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1.
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
2.
Medicago
truncatula, barrel medic, is a forage crop that has been developed into a model legume. The development of new transformation methods is important for functional genomic studies in this species. Based on Agrobacterium tumefaciens-mediated transformation of root explants, we developed an effective system for producing M. truncatula (genotype R108) transgenic plants. Among the four A. tumefaciens strains (AGL1, C58C1, EHA105 and LBA4404) tested, EHA105 and AGL1 were most effective in regenerating transgenics. Callus induction frequency from root explants was 69.8%, and plantlet/shoot regeneration frequency was 41.3% when EHA105 was used. Transgenic nature of the regenerated plants was confirmed by PCR and Southern hybridization analyses. Progeny analysis revealed stable Mendelian meiotic transmission of transgenes. Because M. truncatula is particularly useful for the study of root endosymbiotic associations, we further developed a plant regeneration system from A. rhizogenes-transformed hairy roots of M. truncatula. Fertile true transgenic plants were regenerated from the hairy roots, thus allowing the assessment of gene functions at the whole plant level. Segregation analysis revealed that the hairy root genes could be segregated out in the progenies. By coupling A. rhizogenes-mediated hairy root transformation and the regeneration system reported here, once potential genes of interest are identified, the transformed hairy roots carrying such genes could be directly regenerated into plants for more detailed characterization of the genes. 相似文献
3.
Summary Excised cotyledons from 8-d-old pumpkin ( Cucurbita pepo L.) seedlings were inoculated with Agrobacterium rhizogenes and cultured on hormone-free Murashige and Skoog medium. At the site of inoculation, transformed hairy roots were successfully
induced by using wild strains 8196 (mannopine-type) and 15834 (agropine-type). After a subsequent transfer on a solid MS medium
without hormones, roots obtained by transformation with strain 15834 failed to form stable hairy root cultures, while several
hairy root lines were established with strain 8196. Three hairy root lines, Cp1, Cp2, and Cp31, have spontaneously generated
callus with embryo-like structures after more than 3 yr of growth on the solid medium. The callus proliferation was more frequent
when the autoclaving of nutrient medium, pH 5.7, was prolonged to 30 min. Separated calluses continued to proliferate and
generated embryos with abnormal morphology. The combination of indole-3-acetic acid and benzyladenine had a favorable influence
on embryogenesis and organogenesis in the Cp31 callus line. The Southern analysis of Cp31 root and embryo DNA confirmed the
presence of the T-DNA of Agrobacterium rhizogenes. 相似文献
4.
Axenically grown Saussurea medusa plantlets were inoculated with four Agrobacterium rhizogenes strains, and hairy root lines were established with A. rhizogenes strain R1601 in N6 medium. PCR and Southern hybridization confirmed integration of the T-DNA fragment of the Ri plasmid from A. rhizogenes into the genome of S. medusa hairy roots. In N6 medium, maximum biomass of the hairy root cultures was achieved [8 g (dry weight) per liter; growth ratio 35-fold] after 21 days of culture. The amount of jaceosidin extracted from the hairy root cultures was 46 mg/l (production ratio of 37-fold) after 27 days of culture. The maximum jaceosidin content obtained using N6 medium was higher than that obtained with Modified White, MS or B5 medium. In N6 medium, the tip segments were more efficient for hairy root growth and jaceosidin production than the middle and basal regions of the root.Abbreviations AS Acetosyringone - BA Benzyladenine - cef Cefotaxime sodium - DW Dry weight - FW Fresh weight - HPLC High-performance liquid chromatography - IAA Indole-3-acetic acid - km Kanamycin - NAA -Naphthaleneacetic acid - SDS Sodium dodecyl sulfate 相似文献
5.
Agrobacterium rhizogenes (wild-type strains 8196 and 15834) transformation of pumpkin ( Cucurbita pepo L.) intact seedlings grown in vivo, and 6–8-day-old excised cotyledons cultured in axenic conditions was investigated. Transformed (hairy) roots were successfully induced only on the excised cotyledons with the strain 8196, while intact seedlings failed to form hairy roots with either of the two different bacterial strains. Axenic hairy-root cultures established on MS medium without hormones grew vigorously. Mannopine was detected in all transgenic root clones examined. The peroxidase activity in transformed roots was higher compared with normal roots. Electrophoretic analyses of soluble proteins and isoperoxidases showed substantial differences between transformed and normal pumpkin roots. 相似文献
6.
Infection of Ulmus procera (English elm) cloneSR4 internodal stem explants with Agrobacteriumtumefaciens C58 c1 pRiA4b resulted in callusdevelopment and extensive hairy root production. Shoots which regenerated from hairy roots, followingan extended culture period, were dwarf in stature,with reduced apical dominance and wrinkled leaves whencompared with wild type U. procera SR4. Shootswere rooted successfully and plants with extensiveroot systems have been transferred to soil. Thetransgenic status of regenerants was confirmed by PCRanalysis and DNA sequencing of pRiA4b TL- and TR- DNA rolA (329 bp) and agropine synthase (490 bp)primed amplimers, which were 100% homologous to theexpected sequences. No vir D1 primed PCRproducts were obtained, indicating that the Agrobacterium was successfully removed. Thepotential of Ri plasmid mediated transformation forinducing altered elm xylem structure, restrictedspread of the Dutch elm disease fungus and inphytoremediation is discussed. 相似文献
7.
Summary In wild-type Scopolia parvilfora (Solanaceae) tissues, only the roots express the enzyme putrescine N-methyltransferase (PMT; EC 2.1.1.53), which is the first specific precursor of the tropane alkaloids. Moreover, the tropanane
alkaloid levels were the highest in the root (0.9 mg g −1 on a dry weight basis), followed by the stem and then the leaves. We metabolically engineered S. parviflora by introducing the tobacco pmt gene into its genome by a binary vector system that employs disarmed Agrobacterium rhizogenes. The kanamycin-resistant hairy root lines were shown to bear the pmt gene and to overexpress its mRNA and protein product by at least two-fold, as determined by polymerase chain reaction (PCR)
and Northern and Western blottings, respectively. The transgenic lines also showed higher PMT activity and were morphologically
aberrant in terms of slower growth and the production of lateral roots. The overexpression of pmt markedly elevated the scopolamine and hyoscyamine levels in the transgenic lines that showed the highest pmt mRNA and PMT protein levels. Thus, overexpression of the upstream regulator of the tropane alkaloid pathway enhanced the
biosynthesis of the final product. These observations may be useful in establishing root culture systems that generate large
yields of tropane alkaloids.
These authors contributed equally to this paper (co-first authors). 相似文献
8.
A method is described for producing genetically transformed plants from explants of three scented Pelargonium spp. Transgenic hairy root lines were developed from Pelargonium spp leaf explants and microcuttings after inoculation with Agrobacterium rhizogenes strains derived from the agropine A4 strain. Hairy root lines grew prolifically on growth regulator-free medium. Transgenic
shoots were regenerated from hairy roots and the plants have been successfully transferred to soil. The phenotype of regenerated
plants has been characterized as having abundant root development, more leaves and internodes than the controls, short internodes
and highly branched roots and aerial parts. Southern blot analyses have confirmed the transgenic nature of these plants. 相似文献
9.
Plumbago europaea L. is the main source of plumbagin which is a well-known pharmacological active compound. In this investigation, genetically transformed roots of P. europaea were obtained by improving some factors affecting the efficiency of Agrobacterium rhizoigenes-mediated transformation such as explant type, A. rhizoigenes strain, bacterial infection period, co-cultivation period and acetosyringone concentration. The leaf, hypocotyl and stem explants from in vitro grown plantlets were infected with bacterial strains (A4, ATCC15834, MSU440 and A13). The highest transformation rate of 69.3% was achieved after 7–9 days by inoculating A. rhizogenes MSU440 strain onto the 3-week-old stem explants followed by a co-cultivation period of 2 days on a medium containing 100 μM acetosyringone. To investigate the existence of the rolB gene, polymerase chain reaction was carried out using specific primers. Effects of growth media (MS, 1/2 MS, MS-B5 and ½ MS-B5), different sucrose concentrations and illumination on biomass production and plumbagin biosynthesis in P. europaea hairy root cultures were analyzed using stem explants after infection with MSU440 strain. ½ MS-B5 liquid medium containing 30 g L −1 sucrose incubated in the dark resulted in the efficient biomass production of transformed hairy roots (12.5 g fresh weight, 1.8 g dry weight) with 3.2 mg g −1 DW plumbagin accumulation. This procedure provides a framework for large-scale cultivation of hairy roots for plumbagin production. This is the first report describing the establishment of P. europaea hairy root culture with special emphasis on plumbagin production. 相似文献
10.
Initiation and establishment of hairy root cultures from leaf or seedling hypocotyl explants of Solanum mauritianum Scop., using six strains of Agrobacterium rhizogenes was attempted. Success was only achieved following hypocotyl inoculation with strain LBA 9402. Transformation frequency was very low, with only one instance out of a possible 90 being recorded. Resultant hairy root cultures grew rapidly and could be maintained using a Murashige and Skoog (1962) medium supplemented with 0.1 g L –1
myo-inositol and 3% sucrose, either as a solid or liquid culture. Under these conditions, the roots had a solasodine content of 126 g g –1 DW. Lower levels of solasodine and decreased root growth rates were recorded when the medium strength was reduced by half or 3% glucose substituted for the 3% sucrose.Abbreviations MS
Murashige and Skoog's (1962) medium 相似文献
11.
In this paper we describe the production of transgenic broccoli and cauliflower with normal phenotype using an Agrobacterium rhizogenes-mediated transformation system with efficient selection for transgenic hairy-roots. Hypocotyls were inoculated with Agrobacterium strain A4T harbouring the bacterial plasmid pRiA4 and a binary vector pMaspro::GUS whose T-DNA region carried the gus reporter gene. pRiA4 transfers T L sequences carrying the rol genes that induce hairy root formation. Transgenic hairy-root production was increased in a difficult-to-transform cultivar by inclusion of 2,4-D in the medium used to resuspend the Agrobacterium prior to inoculation. Transgenic hairy roots could be selected from inoculated explants by screening root sections for GUS activity; this method eliminated the use of antibiotic resistance marker genes for selection. Transgenic hairy roots were produced from two cauliflower and four broccoli culivars. Shoots were regenerated from transgenic hairy root cultures of all four cultivars tested and successfully acclimatized to glasshouse conditions, although some plants had higher than diploid ploidy levels. Southern analysis confirmed the transgenic nature of these plants. T 0 plants from seven transgenic lines were crossed or selfed to produce viable seed. Genetic analysis of T 1 progeny confirmed the transmission of traits and revealed both independent and co-segregation of Ri T L-DNA and vector T-DNA. GUS-positive phenotypically normal progeny free of T L-DNA were identified in three transgenic lines out of the six tested representing all the cultivars regenerated including both cauliflower and broccoli. 相似文献
12.
Hairy root cultures of Atropabelladonna were established by transformation with Agrobacterium 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
N 6-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
14.
Hazelnut (Corylus avellana L.), contains a valuable medicinal substance known as Paclitaxel®, which is one of the most effective anticancer drugs. The original plants produce negligible amount of paclitaxel; therefore, tissue culture techniques, especially hairy root culture, could be one of the most practical methods to enhance the amount of paclitaxel. The main goal of this study was to assess the induction of hairy roots in C. avellana. The effects of different strains of Agrobacterium rhizogenes including c58c1pRiA4, K599, and 15834, and six culture media, MS (Murashige and Skoog), half-strength MS, quarter-strength MS, WPM (woody plant media), half-strength WPM, and quarter-strength WPM, were evaluated. The results showed that the maximum amounts of the rooted explants were obtained with c58c1pRiA4 strain in quarter-strength WPM medium. The investigations of explant type (leafstalk, petiole, lamina, and stem) and different propagation media (quarter-strength WPM, half-strength MS, and half-strength SH ((Schenk and Hildebrandt) medium) showed that the leafstalk was the most optimal explant for hairy root induction, and half-strength SH was the best culture medium for growth of the hairy roots in liquid medium. HPLC analyses confirmed the presence of paclitaxel (3.2 μg g−1 (DW)) in hairy root extracts. 相似文献
15.
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 相似文献
16.
Root of Glycyrrhiza uralensis, one of the most important medicinal plants, containing bioactive triterpene saponins (glycyrrhizin). Squalene synthase (SQS)
plays a regulatory role in the biosynthesis of triterpene saponins. In the present investigation, SQS coding sequence from
G. uralensis was cloned by polymerase chain reaction (PCR) and a transgenic system was developed for G. uralensis through Agrobacterium rhizogenes-mediated transformation. The SQS gene placed under a CaMV 35S promoter was transferred into G. uralensis using A. rhizogenes strain ACCC10060. The transformed hairy roots were selected on Murashige and Skoog ( 1962)-containing phosphinothricin (PPT) and root lines were established. The integration of SQS gene was confirmed by PCR and Southern blot. Three transgenic root lines UP1, UP24, UP31 were obtained and their growth rates
were detected. The result showed that transgenic root lines but UP1 line grew faster than control hairy roots; high-performance
liquid chromatography (HPLC) analysis demonstrated the highest glycyrrhizin content of transgenic roots was 2.5 mg/g dry weight
and was about 2.6 times higher than control hairy roots.
The nucleotide sequences GuSQS1 and GUSQS2 reported in this paper appear in the EMBL nucleotide sequence database with the
accession number AM182329 and AM182330, respectively. 相似文献
17.
A procedure for the production of fertile transgenic brassicas via Ri-mediated transformation is reported in this paper. Transgenic hairy root lines were selected for 12 vegetable brassica cultivars and lines representing six varieties: broccoli, Brussels sprouts, cabbage, cauliflower, rapid-cycling (all Brassica oleracea) and Chinese cabbage ( B. campestris). Leaf explants or petioles of intact cotyledons were co-cultivated with Agrobacterium strain A4T harbouring various binary vectors. The T-DNA region of all binary vectors contained a neomycin phosphotransferase II gene for kanamycin resistance, in addition to other genes. Hairy root lines grew prolifically on hormone-free medium containing kanamycin. Transgenic shoots were regenerated from all cultivars either spontaneously or after transfer of hairy roots to a hormone-containing medium. Southern analysis confirmed that the plants were transgenic. Plants from all brassica types were successfully transferred to greenhouse conditions. Plants were fertile and segregation analysis confirmed transmission of traits to progeny.Abbreviations
BA
6-Benzylaminopurine
-
GUS
-Glucuronidase
-
LS
Linsmaier and Skoog medium
-
NAA
I-Naphthaleneacetic acid
-
NPTII
Neomycin phosphotransferase II
-
TDZ
thidiazuron 相似文献
18.
Hairy root cultures of Gentiana macrophylla were established by infecting the different explants four Agrobacterium rhizogenes strains namely A 4GUS, R1000, LBA 9402 and ATCC11325, and hairy root lines were established with A. rhizogenes strain R1000 in 1/2 MS + B 5 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 + B 5 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 + B 5 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 T L-DNA and T R-DNA genes on growth pattern of hairy root, initial root inoculum:media ratio and effect of media composition is discussed. 相似文献
19.
The US Department of Energy recently released a 6.8X draft of the genome sequence for Nisqually-1, a genotype of black cottonwood
( Populus trichocarpa). To improve its utility for functional genomics research, having an efficient means for transformation and regeneration
is necessary. To examine several parameters known to affect the transformation rate, we cocultivated leaf disc and stem explants
with a strain of Agrobacterium tumefaciens harboring a binary plasmid vector containing genes for both neomycin phosphotransferase ( NPTII) and β-glucuronidase ( GUS). Shoot regeneration from stem explants was observed in the presence of kanamycin when thidiazuron was incorporated in the
selection medium. Transformation efficiency was influenced by the level of thidiazuron to which explants were exposed during
the early stages of shoot induction. Histochemical assays revealed expression of the GUS gene in leaf, stem, and root tissues of transgenic plants. Polymerase chain reaction confirmed the presence of both selectable
marker and reporter genes in all lines that stained positive for β-glucuronidase activity. By use of our modified protocol,
transgenic plants were recovered within 6 mo at an efficiency of 6%, adequate to produce a large number of transgenic events
with modest effort. 相似文献
20.
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 KNO 3 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, Cu 2+ 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
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