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

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.