CRISPR/Cas9-Mediated Dual Editing of BcSGR1 and BcSGR2 Exerts Additive Enhancement on Delaying Leaf Senescence in Non-Heading Chinese Cabbage

Abstract

Leaf senescence negatively impacts yield and postharvest quality in non-heading Chinese cabbage (Brassica rapa ssp. chinensis), and the STAY-GREEN (SGR) homologs BcSGR1 and BcSGR2 function as core regulators of chlorophyll degradation. In this study, we constructed CRISPR/Cas9 single- and dual-gene editing vectors using four high-efficiency, high-specificity single-guide RNAs (sgRNAs). By optimizing the transformation system using petiolate cotyledon explants supplemented with 6 mg·L−1 AgNO3, we obtained a PCR-positive transformation efficiency of 26% (this value reflects transformation efficiency; the actual gene editing efficiency for each mutant line is presented in the target sequencing results) and generated BcSGR1 (YB1) and BcSGR2 (YB2) single mutants and a BcSGR1/BcSGR2 double mutant (DKO). All mutant lines exhibited a pronounced stay-green phenotype and delayed leaf senescence. The DKO line produced 3.15-fold higher chlorophyll and only one-quarter of the wild-type MDA level, indicating an additive enhancement effect of the double knockout. Compared with the wild type (3-day shelf life), YB1, YB2 and DKO extended postharvest longevity to 7, 8 and 11 days, with senescence delayed by 15, 18 and 22 days, respectively. Notably, whereas the YB1 and YB2 displayed mild leaf shriveling at later developmental stages, the DKO maintained normal leaf morphology. Collectively, our findings demonstrate that dual editing of BcSGR1 and BcSGR2 exerts an additive effect on delaying leaf senescence and enhances postharvest shelf life without incurring significant yield penalties under the experimental conditions. This study provides a feasible and effective strategy for breeding stay-green cultivars of non-heading Chinese cabbage.