Physiological and molecular mechanisms of radicle development of somatic embryos in Schisandra chinensis cultured in the dark

Abstract

Somatic embryogenesis (SE) is a method for producing plant embryos in vitro and is considered a highly promising approach for micropropagation. As a valuable Chinese herbal medicine, the application of SE in genetic breeding, such as in Schisandra chinensis, faces several technical challenges, including incomplete development of somatic embryos and difficulties in plant regeneration. Here, we established an efficient plant regeneration pathway for somatic embryos in S. chinensis. In this experiment, dark culture conditions were found to significantly improve the plant regeneration rooting rate through SE. To understand the genetic mechanism governing embryogenesis, a comparative transcriptome analysis was performed to elucidate differences between light and dark conditions on somatic embryo development in S. chinensis. Dormant buds of S. chinensis were used as explants, and embryonic calli were cultured in light (16 h/D) or dark conditions for 28 days. The cultivation of explants in darkness has been shown to significantly enhance the production of somatic embryo radicles. Under dark conditions, radicle primordia were initiated at the globular embryo stage and developed from the heart-shaped to the torpedo-shaped embryo stages. To explore the S. chinensis root mechanism, endogenous hormones were quantified, and RNA-seq analysis was performed throughout the process of somatic embryogenesis. The results indicated that from the globular to heart-shaped embryo stages, the levels of IAA and ABA in somatic embryos subjected to the dark treatment were markedly lower (190.965 ng•g− 1 and 525.152 ng•g− 1) than those in somatic embryos exposed to light (597.565 ng•g− 1 and 749.188 ng•g− 1), while the concentrations of GA3 and ZR were lower at all stages under light treatment. Transcriptome sequencing and bioinformatics analysis revealed that the pathways and processes in which the differentially expressed genes in somatic embryos under dark conditions were predominantly enriched were plant hormone signaling, circadian rhythm, and phenylpropanoid biosynthesis. qRT‒PCR was employed to validate the expression of plant hormone signaling transduction-related genes, including GH3, SAUR, ARF1, ARF18, AUX/IAA, MMK1, AHK4, AHK5, and PIF3, and the results were consistent with the transcriptome sequencing results. This work laid the foundation for applied research and could be useful in future reluctant woody plant improvement programs and can even be extended to other species.