共查询到20条相似文献,搜索用时 15 毫秒
1.
M. Van Lijsebettens X. Wang G. Cnops W. Boerjan M. Van Montagu T. Desnos H. Höfte 《Molecular & general genetics : MGG》1996,251(3):365-372
The map positions of a set of eight T-DNA insertions in theArabidopsis genome have been determined by using closely linked visible markers. The insertions are dispersed over four of the five chromosomes. Each T-DNA insert contains one or more of the chimeric marker genes neomycin phosphotransferase (neo), hygromycin phosphotransferase (hpt), phosphinothricin acetyltransferase (bar),-glucuronidase (gusA) and indole-3-acetamide hydrolase (iaaH). Theneo, hpt andbar marker genes are dominant in a selective germination assay or when used as DNA markers in a polymerase chain reaction. These dominant markers will allow recombinants to be discerned in a germinating F2 population, one generation earlier than with a conventional recessive marker. The transgenic marker lines will speed up and simplify the isolation of recombinants in small genetic intervals, a rate-limiting step in positional cloning strategies. The transgenic lines containing thehpt marker will also be of interest for the isolation of deletion mutants at the T-DNA integration sites. 相似文献
2.
New plant binary vectors with selectable markers located proximal to the left T-DNA border 总被引:31,自引:0,他引:31
Detlef Becker Elke Kemper Jeff Schell Robert Masterson 《Plant molecular biology》1992,20(6):1195-1197
Five new binary vectors have been constructed which have the following features: (1) different plant selectable markers including neomycin phosphotransferase (nptII), hygromycin phosphotransferase (hpt), dihydrofolate reductase (dhfr), phosphinothricin acetyl transferase (bar), and bleomycin resistance (ble); (2) selectable markers are located near the T-DNA left border and; (3) selectable marker and -glucuronidase (uidA) reporter genes are divergently organized for efficient expression, and can easily be removed or replaced as needed. 相似文献
3.
4.
Danfeng Long Xueli Wu Zhimin Yang Ingo Lenk Klaus Kristian Nielsen Caixia Gao 《In vitro cellular & developmental biology. Plant》2011,47(6):658-666
A variety of selection systems have been developed for transformation of forage crops. To compare the most frequently used
systems, we tested three selectable marker genes for their selection efficiency under four selection procedures for the production
of transgenic tall fescue. Embryogenic calluses initiated from mature embryos were bombarded with three constructs containing
either the phosphinothricin acetyltransferase (bar) gene, the hygromycin phosphotransferase (hpt) gene or the neomycin phosphotransferase II (nptII) gene. Transformation efficiency was strongly influenced by the selectable marker gene, selection procedure and genotype.
The highest transformation efficiency was observed using the bar gene in combination with bialaphos. Average transformation efficiencies with bialaphos, phosphinothricin (glufosinate), hygromycin
and paromomycin selection across the two callus lines used in the experiments were 9.4%, 4.4%, 5.2% and 1.6%, respectively.
Southern blot analysis revealed the independent nature of the tested transgenic plants and a complex transgene integration
pattern with multiple insertions. 相似文献
5.
Alternative selectable markers for potato transformation using minimal T-DNA vectors 总被引:4,自引:0,他引:4
Barrell P.J. Yongjin Shang Cooper P.A. Conner A.J. 《Plant Cell, Tissue and Organ Culture》2002,70(1):61-68
Alternative selection systems for plant transformation are especially valuable in clonal crops, such as potato (Solanum tuberosum L.), to pyramid transgenes into the same cultivar by successive transformation events. We have modified the pGPTV series of binary vectors to construct pMOA1 to pMOA5, resulting in a series of essentially identical binary vectors except for the presence of different selectable marker genes. These selectable marker genes are tightly inserted between the left and right T-DNA borders and confer resistance to kanamycin (nptII), hygromycin (hpt), methotrexate (dhfr), phosphinothricin (bar), or phleomycin (ble). The T-DNA of all the vectors is based on the minimal features necessary for plant transformation, with no extraneous DNA segments that may be unacceptable to regulatory authorities for general release of transgenic plants. A series of unique restriction sites exists between the right border and each selectable marker gene for subsequent insertion of useful genes. We have also developed improved culture procedures for potato transformation and used the pMOA1 to pMOA5 binary vectors to define stringent selection conditions for each marker gene. Combining these advances improved the frequency of recovering transformed potato plants while maintaining a low frequency of escapes. The relative efficiency of recovering transgenic potato lines with each selectable marker gene can be summarised as: kanamycin resistance>hygromycin resistance>phosphinothricin resistance>phleomycin resistance>methotrexate resistance. 相似文献
6.
A very efficient transformation system, using biolistic bombardment, has been developed for the production of transgenic plants
of Kentucky bluegrass (Poa pratensis L.). Embryogenic calli, initiated from immature embryos, were transformed either with pAct1IHPT-4 containing the hygromycin
phosphotransferase (hpt) gene or with pDM803 containing the phosphinothricin acetyltransferase (bar) gene and the β-glucuronidase (uidA) gene. In total 119 independent transgenic plants were recovered from 153 hygromycin-resistant lines. Bialaphos selection
yielded a total of 99 bialaphos-resistant lines and from these 34 independent transgenic plants were recovered. Southern blot
analysis demonstrated the independent nature of the transgenic plants and also revealed a complex transgene integration pattern
with multiple insertions.
The first two author contributed equally to this work 相似文献
7.
Huixia Wu Caroline A. Sparks Huw D. Jones 《Molecular breeding : new strategies in plant improvement》2006,18(3):195-208
Detailed molecular characterisation of transgene loci is a requirement for gaining regulatory approval for environmental release of genetically modified crops. In cereals, it is generally accepted that Agrobacterium-mediated transformation generates cleaner transgene loci with lower copy number and fewer rearrangements than those generated by biolistics. However, in wheat there has been little detailed analysis of T-DNA insertions at genetic and molecular level. Wheat lines transformed using Agrobacterium tumefaciens with bar and gusA (GUS) genes were subjected to genetic and molecular analysis. Unlike previous studies of transgene loci in wheat, we used functional assays for PAT and GUS proteins, combined with PCR and Southern analysis to detect the presence, copy number, linkage and transmission of two transgenes inserted in the same T-DNA. Thirty-four independent transgenic lines were categorised into three types: type I events (38% of total) where the gusA and bar genes displayed complete genetic linkage, segregating together as a single functional locus at the expected ratio of 3:1; type II events (18%), which possessed two or more transgene loci each containing gusA and bar; and type III events (44%), containing an incomplete T-DNA in which either the gusA or bar gene was lost. Most lines in this last category had lost the bar gene situated near the left T-DNA border. Southern analysis indicated that 30% of all lines possessed a single T-DNA copy containing gusA and bar. However, when data on expression and molecular analysis are combined, only 23% of all lines have single copy T-DNAs in which both gene cassettes are functioning. We also report on the presence of plasmid backbone DNA sequence in transgene loci detected using primer pairs outside the left and right T-DNA borders and within the plasmid selectable marker (NptI) gene. Approximately two thirds of the lines contained some vector backbone DNA, more frequently adjacent to the left border. Taken together, these data imply unstable left border function causing premature T-strand termination or read-through into vector backbone. As far as we are aware, this is the first report revealing near border T-DNA truncation and vector backbone integration in wheat transgenic lines produced by Agrobacterium-mediated transformation. 相似文献
8.
Production of transgenic tall fescue and red fescue plants by particle bombardment of mature seed-derived highly regenerative tissues 总被引:29,自引:0,他引:29
Highly regenerative tissues of tall fescue and red fescue produced from mature seed-derived embryogenic callus were induced
and proliferated on medium containing 2,4-dichlorophenoxyacetic acid (4.5 or 9.0 μM), 6-benzylaminopurine (0, 0.044, 0.44
or 2.2 μM) and cupric sulfate (0.1 or 5.0 μM) under dim-light conditions (10 to 30 μE m–2 s–1, 16 h light). Tall fescue tissues were transformed with three plasmids containing the genes for hygromycin phosphotransferase
(hpt), phosphinothricin acetyltransferase (bar) and β-glucuronidase (uidA;gus), and red fescue with three plasmids containing hpt, uidA and a synthetic green fluorescent protein gene [sgfp(S65T)]. DNA from T0 plants of eight independently transformed lines from tall fescue and 11 from red fescue were analyzed by PCR and DNA blot
hybridization. The co-expression frequency of all three transgenes [hpt/bar/uidA or hpt/uidA/sgfp(S65T)] in transgenic tall fescue and red fescue plants was 25–27%; for two transgenes [hpt/bar or hpt/uidA for tall fescue and hpt/uidA or hpt/sgfp(S65T) for red fescue], the co-expression frequency was 50–75%.
Received: 28 September 1999 / Revision received: 13 March 2000 / Accepted: 16 March 2000 相似文献
9.
Arundhati Mukhopadhyay Reinhard Töpfer Akshay K. Pradhan Yaspal S. Sodhi Hans-Henning Steinbiß Jeff Schell Deepak Pental 《Plant cell reports》1991,10(8):375-379
Summary Efficient regeneration (80%) and high frequency genetic transformation (10–33%) were achieved by culturing protoplasts isolated from hypocotyl tissues of six day old Brassica oleracea seedlings and by subjecting these protoplasts to PEG mediated direct plasmid uptake. Three different plasmid vectors carrying marker genes for resistance to methotrexate (dhfr), hygromycin (hpt) and phosphinotricin (bar) were constructed and used for transformation. Large number of normal, fertile transformants were obtained with vectors carrying hpt and bar genes. No transformants could be regenerated for resistance to methotrexate as it severely suppressed shoot differentiation.Abbreviations
bar/PAT
bialaphos resistance gene/phosphinotricin acetyltransferase
- 2,4-D
2,4-di-chlorophenoxyacetic acid
-
dhfr/DHPR
dihydrofolate reductase gene/enzyme
-
gus/GUS
-glucuronidase gene/enzyme
-
hpt/HPT
hygromycin phosphotransferase gene/enzyme
- Kn
kinetin
- PEG
polyethylene glycol
- RH
relative humidity 相似文献
10.
Hee-Jong Woo Seok-Cheol Suh Yong-Gu Cho 《Biotechnology and Bioprocess Engineering》2011,16(6):1053-1064
The development of marker-free transgenic plants has responded to public concerns over the safety of biotechnology crops.
It seems that continued work in this area will soon remove the question of unwanted marker genes from the debate concerning
the public acceptability of transgenic crop plants. Selectable marker genes are co-introduced with genes of interest to identify
those cells that have integrated the DNA into their genome. Despite the large number of different selection systems, marker
genes that confer resistance to the antibiotics, hygromycin (hpt) and kanamycin (nptII) or herbicide phosphinothricin (bar), have been used in most transgenic research and crop development techniques. The techniques that remove marker gene are
under development and will eventually facilitate more precise and subtle engineering of the plant genome, with widespread
applications in both fundamental research and biotechnology. In addition to allaying public concerns, the absence of resistance
genes in transgenic plants could reduce the costs of developing biotechnology crops and lessen the need for time-consuming
safety evaluations, thereby speeding up the commercial production of biotechnology crops. Many research results and various
techniques have been developed to produce marker-free transgenic plants. This review describes the strategies for eliminating
selectable marker genes to generate marker-free transgenic plants, focusing on the three significant marker-free technologies,
co-transformation, site-specific recombinase-mediated excision, and non-selected transformation. 相似文献
11.
Hitesh Kathuria Amitabh Mohanty Akhilesh K. Tyagi 《Journal of plant biochemistry and biotechnology.》2003,12(2):103-107
Transformation of Oryza sativa subsp indica variety Pusa Basmati 1 with Agrobacterium tumefaciens strain LBA4404(pTOK233) carrying genes coding for neomycin phosphotransferase (nptII), β-glucuronidase (gus) and hygromycin phosphotransferase (hph) under the control of plant-specific promoters (pnos and pCaMV35S) within its T-DNA region produced transgenics with single and multiple copies of T-DNA. Simple Mendelian as well as complex patterns of the inheritance for hygromycin resistance trait were observed in R1 and R2 generations. Non-segregating lines selected in R2 generation did not show further segregation of the resistance trait in R3 and R4 generations accompanied by stabilization of integrated transgenes. One of these lines showed the presence of truncated T-DNA in R1 generation. The single copy transgenics showed high stability of expression of gus gene, whereas multi-copy transgenics were prone to silencing up to R3 generation after which no further reduction in gene expression was observed. 相似文献
12.
Lei Sun Lin Zhou Miao Lu Ming CAI Xi-Wang Jiang Qi-Xiang Zhang 《Plant Molecular Biology Reporter》2009,27(1):102-108
In this study, a superbinary vector was constructed to evaluate the potential of a twin T-DNA system for generating selectable
marker-free transgenic chrysanthemum plants. The first T-DNA of the pCAMBIA 1300 vector contained the hygromycin phosphotransferase
(hpt) selectable marker gene, while the second T-DNA carried the β-glucuronidase gene (uidA) and featuring the gene of interest. The two T-DNA regions were placed adjacent to each other with no intervening region.
This vector was then used to transform transversal thin cell layers (1–2 mm thick) of internodal stem segments of chrysanthemum
via Agrobacterium-mediated transfer. Putative transgenic plants were obtained and analyzed for presence and integration of the transgene using
polymerase chain reaction amplification and Southern blotting. The primary cotransformation frequency was calculated at 38.4%.
A total of 17 hpt-resistant/gus-positive T0 plants were evaluated for segregation in the next generation (T1), and among those
approximately 15.7% carried the transgene. Overall, the two T-DNA system appeared to be a useful approach to generate marker-free
transgenic chrysanthemum plants, thereby eliminating public concerns regarding proliferation of antibiotic and herbicide resistance
genes into the environment. 相似文献
13.
To investigate the various integration patterns of T-DNA generated by infection withAgrobacterium, we developed a vector (pRCV2) for the effective T-DNA tagging and applied it to tobacco (Nicotiana tabacum cv. Havana SR1). pRCV2 was constructed for isolating not only intact T-DNA inserts containing both side borders of T-DNA,
but also for partial T-DNA inserts that comprise only the right or left side. We also designed PCR confirmation primer sets
that can amplify in several important regions within pRCV2 to detect various unpredictable integration patterns. These can
also be used for the direct inverse PCR. Leaf disks of tobacco were transformed withAgrobacterium tumefaciens LBA4404 harboring pRCV2. PCR and Southern analysis revealed the expected 584 bp product for thehpt gene as well as one of 600 bp for thegus gene in all transformants; one or two copies were identified for these integrated genes. Flanking plant genomic DNA sequences
from the transgenic tobacco were obtained via plasmid rescue and then sequenced. Abnormal integration patterns in the tobacco
genome were found in many transgenic lines. Of the 17 lines examined, 11 contained intact vector backbone; a somewhat larger
deletion of the left T-DNA portion was encountered in 4 lines. Because nicking sites at the right border showed irregular
patterns when the T-DNA was integrated, it was difficult to predict the junction regions between the vector and the flanking
plant DNA. 相似文献
14.
Teresa Cegielska-Taras Tomasz Pniewski Laurencja Szała 《Journal of applied genetics》2008,49(4):343-347
Haploid microspore-derived embryos (MDEs) constitute a unique material for the introduction of new traits into winter oilseed
rape (Brassica napus). MDEs have been transformed by usingAgrobacterium tumefaciens strains EHA105 and LBA4404, both carrying the binary vector pKGIB containing theuidA gene encoding β-glucuronidase (GUS) and thebar gene as a marker of resistance to phosphinotricin. Transformed embryos expressed GUS and regenerated plants that were resistant
to herbicide Basta, as confirmed by a leaf-painting test. Progeny plants of the transformant T-39 were all transgenic, as
they inherited T-DNA from their doubled haploid parental plant. Southern-blot analysis confirmed the integration and transmission
of T-DNA into T1 plants. Transformation of MDEs facilitates the obtaining of winter oilseed rape homozygous for the introduced genes. 相似文献
15.
A highly efficient and reproducible transformation system for orchardgrass (Dactylis glomerata L. cv. Rapido, 2n=42=28) was established using microprojectile bombardment of highly regenerative, green tissues derived from mature seeds. These tissues, induced from embryogenic callus, were bombarded with a mixture of three plasmids containing the hygromycin phosphotransferase (hpt), phosphinothricin acetyltransferase (bar) and #-glucuronidase (uidA; gus) genes. From 147 individual explants bombarded, 11 independent hygromycin-resistant lines (7.5%) were obtained after an 8- to 16-week selection period using 30-50 mg/l hygromycin B. Of the 11 independent lines, ten (91%) were regenerable. The presence and integration of the transgene(s) were assessed using PCR and DNA blot hybridization. Coexpression frequency of the three transgenes (hpt/bar/uidA) in T0 plants was 20%, and of two transgenes, either hpt/bar or hpt/uidA, 45-60%. Due to greenhouse conditions optimized for the growth of other species, T1 seed has not been obtained from these plants. While the inability to analyze progeny plants precludes the conclusive demonstration of stable transformation, the results of all molecular and biochemical analyses of T0 plants are consistent with the production of stably transformed plants. Frequent change in ploidy level was observed in transformed T0 orchardgrass plants. Plants from only three of the ten independent lines analyzed had the normal tetraploid number of chromosomes (2n=42=28), while plants from seven lines (70%) were octaploid (2n=82=56). The octaploid plants had abnormal morphological features, such as narrower, thicker and more upright leaves. 相似文献
16.
Ogarkova O. A. Tomilova N. B. Tomilov A. A. Tarasov V. A. 《Russian Journal of Genetics》2001,37(8):899-904
A collection of transgenic Arabidopsis thalianaplants has been obtained by Agrobacterium-mediated transformation. The genomes of the transgenic plants contain insertions of T-DNA of the vector plasmids pLD3 or pPCVRN4. Genes bearing T-DNA insertions were shown to constitute 12–18% of the total number of A. thalianagenes. Seventy-five lines have been chosen from the collection and subjected to genetic and molecular-genetic analysis. Of these, 5 were dominant mutants, and 70, recessive insertion mutants with various morphological defects. Identification of mutant phenotypes and genetic characterization of the transgenic lines have been performed with the use of nutrient media supplemented with exogenous hormones, which revealed five recessive lethal mutants and one dominant sterile mutant. 相似文献
17.
Mature seed‐derived callus from an elite Chinese japonica rice cv. Eyl 105 was transformed with a plasmid containing the selectable marker hygromycin phosphotransferase (hpt) and the reporter β‐glucuronidase (gusA) genes via particle bombardment. After two rounds of selection on hygromycin (30 mg/l)‐containing medium, resistant callus was transferred to hygromycin (30 mg/l)‐containing regeneration medium for plant regeneration. Twenty‐three independent transgenic rice plants were regenerated from 127 bombarded callus with a transformation frequency of 18.1%. All the transgenic plants contained both gusA and hpt genes, revealed by PCR/Southern blot analysis. GUS assay revealed 18 out of 23 plants (78.3%) proliferated on hygromycin‐containing medium had GUS expression at various levels. Genetic analysis confirmed Mendelian segregation of transgenes in progeny. From R2 generations with their R1 parent plants showing 3:1 Mendelian segregation, we identified three independent homozygous transgenic rice lines. The homozygous lines were phenotypically normal and fertile compared to the control plants. We demonstrate that homozygous transgenic rice lines can be obtained via particle bombardment‐mediated transformation and through genetic analysis‐based selection. 相似文献
18.
Kexuan Tang Quanan Hu Xianfen Sun Bingliang Wan Huaxiong Qi Xinggui Lu 《In vitro cellular & developmental biology. Plant》2001,37(3):334-340
Summary Mature seed-derived callus from an elite Chinese japonica rice cv. Ewan 5 was cotransformed with two plasmids, pWRG1515 and
pRSSGNAl, containing the selectable marker hygromycin phosphotransferase gene (hpt), the reporter β-glucuronidase gene (gusA) and the snowdrop (Galanthus nivalis) lectin gene (gna) via particle bombardment. Thirty-five independent transgenic rice plants were regenerated from 177 bombarded calluses. Eighty-three
percent of the transgenic plants contained all three genes, as revealed by Southern blot analysis. Western blot analysis revealed
that 23 out of 29 gna-containing transgenic plants expressed Galanthus nivalis agglutinin (GNA) (79%) at various levels, with the highest expression being approximately 0.5% of total soluble protein.
Genetic analysis confirmed Mendelian segregation of all three transgenes (gna, hpt and gusA) in the R2 progeny. Amongst the R2 generation two independent homozygous lines were identified that expressed all three transgenes.
Insect bioassay and feeding tests showed that these homozygous lines had significant inhibition to rice brown planthopper
(Nilaparvata lugens, BPH) by decreasing the survival, overall fecundity of BPH, retarding development, and decreasing the feeding of BPH. These
BPH-resistant lines have been incorporated into a rice insect resistance breeding program. This is the first report that homozygous
transgenic rice lines expressing GNA, developed by genetic transformation and through genetic analysis-based selection, conferred
enhanced resistance to BPH. 相似文献
19.
Hyemin Lim Hyun-Ju Hwang A-Ram Kim Man-Ho Cho Hyeonso Ji Chang-Kug Kim Seung Uk Ji Jung-Il Cho Soo-Chul Park Gang-Seob Lee 《Plant biotechnology reports》2016,10(1):25-33
We generated 383 independent transgenic lines that contained the PsGPD (Glyceraldehyde-3-Phosphate Dehydrogenase), ArCspA (Cold Shock Protein), BrTSR15 (Triple Stress Resistance 15) and BrTSR53 (Triple Stress Resistance 53) genes under the control of a constitutive (CaMV 35S) promoter to generate genetically modified (GM) rice. TaqMan copy number assay was performed to determine the copy numbers of inserted T-DNA. Flanking sequence tags (FSTs) were isolated from 203 single copy T-DNA lines of transgenic plants, and their sequences were mapped to the rice chromosomes. Of the 157 flanking sequence tags that were isolated from single copy lines, transgenes were found to be integrated into genic regions in 58 lines (36 %), whereas 97 lines (62 %) contained transgene insertions in intergenic regions. Approximately 27 putative homozygous lines were obtained through multi-generations of planting, resistance screening and TaqMan copy number assays. To investigate the transgene expression patterns, quantitative real-time PCR analysis was performed using total RNA from leaf tissue of homozygous T1 plants with a single copy and an intergenic insertion of T-DNA. The mRNA expression levels of the examined transgenic rice were significantly increased in all transgenic plants. In addition, myc-tagged 35S:BrTSR15 and 35S:BrTSR53 transgenic plants displayed higher levels of transgene protein. Using numerical data for the mass production of transgenic plants can reduce the time required to obtain a genetically modified plant. Moreover, the duration, cost, and efforts required for transformation can be deliberately predicted. These results may be useful for the large-scale production of transgenic plants or T-DNA inserted rice mutants. 相似文献
20.
. Transgenic carrot (Daucus carota L.) plants expressing a rice thaumatin-like protein (tlp), phosphinothricin acetyltransferase (bar) and the hygromycin phosphotransferase (hpt) genes were obtained by Agrobacterium-mediated transformation. Petiole and hypocotyl segments of three carrot cultivars were used as the explant sources. Following infection, selection was achieved on Murashige and Skoog medium with 1 mg/l phosphinothricin or 25 mg/l hygromycin B, which was increased after 2 weeks to 10 mg/l phosphinothricin and 100 mg/l hygromycin B. The presence of the tlp and bar transgenes was confirmed by polymerase chain reaction and Southern blot analyses, and the expression of the thaumatin-like protein was demonstrated by Western blot analysis. Among 45 primary transformants, 13 were selected for assessment of herbicide and/or disease tolerance. The transgenic plants showed varying levels of tolerance to the herbicide phosphinothricin, depending on the transformation events in different lines. Four transgenic lines also showed significantly enhanced tolerance to the foliar and root pathogen Botrytis cinerea or Sclerotinia sclerotiorum when inoculated under controlled environment conditions. Two lines had significantly enhanced tolerance to the herbicide phosphinothricin as well as to both pathogens. These results demonstrate the feasibility of introducing two potentially useful agronomic traits into carrot through genetic engineering. 相似文献