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

Abstract

Virus-induced gene silencing (VIGS) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) system 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, the bleaching phenotype was generated through the knockdown of RsPDS using tobacco rattle virus (TRV)- and turnip yellow mosaic virus (TYMV)-mediated gene silencing vector. 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 occurred blue with GUS staining, amongst which four of these adventitious roots were edited at target sequence 1 of RsPDS gene with Sanger sequencing. Furthermore, the 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. The VIGS and CRISPR/Cas9 techniques could be employed to precisely verify the biological functions of genes in radish. Consequently, it would facilitate the genetic improvement of vital horticultural traits in radish breeding programs.