Prolonged culture of Boesenbergia rotunda cells reveals decreased growth and shoot regeneration capacity

Boesenbergia rotunda is an important medicinal herb that contains useful bioactive compounds. Although micropropagation of ginger has undergone spectacular development in recent years, gradual loss of plant regeneration capacity in prolonged cell suspension culture continues to be a significant limitation. In this study, biochemical changes and proteins associated with plant totipotency over 9 months of culture for B. rotunda cell suspension culture were investigated. The established cell line B showed 42.2 and 53.8% decrease in growth after 6 and 9 months of culture respectively compared to 0 month-old cells. About 67, 57 and 47% of cell-derived shoot-like-structures for 0, 6 and 9 month-old cells, respectively, regenerated into shoots. The activities of superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) for 6 month-old cells were 14.40 Unit SOD/mg protein, 74.38 A mu mol H2O2/mg protein, and 2.23 A mu mol AsA/mg protein, respectively, higher than 0-month-old cells. The difference for hydrogen peroxide (H2O2) content between 0 and 6 month-old cells was 5.27 A mu mol H2O2/mg protein. While the activities of SOD, H2O2 and APX decreased from 6 to 9 month-old cells, CAT activity was increased. We applied a gel-based proteomic technique to analyze protein changes for 0, 6 and 9 month-old cells. A total of 13 protein spots showed significant differential expression and 8 protein spots were successfully identified. They were classified as protein synthesis, energy metabolism, defense and stress responses, and catabolic proteins.