Generation and identification of OsCTZFP8-overexpressing rice. (a) Schematic representation of the T-DNA region on the PUbi:OsCTZFP8 construct. The construct contains the bar gene and a maize ubiquitin (Ubi) promoter. The small double arrow bar represents the Ubi-OsCTZFP8 probe used for Southern blot analysis. (b) Agrobacterium-mediated rice seed transformation. The procedures included induction of callus (A), subculture (B), cocultivation and Basta screening (C), differentiation (D), rooting (E), and planet acclimatization (F). (c) PCR analyses for T0 transgenic rice using bar gene-specific primers. PC: plasmid DNA; NT: nontransgenic control plant; 1~19: T0 generation of independent transgenic rice. (d) LibertyLink strip detections of T0 transgenic rice plants; NT: nontransgenic control plant; 1~10: T0 transgenic plants; C: control line; T: test line. (e) Southern blot analysis of OsCTZFP8-overexpressing lines. HindIII-digested genomic DNA from T1 generation was separated on agarose gel and hybridized with DIG-labeled Ubi-OsCTZFP8 probe; NT: nontransgenic control plants; OE-1~OE-8: overexpressing lines; PC: plasmid DNA. (f) mRNA expression of OsCTZFP8 in overexpressing lines. One-step RT-PCR was performed using total RNA extracted from 2-week-old rice leaves. The expression of OsActin1 was used as an internal control; NT: nontransgenic control plants; OE-1~OE-6: single-copy insertion overexpressing lines. (g) Basta resistance segregation assay. Seeds were germinated on 1/2 MS medium (30 mg/L Basta), and Basta resistance were determined at the 5th day. The experiments were performed in two repetitions; NT: nontransgenic control plants; OE-1~6 and OE-3-2: overexpressing lines.