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1.
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
2.
Monique F. van Wordragen Jan de Jong Hans B. M. Huitema Hans J. M. Dons 《Plant cell reports》1991,9(9):505-508
Summary To develop an Agrobacterium mediated transformation protocol for chrysanthemum we studied the transformation efficiency of commonly used A.tumefaciens strains on 14 genotypes by comparing tumour size and frequency. One genotype was analyzed in detail using 14 strains of both A.tumefaciens and A.rhizogenes. Only a few genotype/strain combinations resulted in significant tumour formation. Especially 0-type strains were highly efficient. An 0-type strain was used to transfer genes for neomycine phosphotransferase (NPT II) and ß-glucuronidase (GUS) to a susceptible cultivar. Transfer of the GUS gene was confirmed by using the Polymerase Chain Reaction (PCR). 相似文献
3.
An efficient system for Agrobacterium tumefaciens-mediated transformation of Solanum gilo was established. The marker genes for kanamycin resistance and ß-glucuronidase expression were introduced. A comparison between cotyledon and hypocotyl explants showed that while regeneration was better from hypocotyl explants, cotyledon explants gave better transformation efficiency (46% vs. 32%). Four levels of kanamycin selection (100, 150, 200 and 250 mg/l) were tested for effect on transformation efficiency with each type of explant. Lower levels of kanamycin worked better using cotyledon explants, while higher levels of kanamycin worked better for hypocotyl explants. All nine t0 plants tested for expression of the kan
r
gene were positive. The progeny of three of these plants showed a pattern of classical Mendelian inheritance (3 to 1) for both the kan
r
and the ß-glucuronidase genes.Abbreviations MS
Murashige and Skoog (1962) medium
- 2,4-D
2,4-Dichlorophenoxyacetic acid
- NPTII
neomycin phosphotransferase
- GUS
ß-glucuronidase 相似文献
4.
Antonella Furini Csaba Koncz Francesco Salamini Dorothea Bartels 《Plant cell reports》1994,14(2-3):102-106
Summary An efficient procedure for Agrobacterium tumefaciens- mediated transformation of the desiccation-tolerant plant Craterostigma plantagineum has been developed. Leaf explants were inoculated with A. tumefaciens strain GV3101 carrying the gene for kanamycin- or hygromycin-resistance and the ßglucuronidase reporter gene. Parameters which affected the transformation efficiency were the age of the explant, the degree of wounding and the presence of an antioxidant in the medium. Under optimal conditions, calli originated in more than 80% of leaf explants. Transformed plants were obtained from more than 50% of the cultured calli during regeneration in the presence of a suitable antibiotic. The stable integration of T-DNA was confirmed by Southern blot analysis and its expression by assays for ß-glucuronidase activity.Abbreviations GUS
ß-glucuronidase
- MUG
4-methyl-umbelliferyl ß-D-glucuronide
- ABA
abscisic acid
- NPTII
neomycin phosphotransferase II
- CaMV
cauliflower mosaic virus
- MSAR
modified MS medium
- MS
Murashige and Skoog 相似文献
5.
Agrobacterium-mediated transformation of sweet orange and regeneration of transgenic plants 总被引:1,自引:6,他引:1
Leandro Peña Magdalena Cervera José Juárez Antonio Navarro José A. Pina Nuria Durán-Vila Luis Navarro 《Plant cell reports》1995,14(10):616-619
Summary Transgenic sweet orange (Citrus sinensis L. Osbeck) plants have been obtained by Agrobacterium tumefaciens-mediated gene transfer. An hypervirulent A. tumefaciens strain harboring a binary vector that contains the chimeric neomycin phosphotransferase II (NPT II) and ß-glucuronidase (GUS) genes was cocultivated with stem segments from in vivo grown seedlings. Shoots regenerated under kanamycin selection were harvested from the stem segments within 12 weeks. Shoot basal portions were assayed for GUS activity and the remaining portions were shoot tip grafted in vitro for production of plants. Integration of the GUS gene was confirmed by Southern analysis. This transformation procedure showed the highest transgenic plant production efficiency reported for Citrus.Abbreviations BA
benzyladenine
- CaMV
cauliflowermosaic virus
- GUS
ß-glucuronidase
- LB
Luria Broth
- MS
Murashige and Skoog
- NAA
naphthalenacetic acid
- NOS
nopaline synthase
- NPT II
neomycin phosphotransferase II
- PEG
polyethylene glycol
- RM
rooting medium
- SRM
shoot regeneration medium 相似文献
6.
Kazuhito Akama Hideaki Shiraishi Shozo Ohta Kenzo Nakamura Kiyotaka Okada Yoshiro Shimura 《Plant cell reports》1992,12(1):7-11
Summary The efficiency of Agrobacterium-mediated transformation of Arabidopsis thaliana was compared with different organs, Arabidopsis ecotypes, and Agrobacterium strains. Efficiency of shoot regeneration was examined using hypocotyl, cotyledon and root explants prepared from young seedlings. Hypocotyl expiants had the highest regeneration efficiency in all of the four Arabidopsis ecotypes tested, when based on a tissue culture system of callus-inducing medium (CIM: Valvekens et al. 1988) and shoot-inducing medium (SIM: Feldmann and Marks 1986). Histochemical analysis using the ß-glucuronidase (GUS) reporter gene showed that the gusA gene expression increased as the period of preincubation on CIM was extended, suggesting that dividing cells are susceptible to Agrobacterium infection. In order to obtain transgenic shoots, hypocotyl explants preincubated for 7 or 8 days on CIM were infected with Agrobacterium containing a binary vector which carries two drug-resistant genes as selection markers, and transferred to SIM for selection of transformed shoots. Of four Arabidopsis ecotypes and of three Agrobacterium strains examined, Wassilewskija ecotype and EHA101 strain showed the highest efficiency of regeneration of transformed shoots. By combining the most efficient factors of preincubation period, Arabidopsis ecotype, tissue, and bacterial strain, we obtained a transformation efficiency of about 80–90%. Southern analysis of 124 transgenic plants showed that 44% had one copy of inserted T-DNA while the others had more than one copy.Abbreviations AIM
Agrobacterium infection medium
- CIM
callus-inducing medium
- CTAB
cetyltrimethylammonium bromide
- 2,4-D
2,4-dichlorophenoxy-acetic acid
- GUS
ß-glucuronidase
- hph
hygromycin phosphotransferase
- IAA
indole-3-acetic acid
- IBA
indole-3-butyric acid
- 2ip
N -(2-isopentenyl) adenine
- NPTII
neomycin phosphotransferase II
- RIM
root-inducing medium
- 35S
cauliflower mosaic virus 35S promoter
- SIM
shoot-inducing medium 相似文献
7.
Factors affecting Agrobacterium tumefaciens-mediated transformation in several black poplar clones 总被引:4,自引:0,他引:4
M. Confalonieri A. Balestrazzi S. Bisoffi R. Cella 《Plant Cell, Tissue and Organ Culture》1995,43(3):215-222
Transient expression of the uidA reporter gene was used in preliminary experiments with two oncogenic and two disarmed Agrobacterium tumefaciens strains in order to test the efficiency of T-DNA transfer to N084 x Populus nigra and N107 x P. nigra clones. The oncogenic strain A281 pKIWI105 produced the highest average number of GUS spots per leaf disc. In order to optimize the production of transgenic plantlets from different P. nigra clones (San Giorgio, Jean Pourtet, N084 x P. nigra and N107 x P. nigra, respectively), two A. tumefaciens strains (GV2260 p35S GUS, A281pKIWI105) and bacterial concentrations (7×108; 1.2×09 bacteria ml-1) were used. Following co-cultivation with A281 pKIWI105, the frequency of leaf discs producing kanamycin-resistant calli was not significantly different between the clones and bacteria concentrations used. Transformed shoots were regenerated from all clones, except for Jean Pourtet. Co-cultivation of leaf discs with GV2260 p35S GUS produced very few calli which died when transferred to selective regeneration medium. In addition, the effects of acetosyringone and leaf wounding were evaluated for the San Giorgio and Jean Pourtet clones, using the same strains. Factors which significantly affected the transformation efficiency of leaf explants were the P. nigra clone, the A. tumefaciens strain, and the presence of acetosyringone. Genetic transformation of calli and regenerated plantlets was confirmed by their ability to grow and root on Woody Plant Medium containing kanamycin, by histochemical -glucuronidase assays, and Southern blot hybridization analyses.Abbreviations BA
benzyladenine
- GUS
-glucuronidase
- IBA
indolebutyric acid
- MS
Murashige and Skoog
- NAA
-naphthaleneacetic acid
-
nptII
neomycin phosphotransferase II gene
-
uidA
-glucuronidase gene
- WPM
Woody Plant Medium 相似文献
8.
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
9.
Leaf pieces of in vitro-cultured plantlets of the wild potato species Solanum brevidens Phil. were cocultivated with Agrobacterium tumefaciens that contained nptII and uidA genes on the disarmed plasmid pBI121. Independent transgenic shoots were regenerated from solidified and liquid medium that contained 50 mg l–1 kanamycin. Two Agrobacterium strains were investigated for transformation efficiency. GV2260, which contained p35SGUSINT, resulted in a 11% transformation frequency, compared with 1% using LBA4404. Transformation rates were 7% in liquid culture and 3% on solidified medium. All kanamycinresistant, putatively transformed plantlets were confirmed positive by histochemical GUS assays. GUS activity in 22 independently transformed plants was quantified by fluorometric assay. Southern analysis of randomly selected transgenic plants showed that each transgenic plant contained at least one copy of the uidA gene.Abbreviations GUS
ß-glucuronidase
- MS
Murashige-Skoog medium
- BA
6-benzylaminopurine
- 2ip
6-(, -dimethylallylamino)purine
- IAA
indole-3-acetic acid
- GA3
gibberellic acid
-
npt II
neomycin phosphotransferase II
- NOS
nopaline synthase
- MUG
4-methyl umbelliferyl glucuronide
- MU
7-hydroxy-4-methylcoumarin
- X-gluc
5-bromo-4-chloro-3-indolyl ß-D-glucuronic acid 相似文献
10.
Summary A high frequency shoot regeneration (80%) was developed from callus of leaf discs and stem internodes of Moricandia arvensis. Leaf discs were shown to be a preferable starting material for transformation experiments. Agrobacterium tumefaciens strain GV3101/pMP90 used in this study contained a binary vector with genes for kanamycin resistance, hygromycin resistance and -glucuronidase (GUS). Maximum transformation efficiency (10.3%) was achieved by using kanamycin at the rate of 200 mg/l as a selection agent. Presence of tobacco suspension culture during co-cultivation and a pre-selection period of seven days after co-cultivation was essential for successful transformation. Transgenic plants grew to maturity and exhibited flowering in a glasshouse. GUS activity was evident in all parts of leaf and the presence of GUS gene in plant gemone was confirmed by PCR analysis.Abbreviations GUS
-glucuronidase 相似文献
11.
Summary
Agrobacterium-mediated gene transformation of Populus tremuloides Michx was accomplished by co-cultivation of leaf disks excised from greenhouse plants with Agrobacterium tumefaciens containing a binary Ti-plasmid vector harboring chimeric neomycin phosphotransferase (NPT II) and ß-glucuronidase (GUS) genes. Shoot regeneration in the presence of kanamycin was achieved when thidiazuron (TDZ) was used as a plant growth regulator. Transformation was verified by amplification of NPT II and GUS gene fragments from genomic DNA of transgenic plants with polymerase chain reaction (PCR) and integration of these genes into nuclear genome of transgenic plants was confirmed by genomic Southern hybridization analysis. Histochemical assay revealed the expression of GUS gene in leaf, stem and root tissues of transgenic plants, further confirming the integration and expression of T-DNA in these plants. This protocol allows effective transformation and regeneration of quaking aspen using greenhouse-grown materials as an explant source. Whole plant regeneration from cuttings of fieldgrown mature quaking aspen and hybrid poplar (P. alba x P. grandidentata) was also readily achieved by using this protocol, which represents a potential system for producing transgenic quaking aspen and hybrid poplar of valuable genotypes.Abbreviations AMV RNA4
Alfalfa mosaic virus RNA4
- BA
6-benzyladenine
- CaMV
cauliflower mosaic virus
- 2,4-D
2,4-dichlorophenoxyacetic acid
- EDTA
ethylenediaminetetraacetic acid
- FAA
formalin-acetic acid-alcohol
- GUS
ß-glucuronidase
- NAA
1-naphthylacetic acid
- NPT II
neomycin phosphotransferase II
- PCR
polymerase chain reaction
- SDS
sodium dodecyl sulphate
- TE
Tris-Cl/EDTA
- TDZ
N-phenyl-N-1,2,3-thiadiazol-5-yl-urea (thidiazuron)
- WPM
woody plant medium (Lloyd and McCown 1980)
- X-GLUC
5-bromo-4-chloro-3-indolyl-ß-glucuronic acid 相似文献
12.
Effects of tissue type and promoter strength on transient GUS expression in sugarcane following particle bombardment 总被引:1,自引:0,他引:1
Effects of tissue type and promoter strength on transient GUS expression in the sugarcane (Saccharum spp. hybrids) cultivar NCo 310 were evaluated following microprojectile bombardment of leaf explants. GUS expression was histochemically or fluorometrically measured 48 h after delivery of the uidA gene. High levels of GUS expression were obtained in leaf segments isolated from young, expanding sugarcane leaves cultured for 1, 3, or 6 d prior to bombardment. The promoter derived from the maize ubiquitin 1 gene (Ubi-1) produced significantly more GUS foci and higher GUS activity levels compared to the recombinant Emu, rice actin 1 (Act1), and CaMV 35S promoters. Our transient expression system should facilitate efforts to identify promoters and elements which will regulate desired gene expression patterns in sugarcane and aid in development of an efficient stable transformation system.Abbreviations
Act1
rice actin 1 gene
- CaMV
cauliflower mosaic virus
- GUS
ß-glucuronidase
- Ubi-1
maize ubiquitin 1 gene
-
uidA
GUS gene
- X-Glu
5-bromo-4-chloro-3-indoylglucuronide 相似文献
13.
Summary A reproducible system has been developed for the production of transgenic plants in indica rice using Agrobacterium-mediated gene transfer. Three-week-old scutella calli served as an excellent starting material. These were infected with an Agrobacterium tumefaciens strain EHA101 carrying a plasmid pIG121Hm containing genes for -glucuronidase (GUS) and hygromycin resistnace (HygR). Hygromycin (50 mg/l) was used as a selectable agent. Inclusion of acetosyringone (50M) in the Agrobacterium suspension and co-culture media proved to be indispensable for successful transformation. Transformation efficiency of Basmati 370 was 22% which was as high as reported in japonica rice and dicots. A large number of morphologically normal, fertile transgenic plants were obtained. Integration of foreign genes into the genome of transgenic plants was confirmed by Southern blot analysis. GUS and HygR genes were inherited and expressed in R1 progeny. Mendelian segregation was observed in some R1 progeny.Abbreviations GUS
ß-glucuronidase
- HygR
hygromycin-resistance
- AS
acetosyringone 相似文献
14.
Electroporation was used to evaluate parameters affecting transient gene expression in Glycine max protoplasts. Protoplast viability and reporter enzyme activity for chloramphenicol acetyl transferase (CAT) and ß-glucuronidase (GUS) depended on the field strength employed. Maximum CAT and GUS activity was obtained when a field strength of 500 V/cm at 1000 F and a protoplast concentration of 1–3 × 106/ml was used. Transformation efficiencies up to approximately 1.6% GUS positive protoplasts were obtained. Transient gene expression increased with increasing plasmid DNA concentration and with the time after electroporation, reaching a maximum after 48 hr. Addition of polyethylene glycol at 5.6% and heat shock (5 rain at 45 °C) given to the protoplasts before adding DNA further enhanced the transformation efficiency. Under the optimized experimental conditions, CAT and GUS activity increased simultaneously, thereby indicating that the increased expression is caused by DNA uptake by more cells rather than greater DNA uptake by the same cells. Our results demonstrate that both GUS and CAT can be used as efficient screenable markers for transformation studies in soybean.Abbreviations CAT
chloramphenicol acetyl transferase
- GUS
ß-glucuronidase
- PEG
polyethylene glycol 相似文献
15.
A reliable method of plant regeneration has been achieved from decapitated mature embryo axes (DCMEA) explants. Shoots appear
directly from explants of genotype T-15-15 when cultured on Maheswaran and Williams (EC6) basal medium supplemented with N6-benzylaminopurine (BAP) and indole-3-acetic acid (IAA) at various combinations. The shoots elongated on half strength Murashige
and Skoog (MS) medium fortified with 3 μM gibberellic acid. Elongated shoots were rooted with 80 – 85 % efficiency on half
strength MS medium with 0.5 μM indole-3-butyric acid. Survival of plants in the pots was 75 – 80 %. This protocol was used
in Agrobacterium mediated transformation. The DCMEA explants were treated independently with two A. tumefaciens (LBA 4404) strains harbouring a binary vector carrying the green fluorescent protein (GFP) and β-glucuronidase (GUS) reporter
genes, respectively. Both the strains contained neomycin phosphotransferase selectable marker gene. After co-cultivation,
the explants were cultured on EC6 basal medium supplemented with 5 μM BAP and 1 μM IAA. The selection of putative transformants was on a medium containing
50 mg dm−3 kanamycin. Expression of GUS and GFP gene was confirmed by histochemical assay and fluorescence microscopy, respectively.
The elongated shoots expressing GFP reporter gene were rooted and transferred to pots for hardening. The integration of GFP
gene into the genome of putative transformants was confirmed by Southern blotting.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
16.
Wenhao Dai Zong-Ming Cheng Wayne Sargent 《In vitro cellular & developmental biology. Plant》2003,39(1):6-11
Summary An efficient regeneration and transformation system was developed for two elite aspen hybrid clones (Populus canescens × P. grandidentada and P. tremuloides × P. davidiana). Callus was induced from in vitro leaf explants on modified Murashige and Skoog medium (MSA) and woody plant medium (WPM) containing four different combinations
of cytokinins and auxins. Callus tissues regenerated into shoots on WPM medium supplemented with 2.0 mgl−1 (9.12 μM) zeatin or 0.01 mgl−1 (0.045 μM) thidiazuron. P. canescens × P. grandidentata exhibited the higher callus and shoot production. In vitro leaf explants from the two hybrid clones were cocultivated with Agrobacterium tumefaciens strain EHA105 harboring the binary Ti plasmid pBI121 carrying the uidA gene encoding for β-glucuronidase (GUS) and the npt II gene encoding for neomycin phosphotransferase II. Transformation was confirmed by GUS assays, polymerase chain reaction,
and Southern blot analyses. Agrobacterium concentration, acetosyringone, and pH of the cocultivation medium were evaluated for enhancing transformation efficiency
with the clone P. canescens × P. grandidentata. 相似文献
17.
Epicotyl segments and nodus expiants from etiolated seedlings of Pisum sativum were transformed using Agrobacterium tumefaciens strains GV 2260 (p35S GUS INT) and GV 3850 HPT carrying either a neomycin- or hygromycinphosphotransferase-gene as selectable markers. The transgenic character of hygromycin- or kananamycin-resistant tissue was confirmed by detection of nopaline or neomycinphosphotransferase-II- and ß-glucuronidase activity in crude extracts of resistant tissues. Up to 5 % of developing shoots from shoot proliferating nodi were regenerated via organogenesis to kanamycin-resistant plantlets. Transformation frequency in vitro was found to be influenced by expiant source, A. tumefaciens strain, pea genotype and duration of cocultivation. Acetosyringone did not increase the transformation rate.Abbrevations GUS
ß-glucuronidase
- NAA
1-naphthyl-acetic-acid
- BA
6-benzylaminopurine
- NPT-II
neomycinphosphotransferase-II
- HPT
hygromycinphosphotransferase 相似文献
18.
An De Bondt Kristel Eggermont Philippe Druart Maayke De Vil Inge Goderis Jos Vanderleyden Willem F. Broekaert 《Plant cell reports》1994,13(10):587-593
The factors influencing transfer of an intron — containing -glucuronidase gene to apple leaf explants were studied during early steps of an Agrobacterium tumefaciens-mediated transformation procedure. The gene transfer process was evaluated by counting the number of -glucuronidase expressing leaf zones immediately after cocultivation, as well as by counting the number of -glucuronidase expressing calli developing on the explants after 6 weeks of postcultivation in the presence of 50 mg/l kanamycin. Of three different tested disarmed A. tumefaciens strains, EHA101(pEHA101) was the most effective for apple transformation. Cocultivation of leaf explants with A. tumefaciens on a medium with a high cytokinin level was more conducive to gene transfer than cocultivation on media with high auxin concentrations. Precultivation of leaf explants, prior to cocultivation, slightly increased the number of -glucuronidase expressing zones measured immediately after cocultivation, but it drastically decreased the number of transformed calli appearing on the explants 6 weeks after infection. Other factors examined were: Agrobacterium cell density during infection, bacterial growth phase, nature of the carbon source, explant age, and explant genotype.Abbreviations 2,4-D
2,4-dichlorophenoxyacetic acid
- CaMV35S
35S RNA of cauliflower mosaic virus
- EDTA
ethylenediaminetetraacetate
- FeNaEDTA
ethylenediaminetetraacetate ferric-sodium salt
- GusA
-glucuronidase
-
gusA
ß-glucuronidase gene of Escherichia coli
-
gusA-intron
ß-glucuronidase gene containing an intron in the coding region
- IBA
indole butyric acid
- 2iP
N6-2-isopentenyl adenine
- NAA
naphthaleneacetic acid
-
nptII
neomycinphosphotransferase II gene
- X-Gluc
5-bromo-4-chloro-3-indolyl ß-D-glucuronide 相似文献
19.
High frequency shoot regeneration and Agrobacterium-mediated DNA transfer in Canola (Brassica napus)
Muhammad Ramzan Khan Hamid Rashid Muhammad Ansar Zubeda Chaudry 《Plant Cell, Tissue and Organ Culture》2003,75(3):223-231
Five different varieties of Brassica napus (Cyclone, Dunkled, Oscar, Rainbow and KS75) were tested for their regeneration response. Cyclone showed a very high frequency of regeneration (92%). The use of silver nitrate was a pre-requisite for efficient shoot regeneration. Hypocotyls were selected as the starting material for transformation experiments on the basis of high transient GUS expression. Explants were co-cultivated with Agrobacterium strain EHA101 harboring a binary vector pIG121Hm containing neomycin phosphotransferase II (NPTII) gene, conferring resistance to kanamycin, hygromycin phosphotransferase (HPT) gene, conferring resistance to hygromycin as selectable markers and -glucuronidase (GUS) gene as a reporter. Acetosyringone promoted the transformation but was not an absolute requirement. A pre-selection period of 7 days after co-cultivation was essential for successful transformation. Kanamycin was efficient selective agent for selection and maximum transformation efficiency was 24%. GUS activity was evident in leaf tissues. All the transgenic plants have an expected band of 0.43 kb fragment by PCR analysis confirming the presence of foreign DNA into plant genome. 相似文献
20.
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