培养条件对新疆紫草毛状根生长及紫草素含量的影响

以发根农杆菌诱导的新疆紫草毛状根为试验材料,采用二阶段液体培养法,首次建立了新疆紫草毛状根培养技术体系。结果显示:采用SH无铵培养基、pH 5.8时有利于毛状根的生长。培养12d时毛状根的增殖倍数达最高,平均10.26倍;毛状根生产的继代周期为25～30d;4种树脂吸附的紫草素及其衍生物含量均较对照(不添加树脂)高,以NKA-9所吸附的紫草素及其衍生物含量最高,为2.38%,较对照提高0.97倍。培养10d时添加NKA-9树脂,紫草素及其衍生物含量平均为3.64%,是对照的3.08倍。研究表明,生长阶段采用液体培养可以使新疆紫草毛状根快速增殖,生产阶段添加大孔吸附树脂能够提高紫草素及其衍生物含量。     

Integrated Recovery of Pigments Released from Red Beet Hairy Roots Exposed to Acidic Medium

Hairy root cultures of Beta vulgaris L grown in a bubble column reactor were permeabilised by exposure to B5 medium of pH 2.0. The roots released 39% of their total pigments on a 10 min exposure to B5 medium of pH 2.0 followed by return to standard 135 medium. The pigments released in the extracellular medium were recovered on an adsorption column containing XAD-16 resin. The permeabilised roots regrew and accumulated additional pigments. Comparison of this technique with the previously used techniques like oxygen starvation and temperature shock to permeabilise beet hairy roots suggest that pH mediated release of betalains can be an effective method of releasing betalains from root cultures.     

真菌诱导子与吸附树脂对新疆紫草毛状根中萘醌积累的影响

通过考察真菌诱导子与吸附树脂对新疆紫草毛状根中萘醌积累的影响,获得真菌诱导子与吸附树脂对萘醌类物质积累的最佳处理,为规模化生产提供依据.以新疆紫草毛状根为试验材料,将黑曲霉、米曲霉诱导子及其混合诱导子、大孔吸附树脂添加到M-9培养基中,采用分光光度法测定毛状根总萘醌含量.试验结果表明:在毛状根培养10d时以2.5∶50的比例添加混合诱导子,总萘醌含量是对照的2.28倍;在此结果基础上,在培养第0天添加大孔吸附树脂NKA-9,总萘醌含量最高是对照的3.71倍;黑曲霉诱导子与米曲霉诱导子有协同效应;在生物反应器中添加混合诱导子及大孔吸附树脂NKA-9,其总萘醌含量是对照的4.17倍.米曲霉诱导子、混合诱导子对毛状根增殖有促进作用;同时添加大孔吸附树脂NKA-9及混合诱导子能提高毛状根总萘醌含量.生物反应器培养毛状根为今后利用新疆紫草毛状根规模化生产总萘醌提供了理论参考.     

Transformation of Gynostermma pentaphyllum by Agrobacterium rhizogenes and Saponin Production in Hairy Root Cultures

Authors report here the establishment of an efficient transformation system for Gynosternrna pentaphyllum (Thunb.) Makino using Agrobacteriurn rhizogenes R1600. Hairy roots appeared on leaf explants 10 days after inoculation with the bacteria . Frequency of the explants transformed by R1600 was up to 94%. Transformation was confirmed by Southern analysis. Biomass of hairy root cultures suspended in hormone-free MS medium increased 9 times after 20 days of incubation. There was no callus formation on the hairy roots during suspension culture. Saponin content in the hairy root cultures was about 2 times as much as in the natural roots, saponins of the hairy root cultures were also released into growth medium as well.     

Production and release of pigments by culture of transformed hairy root of red beet

Adventitious roots were induced from red beet (Beta vulgaris L. cv. Detroit dark red) by infecting the plant with a soil bacterium, Agrobacterium rhizogenes. Based on analysis of opines which are uniquely produced in transformed hairy roots, the established clone was proved to be a transformed hairy root. In a shake culture of the beet hairy root clone with a liquid medium, it was found that significant amounts of pigments, mainly betanin and vulgaxanthin-I, were released into the medium by the cessation of culture shaking (temporary limitation of oxygen supply). The hairy root cells were capable of propagation even after the cells were subjected to shaking cessation. Repeated-batch culture of the beet hairy root was performed with the cell growth phases for 9 or 10 d and with pigment leakage phases during shaking cessation for 2 d, and more than 20% of the total intracellular pigments were recovered from the culture broth at a culture time of 35 d. The released pigments were confirmed to be substantially identical to those extracted from the hairy root and original plant cells of red beet.     

蔗糖和光对三裂叶野葛毛状根生长及次生物质产生的影响

研究了蔗糖浓度和光对固体培养的三裂叶野葛毛状根生长及其总异黄酮和葛根素产生的影响。结果表明：在供试的分别添加1%、3%、5%、7%和9%蔗糖的MS固体培养基中,3%蔗糖能促进三裂叶野葛毛状根的生长及其异黄酮类化合物和葛根素的积累;培养20d后,其生物量达到0.48g(DW,干重)/瓶,总异黄酮和葛根素含量分别为25.44mg/g(DW)和11.64mg/g(DW)。与添加3%蔗糖的MS培养基培养的三裂叶野葛毛状根相比,含5%蔗糖的培养基培养的毛状根干重增殖倍数提高了7.0％,而含1％、7％和9％蔗糖的培养基培养的毛状根干重增殖倍数分别下降62.4％、42.8％和65.3％;其总异黄酮含量分别降低574％、13％和33.4％,葛根素含量分别下降47.9％、15.8％和35.1％,但其毛状根培养物的可溶性糖含量则分别增加了0.52、1.45和1.54倍。暗培养30d的毛状根的生物量达到0.83g(DW)/瓶,分别比蓝光和白光培养的毛状根提高37.1%和23.3%。在蓝光和白光下培养的部分毛状根的表面呈淡绿色;但白光处理的毛状根中总异黄酮含量比蓝光和暗培养处理的分别提高了14.7%和19.2%;蓝光抑制毛状根中葛根素含量的积累,白光和暗培养的毛状根培养物中的葛根素含量分别是蓝光处理的1.61倍和1.52倍。     

Umbelliferone released from hairy root cultures of Pharbitis nil treated with copper sulfate and its subsequent glucosylation

Hairy root cultures of Pharbitis nil treated with CuSO4 and methyl jasmonate (MeJA) produced umbelliferone (1) and scopoletin (2) in the culture medium, and skimmin (3), a beta-D-glucopyranoside of 1, was isolated from the hairy roots. While 1 in the medium increased and reached a maximal level 16 h after the treatment with CuSO4, the amount of 3 in the hairy roots decreased, reaching a minimal level after 8 h, before recovering to a level higher than the basal level after 24 h and then continuously increasing. These observations suggest that 1 was released by the hydrolysis of 3. Umbelliferone (1) inhibited hairy root growth, while skimmin (3) did not. This result suggests that, after the release of 1 as a phytoalexin, the hairy roots glycosylated 1 for the detoxification and re-use of 3 as a source of phytoalexin.     

High-yield production of tropane alkaloids by hairy-root cultures of aDatura candida hybrid

Hairy root cultures were obtained following inoculation of the stems of sterile plantlets of aDatura candida hybrid withAgrobacterium rhizogenes. The scopolamine and hyoscyamine content was quantified by HPLC and compared with the non-transformed plants. The alkaloid yield (0.68% dry weight) obtained with the hairy roots was 1.6 and 2.6 times the amount found in the aerial parts and in the roots of the parent plants, respectively. Only a small proportion of alkaloids was released into the growth medium. Scopclamine was the principal alkaloid and the scopolamine/hyoscyamine ratio of ca. 5:1 makes these hairy roct cultures worthy of consideration as a source of scopolamine.     

Production and release of anthraquinone pigments by hairy roots of madder (Rubia tinctorum L.) under improved culture conditions

Carbon and nitrogen sources in the medium were selected for the culture of madder hairy roots producing anthraquinone pigments. The growth and pigment formation of the hairy roots were significantly enhanced by using modified Murashige-Skoog (MS) medium containing fructose and nitrate as the sole carbon and nitrogen sources, respectively, compared with those obtained in conventional MS medium with sucrose. Repeated-batch culture of the hairy roots was carried out, with pigment release into the medium obtained by means of O₂ starvation treatment. In three pigment-release operations during 29-d culture, the total amount of released pigments was 21 mg/dm³, representing an average production rate of 0.72 mg/(dm³·d).     

Characterization of lucidin formation in Rubia tinctorum L.

In order to approach lucidin formation (a strong mutagen or a carcinogen) from a physiological standpoint, hairy roots of Rubia tinctorum L. were established by a transformation of Agrobacterium rhizogenes strain 15834 and cultured in a liquid woody plant medium without plant hormones. The anthraquinone pigment composition of the intact hairy roots was essentially the same as that of the intact non-transformed (normal) roots, in which lucidin O-beta-D-primeveroside (LuP) was one of the major pigments. Lucidin was scarcely detected in the intact hairy roots, but was a main pigment after the squash treatment. The crude protein extract of intact hairy roots exhibited LuP-glycosidase activity (an activity converting LuP to lucidin). This activity was also detected in the roots of the normal plants at a high level, but slightly in the stems and not in the leaves. Methyl jasmonate enhanced the LuP production and LuP-glycosidase activity in the hairy roots. On the other hand, ethephon or salicylic acid had either no effect or rather an inhibitory effect on them. After partial purification of LuP glycosidase, the resultant active fraction producing a major band with an apparent Mr of 68 kDa exhibited the substrate specificity for both aglycon and sugar-moiety. The sugar released from LuP by this fraction was neither D-glucose nor D-xylose and was hydrolyzed into them. These results suggest that LuP specific beta-primeverosidase (EC 3.2.1.149) exists in the roots of R. tinctorum and is involved in the systematic defense system.     

吐温-80对野葛毛状根生长及异黄酮含量的影响

将不同浓度的吐温-80添加到野葛毛状根悬浮培养液中,研究在一定的作用时间内其对毛状根生长及次生代谢物合成与分泌的影响。结果表明,采用2％浓度处理较为适宜,不仅可以提高毛状根内葛根素的含量,而且有利于培养液中葛根素、大豆甙元及总异黄酮的积累,与对照相比,其含量可分别提高24．2％、50％和46．7％。在该浓度下连续处理毛状根72h后,发现毛状根仍生长旺盛,其生长量已是对照的1．5倍。但不同时间的连续处理对毛状根及培养液中几种异黄酮物质的积累与释放作用不同,其中以处理48h最有利于培养液中总异黄酮的累积,其含量是毛状根中的38倍。     

Transformed roots of Artemisia annua exhibit an unusual pattern of border cell release

Summary Border cells from Artemisia annua were examined from hairy roots grown in shake flasks, culture plates, a bubble column reactor, and a nutrient mist (aeroponic) reactor. When well-hydrated roots were subjected to shear, border cells were first released as an agglomerate and did not disperse for several hours. Staining with neutral red and fluorescein diacetate (FDA) showed that both agglomerates and dispersed cells were alive. It was determined that FDA is cleaved by pectin methylesterase (PME) and that PME may not be particularly active in the released agglomerates until the border cells disperse. Untransformed roots isolated from A. annua plants showed no border cell agglomerate formation and border cells readily dispersed. These results suggest that our hairy root clone is deficient in border cell release perhaps resulting from the transformation process.     

Food-grade chemical and biological agents permeabilize red beet hairy roots,assisting the release of betalaines

Hairy root cultures of red beet, Beta vulgaris L., were permeabilized under the functions of food-grade chemical and biological agents cetyl trimethylammonium bromide (CTAB), Triton X-100, Tween-80, Lactobacillus helveticus, Saccharomyces cereviseae, and Candida utilis, as well as cell fractions of L. helveticus, for the recovery of betalaines with or without oxygen stress. Tween-80 (0.15%), Triton X-100 (0.2%), and CTAB (0.05%), in combination with oxygen stress, released 45%, 70%, and 90% pigment into the medium, respectively, with significantly lesser levels in agitated cultures receiving similar treatments. The release was rapid (1 h) in CTAB treatment with a much slower release in Tween-80. CTAB (0.002%) was found to be also useful in effluxing betalaines (80%) from hairy roots grown in a bubble column reactor. Viability of permeabilized hairy roots, tested on agar medium, was not affected by any level of CTAB treatment and was significantly retarded at higher levels of Triton X-100 and Tween-80. An altogether new approach of pigment release using biological agents such as live cells of food-grade microbes was used where C. utilis, L. helveticus, and S. cereviseae released 60%, 85%, and 54% betalaines, respectively, in 24 h, though lower level treatments also released similar levels of pigment by 48 h. Dried whole cell powder of L. helveticus, its total insoluble carbohydrate, and free lipid fractions released 10%, 0%, and 85% pigment, respectively. An extended study with a bubble column reactor using the free lipid fraction of L. helveticus showed 50% and 84% pigment release in 8 and 12 h, respectively, exhibiting good viability when plated on agar medium. Even in the bioreactor, replenishment of medium 8 h after treatment with free lipid of L. helveticus allowed regrowth of hairy roots. The high level of pigment release recorded here, using CTAB or lipid of L. helveticus, appears useful for developing processes for in situ recovery of betalaines. The live microbes, applicable only for batch cultures, are expected to impart improved sensory/nutraceutical effects to the recovered pigment and hence may add value to the product receiving the red beet pigment thus produced.     

Tropane alkaloid production by Agrobacterium rhizogenes transformed hairy root cultures of Atropa baetica Willk. (Solanaceae)

Atropa baetica hairy root cultures were induced after infecting stem segments with Agrobacterium rhizogenes strain ATCC 15834. Accumulation of the tropane alkaloids atropine and scopolamine by hairy roots cultured in half- and full-strength Murashige and Skoog (MS) medium was high, although this was not growth associated. These alkaloids were also released into both liquid media. Higher tropane alkaloids present both in hairy roots and liquid medium occurred in half MS medium, showing a clear relationship between slow growth of cultures and higher product accumulation. The pH of both nutrient media varied as culture progressed, and seemed to be associated with the release of scopolamine. GC-MS analyses showed the presence of a new compound, namely tigloylpseudotropine; moreover, 3α-isobutyryloxytropane, formerly found only in plant leaf tissue, was also identified in the hairy roots. Received: 18 August 1997 / Revision received: 30 November 1997 / Accepted: 20 January 1998     

蔗糖对发根农杆菌C58C1诱导烟草毛状根生长的影响北大核心CSCD

毛状根良好的生长状况是建立毛状根-AM真菌双重培养体系的关键,为优化毛状根培养基成分,确定适宜毛状根生长的蔗糖浓度,改善烟草毛状根的生长状况,该研究以发根农杆菌菌株C58C1诱导2个烟草品种NC82和Va116叶片产生毛状根,经PCR检测证实后,用含有不同蔗糖浓度的1/2MS培养基分别进行固体和液体优化培养,通过测定毛状根的分枝数、鲜重(FW)与干重(DW),研究蔗糖对2个品种烟草毛状根生长的影响。结果表明:(1)C58C1均能诱导两种烟草叶片产生毛状根,但诱导率不同,NC82(87.3%)的诱导率更高,是Va116(38.6%)的2.26倍。(2)培养基蔗糖浓度显著影响毛状根生长,因烟草品种和起始分枝数而异。(3)固体培养基优化培养NC82和Va116的毛状根,分枝数增长的抑制蔗糖浓度分别为25 g·L^(-1)和15 g·L^(-1);液体培养基优化培养分别在25 g·L^(-1)和15 g·L^(-1)时F(D)W达到最大,分别为0.541 g(0.055 g)、0.474 g(0.050 g)。(4)综合分枝数、F(D)W、毛状根生长势考虑,C58C1诱导NC82毛状根最适培养基蔗糖浓度为25 g·L^(-1),Va116毛状根为15 g·L^(-1)。该文优化了烟草毛状根培养基组成的适宜蔗糖浓度及培养方法,为后续毛状根大量扩繁奠定基础,建立了毛状根-AM真菌双重培养体系,解决了关键的寄主生长不良的问题。     

3种农杆菌对茶树发状根诱导的影响

以茶树'福云6号'和'铁观音'成熟种子下胚轴、未成熟种子下胚轴和愈伤组织为材料,以发状根诱导率为指标,探究菌液浓度、农杆菌菌株、外植体类型和预培养时间对发状根诱导的影响.结果表明:(1)菌液浓度OD600在0.4～1.2范围内,'福云6号'成熟种子下胚轴发状根诱导率先升高后降低,ATCC15834在OD 600为0.6...     

Efficient production and recovery of diterpenoid tanshinones in Salvia miltiorrhiza hairy root cultures with in situ adsorption, elicitation and semi-continuous operation

In Salvia miltiorrhiza hairy root cultures, the desired secondary metabolites diterpenoid tanshinones are normally produced at low yields and stored within the roots. To enhance tanshinone production and the secondary product recovery, we employed three means, elicitation with a yeast elicitor (YE), in situ adsorption of tanshinones with a hydrophobic polymeric resin (X-5) and semi-continuous mode of operation. YE treatment stimulated the tanshinone biosynthesis, increasing the total tanshinone (TT) content of root by about two-fold, from 0.46 to 1.37 mg/g dry weight (dw) (TT content=total content of three major tanshinones, cryptotanshinone, tanshinone I and tanshinone IIA). The addition of X-5 resins to the culture only increased the tanshinone yield slightly, but recovered more than 80% of tanshinones from the roots. With the application of a semi-continuous culture process involving repeated medium renewal, elicitor addition and resin replacement, starting at the late exponential growth phase, the root biomass was increased to 30.5g dw/l (versus 8-10g dw/l in batch mode) and the volumetric tanshinone yield to 87.5mg/l (about 15-fold increase), with 76.5% adsorbed to the resin. The volumetric productivity of total tanshinone reached 1.46 mg/lday, more than 7.4 times that of the batch culture. The results demonstrate that the integration of multiple elicitation, in situ adsorption and semi-continuous operation can synergistically enhance tanshinone production in S. miltiorrhiza hairy root cultures.     

In vitro root cultures of Panax ginseng and P. quinquefolium

The paper describes a procedure for the initiation, subculture and continued proliferation of adventitious roots of Panax ginseng and Panax quinquefolium, which resemble hairy roots. The technique took advantage of the high powerful activity of a new synthetic auxin: benzo[b]selenienyl acetic acid (BSAA). Such initiation from root explants was dependent upon the season, the type and concentration of auxin. The hairy-like roots of ginseng could be subcultured by transfer every 4 weeks to fresh liquid medium either in agitated Erlenmeyer flasks or in bioreactors. Optimal conditions for a continued multiplication (up to 14 per month) were determined. The only practical problem was the limitation of the fresh mass as inoculum: the multiplication rate decreased with the increased quantity of roots. It is postulated that a root growth inhibiting substance was released into the media by the proliferating ginseng hairy roots.     

Transformed root cultures for biotechnology

This review is concerned with the application of hairy roots, i.e. plant roots formed from plant cells after transformation by Agrobacterium rhizogenes for the production of bioactive compounds. Transformed root cultures have been established from numerous species of dicotyledonous plants. The plants, as well as the main products accumulated in hairy root cultures derived from these plants, are listed in this paper. Data are presented on novel compounds, hitherto detected only in transformed roots but not occurring in the corresponding intact plants. The possible use of hairy root cultures for the over-production of secondary metabolites and biotransformation of chemicals is discussed. In order to enhance the productivity of hairy root cultures, various methods have been derived, and optimized procedures are proposed. They include selection of high-producing clones, elicitation, composition of growth media, culture conditions and genetic approach. Hairy roots usually store secondary metabolites in vacuoles inside the cells. Therefore, several methods have been used to increase the amount of products released into the medium. Unfortunately, no general procedure is known that works in all cases, and the excretion behaviour of hairy root cultures varies from one species to another and even within one species from one clone to another. Special attention is given to the cultivation methods and bioreactor systems for hairy root cultures. Hairy roots are cultivated usually in shake flasks; however, shake flask culture is not suitable for the complex optimization and continuous control of the culture conditions. In this paper, we are going to present bioreactors proposed for the cultivation of hairy roots under more or less controlled conditions. Modifications of typical bacterial bioreactors, i.e. stirred tanks, airlift loop reactors and other constructions, are presented. A very special type of bioreactor providing good conditions for loose root mass multiplication without oxygen or substrate limitations, is the mist bioreactor. Nowadays, it is practically impossible to select the one best bioreactor type for hairy root culture.