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1.
Islas-Flores I Moreno-Valenzuela O Minero-García Y Loyola-Vargas VM Miranda-Ham Mde L 《Molecular biotechnology》2002,21(3):211-216
Tryptophan decarboxylase (TDC, EC 4.1.1.28) from Catharanthus roseus hairy roots was purified 80-fold. Antibodies against TDC were obtained and they recognized only one protein of 55 kDa in
crude extracts from hairy root cultures. Elicitation of transformed root cultures with macerozyme yielded a marked increase
in TDC activity, which was accompanied by a similar increase in the amount of immunoreactive TDC protein. These results suggest
that the alkaloid accumulation, produced by elicitation, requires the synthesis of new TDC polypeptide in C. roseus root cultures and establishes important differences in the regulatory control of this enzyme in root cultures compared to
developing seedlings, where the posttranslational regulation apparently plays a major role. 相似文献
2.
R. Ciau-Uitz M. L. Miranda-Ham J. Coello-Coello B. Chí L. M. Pacheco V. M. Loyola-Vargas 《In vitro cellular & developmental biology. Plant》1994,30(1):84-88
Summary Ten transformed and two non-transformed root lines ofCatharanthus roseus were established. A systematic study of the growth kinetics and alkaloid content was performed over a culture cycle and showed
significant differences between transformed and non-transformed cultures. Mean doubling times for transformed and normal root
lines were 2.8 and 19.5 days, respectively. Alkaloid content in hairy roots was from two- to threefold higher than in the
non-transformed tissues. The established transformed root lines produced a wide variety of indole alkaloids as can be observed
from their complex thin layer chromatography patterns. A large quantity of serpentine was determined in two of the transformed
root cultures. Alkaloid content, both quantitatively and qualitatively, has been stable in the hairy root cultures for more
than 2 yr of subculturing. 相似文献
3.
4.
Praveen Chandra Verma Laiq ur Rahman Arvind Singh Negi Dharm Chand Jain S. P. S. Khanuja Suchitra Banerjee 《Plant biotechnology reports》2007,1(3):169-174
A protocol for induction and establishment of Agrobacterium rhizogenes-mediated hairy root cultures of Picrorhiza kurroa was developed through optimization of the explant type and the most suitable bacterial strain. The infection of leaf explants
with the LBA9402 strain resulted in the emergence of hairy roots at 66.7% relative transformation frequency. Nine independent,
opine and TL-positive hairy root clones were studied for their growth and specific glycoside (i.e., kutkoside and picroside I) productivities
at different growth phases. Biosynthetic potentials for the commercially desirable active constituents have been expressed
by all the tested hairy root clones, although distinct inter-clonal variations could be noted in terms of their quantity.
The yield potentials of the 14-P clone, both in terms of biomass as well as individual glycoside contents (i.e., kutkoside
and picroside I), superseded that of all other hairy root clones along with the non-transformed, in vitro-grown control roots
of P. kurroa. The present communication reports the first successful establishment, maintenance, growth and selection of superior hairy
root clone of Picrorhiza kurroa with desired phyto-molecule production potential, which can serve as an effective substitute to its roots and thereby prevent
the indiscriminate up-rooting and exploitation of this commercially important, endangered medicinal plant species.
CIMAP Publication No.: 2007-28J 相似文献
5.
Hairy root cultures of Catharanthus roseus var. Prabal were established by infecting the leaves with Agrobacterium rhizogenes agropine-type A4 strain. Two hundred and fifty independent root clones were evaluated for growth, morphology, number of integration of Ri T-DNA genes and alkaloid contents. On the basis of growth pattern, type of branching and number of lateral roots we were able to separate the hairy root clones into four categories. However based on the integration of the Ri TL-DNA and TR-DNA genes, there were only three different categories of independent hairy root clones—C1 (rolA&B
+/ags
+), C2 (rolA&B
-/ags
+) and C3 (rolA&B
+/ags
–). Southern hybridization analysis revealed both single and multiple copies of T-DNA integration in the root clones. The accumulation of considerable amounts of the root-specific alkaloids ajmalicine and serpentine was observed in the presence of both the TL-DNA and TR-DNA genes (C1) and the TL-DNA gene (C3) alone. Two rolA&B
– but ags
+ clones (C2) accumulated much less or only very negligible amounts of ajmalicine. The possible role of the TL-DNA and TR-DNA genes on growth and alkaloid accumulation in these root clones is discussed.Abbreviations
ags
Agropine synthase
-
Ri
Root-inducing
-
T
L
-DNA
Left-terminus DNA
-
T
R
-DNA
Right-terminus DNA
-
TIAs
Terpenoid indole alkaloids 相似文献
6.
F. Vázquez-Flota O. Moreno-Valenzuela M. L. Miranda-Ham J. Coello-Coello V. M. Loyola-Vargas 《Plant Cell, Tissue and Organ Culture》1994,38(2-3):273-279
Two year old, transformed root cultures of Catharanthus roseus accumulate ajmalicine and catharanthine (0.57 and 0.36 mg g-1 DW, or 7.0 and 3.0 mg l-1, respectively). Changes in the concentration of the medium components, as well as the addition of hydrolytic enzymes and biotic elicitors, were used as strategies to increase these alkaloid yields. Regarding the components of the medium, the results obtained, when sucrose was raised from 3 to 4.5%, are noteworthy. The nitrogen source induced differential responses in the individual alkaloid yields. No net change in the alkaloid content was observed either with changes in the concentration of vitamins or macro-and micronutrients. Though the root culture only shows a limited response to elicitors, Aspergillus treatment and the use of macerozyme increased the accumulation of ajmalicine selectively, while the addition of methyl jasmonate increased the yield of both alkaloids.Abbreviations MeJa
methyl jasmonate
- mU
milliunits 相似文献
7.
I. E. Maldonado-Mendoza T. Ayora-Talavera V. M. Loyola-Vargas 《Plant Cell, Tissue and Organ Culture》1993,33(3):321-329
The aim of this paper was the screening of the variability of growth patterns, biomass and tropane alkaloid production of
500 hairy root lines ofDatura stramonium. Data on the long term stability in alkaloid production of these lines for more than 5 years are also provided. In an effort
to obtain high alkaloid-producing root clones, it is demonstrated that systematic selection is necessary. Comparisons are
made, mainly concerning alkaloid production and its stability, with normal root cultures initiated from the same mother plants
when necessary. Hairy root cultures were found to have a hyoscyamine and scopolamine bioproductivity of 2 orders of magnitude
higher than mother plants. 相似文献
8.
Summary Hairy roots of Catharanthus roseus were cultivated in three different types of bioreactors. The best growth and indole alkaloid production was achieved in an airsparged bioreactor with no other mixing. In the stirred bioreactor or in the bioreactor with medium circulation the roots did not grow, suggesting that hairy roots of C. roseus are more sensitive to stress than root cultures of many other plant species. 相似文献
9.
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
10.
Hairy root cultures of Gentiana macrophylla were established by infecting the different explants four Agrobacterium rhizogenes strains namely A4GUS, R1000, LBA 9402 and ATCC11325, and hairy root lines were established with A. rhizogenes strain R1000 in 1/2 MS + B5 medium. Initially, 42 independent hairy root clones were maintained and seven clones belongs to different category were evaluated
for growth, morphology, integration and expression of Ri T-DNA genes, and alkaloid contents in dry root samples. On the basis
of total root elongation, lateral root density and biomass accumulation on solid media, hairy root clones were separated into
three categories. PCR and Southern hybridization analysis revealed both left and right T-DNA integration in the root clones
and RT-PCR analysis confirmed the expression of hairy root inducible gene. GUS assay was also performed to confirm the integration
of left T-DNA. The accumulation of considerable amounts of the root-specific secoiridoid glucosides gentiopicroside was observed
in GM1 ( and ) and the GM2 ( and DNA) type clones in considerably higher amount whether as two but callus-type clones (GM3) accumulated much less or only very negligible amounts of gentiopicroside. Out of four media composition
the 1/2 MS + B5 vitamin media was found most suitable. We found that initial establishment of root cultures largely depends on root:media ratio. Maximum growth rate was recorded in 1:50 root:media ratio. The maximum biomass in terms of fresh weight (33-fold) was achieved in 1/2 MS + B5 media composition after 35 days in comparison to sixfold increase in control. The biomass increase was most abundant maximum
from 15 to 30 days. Influence of A. rhizogenes strains and Ri plasmid of hairy root induction, the possible role of the TL-DNA and TR-DNA genes on growth pattern of hairy root, initial root inoculum:media ratio and effect of media composition is discussed. 相似文献
11.
Light plays an important role in almost all plant developmental processes and provides the fundamental building blocks for
growth, development, primary and secondary metabolism. The effects of light on growth rate and caffeic acid derivative (CADs)
biosynthesis in hairy root cultures of Echinacea purpurea (Moench) were assessed. Light-grown hairy roots accumulated increased levels of anthocyanins, which became visible in outer
cell layer of the cortex as a ring of purple color. The light-grown root cultures also had radially thickened morphology compared
with the dark-grown controls. The growth rate and cell viability of the hairy root cultures in light did not show obvious
difference in comparison with those in dark. However, biosynthesis of CADs including cichoric acid, caftaric acid, chlorogenic
acid and caffeic acid was significantly increased in hairy root cultures grown in the light. The enhanced accumulation of
CADs and anthocyanins in E. purpurea hairy root cultures was correlated to an observed light-stimulated activity of phenylalanine ammonium lyase (PAL). 相似文献
12.
Expression of deacetylvindoline-4-O-acetyltransferase in Catharanthus roseus hairy roots 总被引:3,自引:0,他引:3
Madagascar periwinkle [Catharanthus roseus (L.) G Don] is a pantropical plant of horticultural value that produces the powerful anticancer drugs vinblastine and vincristine that are derived from the dimerization of the monoterpenoid indole alkaloids (MIAs), vindoline and catharanthine. The present study describes the genetic engineering and expression of the terminal step of vindoline biosynthesis, deacetylvindoline-4-O-acetyltransferase (DAT) in Catharanthus roseus hairy root cultures. Biochemical analyses showed that several hairy root lines expressed high levels of DAT enzyme activity compared to control hairy root cultures expressing β-gulucuronidase activity (GUS) activity. Metabolite analysis using high performance liquid chromotagraphy established that hairy root extracts had an altered alkaloid profile with respect to hörhammericine accumulation in DAT expressing lines in comparison to control lines. Further analyses of one hairy root culture expressing high DAT activity suggested that DAT expression and accumulation of hörhammericine (9) were related. It is concluded that expression of DAT in hairy roots altered their MIA profile and suggests that further expression of vindoline pathway genes could lead to significant changes in alkaloid profiles. Evidence is provided that hörhammericine (9) accumulates via a DAT interaction with the root specific minovincinine-19-O-acetyltransferase (MAT) that inhibits the MAT mediated conversion of hörhammericine (9) into 19-O-acetyl-hörhammericine (12). 相似文献
13.
Mondher Jaziri Kayo Yoshimatsu Jacques Homès Koichiro Shimomura 《Plant Cell, Tissue and Organ Culture》1994,38(2-3):257-262
Hairy root cultures of Atropa belladonna L. were established by infection either with Agrobacterium rhizogenes ATCC 15834 or MAFF 03-01724, and transgenic plants were obtained from both hairy root cultures. Doubly transformed roots were induced by re-infection of the leaf segments of transgenic Atropa belladonna plants (A. rhizogenes 15834) with MAFF 03-01724. Shoots and viviparous leaves were regenerated from the doubly transformed roots. The genetic transformation was determined by the opine assay (agropine, mannopine and/or mikimopine) and polymerase chain reaction. Physiological changes and tropane alkaloid biosynthesis in the hairy roots (singly and doubly transformed) were investigated. The alkaloid content in the doubly transformed root strain was intermediate as compared to the root strains which were singly transformed. On the other hand endogenous IAA levels in doubly transformed roots were significantly decreased compared to both singly transformed roots.Abbreviations BA
benzyladenine
- IAA
indoleacetic acid
- NAA
naphthaleneacetic acid
- PCR
polymerase chain reaction
-
t-ZR
trans-zeatin 相似文献
14.
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
15.
Influence of exogenous hormones on the growth and secondary metabolite formation in transformed root cultures 总被引:7,自引:0,他引:7
M. J. C. Rhodes A. J. Parr A. Giulietti E. L. H. Aird 《Plant Cell, Tissue and Organ Culture》1994,38(2-3):143-151
Transformed organ cultures formed following transformation of plant tissues with Agrobacterium species owe their phenotypes to alterations in hormone metabolism. Exogenously supplied hormones have been used to probe the relationship between the growth and morphology of transformed root cultures of a number of species and their ability to accumulate secondary products. Auxins in the presence of low levels of kinetin induce the rapid disorganisation of transformed roots of Nicotiana rustica ultimately toform suspension cultures of transformed cells and this process is associated with a decrease in nicotine content of the cells. This is related to cells in the culture losing competence in alkaloid biosynthesis. In contrast, exogenously supplied GA3 enhanced branching in two transformed root clones of the tropane-alkaloid producing species, Brugmansia candida and so enhanced their typical hairy root phenotype. This growth substance had the effect of reducing the overall alkaloid accumulation but in one case significantly altered the relative concentrations of different tropine esters.In transformed roots of Cucumis sativus, the phenotype of the roots is influenced by the expression of auxin synthesis genes on TR-DNA resulting in roots with two distinct morphologies. The pattern of expression of the enzyme ascorbate oxidase in populations of control roots of different morphologies is described. The significance of these phenotypic variations on the utility of transformed root cultures for the study of secondary metabolic pathways will be discussed.Abbreviations AO
ascorbate oxidase
- DW
dry weight
- FW
fresh weight
- GA3
gibberellic acid 相似文献
16.
Moreno-Valenzuela OA Minero-García Y Chan W Mayer-Geraldo E Carbajal E Loyola-Vargas VM 《Biotechnology letters》2003,25(16):1345-1349
Treatment of Catharanthus roseus hairy roots with antagonists, like verapamil and CdCl2, that block the Ca2+ flux across the plasma membrane enhanced the total alkaloid content by 25% and their secretion 10 times. The specific Ca2+ chelator, EGTA, stimulated 90% of the total alkaloid secretion. Treatment with inhibitors of intracellular Ca2+ movement, like TMB-8 and trapsigargin, enhanced the total alkaloid content by 74% and their secretion into the culture media by 4- to 6-fold. The results suggest that an inhibition of external and internal Ca2+ fluxes induces an increase in the indole alkaloid accumulation and secretion in C. roseus hairy roots. 相似文献
17.
The anticancer agents vinblastine and vincristine are bisindole alkaloids derived from coupling vindoline and catharanthine, monoterpenoid indole alkaloids produced exclusively by the Madagascar periwinkle (Catharanthus roseus). Industrial production of vinblastine and vincristine currently relies on isolation from C. roseus leaves, a process that affords these compounds in 0.0003–0.01% yields. Metabolic engineering efforts to either improve alkaloid content or provide alternative sources of the bisindole alkaloids ultimately rely on the isolation and characterization of the genes involved. Several vindoline biosynthetic genes have been isolated, and the cellular and subcellular organization of the corresponding enzymes has been well studied. However, due to the leaf-specific localization of vindoline biosynthesis, and the lack of production of this precursor in cell suspension and hairy root cultures of C. roseus, further elucidation of this pathway demands the development of reverse genetics approaches to assay gene function in planta. The bipartite pTRV vector system is a Tobacco Rattle Virus-based virus-induced gene silencing (VIGS) platform that has provided efficient and effective means to assay gene function in diverse plant systems. A VIGS method was developed herein to investigate gene function in C. roseus plants using the pTRV vector system. The utility of this approach in understanding gene function in C. roseus leaves is demonstrated by silencing known vindoline biosynthetic genes previously characterized in vitro. 相似文献
18.
Agrobacterium rhizogens mediated transformation of Rauvolfia serpentina: Regeneration and alkaloid synthesis 总被引:1,自引:0,他引:1
Shoot cultures of Rauvolfia serpentina infected with Agrobacterium rhizogenes 15834 produced tumourous tissue at the site of injection, which eventually developed callus with hairy roots. Sporadic shoot formation occurred from the hairy roots. The shoots were grown to maturity in the green house. The mature transformed plants (RST) showed distinct variations in their physiological characteristics. The flowers of the transformed plants were more delicate and less pigmented when compared to the flowers of the mature normal plants. The roots of the transformed plants were hairy with a number of lateral branches, whereas the roots of the normal plant had very few lateral branches. The biomass of the transformed plant was 86.39 g/plant (fresh wt), significantly higher than the normal plant which was 77.3 g/plant (fresh wt). The total alkaloid content in the mature transformed plant (0.073 g per plant) was similar to the normal plant (0.078 g per plant), although the hairy roots contained little alkaloid.Abbreviations MS
Murashige & Skoog's basal medium
- MLS
modified Linsmaier & Skoog's basal medium
- BA
benzyladenine
- NAA
naphthalene acetic acid
- TLC
thin layer chromatography
- HPLC
high performance liquid chromatography 相似文献
19.
Annalisa Giovannini Nicola Pecchioni Mario Rabaglio Andera Allavena 《In vitro cellular & developmental biology. Plant》1997,33(2):101-106
Summary
Datura arborea and D. sanguinea hairy roots were produced by cocultivation of leaf fragments with Agrobacterium rhizogenes strain NCPP 1855. Adventitious buds emerged spontaneously, without exogenous growth regulators, from seven hairy root clones
of D. arborea and from one hairy root clone of D. sanguinea. Regenerated plants were successfully acclimatized in the greenhouse. The integration of the bacterial TL-DNA into the genome
of the putative transformed plants was confirmed by Southern blot analysis. Transgenic plants displayed increased ability
to root in vivo. Morphological traits with relevant ornamental value like plant height, leaf number, size and shape, internode number, and
internode length were also affected. Transformation by wild-type Ri TL-DNA provided the chance to study plant growth and differentiation
and to select improved genotypes. 相似文献
20.
Ignacio Islas Víctor M. Loyola-Vargas María de Lourdes Miranda-Ham 《In vitro cellular & developmental biology. Plant》1994,30(1):81-83
Summary Tryptophan decarboxylase (TDC), the enzyme that catalyzes the decarboxylation of tryptophan to trytamine, was studied in aCatharanthus roseus transformed root culture. Its activity was evaluated through the culture cycle (36 days), along with the variations in the
tryptamine pool as well as the accumulation of alkaloids. Ajmalicine and catharanthine contents in the tissues increased coordinately
with an increase in TDC-specific activity after 18 days of growth. No dramatic shifts were observed for the total alkaloid
and tryptamine profiles. 相似文献