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1.
Summary Hairy root culture of Hyoscyamus albus was established by transformation with Agrobacterium rhizogenes strain A4. The growth and production of five tropane alkaloids were investigated under various culture conditions. Among the four basal culture media tested, Woody Plant medium was the best for growth of the hairy roots, but a high amount of tropane alkaloids was obtained with Gamborg's B5 medium. Sucrose concentration in B5 medium had little effect on the growth, while 3% sucrose was suitable for the alkaloid production. Addition of KNO3 to Woody Plant medium affected the growth, whereas the alkaloid content was not markedly improved. Supplement of some metal ions to B5 medium stimulated the alkaloid production. In particular, Cu2+ remarkably enhanced both the growth and the alkaloid yield. The hairy roots cultured under 16 h/day light survived for more than 32 days compared with those cultured in the dark.Abbreviations EDTA
ethylenediaminetetraacetic acid
- HPLC
high performance liquid chromatography
- MeOH
methanol
- MS medium
Murashige and Skoog medium
- WP medium
McCown's Woody Plant medium
- B5 medium
Gamborg B5 medium
- wt
weight 相似文献
2.
Toshio Aoki Hideki Matsumoto Youichi Asako Yuji Matsunaga Koichiro Shimomura 《Plant cell reports》1997,16(5):282-286
Hairy root cultures ofAtropabelladonna were established by transformation withAgrobacterium rhizogenes 15834. Five clones of them were employed to study the production of hyoscyamine, the main constituent of the plant, together with other tropane alkaloids. The growth and alkaloid production of each clone were differently affected by basal liquid culture media tested. The transgenic plants regenerated from each clone of the hairy roots had different phenotypes and diverse alkaloid productivity both in the cultured condition and in productivitiy both in the cultured condition and in hydroponics.Abbreviations ANOVA
analysis of variance
- B5 medium
Gamborg B5 medium
- BA
N6-benzyladenine
- B.S.
Balanced Solution
- dw
dry weight
- EC
electric conductivity
- fw
fresh weight
- GC/MS
gas chromatography-mass spectrometry
- HPLC
high performance liquid chromatography
- MS medium
Murashige and Skoog medium
- NAA
naphthalene-l-acetic acid
- PCR
polymerase chain reaction
- SDS
sodium dodecyl sulfate
- TMS
trimethylsilyl
- WP medium
Woody Plant medium 相似文献
3.
4.
Li Li Jing Wang Wei Wang Yang Lu Yuliang Wang Genyu Zhou Guoyin Kai 《Biotechnology and Bioprocess Engineering》2008,13(5):606-612
In this study, an efficient transformation system for the medicinal plant Anisodus acutangulus was successfully developed and optimized using Agrobacterium rhizogenes. Three bacterial strains, A4, R1601, and modified C58C1 and three explant types, leaf blade, petiole, and stem, were examined.
The highest transformation efficiency of 94.44% was achieved using strain C58C1 with stem explants. Over 20 independent hairy
root lines were successfully established with strain C58C1 using stem explants, all of which contained the ro/B and ro/C genes as confirmed by polymerase chain reaction (PCR). Out of four media compositions, the liquid 1/2 MS medium was found
the most suitable for hairy root growth. The maximum biomass of one hairy root line increased up to 80 times in liquid 1/2
MS medium after a 30 day culture period. Different hairy root lines displayed a varied capacity for tropane alkaloid production
and the best hairy root line (T4) from the C58C1-stem combination produced up to 10.21 mg/g (dw) of hyoscyamine, which was
about 1.5-fold higher than in the wild type plants. To our knowledge, this is the first report to demonstrate the production
of tropane alkaloids in hairy roots of A. acutangulus. 相似文献
5.
Effects of Oxygen on Nicotine and Tropane Alkaloid Production in Cultured Roots Duboisia myoporoides
《Bioscience, biotechnology, and biochemistry》2013,77(10):1824-1827
The effects of oxygen on nicotine and tropane alkaloid production in root cultures of Duboisia myoporoides were investigated. Duboisia roots cultured in air produced both nicotine and tropane alkaloids equally. However, when roots were cultured in pure oxygen, the metabolic flux to tropane alkaloids increased, and that to nicotine alkaloids decreased. Intermediate product analysis by GC-MS showed an increase in tropine, but decreases in acetyl derivatives of tropane alkaloids and tropine esters with low-class fatty acids. Furthermore, hyoscyamine 6β-hydroxylase (H6H, EC 1.14.11.11, the key enzyme in the pathway from hyosyamine to scopolamine) also increased. These results suggest that pure oxygen contributes to scopolamine production not only by activating the biosynthetic steps for scopolamine, but also by inactivating the biosynthetic steps for nicotine and other tropine derivatives. 相似文献
6.
Summary Hairy root cultures of Hyoscyamus muticus were established using Agrobacterium rhizogenes ATCC 15834. In one out of 8 clones established, an unusual root tip formation was observed after transfer of cultures from half-strength Murashige and Skoog (1962) to White's medium (1939). This phenomenon was associated with the production of a fine brownish cell suspension culture. Hairy root development resumed after transfer of the root tips from White to half-strength Murashige and Skoog medium. After plating the isolated brownish cells on hormone-free half-strength Murashige and Skoog or White solid medium, callus proliferation was observed, and then redifferentiation of hairy roots occurred. The polymerase chain reaction analysis of the H. muticus hairy root (clone Z2) revealed that only the tl region of the T-DNA was integrated. The growth and the production of five tropane alkaloids by this clone were examined.Abbreviations PCR
Polymerase Chain Reaction
- MS medium
Murashige and Skoog Medium
- 1/2 MS medium
half-strength MS medium
- WP medium
Woody Plant medium
- RC medium
Root Culture medium
- WH medium
White medium
- HPLC
High Performance Liquid Chromatography
- wt.
weight 相似文献
7.
Ignacio E. Maldonado-Mendoza Victor M. Loyola-Vargas 《Plant Cell, Tissue and Organ Culture》1995,40(3):197-208
Twelve different lines of Datura stramonium (normal and hairy) root cultures were subjected to conditions which induce photoautotrophy. Two of the hairy root lines responded to induction, showing clearly a diminished growth rate when compared to heterotrophic cultures, an increase in chlorophyll, a net O2 evolution, CO2 fixation, and de novo synthesis of the ribulose 1,5 biphosphate carboxylase enzyme. A time course of growth and tropane alkaloid levels in the tissue and medium, revealed a correlation between the development of the photosynthetic apparatus and the increase in scopolamine. Although normal cultures did not grow photosynthetically, they showed some greening response under the first step of the induction. The correlation between development of photosynthesis and increase in scopolamine synthesis were corroborated with normal root cultures. This experimental model is used for the basic study of the regulatory enzymes involved in the biosynthesis of tropane alkaloids, as well as for the study of their mechanisms of transport. 相似文献
8.
Zhang L Yang B Lu B Kai G Wang Z Xia Y Ding R Zhang H Sun X Chen W Tang K 《Planta》2007,225(4):887-896
The cDNA from Nicotiana tabacum encoding Putrescine N-methyltransferase (PMT), which catalyzes the first committed step in the biosynthesis of tropane alkaloids, has been introduced
into the genome of a scopolamine-producing Hyoscyamus niger mediated by the disarmed Agrobacterium tumefaciens strain C58C1, which also carries Agrobacterium rhizogenes Ri plasmid pRiA4, and expressed under the control of the CaMV 35S promoter. Hairy root lines transformed with pmt presented fivefold higher PMT activity than the control, and the methylputrescine (MPUT) levels of the resulting engineered
hairy roots increased four to fivefold compared to the control and wild-type roots, but there was no significant increase
in tropane alkaloids. However, after methyl jasmonate (MeJA) treatment, a considerable increase of PMTase and endogenous H6Hase
as well as an increase in scopolamine content was found either in the transgenic hairy roots or the control. The results indicate
that hairy root lines over-expressing pmt have a high capacity to synthesize MPUT, whereas their ability to convert hyoscyamine into scopolamine is very limited. Exposure
to MeJA strongly stimulated both polyamine and tropane biosynthesis pathways and elicitation led to more or less enhanced
production simultaneously. 相似文献
9.
Esmail Dehghan Suvi T. H?kkinen Kirsi-Marja Oksman-Caldentey Farajollah Shahriari Ahmadi 《Plant Cell, Tissue and Organ Culture》2012,110(1):35-44
In this study, the effects of ploidy level and culture medium were studied on the production of tropane alkaloids. We have successfully produced stable tetraploid hairy root lines of Hyoscyamus muticus and their ploidy stability was confirmed 30?months after transformation. Tetraploidy affected the growth rate and alkaloid accumulation in plants and transformed root cultures of Egyptian henbane. Although tetraploid plants could produce 200% higher scopolamine than their diploid counterparts, this result was not observed for corresponding induced hairy root cultures. Culture conditions did not only play an important role for biomass production, but also significantly affected tropane alkaloid accumulation in hairy root cultures. In spite of its lower biomass production, tetraploid clone could produce more scopolamine than the diploid counterpart under similar growth conditions. The highest yields of scopolamine (13.87?mg?l?1) and hyoscyamine (107.7?mg 1?1) were obtained when diploid clones were grown on medium consisting of either Murashige and Skoog with 60?g/l sucrose or Gamborg??s B5 with 40?g/l sucrose, respectively. Although the hyoscyamine is the main alkaloid in the H. muticus plants, manipulation of ploidy level and culture conditions successfully changed the scopolamine/hyoscyamine ratio towards scopolamine. The fact that hyoscyamine is converted to scopolamine is very important due to the higher market value of scopolamine. 相似文献
10.
Alejandra B. Cardillo Ana M. Giulietti Javier Palazón Mercè Bonfill 《Plant Cell, Tissue and Organ Culture》2013,114(3):305-312
Hyoscyamine and scopolamine are tropane alkaloids widely applied in medicine. Differences in alkaloid production and growth kinetics have been observed in Argentinian and Colombian ecotypes of Brugmansia candida hairy roots. The aim of this work was to analyze the production of key intermediates in tropane alkaloid synthesis in both ecotypes to determine differences in the biosynthetic pathway. Additionally, rolC gene expression was analyzed to determine its correlation with hairy root growth. The results showed a higher accumulation of polyamines in Colombian hairy roots, suggesting that there may be a rate-limiting enzyme in the last steps of hyoscyamine biosynthesis. Additionally, rolC gene expression was correlated with an improvement in hairy root growth, which supports the function of rol genes as growth modulators and suggests that metabolic engineering approaches involving rolC manipulation may be useful for the development of more efficient B. candida hairy root cultures for biotechnological applications. 相似文献
11.
Fereshte Moharrami Bahman Hosseini Ali Sharafi Manouchehr Farjaminezhad 《In vitro cellular & developmental biology. Plant》2017,53(2):104-111
The medicinal plant Hyoscyamus reticulatus L. is a rich source of hyoscyamine and scopolamine, the tropane alkaloids. The use of hairy root cultures has focused significant attention on production of important metabolites such as stable tropane alkaloid production. Elicitation is an effective approach to induce secondary metabolite biosynthetic pathways. Hairy roots were derived from cotyledon explants inoculated with Agrobacterium rhizogenes and elicited by iron oxide nanoparticles (FeNPs) at different concentrations (0, 450, 900, 1800, and 3600 mg L?1) for different exposure times (24, 48, and 72 h). The highest hairy root fresh and dry weights were found in the medium supplemented with 900 mg L?1 FeNPs. Antioxidant enzyme activity was significantly increased in induced hairy roots compared to non-transgenic roots. The highest hyoscyamine and scopolamine production (about fivefold increase over the control) was achieved with 900 and 450 mg L?1 FeNPs at 24 and 48 h of exposure time, respectively. This is the first report of the effect of FeNP elicitor on hairy root cultures of a medicinal plant. We suggest that FeNPs could be an effective elicitor in hairy root cultures in order to increase tropane alkaloid production. 相似文献
12.
Effect of pmt gene overexpression on tropane alkaloid production in transformed root cultures of Datura metel and Hyoscyamus muticus 总被引:6,自引:0,他引:6
Moyano E Jouhikainen K Tammela P Palazón J Cusidó RM Piñol MT Teeri TH Oksman-Caldentey KM 《Journal of experimental botany》2003,54(381):203-211
In order to increase the production of the pharmaceuticals hyoscyamine and scopolamine in hairy root cultures, a binary vector system was developed to introduce the T-DNA of the Ri plasmid together with the tobacco pmt gene under the control of CaMV 35S promoter, into the genome of Datura metel and Hyoscyamus muticus. This gene codes for putrescine:SAM N-methyltransferase (PMT; EC. 2.1.1.53), which catalyses the first committed step in the tropane alkaloid pathway. Hairy root cultures overexpressing the pmt gene aged faster and accumulated higher amounts of tropane alkaloids than control hairy roots. Both hyoscyamine and scopolamine production were improved in hairy root cultures of D. metel, whereas in H. muticus only hyoscyamine contents were increased by pmt gene overexpression. These roots have a high capacity to synthesize hyoscyamine, but their ability to convert it into scopolamine is very limited. The results indicate that the same biosynthetic pathway in two related plant species can be differently regulated, and overexpression of a given gene does not necessarily lead to a similar accumulation pattern of secondary metabolites. 相似文献
13.
Toshiya Muranaka Hideo Ohkawa Yasuyuki Yamada 《Applied microbiology and biotechnology》1993,40(2-3):219-223
The scopolamine-releasing hairy root clone DL47-1 of Duboisia leichhardtii was cultured in an Amberlite XAD-2 column-combined bioreactor system for continuous production of scopolamine. The medium used was continuously exchanged during culture to maintain the electrical conductivity of the medium constant. After culturing the hairy roots in the system for 11 weeks, 0.5 g/l of scopolamine was obtained in the column. When the roots were cultures in the reactor system containing polyurethane foam or stainless-steel mesh to support the hairy roots, scopolamine recovery was increased. Thereafter, a two-stage culture, the first stage in the medium for hairy root growth and the second stage in the medium for scopolamine release, was carried out in this system by using a turbine-blade reactor with stainless-steel mesh as a support. Under these conditions, 1.3 g/l of scopolamine was recovered during 11 weeks of culture in the medium for scopolamine release. This bioreactor system seems applicable for the production of various plant metabolites by cultures of hairy roots.
Correspondence to: T. Muranaka 相似文献
14.
Zhang Yin-lin 《植物学报(英文版)》1988,30(4)
The steriled plants of Scopolia lurida after being infected by A. rhizogenes were induced to produce hairy roots. They grew well in medium either of hormone free MS agar or of roots suspension culture. As the result, the cultivated hairy root was shown to yield alkaloids such as hyosciamine and scopolamine. Since agropine and mannopine were also detected in the hairy root cultures, the transference of the T-DNA of A. rhizogenes Ri-plasmid into the cell genomic DNA of S. lurida is clearly demonstrated. 相似文献
15.
发根农杆菌的Ri-质粒转化和赛莨菪的发根培养 总被引:5,自引:0,他引:5
张荫鳞 《Acta Botanica Sinica》1988,30(4):368-372
将赛莨菪(Scopolia Iurida)的无菌苗被含有Ri-质粒的发根农村菌(Agrobacterium rhizogenes)感染后,诱导出发根(hariy root)将发根分离,除菌后,在不含激素的琼脂或液体MS培养基上培养,发根能产生正常植物体中含有的莨菪碱和东莨菪碱等生物碱,在发根培养物中检测到agropine和mannopine,说明发根农杆菌Ri-质粒的T-DNA部分已转化到赛菪莨细胞的DNA中。 相似文献
16.
A hairy root clone (M8) of Atropa belladonna, producing high levels of tropane alkaloids, was established by transformation with Agrobacterium rhizogenes (MAFF 03-01724). Littorine, an intermediate of tropane alkaloids, was detected by high-performance liquid chromatography
and gas chromatography-mass spectrometry in the alkaloid fraction of the hairy roots and identified by nuclear magnetic resonance
analysis. Littorine was also detected in the non-transformed root culture of A. belladonna.
Received: 18 March 1998 / Revision received: 15 June 1998 / Accepted: 3 August 1998 相似文献
17.
Philippe Christen Margaret F. Roberts J. David Phillipson William C. Evans 《Plant cell reports》1989,8(2):75-77
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. 相似文献
18.
Coculture of genetically transformed roots and shoots for synthesis, translocation, and biotransformation of secondary metabolites 总被引:3,自引:0,他引:3
Genetically transformed shooty teratomas of Atropa belladonna and a Duboisia leichhardtii x D. myoporoides hybrid were studied for biotransformation of tropane alkaloids in shake flasks and bioreactors. Although de novo synthesis of hyoscyamine and scopolamine was limited, shoots of both species were able to translocate and accumulate significant quantities of exogenous alkaloid. The maximum yield of scopolamine from hyoscyamine fed to the Duboisia hybrid shoots was 35% w/w; the yield of the scopolamine precursor, 6beta-hydroxyhyoscyamine, was 37% w/w. Biotransformation activity was poor in A. belladonna shooty teratomas provided with exogenous hyoscyamine; however, scopolamine levels comparable with those in leaves of the whole plant accumulated in shoots fed with hairy root extract. Coculture of A. belladonna shooty teratomas and hairy roots in the same hormone-free medium was investigated as a means of providing a continuous source of hyoscyamine for conversion to scopolamine. Of the biotransformation systems tested with A. belladonna, coculture produced the highest levels of scopolamine and the highest scopolamine: hyoscyamine ratios. Cocultured shoots accumulated up to 0.84 mg g(-1) dry weight scopolamine, or 3-11 times the average concentrations found in leaves of the whole plant. The scopolamine: hyoscyamine ratio in coculture ranged from 0.07 to 1.9, a significant improvement over levels of 0-0.03 normally found in A. belladonna hairy roots. Addition of Pluronic F-68 or copper sulfate to the medium and variation in initial medium pH did not improve hyoscyamine release from hairy roots. Scopolamine levels were increased using 1 muM copper sulfate or initial medium pH between 6.0 and 8.0; however, results from elicitation of hairy roots could not match the beneficial effect on scopolamine synthesis of root-shoot coculture. Addition of 0.001-1.0% (w/v) Pluronic F-68 to the roots reduced hyoscyamine release but postponed necrosis in the root tissue for up to 60 d. (c) 1996 John Wiley & Sons, Inc. 相似文献
19.
The effects of chitosan, acetic acid and citric acid on production and release of hyoscyamine and scopolamine in hairy root
cultures of Brugmansia candida were studied. Chitosan and acetic acid were tested at different concentrations and also at different media pH values. At
pH 5.5, and at certain concentrations, acetic acid and chitosan increased the content of root scopolamine and hyoscyamine,
and promoted the release of both alkaloids. Lowering the pH to 3.5 and 4.5 reduced the accumulation of both alkaloids in the
roots, but at a pH of 4.5, their release increased significantly. Acetic and citric acid stimulated the release of scopolamine
and hyoscyamine.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
20.
Kamal Rashidi Asl Bahman Hosseini Ali Sharafi Javier Palazon 《Engineering in Life Science》2019,19(1):73-89
The use of nanotechnology and biotechnology to improve the production of plant bioactive compounds is growing. Hyoscyamus reticulatus L. is a major source of tropane alkaloids with a wide therapeutic use, including treatment of Parkinson's disease and to calm schizoid patients. In the present study, hairy roots were obtained from two‐week‐old cotyledon explants of H. reticulatus L. using the A7 strain of Agrobacterium rhizogenes. The effects of different concentrations of the signaling molecule nano‐zinc oxide (ZnO) (0, 50, 100 and 200 mg/L), with three exposure times (24, 48 and 72 h), on the growth rate, antioxidant enzyme activity, total phenol contents (TPC), tropane alkaloid contents and hyoscyamine‐6‐beta‐hydroxylase (h6h) gene expression levels were investigated. Growth curve analysis revealed a decrease in fresh and dry weight of ZnO‐treated hairy roots compared to the control. ANOVA results showed that the antioxidant activity of the enzymes catalase, guaiacol peroxidase and ascorbate peroxidase was significantly higher in the ZnO‐treated hairy roots than in the control, as was the TPC. The highest levels of hyoscyamine (37%) and scopolamine (37.63%) were obtained in hairy roots treated with 100 mg/L of ZnO after 48 and 72 h, respectively. Semi‐quantitative RT‐PCR analysis revealed the highest h6h gene expression was in hairy roots treated with 100 mg/L of ZnO after 24 h. It can be concluded that ZnO is as an effective elicitor of tropane alkaloids such as hyoscyamine and scopolamine due to its enhancing effect on expression levels of the biosynthetic h6h gene. 相似文献