*2.1. Plant Material and Growth Condition*

Herbaceous peony hybrid seeds ('Fen Yu Nu' × 'Fen Yu Lou') were harvested in the Shenyang Agricultural University germplasm resources nursery (Shenyang, Liaoning, China) in August 2019. Filled hybrid seeds were used for variable temperature stratification using a previous method [29]. According to the observation of the seed anatomical structure [29], seeds in six key dormancy release stages were collected: stage 1 (S1: dry seed), stage 2 (S2: imbibition seed), stage 3 (S3: the radicle breaking of seed coat), stage 4 (S4: the length of the seed root is 3–4 cm), stage 5 (S5: the basal part of the seed root turns red), and stage 6 (S6: seed germ breakout) (Figure 1). The cotyledons used as explants were obtained using the conventional embryo induction method [30]. Then, the explants were transferred to an MS callus induction and proliferation medium containing 0.5 mg/L 2,4-dichlorophenoxyacetic acid, 0.5 mg/L α-naphthalene acetic acid, 0.5 mg/L thidiazuron, and 1 g/L polyvinyl pyrrolidone (PVP). The *nced5-2* (GK\_328D05) and *nced9-1* (SALK\_033388) genes, which are in the Col-0 background, were obtained from the Arabidopsis Biological Resource Center (ABRC, http://abrc.osu.edu). Homozygous mutants were screened and validated by PCR using the left and right genomic primers (LP and RP) and the T-DNA left border primer (LB) (Supplementary Table S1). Seeds of *A. thaliana* WT (Col-0) and mutants were grown following previously reported methods [31].

**Figure 1.** Seeds of herbaceous peony at six different dormancy release stages.
