共查询到20条相似文献,搜索用时 156 毫秒
1.
Dominant expression of a gene amplification-related herbicide resistance in medicago cell hybrids 总被引:2,自引:0,他引:2
Protoplasts from phosphinotricin resistant M. sativa and M. varia cell lines carrying an amplified glutamine synthethase gene were fused with leaf protoplasts of kanamycin resistant M. varia transformants. The dominant nature of both PPT and kanamycin resistant traits was shown by the double resistant phenotype of the intra- and interspecific cell hybrids obtained. The presence of amplified GS gene in the hybrid genomes and the expression of chimeric neomycin phosphotransferase II gene was detected. The highly embryogenic character of the M. varia parent was not expressed after cell fusion. All hybrid cell lines with the double resistant phenotype showed non-morphogenic growth similarly to the PPT resistant parent. The possible role of GS gene amplification and other factors in the dominant behaviour of unorganized cell growth in alfalfa somatic hybrids is discussed. 相似文献
2.
Mireille Chabaud Joan E. Passiatore Frank Cannon Vicky Buchanan-Wollaston 《Plant cell reports》1988,7(7):512-516
Kanamycin resistant plants of Medicago varia A2 were obtained by an optimized procedure for high frequency transformation using Agrobacterium tumefaciens infection of leaf and petiole tissue. Parameters which affected the frequency were explant type, the Agrobacterium strain used and the time allowed for cocultivation. Under optimum conditions, i.e., using the Agrobacterium strain A281 and a 4 day cocultivation period, the frequency of transformed leaflets obtained was greater than 70%. 相似文献
3.
Plants of Medicago arborea have been infected with Agrobacterium rhizogenes strain LBA9402 harbouring the plasmids Ri 1855 and AGS125 carrying a gene conferring resistance to the antibiotic hygromycin. About 7056 of the hairy roots showed callus formation on hygromycin-supplemented medium. Regeneration took place on antibiotic free medium only. Plantlets suitable for transfer to soil were obtained after the manual removal of most of the leaves. Plant morphology showed the usual alterations induced by the Ri plasmid; moreover, two years after soil-transfer, transformants have not flowered. Molecular analysis indicates the presence of T-DNA from both pAGS 125 and p1855. The expression of the hygromycin phosphotransferase gene allowed callus and protoplasts of transformed plants to grow on media supplemented with the antibiotic. This trait will be utilized as a marker in protoplast fusion between Medicago arborea and Medicago sativa (alfalfa).Abbreviations 2,4-D
2,4-dichlorophenoxyaceticacid
- kin
kinetin
- GA3
Gibberellic acid
- IAA
Indole-3-acetic acid
- HPT
hygromycin phosphotransferase
- NOS
nopaline synthase
- MS
Murashige and Skoog (1962)
- B5
Gamborg et al. (1968)
- B5hy
B5 supplemented with 20 mg 1-1 of hygromycin
- YMB
yeast mannitol broth 相似文献
4.
Summary Kanamycin resistant plants of Solarium melongena L. (eggplant) cv. Picentia were obtained following the cocultivation of leaf explants with Agrobacterium tumefaciens. A disarmed binary vector system containing the neomycin phosphotransferase (NPTII) gene as the selectable marker and chloramphenicol acetyltransferase (CAT) as a reporter gene was utilized. In vitro grown plants were used as sources of explants to produce transgenic plants on selective medium containing 100 mg/l kanamycin. The transformation and expression of the foreign genes was confirmed by DNA hybridizations, leaf disc assays, and by measuring NPTII and CAT enzyme activities. This technique is simple, rapid, efficient, and transgenic eggplants of this commercial cultivar have been transferred to soil where they have flowered and set seed.Abbreviations CAT
chloramphenicol acetyltransferase
- MS
Murashige and Skoog
- NPTII
neomycin phosphotransferase
- NOS
nopaline synthase
- ZEA
zeatin 相似文献
5.
Transgenic tobacco (Nicotiana tabacum L.) plants, carrying the neomycin phosphotransferase (NPT II) gene from E. coli, are resistant to kanamycin when grown from seeds on kanamycin containing medium. Tissue and cell cultures derived from those transformants also express resistance and regenerate complete plantlets in the presence of the antibiotic. This unspecific response to the selective condition has led to the belief that the foreign gene is continuously active or uniformly inducible in all cells of the transgenic plant. However, our experiments show that this view is not true for pollen grains during in vitro germination. Pollen grains isolated from kanamycin resistant tobacco plants carry and transmit the foreign gene but do not express resistance when germinating in vitro. This data presents evidence for differential silencing of a foreign gene in a mature gamete. On the other hand, immature pollen grains (microspores) appear to express resistance. The point of the downregulation of the neomycin transferase gene during pollen maturation is discussed.Abbreviations kan
kanamycin sulfate
- NPT II
neomycin phosphotransferase II
- sr
streptomycin sulfate 相似文献
6.
A chimaeric neomycin phosphotransferase II (NPT II) gene was introduced in Brassica oleracea using an oncogenic strain of Agrobacterium tumefaciens harbouring Ti plasmid which contains Nos/NPTII in its T-DNA. The transformation of B. oleracea with the oncogenic Ti plasmid, resulted in regeneration of shoots and roots without any exogenous requirement of phytohormones. The presence of NPT II gene was determined by hybridization of Tn5 encoded NPT II gene with DNA of kanamycin resistant regenerated plants. The expression of NPT II was demonstrated by kanamycin phosphorylation assay. Several regenerated plants were obtained, a few of them were found to be morphological variants and a chlorophyll deficient mutant plant was also obtained. 相似文献
7.
Herbicide resistant transgenic papaya plants produced by an efficient particle bombardment transformation method 总被引:7,自引:0,他引:7
José L. Cabrera-Ponce Ariadne Vegas-Garcia Luis Herrera-Estrella 《Plant cell reports》1995,15(1-2):1-7
A system for the production of transgenic papaya (Carica papaya L.) plants using zygotic embryos and embryogenic callus as target cells for particle bombardment is described. Phosphinothricin (bar ) and kanamycin (npt II) resistance genes were used as selectable markers, and the gus gene (uidA) as a reporter gene. Selection with 100 mg/l kanamycin and 4 mg/l phosphinothricin (PPT) yielded a total of over 90 resistant embryogenic colonies from three independent experiments using embryogenic callus as a target tissue. This represents an efficiency of 60 transgenic clones per gram of fresh weight callus bombarded. The efficiency of genetic transformation using zygotic embryos was lower, as only 8 independent resistant clones were recovered out of 645 bombarded zygotic embryos, giving a efficiency of 1.24%. Subsequent subculture of transgenic somatic embryos both from zygotic embryos and embryogenic callus led to the development of plants with apparently normal morphology. Histological, fluorimetric assay for GUS, NPT II assay and DNA analysis (Southern hybridization) showed that kanamycin /PPT resistant plants carried and expressed the transgenes.Abbreviations Gus
-glucuronidase
- NPTII
neomycin phophotransferase II
-
bar
phophinothricin acetyl transferase gene
- Pat
phosphinothricin acetyl transferase
- PPT
phosphinothricin
- Km
kanamycin
- 2,4-D
2,4-dichlorophenoxyacetic acid
- K
kinetin
- BAP
benzylaminopurine
- IBA
indolbutyric acid 相似文献
8.
Improved efficiency of the walnut somatic embryo gene transfer system 总被引:11,自引:0,他引:11
Gale H. McGranahan Charles A. Leslie Sandra L. Uratsu Abhaya M. Dandekar 《Plant cell reports》1990,8(9):512-516
Summary AnAgrobacterium-mediated gene transfer system which relies on repetitive embryogenesis to regenerate transgenic walnut plants has been made more efficient by using a more virulent strain ofAgrobacterium and vectors containing genes for both kanamycin resistance and beta-glucuronidase (GUS) activity to facilitate early screening and selection. Two plasmids (pCGN7001 and pCGN7314) introduced individually into the disarmedAgrobacterium host strain EHA101 were used as inoculum. Embryos maintained on medium containing 100 mg/l kanamycin after co-cultivation produced more transformed secondary embryos than embryos maintained on kanamycin-free medium. Of the 186 GUS-positive secondary embryo lines identified, 70% were regenerated from 3 out of 16 primary embryos inoculated with EHA101/pCGN7314 and grown on kanamycin- containing medium, 28% from 4 out of 17 primary embryos inoculated with EHA101/ pCGN7001 and grown on kanamycin medium, and 2% from one out of 13 primary embryos inoculated with EHA101/pCGN7001 but not exposed to kanamycin. Because kanamycin inhibits but does not completely block new embryo formation in controls, identification of transformants formerly required repetitive selection on kanamycin for several months. Introduction of the GUS marker gene allowed positive identification of transformant secondary embryos as early as 5–6 weeks after inoculation. DNA analysis of a representative subset of lines (n=13) derived from secondary embryos confirmed transformation and provided evidence for multiple insertion events in single inoculated primary embryos. 相似文献
9.
Summary AnEscherichia
coli/Corynebacterium
glutamicum chimeric plasmid has been constructed containing the tetracycline resistance (TcR) determinant from pAM1 and the kanamycin resistant (KmR) determinant from pBD10. The paAM1 tetracycline resistance determinant is expressed inC.
glutamicum although the transformed population segregates readily into tetracycline and kanamycin resistant populations. The segregation event is the result of an intramolecular recombination between the pAM1 and pUB110 regions of the chimera. 相似文献
10.
Transgenic celery plants were obtained following co-cultivation of petiole explants with Agrobacterlum tumefaciens containing pMON200, a cointegrate vector carrying genes for kanamycin resistance and nopaline synthase. Transformants were selected by ability of callus to grow in the presence of 50mg/l kanamycin. Transformation was confirmed either by the presence of nopaline or by Southern blots. Cytological analysis of 14 transformed plants revealed chromosomal aberrations, both in structure and number. Only 20% of the regenerated plants had the normal karyotype. Kanamycin resistance behaved as a monogenic, dominant trait, segregating in a 3:1 ratio in three families derived from plants with normal karyotypes.Abbreviations KB
Kilobases
- 2-4D
2,4-diphenoxyacetic acid 相似文献
11.
Nineteen accessions of diploid Medicago sativa L. belonging to the four subspecies sativa, caerula, falcata and xvaria were screened for their ability to produce somatic embryos on hypocotyl-derived callus. Two medium protocols were used in this study, a three-step sequence with exposure of the callus cultures to a high 2,4-D concentration and a two-step sequence without exposure to a high 2,4-D concentration. Considerable variation for callus proliferation was observed. In general, the diploid M. sativa accessions showed poor regenerability and it was not possible to correlate high regeneration frequencies with a particular germplasm source. It was, however, possible to identify regenerable genotypes in all four subspecies. One falcata accession produced somatic embryos on the callus induction media at high frequencies. This response was also obtained with a few genotypes from one xvaria accession. All regenerable plants were maintained as shoot cultures and were able to form somatic embryos on petiole-derived calli.Abbreviations BA
6-benzyladenine
- 2,4-D
2,4-dichlorophenoxyacetic acid
- IAA
indole-3-acetic acid
- 2iP
iso-pentyladenine
- NAA
-naphthaleneacetic acid
Contribution No. 772 Ottawa Research Station 相似文献
12.
Hypocotyl protoplasts of German winter oilseed, rape (Brassica napus) lines of double-low quality were transformed using Agrobacterium tumefaciens harbouring pGV 38501103 neo (dimer) containing chimaeric kanamycin resistance reporter genes. Transformed protoplasts were regenerated to fertile and phenotypically normal plants. Transformation was confirmed by kanamycin resistance, nopaline production, neomycinphosphotransferase II activity, and Southern blot hybridization. Seed progeny from self-pollinated transformants expressed the introduced kanamycin resistance as a Mendelian trait.Abbreviations BAP
6-benzylaminopurine
- Cf
ClaforanR
- 2.4D
2,4-dichlorophenoxy acetic acid
- Km
kanamycin
- MS
Murashige and Skoog (1962)
- NAA
-naphthalene acetic acid
- NPT II
neomycinphosphotransferase
- npt II
neomycinphosphotransferase II gene
- NOS
nopaline synthase
- nos
nopaline synthase gene
- ocs
octopine synthase gene
- IAA
indole-3-acetic acid 相似文献
13.
Recovery of primary transformants of soybean 总被引:17,自引:0,他引:17
W. A. Parrott L. M. Hoffman D. F. Hildebrand E. G. Williams G. B. Collins 《Plant cell reports》1989,7(8):615-617
Three transformants of soybean, Glycine max (L.) Merr., have been recovered among a total of 18 plants regenerated by somatic embryogenesis from immature cotyledon tissues after cocultivation with Agrobacterium strains carrying a 15 kD zein gene (pH5PZ3D). DNA from upper leaves hybridized to a synthetic RNA probe specific for the zein sequence at a level equivalent to at least one copy per haploid genome. Hybridization to a vir G/C probe, however, was negligible, indicating that sequestration of whole bacteria or even persistence of plasmids within the tissues could not account for the zein hybridization signals. Progeny of all plants were uniformly untransformed. Since most somatic embryos have a multicellular origin in the regeneration system used, it is believed that the primary transformants were chimeric. The results indicate that somatic embryogenesis may be adaptable to Agrobacterium-mediated transformation in soybean, but that greater numbers of mitotic cycles under selection before embryo initiation will be required if somatic embryogenesis is to be used efficiently for production of plants with transformed germ-line cells.Abbreviations NAA
1-naphthaleneacetic acid
- 2,4-D
2,4-dichlorophenoxyacetic acid
This paper (No. 88-3-267) is published with the approval of the Director of the Agricultural Experiment Station. 相似文献
14.
Nicolay Kuchuk Igor Komarnitski Anatoliy Shakhovsky Yuri Gleba 《Plant cell reports》1990,8(11):660-663
Shoot and leaf segments of a non-regenerable Medicago sativa L. genotype were cocultivated with the shooty mutant of Agrobacterium tumefaciens carrying the pGV 2206 plasmid. Transformed callus lines were selected and regenerated on the hormone free B5 medium. Southern blot analysis demonstrated integration of T-DNA in to the genome of the regenerated plants.Transgenic plants resistant to kanamycin were obtained by electroporation of Medicago borealis protoplasts with the pGA 472 plasmid DNA.Abbreviations 2.4 D
2.4 dichlorophenoxyacetic acid
- BAP
6-benzyladenine
- T-DNA
transferred DNA into plants from Ti-plasmid of A. tumefaciens 相似文献
15.
Paul M. Pechan 《Plant cell reports》1989,8(7):387-390
Brassica napus microspores and microspore-derived proembryos were cocultivated with Agrobacterium tumefaciens harbouring a binary vector. The vector contained selectable genes for kanamycin and hygromycin antibiotic resistance. Microspores and proembryos survived the cocultivation procedure and subsequent antibiotic selection. Thousands of plantlets can be regenerated from a single experiment. Biochemical analysis indicated up to 7.3% of plants exhibited neomycin phosphotransferase II enzyme activity. Success of the cocultivation procedure depended largely on choosing the proper coculture conditions while allowing microspore embryogenesis to proceed.Abbreviations Hm
hygromycin
- Km
kanamycin 相似文献
16.
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
17.
A new approach to direct somatic embryogenesis in Medicago 总被引:2,自引:0,他引:2
A highly efficient system for direct somatic embryogenesis is described. Leaf sections originating from young trifoliate leaves of Medicago falcata line 47/1–5 and Medicago sativa line No2/9R, directly produced embryos after cultivation in liquid B5IV induction medium. In comparison with indirect somatic embryogenesis the system omits the callus stage and thus allows shortening of the process of somatic embryogenesis in alfalfa by 35–40 days. It permits the avoidance of secondary changes occurring during the process of dedifferentiation. A modified B5/3H medium containing Polyethylene Glycol 6000 promoted embryo development from globular up to torpedo stage. It was clearly shown that 2.5% Polyethylene Glycol stimulated this process for both H. falcata 47/1–5 and M. sativa No 2/9R. Maturation of torpedo stage embryos was carried out on solidified or liquid abscisic acidcontaining medium. A 30M abscisic acid concentration was optimal in allowing one embryo to yield one plant. Somatic embryo conversion to plants and plant regeneration was performed on Murashige and Skoog medium. Regenerated plants showed a normal morphology.Abbreviations ABA
Abscisic acid
- B5
Medium of Gamborg et al.(1968)
- COT
Cotyledone stage embryos
- 2,4-D
2,4-dichlorphenoxyacetic acid
- FW
Fresh weight
- GA3
Gibberellin A3
- MS
Medium of Murashige and Skoog (1962)
- PEG
Polyethylene Glycol
- POLY
Polyembryos 相似文献
18.
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
19.
Surface sterilized seeds and mesocotyls from sterile seedlings from Panicum bisulcatum Thumb., as well as basal parts of leaves and mesocotyls from sterile seedlings, and seeds from Panicum milioides Nees ex. Trin were used as explants to induce callus on a Murashige and Skoog medium supplemented with 2.5 to 10 mg/l of 2,4-D. Subculturing of the white callus from P. milioides and of the brown callus from P. bisulcatum on a medium containing 0.1 mg/l 2,4-D and 10 g/l sucrose led in both species to the appearance of green structures from which plants could be regenerated. Plants were regenerated by an organogenetic process in P. milioides, while P. bisulcatum plants were regenerated both via organogenesis and somatic embryogenesis. 1032 and 94 plants, from P. bisulcatum and P. milioides, respectively, were transferred into soil, and about 90% of them were grown to maturity and set seeds.Abbreviations MS
Murashige and Skoog medium (15)
- 2,4-D
2,4-dichlorophenoxyacetic acid
- IAA
indoleacetic acid 相似文献
20.
Jovanka Miljuš-Djukić Mirjana Nešković Slavica Ninković Radomir Crkvenjakov 《Plant Cell, Tissue and Organ Culture》1992,29(2):101-108
Genetic transformation of buckwheat (Fagopyrum esculentum Moench.) and regeneration of transgenic plants were obtained by using Agrobacterium tumefaciens strains as vectors. Buckwheat cotyledons were excised from imbibed seeds, co-cultivated with A. tumefaciens and subjected to previously reported protocols for callus and shoot regeneration. The transformation with oncogenic strains was confirmed by opine and DNA analyses of tumour tissue extracts. Plants were regenerated on cotyledon fragments incubated with strain A281, harboring pGA472, which carries the neomycin phosphotransferase II gene for kanamycin resistance. The transformation of resistant shoot clones was confirmed by NPTII enzyme assay and DNA hybridization. A large number of transformed shoots were rooted and fertile plantlets were raised in the greenhouse. Transgenic plants comprised pin and thrum clones, which were allowed to cross-pollinate. In about 180 R2 seeds tested for kanamycin resistance, the ratio of resistant to sensitive seedlings was roughly 3:1.Abbreviations BAP
6-benzylaminopurine
- 2,4-D
dichloro-phenoxyacetic acid
- 2iP
6-(, ,-dimethylallyl-amino)-purine
- IBA
indole-3-butyric acid
- IAA
indole-3-acetic acid
- Km
kanamycin
- NPTII
neomycin phosphotransferase II 相似文献