Long-term culture with 2,4-dichlorophenoxyacetic acid affects embryogenic competence in sugarcane callus via changes in starch, polyamine and protein profiles

Abstract

Somatic embryogenesis is a biotechnological tool with high application potential in the in vitro propagation and regeneration of crop plants, such as sugarcane. However, decreasing embryogenic competence is observed in embryogenic callus with successive subcultures, especially when the callus are maintained in the presence of 2,4-dichlorophenoxyacetic acid (2,4-d). Thus, the aim of this work was to investigate the morphological and molecular changes associated with the decreasing embryogenic competence in sugarcane embryogenic callus under long-term culture with or without 2,4-d. Sugarcane embryogenic callus were subcultured with 10 μM 2,4-d every 21 days for eight subcultures, and during each subculture, the callus were matured. In addition, the embryogenic callus from the first subculture after induction (S1) and after six subcultures with (S6) or without 2,4-d [S6(-)] were matured, and their histomorphological and histochemical features, endogenous polyamine levels and proteomic profiles were examined. The callus cultured with 2,4-d for a long time period showed reduced embryogenic competence after six subcultures, while callus cultivated in culture medium without 2,4-d maintained high embryogenic competence. In contrast to the callus in S6, those in the first subculture (S1) and S6(-) presented prominent nuclei, a high nucleus/cytoplasm ratio. Long-term culture with 2,4-d also affected polyamine (PA) metabolism, leading to high concentrations of putrescine (Put) and spermidine (Spd) in particular, and the synthesis and regulation of proteins, such as late embryogenesis abundant protein, chitinase, oleosin, and heat shock proteins. Therefore, we demonstrate that long-term culture with 2,4-d decreases embryogenic competence in sugarcane embryogenic callus. Loss in embryogenic competence in sugarcane callus by the long exposure to 2,4-d, is induced via changes in the synthesis/mobilization of reserves and protein abundance.