Transcriptome and physiological analysis highlights the hormone, phenylpropanoid, and photosynthesis effects on early somatic embryogenesis in Ginkgo biloba

The somatic embryogenesis (SEs) system in Ginkgo biloba L. has not been fully established due to the recalcitrance of this species, and it has seriously limited the application of SEs technology, such as elite varieties large-scale production, gene editing, or transformation. Recently, we have induced the cotyledon-shaped somatic em-bryos (SE) of G. biloba, which was so far observed the closest to mature somatic embryos. Here, we performed the physiological analysis in six cultured stages during SEs formation, including D-NEC (non-embryogenic callus), D- EC (embryogenic callus), D-SE, D-WHC (while hairy callus), L-EC (EC in light), and L-SE (SE in light). The transcriptomic analysis in D-NEC, D-EC, L-EC, and D-WHC was also carried out. We found that the higher (IAA+ZR+iPA)/ABA and the lower IAA/(iPA+ZR) levels were critical in the somatic embryogenesis of G. biloba. The higher levels of soluble sugar, total flavonoid, terpene lactone, PAL, F3'H, and CCR were beneficial to so-matic embryogenesis. In addition, the peroxidase (POD) might be related to the cell wall formation of somatic embryos in G. biloba. A total of 36517 unigenes were identified in four groups by transcriptome analysis, and genes differentially expressed (DEGs) were related to hormone homeostasis i.e., SAUR50, Jasmonic acid syn-thesis i.e., OPR3, phenylpropanoid biosynthesis i.e., CCR1, POD43 and C4H, terpenoid backbone biosynthesis i. e., HMGS, starch and sucrose metabolism, i.e., coniferin & beta;-glucosidase, and photosynthesis, i.e., D1 protein. Furthermore, the majority of identified up-regulated transcription factors (TFs) such as MYB4, WRKY43, EFR8, and WUS might be responsible for the acquisition of SEs. The light was helpful to the early formation of somatic embryos, and the gene GbWUS1 and GbCLV3 were detected with higher expression in early SE induction of G. biloba. This finding will provide important theoretical and technical support for gene editing, cell totipotency research, and rapid propagation in G. biloba.