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1.
Julie R. Kikkert Dominique Hébert-Soulé Patricia G. Wallace Michael J. Striem Bruce I. Reisch 《Plant cell reports》1996,15(5):311-316
Transgenic plantlets of Chancellor grapevine (Vitis L. complex interspecific hybrid) were produced via biolistic transformation. Embryogenic cell suspensions were bombarded with 1 m tungsten particles coated with pBI426 which encodes a fusion peptide between -glucuronidase (GUS) and neomycin phosphotransferase II (NPTII). The fusion peptide is under the control of a double 35S Cauliflower Mosaic Virus promoter and a leader sequence from Alfalfa Mosaic Virus. The cells were placed on kanamycin-containing media (10, 25 or 50 mg/l) 2 d after bombardment. Activated charcoal reduced cell browning. Embryos were first observed on selective media 14–29 weeks after bombardment. More than 1600 clusters of embryos were germinated and/or assayed for GUS. Of 621 embryos assayed for GUS expression, 182 (29.3%) were positive. PCR confirmed the presence of the NPTII gene in all 5 GUS-positive and 2 GUS-negative (bombarded) embryos tested. In germination experiments, 15% of the embryo clusters produced at least one plant with normal shoot growth. Of 164 normal plants assayed for GUS expression, 37 (22.6%) were positive. The NPTII gene was amplified by PCR in 1 (of 1) GUS-positive and 4 (of 5) GUS-negative bombarded plants, but not in non-bombarded control plants. Southern blotting confirmed integration of the NPTII gene in all 3 of the GUS and PCR-NPTII positive plants tested. Biolistics is an efficient method for transformation of Chancellor and should be applicable to other important grape cultivars.Abbreviations AC
activated charcoal
- GUS
-glucuronidase
- 2,4-D
2,4-dichlorophenoxyacetic acid
- BA
6-benzylaminopurine
- NAA
-naphthalene acetic acid
- TDZ
thidiazuron
- NPTII
neomycin phosphotransferase II
- Km
kanamycin
- MS
Murashige and Skoog (1962) medium
- WPM
Woody Plant Medium of Lloyd and McCown (1980) 相似文献
2.
Summary A procedure for the regeneration of fertile transgenic white mustard (Sinapis alba L.) is presented. The protocol is based on infection of stem explants of 7–9 day old plants with an Agrobacterium tumefaciens strain harboring a disarmed binary vector with chimeric genes encoding neomycin phosphotransferase and -glucuronidase. Shoots are regenerated from callus-forming explants within 3–4 weeks. Under selection, 10% of the explants with transgenic embryonic callus develop into fertile transgenic plants. Rooting shoots transferred to soil yield seeds within 14–16 weeks following transformation. Integration and expression of the T-DNA encoded marker genes was confirmed by histochemical glucuronidase assays and Southern-DNA hybridization using primary transformants and S1-progeny. The analysis showed stable integration and Mendelian inheritance of trans-genes in transformed Sinapis lines.Abbreviations BAP
6-benzylaminopurine
- CaMV
cauliflower mosaic virus
- GUS
-glucuronidase
- IBA
indole-3-butyric acid
- IM
infection medium
- NAA
1-naphthalene acetic acid
-
neo
gene encoding NPTII
- NPTII
neomycin phosphotransferase
- RIM
root-inducing medium
- SEM
shoot-elongation medium
- SIM
shoot-inducing medium
- t-nos
polyadenylation site of the nopaline synthase gene
-
uidA
gene encoding GUS
- WM
wash medium
- X-Gluc
5-bromo-4-chloro-3-indolyl -D-glucuronide 相似文献
3.
Highly efficient transformation and regeneration of aspen plants through shoot-bud formation in root culture 总被引:5,自引:0,他引:5
The natural capacity of aspen (Populus tremula L.) roots for direct shoot-bud regeneration was harnessed to establish a highly efficient transformation and regeneration procedure that does not require a pre-selection stage on antibiotics. Aspen stem segments were transformed using wildtype Agrobacterium rhizogenes (LBA9402) with the binary p35SGUSINT plasmid carrying the genes coding for -glucuronidase (GUS) and neomycin phosphotransferase II. High levels of transient GUS expression were found in the basal cut surface of 87% of the segments, and 98% of these formed well-developed adventitious roots. Proliferating root cultures were established in liquid culture, and GUS expression was found in 75% of the roots. Shoot-bud regeneration in root cultures was very high: 99% of the roots yielded shoot-buds (4.3 buds per root), of which 91% expressed GUS. Southern blot analysis and polymerase chain reaction confirmed the transgenic nature of the plants expressing GUS. Kanamycin resistance of transformants was tested with respect to callus growth and bud regeneration. Callus from transgenic plants exhibited a high growth rate in the presence of up to 100 g/l kanamycin, and bud regeneration from transformed roots occurred in the presence of up to 30 g/l kanamycin. Callus and buds from control (non-transformed) plants failed to proliferate or regenerate, respectively, in the presence of kanamycin at concentrations above 10 g/l. Ninety-four independent clones from different transformation events were established, of which 52 were phenotypically true-to-type.Abbreviations NAA
-naphthaleneacetic acid
- BA
6-benzylaminopurine
- GUS
-glucuronidase
- NPTII
neomycin phosphotransferase II
- PCR
polymerase chain reaction
- EtOH
ethanol
- CTAB
cetyltrimethylammonium bromide
- SDS
sodium dodecyl sulfate
- NOS
nopaline synthase
- CaMV
cauliflower mosaic virus 相似文献
4.
A. Ritala L. Mannonen K. Aspegren M. Salmenkallio-Marttila U. Kurtén R. Hannus J. Mendez Lozano T. H. Teeri V. Kauppinen 《Plant cell reports》1993,12(7-8):435-440
Summary Suspension culture cells of barley (Hordeum vulgare L. cv Pokko) were stably transformed with two separate plasmids containing genes coding for neomycin phosphotransferase II and -glucuronidase, respectively. Transformed cultures were selected with the antibiotic GeneticinR. Enzymatic activity was tested in the GeneticinR resistant cultures, and in 96% of them neomycin phosphotransferase could be detected. The non-selected marker, detected as -glucuronidase activity, was expressed in 40% of the resistant cultures. Stable transformation was confirmed with Southern blot hybridization.Abbreviations GUS
-glucuronidase of E. coli
-
uidA
gene coding for
- GUS
G418
- GeneticinR
O-2-amino-2,7-dideoxy-D-glycero--D-glucoheptopyranosyl[1-4]-O-3-deoxy-4C-methyl-3-[methylamino]--L-arabinopyranosyl-D-streptamine disulfate salt
- NPTII
neomycin phosphotransferase II of Tn5
-
nptII
gene coding for NPTII
- 2,4-D
2,4-dichlorophenoxyacetic acid
- X-Gluc
5-bromo-4-chloro-3-indolyl-D-glucuronic acid cyclohexylammonium salt 相似文献
5.
The GUS gene of E. coli, encoding -glucuronidase, has been widely used as a reporter gene in plant transformation. However, -glucuronidase activity in transgenic wheat leaf or root tissue is rarely observed or reported. To address this question, we investigated three wheat lines transformed with the GUS reporter gene. We found all three lines expressed GUS mRNA as well as -glucuronidase protein in their leaf and root tissues as detected by RNA gel blot, ELISA, and immunoblot analyses. However, -glucuronidase enzyme activity was only detected in pollen grains from the transgenic plants. Fluorometric and histochemical assays performed in the presence of wheat tissue extracts indicated that wheat leaf and root tissues contain inhibitor(s) of -glucuronidase activity, but pollen grains contain much lower concentrations. Further characterizations indicated that the inhibitor(s) is of low molecular weight (<10 kDa) and is non-proteinaceous. 相似文献
6.
Expression and inheritance of foreign genes in transgenic peanut plants generated byAgrobacterium-mediated transformation 总被引:11,自引:0,他引:11
To evaluate and characterize the stability of traits transferred viaAgrobacterium transformation, foreign gene expression must be examined in sexually derived progeny. The objective of this study was to analyze three transgenic peanut plants, 1-10, 12-1, and 17-1, for the inheritance and expression of their foreign genes. Segregation ratios for the introduced genes in T2 plants gave either 100% or 3:1 expression of the -glucuronidase (GUS) gene, demonstrating recovery of both homozygous and heterozygous T1 plants. Fluorometric GUS assay in T1 and T2 generations of all three plants showed that the GUS gene was stably expressed in the progeny. DNA analyses showed 100% concordance between the presence of the foreign gene and enzyme activity. Our results demonstrate that transgenes in peanut introduced byAgrobacterium can be inherited in a Mendelian manner.Abbreviations
GUS
-Glucuronidase
-
MS
Murashige and Skoog
-
MU
4-Methylumbelliferone
-
NPTII
Neomycin phosphotransferase II 相似文献
7.
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
8.
An efficient system for gene transfer into plants of Brassica juncea var. India Mustard, mediated by Agrobacterium tumefaciens. was developed through the manipulation of the culture medium and the use of the appropriate Agrobacterium strain. High frequency shoot regeneration (90–100%) was obtained from hypocotyl explants grown on medium containing 0.9% agarose, 3.3 mg/L AgNO3 and 0.5–2 mg/L BA in combination with 0.01–0.05 mg/L 2,4-D or 0.1–1 mg/L NAA. Of all the Agrobacterium strains tested, A. tumefaciens A208-SE, carrying the disarmed Ti plasmid and a binary vector pROA93, was the most effective for B. juncea transformation. pROA93 carries the coding sequences of the NPTII and the GUS genes, both driven by a common CaMV 35S promoter in two divergent directions. Inoculated explants grown on the selection medium in the presence of 0.5 mg/L BA and 0.1 mg/L NAA gave rise to transgenic shoots at the highest frequency (9%). All Ro transgenic plants were phenotypically normal, but variation in expression patterns of the GUS gene occurred among the transgenic plants in an organ- and tissue-specific manner. Both the NPTII and the GUS genes were transmitted to the R1 seed progeny and showed co-segregation.Abbreviations BA
benzyladenine
- 2,4-D
2,4-dichlorophenoxyacetic acid
- NAA
naphthaleneacetic acid
- NPTII
neomycin phosphotransferase type II
- GUS
-glucuronidase
- CaMV
cauliflower mosaic virus
- MS
Murashige and Skoog
- X-Gluc
5-bromo-4-chloro-3-indolyl-D--glucuronic acid
- IBA
indolebutyric acid
- SDS
sodium dodecyl sulfate 相似文献
9.
The 5-upstream region of the pea plastocyanin gene (petE) directed 5–10-fold higher levels of -glucuronidase (GUS) activity than the cauliflower mosaic virus 35S promoter in transgenic tobacco plants, although the levels of GUS mRNA were similar. The sequence (AAAAAUGG) around the translation initiation codon of petE enhanced translation of the GUS mRNA 10-fold compared to translation from the GUS translation initiation codon in transgenic tobacco plants and transfected protoplasts. 相似文献
10.
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
11.
Agrobacterium-mediated transformation of Citrus stem segments and regeneration of transgenic plants 总被引:1,自引:0,他引:1
G. A. Moore C. C. Jacono J. L. Neidigh S. D. Lawrence K. Cline 《Plant cell reports》1992,11(5-6):238-242
Summary A method for Agrobacterium-mediated transformation of Citrus and organogenic regeneration of transgenic plants is reported. Internodal stem segments were co-cultured with Agrobacterium harboring binary vectors that contained the genes for the scorable marker ß-glucuronidase (GUS) and the selectable marker NPT-II. A low but significant percentage ( 5%) of the shoots regenerated in the presence of 100 g/ml kanamycin were GUS+. Polymerase chain reaction (PCR) analysis confirmed that GUS+ shoots contained T-DNA. Two plants established in soil were shown to be transgenic by Southern analysis. 相似文献
12.
Masahiro Nishihara Motoaki Seki Masaharu Kyo Kohei Irifune Hiromichi Morikawa 《Transgenic research》1995,4(5):341-348
Immature pollen fromNicotiana rustica was bombarded with gold particles coated with plasmid DNA encoding neomycin phosphotransferase II (NPTII) and -glucuronidase (GUS) genes which, respectively, are under the control of the cauliflower mosaic virus (CaMV) 35S promoter and nopaline synthase (NOS) terminator in the plasmid. Kanamycin-resistant pollen embryoids were selected from the bombarded pollen cells and two independent lines of transgenic plants were regenerated. Enzyme assays showed that one has both NPTII and GUS activities and the other only weak NPTII activity. Southern blot analyses indicated that the former has a DNA fragment corresponding to the intact expression cassettes for both genes in its genome; whereas the latter lacks intact expression cassettes for both genes and has only the intactnptII coding sequence in its genome. The transgenic plants of both lines have 24 chromosomes, confirming haploidy, and they are infertile. These results indicate that transgenic haploid plants can be produced directly by the bombardment-mediated transformation of immature pollen. 相似文献
13.
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
14.
Jeffrey L. Carpenter Steven D. Kopczak D. Peter Snustad Carolyn D. Silflow 《Plant molecular biology》1993,21(5):937-942
In Arabidopsis tissues, the pool of tubulin protein is provided by the expression of multiple -tubulin and -tubulin genes. Previous evidence suggested that the TUA2 -tubulin gene was expressed in all organs of mature plants. We now report a more detailed analysis of TUA2 expression during plant development. Chimeric genes containing TUA2 5-flanking DNA fused to the -glucuronidase (GUS) coding region were used to create transgenic Arabidopsis plants. Second-generation progeny of regenerated plants were analyzed by histochemical assay to localize GUS expression. GUS activity was seen throughout plant development and in nearly all tissues. The blue product of GUS activity accumulated to the highest levels in tissues with actively dividing and elongating cells. GUS activity was not detected in a few plant tissues, suggesting that, though widely expressed, the TUA2 promoter is not constitutively active. 相似文献
15.
E. Cuzzoni L. Ferretti C. Giordani S. Castiglione F. Sala 《Molecular & general genetics : MGG》1990,222(1):58-64
Summary The regulation in tobacco of the rolB and rolC promoters of Agrobacterium rhizogenes pRi 1855 TL-DNA was studied by using the -glucuronidase (GUS) reporter system in transgenic plants. A 20- to 100-fold increase of GUS activity was selectively induced by auxin in rolB-GUS transformed mesophyll protoplasts, whereas this auxin-dependent increase was only 5-fold in rolC-GUS protoplasts. Moreover, both gene fusions exhibited similar tissue-specific expression in aerial parts but different patterns in roots. The spatial pattern of rolBGUS expression could be strongly modified by the addition of exogenous auxin, further suggesting that auxin plays a central role in the regulation of the rolB promoter in tobacco. The tissue-specific and auxin-dependent regulation of the rolB promoter is discussed in relation to the effects of the rolB gene on rhizogenesis and on cellular responses to auxin.Abbreviations BA
benzoic acid
- 6-BAP
benzylaminopurine
- 2,4-D
2,4-dichlorophenoxyacetic acid
- GUS
-glucuronidase
- 2,4,5-T
2,4,5,-trichlorophenoxyacetic acid
- 2,4,6-T
2,4,6-trichlorophenoxyacetic acid
- IAA
indoleacetic acid
- NAA
naphthaleneacetic acid
- MU
4-methyl umbelliferone
- 35S CaMV
cauliflower mosaic virus 35S (promoter)
- TCA
trichloroacetic acid
- X-Glu
5-bromo-4chloro-3-indolyl -d-glucuronic acid 相似文献
16.
Heat shock elements are involved in heat shock promoter activation during tobacco seed maturation 总被引:5,自引:0,他引:5
The soybean Gmhsp17.3-B heat shock promoter is developmentally regulated in transgenic tobacco, as indicated by the constitutive expression of a -glucuronidase reporter in seeds [16]. In this paper, we show that both the heat shock promoter-driven -glucuronidase activity and the mRNA of the endogenous Nthsp18P gene accumulate coincident with the onset of seed desiccation. Deletions of the soybean Gmhsp17.3-B promoter, encompassing the heat shock element (HSE)-containing regions, revealed a co-localization of sequences responsible for heat induction and developmental expression. Moreover, synthetic HSEs fused to a TATA box sequence had the potential to stimulate the developmental expression of a GUS reporter gene in seeds of transgenic plants. 相似文献
17.
The rice metallothionein gene promoter does not direct foreign gene expression in seed endosperm 总被引:3,自引:0,他引:3
We generated transgenic tobacco and rice plants harboring a chimeric gene consisting of the 5-upstream sequence of the rice metallothionein gene (ricMT) fused to the -glucuronidase (GUS) gene. The activity and tissue-specific expression of the ricMT promoter were demonstrated in these transgenic plants. In the transgenic rice plants, despite substantial levels of GUS activity in the shoot and root, almost no GUS signal was detected in the endosperm. Thus, the ricMT promoter could be useful in avoiding accumulation of undesired proteins in the seed endosperm. 相似文献
18.
O. M. -F. Zaghmout N. L. Trolinder 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1993,86(6):721-730
Protoplasts isolated from embryogenic (Mustang and Chinese Spring) and non-embryogenic (Mit) calli of wheat (Triticum aestivum L.) genotypes transiently expressed -glucuronidase (GUS) activity when electroporated with a plasmid containing the GUS gene and driven by an enhanced 35S promoter and a TMV leader sequence. Conditions for the maximum expression of GUS activity were: electroporation of the freshly isolated protoplasts at 250 Vcm-1 and 250 F for 2 s using 50 g/ml of plasmid DNA; incubation of the protoplasts with the plasmid before the pulse for 2 h; and a 15-min recovery period on ice after the pulse. In general, a higher GUS activity was obtained in protoplasts of non-embryogenic (NE) callus origin than in those of embryogenic (E) callus origin. Only GUS constructs containing a duplicate 35S promoter derivative resulted in a significant level of GUS expression. The presence of the TMV viral leader sequence in the pAGUS1-TN2 plasmid construct resulted in a significant increase of GUS activity in the electroporated protoplasts of both callus types. On the other hand, protoplasts electroporated with the Adh1 promoter and intron showed a threefold less GUS activity than those electroporated with pAGUS1-TN2. Optimized conditions for DNA uptake and expression were very similar for protoplasts of both callus types. The importance of these findings for the successful regeneration of transgenic and fertile wheat plants is discussed. 相似文献
19.
Transgenic creeping bentgrass (Agrostis palustris Huds., cv. Pencross; Poaceae) plants have been obtained by microprojectile bombardment of and regeneration from embryogenic calli with a vector designed to deliver the -glucuronidase (GUS) gene under the control of rice actin 1 5' regulatory sequences. Southern analysis of polymerase chain reaction (PCR)-amplified and restriction-digested genomic DNA of four transgenic plants regenerated from these cultures showed the unscrambled integration of the gus fragment. Northern blot analysis confirmed the expression of gus mRNA in one of the transgenic plants. Western blot analysis revealed a high level of accumulation of gus protein. Histochemical assays showed enzymatic activity of -glucuronidase in all parts of the transgenic turfgrass plant. The order of gus expression level in different tissues of the transgenic plant is as follows: stem node > first young leaf > root tip > second / third / fourth young leaf > stem internode > root hair-zone.Abbreviation GUS
-glucuronidase
- MS
Murashige and Skoog(1962) medium
- BA
6-benzyladenine
- dicamba
3, 6 -dichloro-o-anisic acid
- PCR
polymerase chain reaction 相似文献
20.
Stable transformation of papaya via microprojectile bombardment 总被引:27,自引:0,他引:27
Maureen M. M. Fitch Richard M. Manshardt Dennis Gonsalves Jerry L. Slightom John C. Sanford 《Plant cell reports》1990,9(4):189-194
Summary Stable transformation of papaya (Carica papaya L.) has been achieved following DNA delivery via high velocity microprojectiles. Three types of embryogenic tissues, including immature zygotic embryos, freshly explanted hypocotyl sections, and somatic embryos derived from both, were bombarded with tungsten particles carrying chimeric NPTII and GUS genes. All tissue types were cultured prior to and following bombardment on half-strength MS medium supplemented with 10 mg 1–1 2,4-D, 400 mg 1–1 glutamine, and 6% sucrose. Upon transfer to 2,4-D-free medium containing 150 mg 1–1 kanamycin sulfate, ten putative transgenic isolates produced somatic embryos and five regenerated leafy shoots. Leafy shoots were produced six to nine months following bombardment. Tissues from 13 of these isolates were assayed for NPTII activity, and 10 were positive. Six out of 15 isolates assayed for GUS expression were positive. Three isolates were positive for both NPTII and GUS,Abbreviations 2,4-D
2,4-dichlorophenoxyacetic acid
- GUS
-glucuronidase
- X-gluc
5-Br-4-Cl-3-indolyl--D-glucuronic acid
- CaMV
cauliflower mosaic virus
- NOS
nopaline synthase
- NPTII
neomycin phosphotransferase II
Journal Series no. 3448 of the Hawaii Institute of Tropical Agriculture and Human Resources 相似文献