM) evolved more methane. Callus forming explants like cotyledonary nodes and hypocotyls evolve more ethylene than shoot tips. This revised version was published online in August 2006 with corrections to the Cover Date.     

Bioprocess considerations for production of secondary metabolites by plant cell suspension cultures

Plant cell culture provides a viable alternative over whole plant cultivation for the production of secondary metabolites. In order to successfully cultivate the plant cells at large scale, several engineering parameters such as, cell aggregation, mixing, aeration, and shear sensitivity are taken into account for selection of a suitable bioreactor. The media ingredients, their concentrations and the environmental factors are optimized for maximal synthesis of a desired metabolite. Increased productivity in a bioreactor can be achieved by selection of a proper cultivation strategy (batch, fed-batch, two-stageetc.), feeding of metabolic precursors and extraction of intracellular metabolites. Proper understanding and rigorous analysis of these parameters would pave the way towards the successful commercialization of plant cell bioprocesses.     

培养基及培养条件对杜仲愈伤组织生长及次生代谢产物含量的影响

以MS、LS、B5、N6、H、Nitsch、White、1／2MS为基本培养基,分别添加0．5mg／L NAA和0．5mg／L BA,分析不同类型培养基对杜仲愈伤组织生长及次生代谢产物含量的影响,并以B5培养基进行光照条件、碳源、蔗糖浓度试验。结果表明：B5培养基不仅有利于愈伤组织生长,也有利于总黄酮的形成,而1／2MS培养基有利于绿原酸的积累;12h／d光照对愈伤组织的生长及绿原酸和总黄酮的合成有明显的促进作用,黑暗不影响愈伤组织的生长,但却抑制绿原酸和总黄酮的形成;3种碳源中,愈伤组织的增长量、绿原酸和总黄酮的含量均以蔗糖为碳源时最高,葡萄糖最低;蔗糖浓度在10～50g／L范围内绿原酸的含量随着糖浓度的升高而升高,40g／L时愈伤组织的增长量和总黄酮的含量最高。