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1.
Summary An efficient technique was developed for the isolation, culture, transformation and regeneration of protoplasts derived from auxin conditioned Arabidopsis root cultures. On an average 30% of root protoplasts underwent cell division in liquid culture and formed somatic embryolike structures which regenerated to plants without embedding in Ca2+-alginate. The protoplast protocol was applicable to different landraces of Arabidopsis thaliana (L.) Heynh., such as RLD, Columbia or C24. PEG-mediated DNA uptake into protoplasts using different uidA reporter gene constructs yielded transient gene expression in over 25% of treated cells indicating that root-derived protoplasts are suitable recipients for transformation.Abbreviations BA
6-benzylaminopurine
- 2,4D
2,4dichlorophenoxyacetic acid
- IAA
indole-3-acetic acid
- ISA
indole-3buryric acid
- IPAR
6-(,-dimethylallylamino)purine riboside
- NAA
naphthaleneacetic acid
- uidA
ß-glucuronidase gene
- GUS
ß-glucuronidase enzyme
- CaMV
Cauliflower Mosaic Virus
- nos
nopaline synthase
- MES
2[N-morpholino]ethane-sulfonicacid
- PEG
polyethylene glycol
- X-gluc
5bromo-4-chloro-3-indolyl glucuronide
- MUG
4-methyl umbelliferyl glucuronide
- MU
4-methylumbelliferone 相似文献
2.
Elke Kemper Christoph Grevelding Jeff Schell Robert Masterson 《Plant cell reports》1992,11(3):118-121
Summary A modified root explant transformation method has been developed that is effective in producing transgenic Arabidopsis thaliana plants which are methotrexate resistant due to the integration of T-DNA vectors containing a chimeric dihydrofolate reductase gene. Molecular analysis shows that transformed methotrexate resistant plants contain the expected T-DNA construct with the chimeric gene. This transformation method also works well with other plant selectable markers, including hygromycin phosphotransferase and neomycin phosphotransferase II.Abbreviations DHFR
(dihydrofolate reductase)
- HPT
(hygromycin phosphotransferase)
- NPTII
(neomycin phosphotransferase)
- CaMV
(Cauliflower Mosaic Virus)
- MES
(2-[N-morpholino]ethanesulfonic acid)
- BAP
(N6-benzylaminopurine)
- NAA
(naphthaleneacetic acid)
- 2,4-D
(2,4 dichlorophenoxyacetic acid)
- 2iP
(6-(dimethylallylamino)-purine)
- 2iPAde
(6-(dimethylallylamino)-ade-nine)
- IAA
(indole-3-acetic acid)
- IBA
(indole-3-butyric acid) 相似文献
3.
Summary Growth characteristics of tobacco protoplasts containing rolA linked to its own promoter, or the rolB, or rolC genes of Agrobacterium rhizogenes linked to the Cauliflower Mosaic Virus 35S RNA promoter were compared with those from untransformed plants. RolA protoplasts require auxin and cytokinin for callus formation. Protoplasts overexpressing rolB and C form callus in the absence of exogenously applied auxin and cytokinin, respectively. Long term callus growth requires auxin, but the requirement for cytokinin is not critical. Optimal transient expression of an auxin responsive promoter element occurred at lower external levels of auxin in rolB and rolC protoplasts compared with untransformed protoplasts. Addition of putrescine was required for auxin responsive transient gene expression in rolA protoplasts suggesting that polyamines, or their products affect gene expression in rolA plants.Abbreviations T-DNA
transferred DNA
- TL-DNA
left transferred DNA
- NAA
naphthalene acetic acid
- PEG
polyethylene glycol
- GUS
glucuronidase
- CaMV
cauliflower mosaic virus 相似文献
4.
Ferdinand Regner Artur da Câmara Machado Margit Laimer da Câmara Machado Herta Steinkellner Diethard Mattanovich Veronika Hanzer Hans Weiss Hermann Katinger 《Plant cell reports》1992,11(1):30-33
Transgenic Nicotiana benthamiana and N. clevelandii plants expressing the coat protein of Plum Pox Virus under the control of the 35S promoter from Cauliflower Mosaic Virus were engineered by Agrobacterium tumefaciens mediated transformation. The phenomenon of virus resistance was observed at different levels when transgenic plants, expressing the coat protein and control plants were compared after challenge infection with Plum Pox Virus. N. clevelandii coat protein transgenic plants circumvent virus accumulation. After an initial increase in virus titer similar to the control plants, some coat protein expressing plants showed a reduced accumulation of virus and inhibition of the systemic spread, characterized by decrease of the virus titer and formation of new symptomless leaves. In other N. clevelandii coat protein expressing plants virus accumulation was inhibited and disease symptoms never appeared. N. benthamiana coat protein expressing plants were also protected. After a temporary virus accumulation, virus titer decreased without the appearance of symptoms with the exception of a few plants, which showed a delay of thirty days in the development of symptoms post challenge infection.Abbreviations PPV
Plum Pox Virus
- CP
coat protein
- CaMV
Cauliflower Mosaic Virus
- CP+
coat protein expressing plant
- CP–
control plant = non coat protein expressing plant
- TMV
Tobacco Mosaic Virus
- NPTII
neomycin phosphotransferaseII
- IBA
indole-3-butyric acid
- BAP
6-benzylaminopurine;
- MS
Murashige Skoog
- ELISA
enzyme linked immunosorbent assay 相似文献
5.
Movement proteins (MPs) are non-cell autonomous viral-encoded proteins that assist viruses in their cell-to-cell movement. The MP encoded by Tobamoviruses is the best characterized example among MPs of non-tubule-inducing plant RNA viruses. The MP of Oilseed Rape Mosaic Tobamovirus (ORMV) was transgenically expressed in Arabidopsis thaliana, ecotype RLD, under the expression of the 35S promoter from Cauliflower Mosaic Virus. Transgenic lines were obtained in sense and antisense orientations. One of the sense transgenic lines was further characterized turning out to carry one copy of the transgene inserted in the terminal region of the right arm of chromosome 1. The constitutive expression of ORMV-MP induced mild physiological effects in Arabidopsis. Plants of the transgenic line allowed a faster systemic movement of the phloem tracer carboxyfluorescein. The tracer was unloaded differentially in different flower parts, revealing differential effects of ORMV-MP on phloem unloading in sink organs. On the other hand, transgenic Arabidopsis did not show any effect on biomass partitioning or sugar availability, effects reported for equivalent transgenic solanaceous plants expressing the MP of Tobacco Mosaic Virus, another Tobamovirus. Finally, the transgenic Arabidopsis plants were susceptible to ORMV infection, although showing milder overall symptoms than non-transgenic controls. The results highlight the relevance of the specific host-virus system, in the physiological outcome of the molecular interactions established by MPs.C. Mansilla and I. Aguilar contributed equally. 相似文献
6.
Two clones of Populus nigra L. were tested in vivo and in vitro for their susceptibility to three different Agrobacterium tumefaciens wild-type strains evaluating number and size of resulting calluses. Strain C58 proved to be the most virulent.Various parameters affecting Agrobacterium-mediated transformation of P. nigra clones were further analyzed using ß-glucuronidase gene transient expression. The clone Jean Pourtet proved to be more susceptible than the clone San Giorgio. A. tumefaciens strain A281 pKIWI105 proved to be the most virulent. The optimal procedure involved dipping of leaf discs into a bacterial suspension (7×108 cells/ml) for 20 min, followed by a 48 h co-cultivation period on semi-solid regeneration medium.Leaf explants were co-cultivated with two disarmed A. tumefaciens strains. Plantlets of San Giorgio were regenerated, tested for ß-glucuronidase activity and rooted on selective medium containing kanamycin. Polymerase chain reaction analysis and Southern blot hybridization confirmed the integration of the neomycin phosphotransferase II gene into the poplar genome.Abbreviations BAP
6-benzyl-aminopurine
- CaMV
Cauliflower Mosaic Virus
- 2,4-D
2,4-dichlorophenoxyacetic acid
- GUS and gus
ß-glucuronidase
- hpt
hygromycin phosphotransferase
- IBA
indole-3-butyric acid
- KIN
kinetin
- LB
Luria Bertani
- MS
Murashige and Skoog
- NAA
ßnaphthaleneacetic acid
- NOS
Nopaline synthase
- NPTII and nptII
neomycin phosphotransferase II
- PCR
Polymerase chain reaction
- PVC
poly-vinyl-cloride
- SDS
sodium dodecyl sulfate
- SSC
sodium cloride-sodium citrate
- Tris
tris(hydroxymethyl)amino-methane
- WPM
Woody Plant Medium 相似文献
7.
Summary Direct delivery of DNA into embryogenic pollen was used to produce transgenic plants in tobacco. A plasmid bearing the ß-glucuronidase (GUS) marker gene in fusion with the 35S-promoter was introduced by microprojectile bombardment into mid-binucleate pollen of Nicotiana tabacum that had been induced to form embryos by a starvation treatment. In cytochemical expression assays, 5 out of 104 pollen grains were GUS+. Visual selection by staining with a non-lethal substrate for GUS was used to manually isolate transformed embryos. From the initial population of embryogenic GUS+ pollen, 1–5% developed into multicellular structures and 0.02% formed regenerable embryos. Two haploid transformants were regenerated. GUS expression was detected in different parts of the plants, and Southern analysis confirmed stable integration of the foreign DNA. Diploidisation was induced by injection of colchicine into the stem near adventitious buds. Offspring from selfings and backcrosses of one transformant were tested for GUS expression and by Southern blots. All F1-plants were transgenic, in accordance with Mendelian inheritance.Abbreviations GUS
ß-glucuronidase
- CaMV
Cauliflower Mosaic Virus
- MCS
multicellular structure
- NPTII
neomycin phosphotransferase
- PEG
polyethylene glycol
- X-gluc
5-bromo-4-chloro-3-indolyl glucuronide
- DAPI
4,6-diamidino-2-phenylindole
- Tris
Tris(hydroxymethyl)aminomethane hydrochloride
- EDTA
ethylenedinitrilo tetraacetic acid, disodium salt dihydrate 相似文献
8.
Nacyra Assad-García Neftali Ochoa-Alejo Enrique García-Hernández Luis Herrera-Estrella June Simpson 《Plant cell reports》1992,11(11):558-562
A protocol for the Agrobacterium-mediated transformation of tomatillo was developed. Up to 40 transgenic plants could be obtained in experiments using 60 cotyledon expiants. The transformed nature of the regenerated plants was confirmed by NPT II and Southern blot hybridization analysis. Using the b-glucuronidase system the tissue specific and developmental patterns of expression of the Cauliflower Mosaic Virus 35S promoter were determined in transgenic tomatillo plants. It was found that this promoter is developmentally regulated during fruit and seed formation. 相似文献
9.
Cotyledon explants of muskmelon (Cucumis melo L., cv. Amarillo Oro) seedlings were co-cultivated with disarmed Agrobacterium tumefaciens strain LBA4404 that contained the binary vector plasmid pBI121.1. The T-DNA region of this binary vector contains the Nopaline synthase/neomycin phosphotransferase II (NPTII) chimeric gene for kanamycin resistance and the Cauliflower Mosaic Virus 35S/-glucuronidase (GUS) chimeric gene. After infection, the cotyledon pieces were placed in induction medium containing 100 mg/l kanamycin. Putative transformed shoots were obtained, followed by the development of morphologically normal plantlets. The transgenic nature of regenerants was demonstrated by polymerase chain reaction, Southern blot analysis, plant growth on medium selective for the transgene (NPTII) and expression of the co-transformed GUS gene. Factors affecting the transformation procedure are discussed.Abbreviations CaMV
Cauliflower Mosaic Virus
- Cf
Cefotaxime
- GUS
-glucuronidase
- Km
Kanamycin
- MS
Murashige and Skoog
- NOS
nopaline synthase
- NPTII
neomycin phosphotransferase II
- PCR
polymerase chain reaction 相似文献
10.
High voltage electrical pulses were used to introduce the CAT reporter gene into cultured protoplasts of breadwheat,Triticum aestivum. Four DNA constructs harboring the CAT gene and the 35S or mannipine synthase promoter were tested for levels of CAT activity 40–45 hr after electroporation of protoplasts. One construct, containing a maize intron sequence between 35S and CAT sequences, conferred 30 to 185 fold greater CAT activity over the other three constructs. Data from these experiments suggest that a maize intron or sequences with similar effects may be required in DNA constructs for efficient heterologous gene expression in cultured cells of breadwheat.Abbreviations CAT
Chloramphenicol acetyl transferase
- NPT II
neomycin phosphotransferase
- 35S
the 35S promoter of Cauliflower Mosaic Virus
- PEG
Polyethylene glycol
- MES
2-[N-morpholino] ethanesulfonic acid 相似文献
11.
Summary The pat gene, coding for phosphinothricin acetyltransferase (PAT) from Streptomyces viridochromogenes, was cloned behind the par promoter of the hemoglobin gene from Parasponia andersonii, Introduction into tobacco (Nicotiana tabacum) resulted in predominantly root specific PAT expression. Application of 5 l/ha BASTA® (herbicidal component: phosphinothricin) did not effect growth morphology and vigor of the plants. After application of 20 l/ha BASTA® the plants showed herbicide damage. Nevertheless, they all recovered by forming new undamaged leaves and resumed full growth despite virtually non-detectable expression of the PAT enzyme in the leaves.Abbreviations BAP
6-benzylaminopurine
- CaMV
Cauliflower Mosaic Virus
- IAA
indole-3-acetic acid
- kb
kilobases
- LB
Luria-Bertani
- MS
Murashige and Skoog
- par
Parasponia andersonii
- PAT
phosphinothricin acetyltransferase
- ppt
phosphinothricin
- TCA
trichloric acid 相似文献
12.
Summary We report here an efficient Arabidopsis leafdisc transformation protocol yielding an average transformation frequency of 1.6 transgenic shoots per leaf explant 4 weeks after the bacterial infection period. Subsequent cultivation in vitro is such that a high percentage (85–90%) of the primary transformants produces seeds with an average seed yield of 100–300 seeds per plant. This improved transformation protocol yields mainly (70%) transformants segregating for a single T-DNA locus of which 68% actually contain one T-DNA insert. The objective is to generate a pool of independent transformants harboring an activator T-DNA construct in a gene tagging approach to isolate genes involved in morphogenesis and auxin signal transduction.Abbreviations
2,4-D
2,4-dichlorophenoxyacetic acid
-
AGM
Arabidopsis growth medium
-
BAP
benzylaminopurine
-
CaMV
Cauliflower Mosaic Virus
-
CTAB
Hexadecyltrimethylammoniumbromide
-
DIG
digoxigenin
-
FeNaEDTA
Iron-sodium-ethylenedinitrilo tetraacetic acid complex
-
GUS
ß-Glucuronidase
-
IBA
indole-3-butyric acid
-
LB
left T-DNA border
-
MES
2-(N-morpholino) ethane sulfonic acid
-
MS
Murashige and Skoog medium
-
NAA
-naphthaleneacetic acid
-
RB
right T-DNA border 相似文献
13.
Laszlo Sagi Serge Remy Bart Panis Rony Swennen Guido Volckaert 《Plant cell reports》1994,13(5):262-266
Summary Electroporation conditions were established for transient expression of introduced DNA in banana (Musa spp., cv. Bluggoe) protoplasts isolated from regenerable embryogenic cell suspensions. The following parameters were found to be highly influential: electroporation buffer, polyethylene glycol treatment and its duration before electroporation, use of a heat shock, and chimaeric gene constructs. The maximum frequency of DNA introduction as detected by an in situ assay for transient expression of the uidA gene, amounted to 1.8% of total protoplasts. Since plants have recently been regenerated from banana protoplasts at a high frequency, the present results may contribute to the production of transgenic banana.Abbreviations AMV
alfalfa mosaic virus
- CaMV
cauliflower mosaic virus
- 2,4-D
2,4-dichlorophenoxyacetic acid
- EGTA
ethylene glycol-O-O'-bis(2-aminoethyl)-N,N,N',N'-tetraacetic acid
- GUS
glucuronidase
- HEPES
4-(2-nydroxyethyl)piperazine-1-etnanesulfonic acid
- MES
2-morpholinoethanesulfonic acid
- MS
Murashige-Skoog
- NOS
nopaline synthase
- NFTII
neomycin phosphotransferase
- PEG
polyethylene glycol
- TGE
transient GUS expression
- X-Gluc
5-bromo-4-chloro-3-indolyl -D-glucuronic acid 相似文献
14.
D. M. Lonsdale R. L. Allen D. Belostotsky T. K. Ghose A. J. Harvey H. J. Rogers S. J. Tebbut M. Trick 《Plant cell reports》1995,15(1-2):154-158
Summary The promoters of a tobacco actin gene, a tobacco pectate lyase, a tobacco and maize polygalacturonase and aBrassica S-locus related gene have been fused to the-glucuronidase reporter gene and their activities determined by biolistic transient assay in tobacco pollen. In stably transformed tobacco all the transgenes with the exception of Cauliflower Mosaic Virus-35S--glucuronidase appear to express efficiently in maturing pollen. Transient assay analysis showed that the tobacco pectate lyase and the polygalacturonase constructs were 8x more active than the tobacco actin construct, and that the tobacco polygalacturonase construct was some 33x more active than the maize polygalacturonase construct. Constructional manipulations that altered the lengths of the 5-untranslated leaders including one which resulted in the removal of a 490 bp leader intron had little effect on the observed level of expression. However, the alteration of the context of the ATG from A/TnnATGG to CnnATGT resulting in a 70% reduction in the observed levels of activity, was obtained with the pectate lyase and polygalacturonase promoters. An identical reductional was also observed in transgenic plant populations transformed with the polygalacturonase transgenes.Abbreviations GUS
-glucuronidase
- LUC
luciferase
- NosTer
nopaline synthase terminator
- CaMV
Cauliflower Mosaic Virus
- UTL
untranslated leader
- PCR
polymerase chain reaction
- PG
polygalacturonase
- Npg
tobacco polygalacturonase
- Pl
pectate lyase
- Ac
actin 相似文献
15.
16.
Wissam A. Abou-Alaiwi Shobha D. Potlakayala Stephen L. Goldman Puthiyaparambil C. Josekutty Deepkamal N. Karelia Sairam V. Rudrabhatla 《Plant Cell, Tissue and Organ Culture》2012,109(1):1-8
An efficient transformation system was developed for Centaurea montana by co-cultivation of leaf explants with Agrobacterium tumefaciens strain AGL1 that contained a plasmid harboring the isopentenyl transferase gene under the control of the developmentally
regulated Atmyb32 promoter of Arabidopsis thaliana and the gene encoding for hygromycin resistance under the control of the Cauliflower Mosaic Virus 35S (CaMV35S) promoter.
A total of 990 explants were infected with Agrobacterium, and 18 shoots were regenerated resulting in an overall transformation efficiency of 1.8%. Molecular analyses, including
PCR, Southern blotting and RT-PCR, were performed on T0 and T1 plants to confirm chromosomal integration and expression of the transgene in the phenotypically normal transformed plants.
Transformation of C. montana was also performed using A. tumefaciens supervirulent strain EHA105 harboring the β-glucuronidase (GUS) reporter gene. Expression of the GUS gene in the putative transgenics was confirmed using a histochemical GUS assay. 相似文献
17.
Leonne M. van der Leede-Plegt Bernadette C. E. van de Ven Raoul J. Bino Theo P. M. van der Salm Arjen J. van Tunen 《Plant cell reports》1992,11(1):20-24
Summary As part of our research to develop an alternative system for the transformation of recalcitrant plant species we investigated the use of the male gametophyte as a transformation vector. Therefore the activity of four different promoters (CaMV 35S, LAT52, chiA PA2 and TR2') was analyzed in pollen of a dicot (Nicotiana glutinosa) and a monocot (Lilium longiflorum) plant species. Gene constructs in which the ß-glucuronidase (GUS) gene was placed under the control of these promoters were introduced in pollen using a particle delivery system. No activity of the Cauliflower Mosaic Virus (CaMV) 35S promoter was detected in pollen of both N. glutinosa and L. longiflorum. The promoter of the tomato flower-specific LAT52 gene was highly active in N. glutinosa pollen but remained silent in L. longiflorum pollen. A similar expression pattern was observed for the pollen-specific Chalcone Flavanone Isomerase chiA PA2 promoter originally isolated from petunia. The TR2 mannopine synthase promoter of Agrobacterium tumefaciens, however, was active in pollen from Solanaceous species and also in pollen from the monocot L. longiflorum. This suggests that the TR2' promoter is active in vegetative and sporogenous tissues of dicot and monocot plant species.Abbreviations
ADH1
Alcohol Dehydrogenase 1
-
A. tumefaciens
Agrobacterium tumefaciens
-
CaMV
Cauliflower Mosaic Virus
-
ChiA
Chalcone Flavanone Isomerase A
-
L. longiflorum
Lilium longiflorum
- N. glutinosa
Nicotiana glutinosa
- Nos
Nopaline Synthase
- N. tabacum
Nicotiana tabacum 相似文献
18.
Reinhard Töpfer Markus Pröls Jozef Schell Hans-Henning Steinbiß 《Plant cell reports》1988,7(4):225-228
The reporter genes for Chloramphenicolacetyltransferase (CAT), Neomycinphosphotransferase-(NPT)-II and -Glucuronidase (GUS) were compared in transient gene expression experiments in tobacco mesophyll protoplasts. For this purpose, nearly identical chimeric genes controlled by the CaMV 35 S promoter were constructed. The detection level of each system was determined yielding the following order of relative sensitivity: CAT相似文献
19.
20.
Regeneration of transgenic plants of Prunus armeniaca containing the coat protein gene of Plum Pox Virus 总被引:1,自引:0,他引:1
Margit Laimer da Câmara Machado Artur da Câmara Machado Veronika Hanzer Hans Weiss Ferdinand Regner Herta Steinkellner Diethard Mattanovich Regina Plail Elisabeth Knapp Birgit Kalthoff Hermann Katinger 《Plant cell reports》1992,11(1):25-29
Summary A system was developed which allows the transfer of foreign genes into apricot cultivars. We report the transformation and regeneration of Prunus armeniaca plants with Agrobacterium tumefaciens strain LBA 4404 containing various binary plasmids, pBinGUSint, carrying the marker gene ß-glucuronidase (GUS) and pBinPPVm, carrying the coat protein gene of Plum Pox Virus (PPV). The marker gene GUS was used for optical evaluation of the efficiency of the transformation system. The coat protein gene of PPV was used to introduce coat protein mediated resistance against one of the most important pathogens of stone fruit trees in Europe and the whole Mediterranean area. This is the first report of the successful integration of a viral coat protein gene into a fruit tree species, opening a new perspective on the control of the disease.Abbreviations GUS
ß-glucuronidase
- PPV
Plum Pox Virus
- BA
6-benzylaminopurine
- NPTII
neomycin phosphotransferase II
- CP
coat protein
- CaMV
Cauliflower Mosaic Virus
- P35S 35S
promoter
- MS
Murashige and Skoog
- PCR
polymerase chain reaction
- P/C/I
phenol/chloroform/isoamylalcohol
- RNase
ribonuclease
- dNTP
deoxyribonucleosidetriphosphate
- DMSO
dimethyl sulfoxide 相似文献