T-DNA insertion mutagenesis in Arabidopsis: going back and forth

Approximately 13 000 independent transformants of Arabidopsis have been generated utilising the Agrobacterium-mediated seed infection/transformation procedure. The average number of functional independent T-DNA insertion events per transformed line is 1.5, thus constituting a population with 19 500 independent inserts. These transformed lines have been screened under two environmental regimes for visible alterations in phenotype. In one screen the lines were grown under greenhouse conditions and examined at weekly intervals. In the second, seeds from each line were plated under axenic conditions in vertically oriented square petri plates and examined 4 and 9 days after germination. The mutants observed were placed into 7 distinct phenotypic classes: seedling-lethal, size variant, reduced-fertility, pigment defect, embryo-defective, dramatic morphological, and physiological. The mutational spectrum of the first 8000 transformed lines has been reported earlier. The mutational spectrum for a further 5000 transformed lines is reported here and, with several notable exceptions, is similar to that observed previously.

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T-DNA insertion mutagenesis.

Plant mutation breeding and biotechnology ◽

10.1079/9781780640853.0489 ◽

2012 ◽

pp. 489-506

Author(s):

R. S. Sangwan ◽

S. Ochatt ◽

J. E. Nava-Saucedo ◽

B. Sangwan-Norreel

Keyword(s):

Insertion Mutagenesis ◽

Dna Insertion

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T-DNA Insertion Mutagenesis in Arabidopsis: A Procedure for Unravelling Plant Development

Plant Molecular Biology 2 ◽

10.1007/978-1-4615-3304-7_55 ◽

1991 ◽

pp. 563-574 ◽

Cited By ~ 1

Author(s):

Kenneth A. Feldmann ◽

Anna M. Wierzbicki ◽

Robert S. Reiter ◽

Shirley A. Coomber

Keyword(s):

Plant Development ◽

Insertion Mutagenesis ◽

Dna Insertion

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Large scale genomic rearrangements in selected Arabidopsis thaliana T-DNA lines are caused by T-DNA insertion mutagenesis

BMC Genomics ◽

10.1186/s12864-021-07877-8 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Boas Pucker ◽

Nils Kleinbölting ◽

Bernd Weisshaar

Keyword(s):

Arabidopsis Thaliana ◽

Large Scale ◽

Reverse Genetics ◽

Chromosomal Rearrangements ◽

Insertion Mutagenesis ◽

Flanking Sequence ◽

Experimental Proof ◽

Long Read ◽

Local Assembly ◽

Dna Insertion

Abstract Background Experimental proof of gene function assignments in plants is based on mutant analyses. T-DNA insertion lines provided an invaluable resource of mutants and enabled systematic reverse genetics-based investigation of the functions of Arabidopsis thaliana genes during the last decades. Results We sequenced the genomes of 14 A. thaliana GABI-Kat T-DNA insertion lines, which eluded flanking sequence tag-based attempts to characterize their insertion loci, with Oxford Nanopore Technologies (ONT) long reads. Complex T-DNA insertions were resolved and 11 previously unknown T-DNA loci identified, resulting in about 2 T-DNA insertions per line and suggesting that this number was previously underestimated. T-DNA mutagenesis caused fusions of chromosomes along with compensating translocations to keep the gene set complete throughout meiosis. Also, an inverted duplication of 800 kbp was detected. About 10 % of GABI-Kat lines might be affected by chromosomal rearrangements, some of which do not involve T-DNA. Local assembly of selected reads was shown to be a computationally effective method to resolve the structure of T-DNA insertion loci. We developed an automated workflow to support investigation of long read data from T-DNA insertion lines. All steps from DNA extraction to assembly of T-DNA loci can be completed within days. Conclusions Long read sequencing was demonstrated to be an effective way to resolve complex T-DNA insertions and chromosome fusions. Many T-DNA insertions comprise not just a single T-DNA, but complex arrays of multiple T-DNAs. It is becoming obvious that T-DNA insertion alleles must be characterized by exact identification of both T-DNA::genome junctions to generate clear genotype-to-phenotype relations.

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