During the 3-week-long induction phase, when M. truncatula cells leaf explants from non-embryogenic genotype (M9) and embryogenic variant (M9-10a) were forming the callus, biosynthesis and degradation of ABA, Gas and IAA proceeded at different levels. Induction of embryo formation is related to a lower ABA content, compared to the content of IAA and that of total bioactive GAs. Endogenous phytohormones are involved in the regulation of zygotic embryogenesis, but their role, especially of ABA, a plant growth inhibitor, in inducing somatic embryogenesis (SE) in angiosperms is still incompletely known. To arrive a better understanding of the ABA role in the process, we analyzed simultaneously and in detail changes in the contents of both ABA and five bioactive GAs (GA4, GA7, GA1, GA3, GA6) and IAA in M. truncatula non-embryogenic M9 (NE) and embryogenic M9-10a (E) genotypes. The initial leaf explants of both genotypes, and particularly NE, contained many times more ABA compared to the total bioactive GAs or IAA. In tissues during the entire 21-day induction all the hormones mentioned and their metabolites or conjugates were present; however, their contents were found to differ between the lines tested. The ABA level in primary explants of NE genotype was more than two times higher than that in E genotype. An even larger difference in the ABA content was found on the last day (day 21) of the induction phase (IP); the ABA content in E callus was over six times lower than in NE callus. In contrast, the IAA and GAs contents in primary explants of both genotypes in relation to ABA were low, but the contents of IAA and GAs exceeded that of ABA in the M9-10a tissues on the last day of IP. It is shown for the first time that endogenous ABA together with endogenous bioactive GAs and IAA is involved in acquisition of embryogenic competence in Medicago truncatula leaf somatic cells. These findings have a strong functional implication as they allow to improve the SE induction protocol.
Keywords: Abscisic acid; Bioactive gibberellins; Indole-3-acetic acid; Legume; Somatic embryogenesis.