Temporal morphological characterization of embryogenic cultures of sugi (Cryptomeria japonica, Japanese cedar)

Somatic embryogenesis (SE) is a technology used for clonal propagation of Cryptomeria japonica (sugi), yet line specific differences in proembryogenic mass (PEM) developmental stages complicate protocol standardization. In this study, we provide a temporal analysis of PEM development in two male-sterile sugi lines (SSD18 and SSD100) from the same family and a fertile one (ST4‑11‑51) from a different family. Cultures were imaged at seven time points after subculture (3, 6, 9, 12, 15, 18, and 21 days). PEM stages were distinguished and classified. Biomass accumulation was also quantified as fold increase (x) in fresh weight (FW) and dry weight (DW). ST4‑11‑51 exhibited the highest proliferation (over 4x FW and over 15x DW), exceeding SSD18 (over 2x FW and over 8x DW) and SSD100 (over 1x FW and over 7x DW), confirming line dependence. Morphologically, SSD18 produced compact PEMs with long suspensor cells, SSD100 formed larger PEMs with short suspensors and abundant single cells, and ST4‑11‑51 displayed thicker suspensor cells and bigger embryogenic cells area. Temporally, Stage III PEMs peaked at days 9, 12, and 15 in SSD18 and SSD100 but earlier and more transiently in ST4‑11‑51. Single cells increased in later time points (days 18–21), indicating a loss of embryogenic tissue organization. Periods characterized by high percentage of Stage III embryos and minimal accumulation of single cells were identified as days 9–12 for SSD18 and SSD100, and days 3–12 for ST4‑11‑51. Together, these results provide practical benchmarks for synchronizing embryo development and for timing maturation treatments in sugi SE pipelines. We provide the first detailed temporal characterization of proembryogenic mass morphology and composition in Cryptomeria japonica, clarifying developmental phases essential for improving somatic embryogenesis.