Oligonucleotide-directed gene repair in wheat using a transient plasmid gene repair assay system

Abstract

Oligonucleotide-directed gene repair is a potential technique for agricultural trait modification in economically important crops. However, large variation in the repair frequencies among the scientific reports indicates that there are many factors influencing the repair process. We report here a transient assay system using GFP as a reporter for testing the efficiency of plasmid DNA repair in cultured wheat cells. This assay showed that osmotic medium supplemented with 2,4-D increased the oligo-targeting frequency, and that the repair of a point mutation was more efficient than repair of a single base deletion mutation in cultured scutellum cells of immature wheat embryos. This study provides the first evidence that oligonucleotide-directed mutagenesis is applicable to regenerable cultured wheat scutellum cells.

Commentary: Genetic modification through oligonucleotide-mediated mutagenesis. A GMO regulatory challenge?

“…Successful in vivo gene modification has been demonstrated notably in maize, rice, tobacco and wheat, e.g. to create plants insensitive to the action of a specific herbicide (Dong et al, 2006;Iida and Terada, 2005;Kochevenko and Willmitzer, 2003;Okuzaki and Toriyama, 2004;Zhu et al, 2000). Altered genes have been shown to be stably maintained during mitosis (Beetham et al, 1999;Kochevenko and Willmitzer, 2003), and transmitted in a Mendelian fashion to subsequent generations (Zhu et al, 1999(Zhu et al, , 2000.…”

Relaxed chromatin induced by histone deacetylase inhibitors improves the oligonucleotide-directed gene editing in plant cells

“…The modified pAHC25 plasmid was used to generate a transgenic maize line with mutated Green Fluorescent Protein gene (mGFP) (Dong et al 2006). The original pAHC25 plasmid (Christensen and Quail 1996;Yao et al 2006) was modified by removing the GUS gene using SacI and SmaI restriction enzymes, and the transformation plasmid pAHC25::mGFP was constructed by inserting the mGFP gene between the SacI and SmaI restriction sites.…”

“…A single-stranded DNA oligonucleotide named SDO GFP (5'-ccaCCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGggtG-3', lowercase letters stand for phosphorothioates) was used for correcting the mutation in the GFP gene (Dong et al 2006). The synthesis of the SDO GFP molecule was performed using an Expedite 8909 DNA synthesizer (Applied Biosystems) by standard β-cyanoethyl phosphoramidite chemistry at a nominal scale of 0.2 µmol.…”

“…For the present optimization of protocols for plant gene editing, we first generated an assay system that followed a strategy similar to that published earlier by Zhu et al (1999). Using the vector construct with the mutated GFP gene from Dong et al (2006) we produced transgenic maize cell lines and they were bombarded with synthetic GFP oligonucleotides for correction of the GFP function. Pretreatment of transgenic cells with NaBt or NA generated more relaxed chromatin in the region of the GFP transgenes and increased the potential for oligonucleotide-targeted nucleotide exchange.…”

“…Sauer et al (2016a) reported that oligonucleotide length had a positive correlation with the frequency of targeted edits as monitored by changing the blue fluorescent protein to green fluorescent protein. Dong et al (2006) developed a transient assay system based on cobombardment of mutated GFP plasmid constructs with targeting SDO GFP to the scutellum cells of immature wheat embryos. In this experiment, the restoration of green fluorescence in the cells served as a visual marker for transient plasmid gene repair.…”