共查询到20条相似文献,搜索用时 15 毫秒
1.
The chimerical gene, Arabidopsis thaliana sHSP18.2 promoter fused to E. coli gusA gene, was Agrobacterium rhizogenes-mediated transformed into Nicotiana tabacum as a heat-regulatable model, and the thermo-inducible expression of GUS activity in N. tabacum transgenic hairy roots was profiled. An activation of A. rhizogenes with acetosyringone (AS) before cocultured with tobacco's leaf disc strongly promoted transgenic hairy roots formation. Transgenic hairy roots formation efficiency of A. rhizogenes precultured with 200 μM AS supplementation was 3.1-fold and 7.5-fold, respectively, compared to the formation efficiency obtained with and without AS supplementation in coculture. Transgenic hairy roots transformed with different AS concentration exhibited a similar pattern of thermo-inducibility after 10 min to 3 h heat treatments detected by GUS expression. The peak of expressed GUS specific activity, 399,530 pmol MUG per mg total protein per min, of the transgenic hairy roots was observed at 48 h after 3 h of 42°C heat treatment, and the expressed GUS specific activity was 7–26 times more than that reported in A. thaliana, tobacco BY-2 cells and Nicotiana plumbaginifolia. Interference caused by AS supplementation on the growth of transgenic hairy roots, time-course of GUS expression and its expression level were not observed. 相似文献
2.
Parasitic plants in the Orobanchaceae invade roots of neighboring plants to rob them of water and nutrients. Triphysaria is facultative parasite that parasitizes a broad range of plant species including maize and Arabidopsis. In this paper we describe transient and stable transformation systems for Triphysaria
versicolor Fischer and C. Meyer. Agrobacterium
tumefaciens and Agrobacterium
rhizogenes were both able to transiently express a GUS reporter in Triphysaria seedlings following vacuum infiltration. There was a correlation between the length of time seedlings were conditioned in
the dark prior to infiltration and the tissue type transformed. In optimized experiments, nearly all of the vacuum infiltrated
seedlings transiently expressed GUS activity in some tissue. Calluses that developed from transformed tissues were selected
using non-destructive GUS staining and after several rounds of in vivo GUS selection, we recovered uniformly staining GUS
calluses from which roots were subsequently induced. The presence and expression of the transgene in Triphysaria was verified using genomic PCR, RT PCR and Southern hybridizations. Transgenic roots were also obtained by inoculating A. rhizogenes into wounded Triphysaria seedlings. Stable transformed roots were identified using GUS staining or fluorescent microscopy following transformation
with vectors containing GFP, dsRED or EYFP. Transgenic roots derived from both A.
tumefaciens and A.
rhizogenes transformations were morphologically normal and developed haustoria that attached to and invaded lettuce roots. Transgenic
roots also remained competent to form haustoria in response to purified inducing factors. These transformation systems will
allow an in planta assessment of genes predicted to function in plant parasitism.
Alexey Tomilov and Natalya Tomilova made an equal contribution in the paper. 相似文献
3.
Vincent P. Klink Margaret H. MacDonald Veronica E. Martins Soo-Chul Park Kyung-Hwan Kim So-Hyeon Baek Benjamin F. Matthews 《Plant Cell, Tissue and Organ Culture》2008,92(2):183-195
We developed Glycine max cv MiniMax (PI643148) that has a rapid life cycle, short stature and characteristic simple sequence repeat (SSR) markers
that could make it useful for mutant screening, functional genomics, genetic mapping and other studies involving soybeans.
We demonstrate that MiniMax is able to make somatic embryos (SEs) that rapidly develop into plantlets. Thus, the rapid cycling
habit carries over into aspects of plant regeneration. Chimaeras (having transformed roots with untransformed aerial stocks)
have been produced rapidly under non-axenic conditions using Agrobacterium rhizogenes-mediated transformation. Part of these experiments involved the engineering an enhanced green fluorescent protein (eGFP)
reporter cassette outside the multi-cloning site of a plant expression vector, permitting non-invasive visual screening of
the transformed roots. The rapid cycling growth habit of MiniMax, its ability to efficiently generate SEs and ability to be
transformed should prove useful for basic aspects of G. max molecular and genetic research. 相似文献
4.
Agrobacterium rhizogenes was used for efficient transformation of chrysanthemum. Two types of Agrobacterium, A. rhizogenes (A-13) and A. tumefaciens (LBA4404), which harbor pIG121-Hm, were employed for infection. In the A. rhizogenes-infected explants, hairy roots were not observed on any tested medium or culture condition. When explants were cultured on shoot induction medium, calli were formed at the cutting edge within 4–6 weeks of culture, and shoot primordia were observed on the callus surface after 2 weeks of callus formation. Consequently, with gus introduction, a significantly higher transformation rate was observed for A. rhizogenes (6.0%) compared with A. tumefaciens (3.3%). However, only 0.6% of the frequency of rol insertion was exhibited in A. rhizogenes mediation. These results indicate that A. rhizogenes effectively introduces T-DNA of the binary plasmid into the chrysanthemum genome by introducing Ri T-DNA at a low frequency. It also indicates that the system is a useful alternative for the transformation of chrysanthemum. 相似文献
5.
Hairy roots of Plumbago indica were established at high frequency (90 %) by infecting leaf explants with Agrobacterium rhizogenes strain ATCC 15834. The axenic root cultures were established under darkness in hormone-free liquid Murashige and Skoog medium
containing 3 % sucrose. The highest plumbagin content was found to accumulate in roots at their exponential phase of growth.
A low pH (4.6) and a low concentration of sucrose (1 %) were beneficial for root growth in darkness, while pH 5.6 and 3 %
sucrose under continuous irradiance enhanced plumbagin accumulation in roots up to 7.8 mg g−1(d.m.). Direct shoot regeneration from hairy root culture was also achieved under continuous irradiance, thus indicated an
easy way of obtaining transformed P. indica plants. 相似文献
6.
Agrobacterium tumefaciens has the ability to transfer its T-DNA to plants, yeast, filamentous fungi, and human cells and integrate it into their genome. Conidia of the maize pathogen Helminthosporium turcicum were transformed to hygromycin B resistance by a Agrobacterium-tumefaciens-mediated transformation system using a binary plasmid vector containing the hygromycin B phosphotransferase (hph) and the enhanced green fluorescent protein (EGFP) genes controlled by the gpd promoter from Agaricus bisporus and the CaMV 35S terminator. Agrobacterium-tumefaciens-mediated transformation yielded stable transformants capable of growing on increased concentrations of hygromycin B. The presence of hph in the transformants was confirmed by PCR, and integration of the T-DNA at random sites in the genome was demonstrated by Southern blot analysis. Agrobacterium-tumefaciens-mediated transformation of Helminthosporium turcicum provides an opportunity for advancing studies of the molecular genetics of the fungus and of the molecular basis of its pathogenicity on maize. 相似文献
7.
A cell line of Taxus cuspidata has been transformed with wild-type Agrobacterium rhizogenes ATCC strain 15834 containing binary vector pCAMBIA1301 and, separately, with A. tumefaciens strain EHA105 containing binary vector pCAMBIA1305.2. Additionally, a cell line of T. chinensis has been transformed with wild-type A. rhizogenes ATCC strain 25818 containing binary vector pCAMBIA1301. The two transgenic T. cuspidata cell lines have been maintained in culture for more than 20 months, and the transgenic T. chinensis cell line for more than 9 months, with no loss of reporter gene expression or antibiotic resistance. The introduced genes
had no discernable effect on growth or Taxol production in the transgenic cell lines when compared to the parent control.
The methods for transforming non-embryogenic Taxus suspension cultures are described. 相似文献
8.
Hairy roots were induced from androgenic embryos of horse chestnut (Aesculus hippocastanum L.) by infection with Agrobacterium rhizogenes strain A4GUS. Single roots were selected according to their morphology in the absence of antibiotic or herbicide resistance markers. Seventy-one putative transformed hairy root lines from independent transformation events were established. Regeneration was induced in MS liquid medium supplemented with 30 6-benzylaminopurine (BA), and the regenerants were multiplied on MS solid medium containing 10 M BA. Following elongation on MS medium supplemented with 1 M BA and 500 mg/l polyvinylpyrrolidone, the shoots were subjected to a root-inducing treatment. Stable integration of TL-DNA within the horse chestnut genome was confirmed by Southern hybridization. The copy number of transgenes was estimated to be from two to four.Communicated by E.D. Earle 相似文献
9.
Ok-Sun?Lee Young-Min?Kang Hee-Young?Jung Ji-Yun?Min Seung-Mi?Kang Chandrakant?S.?Karigar D.?Theertha?Prasad Jung-Dong?Bahk Myung-Suk?Choi
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
10.
Developmental variability was introduced into Withania somnifera using genetic transformation by Agrobacterium rhizogenes, with the aim of changing withasteroid production. Inoculation of W. somnifera with A. rhizogenes strains LBA 9402 and A4 produced typical transformed root lines, transformed callus lines, and rooty callus lines with simultaneous
root dedifferentiation and redifferentiation. These morphologically distinct transformed lines varied in T-DNA content, growth
rates, and withasteroid accumulation. All of the lines with the typical transformed root morphology contained the TL T-DNA, and 90% of them carried the TR T-DNA, irrespective of the strain used for infection. Accumulation of withaferin A was maximum (0.44% dry weight) in the
transformed root line WSKHRL-1. This is the first detection of withaferin A in the roots of W. somnifera. All of the rooty callus lines induced by strain A4 contained both the TL and the TR-DNAs. In contrast, 50% of the rooty-callus lines obtained with strain LBA 9402 contained only the TR T-DNA. All the rooty callus lines accumulated both withaferin A and withanolide D. The callusing lines induced by LBA 9402
lacked the TL T-DNA genes, while all the callusing lines induced by strain A4 contained the TL DNA. Four of these callus lines produced both withaferin A (0.15–0.21% dry weight) and withanolide D (0.08–0.11% dry weight),
and they grew faster than the transformed root lines. This is the first report of the presence of withasteroids in undifferentiated
callus cultures of W. somnifera. 相似文献
11.
Summary Protoplasts were isolated from Agrobacterium rhizogenes A4-transformed cell line of Medicago sativa L. The highest yield of protoplasts (4.2×106 per g fresh weight) was obtained from 12-d-old calluses after being subeultured on fresh medium. The viability of protoplasts
reached over 80%. Protoplasts were induced to undergo sustained divisions when cultured in Durand et al. (DPD) medium supplemented
with 2 mgl−1 (9.05 μM) 2,4-dichlorophenoxyacetic acid, 0,2mgl−1 (0.93 μM) kinetin, 0.3 M mannitol, 2% (w/v) sucrose, and 500 mgl−1 casein hydrolyzate at a plating density of 1.0×105 per ml. An agarose-beads culture method was appropriate for protoplast division of transformed alfalfa. The division frequency
was about 30%. Numerous hairy roots were induced from protocalluses on Murashige and Skoog medium without growth regulators.
Paper electrophoresis revealed that all of the regenerated hairy roots tested synthesized the corresponding opines. This protoplast
culture system would be valuable for further somatic hybridization in forage legumes. 相似文献
12.
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
13.
Veena Veena Christopher G. Taylor 《In vitro cellular & developmental biology. Plant》2007,43(5):383-403
Agrobacterium rhizogenes is the etiological agent for hairy-root disease (also known as root-mat disease). This bacterium induces the neoplastic growth
of plant cells that differentiate to form “hairy roots.” Morphologically, A. rhizogenes-induced hairy roots are very similar in structure to wild-type roots with a few notable exceptions: Root hairs are longer,
more numerous, and root systems are more branched and exhibit an agravitropic phenotype. Hairy roots are induced by the incorporation
of a bacterial-derived segment of DNA transferred (T-DNA) into the chromosome of the plant cell. The expression of genes encoded
within the T-DNA promotes the development and production of roots at the site of infection on most dicotyledonous plants.
A key characteristic of hairy roots is their ability to grow quickly in the absence of exogenous plant growth regulators.
As a result, hairy roots are widely used as a transgenic tool for the production of metabolites and for the study of gene
function in plants. Researchers have utilized this tool to study root development and root–biotic interactions, to overexpress
proteins and secondary metabolites, to detoxify environmental pollutants, and to increase drought tolerance. In this review,
we provide an up-to-date overview of the current knowledge of how A. rhizogenes induces root formation, on the new uses for A. rhizogenes in tissue culture and composite plant production (wild-type shoots with transgenic roots), and the recent development of
a disarmed version of A. rhizogenes for stable transgenic plant production. 相似文献
14.
Watercress (Nasturtium officinale) is a member of the Brassicaceae family and a rich source of glucosinolate, which has been shown to possess anticancer properties.
To extract these compounds from N. officinale for study, a method was developed in which Agrobacterium rhizogenes was used to transfer DNA segments into plant genomes in order to produce hairy root cultures, which are a reliable source
of plant compounds. The A. rhizogenes strain R1000 had the highest infection frequency and induces the most hairy roots per explant. Polymerase chain reaction
and cytohistochemical staining methods were used to validate transgenic hairy roots from N. officinale. Glucosinolate from watercress hairy roots was separated and analyzed using high-performance liquid chromatography coupled
to electrospray ionization mass spectrometry. Indolic glucosinolates, including glucobrassicin (0.01–0.02 μmol/g of DW) and
4-methoxyglucobrassicin (0.06–0.18 μmol/g of DW), as well as aromatic glucosinolate (gluconasturtiin) (0.06–0.21 μmol/g of
DW), were identified virtually identical or more in transformed than wild type roots of N. officinale. Hairy root culture of watercress is a valuable approach for future efforts in the metabolic engineering of glucosinolate
biofortification in plants, particularly, because indolic glucosinolates are the precursors of a potent cancer chemopreventive
agent (indole-3-carbinol). 相似文献
15.
Aarrouf J Castro-Quezada P Mallard S Caromel B Lizzi Y Lefebvre V 《Plant cell reports》2012,31(2):391-401
Pepper is known to be a recalcitrant species to genetic transformation via Agrobacterium tumefaciens. A. rhizogenes-mediated transformation offers an alternative and rapid possibility to study gene functions in roots. In our study, we developed
a new and efficient system for A. rhizogenes transformation of the cultivated species Capsicum annuum. Hypocotyls and foliar organs (true leaves and cotyledons) of Yolo Wonder (YW) and Criollo de Morelos 334 (CM334) pepper
cultivars were inoculated with the two constructs pBIN-gus and pHKN29-gfp of A.
rhizogenes strain A4RS. Foliar explants of both pepper genotypes infected by A4RS-pBIN-gus or A4RS-pHKN29-gfp produced transformed roots. Optimal results were obtained using the combination of the foliar explants with A4RS-pHKN29-gfp. 20.5% of YW foliar explants and 14.6% of CM334 foliar explants inoculated with A4RS-pHKN29-gfp produced at least one root expressing uniform green fluorescent protein. We confirmed by polymerase chain reaction the presence
of the rolB and gfp genes in the co-transformed roots ensuring that they integrated both the T-DNA from the Ri plasmid and the reporter gene.
We also demonstrated that co-transformed roots of YW and CM334 displayed the same resistance response to Phytophthora capsici than the corresponding untransformed roots. Our novel procedure to produce C. annuum hairy roots will thus support the functional analysis of potential resistance genes involved in pepper P.
capsici interaction. 相似文献
16.
Transformation of <Emphasis Type="Italic">Saussurea medusa</Emphasis> for hairy roots and jaceosidin production 总被引:2,自引:0,他引:2
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
17.
18.
The Perilla (Perilla frutescens L. cv. Okdong) oleosin gene, PfOle19, produces a 19-kDa protein that is highly expressed only in seeds. The activity of the −2,015 bp 5′-upstream promoter region
of this gene was investigated in transgenic Arabidopsis plants using the fusion reporter constructs of enhanced green fluorescent protein (EGFP) and β-glucuronidase (GUS). The PfOle19 promoter directs Egfp expression in developing siliques, but not in leaves, stems or roots. In the transgenic Arabidopsis, EGFP fluorescence and histochemical GUS staining were restricted to early seedlings, indehiscent siliques and mature seeds.
Progressive 5′-deletions up to the −963 bp position of the PfOle19 promoter increases the spatial control of the gene expression in seeds, but reduces its quantitative levels of expression.
Moreover, the activity of the PfOle19 promoter in mature seeds is 4- and 5-fold greater than that of the cauliflower mosaic virus 35S promoter in terms of both
EGFP intensity and fluorometric GUS activity, respectively. 相似文献
19.
Ana Sofia Duque Susana de Sousa Araújo Matilde Ataíde Cordeiro Dulce Maria Santos Manuel Pedro Fevereiro 《Plant Cell, Tissue and Organ Culture》2007,90(3):325-330
We developed an alternative methodology for in vitro selection of transgenic Medicago truncatula cv. Jemalong plants using a bifunctional construct in which the coding sequences for the green fluorescent protein (GFP)
and the β-glucuronidase protein (GUS) are fused. An Agrobacterium-mediated transformation protocol was used followed by regeneration via somatic embryogenesis in the dark, to avoid the synthesis and the consequent autofluorescence of chlorophyll. This method
is a clear advantage over antibiotic and herbicide selection in which survival of non-transformed tissue is commonly reported,
with the reassurance that all the somatic embryos selected as GFP positive are transformed. This was subsequently corroborated
by the detection of GUS activity in leaves, stems and roots of the regenerated plants. Without antibiotic selection, and performing
the embryo induction in the dark, it was possible to attest the advantage of using GFP as an in vivo detectable reporter for
early embryo selection. The fusion with the GUS coding sequence provided additional evidence for the transformation of the
previously selected embryos. 相似文献
20.
Shi He-Ping Long Yong-Yue Sun Tie-Shan Tsang Po Keung Eric 《Plant Cell, Tissue and Organ Culture》2011,107(2):251-260
An efficient transformation system for the medicinal and aromatic plant, Pogostemon cablin Benth was developed by using agropine-type Agrobacterium rhizogenes ATCC15834. Hairy roots formed directly from the cut edges of leaf explants or via callus stage 8 days after inoculation with
the bacterium. The highest frequency of leaf explant transformation by Agrobacterium rhizogenes ATCC15834 was about 80% after infection for 25 days. Hairy roots grew rapidly on plant growth regulators (PGRs)-free Murashige
and Skoog (MS) or 6,7-V medium and had characteristics of transformed roots such as fast growth and high lateral branching.
The PCR amplification showed that rol genes of Ri plasmid of A. rhizogenes were integrated and expressed into the genome of transformed hairy roots. The hairy root line, PL6, grew very slowly in the
first 8 days, then grew very quickly between day 8 and day 24. The optimum medium for callus induction of hairy roots consisted
of 2.0 mg l−1 benzyladenine (BA) and 0.1 mg/l α-naphthaleneacetic acid (NAA); while optimum medium for adventitious shoot regeneration
from these cultures consisted of 0.1 mg l−1 BA and 0.1 mg l−1 NAA. Adventitious shoots could be rooted on 1/2MS. Southern blot analysis confirmed that rol genes of TL-DNA of Ri plasmid was integrated with at least three copies into the genome of hairy roots- regenerated P. cablin plants. The results presented provide a solid foundation for production of patchouli essential oil from hairy roots or its
regenerated plants and also provide possibilities for utilization of artifical polyploidization or chemical mutation of hairy
roots for improving germplasm and breeding of a new cultivar of P. cablin. 相似文献