Establishing VIGS and CRISPR/Cas9 techniques to verify RsPDS function in radish

Abstract

Virus-induced gene silencing (VIGS) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) systems are effective technologies for rapid and accurate gene function verification in modern plant biotechnology. However, the investigation of gene silencing and editing in radish remains limited. In this study, a bleaching phenotype was generated through the knockdown of RsPDS using tobacco rattle virus (TRV)- and turnip yellow mosaic virus (TYMV)-mediated gene silencing vectors. The TYMV-mediated gene silencing efficiency was higher than the TRV-based VIGS system in radish. The expression level of RsPDS was significantly inhibited using VIGS in ‘NAU-067’ radish leaves. The rootless seedlings of ‘NAU-067’ were infected with Agrobacterium rhizogenes using the 2300GN-Ubi-RsPDS-Cas9 vector with two target sequences. Nine adventitious roots were blue with GUS staining, and four of these adventitious roots were edited at target sequence 1 of the RsPDS gene as indicated by Sanger sequencing. Furthermore, albino lines were generated with A. tumefaciens-mediated transformation of radish cotyledons. Five base substitutions and three base deletions occurred at target sequence 2 in Line 1, and three base insertions and three base substitutions occurred at target sequence 1 in Line 2. This study shows that VIGS and CRISPR/Cas9 techniques can be employed to precisely verify the biological functions of genes in radish, which will facilitate the genetic improvement of vital horticultural traits in radish breeding programs.

Keywords

• Raphanus sativus L., VIGS
• CRISPR/Cas9
• Agrobacterium rhizogenes
• A. tumefaciens
• RsPDS