Hormonal homeostasis associated with effective induction of triticale microspore embryogenesis

Abstract

The endogenous level of cytokinins (CKs) and auxins (Auxs) was analyzed in isolated microspores and ovaries of two doubled haploid (DH) lines of triticale (× Triticosecale Wittm.) to better understand the mechanism of microspore embryogenesis (ME)—the most efficient and widely applied method of producing DHs. The responsiveness of the studied lines to ME significantly varied. ME was induced by pre-treating tillers with low temperature (4 °C for 3 weeks) alone or in combination with synthetic auxin (2,4-D), auxin inhibitor (PCIB) or melatonin (MEL) applied for 4 days before microspore isolation. Hormonal profile analyses, accompanied by ME effectiveness evaluation confirmed the multi-level crosstalk of Auxs/CKs and the specific hormonal homeostasis required for effective microspore reprogramming. It was found that triticale microspores contained mainly cis zeatin derivatives: cis-zeatin-O-glucoside (cZOG), cis-zeatin riboside (cZR) and cis-zeatin (cZ), as well as indole-3-acetic acid (IAA) and IAA-aspartate (IAAsp). Increased ME efficiency was associated with higher contents of most of the identified CKs and Auxs, as well as the higher active Aux/active CK ratio. Trans CK isoforms were detected only in ovaries, confirming their importance as a source of bioactive molecules stimulating embryogenic development. Two of the pre-treatments tested: 12.5 μM PCIB and 50 μM MEL decreased the active Aux/active CK ratio, which was accompanied by an increase in the regeneration efficiency.