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1.
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
2.
Narender S. Nehra Ravindra N. Chibbar Nick Leung Karen Caswell Cliff Mallard Lee Steinhauer Monica Baga Kutty K. Kartha 《The Plant journal : for cell and molecular biology》1994,5(2):285-297
A system for enhanced induction of somatic embryo-genesis and regeneration of plants from isolated scutellar tissue of wheat has been developed. This system has been successfully used in the development of a simple and reproducible protocol for the production of self-fertile transgenic wheat plants. The procedure is rapid resulting in the production of transgenic plantlets within 12 weeks from initiation of cultures and it avoids the need for establishing long-term callus, cell suspension or protoplast cultures. Somatic embryos regenerated from scutella bombarded with plasmid pBARGUS were selected on L-phosphinothricin (L-PPT) to obtain herbicide-resistant self-fertile transgenic plants. Phosphinothricin acetyltransferase (PAT) activity was observed at varying levels in 50% of the plants selected on L-PPT whereas none of the plants showed β-glucuronidase (GUS) activity. Molecular analysis of PAT-positive plants confirmed stable integration of both bar and gus genes in R0 and R1 progeny plants. Segregation of the PAT activity and herbicide resistance in R1 progeny plants confirmed the Mendelian inheritance of the bar gene. Additionally, isolated scutella bombarded with plasmid DNA containing a gus::nptII fusion gene driven by a rice actin promoter and its first intron were selected in the presence of geneticin to obtain fully fertile transgenic plants. Functional expression of the fusion gene was demonstrated in transgenic plants by GUS and neomycin phospho-transferase (NPTII) enzyme assays. Southern blot analysis confirmed the integration of transgenes into the wheat genome. Histochemical GUS staining showed transmission of the fusion gene to floral organs of primary transformants and confirmed Mendelian segregation of the transgene in R1 progeny. 相似文献
3.
Transgenic radish (Raphanus sativus L. longipinnatus Bailey) plants were produced from the progeny of plants which were dipped into a suspension of Agrobacterium carrying both the -glucuronidase (gusA) gene and a gene for resistance to the herbicide Basta (bar) between T-DNA border sequences. The importance of development of the floral-dipped plant and presence of surfactant in the inoculation medium were evaluated in terms of transgenic plant production. Plants dipped at the primary bolt stage of growth, into a suspension of Agrobacterium containing 0.05% (v/v) Silwet L-77 resulted in optimum transformation efficiency, with 1.4% from 1110 seeds. The presence of Pluronic F-68 or Tween 20 in the inoculation medium was beneficial towards transgenic plant output compared to treatments without surfactant. Putative transformed T1 plants were efficiently selected by spraying with 0.03% (v/v) Basta and all herbicide-resistant plants tested positive for GUS activity when analysed both histochemically and fluorometrically. Southern analysis revealed that both the gusA and bar genes integrated into the genome of transformed plants and segregated as dominant Mendelian traits. These results demonstrate that radish can be genetically modified for the improvement of this important vegetable crop. 相似文献
4.
Generation of marker‐free transgenic hexaploid wheat via an Agrobacterium‐mediated co‐transformation strategy in commercial Chinese wheat varieties
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Genotype specificity is a big problem lagging the development of efficient hexaploid wheat transformation system. Increasingly, the biosecurity of genetically modified organisms is garnering public attention, so the generation of marker‐free transgenic plants is very important to the eventual potential commercial release of transgenic wheat. In this study, 15 commercial Chinese hexaploid wheat varieties were successfully transformed via an Agrobacterium‐mediated method, with efficiency of up to 37.7%, as confirmed by the use of Quickstix strips, histochemical staining, PCR analysis and Southern blotting. Of particular interest, marker‐free transgenic wheat plants from various commercial Chinese varieties and their F1 hybrids were successfully obtained for the first time, with a frequency of 4.3%, using a plasmid harbouring two independent T‐DNA regions. The average co‐integration frequency of the gus and the bar genes located on the two independent T‐DNA regions was 49.0% in T0 plants. We further found that the efficiency of generating marker‐free plants was related to the number of bar gene copies integrated in the genome. Marker‐free transgenic wheat plants were identified in the progeny of three transgenic lines that had only one or two bar gene copies. Moreover, silencing of the bar gene was detected in 30.7% of T1 positive plants, but the gus gene was never found to be silenced in T1 plants. Bisulphite genomic sequencing suggested that DNA methylation in the 35S promoter of the bar gene regulatory region might be the main reason for bar gene silencing in the transgenic plants. 相似文献
5.
Girgi Maram O'Kennedy Maretha M. Morgenstern Anika Mayer Gillian Lörz Horst Oldach Klaus H. 《Molecular breeding : new strategies in plant improvement》2002,10(4):243-252
Four different pearl millet breeding lines were transformed and led to the regeneration of fertile transgenic plants. Scutellar tissue was bombarded with two plasmids containing the bar selectable marker and the -glucuronidase reporter gene (gus or uidA) under control of the constitutive CaMV 35S promoter or the maize Ubiquitin1 promoter (the CaMV 35S is not a maize promoter). For the delivery of the DNA-coated microprojectiles, either the particle gun PDS 1000/He or the particle inflow gun was used. The calli and regenerants were selected for their resistance to the herbicide Basta (glufosinate ammonium) mediated by the bar gene. Putative transformants were screened for enzyme activity by painting selected leaves or spraying whole plants with an aqueous solution of the herbicide Basta and by the histochemical GUS assay using cut leaf segments. PCR and Southern blot analysis of genomic DNA indicated the presence of introduced foreign genes in the genomic DNA of the transformants. Five regenerated plants represent independent transformation events and have been grown to maturity and set seed. The integration of the bar selectable and the gus reporter gene was confirmed by genomic Southern blot analysis in all five plants. All five plants had multiple integrations of both marker genes. To date, the T1 progeny of three out of four lines generated by the PDS particle gun shows co-segregating marker genes, indicating an integration of the bar and the gus gene at the same locus in the genome. 相似文献
6.
7.
Agrobacterium-mediated Japonica rice transformation: a procedure assisted by an antinecrotic treatment 总被引:5,自引:0,他引:5
Enríquez-Obregón Gil A. Prieto-Samsónov Dmitri L. de la Riva Gustavo A. Pérez Marlene Selman-Housein Guillermo Vázquez-Padrón Roberto I. 《Plant Cell, Tissue and Organ Culture》1999,59(3):159-168
An Agrobacterium-mediated transformation protocol for Japonica rice (cv. R321), using conventional genetic vectors and explants pretreated
with antinecrotic compounds is presented. We evaluated the effect of two compounds with known antioxidant activity (ascorbic
acid and cysteine) and silver nitrate on the viability of stem sections taken from in vitro rice plantlets, and on their interaction with Agrobacterium tumefaciens (At 2260) containing a shuttle vector bearing the gusand bar genes. After co-culture, calli formed on the callus-induction medium were supplemented with phosphinotricin and cefotaxime;
putative transgenic plants were recovered on the regeneration medium after three months. All recovered plants were challenged
with the herbicide BASTA under greenhouse conditions, and some resistant individuals were analyzed using PCR and a histochemical
GUS test. Southern blot analysis of several R1 transgenic plants indicated the presence of at least two intact bar gene copies per genome. Inheritance of the bar gene at the R2 generation was confirmed. Antinecrotic pretreatment of the explants provides an adequate environment for the interaction
of A. tumefacienswith the plant cells, thus allowing satisfactory transformation performance without the need of super-binary vectors and hyperinfective
A. tumefaciens strains.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
8.
Stable transformation of Coffea canephora P. was obtained by particle bombardment of embryogenic tissue. Leaf explants were cultured on medium supplemented with 5 µM isopentenyl-adenosine to induce direct embryogenesis. Explants with somatic embryos were transferred to half strength MS medium with 9 µM 2,4 dichlorophenoxyacetic acid. After 2 weeks, the explants with somatic embryos and embryogenic tissue were bombarded with tungsten particles (M-25) carrying the plasmid pCambia3301 (containing the bar and uidA genes) using a high pressure helium microprojectile device. The bombarded explants were submitted to selection on medium containing 5 µM ammonium glufosinate herbicide as selective agent. After 6 months, putative transgenic embryos were transferred to a growth regulator-free medium for germination. The regenerated plantlets were β-glucuronidase (GUS) positive whereas no GUS activity was observed in non-transgenic controls. Incorporation of the bar gene into the genome was confirmed by PCR and Southern blot analysis of the regenerated transformed plants. Greenhouse grown transgenic coffee plants were found to withstand the recommended level of the herbicide Finale™ for weed control.This research was supported by the Consorcio Brasileiro de Pesquisa e Desenvolvimento do Cafe (CBP&D-Cafe). 相似文献
9.
Stable transformation of mature zygotic embryos and regeneration of transgenic plants of chir pine (Pinus roxbughii Sarg.) 总被引:1,自引:0,他引:1
A particle inflow gun was used to transfer the plasmid pAHC25 containing the bar gene conferring resistance to glufosinate and the gusA reporter gene, each driven by the maize ubiquitin promoter, to mature embryos of Pinus roxburghii (chir pine). High levels of transient expression were obtained when embryos were cultured for 6 days on 10 μM benzyl adenine-containing medium and then exposed to high osmoticum (0.5 M sucrose) before and after bombardment. Selection on medium containing Basta enabled recovery of stably transformed shoots, both from the epicotyl and from adventitious buds. The primary transformed shoots from the epicotyl were multiplied via axillary shoots. Transformation was confirmed by histochemical staining for β-glucuronidase (GUS) activity, by polymerase chain reaction (PCR) amplification of fragments of gusA and nos terminator, and by the resistance of needles to Basta. 相似文献
10.
Production of fertile transgenic maize plants by silicon carbide whisker-mediated transformation 总被引:10,自引:1,他引:9
Bronwyn R. Frame Paul R. Drayton Susan V. Bagnall Carol J. Lewnau W. Paul Bullock H. Martin Wilson James M. Dunwell John A. Thompson Kan Wang 《The Plant journal : for cell and molecular biology》1994,6(6):941-948
A simple and inexpensive system for the generation of fertile, transgenic maize plants has been developed. Cells from embryogenic maize suspension cultures were transformed using silicon carbide whiskers to deliver plasmid DNA carrying the bacterial bar and uidA (gus) genes. Transformed cells were selected on medium containing the herbicide bialaphos. Integration of the bar gene and activity of the enzyme phosphinothricin acetyl transferase (PAT) were confirmed in all bialaphos-resistant callus lines analysed. Fertile transgenic maize plants were regenerated. Herbicide spraying of progeny plants revealed that the bar gene was transmitted in a Mendelian fashion. 相似文献
11.
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
12.
Transformation of oat and inheritance of bar gene expression 总被引:2,自引:0,他引:2
Kuai B. Perret S. Wan S. M. Dalton S. J. Bettany A. J. E. Morris P. 《Plant Cell, Tissue and Organ Culture》2001,66(2):79-88
Fertile transgenic plants of oat (Avena sativa L. var. Melys) were produced following microprojectile bombardment of primary embryogenic calli from immature embryos with
two plasmids containing the bar gene or the β-glucuronidase (uidA) gene, after selection with glufosinate ammonium. Eleven plants were regenerated from phosphinothricin resistant callus,
with three of the eleven plants containing either intact or rearranged copies. No plants co-transformed with the non-selected
uidA gene were detected. Stable transmission and expression of the bar gene in the T1 inbred progenies occurred in a Mendelian manner in one line, which contained an intact bar gene, and in all six T2 lines tested from this transformant.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
13.
High-efficiency,microprojectile-mediated cotransformation of sugarcane,using visible or selectable markers 总被引:3,自引:0,他引:3
Robert Bower Adrian R. Elliott Bernard A. M. Potier Robert G. Birch 《Molecular breeding : new strategies in plant improvement》1996,2(3):239-249
Transient expression of the maize anthocyanin regulatory elements,R andC1, was used to optimise parameters for microprojectile-mediated delivery of DNA into sugarcane embryogenic callus. Osmotic treatment of target tissues and particle acceleration in a high-pressure helium pulse increased the frequency of transient expression to 5–8×103 cells per bombardment, with minimal tissue damage. An average of 0.34% of transiently expressing cells developed into stably transformed, anthocyanin-pigmented proembryoids which subsequently regenerated into plantlets. However, constitutive expression ofR andC1 proved deleterious, and no anthocyanin-pigmented plant survived beyond 3 cm in height. We also compared selective subculture of callus portions showing luciferase activity with antibiotic selection on medium containing G418 or phosphinothricin, upon bombardment of callus with constructs driving strong expression ofluc, aphA orbar genes. Selective subculture based on luciferase activity enabled recovery of 1.4±0.5 independent transgenic plants per bombardment, compared to 19.8±3.7 independent transgenic plants per bombardment from an optimised G418 selection regimen, and no transformed plants from phosphinothricin selection. Whenluc andaphA on separate plasmids were coprecipitated onto microprojectiles before bombardment, 67–79% of callus lines selected for G418 resistance also showed luciferase activity detectable under a low-light camera. Southern analysis confirmed a very high cotransformation frequency, with variable copy numbers of introduced genes. The high efficiencies of gene transfer, selection and cotransformation in the optimised system, coupled with the simple initiation and regeneration of embryogenic callus, provide an effective tool for practical genetic transformation of sugarcane. 相似文献
14.
Turmeric (Curcuma longa L.) is an important spice crop plant that is sterile and cannot be improved by conventional breeding. An efficient method
for stable transformation for turmeric, C. longa L., was developed using particle bombardment. Callus cultures initiated from shoots were bombarded with gold particles coated
with plasmid pAHC25 containing the bar and gusA genes each driven by the maize ubiquitin promoter. Transformants were selected on medium containing glufosinate. Transgenic
lines were established on selection medium from 50% of the bombarded calluses. Transgenic shoots regenerated from these were
multiplied and stably transformed plantlets were produced. Polymerase chain reaction (PCR) and histochemical GUS assay confirmed
the stable transformation. Transformed plantlets were resistant to glufosinate. 相似文献
15.
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
16.
N. Bohorova W. Zhang P. Julstrum S. McLean B. Luna R. M. Brito L. Diaz M. E. Ramos P. Estanol M. Pacheco M. Salgado D. Hoisington 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1999,99(3-4):437-444
To enhance the level of resistance to insects in tropical maize germplasm we have developed techniques to successfully transform
elite tropical maize inbred based on the activity of specific cryI proteins against four major maize pests – corn earworm, fall armyworm, southwestern corn borer and sugarcane borer. Constructs
containing cryIAb or cryIAc synthetic genes were used. To generate transgenic plants we have established methods for biolistic bombardment and the selection
and regeneration of immature embryos and calli from the elite tropical lines CML72, CML216, CML323, CML327 and hybrids. Transgenic
plants resistant to the herbicide BastaTM contained the bands for the cry, bar and gus genes as detected by Southern blot analyses. A simple leaf bioassay presented varying levels of resistance to Southwestern
corn borer of transgenic tropical maize carrying the cryIAc gene. Analyses of the progenies confirmed the sexual transmission of the introduced genes and their stable expression.
Received: 25 September 1998 / Accepted: 27 October 1998 相似文献
17.
Three constructs harbouring novel Bacillus thuringiensis genes (Cry1C, Cry2A, Cry9C) and bar gene were transformed into four upland cotton cultivars, Ekangmian10, Emian22, Coker201 and YZ1 via Agrobacterium-mediated transformation. With the bar gene as a selectable marker, about 84.8 % of resistant calli have been confirmed positive by polymerase chain reaction (PCR)
tests, and totally 50 transgenic plants were regenerated. The insertions were verified by means of Southern blotting. Bioassay
showed 80 % of the transgenic plantlets generated resistance to both herbicide and insect. We optimized conditions for improving
the transformation efficiency. A modified in vitro shoot-tip grafting technique was introduced to help entire transplantation. This result showed that bar gene can replace antibiotic marker genes (ex. npt II gene) used in cotton transformation. 相似文献
18.
Nadolska-Orczyk A Pietrusinska A Binka-Wyrwa A Kuc D Orczyk W 《Cellular & molecular biology letters》2007,12(2):206-219
This paper presents a method of Agrobacterium-mediated transformation for two diploid breeding lines of potato, and gives a detailed analysis of reporter gene expression.
In our lab, these lines were also used to obtain tetraploid somatic hybrids. We tested four newly prepared constructs based
on the pGreen vector system containing the selection gene nptII or bar under the 35S or nos promoter. All these vectors carried gus under 35S. We also tested the pDM805 vector, with the bar and gus genes respectively under the Ubi1 and Act1 promoters, which are strong for monocots. The selection efficiency (about 17%)
was highest in the stem and leaf explants after transformation with pGreen where nptII was under 35S. About half of the selected plants were confirmed via PCR and Southern blot analysis to be transgenic and,
depending on the combination, 0 to 100% showed GUS expression. GUS expression was strongest in multi-copy transgenic plants
where gus was under Act1. The same potato lines carrying multi-copy bar under Ubi1 were also highly resistant to the herbicide Basta. The suggestion of using Agrobacterium-mediated transformation of diploid lines of potato as a model crop is discussed herein. 相似文献
19.
A. Nadolska-Orczyk A. Przetakiewicz K. Kopera A. Binka W. Orczyk 《Journal of Plant Growth Regulation》2005,24(1):2-10
Transgenic plants of triticale cv. Wanad were obtained after transformation using three combinations of strain/vectors. Two
of them were hypervirulent Agrobacterium tumefaciens strains (AGL1 and EHA101) with vectors containing bar under maize ubiquitin 1 promoter (pDM805), and both hpt under p35S and nptII under pnos (pGAH). The third one was a regular LBA4404 strain containing super-binary plasmid pTOK233 with selection genes
the same as in pGAH. The efficiency of transformation was from 0 to 16% and it was dependent on the selection factor, auxin
pretreatment, and the strain/vector combination. The highest number of transgenic plants was obtained after transformation
with LBA4404(pTOK233) and kanamycin selection. Pretreatment of explants with picloram led to the highest number of plants
obtained after transformation with both Agrobacterium/vector systems LBA4404(pTOK233) and EHA101(pGAH) and selected with kanamycin. Transgenic character of selected plants was
examined by PCR using specific primers for bar, gus, nptII, and hpt and confirmed by Southern blot hybridization analysis. There was no GUS expression in T0 transgenic plants transformed with gus under p35S. However the GUS expression was detectable in the progeny of some lines. Only 30% of 46 transgenic lines showed
Mendelian segregation of GUS expressing to GUS not expressing plants. In the remaining 70% the segregation was non-Mendelian
and the rate was much lower than 3:1. Factors that might effect expression of transgenes in allohexaploid monocot species
are discussed. 相似文献
20.
Anther culture–derived haploid embryos were used as explants for Agrobacterium‐mediated genetic transformation of bread wheat (Triticum aestivum L. cv CPAN1676) using barley HVA1 gene for drought tolerance. Regenerated plantlets were checked for transgene integration in T0 generation, and positive transgenic haploid plants were doubled by colchicine treatment. Stable transgenic doubled haploid plants were obtained, and transgene expression was monitored till T4 generation, and no transgene silencing was observed over the generations. Doubled haploid transgenic plants have faster seed germination and seedling establishment and show better drought tolerance in comparison with nontransgenic, doubled haploid plants, as measured by per cent germination, seedling growth and biomass accumulation. Physiological evaluation for abiotic stress by assessing nitrate reductase enzyme activity and plant yield under post‐anthesis water limitation revealed a better tolerance of the transgenics over the wild type. This is the first report on the production of double haploid transgenic wheat through anther culture technique in a commercial cultivar for a desirable trait. This method would also be useful in functional genomics of wheat and other allopolyploids of agronomic importance. 相似文献