Fluorescence <i>in situ</i> hybridization on extended DNA fibres as a tool to analyse complex T‐DNA loci in potato

Wolters, Anne‐Marie A.; Trindade, Luisa M.; Jacobsen, E.; Visser, Richard G. F.

doi:10.1046/j.1365-313x.1998.00079.x

Cited by 48 publications

(27 citation statements)

References 36 publications

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“…T-DNA repeats are a frequent pattern found at insertion sites following A. tumefaciens-mediated transformation (Cluster et al, 1996;Krizkova and Hrouda, 1998;Wolters et al, 1998;De Paepe et al, 2009). Moreover, following cotransformation using different T-DNA constructs, cointegration of the T-DNAs often occurs into the same site (De Buck et al, 2000;Radchuk et al, 2005).…”

Section: T-dna Integration and The Formation Of Complex T-dna Insertionsmentioning

confidence: 99%

Formation of Complex Extrachromosomal T-DNA Structures in Agrobacterium tumefaciens-Infected Plants

Singer

Shiboleth

et al. 2012

Plant Physiology

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Agrobacterium tumefaciens is a unique plant pathogenic bacterium renowned for its ability to transform plants. The integration of transferred DNA (T-DNA) and the formation of complex insertions in the genome of transgenic plants during A. tumefaciensmediated transformation are still poorly understood. Here, we show that complex extrachromosomal T-DNA structures form in A. tumefaciens-infected plants immediately after infection. Furthermore, these extrachromosomal complex DNA molecules can circularize in planta. We recovered circular T-DNA molecules (T-circles) using a novel plasmid-rescue method. Sequencing analysis of the T-circles revealed patterns similar to the insertion patterns commonly found in transgenic plants. The patterns include illegitimate DNA end joining, T-DNA truncations, T-DNA repeats, binary vector sequences, and other unknown "filler" sequences. Our data suggest that prior to T-DNA integration, a transferred single-stranded T-DNA is converted into a doublestranded form. We propose that termini of linear double-stranded T-DNAs are recognized and repaired by the plant's DNA double-strand break-repair machinery. This can lead to circularization, integration, or the formation of extrachromosomal complex T-DNA structures that subsequently may integrate.

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Section: T-dna Integration and The Formation Of Complex T-dna Insertionsmentioning

confidence: 99%

Formation of Complex Extrachromosomal T-DNA Structures in Agrobacterium tumefaciens-Infected Plants

Singer

Shiboleth

et al. 2012

Plant Physiology

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“…T-DNA is frequently inserted into chromosomes as a tandem or invert repeat (Krizkova and Hrouda, 1998;Jeon et al, 2000;Kumar and Fladung, 2000). The vector backbone is also commonly cotransferred with the T-DNA (Kononov et al, 1997;Wolters et al, 1998;de Buck et al, 2000;Kim et al, 2003). In our study, the nucleotide sequence of the amplified DNA fragments was analyzed by the BLASTN homology search program, using the rice genomic sequence databases in National Center for Biotechnology Information (NCBI) and RiceGD.…”

Section: Isolation Of Sequences Flanking T-dnamentioning

confidence: 99%

Generation and Analysis of End Sequence Database for T-DNA Tagging Lines in Rice

et al. 2003

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We analyzed 6,749 lines tagged by the gene trap vector pGA2707. This resulted in the isolation of 3,793 genomic sequences flanking the T-DNA. Among the insertions, 1,846 T-DNAs were integrated into genic regions, and 1,864 were located in intergenic regions. Frequencies were also higher at the beginning and end of the coding regions and upstream near the ATG start codon. The overall GC content at the insertion sites was close to that measured from the entire rice (Oryza sativa) genome. Functional classification of these 1,846 tagged genes showed a distribution similar to that observed for all the genes in the rice chromosomes. This indicates that T-DNA insertion is not biased toward a particular class of genes. There were 764, 327, and 346 T-DNA insertions in chromosomes 1, 4 and 10, respectively. Insertions were not evenly distributed; frequencies were higher at the ends of the chromosomes and lower near the centromere. At certain sites, the frequency was higher than in the surrounding regions. This sequence database will be valuable in identifying knockout mutants for elucidating gene function in rice. This resource is available to the scientific community at http://www.postech.ac.kr/life/pfg/risd.Insertional mutagenesis is one of the most useful methods for analyzing gene function. When foreign DNA is inserted into a gene, it not only creates a mutation but also tags the affected gene, facilitating its isolation and characterization (Azpiroz-Leehan and Feldmann, 1997). Transposons and T-DNA have been used most widely as an insertional mutagen (Mathur et al., 1998;Wisman et al., 1998; Krysan et al., 1999;Parinov et al., 1999;Speulman et al., 1999;Tissier et al., 1999). It is believed that T-DNA insertion is a random event and that the inserted sequences are stable through multiple generations (Azpiroz- Leehan and Feldmann, 1997;Parinov and Sundaresan, 2000). Insertional mutant pools have been constructed in Arabidopsis and used for functional analysis of a number of genes (Feldmann, 1991; Koncz et al., 1992; Azpiroz-Leehan and Feldmann, 1997; Bechtold and Pelletier, 1998; Krysan et al., 1999; Galbiati et al., 2000;Parinov and Sundaresan, 2000; Bouché and Bouchez, 2001;Sessions et al., 2002;Szabados et al., 2002). The procedure for T-DNA insertional mutagenesis has also been applied to rice (Oryza sativa) using the Agrobacterium tumefaciensmediated transformation method (Hiei et al., 1994). Jeon et al. (2000) have reported the construction of over 20,000 T-DNA-tagged rice lines. A T-DNA insertional mutagen can be modified to trap a gene by inserting a reporter gene, such as gus (␤-glucuronidase), next to the T-DNA border (Sundaresan et al., 1995; Jeon et al., 2000;Springer, 2000). Approximately 5% to 10% of the mutagenized lines are GUS positive, demonstrating the efficiency of this gene-trapping system (Chin et al., 1999; Jeon et al., 2000).Completion of the genome sequencing for both Arabidopsis and rice has provided new reverse genetic means for assigning biological functions to sequenced genes (Kumar...

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“…Lipids are an essential component needed for pollen tube growth and penetration (Wolters et al 1998). It provides guidance and an adequate environment for pollen tube development and nutrition over a long distance through the style from the stigma toward the ovary (Erbar 2003).…”

Section: Transmitting Tissuementioning

confidence: 99%

Structural features of Rhododendron luteum flower

Sawidis¹,

Theodoridou²,

Weryszko-Chmielewska³

et al. 2011

Biologia

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The flower of Rhododendron luteum (L.) Sweet has a pentamerous structure with radial symmetry. The anthers filament surface is covered by dense non-glandular hairs to the half of the height. The tubular anther dehisces along creating two openings in the anther-sac walls and the viscous pollen is released through two splits along the anther lobes. The pistil is pentamerous and the axial channel is filled with a mucilaginous secretion product which is continuous with the exudate on the stigma surface. The stigmatic papillae are densely packed and their exudate is stained intensively red for carbohydrates, while pollen grains are stained positively for lipids. The five-locular ovary has isomerous carpels (syncarpous gynoecium) and the ovary surface is covered by numerous, densely-packed glandular and non-glandular hairs protecting the nectar against transpiration. Numerous ovules per locule occur with one integument and a thin-walled megasporiangium. In carpels, oil cells occur sporadically as solitary idioblasts, located around the vascular bundles. Transmitting tissue cells contain a large central, electron translucent vacuole, filling most of the cell containing dark osmiophilic bodies homogenous or granular in appearance.

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Fluorescence in situ hybridization on extended DNA fibres as a tool to analyse complex T‐DNA loci in potato

Cited by 48 publications

References 36 publications

Formation of Complex Extrachromosomal T-DNA Structures in Agrobacterium tumefaciens-Infected Plants

Formation of Complex Extrachromosomal T-DNA Structures in Agrobacterium tumefaciens-Infected Plants

Generation and Analysis of End Sequence Database for T-DNA Tagging Lines in Rice

Structural features of Rhododendron luteum flower

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