Abstract
2,4-dichlorophenoxyacetic acid (2,4-D), an auxinic herbicide, in many in vitro somatic embryogenesis systems is used to induce embryogenesis. This compound present at a low concentration (0.5 µM) during the induction phase, is necessary for M. truncatula embryogenic callus formation and, consequently, for embryo development. 2,4-D in higher concentration (5 µM) causes an increase in O2·− accumulation which disturbs the callus and embryos formation. However, above processes are disturbed by DPI, an inhibitor of the NADPH oxidase responsible for the O2·− production. Thus, the aim of this study was to elucidate whether during first week of induction phase the change in the level of O2·− affects the expression of genes encoding Polycomb and Trithorax group proteins and the transcription level of some marker genes for SE. The expression of Polycomb and Trithorax group genes, and coding the transcription factors such as: LEC1, L1L, WUS, WOX5 and STM depends on the O2·− accumulation in explant cells. Disorder of its level by manipulation of the concentration of 2,4-D or DPI disrupts expression of the above-mentioned genes and causes disturbances in the callus growth and development of somatic embryos.
Highlights
Plants have the capacity of reproduction without egg fertilization from different kinds of somatic cells in the process known as somatic embryogenesis (SE)
It has been shown that the acquisition of competence for embryogenesis by somatic cells of M. truncatula leaves is probably associated with a lower level of the Polycomb and Trithorax groups gene expression in primary explants of the embryogenic line compared with the expression in the non-embryogenic line (Orłowska et al 2017; Orłowska and Kępczyńska 2018)
O2·− is present in explants during the first week of the SE induction phase and 2,4-D and diphenylene iodonium (DPI) are modulators of its accumulation (Fig. 1), which significantly affects the callus growth of M. truncatula M9-10a line and somatic embryo formation (Fig. 2)
Summary
Plants have the capacity of reproduction without egg fertilization from different kinds of somatic cells in the process known as somatic embryogenesis (SE). It was commonly believed that in addition to genotype cellular stress caused by exposing plant explants to different stresses including supra-optimal growth regulators concentrations (e.g. 2,4-D) plays an important role in the cell fate switch, which lead to embryo development and suggest that SE is a developmental stress response (Dudits et al 1991). This process starts with mechanical excision of the explant from the mother plant (wounding stress), subjecting it to chemical surface sterilization (oxidative stress) and putting it on artificial medium (dehydratation/osmotic stress) (Nolan et al 2006). Other genes are important for encoding transcription factors of the LAFL pathway: LEAFY COTYLEDON1 (LEC1), LEAFY COTYLEDON1-LIKE (L1L) (Nolan et al.2014; Orłowska et al 2017), LEC2, FUSCA3 (FUS3) and ABSCISIC ACID-INTENSIVE3 (ABI3)(Barreto et al 2019)
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