Development of late-bolting plants by CRISPR/Cas9-mediated genome editing from mesophyll protoplasts of lettuce.

CRISPR/Cas9-mediated introduction of a single base mutation in SOC1, a transcription factor that regulates flowering time, results in late-bolting phenotypes in lettuce. Lettuce is a widely consumed leafy vegetable crop. One of the molecular approaches that can increase leaf yield of lettuce is to delay the onset of flowering. Flowering time or time-to-bolting is not only a valuable trait for lettuce, but also a sought-after phenotype for other leafy vegetable crops. This is because delayed flowering enables more extensive vegetative growth, which leads to higher leaf numbers, and possibly larger leaves. Here, we deployed the most recent gene-editing technique to reduce the expression of SOC1, which is a gene that encodes one of several transcription factors that regulate the onset of flowering in plants. By inducing a single base mutation in SOC1 through Cas9 protein-gRNA ribonucleoproteins (RNPs) system, we showed that the time to first flower bud formation in lettuce is longer than that of wild type. In addition, expression of the floral regulatory genes including LsLFY, LsFUL, LsAPL1, and LsAPL2, was lower in the SOC1 gene edited plants than that of the wild type. The gene-editing technique established in this study could be directly applied for diverse quality improvement of lettuce by direct RNP transfer from protoplasts. Furthermore, it is expected that direct RNP transfer from protoplasts can be used as a useful mean for developing various gene edited crops.