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1.
Transgenic peanut plants were produced using Agrobacterium mediated gene transfer. Primary leaf explants of peanut were co-cultivated with Agrobacterium tumefaciens LBA 4404 harbouring the binary plasmid pBI 121 (conferring -glucuronidase activity and resistance to kanamycin) and cultured on regeneration medium supplemented with kanamycin to select putatively transformed shoots. They were rooted and plants were transferred to soil. Stable integration and expression of the transgenes were confirmed by NPT II assay, Southern blot hybridization and GUS assay.Abbreviations BA
6-benzyladenine
- GUS
-glucuronidase
- IAA
indole-3-acetic acid
- NAA
-naphthaleneacetic acid
- NOS
nopaline synthase
- NPT II
neomycin phosphotransferase II
- SDS
Lauryl sulfate 相似文献
2.
Willem J. Stiekema Freek Heidekamp Jeanine D. Louwerse Harrie A. Verhoeven Paul Dijkhuis 《Plant cell reports》1988,7(1):47-50
Tuber discs of Solanum tuberosum cv Bintje and Désirée were cocultivated with an Agrobacterium tumefaciens binary vector, carrying both the neomycine phosphotransferase and the E. coli -glucuronidase gene fused to resp. the nopaline synthase and Cauliflower mosaic virus 35S promotor.Inoculated tuber discs produce transgenic shoots in selective media containing kanamycin. The transgenic plants are phenotypically normal and contain the euploid number of chromosomes. Both the neomycin phosphotransferase as well as the -glucuronidase gene are expressed conferring resp. kanamycin resistance and -glucuronidase activity to the plants.Abbreviations GUS
-glucuronidase
- NPT
neomycin phosphotransferase
- CaMV
Cauliflower Mosaic Virus
- BAP
6-benzylaminopurine
- GA3
gibberellic acid
- NAA
naphthalineacetic acid
- LB
Luria Broth
- MU
methylumbelliferone 相似文献
3.
Induced expression of a chimeric gene construct in transgenic lettuce plants using tobacco pathogenesis-related protein gene promoter region 总被引:3,自引:0,他引:3
The expression of a stress- and salicylic acidinducible protein gene from tobacco, PR1a protein gene, was determined after its Introduction to lettuce (Lactuca sativa L.) plants. The 5 flanking 2.4 Kb fragment from PR1a gene was joined to the bacterial -glucuronidase (GUS) gene (PR-GUS) and introduced into lettuce cotyledons by Agrobacterium-mediated gene transfer using a binary vector containing a kanamycin-resistance gene as a selectable marker. As a control with constitutive expression, the chimeric gene consisting of CaMV 35S RNA promoter and GUS gene (35S-GUS) was used. An improved method for shoot formation directly from lettuce cotyledons was used effectively for transformation, shortening the time for regeneration. In 70% or more of kanamycin-resistant regenerated lettuce plants, into which PR-GUS or 35S-GUS was introduced, high GUS activity and integration of the chimeric gene into the lettuce genome were detected. By treatment with salicylic acid, GUS activity increased 3- to 50-fold in PR-GUS transformants, however, no increase was detected in 35S-GUS plants. These results showed that the promoter of the stress-inducible tobacco PR1a protein gene was introduced into lettuce plants, and the introduced chimeric gene was expressed normally under the regulated control of the PRla promoter.Abbreviations BA
N6-benzyladenine
- GUS
-glucuronidase
- NAA
-naphthaleneacetic acid
- Km
kanamycin
- Kms
kanamycin resistant
- Km0
kanamycin sensitive
- NPT- II
neomycin phosphotransferase II
- PR
pathogenesis-related
- SA
salicylic acid
- MS
Murashige and Skoog medium
- NOS
nopaline synthase 相似文献
4.
Transformation of sugarcane protoplasts by direct uptake of a selectable chimaeric gene 总被引:6,自引:0,他引:6
W. H. Chen K. M. A. Gartland M. R. Davey R. Sotak J. S. Gartland B. J. Mulligan J. B. Power E. C. Cocking 《Plant cell reports》1987,6(4):297-301
Sugarcane protoplasts were transformed to kanamycin resistance at a frequency of approximately 8 in 107 following PEG-induced uptake of Sma1 linearised pABD1 plasmid. DNA-treated protoplasts were cultured in agarose droplets, and protoplast-derived transformants selected on 80 g ml–1 kanamycin. Transformed tissues maintained on this level of antibiotic expressed APH(3)II activity, and contained DNA that hybridised to a probe with the APH(3)II gene.Abbreviations APH(3)II
aminoglycoside phosphotransferase
- PEG
polyethylene glycol
- 2,4-D
2,4-dichlorophenoxyacetic acid 相似文献
5.
Intergeneric somatic hybrid plants between Hamlin sweet orange [Citrus sinensis (L.) Osbeck] and Flying Dragon trifoliate orange (Poncirus trifoliata Raf.) were regenerated following protoplast fusion. Hamlin protoplasts, isolated from an habituated embryogenic suspension culture, were fused chemically with Flying Dragon protoplasts isolated from juvenile leaf tissue. The hybrid selection scheme was based on complementation of the regenerative ability of the Hamlin protoplasts with the subsequent expression of the trifoliate leaf character of Flying Dragon. Hybrid plants were regenerated via somatic embryogenesis and multiplied organogenically. Hybrid morphology was intermediate to that of the parents. Chromosome counts indicated that the hybrids were allotetraploids (2n=4x=36). Malate dehydrogenase (MDH) isozyme patterns confirmed the hybrid nature of the regenerated plants. These genetically unique somatic hybrid plants will be evaluated for citrus rootstock potential. The cell fusion, selection, and regeneration scheme developed herein should provide a general means to expand the germplasm base of cultivated Citrus by intergeneric hybridization with related sexually incompatible genera.Abbreviations MDH
malate dehydrogenase
- CTV
citrus tristeza virus
- MT
Murashige and Tucker basal medium
- BH3
protoplast culture medium, Grosser and Chandler, 1987
- PEG
polyethylene glycol
- GA3
giberellic acid
- BA
N-(phenylmethyl)-1 H-purin-6-amine
- HCl
hydrochloric acid
Florida Agricultural Experiment Station Journal Series No. 7972 相似文献
6.
Protoplasts were isolated from cell cultures of oil palm (Elaeis Guineensis). The protoplasts were cultured on a nurse medium containing oil palm cells in the presence of which protoplasts formed cell walls and divided to form cell cultures.Abbreviations NAA
-naphthalene acetic acid
- 2,4-D
2,4-dichlorophenoxyacetic acid
- FDA
fluorescein diacetate
- TEM
thin-section electron microscopy 相似文献
7.
J. Zhang V. K. Tiwari T. J. Golds N. W. Blackhall E. C. Cocking B. J. Mulligan J. B. Power M. R. Davey 《Plant Cell, Tissue and Organ Culture》1995,41(2):125-138
Cat gene expression has been investigated following PEG-mediated plasmid uptake into barley protoplasts. The uptake conditions optimised for transient expression were employed for stable transformation. Transformed protoplast-derived calli of the cvs. Dissa and Igri, were selected on medium containing G418 at 40 g ml–1 or kanamycin sulphate at 250 g ml–1. Absolute transformation frequencies of 28.9×10–5 and 21.3×10–5 were recorded for Dissa with kanamycin sulphate and G418 selection, respectively. The frequency for Igri was 11.5×10–5 with G418 selection. Antibiotic resistant protoplast-derived colonies expressed NPTII activity; Southern hybridisation confirmed integration of the nptII gene into barley genomic DNA.Abbreviations ABA
abscisic acid
- AC-CAP
acetylated chloramphenicol
- BAP
6-benzylaminopurine
-
cat
chloramphenicol acetyltransferase gene
- CAT
chloramphenicol acetyltransferase activity
- CaMV
cauliflower mosaic virus
- CAP
chloramphenicol, 2,4-d-2,4-dichlorophenoxyacetic acid
- GA3
gibberellic acid
- G418
Geneticin
-
gus
-glucuronidase gene
- HEPES
(N[2-hydroxyethyl] piperazine-N-[2-ethanesulphonic acid])
- IAA
indole acetic acid
- MES
2-N-morpholinoethane sulphonic acid
- NAA
-naphthaleneacetic acid
-
npt II
neomycin phosphotransferase gene
- NPTH
neomycin phosphotransferase activity
- PEG
polyethylene glycol
- SCV
settled cell volume 相似文献
8.
A protocol for regenerating plants from callus-derived protoplasts of Pelargonium x domesticum (rega l geranium cv. Melissa) has been developed. Protoplasts were isolated from leaf-derived callus tissue on MS medium supplemented with 3.0 mg/l naphthalene acetic acid, 2.0 mg/l 6- benzylaminopurine, and 3.0% sucrose. This callus yielded 2.7×105 protoplasts/gram of tissue after a 6 hr incubation in an enzyme solution consisting of 2.0% cellulysin, 0.5% macerase, and 0.5 M sucrose. Protoplasts were plated at 1×105 protoplasts/ml in a mixture (11 v/v) of KMP8/KP liquid medium layered on the same medium solidified with 0.6% agarose. Protoplast division was initiated within 2 days, and colonies of 15 to 50 cells developed 8 wk after plating. P-calli 1–2 mm3 developed 15 wk after plating, and plants regenerated from the p-calli have been transferred to the greenhouse.Abbreviations NAA
naphthaleneacetic acid
- 6-BAP
6-benzylaminopurine
- CW
Calcofluor White
- FDA
fluorescein diacetate 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.
John C. Thomas Mark J. Guiltinan Silvia Bustos Terry Thomas Craig Nessler 《Plant cell reports》1989,8(6):354-357
Summary
Daucus carota hypocotyl sections were transformed withAgrobacterium tumefaciens LBA4404 containing CaMV 35S promoter, -glucuronidase coding sequence and the nopaline synthase (Nos) poly adenylation sequences in Bin 19. Sliced sterile seedling hypocotyl segments were preincubated for 2 days, co-cultivated withAgrobacterium for an additional 2 days, and then transferred to medium containing 100ug/ml of kanamycin and 400ug/ml carbenicillin. In 6 weeks kanamycin resistant calli were obtained in 5.8% of the explants from one variety. Calli were subcultured on solid medium, and in 4 weeks introduced into suspension culture. NPTII and Southern blot analysis confirmed that three selected lines were transformed with 1–3 copies of the GUSII construction. GUS activity in transformants was 5 to 250 fold over background.Abbreviations NPT II
neomycin phosphotransferase II
- Nos
nopaline synthase
- GUS
-glucuronidase
- CaMV
cauliflower mosaic virus
- 2,4-D
2,4 dichlorophenoxyacetic acid
- PMSF
phenylmethylsulfonyl fluoride 相似文献
11.
Tobacco plants were transformed with derivatives of a binary vector pMON505 and two kanamycin resistant lines that were nopaline positive were selected for second transformation. The plasmids used for the second transformation were derivatives of pMON850 which carries the nopaline synthase gene in addition to a gene for gentamicin resistance. Insertion of each transgene was confirmed by Southern hybridization. Surprisingly, we found that more than 50% of the doubly transformed tobacco plants were nopaline negative. Tobacco plants that were transformed only by the second vector exhibited nopaline accumulation. DNA methylation patterns at the HpaII site in the promoter region of the nopaline synthase gene did not correlate with the nopaline phenotype. In some plant lines, seedlings of the R1 generation which segregated out the second T-DNA insertion recovered the nop+ phenotype. These results indicate that nopaline accumulation was inhibited by the presence of the second T-DNA.Abbreviations T-DNA
transferred DNA
- NPTII
neomycin phosphotransferase II
-
uidA
-glucuronidase
- Km
kanamycin
- Gm
gentamicin
- nop+
nopaline positive
- nop–
nopaline negative
- MS
medium, Murashige-Skoog medium 相似文献
12.
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
13.
Large yields (1.85 × 107/g.f.wt.) of viable protoplasts were obtained from leaves of axenic shoot cultures of Malus Xdomestica Borkh. cv. Greensleeves. Protoplasts cultured in liquid or agarose semi-solidified KM8P medium underwent cell wall regeneration and colony formation.Protoplast-derived cell colonies developed to callus on semi-solid KM8 medium. This is the first report of callus formation from mesophyll protoplasts of apple.Abbreviations BAP
6-benzylaminopurine
- K
kinetin
- Z
zeatin
- GA3
gibberellic acid
- IBA
3-indole butyric acid
- NAA
1-naphthalene acetic acid
- IAA
3-indole acetic acid
- ABA
abscisic acid
- f.wt.
fresh weight
- MS
Murashige and Skoog (1962) 相似文献
14.
The cucumber mosaic virus coat protein (CMV-CP) gene and a modified Bacillus thuringiensis -endotoxin (Bt toxin) gene were cloned into plant expression vector pE3. Tobacco (Nicotiana tabacum cv. G28) leaf discs were transformed with Agrobacterium tumefaciens A12 carrying recombinant pE14. Transgenic r0 and R1 tobacco plants expressing CMV-CP and Bt toxin genes were protected from CMV infection as well as feeding damage of Manduca Sexta (tobacco hornworm) larvae. These results demonstrate that it is feasible to breed new cultivars with multiple resistances via genetic engineering.Abbreviations CMV
cucumber mosaic virus
- Bt toxin
Bacillus thuringiensis -endotoxin
- CaMV
cauliflower mosaic virus
- NOS
nopaline synthase
- Kan
Kanamycin
- Spe
spectinomycin
- Carb
Carbenicillin 相似文献
15.
Dominique Hébert Julie R. Kikkert Franzine D. Smith Bruce I. Reisch 《Plant cell reports》1993,12(10):585-589
Embryogenic suspensions of Chancellor (Vitis L. complex interspecific hybrid) were bombarded with tungsten particles coated with plasmid pBI426 encoding ß-glucuronidase (GUS) and neomycin phosphotransferase (NPTII) which results in kanamycin resistance. Two d after bombardment, cultures were placed on semi-solid medium containing either 8.6 or 17.2 M kanamycin. Factors that affect biolistic transformation rates were studied. Tungsten microprojectiles with a mean diameter of 1.07 m (M10) resulted in more transient gene expression than 0.771 m diameter particles. Using M10 particles, helium pressures of 1000 and 1200 psi yielded more GUS-expressing colonies per plate than did 800 psi 2 d following bombardment. The number of transformants present after 34 d was not affected by the helium pressure. The distance between the particle launch site and the target cells, and the number of days between the last cell subculture and bombardment, did not affect the numbers of transient and long term GUS expressing colonies. The addition of 3 g/l of activated charcoal to the post-bombardment medium increased long term GUS expression four fold. Wrapping the plates after bombardment with Parafilm increased long term GUS expression three fold compared with plates wrapped with a porous venting tape. With up to 850 transformed callus colonies per plate 23 d after bombardment, the biolistic device holds much promise as a method to achieve stable transformation of grapevines.Abbreviations AC
activated charcoal
- GUS
ß-glucuronidase
- 2,4-D
2,4-dichlorophenoxyacetic acid
- BA
6-benzylaminopurine
- IAA-L-alanine
indole-3 acetic acid L-alanine
- MS
Murashige and Skoog
- CH
casein hydrolysate
- Km
kanamycin
- NPTII
neomycin phosphotransferase II 相似文献
16.
Genetic transformation of flax (Linum usitatissimum) by Agrobacterium tumefaciens: regeneration of transformed shoots via a callus phase 总被引:1,自引:0,他引:1
Genetic transformation of flax (Linum usitatissimum) has been achieved using an A. tumefaciens strain carrying a non-oncogenic Ti plasmid-derived vector containing a chimaeric npt-II gene and a wild type nopaline synthase gene. Fertile, transformed shoots were most easily obtained from Kmr callus developing on hypocotyl sections. The totipotency of the Kmr callus was dependent upon its origin. T-DNA was visualised by Southern blotting in all Kmr tissues. Efficient expression of nopaline synthase and the chimaeric npt-II gene was found in transformed Kmr callus and regenerated shoots.Abbreviations
npt-II
neomycin phosphotransferase II gene
- NPT-II
neomycin phosphotransferase II
- nos
nopaline synthase gene promoter
- Kmr
kanamycin resistant
- BAP
6-benzylaminopurine
- NAA
-naphthaleneacetic acid
- MSD4×2
medium D4×2 based on Murashige & Skoog medium (see Scott & Draper, 1987) 相似文献
17.
Mohamed Kouider Randal Hauptmann Jack M. Widholm Robert M. Skirvin Safi S. Korban 《Plant cell reports》1984,3(4):142-145
Protoplasts could be successfully isolated and cultured from callus and suspension cultures of Malus xdomestica cv. Jonathan. Protoplast-derived colonies were recovered when the osmoticum (glucose) was gradually reduced in semi-solid 8p medium or by the use of feeder plates. Formation of embryo-like structures was induced from the protoplast-derived callus on media supplemented with IAA and BA. These structures formed roots but plants failed to develop. Protoplasts could be isolated from leaves, but not from stems or petioles. The leaf protoplasts failed to divide.List of abbreviations BA
benzyladenine
- 2,4-D
2,4-dichlorophenoxyacetic acid
- ABA
abscisic acid
- IAA
indole acetic acid 相似文献
18.
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
19.
Propagation by axillary and multiple axillary bud development was achieved in three native Leptospermum spp. when axillary buds derived from nodal tissues ex mature plants were placed in benzylaminopurine media (0.04–1.0 M) containing macro- and micro-nutrients, sucrose (0.06 M) and a vitamin/amino acid supplement. Reduction of agar concentration from 0.8 to 0.2% greatly stimulated axillary bud development and growth in L. flavescens and L. brachyandrum. Rooting of axillary shoots was stimulated by 2,4-dichlorophenoxyacetic acid and p-chlorophenoxy acetic acid in L. flavescens at concentrations of 5 and 1 M respectively. In L. petersonii ssp. root initiation and development was favoured by -naphthoxyacetic acid (1 M) and in L. brachyandrum indole butyric acid and -naphthalene acetic acid (1 M) were almost equally effective. 相似文献
20.
An De Bondt Kristel Eggermont Iris Penninckx Inge Goderis Willem F. Broekaert 《Plant cell reports》1996,15(7):549-554
We have previously developed a protocol for efficient gene transfer and regeneration of transgenic calli following cocultivation of apple (cv. Jonagold) explants with Agrobacterium tumefaciens (De Bondt et al. 1994, Plant Cell Reports 13: 587–593). Now we report on the optimization of postcultivation conditions for efficient and reproducible regeneration of transgenic shoots from the apple cultivar Jonagold. Factors which were found to be essential for efficient shoot regeneration were the use of gelrite as a gelling agent and the use of the cytokinin-mimicing thidiazuron in the selective postcultivation medium. Improved transformation efficiencies were obtained by combining the hormones thidiazuron and zeatin and by using leaf explants from in vitro grown shoots not older than 4 weeks after multiplication. Attempts to use phosphinothricin acetyl transferase as a selectable marker were not successful. Using selection on kanamycin under optimal postcultivation conditions, about 2% of the leaf explants developed transgenic shoots or shoot clusters. The presence and expression of the transferred genes was verified by -glucuronidase assays and Southern analysis. The transformation procedure has also been succesfully applied to several other apple cultivars.Abbreviations BAP
benzylaminopurine
- CTAB
hexadecyltrimethylammoniumbromide
- Na2EDTA
ethylenediamine-tetra-acetate ferric-sodium salt
- FeNaEDTA
ethylenediamine-tetra-acetate ferric-sodium salt
- GA3
gibberellic acid 3
- GusA
-glucuronidase
-
gusA
-glucuronidase gene of Escherichia coli
- IAA
indole acetic acid
- IBA
indole butyric acid
- 2iP
N6-2-isopentenyl adenine
- NAA
naphthalene acetic acid
-
nptII
neomycinphosphotransferase II gene
-
bar
phosphinothricin acetyl transferase gene
- PCR
polymerase chain reaction
- PPT
phosphinothricin
- STS
silver thiosulphate
- T-DNA
transferred DNA
- TDZ
thidiazuron
- X-Gluc
5-bromo-4-chloro-3-indolyl -D-glucuronide
- Zea
trans-Zeatin 相似文献