PMC

Expression profiling by qRT-PCR of MtCLV3 in different tissues of 2HA and Jemalong. Jemalong (grey bricks) and 2HA (white). Expression normalised to the expression level in somatic embryos. Values are ± SE (n = 3)

Phylogram for CLEs based on the CLE domain sequence. Fifty-five genes were analysed and are detailed in Oelkers et al. () [except AC151522]. Black arrow indicates the location of MtCLV3

Transgenic calli transformed with dexamethasone-induced RNAi for MtWUS (a) and empty vector control (b) developed in auxin plus cytokinin culture. Somatic embryos can be seen in the control, but not in MtWUS RNAi transformed callus

Phylogenetic tree of WOX genes. Dendrogram based on the sequence of the homeodomains. Bootstrap (1,000 rounds) was applied and the tree drawn using Dendroscope (Huson et al. ) with “majority” settings for consensus. Numbers and names are the same as on Fig. 

The effect of dexamethasone-induced RNAi expression for MtWUS in tissue cultures (as well as an empty vector control) developed in auxin plus cytokinin medium. The callus sizes were investigated by callus imaging and are normalised with 0 day as 100. Values are ± SE (n = 3)

MtWOX5::GUS expression in roots induced on auxin medium. The GUS signals in the root tip (a) and root maturation zone (b) in 8 μm paraffin-embedded sections. Two strong areas of GUS signal are indicated by red (distal) and black (proximal) arrows in a. In b, the GUS signal is indicated by a red arrow. Bar 80 μm

MtWUS::GUS expression at the early stages of somatic embryo induction in tissue culture. Blue-green colouring indicates the GUS signal. The signals were investigated in 3 days (a), and 14 days (b) cultured explants, 28 days callus (c), and older callus (d) with somatic embryos (white arrow). Bar 500 μm

MtWUS RNA in-situ hybridisation in heart stage zygotic embryos (a, b), apical meristem (c) and ovules (d) at early stage of development of wild-type Jemalong. MtWOX5 RNA in-situ hybridisation in the root meristem of a seedling root (e). VC vascular cylinder (red arrows), RC root cap and black arrow indicates the quiescent centre. Bar 100 μm

Expression profiling by qRT-PCR of MtWUS (a) and MtWOX5 (b) in tissue culture with different hormones. The expression was investigated in auxin plus cytokinin (Aux + Cyt, square-line), auxin alone (Aux, diamond-dash line), and cytokinin (Cyt, triangle-dot line) treatments in 35 days in 2HA. Expression calibrated to the expression level of 0 day of 2HA. Values are ± SE (n = 3)

MtWUS RNA in-situ hybridisation in embryogenic callus of 2HA. The signals were investigated in whole globular stage somatic embryos. Anti-sense probe indicating the MtWUS signals (a, c). Sense probe controls (b, d). a and b are 40 μm vibratome sections while c and d are 8 μm paraffin-embedded sections. The somatic embryo (c, d) has a suspensor like structure. Bar 80 μm

Expression of MtWUS and MtCLV3 in tissue culture of Jemalong and 2HA lines with auxin plus cytokinin in the medium. The expressions of MtWUS in 2HA (square-line) and MtCLV3 in 2HA (diamond-dash line) and Jemalong (MtCLV3-Jem, triangle-dot line) were investigated over 77 days. MtCLV3 expression only occurs in the highly embryogenic 2HA line as wild-type Jemalong does not produce somatic embryos. Expression normalised to the expression level of 0 day of 2HA. Values are ± SE (n = 3)

Expression profiling by qRT-PCR of MtWUS (a) and MtWOX5 (b) in different M. truncatula tissues by qRT-PCR. The expression was investigated in the shoot apices, developing flowers (buds) and mature leaves of normally-grown plants, tips of cultured roots and somatic embryos (latter one in 2HA). Expression was related to the expression level of somatic embryos. Values are ± SE (n = 3)

MtWOX5 RNA in-situ hybridisation during auxin-induced de novo root formation. The anti-sense probe (a–c). Sense probe for controls (b, d, f). The arrow labelled “1” shows centres of expression in what we have called “vein-derived” cells that emanate from the procambial cells (Rose et al. ). The arrow labelled “2” is pointing to the root primordium. The arrow “3” indicates the signal in the root meristem and the arrow “4” in the vascular tissue. Bar 80 μm

Alignment of the WOX homeodomain protein sequences. Fifty-one peptide sequences from dicotyledonous species were used. AtArabidopsis thaliana, MtMedicago truncatula MtWOX1(AC137078), MtWOX3(AC169182), MtWOX4(AC148486), MtWOX5(CU326389), MtWUS(CT009654/FJ477681), MtWOX9(AC199760), Mt64(AC141864), Mt80(TC104580), Mt47(BG581947), Mt96(AC232696), Mt72(AC157472), Mt05(AC198005), Petunia (Ph) (2-EF187281, 6-PhWUS), Populus trichocarpa (P. tr)(4-AM234761, 23-AM234764, 22-AM234762, 11-AM234756, 12-AM234757, 14-AM234759, 17-AM234766, 18-AM234765), Vitis Vinifera (V. vi) (13-AM439847, 10-AM463144, 9-AM429035, 8-AM488389, 15-AM447494, 19-CAAP02003786, 1-AM488026, 21-AM463736, 20-AM486367, 24-AM435207), 3-Solanum lycopersicum (S. lyc) (FJ190667), 7-tomato(Le) LeWUS), 16-Glycine max (Gm) (DQ336954), 5-Citrus sinensis (Cs) (EU032533). Numbers at the beginning of the gene accession refer to the corresponding genes for the phylogenetic tree in Fig.