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

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.

Keywords

• <i>Brassica rapa</i>
• CRISPR/Cas9
• floral identity
• late bolting
• <i>LEAFY</i> gene