Fig. 4

Characterization of osbag6 mutant lines under saline-alkaline stress conditions. A Sequence chromatograms showing mutations introduced in the OsBAG6 gene in the osbag6-1 and osbag6-2 mutants using CRISPR/Cas9 technology, as revealed by Sanger sequencing. B Isolation of Cas9-free mutants. The indicated genotypes were sown in media including 50 mg L⁻¹ hygromycin. C Immunoblot analysis of OsBAG6-FLAG in Kitaake, osbag6-1, osbag6-2, and 2 independent complementation lines (Com#1 and Com#2) using anti-FLAG antibody. Histone H3 was used as a loading control. Image (D) and survival rate (E) of Kitaake, 2 independent osbag6 mutant lines, Com#1 and Com#2 before and after recovery from saline-alkaline stress condition (25 mM Na₂CO₃, pH = 10.0). In D, scale bar = 8 cm. In E, Data represent mean ± SD of 5 replicates (n = 5, 32 plants per genotype were used to calculate survival rate per replicate). Significant differences were evaluated by two-way ANOVA, followed by Tukey’s multiple comparison test