Fig. 4

Overexpression of VaPYL4 enhances cold tolerance of grape calli. A Grape calli induced from V. amurensis petioles on selection medium. The V. amurensis petioles were used as explants for co-culture with Agrobacterium cells that contain the VaPYL4 overexpression vector. After co-culture, the V. amurensis petioles were placed on Gamborg medium supplemented with 50 mg/L kanamycin. The induced kanamycin-resistant callus is indicated by red arrow. B Detection of EGFP fluorescence in induced grape calli. C Knockout (KO) of VaPYL4 in grape calli. Two sgRNAs targeting the exon of VaPYL4 were cloned into the pCACRISPR/Cas9 vector. The results of targeted mutagenesis in VaPYL4 in two independent calli (KO-1 and KO-2) were shown. The sequences of sgRNAs are shown in red, while the PAM (protospacer-adjacent motif) sequences are indicated in green. WT, wild-type sequence; Mut, mutated sequence. The number of analyzed amplicons and mutation efficiencies are shown on the right. D Values of low-temperature exotherms (LTEs) of transgenic grape calli. Data are shown as means ± SD collected from at least five biological replicates. **P < 0.01