The T‐DNA integration pattern in Arabidopsis transformants is highly determined by the transformed target cell

Buck, Sylvie De; Podevin, Nancy; Nolf, Jonah; Jacobs, Anni; Depicker, Anna

doi:10.1111/j.1365-313x.2009.03942.x

Cited by 70 publications

(44 citation statements)

References 60 publications

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“…In contrast to other transformation methods, Agrobacteriummediated transformation generally results in lower transgene copy numbers (Kohli et al 2003;Shou et al 2004). However, multiple integrations and complex integration structures frequently occur during Agrobacterium-mediated transformation (Jorgensen et al 1996;Shou et al 2004;De Buck et al 2009). After investigation of 177 rice T-DNA transformants, Kim et al (2003) estimated that approximately 33% of the transformants carried direct repeats, 21% had inverted repeats with the 5′-end junctions, and 7% carried inverted repeats with the 3′-end junctions.…”

Section: Discussionmentioning

confidence: 99%

Molecular Characterization of Transgenic Rice Producing Resveratrol

Yang¹,

Ahn²,

Kweon³

et al. 2013

Plant Breed. Biotech.

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This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. ABSTRACT Resveratrol, a plant phenolic compound, has potential therapeutic benefits due to its antioxidant properties. This is substantiated by previous studies that show that resveratrol derived from rice grains is an effective treatment agent for metabolic syndrome. Here, we characterized the T-DNA sequence, inserted T-DNA structure, copy number, integrity of the transgene locus, resveratrol synthase gene expression and resveratrol contents in the grains of two resveratrol transgenic rice lines, Iksan515 and Iksan526. The T-DNA transformation vector contained two expression cassettes of the resveratrol synthase gene under the control of the ubiquitin promoter and the bar selection marker gene under the control of the CaMV35S promoter. Flanking sequence analysis indicated that the T-DNAs were inserted into intergenic regions of chromosome 4 for Iksan515 and chromosome 12 for Iksan526. Two T-DNAs connected in an inverted repeat structure at a single locus of the rice genome were identified by whole genome sequencing and Southern blot hybridization in both Iksan515 and Iksan526. No novel open reading frames (ORFs) around insertion sites, sequences encoding allergenic or toxic protein, or other unintended effects by T-DNA insertion were found in either case. In addition, resveratrol synthase gene expression in leaves and resveratrol detection in brown rice grains suggested the successful expression of the inserted foreign resveratrol synthase gene in two transgenic rice lines.

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Section: Discussionmentioning

confidence: 99%

Molecular Characterization of Transgenic Rice Producing Resveratrol

Yang¹,

Ahn²,

Kweon³

et al. 2013

Plant Breed. Biotech.

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“…Most transgenic plants produced by a leaf-disc inoculation method contained multiple T-DNA insertions, whereas root transformation resulted mostly in single T-DNA insertions. Therefore, the Agrobacterium strain, transformation method, and plant target tissue may influence the number of integrated T-DNA molecules (De Buck et al, 2009).…”

Section: Discussionmentioning

confidence: 99%

“…Agrobacterium-mediated transformation generally results in lower transgene copy numbers than do other transformation methods such as particle bombardment or polyethylene glycolmediated transformation (Kohli et al, 1998;Shou et al, 2004). On the other hand, transformation frequently results in unwanted high copy number T-DNA integration events (Jorgensen et al, 1987;Deroles and Gardner, 1988;Shou et al, 2004;De Buck et al, 2009). Multiple integration events, often coupled with inverted repeat T-DNA integration patterns, may affect the stability of transgene expression by silencing mechanisms (Jorgensen et al, 1996).…”

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confidence: 99%

Generation of Backbone-Free, Low Transgene Copy Plants by Launching T-DNA from theAgrobacteriumChromosome

et al. 2009

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In both applied and basic research, Agrobacterium-mediated transformation is commonly used to introduce genes into plants. We investigated the effect of three Agrobacterium tumefaciens strains and five transferred (T)-DNA origins of replication on transformation frequency, transgene copy number, and the frequency of integration of non-T-DNA portions of the T-DNA-containing vector (backbone) into the genome of Arabidopsis (Arabidopsis thaliana) and maize (Zea mays). Launching T-DNA from the picA locus of the Agrobacterium chromosome increases the frequency of single transgene integration events and almost eliminates the presence of vector backbone sequences in transgenic plants. Along with novel Agrobacterium strains we have developed, our findings are useful for improving the quality of T-DNA integration events.

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“…Combining our results with previously suggested T-DNA integration models 12 , truncation of T-DNA likely occurs during its conversion to double-stranded form, as shown here, and/or during integration, at time when plant DNA processing factors are activated. Studies have shown that two T-DNA molecules deriving from different Agrobacterium cells often integrate at the same genomic location 29 , and that various host proteins may mediate the interaction of T-complexes with plant nucleosomes 7 and aid in stripping T-strands of their coating proteins. It would be intriguing to test whether T-strands can be actively directed to the integration sites where active DNA synthesis and repair are occurring and reagents (that is, DNA or RNA fragments and dNTPs) are abundant for the conversion process.…”

Section: Discussionmentioning

confidence: 99%

In vivo formation of double-stranded T-DNA molecules by T-strand priming

Liang

Tzfira

2013

Nat Commun

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During plant genetic transformation, Agrobacterium transfers a single-stranded DNA (T-strand) into the host cell. Increasing evidence suggests that double-stranded (ds) T-DNA, converted from T-strands, are potent substrates for integration. Nevertheless, the molecular mechanism governing T-strand conversion to dsT-DNA is unknown. Integrated T-DNA molecules typically exhibit deletions at their 3 0 end as compared with their 5 0 end. We hypothesize that this may result from asymmetric polymerization of T-DNA's ends. Here we show that b-glucuronidase (GUS) expression from sense T-strands is more efficient than from antisense T-strands, supporting asymmetric conversion. Co-transfection with two partially complementary, truncated GUS-encoding T-strands results in GUS expression, which suggests functional hybridization of the T-strands via complementary annealing and supports the notion that T-strands can anneal with primers. Indeed, red fluorescent protein (RFP) expression from mutated T-strand can be restored by delivery of synthetic DNA and RNA oligonucleotides with partial wild-type RFP sequence, implying the involvement of plant DNA repair machinery.

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The T‐DNA integration pattern in Arabidopsis transformants is highly determined by the transformed target cell

Cited by 70 publications

References 60 publications

Molecular Characterization of Transgenic Rice Producing Resveratrol

Molecular Characterization of Transgenic Rice Producing Resveratrol

Generation of Backbone-Free, Low Transgene Copy Plants by Launching T-DNA from theAgrobacteriumChromosome

In vivo formation of double-stranded T-DNA molecules by T-strand priming

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