共查询到20条相似文献,搜索用时 15 毫秒
1.
Summary Co-transformation experiments were carried out onPetunia hybrida protoplasts. The method used was electroporation with two plasmids: one confering kanamycin resistance, and the other harbouring a phosphoenolpyruvate carboxylase (PEPC) cDNA fromSorghum vulgare leaves. Southern blot analysis of the selected lines demonstrated a high co-transformation frequency.Abbreviations PEPC
phosphoenolpyruvate carboxylase
- NPT
neomycin phosphotransferase
- ATF
absolute transformation frequency
- PEG
polyethylenglycol
- BA
6-benzyl-aminopurine
- IAA
3-indoleacetic acid
- 2,4-D
2,4-dichlorophenoxyacetic acid 相似文献
2.
Genetic transformation of cauliflower (Brassica oleracea var. botrytis) by direct DNA uptake into mesophyll protoplasts 总被引:1,自引:0,他引:1
Mesophyll protoplasts of Brassica oleracea var. botrytis were successfully transformed using polyethylene glycol (PEG). The success of plant transformation depended on both gene transfer and plant regeneration. Parameters, such as PEG and vector concentrations and heat shock conditions were tested in experiments on transient expression of the β -glucuronidase (EC 3.2.1.31) gene and the most suitable conditions for DNA uptake were determined. Two antibiotic resistance marker genes for neomycin phosphotransferase (EC 2.7.1.95) and hygromycin phosphotransferase (EC 2.7.1.104), and three vector plasmids with different lengths were used to obtain stable transformants. 相似文献
3.
We cryopreserved whole rice calli (Oryza sativa L cv Taipei 309) to investigate the ability of the surviving cells to regenerate plants and yield protoplasts competent for genetic transformation. Four out of six callus lines cryopreserved after four months in culture contained small sectors able to continue cell division and subsequently regenerate fertile plants. Both cryopreservation efficiency and regeneration ability decreased when using eight month old cultures. High yields of protoplasts were obtained from different cryopreserved callus lines. Protoplasts were transfected with chimeric genes consisting of the maize ubiquitin 1 promoter, first exon and first intron fused to the coding region of either the GUS or BAR marker genes. Levels of transient gene expression from both marker genes were similar to those previously obtained using protoplasts derived from callus that had not been frozen. Stable transformants were selected by their resistance to Bialaphos and could be identified with the pH indicator chlorophenol red. Southern blot analysis confirmed the integration of the BAR gene into the rice genome. Therefore, cryopreservation does not affect the ability of rice cells to integrate and express foreign genes.Abbreviations BA
6-benzylaminopurine
- BAR
Bialaphos-resistance
- CaMV
cauliflower mosaic virus
- CPS
cryoprotectant solution
- CR
chlorophenol red 2,4-D 2,4-dichlorophenoxyacetic acid
- DMSO
dimethyl sulfoxide
- FW
fresh weight
- GUS
-glucuronidase
- IOD
interoptical density
- MS
Murashige and Skoog
- MU
methyl umbelliferone
- NAA
naphthaleneacetic acid
- PAT
phosphinothricin acetyl transferase
- PEG
polyethylene glycol
- TTC
2,3,5, triphenyltetrazolium chloride
- UBI
maize ubiquitin 1 promoter, first exon and first intron 相似文献
4.
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
5.
Yang Jin-shui Ge Kou-lin Luo Min Wang Yun-zhu Wang Bei Hu Rong-xia Qian Min Tan Jia-zhen Nancy Lee Douglas Testa 《植物学报(英文版)》1991,33(11)
Suspension cultures of small cell groups (SCG; ca. 50–100 cells per group) were established from calli of Japonica rice Fang 7 and Hl24. The SCG were partially digested and transformed by plasmid pBll21 harboring the NPT-II (neomycin phosphotransferase) and GUS (betaglucuronidase) genes. Plasmid DNA was introduced into cells' by PEG, electroporation and PEG plus electroporation. NTP-II and GUS activity assay showed that the report genes were expressed in transformed cells. Transgenic plants were regeneiated possessing GUS activity due to the integration of intact foreign DNA into their genome as evidanced by hybridization. The results prove that the partially digested SCG is a potential, feasible system as receptor for gene transfer, especially for plants which are difficult for protoplast culture and plant regeneration from protoplasts. 相似文献
6.
Polyethylene glycol (PEG)-mediated transient gene expression and silencing in protoplasts is widely applied in model plants such as Arabidopsis thaliana and rice. Here, we developed an efficient transient gene expression system based on the PEG-mediated method both in etiolated and green maize mesophyll protoplasts. The results showed that both yellow fluorescent protein encoding gene and glucuronidase encoding gene were efficiently expressed in maize protoplasts. More importantly, double-stranded RNAs (dsRNAs) can also be transfected into maize protoplasts by the PEG-mediated method to specifically silence exogenous and endogenous genes. Our results showed that dsRNA can be used to knockdown both exogenous and endogenous gene expression. Furthermore, bimolecular fluorescence complementation system for the detection of protein–protein interactions in maize protoplasts was developed. We also overexpressed and knockdowned the mitogen-activated protein kinase encoding gene ZmMPK5 to investigate the role of ZmMPK5 in abscisic acid (ABA)-induced antioxidant defense in maize protoplasts. This method here we reported will be valuable for signal transduction study in maize. 相似文献
7.
Transgenic rice plants produced by electroporation-mediated plasmid uptake into protoplasts 总被引:13,自引:0,他引:13
H. M. Zhang H. Yang E. L. Rech T. J. Golds A. S. Davis B. J. Mulligan E. C. Cocking M. R. Davey 《Plant cell reports》1988,7(6):379-384
Transgenic rice plants have been regenerated by somatic embryogenesis from cell suspension derived protoplasts electroporated with plasmid carrying the NPTII gene under the control of the 35S promoter from cauliflower mosaic virus. Heat shock of protoplasts prior to electroporation maximised the throughput of kanamycin resistant colonies. Omission of kanamycin from the medium for plant regeneration was essential for the recovery of transgenic rice plants carrying the NPTII gene. This report of the production of kanamycin resistant transgenic rice plants establishes the use of protoplasts for rice genetic engineering.Abbreviations NPTII
neomycin phosphotransferase
- SDS
sodium dodecyl sulphate 相似文献
8.
Thilo Schmidt-Rogge Martin Meixner Vibha Srivastava Sipra Guha-Mukherjee Otto Schieder 《Plant cell reports》1993,12(7-8):390-394
We developed a highly efficient transformation protocol for the PEG-mediated direct transfer of plasmid DNA into protoplasts of haploid Datura innoxia. Vectors harbouring a neomycin phosphotransferase II gene or a hygromycin B phosphotransferase gene under the control of different promoters were used in the transformation experiments. Various amounts of plasmid DNA were applied without any carrier DNA to show the direct influence of the plasmid DNA concentration on the transformation efficiency. Approximately 95% of the selected calli were regenerated to plants; 20% of them remained haploid. Total DNA of different transgenic plants was analysed with regard to the integration pattern of the plasmid DNA. Plants carrying only one or two copies of the vector DNA were observed as well as individuals with multi-copy integration (up to ten or more copies).Abbreviations ATF/RTF
absolute/relative transformation frequency
- BAP
6-benzylaminopurine
- CaMV
cauliflower mosaic virus
- CTAB
N-cetyl-N,N,N-trimethyl-ammonium bromide
- HPT
hygromycin B phosphotransferase gene
- PEG
polyethyleneglycol
- MES
2-(N-morpholino) ethanesulfonic acid
- NPT II
neomycin phosphotransferase II gene 相似文献
9.
N. M. Antonelli J. Stadler 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1990,80(3):395-401
Summary Efficient delivery of genomic DNA fragments to maize protoplasts was obtained by new methods using the polycation Polybrene or Lipofectin cationic liposomes. Stable kanamycin-resistant secondary transformants were recovered after transfection with genomic DNA from a maize cell line that had previously been tagged with the bacterial gene neomycin phosphotransferase (nptII) in a first-round transformation. The frequency of secondary transformants with nptII-homologous DNA sequences was 3% or 6% of all randomly picked microcalli after Polybrene or Lipofectin-mediated transfection, respectively. Transformation with genomic DNA by these methods may allow easy transfer of uncloned genes encoding desirable characteristics to crop species that can be regenerated from protoplasts. 相似文献
10.
Conditions were optimized for the culture, antibiotic selection and stable transformation by electroporation of suspension culture protoplasts of sugarbeet,Beta vulgaris L.. Highest plating efficiencies (up to 65% at day 21) were obtained if protoplasts were cultured in PGO salts (de Greef and Jacobs, 1979) supplemented with 0.1 mg/1 2,4-D, 0.01 mg/l BAP and 9% mannitol, and in 0.6% agarose rather than in liquid medium. Sensitivity to kanamycin also depended on whether protoplasts were cultured in liquid or agarose medium. Stable transformation of protoplast-derived colonies, as determined by resistance to kanamycin and Southern blot analysis, was achieved by electroporation using both rectangular and exponentially-decaying pulses.Abbreviations 2,4-D
2,4-dichlorophenoxyacetic acid
- BAP
6-benzylaminopurine
- npt-II
neomycin phosphotransferase
- EDTA
ethylenediaminetetracetic acid
- SDS
sodium dodecyl sulphate 相似文献
11.
Irradiation of protoplasts with X-rays or ultraviolet light does not seem to influence the level of transient expression of
foreign DNA inPetunia protoplasts, whereas the number of stably transformed colonies is significantly raised. This may indicate that irradiation
influences integration and/or the expression of marker genes and does not result in enhanced uptake rates of plasmids into
protoplasts and cell nuclei. Co-transformation with plasmids carrying a gene for kanamycin resistance (neomycin phosphotransferase
II) and a gene for hygromycin resistance (hygromycin phosphotransferase) revealed that the cotransformation rates were not
stimulated by irradiation when measuring expression. Twenty-five kanamycin resistant but hygromycin sensitive colonies were
examined with Southern or slot blotting and all were found to contain the coding sequence for the hygromycinphosphotransferase
gene in their genomes. No obvious differences regarding copy number of integrated genes were observed when comparing transformed
colonies derived from irradiated and non-irradiated protoplasts. 相似文献
12.
Anne-Laure Moyne Denis Tagu Véronique Thor Catherine Bergounioux Georges Freyssinet Pierre Gadal 《Plant cell reports》1989,8(2):97-100
Helianthus annuus protoplasts were transformed with the plasmid pCaMVNEO (Frommet al. 1986) conferring kanamycin resistance to plant. Transformed calli were selected with a frequency of 4 calli for 106 treated protoplasts. DNA was extracted from kanamycin resistant calli. Analysis of this DNA shows the presence of the NPTII gene.Abbreviations BAP
6-benzylaminopurine
- 2,4-D
2,4 dichlorophenoxyacetic acid
- IAA
Indole-3-acetic acid
- NAA
1-naphtalenoacetic acid
- NPT
Neomycin phosphotransferase
- PEG
Polyethyleneglycol 相似文献
13.
14.
Joo Mi Jeon Nam Young Ahn Bo Hwa Son Cha Young Kim Chang-deok Han Gun-Do Kim Sang Wan Gal Sung-Ho Lee 《Plant Cell, Tissue and Organ Culture》2007,88(2):225-232
PEG-mediated transformation was used for gene delivery and evaluation of various parameters affecting the transient expression of a gene for ß-glucuronidase (gus) in mesophyll protoplasts of Capsicum annuum. Transient expression was found to be dependent on PEG concentration and exposure time of plasmid DNA to protoplasts as well as the amount of plasmid DNA. Maximum GUS activity was obtained when protoplasts were applied to 40% concentration and molecular weight was 6,000 of PEG solution with 30 min of exposure time. Protoplasts of pepper were transformed with a vector, pCAMBIA::Ac, which contained a pCAMBIA1302 T-DNA vector carrying a maize transposable element, Ac (activator), a selection marker HPT (hygromycin phosphotransferase), and a GFP-coding region driven by the 35S promoter in the presence of PEG. Approximately 30% of the protoplasts expressed GFP. Visibly transformed colonies were obtained from protoplasts after 2 months of culture and GFP was expressed. Southern hybridization confirmed the presence of Ac in the pepper genome. 相似文献
15.
Arundhati Mukhopadhyay Reinhard Töpfer Akshay K. Pradhan Yaspal S. Sodhi Hans-Henning Steinbiß Jeff Schell Deepak Pental 《Plant cell reports》1991,10(8):375-379
Summary Efficient regeneration (80%) and high frequency genetic transformation (10–33%) were achieved by culturing protoplasts isolated from hypocotyl tissues of six day old Brassica oleracea seedlings and by subjecting these protoplasts to PEG mediated direct plasmid uptake. Three different plasmid vectors carrying marker genes for resistance to methotrexate (dhfr), hygromycin (hpt) and phosphinotricin (bar) were constructed and used for transformation. Large number of normal, fertile transformants were obtained with vectors carrying hpt and bar genes. No transformants could be regenerated for resistance to methotrexate as it severely suppressed shoot differentiation.Abbreviations
bar/PAT
bialaphos resistance gene/phosphinotricin acetyltransferase
- 2,4-D
2,4-di-chlorophenoxyacetic acid
-
dhfr/DHPR
dihydrofolate reductase gene/enzyme
-
gus/GUS
-glucuronidase gene/enzyme
-
hpt/HPT
hygromycin phosphotransferase gene/enzyme
- Kn
kinetin
- PEG
polyethylene glycol
- RH
relative humidity 相似文献
16.
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
17.
Wheat dwarf virus vectors replicate and express foreign genes in cells of monocotyledonous plants. 总被引:12,自引:2,他引:10
下载免费PDF全文
![点击此处可从《The Plant cell》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Wheat dwarf virus (WDV) is a geminivirus that infects monocotyledonous plants. To exploit the potential of WDV as a replicative gene vector, we developed a transient replication and expression system based on the transfection of protoplasts derived from Triticum monococcum suspension culture cells. Cloned genomic copies of various WDV isolates as well as mutants constructed in vitro were introduced into the protoplasts and assayed for their ability to replicate. As a result, regions of the WDV genome necessary or dispensable for the viral DNA replication could be defined. In addition, the gene encoding the viral capsid protein was replaced by three different bacterial marker genes, neomycin phosphotransferase, chloramphenicol acetyltransferase, and beta-galactosidase. The beta-galactosidase gene doubled the size of the WDV genome. The replication of the recombinant WDV genomes and the expression of these genes were monitored in suspension culture cells of T. monococcum. The potential of replicative expression vectors based on the WDV genome is discussed. 相似文献
18.
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
19.
20.
Summary The availability of a substantial amount of high molecular weight DNA is an essential prerequisite for the construction of yeast artificial chromosome (YAC) libraries. Parameters concerning protoplast isolation and DNA extraction have been systematically analyzed. Conditions have been established for the obtainment of high molecular weight DNA from Arabidopsis thaliana and Nicotiana plumbaginifolia protoplasts either embedded in agarose plugs or in liquid suspension. Restriction fragments were obtained by partial and total digestion with different endonucleases, and separated by pulsed-field gel electrophoresis. Ligation of partially EcoRI-digested DNA (range 30–300 kbp) followed by transformation of yeast spheroplasts gave rise to YACs with an average size of 60 kbp. The introduction of a DNA size-selection step before ligation led to production of YACs in the range of 100–200 kbp. Clones of up to 460 kbp were obtained by blunt-end ligation of pre-selected unrestricted DNA.Abbreviations 2,4-D
2,4-dichloro phenoxyacetic acid
- 6BAP
6-benzylaninopurine
- BFP
bovine serum albumin 0.1%, Ficoll 400 0.1%; polyvinylpyrrolidone 0.1%
- CHEF
clumped homogeneous electric field
- DTT
dithiothreitol
- EDTA
ethylenediaminetetraacetic acid
- HMW
high molecular weight
- km
kanamycin
- LMP agarose
low melting point agarose
- MS
Murashige and Skoog mediun
- npt
neomycin phosphotransferase
- PEG
polyethyleneglycol
- PFGE
pulsed-field gel electrophoresis
- RFLP
restriction fragrant lenght polymorphism
- SDS
sodium dodecyl sulphate
- SSC
sodium chloride 150 mM, sodiun citrate 15 nM, pH 7
- TAE
TRIS-Acetate pH 8 40 mM, EDTA 2 mM
- TE
TRIS-HCl pH 8 10 mM, EDTA 1 mM
- YAC
yeast artificial chromosome 相似文献