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1.
Agrobacterium-mediated engineering for sheath blight resistance of indica rice cultivars from different ecosystems 总被引:3,自引:0,他引:3
K. Datta Z. Koukolíková-Nicola N. Baisakh N. Oliva S.K. Datta 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2000,100(6):832-839
A concise T-DNA element was engineered containing the rice class-I chitinase gene expressed under the control of CaMV35S and the hygromycin phosphotransferase gene (hph) as a selectable marker. The binary plasmid vector pNO1 with the T-DNA element containing these genes of interest was mobilized
to Agrobacterium
tumefaciens strain LBA4404 to act as an efficient donor of T-DNA in the transformation of three different indica rice cultivars from
different ecosystems. Many morphologically normal, fertile transgenic plants from these rice cultivars were generated after
Agrobacterium-mediated transformation using 3-week-old scutella calli as initial explants. Stable integration, inheritance and expression
of the chimeric chitinase gene were demonstrated by Southern blot and Western blot analysis of the transformants. Bioassay data showed that transgenic
plants can restrict the growth of the sheath blight pathogen Rhizoctonia solani. Bioassay results were correlated with the molecular analysis. Although we obtained similar results upon DNA-mediated transformation,
this report shows the potential of the cost-effective, simple Agrobacterium system for genetic manipulation of rice cultivars with a pathogenesis-related (PR) gene.
Received: 26 July 1999 / Accepted: 27 August 1999 相似文献
2.
Number and Accuracy of T-DNA Insertions in Transgenic Banana (Musa spp.) Plants Characterized by an Improved Anchored PCR Technique 总被引:3,自引:0,他引:3
Nineteen transgenic banana plants, produced via Agrobacterium-mediated transformation, were analyzed for the integration of T-DNA border regions using an improved anchored PCR technique.
The method described is a relatively fast, three-step procedure (restriction digestion of genomic DNA, ligation of ‘vectorette’-type
adaptors, and a single round of suppression PCR) for the amplification of specific T-DNA border-containing genomic fragments.
Most transgenic plants carried a low number of inserts and the method was suitable for a detailed characterization of the
integration events, including T-DNA border integrity as well as the insertion of non-T-DNA vector sequences, which occurred
in 26% of the plants. Furthermore, the particular band pattern generated by four enzyme/primer combinations for each individual
plant served as a fingerprint, allowing the identification of plants representing identical transformation events. Genomic
Southern hybridization and nucleotide sequence analysis of amplification products confirmed the data obtained by anchored
PCR. Sequencing of seven right or left border junction regions revealed different T-DNA processing events for each plant,
indicating a relatively low frequency of precisely nicked T-DNA integration among the plants studied. 相似文献
3.
pBECKS2000: a novel plasmid series for the facile creation of complex binary vectors, which incorporates "clean-gene" facilities 总被引:5,自引:0,他引:5
A new plasmid series has been created for Agrobacterium-mediated plant transformation. The pBECKS2000 series of binary vectors exploits the Cre/loxP site-specific recombinase system to facilitate the construction of complex T-DNA vectors. The new plasmids enable the rapid
generation of T-DNA vectors in which multiple genes are linked, without relying on the availability of purpose-built cassette
systems or demanding complex, and therefore inefficient, ligation reactions. The vectors incorporate facilities for the removal
of transformation markers from transgenic plants, while still permitting simple in vitro manipulations of the T-DNA vectors.
A `shuttle' or intermediate plasmid approach has been employed. This permits independent ligation strategies to be used for
two gene sets. The intermediate plasmid sequence is incorporated into the binary vector through a plasmid co-integration reaction
which is mediated by the Cre/loxP site-specific recombinase system. This reaction is carried out within Agrobacterium cells. Recombinant clones, carrying the co-integrative binary plasmid form, are selected directly using the antibiotic resistance
marker carried on the intermediate plasmid. This strategy facilitates production of co-integrative T-DNA binary vector forms
which are appropriate for either (1) transfer to and integration within the plant genome of target and marker genes as a single
T-DNA unit; (2) transfer and integration of target and marker genes as a single T-DNA unit but with a Cre/loxP facility for site-specific excision of marker genes from the plant genome; or (3) co-transfer of target and marker genes
as two independent T-DNAs within a single-strain Agrobacterium system, providing the potential for segregational loss of marker genes.
Received: 30 July 1998 / Accepted: 2 November 1998 相似文献
4.
Massimo Galbiati Maria A. Moreno Gregory Nadzan Melina Zourelidou Stephen L. Dellaporta 《Functional & integrative genomics》2000,1(1):25-34
In planta Agrobacterium-mediated transformation combined with a soil-based herbicide selection for transgenic plants was used to recover large numbers
of transgenic Arabidopsis plants for functional genomic studies. A tissue-culture-free system for generating transgenic plants was achieved by infiltrating Arabidopsis plants with Agrobacterium tumefaciens harboring a binary T-DNA vector containing the phosphinothricin acetyltransferase gene from Streptomyces hygroscopicus, and by selecting transgenic Arabidopsis growing in soil by foliar application of the herbicide Finale (phosphinothricin). Analysis of herbicide-resistant plants
indicated that all were transgenic and that the T-DNA transformation process occurred late during flower development, resulting
in a preponderance of independently derived T-DNA insertions. T-DNA insertions were usually integrated in a concatenated,
rearranged form, and using linkage analysis, we estimated that T1 plants carried between one and five T-DNA loci. Using pooling
strategies, both DNA and seed pools were generated from about 38,000 Arabidopsis plants representing over 115,000 independent T-DNA insertions. We show the utility of these transgenic lines for identifying
insertion mutations using gene sequence and PCR-based screening.
Electronic Publication 相似文献
5.
Nakano A Suzuki G Yamamoto M Turnbull K Rahman S Mukai Y 《Molecular genetics and genomics : MGG》2005,273(2):123-129
Introduction of large-DNA fragments into cereals by Agrobacterium-mediated transformation is a useful technique for map-based cloning and molecular breeding. However, little is known about the organization and stability of large fragments of foreign DNA introduced into plant genomes. In this study, we produced transgenic rice plants by Agrobacterium-mediated transformation with a large-insert T-DNA containing a 92-kb region of the wheat genome. The structures of the T-DNA in four independent transgenic lines were visualized by fluorescence in situ hybridization on extended DNA fibers (fiber FISH). By using this cytogenetic technique, we showed that rearrangements of the large-insert T-DNA, involving duplication, deletion and insertion, had occurred in all four lines. Deletion of long stretches of the large-insert DNA was also observed in Agrobacterium. 相似文献
6.
Agrobacterium-mediated barley transformation promises many advantages compared to alternative gene transfer methods, but has so far been established in only a few laboratories. We describe a protocol that facilitates rapid establishment and optimisation of Agrobacterium-mediated transformation for barley by instant monitoring of the transformation success. The synthetic green fluorescent protein (sgfpS65T) reporter gene was introduced in combination with thehpt selectable marker gene into immature embryos of barley (Hordeum vulgare L.) by cocultivation with Agrobacterium tumefaciens strain AGLO harboring binary vector pYF133. Using green fluorescent protein (GFP) as a non-destructive visual marker allowed us to identify single-cell recipients of T-DNA at an early stage, track their fate and evaluate factors that affect T-DNA delivery. GFP screening was combined with a low level hygromycin selection. Consequently, transgenic plantlets ready to transfer to soil were obtained within 50 days of explant culture. Southern blot- and progeny segregation analyses revealed a single copy T-DNA insert in more than half of the transgenic barley plants. T-DNA/barley genomic DNA junctions were amplified and sequenced. The right T-DNA ends were highly conserved and clustered around the first 4 nucleotides of the right 25 bp border repeat, while the left T-DNA ends were more variable, located either in the left 25 bp border repeat or within 13 bp from the left repeat. T-DNAs were transferred from Agrobacterium to barley with exclusion of vector sequence suggesting a similar molecular T-DNA transfer mechanism as in dicotyledonous plants. 相似文献
7.
A series of binary T-DNA vectors (pBECKS) has been created for use in theAgrobacterium-mediated genetic transformation of plants. The pBECKS series has corrected the undesirable features of the popular pBIN19
vector; the deleterious mutation within the coding sequence ofnptII has been amended and the cloning sites are now adjacent to the right border repeat in order to reduce the possibility of
producing truncated sequences of novel genes within transformants. One set of vectors incorporates various combiantions of
the marker genesgusA,C1/Lc,nptII,hph, andbar, for pursuit of early and stable transformation events. A set of constructs which contain deleted T-DNA borders in various
combinations and display predictably altered efficacies for gene transfer has also been created. A modular set of vectors
has been designed to facilitate the insertion and transfer of novel gene sequences by providing anptII-linked plant expression cassette orlacZ-multiple cloning site. A range of antibiotic resistance genes has been incorporated into the non-T-DNA part of the vectors
in order to facilitate their selection across the range ofAgrobacterium virulence strains. 相似文献
8.
Vikrant Nain Rajani Jaiswal Monika Dalal Bandarupalli Ramesh Polumetla A. Kumar 《Plant Molecular Biology Reporter》2005,23(1):59-65
Agrobacterium-mediated genetic transformation is a method of choice for the development of transgenic plants. The presence of latentAgrobacterium that multiplies in the plant tissue in spite of antibiotic application confounds the results obtained by polymerase chain
reaction (PCR) analysis of putative transgenic plants. The presence ofAgrobacterium can be confirmed by amplification of eitherAgrobacterium chromosomal genes or genes present out of transfer DNA (T-DNA) in the binary vector. However, the transgenic nature ofAgrobacterium-contaminated transgenic plants cannot be confirmed by PCR. Here we report a simple protocol for PCR analysis ofAgrobacterium-contaminated transgenic plants. This protocol is based on denaturation and renaturation of DNA. The contaminating plasmid
vector becomes double-stranded after renaturation and is cut by a restriction enzyme having site(s) within the PCR amplicon.
As a result, amplification by PCR is not possible. The genomic DNA with a few copies of the transgene remains single-stranded
and unaffected by the restriction enzyme, leading to amplification by PCR. This protocol has been successfully tested with
4 different binary vectors and 3Agrobacterium tumefaciens strains: EHA105, LBA4404, and GV3101. 相似文献
9.
Marker gene elimination from transgenic barley,using co-transformation with adjacent `twin T-DNAs' on a standard Agrobacterium transformation vector 总被引:14,自引:0,他引:14
Matthews Peter R. Wang Ming-Bo Waterhouse Peter M. Thornton Sarah Fieg Sarah J. Gubler Frank Jacobsen John V. 《Molecular breeding : new strategies in plant improvement》2001,7(3):195-202
We have tested a methodology for the elimination of the selectable marker gene after Agrobacterium-mediated transformation of barley. This involves segregation of the selectable marker gene away from the gene of interest following co-transformation using a plasmid carrying two T-DNAs, which were located adjacent to each other with no intervening region. A standard binary transformation vector was modified by insertion of a small section composed of an additional left and right T-DNA border, so that the selectable marker gene and the site for insertion of the gene of interest (GOI) were each flanked by a left and right border. Using this vector three different GOIs were transformed into barley. Analysis of transgene inheritance was facilitated by a novel and rapid assay utilizing PCR amplification from macerated leaf tissue. Co-insertion was observed in two thirds of transformants, and among these approximately one quarter had transgene inserts which segregated in the next generation to yield selectable marker-free transgenic plants. Insertion of non-T-DNA plasmid sequences was observed in only one of fourteen SMF lines tested. This technique thus provides a workable system for generating transgenic barley free from selectable marker genes, thereby obviating public concerns regarding proliferation of these genes. 相似文献
10.
Sha Y Li S Pei Z Luo L Tian Y He C 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2004,108(2):306-314
Insertional mutagenesis provides a rapid way to clone a mutated gene. Transfer DNA (T-DNA) of Agrobacterium tumefaciens has been proven to be a successful tool for gene discovery in Arabidopsis and rice (Oryza sativa L. ssp. japonica). Here, we report the generation of 5,200 independent T-DNA tagged rice lines. The T-DNA insertion pattern in the rice genome was investigated, and an initial database was constructed based on T-DNA flanking sequences amplified from randomly selected T-DNA tagged rice lines using Thermal Asymmetric Interlaced PCR (TAIL-PCR). Of 361 T-DNA flanking sequences, 92 showed long T-DNA integration (T-DNA together with non-T-DNA). Another 55 sequences showed complex integration of T-DNA into the rice genome. Besides direct integration, filler sequences and microhomology (one to several nucleotides of homology) were observed between the T-DNA right border and other portions of the vector pCAMBIA1301 in transgenic rice. Preferential insertion of T-DNA into protein-coding regions of the rice genome was detected. Insertion sites mapped onto rice chromosomes were scattered in the genome. Some phenotypic mutants were observed in the T1 generation of the T-DNA tagged plants. Our mutant population will be useful for studying T-DNA integration patterns and for analyzing gene function in rice.Electronic Supplementary Material Supplementary material is available in the online version of this article at .Communicated by D. Mackill 相似文献
11.
Summary Fertile transgenic plants of the annual pasture legume Medicago truncatula were obtained by Agrobacterium-mediated transformation, utilising a disarmed Ti plasmid and a binary vector containing the kanamycin resistance gene under the control of the cauliflower mosaic virus 35S promoter. Factors contributing to the result included an improved plant regeneration protocol and the use of explants from a plant identified as possessing high regeneration capability from tissue culture. Genes present on the T-DNA of the Ri plasmid had a negative effect on somatic embryogenesis. Only tissue inoculated with Agrobacterium strains containing a disarmed Ti plasmid lacking the T-DNA region or a Ri plasmid with an inactivated rol A gene regenerated transgenic plants. Fertile transgenic plants were only obtained with disarmed A. tumefaciens, and the introduced NPT II gene was transmitted to R1 progeny.Abbreviations BAP
6-benzylaminopurine
- NAA
1-naphthaleneacetic acid
- NPT
neomycin phosphotransferase 相似文献
12.
13.
T-DNA vector backbone sequences are frequently integrated into the genome of transgenic plants obtained by Agrobacterium-mediated transformation 总被引:5,自引:0,他引:5
De Buck Sylvie De Wilde Chris Van Montagu Marc Depicker Ann 《Molecular breeding : new strategies in plant improvement》2000,6(5):459-468
Transgenic Arabidopsis and tobacco plants (125) derived from seven Agrobacterium-mediated transformation experiments were screened by polymerase chain reaction and DNA gel blot analysis for the presence of vector `backbone' sequences. The percentage of plants with vector DNA not belonging to the T-DNA varied between 20% and 50%. Neither the plant species, the explant type used for transformation, the replicon type nor the selection seem to have a major influence on the frequency of vector transfer. Only the border repeat sequence context could have an effect because T-DNA vector junctions were found in more than 50% of the plants of three different transformation series in which T-DNAs with octopine borders without inner border regions were used. Strikingly, many transgenic plants contain vector backbone sequences linked to the left T-DNA border as well as vector junctions with the right T-DNA border. DNA gel blots indicate that in most of these plants the complete vector sequence is integrated. We assume that integration into the plant genome of complete vector backbone sequences could be the result of a conjugative transfer initiated at the right border and subsequent continued copying at the left and right borders, called read-through. This model would imply that the left border is not frequently recognized as an initiation site for DNA transfer and that the right border is not efficiently recognized as a termination site for DNA transfer. 相似文献
14.
Yasunori Machida Shigehisa Okamoto Shogo Matsumoto Shoji Usami Akiko Yamamoto Yasuo Niwa Soo Doo Jeong Jun Nagamine Nobuyoshi Shimoda Chiyoko Machida Motoko Iwahashi 《Journal of plant research》1989,102(2):331-350
Agrobacterium tumefaciens harbouring the Ti plasmid incites crown gall tumor on dicotyledonous species. Upon infection of these plants, T-DNA in the
Ti plasmid is transferred by unknown mechanisms to plant cells to be integrated into nuclear DNA. WhenAgrobacterium is incubated with protoplasts or seedlings of dicotyledonous plants, circulation of T-DNA and expression ofvir (virulence) genes on the Ti plasmid are induced. The circularization event is efficiently induced by mesophyll protoplasts
of tobacco which are highly competent for transformation by the T-DNA, and is also induced by diffusible phenolic compounds
excreted from the protoplasts. The circularization and formation of crown gall both require the expression of thevirD locus, one of the induciblevir genes. These results suggest that the circularization of T-DNA reflects one of steps of the T-DNA transfer during formation
of crown gall. In contrast to dicotyledonous plants, monocotyledonous plants are thought to be unresponsive to infection byAgrobacterium. We showed that monocotyledonous plants do not excrete diffusible inducers for the expression ofvir genes, while they contain a novel type of a signal substance(s). This inducer is not detected in the exudates of seedlings
of monocotyledonous plants, but is found in the extracts from the seedlings, and also those from the seeds, bran and germ
of wheat and oats. This finding suggests that T-DNA processing, and possibly its transfer, should take place whenAgrobacterium invades seedlings and seeds of monocotyledonous plants.
Recipient of the Botanical Society Award for Young Scientists, 1987. 相似文献
15.
Christoph Grevelding Verena Fantes Elke Kemper Jeff Schell Robert Masterson 《Plant molecular biology》1993,23(4):847-860
Different patterns of T-DNA integration in Arabidopsis were obtained that depended on whether a root or a leaf-disc transformation method was used. An examination of 82 individual transgenic Arabidopsis plants, derived from 15 independent Agrobacterium-mediated transformations in which different cointegrate and binary constructs were used, indicated that the transformation method had a significant influence on the type and copy number of T-DNA integration events. Southern hybridizations showed that most of the transgenic plants produced by a leaf-disc method contained multiple T-DNA insertions (89%), the majority of which were organized as right-border inverted repeat structures (58%). In contrast, a root transformation method mostly resulted in single T-DNA insertions (64%), with fewer right-border inverted repeats (38%). The transformation vectors, including cointegrate and binary types, and the plant selectable markers, hygromycin phosphotransferase and dihydrofolate reductase, did not appear to influence the T-DNA integration patterns. 相似文献
16.
Summary We describe in this paper the construction and use of a set of novel Ti plasmid-derived vectors that can be used to produce transgenic plants. These vectors are based on one of two strategies: 1) double recombination into the wild-type Ti plasmid of genetic information flanked by two T-DNA fragments on a wide-host range plasmid; 2) the binary vector strategy. The vector based on the double recombination principle contains a kanamycin resistance gene for use as a plant selectable marker, a polylinker for the insertion of foreign genes, and a nopaline synthase gene. The vector was constructed such that a disarmed T-DNA results from the double recombination event. The binary vector combines several advantageous features including an origin of replication that is stable in Agrobacterium in the absence of selection, six unique sites for insertion of foreign genes, an intact nopaline synthase gene, and a kanamycin resistance marker for selection of transformed plant cells. All of these vectors have been used to produce tobacco plants transformed with a variety of foreign genes. 相似文献
17.
Pooja Jha Shashi Anjana Rustagi Pankaj Kumar Agnihotri Vishvas M. Kulkarni Vishnu Bhat 《Plant Cell, Tissue and Organ Culture》2011,107(3):501-512
A critical step in the development of a reproducible Agrobacterium tumefaciens mediated transformation system for a recalcitrant species, such as pearl millet, is the establishment of optimal conditions
for efficient T-DNA delivery into target tissue from which plants can be regenerated. A multiple shoot regeneration system,
without any intervening callus phase, was developed and used as a tissue culture system for Agrobacterium-mediated transformation. Agrobacterium super virulent strain EHA105 harboring the binary vector pCAMBIA 1301 which contains a T-DNA incorporating the hygromycin
phosphotransferase (hpt II) and β-glucuronidase (GUS) genes was used to investigate and optimize T-DNA delivery into shoot apices of pearl millet. A
number of factors produced significant differences in T-DNA delivery; these included optical density, inoculation duration,
co-cultivation time, acetosyringone concentration in co-cultivation medium and vacuum infiltration assisted inoculation. The
highest transformation frequency of 5.79% was obtained when the shoot apex explants were infected for 30 min with Agrobacterium O.D.600 = 1.2 under a negative pressure of 0.5 × 105 Pa and co-cultivated for 3 days in medium containing 400 μM acetosyringone. Histochemical GUS assay and polymerase chain
reaction (PCR) analysis confirmed the presence of the GUS gene in putative transgenic plants, while stable integration of
the GUS gene into the plant genome was confirmed by Southern analysis. This is the first report showing reproducible, rapid
and efficient Agrobacterium-mediated transformation of shoot apices and the subsequent regeneration of transgenic plants in pearl millet. The developed
protocol will facilitate the insertion of desirable genes of useful traits into pearl millet. 相似文献
18.
Transfer of non-T-DNA portions of the Agrobacterium tumefaciens Ti plasmid pTiA6 from the left terminus of TL-DNA 总被引:3,自引:0,他引:3
We introduced a plant selection marker, nptII, to the left of border A in the Agrobacterium Ti plasmid pTiA6. Infection of tobacco leaf discs with the modified Agrobacterium strain gave rise to kanamycin-resistant calli which grew in a hormone-dependent manner. Southern hybridization analysis of DNA isolated from four transformants indicated initiation of DNA transfer at or near border A and absence of T-DNA sequences. These results demonstrate that DNA transfer events starting at a left border on a native Ti plasmid and moving away from the T-DNA region occur and that they can be detected by designing a suitable selection strategy. 相似文献
19.
A new plasmid series has been created for Agrobacterium-mediated plant transformation. The pBECKS2000 series of binary vectors exploits the Cre/loxP site-specific recombinase system to facilitate the construction of complex T-DNA vectors. The new plasmids enable the rapid generation of T-DNA vectors in which multiple genes are linked, without relying on the availability of purpose-built cassette systems or demanding complex, and therefore inefficient, ligation reactions. The vectors incorporate facilities for the removal of transformation markers from transgenic plants, while still permitting simple in vitro manipulations of the T-DNA vectors. A `shuttle' or intermediate plasmid approach has been employed. This permits independent ligation strategies to be used for two gene sets. The intermediate plasmid sequence is incorporated into the binary vector through a plasmid co-integration reaction which is mediated by the Cre/loxP site-specific recombinase system. This reaction is carried out within Agrobacterium cells. Recombinant clones, carrying the co-integrative binary plasmid form, are selected directly using the antibiotic resistance marker carried on the intermediate plasmid. This strategy facilitates production of co-integrative T-DNA binary vector forms which are appropriate for either (1) transfer to and integration within the plant genome of target and marker genes as a single T-DNA unit; (2) transfer and integration of target and marker genes as a single T-DNA unit but with a Cre/loxP facility for site-specific excision of marker genes from the plant genome; or (3) co-transfer of target and marker genes as two independent T-DNAs within a single-strain Agrobacterium system, providing the potential for segregational loss of marker genes. 相似文献
20.
M. V. Ramana Rao K. S. Behera N. Baisakh S. K. Datta G. J. N. Rao 《Plant Cell, Tissue and Organ Culture》2009,99(3):277-285
Transgenic rice was developed from ‘Swarna’, the most popular indica rice cultivar (Oryza sativa L.) in South East Asia, with a potato chymotrypsin inhibitor gene (pin2) through Agrobacterium-mediated transformation. Four out of nine primary transgenic plants had a single-copy T-DNA insertion while other five plants
had two copies. Mendelian pattern of inheritance of the transgene (pin2) was observed in the T1 generation progeny plants. Whole plant bioassays conducted at both vegetative and reproductive stages and cut stem assays
showed enhanced levels of resistance of transgenic rice against yellow stem borer. The transgenic rice lines with plant derived
proteinase inhibitor genes would develop into resistant cultivars to fit into resistance breeding strategies as an important
component of integrated pest management in rice. 相似文献