CRISPR/Cas9-Mediated Mutagenesis of BrLEAFY Delays the Bolting Time in Chinese Cabbage (Brassica rapa L. ssp. pekinensis)

Abstract

Chinese cabbage has unintended bolting in early spring due to sudden climate change. In this study, late-bolting Chinese cabbage lines were developed via mutagenesis of the BrLEAFY (BrLFY) gene, a transcription factor that determines floral identity, using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system. Double-strand break of the target region via gene editing based on nonhomologous end joining (NHEJ) was applied to acquire useful traits in plants. Based on the 'CT001' pseudomolecule, a single guide RNA (sgRNA) was designed and the gene-editing vector was constructed. Agrobacterium-mediated transformation was used to generate a Chinese cabbage line in which the sequence of the BrLFY paralogs was edited. In particular, single base inserted mutations occurred in the BrLFY paralogs of the LFY-7 and LFY-13 lines, and one copy of T-DNA was inserted into the intergenic region. The selected LFY-edited lines displayed continuous vegetative growth and late bolting compared to the control inbred line, 'CT001'. Further, some LFY-edited lines showing late bolting were advanced to the next generation. The T-DNA-free E1LFY-edited lines bolted later than the inbred line, 'CT001'. Overall, CRISPR/Cas9-mediated mutagenesis of the BrLFY gene was found to delay the bolting time. Accordingly, CRISPR/Cas9 is considered an available method for the molecular breeding of crops.