Integration of T-DNA binary vector 'backbone' sequences into the tobacco genome: evidence for multiple complex patterns of integration

During the process of crown gall tumorigenesis, Agrobacterium tumefaciens transfers part of the tumor-inducing (Ti) plasmid, the T-DNA, to a plant cell where it eventually becomes stably integrated into the plant genome. Directly repeated DNA sequences, called T-DNA borders, define the left and the right ends of the T-DNA. The T-DNA can be physically separated from the remainder of the Ti-plasmid, creating a 'binary vector' system; this system is frequently used to generate transgenic plants. Scientists initially thought that only those sequences located between T-DNA left and right borders transferred to the plant. More recently, however, several reports have appeared describing the integration of the non-T-DNA binary vector 'backbone' sequences into the genome of transgenic plants. In order to investigate this phenomenon, we constructed T-DNA binary vectors containing a nos-nptll gene within the T-DNA and a mas2'-gusA (β-glucuronidase) gene outside the T-DNA borders. We regenerated kanamycin-resistant transgenic tobacco plants and analyzed these plants for the expression of the vector-localized gusA gene and for the presence of binary vector backbone sequences. Approximately one-fifth of the plants expressed detectable GUS activity. PCR analysis indicated that approximately 75% of the plants contained the gusA gene. Southern blot analysis indicated that the vector backbone sequences could integrate into the tobacco genome linked either to the left or to the right T-DNA border. The vector backbone sequences could also integrate into the plant genome independently of (unlinked to) the T-DNA. Although we could readily detect T-strands containing the T-DNA within the bacterium, we could not detect T-strands containing only the vector backbone sequences or these vector sequences linked to the T-DNA.     

利用双T-DNA载体系统培育无选择标记转基因烟草

建立了一种利用双T-DNA载体培育无选择标记转基因植物的方法.通过体外重组构建了双T-DNA双元载体pDLBRBbarm.载体中,选择标记nptⅡ基因和另一代表外源基因的bar基因分别位于2个独立的T-DNA.利用农杆菌介导转化烟草(Nicotiana tabacum L.),在获得的转化植株中,同时整合有nptⅡ基因和bar基因的频率为59.2%.对4个同时整合有nptⅡ和bar基因植株自交获得的T1代株系进行检测分析,发现在3个T1代株系2个T-DNA可以发生分离,其中约19.5%的转基因T1代植株中只存在bar基因而不带选择标记nptⅡ.这一结果说明双T-DNA载体系统能有效地用于培育无选择标记的转基因植物.研究还利用位于2个不同载体上的nptⅡ基因与 bar基因通过农杆菌介导共转化烟草,获得共转化植株的频率为20.0%～47.4%,低于使用双T-DNA转化的共转化频率.     

Marker gene elimination from transgenic barley,using co-transformation with adjacent `twin T-DNAs' on a standard Agrobacterium transformation vector

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.     

Alternative selectable markers for potato transformation using minimal T-DNA vectors

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.     

Evidence of multiple complex patterns of T-DNA integration into the rice genome

The transfer of the long T-DNA (T-DNA and non-T-DNA) of a binary plasmid from Agrobacterium into the rice genome was investigated at both molecular and genetic levels. Out of 226 independent transgenic plants, 33% of the transformants contained non-T-DNA sequences. There was no major difference in the frequency of non-T-DNA transfer among three Agrobacterium tumefaciens strains.Four T1 plants containing a single putative long T-DNA insertion were selected for Southern analysis. Three of them were confirmed to have a long T-DNA insertion with a size of greater-than-unit-length of the binary plasmid. This was further confirmed by rescuing the intact binary plasmid from these plants. Our results suggest that long T-DNA transfer by rolling-circle replication from Agrobacterium to rice occurs frequently, and that the high frequency of non-T-DNA transfer should be considered when producing transgenic rice for commercial production. Received: 22 April 1999 / Accepted: 22 June 1999     

pBECKS2000: a novel plasmid series for the facile creation of complex binary vectors, which incorporates "clean-gene" facilities

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     

A set of novel Ti plasmid-derived vectors for the production of transgenic plants

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.     

Development of DNA-mediated transformation systems for plants

The genetic engineering of plants by DNA-mediated gene transfer requires that efficient transformation systems be developed. Considerable progress has been made in manipulating the Ti plasmid of Agrobacterium tumefaciens as a vehicle for delivery of foreign genes into protoplasts of dicotyle-donous plants. Part of the Ti plasmid, the T-DNA, can be incorporated into the genome of the host cell; the T-DNA can carry a foreign DNA sequence which co-integrates with it; under normal conditions, the tumorigenic-causing portion of the T-DNA can be inactivated so that transformed protoplasts can be regenerated and T-DNA with an inserted foreign gene can be stably maintained during regeneration, meiosis and gamete formation. A foreign gene has yet to be expressed in regenerated plants although a T-DNA gene for opine synthesis can function in regenerates. Developing a more ubiquitous transformation system for monocotyledons is further from fruition. Based on transformation systems for simple eukaryotic organisms, it is reasonable to expect that a DNA vector which is capable of amplifying a novel plant gene and which contains both a drug resistance marker to facilitate the selection of transformed plant protoplasts and a species-specific autonomously replicating sequence to ensure the stable maintenance of the input gene in the recipient cell can be constructed.     

一种马铃薯高效无标记转基因技术的建立

用农杆菌介导法转化马铃薯栽培品种紫花白的叶盘,通过1/4 MS培养基预培养、热激处理、低pH、高糖培养基共培养,之后利用PCR直接检测转化体,结果表明遗传转化效率可达5.1%,建立了马铃薯无标记转基因技术.该技术受基因型的限制小,用于其它3个不同的栽培品种东北白、晋薯7号和早大白,遗传转化效率亦达到了4.1%~8.3%.利用这项无标记转基因技术,在载体构建时就剔除了标记基因,遗传转化后直接分化培养,不必对转化细胞进行抗性筛选,缩短了遗传转化周期,省去了费时费力的标记基因剔除步骤,亦为重复转化聚合多个优良基因提供了便利.     

A novel double T-DNA system for producing stack and marker-free transgenic plants

This study aimed to develop a new vector system to remove selection genes and to introduce two or more genes of interest into plants in order to express them in a coordinated manner. A multigene expression vector was established based on pCamBIA2300 using a selectable marker gene (SMG)-free system based on the combination of the isocaudamer technique and double T-DNA. The vector DT7 containing seven target genes was constructed and introduced into tobacco using Agrobacterium-mediated transformation. Twenty-one of 27 positive transgenic plants contained both T-DNA regions. The co-transformation frequency was 77.8 %. The frequency of unlinked integration of two intact T-DNAs was 22.22 % (6/27). The frequency of removal of SMG from transgenic T1 plants was 19.10 %. These results suggest that this vector system was functional and effective for multigene expression and SMG-free transgenic plant cultivation. At least seven target genes can be co-expressed using this system. Overall, these findings provide a new and highly effective platform for multigene and marker-free transgenic plant production.     

A mini binary vector series for plant transformation

A streamlined mini binary vector was constructed that is less than 1/2 the size of the pBIN19 backbone (3.5 kb). This was accomplished by eliminating over 5 kb of non-T-DNA sequences from the pBIN19 vector. The vector still retains all the essential elements required for a binary vector. These include a RK2 replication origin, the nptIII gene conferring kanamycin resistance in bacteria, both the right and left T-DNA borders, and a multiple cloning site (MCS) in between the T-DNA borders to facilitate cloning. Due to the reduced size, more unique restriction sites are available in the MCS, thus allowing more versatile cloning. Since the traF region was not included, it is not possible to mobilize this binary vector into Agrobacterium by triparental mating. This problem can be easily resolved by direct transformation. The mini binary vector has been demonstrated to successfully transform Arabidopsis plants. Based on this mini binary vector, a series of binary vectors were constructed for plant transformation.     

Green fluorescent protein as a vital elimination marker to easily screen marker-free transgenic progeny derived from plants co-transformed with a double T-DNA binary vector system

We investigated the potential of a novel double T-DNA vector for generating marker-free transgenic plants. Co-transformation methods using a double T-DNA vector or using mixture of two Agrobacterium tumefaciens strains were compared, and showed that the double T-DNA vector method could produce marker-free transgenic tobacco (Nicotiana tabacum L.) plants more efficiently. A dual marker double T-DNA vector was then constructed by assembling the green fluorescent protein (GFP) gene mgfp5 and the neomycin phosphotransferase gene nptII into the same T-DNA. The frequency of co-transformants produced by this vector was 56.3%. Co-expression of mgfp5 and nptII was found in 28 out of 29 T₁ lines, and segregation of the reporter -glucuronidase gene, gusA, from mgfp5 to nptII was found in 12 out of 29 T₁ lines. Therefore, GFP could be used as a vital marker to improve the transformation efficiency and to easily monitor the segregation of marker genes, thus facilitating screening of marker-free progeny.     

Agrobacterium-mediated barley (Hordeum vulgare L.) transformation using green fluorescent protein as a visual marker and sequence analysis of the T-DNA∝barley genomic DNA junctions

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.     

T-DNA locus structure in a large population of soybean plants transformed using the Agrobacterium-mediated cotyledonary-node method

Designing transformation experiments for either functional genomics or crop improvement requires knowledge of the transgene locus structure, number, transmission and expression resulting from a specific transformation method. We recently reported an improvement to the soybean [Glycine max (L.) Merrill] cotyledonary-node transformation method that resulted in the efficient production of transgenic plants. To characterize the transgene loci resulting from this method, we analysed 270 independent T0 plants and 95 randomly selected T1 progenies for T-DNA locus complexity using Southern analysis. The lines were transformed with Agrobacterium tumefaciens strains LBA4404 or EHA105 carrying the binary plasmids pGPTV, pTOK233, pCAMBIA1303 or pCAMBIA1309, and regenerated in medium supplemented with or without silver nitrate (AgNO3). Analysis in the T0 generation showed that the number of hpt-hybridizing fragments per plant ranged from 1-15, with 31.5% of the lines having a single hpt-hybridizing fragment. Each primary soybean transformant had, on average, 2.0 unlinked transgene loci and that half of the segregating loci in the T1 progenies were single, simple T-DNA insertions. Of the loci containing multiple T-DNA fragments, a low frequency had tandem and inverted repeat T-DNA structures. Integration of binary plasmid backbone sequences occurred in 37% of primary transformants. A. tumefaciens strain, binary plasmid and thiol treatment had no significant effect on transgene locus structure, numbers or expression. Interestingly, exposure of soybean explants to AgNO3 throughout shoot induction and elongation increased T-DNA locus complexity in the primary transformants and decreased silencing of gusA expression in the T1 generation.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation system for large-scale producion of transgenic chinese cabbage (<Emphasis Type="Italic">Brassica rapa</Emphasis> L. ssp.<Emphasis Type="Italic">pekinensis</Emphasis>) plants for insertional mutagenesis

In order to better utilize insertional mutagenesis and functional genomics in Chinese cabbage, we have developed an improved transformation system that more efficiently produces a large number of transgenic plants. Hypocotyl explants were inoculated withAgrobacterium tumefaciens LBA4404. This strain harbors tagging vector pRCV2, which contains a hygromycin-resistance gene, an ampicillin resistance gene, and a bacterial replication origin within the T-DNA. Transformation efficiency was highest when the explants were first co-cultivated for 3 d in a medium supplemented with 5 mg L^-1 acetosyringone, then transferred to a 0.8% agar selection medium containing 10 mg L^-1 hygro-mycin. In addition, maintaining a low pH in the co-cultivation medium was critical to enhancing transformation frequency. A total of 3369 transgenic plants were obtained, with efficiencies ranging from 2.89% to 5.00%. Southern blot analysis and T, progeny tests from 120 transgenic plants confirmed that the transgenes were stably inherited to the next generation. We also conducted plasmid rescue and inverse PCR with some transformants, based on their phenotype, to demonstrate the applicability of T-DNA tagging in Chinese cabbage. The tagged sequences were then analyzed.     

A plant transformation vector with a minimal T-DNA

Plant transformation, viaAgrobacterium tumefaciens, is usually performed with binary vectors. Most of the available binary vectors contain within the T-DNA (which is transferred to the plant genome) components not required for the intended modification. These additional sequences may cause potential risks during field testing of the transgenic plants or even more in the case of commercialization. The aim of this study was to produce a plant transformation vector which only contains a selectable and screenable marker gene and a multiple cloning site for insertion of promoter::foreign gene::terminator cassettes from other plasmids.     

Excision of a selectable marker in transgenic rice (Oryza sativa L.) using a chemically regulated Cre/loxP system

Removal of a selectable marker gene from genetically modified (GM) crops alleviates the risk of its release into the environment and hastens the public acceptance of GM crops. Here we report the production of marker-free transgenic rice by using a chemically regulated, Cre/loxP-mediated site-specific DNA recombination in a single transformation. Among 86 independent transgenic lines, ten were found to be marker-free in the T₀ generation and an additional 17 lines segregated marker-free transgenic plants in the T₁ generation. Molecular and genetic analyses indicated that the DNA recombination and excision in transgenic rice were precise and the marker-free recombinant T-DNA was stable and heritable.The first two authors contributed equally to the work     

Superfluous Transgene Integration in Plants

Referee: Dr. Paul Hooykass, Institut of Molecular Plant Sciences, Leiden University, Clusius Laboratory, Wassenaarseweg 64, 2333, Al Leiden, Netherlands Recent reports suggest the transfer of superfluous DNA sequences to plant genomes during transformation processes. This review investigates the evidence from the published literature for the prevalence of this phenomenon and highlights methods to limit or prevent DNA transfer and subsequent potentially detrimental evolutionary consequences. Evidence for superfluous foreign DNA transfer using both Agrobacterium-mediated transformation and direct DNA transfer methods such as microprojectile bombardment and PEG-mediated transformation of protoplasts is reported. In the case of Agrobacterium-mediated transformation, the lack of information on the integration of sequences from outside of the T-DNA borders has been due to the general belief by researchers that T-DNA processing is precise. This assumption was based on analysis of T-DNA in tumors and as a result the majority of T-DNA integration events have been identified exclusively using DNA probes, which are homologous only to DNA from within the T-DNA borders. Where direct gene transfer protocols are employed, any part of the transforming plasmid and indeed accompanying carrier DNA may become integrated into the plant genome. The main body of evidence proving that superfluous vector DNA sequences are present in plant genomes transformed using direct transfer methods is confined to the identification of plasmid concatamers integrated into plant genomes. The limited amount of recorded evidence pertaining to superfluous vector DNA integration in transgenic plants and transformed tissues makes it impossible to draw definitive conclusions as to the factors involved in promoting this phenomenon. However, there are methods available for removing superfluous sequences from transgenic plants. These have been developed for the removal of selectable marker genes, whose presence in transgenic plants has been a source of much controversy, but can equally be applied to other DNA sequences. Suggestions have been made in the review that might limit or prevent the integration of superfluous vector sequences during transformation procedures; however, these are not proven and further research is required.