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

  • Lucas Z. Passamani
  • Ricardo S. Reis
  • Ellen M. Vale
  • Kariane R. Sousa
  • Victor P. M. Aragão
  • Claudete Santa-Catarina
  • Vanildo SilveiraEmail author
Original Article


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.

Key message

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.


Somatic embryogenesis Sugarcane Histomorphology Histochemistry Polyamines Proteomic analysis 



This research was supported by funds from the Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro-FAPERJ (Proc. E26/211.690/2015; Proc. E26/203.311/2017) and Conselho Nacional de Desenvolvimento Científico e Tecnológico-CNPq (305415/2016-6) to VS. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brasil (CAPES)—Finance Code 001. LZP, VPMA and EMV thank CAPES for a fellowship, and RSS and KSR thank FAPERJ for a fellowship.

Author contributions

LZP and VS designed the study; LZP and RSR carried out the experiments; LZP, EMV, KRS and CSC performed the histomorphological and histochemical analyses; LZP, VPMA and CSC performed the free polyamine determination; LZP, RSR and VS performed the proteomic analysis; and all authors read, reviewed and approved the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.

Supplementary material

11240_2019_1737_MOESM1_ESM.tif (820 kb)
Fig. S1 Number of somatic embryos (SEs) formed by callus in the different subcultures in the presence of 2,4-d after 28 days of maturation. Means followed by different letters are significantly different (P < 0.05) according to the Student–Newman–Keuls test. S1–S8 are callus from the first to eighth subculture with 2,4-d (n = 9, coefficient of variation = 19.35%) (TIFF 819 kb)
11240_2019_1737_MOESM2_ESM.xlsx (1.7 mb)
Table S1 Complete list of identified proteins (XLSX 1698 kb)


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© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Laboratório de BiotecnologiaCentro de Biociências e Biotecnologia (CBB), Universidade Estadual do Norte Fluminense Darcy Ribeiro (UENF)Campos dos GoytacazesBrazil
  2. 2.Unidade de Biologia Integrativa, Setor de Genômica e Proteômica, UENFCampos dos GoytacazesBrazil
  3. 3.Laboratório de Biologia Celular e TecidualCBB-UENFCampos dos GoytacazesBrazil

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