Pretreatment free of 2,4-dichlorophenoxyacetic acid improves the differentiation of sugarcane somatic embryos by affecting the hormonal balance and the accumulation of reserves

Several factors influence culture conditions and somatic embryogenesis responses, such as the role of plant growth regulators (PGRs) during the establishment of in vitro cultures. For instance, auxin is required for callus induction and the acquisition of embryogenic capacity in different species, but the removal of auxin is needed for the further differentiation of somatic embryos. Thus, we aimed to evaluate the effects of residual 2,4-dichlorophenoxyacetic acid (2,4-D) on the differentiation of sugarcane somatic embryos. First, embryogenic calli (EC) were separated into two groups: (a) the PGR-free group was cultured without 2,4-D, and (b) the control group was maintained on proliferation culture medium containing 2,4-D. After 21 days of culture in the dark, both groups were transferred to differentiation culture medium under light. Our results showed that calli grown in PGR-free culture medium yielded more somatic embryos and exhibited a higher accumulation of protein and starch reserves. Proteomic analysis showed a decrease in the abundance of abscisic acid (ABA)-induced proteins in the control group. The levels of residual 2,4-D and endogenous 1-aminocyclopropane-1-carboxylic acid (ACC), an ethylene precursor, were higher in the control group than in the PGR-free group. Conversely, the level of ABA was higher in the PGR-free group than in the control group. A disruption in the ABA and ethylene levels might be responsible for the observed delay in the accumulation of storage reserves in the EC of the control group, which might have affected the development of somatic embryos. Our results also showed that the efficient development of sugarcane somatic embryos appears to be preceded by efficient accumulation of storage reserves in calli, which is related to hormone homeostasis. [GRAPHICS] Key message We demonstrated that sugarcane embryogenic calli need to reduce their residual 2,4-Dlevel to efficiently differentiate into somatic embryos.

Automated summary

This study demonstrated that reducing residual 2,4-D levels in embryogenic calli of sugarcane is crucial for efficient differentiation into somatic embryos. The balance of hormones, including ABA, ethylene, and auxin, plays a significant role in the accumulation of storage reserves and the development of somatic embryos in vitro.