Synergistic Promotion of Somatic Embryogenesis in Pinus elliottii by Reduced Glutathione and Riboflavin

Somatic embryogenesis (SE) is a core technology for the clonal propagation of forest tree species. However, the low frequency of normal embryos and high rate of abnormal embryos during SE maturation induction in slash pine (Pinus elliottii) severely constrain its commercial application. In this study, comparative transcriptome analysis between normal embryos (NE) and abnormal embryos (AE) revealed that glutathione metabolism and riboflavin metabolism pathways may be involved in the regulation of somatic embryo maturation. Based on these findings, different concentration gradients of reduced glutathione (GSH) and riboflavin were supplemented into the somatic embryo maturation medium to verify their effects. Single-factor experiments showed that the 0.5 mM GSH treatment yielded 158.33 normal embryos per gram of embryogenic callus with a normal embryo ratio of 42.20%, while the 7.5 µM riboflavin treatment yielded 100 normal embryos per gram with a normal embryo ratio of 18.27%. Further 2 × 2 orthogonal combination experiments demonstrated that the synergistic treatment of 0.5 mM GSH and 7.5 µM riboflavin achieved the optimal effect, producing 333.33 normal embryos per gram of embryogenic callus with a normal embryo ratio of 72.88%. Both maturation rate and induction efficiency were significantly superior to single treatments and other combinations. These results demonstrate that GSH and riboflavin can synergistically improve the normal embryo ratio and accelerate somatic embryo maturation in slash pine. The underlying mechanisms involving metabolic and hormonal pathways warrant further physiological and biochemical investigation. This study provides a technical reference for optimizing the somatic embryogenesis system of slash pine.