Somatic embryogenesis (SE), which leads to the formation of embryonic callus (EC) tissue, is the most promising method for large-scale production and selective breeding of woody plants. However, in many species, SE suffers from low proliferation rates, hindering the production of improved plant materials. One way of improving proliferation rates is achieved by improving the redox status of the culture medium. In this study, we investigated the effects of exogenous glutathione (GSH) and L-buthionine sulfoximine (BSO, the inhibitor of glutathione synthase) on the EC proliferation rate in Korean pine (Pinus koraiensis), using cell lines with both high (F: 001#-001) and low (S: 001#-010) proliferation potential. We found that exogenous GSH promoted cell proliferation in both cell lines, while exogenous BSO inhibited proliferation in both cell lines. At 35 d with exogenous GSH treatment, the fresh weight of F and S cell lines increased by 35.48% and 48.39%, respectively, compared with the control. The exogenous application of GSH increased the intracellular levels of GSH, total GSH (T-GSH), oxidized glutathione (GSSG), ascorbic acid (ASA), total ASA (T-ASA), and the ratios of GSH:T-GSH and ASA:T-ASA in both F and S cell lines. Furthermore, exogenous GSH increased the activity of both glutathione reductase (GR) and dehydroascorbate reductase (DHAR) while decreasing the activity of ascorbate peroxidase (APX) in both cell lines. It appears that the application of exogenous GSH promotes a reducing cultural environment, which is conducive to EC proliferation in Korean pine. By helping to reveal the mechanism whereby GSH regulates redox homeostasis in Korean pine EC cells, we have laid the foundation for a large-scale breeding of Korean pine somatic embryogenesis technology system.

Exogenous glutathione (GSH) promotes the proliferation of embryogenic callus (EC) cells in Korean pine in the course of somatic embryogenesis, and reactive oxygen species (ROS) may play an important role in regulating the proliferation of EC cells by exogenous GSH. However, the concrete metabolic response of ROS is unclear. In this study, two cell lines of Korean pine with high proliferative potential 001#-001 (F, Fast proliferative potential cell line is abbreviated as F) and low proliferative potential 001#-010 (S, Slow proliferative potential cell line is abbreviated as S) were used as test materials. The responses of ROS-related enzymes and substances to exogenous GSH and L-Buthionine-sulfoximine (BSO) were investigated in EC cells. The results showed that the exogenous addition of GSH increased the number of early somatic embryogenesis (SEs) in EC cells of both F and S cell lines, decreased the amount of cell death in both cell lines. Exogenous addition of GSH promoted cell division in both cell lines, increased intracellular superoxide dismutase (SOD) and catalase (CAT) activities, inhibited intracellular hydrogen peroxide (H2O2), malondialdehyde (MDA) and nitric oxide (NO) production, and increased NO/ROS ratio. In conclusion, the exogenous GSH promoting the proliferation of Korean pine EC cells, the activity of intracellular antioxidant enzymes was enhanced, the ROS level was reduced, and the resistance of cells to stress was enhanced.

Somatic embryogenesis (SE), which leads to the formation of embryonic callus (EC) tissue, is the most promising method for large-scale production and selective breeding of woody plants. However, in many species, SE suffers from low induction and proliferation rates, hindering the production of improved plant materials. We investigated the effects of the explant sterilization method, 4 °C cryopreservation, basal medium, ethylene removal, liquid medium supplementation, and a combination of PGRs on embryogenic callus (EC) induction of Korean pine, using immature embryos of Korean pine as explants. The effects of sucrose and maltose on EC proliferation and maturation were investigated. The differences in the maturation ability of EC somatic embryos before and after cryopreservation were evaluated using the induced embryonic cell lines. The results showed that zygotic embryos (ZEs) performed better than megagametophytes (MGs) as explants. The induction rate of EC was significantly increased after 28 days of cryopreservation at 4 °C. The induction rate of EC in the #5 family increased from 10.00% to 62.8%. The EC induction rate of the five families cultured with the DCR basal medium was higher than that with the mLV basal medium. Among them, the induction rate of the #5 family cultured with the mLV basal medium was 23.3%, while that with the DCR basal medium was 60.9%, an increase of 2.6 times. There was no significant difference in the maturation ability of EC somatic embryos before and after cryopreservation. In conclusion, this study provides a method to improve the EC induction rate and maturation ability of Korean pine.