Repositorium

What is a repositorium?

The repositorium is a searchable database that provides data on relevant articles from journals, company web pages and web pages of governmental agencies about studies/applications of genome-editing in model plants and agricultural crops in the period January 1996 to May 2018. Search options are article type, technique, plant, traits or free text. The repositorium is based on the systematic map of Dominik Modrzejewski et al., published in the journal environmental evidence. (Download article PDF).

ZFN-mediated gene targeting of the Arabidopsis protoporphyrinogen oxidase gene through Agrobacterium-mediated floral dip transformation


Typ / Jahr

Journal Article / 2013

Autoren

Pater, Sylvia de; Pinas, Johan E.; Hooykaas, Paul J. J.; van der Zaal, Bert J.

Abstract

Previously, we showed that ZFN-mediated induction of double-strand breaks (DSBs) at the intended recombination site enhanced the frequency of gene targeting (GT) at an artificial target locus using Agrobacterium-mediated floral dip transformation. Here, we designed zinc finger nucleases (ZFNs) for induction of DSBs in the natural protoporphyrinogen oxidase (PPO) gene, which can be conveniently utilized for GT experiments. Wild-type Arabidopsis plants and plants expressing the ZFNs were transformed via floral dip transformation with a repair T-DNA with an incomplete PPO gene, missing the 5' coding region but containing two mutations rendering the enzyme insensitive to the herbicide butafenacil as well as an extra KpnI site for molecular analysis of GT events. Selection on butafenacil yielded 2 GT events for the wild type with a frequency of 0.8 × 10⁻³ per transformation event and 8 GT events for the ZFNs expressing plant line with a frequency of 3.1 × 10⁻³ per transformation event. Molecular analysis using PCR and Southern blot analysis showed that 9 of the GT events were so-called true GT events, repaired via homologous recombination (HR) at the 5' and the 3' end of the gene. One plant line contained a PPO gene repaired only at the 5' end via HR. Most plant lines contained extra randomly integrated T-DNA copies. Two plant lines did not contain extra T-DNAs, and the repaired PPO genes in these lines were transmitted to the next generation in a Mendelian fashion.

Keywords
Agrobacterium/genetics; Arabidopsis/enzymology/genetics; DNA Breaks, Double-Stranded; gene targeting; Plants, Genetically Modified/enzymology/genetics; Protoporphyrinogen Oxidase/genetics
Periodical
Plant biotechnology journal
Periodical Number
4
Page range
510–515
Volume
11
DOI
10.1111/pbi.12040

Techniques

ID Corresponding Author
Country
Plant Species GE Technique
Sequence Identifier
Trait
Type of Alteration
Progress in Research
Key Topic
356 De Pater, Sylvia
Netherlands
Arabidopsis thaliana Zinc-finger nucleases
PPO
herbicide tolerance
SDN2
Basic research
Basic research