Plant Cell Reports

, Volume 35, Issue 1, pp 77–89 | Cite as

Hormonal and epigenetic regulation during embryogenic tissue habituation in Cucurbita pepo L.

  • Dunja Leljak-Levanić
  • Mihaela Mrvková
  • Veronika Turečková
  • Aleš Pěnčík
  • Jakub Rolčík
  • Miroslav Strnad
  • Snježana MihaljevićEmail author
Original Article


Key message

Habituated embryogenic line of pumpkin contained more CKs and IAA, but less ABA than the non-habituated line. Pronounced hypomethylation correlated with the absence of 2,4-D, addition of 5-azaC, and the process of habituation.


A comparative analysis between habituated and non-habituated embryogenic cultures of pumpkin (Cucurbita pepo L.) in relation to endogenous phytohormones, global DNA methylation, and developmental and regeneration capacities of the cultures was conducted. The analysis revealed more cytokinins (CKs) and indole-3-acetic acid (IAA), but less abscisic acid (ABA) in the habituated HEC line than in the non-habituated DEC line. Ribosides and ribotides were the most abundant CK forms in both HEC and DEC lines (75.9 and 57.6 %, respectively). HEC contained more free-base CKs (5.8 vs. 3.2 %), whereas DEC contained considerably more O-glycosides (39.1 vs. 18.3 %). Although prevalence of IAA was common for both lines, relative ratio of CKs and ABA differed between DEC and HEC lines. ABA was prevailing over CKs in DEC, while CKs prevailed over ABA in HEC line. Taking into account the importance of ABA for embryo maturation, the reduced endogenous ABA content in HEC line might be the reason for a 5-fold reduction in regeneration capacity compared to DEC. Both habituated and non-habituated embryogenic lines were highly methylated in the presence of 2,4-dichlorophenoxyacetic acid (2,4-D). Pronounced hypomethylation correlated with the absence of 2,4-D, addition of 5-azacytidine (5-azaC), but also with the process of habituation. The habituated line was resistant to the effect of hypomethylation drug 5-azaC and remained highly methylated even after the addition of 5-azaC. Also, 5-azaC did not change the developmental pattern in the habituated line, indicating the existence of separate mechanisms by which 2,4-D influences global DNA methylation in comparison to habituation-related global DNA methylation.


ABA CKs DNA methylation Habituation IAA Somatic embryogenesis 



This research was funded by the Ministry of Science, Education and Sport of the Republic of Croatia (Projects no. 098-0982913-2829 and 119-1191196-1225), and the Ministry of Education, Youth and Sports of the Czech Republic, NPU I program with project LO1204. AP was partly supported by the Operational Program Education for Competitiveness, European Social Fund (Project CZ.1.07/2.3.00/20.0165).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


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Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Dunja Leljak-Levanić
    • 1
  • Mihaela Mrvková
    • 2
  • Veronika Turečková
    • 2
  • Aleš Pěnčík
    • 2
  • Jakub Rolčík
    • 2
  • Miroslav Strnad
    • 2
  • Snježana Mihaljević
    • 3
    Email author
  1. 1.Faculty of ScienceUniversity of ZagrebZagrebCroatia
  2. 2.Laboratory of Growth Regulators, Centre of the Region Haná for Biotechnological and Agricultural ResearchPalacký University and Institute of Experimental Botany ASCROlomoucCzech Republic
  3. 3.Institute Ruđer BoškovićZagrebCroatia

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