This study describes a plantlet regeneration protocol of somatic embryos in Rosa 'John F. Kennedy' (hybrid tea rose). Different somatic embryo sizes exhibited significant differences in the single bud (SB type) regeneration rate and multiple bud (MB type) regeneration rate. The highest single bud (SB type) regeneration rate (27.10%) was obtained from the large size (4 mm × 5 mm). The multiple bud regeneration rate was highest at 39.60% for the medium size (3 mm × 4 mm). Changes in the endogenous hormone content and ratios of various types of embryogenic cultures were clearly diverse: higher contents of abscisic acid (ABA) and indole-3-acetic acid (IAA) occurred in the SPC explant (single-piece cotyledonary somatic embryo) with a regenerated single bud (SB type). In a MW-type somatic embryo (milky-white single-piece-cotyledon explant), the gibberellic acid (GA3)/ABA ratio was the highest (1.807), and the IAA/GA3 ratio was the lowest (0.902). However, the highest ratios of IAA/GA3 (6.159) and the lowest ratios of GA3/ABA (0.383) appeared in SB-type cultures. Additionally, the highest IAA/ABA ratios (6.535) and higher ratios of GA3/ABA (1.729) were found in MB-type cultures. This indicated that ways to regulate plant cell totipotency in Rosa 'John F. Kennedy' somatic embryos differed between single bud (SB type) regeneration and multiple bud (MB type) regeneration. Finally, this study classified and summarized common intermediate materials in in vitro culture based on morphological characteristics and plantlet regeneration pathways. © The Society for In Vitro Biology 2024.