A quick method to estimate the T-DNA copy number in transgenic plants at an early stage after transformation,using inverse PCR

Agrobacterium-mediated transformation of plants is known to result in transgenic plants with a variable number of integrated T-DNA copies [1, 2, 3, 7]. Our aim was to obtain transgenic tobacco plants containing one integrated T-DNA copy per genome. Therefore, a quick method was developed to estimate the T-DNA copy number of young transgenic plantlets within 10 weeks after transformation. Inverse polymerase chain reaction (IPCR) was used to amplify junction fragments, i.e. plant genomic DNA sequences flanking the known T-DNA sequences [5].     

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.     

利用Inverse PCR快速筛选单个T—DNA拷贝转基因水稻植株的方法

In order to obtain single T-DNA copy transgenic rice, we have established a quick method to estimate the T-DNA copy number in transgenic rice using inverse PCR (IPCR). IPCR was used to amplify junction fragments, i.e. plant genomic DNA sequences flanking the known T-DNA sequences, which will help to estimate the T-DNA copy number in transgenic rice. We have analyzed 20 transgenic plants of 15 transgenic lines. Most plants (12) contain one integrated T-DNA copy per genome, 3 plants contain two and 1 plant contains 3 copies. In 4 transgenic plants no T-DNA copies could be detected using this method. The IPCR results were further tested by Southern analysis and sequence analysis.     

Agrobacterium T-DNA integration in Arabidopsis is correlated with DNA sequence compositions that occur frequently in gene promoter regions

Mobile insertion elements such as transposons and T-DNA generate useful genetic variation and are important tools for functional genomics studies in plants and animals. The spectrum of mutations obtained in different systems can be highly influenced by target site preferences inherent in the mechanism of DNA integration. We investigated the target site preferences of Agrobacterium T-DNA insertions in the chromosomes of the model plant Arabidopsis thaliana. The relative frequencies of insertions in genic and intergenic regions of the genome were calculated and DNA composition features associated with the insertion site flanking sequences were identified. Insertion frequencies across the genome indicate that T-strand integration is suppressed near centromeres and rDNA loci, progressively increases towards telomeres, and is highly correlated with gene density. At the gene level, T-DNA integration events show a statistically significant preference for insertion in the 5 and 3 flanking regions of protein coding sequences as well as the promoter region of RNA polymerase I transcribed rRNA gene repeats. The increased insertion frequencies in 5 upstream regions compared to coding sequences are positively correlated with gene expression activity and DNA sequence composition. Analysis of the relationship between DNA sequence composition and gene activity further demonstrates that DNA sequences with high CG-skew ratios are consistently correlated with T-DNA insertion site preference and high gene expression. The results demonstrate genomic and gene-specific preferences for T-strand integration and suggest that DNA sequences with a pronounced transition in CG- and AT-skew ratios are preferred targets for T-DNA integration.Electronic Supplementary Material Supplementary material is available for this article at .This revised version was published online in March 2005 with corrections to Dr. Tatarinovas name.     

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.     

利用Inverse PCR快速筛选单个T-DNA拷贝转基因水稻植株的方法

为了获得单个T-DNA插入拷贝的植株, 我们建立了一套利用Inverse PCR(IPCR)快速检测转基因水稻中T-DNA拷贝数的方法。用IPCR的方法可以扩增出与已知T-DNA序列相邻的水稻基因组DNA未知序列,由此推测转基因水稻植株中T-DNA的拷贝数。我们共对15个转化株系20棵不同植株的DNA进行了IPCR检测。其中12株表现为T-DNA单拷贝插入,3株为双拷贝插入,1株为三拷贝插入。另外4株未检测到T-DNA插入拷贝。IPCR分析结果经过Southern杂交和测序的验证。     

Number and Accuracy of T-DNA Insertions in Transgenic Banana (Musa spp.) Plants Characterized by an Improved Anchored PCR Technique

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.     

转基因水稻T—DNA侧翼序列的扩增与分析

利用现有的转抗白叶枯病基因Xa21的水稻材料,通过TAIL－PCR技术扩增出携带Xa21基因的T－DNA的侧翼序列,对24个有效扩增片段的序列分析结果表明,其中14个侧翼序列是水稻DNA,9个含载体主干序列,1个是外源基因Xa21片段,14个T－DNA侧翼的水稻DNA序列与直接转化法外源基因整合位点的基因组序列具有不同的特点,这些T－DNA在水稻染色体上整合后其两端序列的特点类似于在转基因双子叶植物中观察到的现象,在含主干序列的侧翼序列（37．5％,9／24）,中,载体主干序列是以不同的类型出现的。     

T-DNA integration patterns in transgenic tobacco plants

To investigate the various integration patterns of T-DNA generated by infection withAgrobacterium, we developed a vector (pRCV2) for the effective T-DNA tagging and applied it to tobacco (Nicotiana tabacum cv. Havana SR1). pRCV2 was constructed for isolating not only intact T-DNA inserts containing both side borders of T-DNA, but also for partial T-DNA inserts that comprise only the right or left side. We also designed PCR confirmation primer sets that can amplify in several important regions within pRCV2 to detect various unpredictable integration patterns. These can also be used for the direct inverse PCR. Leaf disks of tobacco were transformed withAgrobacterium tumefaciens LBA4404 harboring pRCV2. PCR and Southern analysis revealed the expected 584 bp product for thehpt gene as well as one of 600 bp for thegus gene in all transformants; one or two copies were identified for these integrated genes. Flanking plant genomic DNA sequences from the transgenic tobacco were obtained via plasmid rescue and then sequenced. Abnormal integration patterns in the tobacco genome were found in many transgenic lines. Of the 17 lines examined, 11 contained intact vector backbone; a somewhat larger deletion of the left T-DNA portion was encountered in 4 lines. Because nicking sites at the right border showed irregular patterns when the T-DNA was integrated, it was difficult to predict the junction regions between the vector and the flanking plant DNA.     

Centrifugation Assisted Agrobacterium tumefaciens-mediated Transformation (CAAT) of embryogenic cell suspensions of banana (Musa spp. Cavendish AAA and Lady finger AAB)

Centrifugation-assisted Agrobacterium-mediated transformation (CAAT) protocol, developed using banana cultivars from two economically important genomic groups (AAA and AAB) of cultivated Musa, is described. This protocol resulted in 25-65 plants/50mg of settled cell volume of embryogenic suspension cells, depending upon the Agrobacterium strain used, and gave rise to hundreds of morphologically normal, transgenic plants in two banana cultivars from the two genomic groups. Development of a highly efficient Agrobacterium-mediated transformation protocol for a recalcitrant species like banana, especially the Cavendish group (AAA) cultivars, required the identification and optimisation of the factors affecting T-DNA delivery and subsequent plant regeneration. We used male-flower-derived embryogenic cell suspensions of two banana cultivars (Cavendish and Lady Finger) and Agrobacterium strains AGL1 and LBA4404, harbouring binary vectors carrying hpt (hygromycin phosphotransferase) and gusA (-glucuronidase) or nptII (neomycin phosphotransferase) and a modified gfp (green fluorescent protein) gene in the T-DNA, to investigate and optimise T-DNA delivery and tissue culture variables. Factors evaluated included pre-induction of Agrobacterium, conditions and media used for inoculation and co-cultivation, and the presence of acetosyringone and Pluronic F68 in the co-cultivation media. One factor that led to a significant enhancement in transformation frequency was the introduction of a centrifugation step during co-cultivation. Post co-cultivation liquid-media wash and recovery step helped avoid Agrobacterium overgrowth on filters supporting suspension culture cells. Marker-gene expression and molecular analysis demonstrated that transgenes integrated stably into the banana genome. T-DNA:banana DNA boundary sequences were amplified and sequenced in order to study the integration profile.     

A comprehensive characterization of single-copy T-DNA insertions in the Arabidopsis thaliana genome

T-DNA flanking sequences were isolated from 112 Arabidopsis thaliana single-copy T-DNA lines and sequence mapped to the chromosomes. Even though two T-DNA insertions mapped to a heterochromatic domain located in the pericentromeric region of chromosome I, expression of reporter genes was detected in these transgenic lines. T-DNA insertion did not seem to be biased toward any of Arabidopsis' five chromosomes. The observed distribution of T-DNA copies in intergenic sequence versus gene sequence (i.e. 5-upstream regions, coding sequences and 3-downstream regions) appeared randomly. An evaluation of T-DNA insertion frequencies within gene sequence revealed that integration into 5-upstream regions occurred more frequently than expected, whereas insertions in coding sequences (exons and introns) were found less frequently than expected based on random distribution predictions. In the majority of cases, single-copy T-DNA insertions were associated with small or large rearrangements such as deletions and/or duplications of target site sequences, deletions and/or duplications of T-DNA sequences, and gross chromosomal rearrangements such as translocations. The accuracy of integration was similarly high for both left- and right-border sequences. These results may be called upon when making detailed molecular analyses of transgenic plants or T-DNA induced mutants.     

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.     

Inhibition of cytosine methylation allows efficient cloning of T-DNA tagged plant DNA ofArabidopsis thaliana by plasmid rescue

Summary Plasmid rescue can provide an efficient way of cloning T-DNA-tagged genomic DNA of plants. However, rescue has often been hampered by extensive rearrangements in the cloned DNA. We have demonstrated using a transgenic line ofArabidopsis thaliana that the plant DNA flanking the T-DNA tag was heavily cytosine methylated. This methylation could be completely inhibited by growing the plants in the presence of azacytidine. Rescue of the T-DNA tag together with the flanking plant genomic DNA sequences from nontreated control plants into an modified cytosine restriction (mcr) proficient strain ofEscherichia coli resulted in rearrangements of the majority of the rescued plasmids. These rearrangements could be avoided if the methylation was inhibited in the transgenic plants by azacytidine treatment or by cloning into anmcr-deficient strain ofE. coli. The results indicate that cytosine methylation of the DNA in the transgenic plants is the main cause of the DNA rearrangements observed during plasmid rescue and suggest efficient strategies to eliminate such artifacts.     

Agrobacterium-mediated transformation of quaking aspen (Populus tremuloides) and regeneration of transgenic plants

Summary Agrobacterium-mediated gene transformation of Populus tremuloides Michx was accomplished by co-cultivation of leaf disks excised from greenhouse plants with Agrobacterium tumefaciens containing a binary Ti-plasmid vector harboring chimeric neomycin phosphotransferase (NPT II) and ß-glucuronidase (GUS) genes. Shoot regeneration in the presence of kanamycin was achieved when thidiazuron (TDZ) was used as a plant growth regulator. Transformation was verified by amplification of NPT II and GUS gene fragments from genomic DNA of transgenic plants with polymerase chain reaction (PCR) and integration of these genes into nuclear genome of transgenic plants was confirmed by genomic Southern hybridization analysis. Histochemical assay revealed the expression of GUS gene in leaf, stem and root tissues of transgenic plants, further confirming the integration and expression of T-DNA in these plants. This protocol allows effective transformation and regeneration of quaking aspen using greenhouse-grown materials as an explant source. Whole plant regeneration from cuttings of fieldgrown mature quaking aspen and hybrid poplar (P. alba x P. grandidentata) was also readily achieved by using this protocol, which represents a potential system for producing transgenic quaking aspen and hybrid poplar of valuable genotypes.Abbreviations AMV RNA4 Alfalfa mosaic virus RNA4 - BA 6-benzyladenine - CaMV cauliflower mosaic virus - 2,4-D 2,4-dichlorophenoxyacetic acid - EDTA ethylenediaminetetraacetic acid - FAA formalin-acetic acid-alcohol - GUS ß-glucuronidase - NAA 1-naphthylacetic acid - NPT II neomycin phosphotransferase II - PCR polymerase chain reaction - SDS sodium dodecyl sulphate - TE Tris-Cl/EDTA - TDZ N-phenyl-N-1,2,3-thiadiazol-5-yl-urea (thidiazuron) - WPM woody plant medium (Lloyd and McCown 1980) - X-GLUC 5-bromo-4-chloro-3-indolyl-ß-glucuronic acid     

Transgenic and herbicide resistant pearl millet (Pennisetum glaucum L.) R.Br. via microprojectile bombardment of scutellar tissue

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.     

Transformation of cucumber (Cucumis sativus L.) plants with Agrobacterium rhizogenes

Summary Transgenic cucumber plants (Cucumis sativus L., cv. Straight Eight) were regenerated from roots induced by inoculation of inverted hypocotyl sections with Agrobacterium rhizogenes containing the vector pARC8 in addition to the resident Ri-plasmid. The DNA transferred to the plant from the vector (T-DNA) included a gene which encoded the enzyme neomycin phosphotransferase II, and thus conferred on the plant cells resistance to kanamycin. The transgenic plants looked normal and were positive for the neomycin phosphotransferase II. Southern blot analysis of the transgenic plants revealed that all plants contained vector DNA, but only some of them contained DNA from the Ri plasmid.     

T-DNA vector backbone sequences are frequently integrated into the genome of transgenic plants obtained by Agrobacterium-mediated transformation

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.     

Label-free amperometric immunosensor for the detection of human serum chorionic gonadotropin based on nanoporous gold and graphene

Identifying a good transgenic event from the pool of putative transgenics is crucial for further characterization. In transgenic plants, the transgene can integrate in either single or multiple locations by disrupting the endogenes and/or in heterochromatin regions causing the positional effect. Apart from this, to protect the unauthorized use of transgenic plants, the signature of transgene integration for every commercial transgenic event needs to be characterized. Here we show an affinity-based genome walking method, named locus-finding (LF) PCR (polymerase chain reaction), to determine the transgene flanking sequences of rice plants transformed by Agrobacterium tumefaciens. LF PCR includes a primary PCR by a degenerated primer and transfer DNA (T-DNA)-specific primer, a nested PCR, and a method of enriching the desired amplicons by using a biotin-tagged primer that is complementary to the T-DNA. This enrichment technique separates the single strands of desired amplicons from the off-target amplicons, reducing the template complexity by several orders of magnitude. We analyzed eight transgenic rice plants and found the transgene integration loci in three different chromosomes. The characteristic illegitimate recombination of the Agrobacterium sp. was also observed from the sequenced integration loci. We believe that the LF PCR should be an indispensable technique in transgenic analysis.     

Chromosomal localization of foreign genes in Petunia hybrida

Summary A F1 hybrid of Petunia hybrida, heterozygous for at least one marker on each of the seven chromosomes, was transformed with a modified strain of Agrobacterium tumefaciens in which the phytohormone biosynthetic genes in the transferred DNA (T-DNA) were replaced with a NOS/NPTII/NOS chimeric gene and a wildtype nopaline synthase (NOS) gene. The chimeric gene, which confers kanamycin resistance, was used as selectable marker during the transformation process and the NOS gene was used as a scorable marker in the genetic studies. After plants had been regenerated from the transformed tissues, the transgenic plants that expressed both of these markers were backcrossed to the parental lines. The offspring were examined for the segregation of the NOS gene and the Petunia markers. Genetic mapping was thus accomplished in a single generation.By Southern hybridization analysis we confirmed the presence of the expected T-DNA fragments in the transformed plants. Four out of the six plants presented here, had just one monomeric T-DNA insertion. The sizes of the plant/T-DNA junction fragments suggest that the integration occurred in different sites of the Petunia genome. One transformant gave a more complicated hybridization pattern and possibly has two T-DNA inserts. Another transgenic plant was earlier reported (Fraley et al. 1985) to have two, possibly tandemly repeated T-DNAs.Data is presented on the genetic localization of the T-DNA inserts in six independently obtained transgenic plants. The T-DNA inserts in three plants were mapped to chromosome I. However, the distances between the NOS gene and the marker gene on this chromosome were significantly different. In another transgenic plant the NOS gene was coinherited with the marker on chromosome IV. Two other transgenic plants have the T-DNA insert on chromosome III. A three point cross enabled us to determine that both plants have the NOS gene distally located from the peroxidaseA (prxA) marker and both plants showed about 18% recombination. However, Southern hybridization analysis shows that the sizes of the plant/T-DNA junction fragments in these transgenic plants are different, thus suggesting that the integrations occurred in different sites.     

Rapid and accurate early-stage detection of T-DNA/plant flanking sequences of resistant kumquats

Agrobacterium-mediated genetic transformation is a widely applied tool in plant biotechnology. In this process, genes of interest are integrated into plant genomes via T-DNAs present on plasmids in Agrobacteria. Classical and standard methods for screening transformants, such as Southern blot, are inconvenient for most woodland plants because of extremely low transformation efficiency. For the purpose of identifying transgenic woody lines at early selection stages, a right-border T-DNA/plant conjunction sequence analysis was carried out. By analyzing these sequences, 15 out of 17 kanamycin-resistant kumquats were found to be integrated with foreign genes, and two or more copies were present in 33.3% of the transgenic lines, which is completely concordant with Southern blots. Moreover, T-DNA integration into plant nuclear DNA was random without any sequence hotspots, and cleavage sites are any base of the sequence ‘TGAC’. These results showed that this screening method could not only detect resistant woodland plants rapidly at the early selection stage, but unequivocally detect copy numbers. Compared with other screening technique, this method could save time and effort for conducting genetic transformation in woody plants, and also provides accurate integration information for transgenic plants.