*4.2. Mutation Detection in T0 Plants*

For detection of transgenes and mutations in regenerated T0 plants, total genomic DNA was extracted from leaf tissues following a modified cetyltrimethylammonium bromide (CTAB) method [41]. The presence of the *HPT* gene was assessed by PCR using the primers HygR-F (5--AGAAGAAGATGTTGGCGACCT-3-) and HygR-R (5--GTCCTGCGGGTAAATAGCT-3-) [42]. Site-specific mutations were detected by PCR amplification using primer pairs flanking the designated target sites in *OsITPK6*, i.e., P6-F (5--CTCGACCCATCCGGTGTTAC-3-) and P6-R (5--AAATCGCAGGGGAGAGATCG-3-) (Figure 1A). The following generalized PCR program was used: 5 min at 94 ◦C, followed by 35 cycles of 30 s at 94 ◦C, 1 min at 60 ◦C, and 1 min at 72 ◦C, with a final extension of 10 min at 72 ◦C.

The PCR products were first subjected to HRM analysis for mutations according to reference [43], putative mutants were sequenced (TSINGKE, Hangzhou, China), and mutated sequences were decoded using the DSDecode program (http://skl.scau.edu.cn/dsdecode/) [44]. Mutations of a few selected plants were further confirmed by clone sequencing.
