Abstract
Indirect somatic embryogenesis (ISE) establishment for Coffea species started in the 1970s. Since then, intraspecific variations in the morphogenic pathway have been reported, even in the common environmental condition in vitro. Several authors have suggested that these variations are the result of genetic, epigenetic, and/or physiological events, highlighting the need for investigations to know the causes. Along these lines, this study aimed to investigate and describe, for the first time, the global 5-methylcytosine and physiological changes that occur in the cells of the aggregate suspensions of Coffea canephora during proliferation and somatic embryo regeneration steps. The cell proliferation step was characterized by increase in cell mass in all subcultures; relatively low mean values of global 5-methylcytosine (5-mC%), abscisic acid (ABA), and indole-3-acetic acid (IAA); high mean value of 1-aminocyclopropane-1-carboxylic acid (ACC, an ethylene precursor); and increase followed by decrease in spermidine (Spd, a polyamine) level. Therefore, these epigenetic and physiologic aspects promoted the cell proliferation, which is fundamental for ISE. In turn, the somatic embryo regeneration was correlated with global 5-mC% and physiological changes. The competence acquisition, determination, and cell differentiation steps were marked by increases in mean values of 5-mC%, IAA and ABA, and decreases in ACC and Spd, evincing that these changes are the triggers for regeneration and maturation of somatic embryos. Therefore, dynamic and coordinated epigenetic and physiologic changes occur in the cells of the aggregate suspensions during the C. canephora ISE in liquid system.
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Acknowledgments
We are grateful to Dr. Camilo Elber Vital and Dr. Jenny Gomez Arrieta for their support during the HPLC/MS analyses.
Funding
We would like to thank Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brasília—DF, Brazil, grant: 443801/2014-2), Fundação de Amparo à Pesquisa do Espírito Santo (FAPES, Vitória—ES, Brazil, grants: 65942604/2014 and 82/2017), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brasília—DF, Brazil) for the financial support. We also thank Financiadora de Estudos e Projetos (FINEP) and Fundo Nacional de Desenvolvimento Científico e Tecnológico (FNDCT) for the financial support that enabled equipment acquisition at the Núcleo de Análise de Biomoléculas (NuBioMol—Universidade Federal de Viçosa—MG, Brazil).
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The authors Amaral-Silva PM, Sanglard NA, and Clarindo WR conceived, designed, and conducted the tissue culture experiments. Amaral-Silva PM and Passos ABRJ carried out the epigenetic and physiological analyses. The authors Clarindo WR, Guilhen JHS, and Ferreira A performed the statistical analyses. All authors equally contributed to the manuscript editing and revision and approved the final manuscript version for submission.
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Key message
Dynamic and coordinated changes in global cytosine methylation, abscisic acid, indole-3-acetic acid, 1-aminocyclopropane-1-carboxylic acid and spermidine trigger the indirect somatic embryogenesis pathway in Coffea canephora propagated in liquid system.
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Supplement 1
Surplus mass and global 5-mC% values and SE recovered during C. canephora ISE. (a) Surplus mass values measured every 15 days for each repetition in each subculture during the two morphogenic development stages of ISE. The numbers to the right of each colored line indicate the number of SE identified and separately counted up according to the morphogenic development stage: globular and heart/torpedo/cotyledonary. (b) Global 5-mC% values measured for each repetition in each subculture during the two ISE stages. (PNG 1594 kb)
Supplement 2
Mean number of SE in globular/heart-shaped, torpedo-shaped or cotyledonary stage. The mean SE number was higher for globular/heart-shaped SE, followed by torpedo-shaped and cotyledonary ones. (PNG 51 kb)
Supplement 3
Identification and quantification (ng g−1) of abscisic acid (ABA), 1-aminocyclopropane-1-carboxylic acid (ACC), indole-3-acetic acid (IAA), zeatin (Z), putrescine (Put), spermidine (Spd) and spermine (Spm) during C. canephora ISE for (a) the lower, (b) intermediate and (c) higher global 5-mC% classes. (PNG 2153 kb)
Supplement 4
(a) Zeatin (Z), (b) spermidine (Spd), (c) putrescine (Put), in ng g−1, during each subculture of the C. canephora ISE: cell proliferation from the first to the seventh subculture, and SE regeneration from the eighth to the ninth subculture. Boxplots followed by the same letter are not different by Scott-Knott test (P ≤ 0.05). (PNG 212 kb)
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Amaral-Silva, P.M., Clarindo, W.R., Guilhen, J.H.S. et al. Global 5-methylcytosine and physiological changes are triggers of indirect somatic embryogenesis in Coffea canephora. Protoplasma 258, 45–57 (2021). https://doi.org/10.1007/s00709-020-01551-8
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DOI: https://doi.org/10.1007/s00709-020-01551-8