Integration,expression and inheritance of two linked T-DNA marker genes in transgenic lettuce

T-DNA integration and stability were assessed in Agrobacterium-derived transgenic lettuce lines carrying a chimaeric CaMV 35S promoter-driven gus-intron gene and a chimaeric nos.nptII.nos gene. T-DNA integration was predominantly complex in transgenic plants derived from an A. tumefaciens strain carrying the supervirulent plasmid ToK47. Truncation of the right side of the T-DNA was observed in first seed generation R₁ plants from one line. Complex T-DNA integration patterns did not always correlate with low transgene expression. Despite a high T-DNA copy number, ca. 30% of the lines analysed showed high transgene expression in the R₁ generation. High transgene expression was stable at least to the R₄ seed generation in selected high-expressing lines. Transgene expression was lost in the R₂ generation in a low expressing line, while complete, heritable transgene silencing from the R₀ to R₂ generations was also observed in another line. A 50-fold variation in -glucuronidase (GUS) activity and a 16-fold variation in NPTII protein content were observed between R₁ plants derived from different R₀ parents. Reactivation of transgene expression with 5-azacytidine in partially silenced lines indicated that low expression was associated with DNA methylation.     

A large-scale study of rice plants transformed with different T-DNAs provides new insights into locus composition and T-DNA linkage configurations

Transgenic locus composition and T-DNA linkage configuration were assessed in a population of rice plants transformed using the dual-binary vector system pGreen (T-DNA containing the bar and gus genes)/pSoup (T-DNA containing the aphIV and gfp genes). Transgene structure, expression and inheritance were analysed in 62 independently transformed plant lines and in around 4,000 progeny plants. The plant lines exhibited a wide variety of transgenic locus number and composition. The most frequent form of integration was where both T-DNAs integrated at the same locus (56% of loci). When single-type T-DNA integration occurred (44% of loci), pGreen T-DNA was preferentially integrated. In around half of the plant lines (52%), the T-DNAs integrated at two independent loci or more. In these plants, both mixed and single-type T-DNA integration often occurred concurrently at different loci during the transformation process. Non-intact T-DNAs were present in 70–78% of the plant lines causing 14–21% of the loci to contain only the mid to right border part of a T-DNA. In 53–66% of the loci, T-DNA integrated with vector backbone sequences. Comparison of transgene presence and expression in progeny plants showed that segregation of the transgene phenotype was not a reliable indicator of either transgene inheritance or T-DNA linkage, as only 60–80% of the transgenic loci were detected by the expression study. Co-expression (28% of lines) and backbone transfer (53–66% of loci) were generally a greater limitation to the production of marker-free T₁ plants expressing the gene of interest than co-transformation (71% of lines) and unlinked integration (44% of loci).     

Transgene integration and organization in Cotton (Gossypium hirsutum L.) genome

While genetically modified upland cotton (Gossypium hirsutum L.) varieties are ranked among the most successful genetically modified organisms (GMO), there is little knowledge on transgene integration in the cotton genome, partly because of the difficulty in obtaining large numbers of transgenic plants. In this study, we analyzed 139 independently derived T0 transgenic cotton plants transformed by Agrobacterium tumefaciens strain AGL1 carrying a binary plasmid pPZP-GFP. It was found by PCR that as many as 31% of the plants had integration of vector backbone sequences. Of the 110 plants with good genomic Southern blot results, 37% had integration of a single T-DNA, 24% had two T-DNA copies and 39% had three or more copies. Multiple copies of the T-DNA existed either as repeats in complex loci or unlinked loci. Our further analysis of two T1 populations showed that segregants with a single T-DNA and no vector sequence could be obtained from T0 plants having multiple T-DNA copies and vector sequence. Out of the 57 T-DNA/T-DNA junctions cloned from complex loci, 27 had canonical T-DNA tandem repeats, the rest (30) had deletions to T-DNAs or had inclusion of vector sequences. Overlapping micro-homology was present for most of the T-DNA/T-DNA junctions (38/57). Right border (RB) ends of the T-DNA were precise while most left border (LB) ends (64%) had truncations to internal border sequences. Sequencing of collinear vector integration outside LB in 33 plants gave evidence that collinear vector sequence was determined in agrobacterium culture. Among the 130 plants with characterized flanking sequences, 12% had the transgene integrated into coding sequences, 12% into repetitive sequences, 7% into rDNAs. Interestingly, 7% had the transgene integrated into chloroplast derived sequences. Nucleotide sequence comparison of target sites in cotton genome before and after T-DNA integration revealed overlapping microhomology between target sites and the T-DNA (8/8), deletions to cotton genome in most cases studied (7/8) and some also had filler sequences (3/8). This information on T-DNA integration in cotton will facilitate functional genomic studies and further crop improvement.     

Reduced Position Effect in Mature Transgenic Plants Conferred by the Chicken Lysozyme Matrix-Associated Region

Matrix-associated regions may be useful for studying the role of chromatin architecture in transgene activity of transformed plants. The chicken lysozyme A element was shown to have specific affinity for tobacco nuclear matrices, and its influence on the variability of transgene expression in tobacco plants was studied. T-DNA constructs in which this element flanked either the [beta]-glucuronidase (GUS) reporter gene or both reporter and selection gene were introduced in tobacco. The variation in GUS gene activity was reduced significantly among mature first-generation transgenic plants carrying the A element. Average GUS activity became somewhat higher, but the maximum attainable level of gene expression was similar for all three constructs. Transient gene expression assays showed that the A element did not contain general enhancer functions; therefore, its presence seemed to prevent the lower levels of transgene expression. The strongest reduction in variability was found in plants transformed with the construct carrying the A elements at the borders of the T-DNA. In this population, expression levels became copy number dependent. The presence of two A elements in the T-DNA did not interfere with meiosis.     

Segregation of genes transferred to one plant cell from two separate Agrobacterium strains

Agrobacterium tumefaciens and Agrobacterium rhizogenes are soil bacteria which transfer DNA (T-DNA) to plant cells. Two Agrobacterium strains, each with a different T-DNA, can infect plants and give rise to transformed tissue which has markers from both T-DNAs. Although marker genes from both T-DNAs are in the tissue, definitive proof that the tissue is a cellular clone and that both T-DNAs are in a single cell is necessary to demonstrate cotransformation. We have transferred two distinguishable T-DNAs, carried on binary vectors in separate Agrobacterium rhizogenes strains, into tomato cells and have recovered hairy roots which received both T-DNAs. Continued expression of marker genes from each T-DNA in hairy roots propagated from individual root tips indicated that both T-DNAs were present in a single meristem. Also, we have transferred the two different T-DNAs, carried on identical binary vector plasmids in separate Agrobacterium tumefaciens strains, into tobacco cells and recovered plants which received both T-DNAs. Transformed plants with marker genes from each T-DNA were outcrossed to wild-type tobacco plants. Distribution of the markers in the F1 generation from three cotransformed plants of independent origin showed that both T-DNAs in the plants must have been present in the same cell and that the T-DNAs were genetically unlinked. Cotransformation of plant cells with T-DNAs from two bacterial strains and subsequent segregation of the transferred genes should be useful for altering the genetic content of higher plants.     

Transgene behaviour in populations of rice plants transformed using a new dual binary vector system: pGreen/pSoup

Transgene integration, expression level and stability have been studied, across two generations, in a population of rice plants transformed using a new dual binary vector system: pGreen/pSoup. pGreen is a small Ti binary vector unable to replicate in Agrobacterium without the presence of another binary plasmid, pSoup, in the same strain. We engineered both pGreen and pSoup to contain each a different T-DNA. Transformation experiments were conducted using a pGreen vector containing the bar and gusA expression units (no transgene in pSoup) or with a pSoup vector containing an aphIV and gfp expression units (no transgene in pGreen). High plant transformation frequencies (up to 40%) were obtained using herbicide resistance ( bar) or antibiotic resistance ( aphIV) genes. Around 80% of the independently transformed plants expressed unselected reporter genes ( gusA or gfp) present in the vectors. Backbone sequences transfer was frequent (45% of lines) and occurred often in multicopy lines. Around 15-20% of the rice plant lines contained a single T-DNA integration without backbone. Integration of additional transgene copies did not improve expression levels in either T(0) plants or T(1) progenies. Nearly all multicopy lines contained transgenes integrated at several loci in the plant genome, showing that T-DNAs from either pGreen or pSoup frequently integrated at unlinked loci. Precise determination of loci number required the analysis of transgene presence in progeny. Segregation of transgene phenotype was generally misleading and tended to underestimate the real number of transgenic loci. The contribution of this new dual-binary vector system to the development of high-throughput rice transformation systems and to the production of marker-free transgenic rice plants is discussed.     

Efficient production of transgenic citrus plants expressing the coat protein gene of citrus tristeza virus

 The coat protein gene of citrus tristeza virus (CTV) has been introduced into Mexican lime (Citrus aurantifolia Swing.) plants by using an improved Agrobacterium-mediated genetic transformation system. Internodal stem segments from greenhouse-grown seedlings were co-cultivated with A. tumefaciens strain EHA 105 carrying the binary plasmid pBI 121/CTV-CP in a medium rich in auxins that provided the explant cells with the proper treatment to shift them to a competent state for transformation. The transformation frequency was enhanced, and this allowed us to recover 42 transgenic plants from 1200 explants. Regenerated shoots were identified as transformants by performing β-glucuronidase (GUS) assays and subsequently by PCR amplifications of the CTV-CP transgene. Southern analyses revealed that at least one copy of the CTV-CP gene was integrated in all PCR positive plants. Interestingly, 70% of them had linked T-DNAs arranged at one locus. Copy number of the CTV-CP gene varied from one to six among the transgenic lines. Half of them showed truncated T-DNAs in which the left border was lost. Expression of the CTV-CP transgene was demonstrated in 38 out of 42 plants by western analysis and DASI-ELISA. No correlation was found between coat protein expression and transgene copy number or integration pattern. Received: 7 April 1999 / Revision received: 17 June 1999 · Accepted: 24 June 1999     

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.     

Analysis of T-DNA-<Emphasis Type="Italic">Xa21</Emphasis> loci and bacterial blight resistance effects of the transgene <Emphasis Type="Italic">Xa21</Emphasis> in transgenic rice

The genetic loci and phenotypic effects of the transgene Xa21, a bacterial blight (BB) resistance gene cloned from rice, were investigated in transgenic rice produced through an Agrobacterium-mediated transformation system. The flanking sequences of integrated T-DNAs were isolated from Xa21 transgenic rice lines using thermal asymmetric interlaced PCR. Based on the analysis of 24 T-DNA- Xa21 flanking sequences, T-DNA loci in rice could be classified into three types: the typical T-DNA integration with the definite left and right borders, the T-DNA integration linked with the adjacent vector backbone sequences and the T-DNA integration involved in a complicated recombination in the flanking sequences. The T-DNA integration in rice was similar to that in dicotyledonous genomes but was significantly different from the integration produced through direct DNA transformation approaches. All three types of integrated transgene Xa21 could be stably inherited and expressed the BB resistance through derived generations in their respective transgenic lines. The flanking sequences of the typical T-DNA integration consisted of actual rice genomic DNA and could be used as probes to locate the transgene on the rice genetic map. A total of 15 different rice T-DNA flanking sequences were identified. They displayed restriction fragment length polymorphisms (RFLPs) between two rice varieties, ZYQ8 and JX17, and were mapped on rice chromosomes 1, 3, 4, 5, 7, 9, 10, 11 and 12, respectively, by using a double haploid population derived from a cross between ZYQ8 and JX17. The blast search and homology comparison of the rice T-DNA flanking sequences with the rice chromosome-anchored sequence database confirmed the RFLP mapping results. On the basis of genetic mapping of the T-DNA- Xa21 loci, the BB resistance effects of the transgene Xa21 at different chromosome locations were investigated using homozygous transgenic lines with only one copy of the transgene. Among the transgenic lines, no obvious position effects of the transgene Xa21 were observed. In addition, the BB resistance levels of the Xa21 transgenic plants with different transgene copy numbers and on different genetic backgrounds were also investigated. It was observed that genetic background (or genome) effects were more obvious than dosage effects and position effects on the BB resistance level of the transgenic plants.     

Agrobacterium-mediated transformation of Bangladeshi Indica rices

Morphologically normal, fertile transgenic plants were obtained by co-culturing embryogenic calli of the Bangladeshi Indica rice cultivars BR26 and Binni with Agrobacterium tumefaciens strain LBA4404 carrying the super binary vector pTOK233. Acetosyringone (100 microM) in the medium during co-culture (25-28 degrees C) and selection on hygromycin B (50 mg l(-1)) were essential for efficient transformation. Stable integration and expression of beta-glucuronidase, neomycin phosphotransferase and hygromycin phosphotransferase genes in regenerated plants were confirmed by histochemical and fluorometric assays, ELISA and Southern analysis. Two to 3 copies of T-DNA were integrated into regenerated plants; transgene expression did not correlate with gene copy number. Mendelian segregation of transgenes occurred in T1 seed progeny.     

Assessment of transgenic maize events produced by particle bombardment or Agrobacterium-mediated transformation

Particle bombardment and Agrobacterium-mediated transformation are two popular methods currently used for producing transgenic maize. Agrobacterium-mediated transformation is expected to produce transformants carrying fewer copies of the transgene and a more predictable pattern of integration. These putative advantages, however, tradeoff with transformation efficiency in maize when a standard binary vector transformation system is used. Using Southern, northern, real-time PCR, and real-time RT-PCR techniques, we compared transgene copy numbers and RNA expression levels in R₁ and R₂ generations of transgenic maize events generated using the above two gene delivery methods. Our results demonstrated that the Agrobacterium-derived maize transformants have lower transgene copies, and higher and more stable gene expression than their bombardment-derived counterparts. In addition, we showed that more than 70% of transgenic events produced from Agrobacterium-mediated transformation contained various lengths of the bacterial plasmid backbone DNA sequence, indicating that the Agrobacterium-mediated transformation was not as precise as previously perceived, using the current binary vector system.     

Effect of ploidy and homozygosity on transgene expression in primary tobacco transformants and their androgenetic progenies

Expression of a transgene is rarely analysed in the androgenetic progenies of the transgenic plants. Here, we report differential transgene expression in androgenetic haploid and doubled haploid (DH) tobacco plants as compared to the diploid parental lines, thus demonstrating a gene dosage effect. Using Agrobacterium-mediated transformation, and bacterial reporter genes encoding neomycin phosphotransferase (nptII) and β-glucuronidase (uidA/ GUS), driven respectively by the mas 1′ and mas 2′ promoters, we have generated more than 150 independent transgenic (R₀) Nicotiana tabacum plants containing one or more T-DNA copies. Transgene analyses of these R₀, their selfed R₁ lines and their corresponding haploid progenies showed an obvious position effect (site of T-DNA insertion on chromosome) on uidA expression. However, transgene (GUS) expression levels were not proportional to transgene copy number. More than 150 haploids and doubled haploids, induced by treatment with colchicine, were produced from 20 independent transgenic R₀ plants containing single and multiple copies of the uidA gene. We observed that homozygous DH plants expressed GUS at approximately 2.9-fold the level of the corresponding parental haploid plants. This increase in transgene expression may be attributed mainly to the increase (2-fold) in chromosome number. Based on this observation, we suggest a strong link between chromosome number (ploidy dosage effect) and transgene expression. In particular, we demonstrate the effect on its expression level of converting the transgene from the heterozygous (in R₀ plants) to the homozygous (DH) state: e.g. an increase of 50% was observed in the homozygous DH as compared to the original heterozygous diploid plants. We propose that ploidy coupled with homozygosity can result in a new type of gene activation, creating differences in gene expression patterns. Received: 27 April 1998 / Accepted: 12 August 1998     

Transgenic tomato (Lycopersicon esculentum L.) transformed with a binary vector in Agrobacterium rhizogenes: Non-chimeric origin of callus clone and low copy numbers of integrated vector T-DNA

Summary We transformed tomato (Lycopersicon esculentum L.) by using Agrobacterium rhizogenes containing two independent plasmids: the wild-type Ri-plasmid, and the vector plasmid, pARC8. The T-DNA of the vector plasmid contained a marker gene (Nos/Kan) encoding neomycin phosphotransferase which conferred resistance to kanamycin in transformed plant cells. Transgenic plants (R ₀) with normal phenotype were regenerated from transformed organogenic calli by the punctured cotyledon transformation method. Southern blot analysis of the DNA from these transgenic plants showed that one or two copies of the vector plasmid T-DNA, but none of the Ri-plamid T-DNA, were integrated into the plant genome. Different transgenic plants derived from the same callus clone showed an identical DNA banding pattern, indicating the non-chimeric origin of these plants. We also transformed tomato by using A. tumefaciens strain LBA4404 containing a disarmed Ti-plasmid (pAL4404), and a vector plasmid (pARC8). Transgenic plants derived via A. tumefaciens transformation, like those via A. rhizogenes, contained one to two copies of the integrated vector T-DNA. The kanamycin resistance trait in the progeny (R ₁) of most transgenic plants segregated at a ratio of 3:1, suggesting that the vector T-DNAs were integrated at a single site on a tomato chromosome. In some cases, the expression of the marker gene (Nos/Kan) seemed to be suppressed or lost in the progeny.     

Cotransformation of aspen and birch with three T-DNA regions from two different replicons in one <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> strain

The cointegration rate into the aspen and birch genomes of foreign genes from a binary vector and a disarmed Ti plasmid pCBE21 carried by the same Agrobacterium tumefaciens strain was studied. The cotransformation rate for the genes within the Ti plasmid varied from 30 to 100%; while the transformation rate for the gene from T_L region was twofold higher as compared with the T_R region. On the average, the gene transfer from all three T-DNAs was recorded in 10.9% of the transgenic lines. For the vector pBI121, the cotransformation rates for the genes from both regions of pCBE21 T-DNA were higher as compared with the vector pGS. In addition, a concurrent transfer of the genes from the Ti plasmid T_L and T_R regions was recorded only after the transformation with the vector pBI121. These results can be used for constructing woody plants containing several genes.     

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.     

T-DNA tagging in the model legume Medicago truncatula allows efficient gene discovery

The annual legume Medicago truncatula has been proposed as a model plant to study various aspects of legume biology including rhizobial and mycorrhizal symbiosis because it is well suited for the genetic analysis of these processes . To facilitate the characterization of M. truncatula genes participating in various developmental processes we have initiated an insertion mutagenesis program in this plant using three different T-DNAs as tags. To investigate which type of vector is the most suitable for mutagenesis we compared the behavior of these T-DNAs. One T-DNA vector was a derivative of pBin19 and plant selection was based on kanamycin resistance. The two other vectors carried T-DNA conferring Basta resistance in the transgenic plants. For each T-DNA type, we determined the copy number in the transgenic lines, the structure of the T-DNA loci and the sequences of the integration sites. The T-DNA derived from pBin19 generated complex T-DNA insertion patterns. The two others generally gave single copy T-DNA inserts that could result in gene fusions for the pGKB5 T-DNA. Analysis of the T-DNA borders revealed that several M. truncatula genes were tagged in these transgenic lines and in vivo gus fusions were also obtained. These results demonstrate that T-DNA tagging can efficiently be used in M. truncatula for gene discovery.     

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.     

Analysis of the T-DNA structure in a large number of transgenic petunias generated by Agrobacterium-mediated transformation

Southern hybridisation was performed on ninety-six transgenic petunias that had been selected for resistance to kanamycin. Just over half of the plants contained intact copies of the T-DNA. The most common rearrangements (at least 24 plants out of 96) were simple deleted derivatives that had lost one or both ends of the T-DNA. T-DNAs lacking the left border occurred at a frequency of 20%, and estimates of the frequency of T-DNAs lacking the right border were at least this high. Three plants contained grossly rearranged T-DNAs, of which all expressed the kanamycin resistance gene but only one transmitted the gene to progeny. Two plants lacked T-DNA homology altogether and did not express kanamycin resistance in their leaves or their progeny. Circumstantial evidence suggests that plants containing a chimaeric kanamycin resistance gene driven by the ocs promoter do not root efficiently in the presence of kanamycin. There was no correlation between intactness of the T-DNA and Mendelian inheritance of the kanamycin-resistance phenotype. However, a disproportionate number of plants showing non-Mendelian inheritance had a high copy number of their T-DNA.