title: Hormonal and epigenetic regulation during embryogenic tissue habituation in Cucurbita pepo L.
abstract: 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.
authors.affiliation: Faculty of Science, University of Zagreb, Horvatovac 102a, 10000, Zagreb, Croatia.
authors.forename: Dunja
authors.initials: D
authors.lastname: Leljak-Levanić
authors.affiliation: Laboratory of Growth Regulators, Centre of the Region Haná for Biotechnological and Agricultural Research, Palacký University and Institute of Experimental Botany ASCR, Šlechtitelů 11, 783 71, Olomouc, Czech Republic.
authors.forename: Mihaela
authors.initials: M
authors.lastname: Mrvková
authors.affiliation: Laboratory of Growth Regulators, Centre of the Region Haná for Biotechnological and Agricultural Research, Palacký University and Institute of Experimental Botany ASCR, Šlechtitelů 11, 783 71, Olomouc, Czech Republic.
authors.forename: Veronika
authors.initials: V
authors.lastname: Turečková
authors.affiliation: Laboratory of Growth Regulators, Centre of the Region Haná for Biotechnological and Agricultural Research, Palacký University and Institute of Experimental Botany ASCR, Šlechtitelů 11, 783 71, Olomouc, Czech Republic.
authors.forename: Aleš
authors.initials: A
authors.lastname: Pěnčík
authors.affiliation: Laboratory of Growth Regulators, Centre of the Region Haná for Biotechnological and Agricultural Research, Palacký University and Institute of Experimental Botany ASCR, Šlechtitelů 11, 783 71, Olomouc, Czech Republic.
authors.forename: Jakub
authors.initials: J
authors.lastname: Rolčík
authors.affiliation: Laboratory of Growth Regulators, Centre of the Region Haná for Biotechnological and Agricultural Research, Palacký University and Institute of Experimental Botany ASCR, Šlechtitelů 11, 783 71, Olomouc, Czech Republic.
authors.forename: Miroslav
authors.initials: M
authors.lastname: Strnad
authors.affiliation: Institute Ruđer Bošković, Bijenička cesta 54, 10000, Zagreb, Croatia. mihaljev@irb.hr.
authors.forename: Snježana
authors.initials: S
authors.lastname: Mihaljević
mesh_terms: D015084:2,4-Dichlorophenoxyacetic Acid / Q000494:pharmacology; D000040:Abscisic Acid / Q000378:metabolism; D001374:Azacitidine / Q000494:pharmacology; D028464:Cucurbita / Q000187:drug effects / Q000196:embryology / Q000235:genetics*; D003583:Cytokinins / Q000378:metabolism; D019175:DNA Methylation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D044127:Epigenesis, Genetic / Q000187:drug effects*; D006540:Herbicides / Q000494:pharmacology; D007210:Indoleacetic Acids / Q000378:metabolism*; D010937:Plant Growth Regulators / Q000378:metabolism*; D058439:Plant Somatic Embryogenesis Techniques
chemical_list: D003583:Cytokinins; D004791:Enzyme Inhibitors; D006540:Herbicides; D007210:Indoleacetic Acids; D010937:Plant Growth Regulators; D015084:2,4-Dichlorophenoxyacetic Acid; C030737:indoleacetic acid; D000040:Abscisic Acid; D001374:Azacitidine
keywords: ABA; CKs; DNA methylation; Habituation; IAA; Somatic embryogenesis