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The WUSCHEL-related homeobox transcription factor MtWOX9-1 stimulates somatic embryogenesis in Medicago truncatula


Somatic embryogenesis is widely used in plant biotechnology, but regulatory networks underlying this process are not fully understood. This process is regulated by many protein and hormonal factors, among which WUSCHEL-related homeobox proteins are believed to play key roles. In this study, we aimed to identify the role of Medicago truncatula WOX9-1 (MtWOX9-1), an Arabidopsis thaliana WOX9 (STIMPY) homolog, in somatic embryogenesis. We demonstrated that the MtWOX9-1 promoter is active in somatic embryos, and overexpression of MtWOX9-1 leads to the increase in embryogenic capacity, as well as to the changes in expression levels of two MADS-box genes (AGL15 and AGL8 homologs), associated with embryogenesis. The data obtained suggest that stimulation of MtWOX9-1 expression may have positive impact on plant biotechnology in improving the transformation and regeneration capacity of recalcitrant plants.

Key message

MtWOX9-1 participates in somatic embryogenesis and can stimulate this process in tissue culture changing the expression level of different embryogenesis-associated genes.

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This work was supported by Russian Science Foundation Project No. 16-16-10011, the grant from Russian Foundation for Basic Research No. 17-04-01708 and the grant of SPbU Alumni Association. Authors acknowledge the Research Resource Center for Molecular and Cell Technologies of Saint-Petersburg State University for DNA sequencing and the equipment used in this study. Authors are grateful to Andrew Matveenko for discussing the data in the process of preparing this paper.

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VT, YF, TW, FZ, MT, and LL designed the experiments. VT, YF, EP, AK, EE, VK, TW, and FZ performed the experiments. VT, YF, and MT analyzed the data. VT, YF, VK, TW, FZ, and LL wrote the paper. All authors read and approved the final manuscript.

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Correspondence to Varvara E. Tvorogova.

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The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Communicated by Sergio J. Ochatt.

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Supplementary material 1 (PDF 45 kb)

Sup. Figure 1. Summary of cultivation methods and lines used in the study. Supplementary material 2 (EPS 60 kb)


Sup. Figure 2. (a-d) Expression of pMtWOX9-1:GUS construction in de novo transformed T0 embryogenic calli at 68th d.o.c. Blue staining indicates the expression of GUS reporter gene. White arrows indicate somatic embryos at torpedo and cotyledonary stages, black arrow indicates somatic embryo at globular stage. The results were observed in 4 out of 4 calli. Supplementary material 3 (TIFF 6835 kb)


Sup. figure 3. (a) Boxplot reflecting SE intensity in liquid medium for wt and w9OE lines. Number of somatic embryos per explant is demonstrated. For wt and w9OE, 5 and 8 explants were analysed, respectively. 1,5 ml of liquid medium and 2-3 explants per bottle were used. (b) MtWOX9-1 wildtype frameshift isoform expression dynamics during in vitro cultivation of explants of embryogenic 2HA (full line) and non-embryogenic A17 (dashed line) lines. The graphs show the results of one biological repeat, representative for two additional independent experiments. The error bars represent sd of three technical repeats. (c) Expression levels of MtAGL8 in different organs according to M. truncatula Gene Expression Atlas (Benedito et al. 2008). (d) Expression levels of MtWOX9-1 in individual wt and w9OE callus samples. The mean for three technical repeats is demonstrated. Supplementary material 4 (EPS 57 kb)

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Tvorogova, V.E., Fedorova, Y.A., Potsenkovskaya, E.A. et al. The WUSCHEL-related homeobox transcription factor MtWOX9-1 stimulates somatic embryogenesis in Medicago truncatula. Plant Cell Tiss Organ Cult 138, 517–527 (2019).

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  • WOX genes
  • Somatic embryogenesis
  • Regeneration
  • In vitro culture