The embryogenic cell line 61:21 of Norway spruce (Picea abies L. Karst) has been used in this study. The cell line was established as described by Högberg et al. . The cell line was stored in liquid nitrogen and thawed a couple of months before the start of experiments. After thawing the cell cultures were maintained as described previously . Briefly, proembryogenic masses (PEMs) were maintained on solidified proliferation medium containing the plant growth regulators (PGRs) auxin and cytokinin. To stimulate development of somatic embryos the cultures were first transferred to pre-maturation medium lacking PGRs for one week and then to maturation medium containing 30 μM abscisic acid (ABA). Early embryos (EEs) differentiated after one week on maturation medium; early late embryos (LE1s) and LE2s developed after two and three weeks on maturation medium, respectively; maturing embryos (ME1s), characterized by the initiation of cotyledons, developed after five weeks on maturation medium. ME2s (almost fully matured embryos) and ME3s (fully matured embryos) developed after about eight weeks on maturation medium.
RNA extraction, cDNA synthesis and quantitative real-time PCR
Samples for analyzing the mRNA level of PaWOX2 (accession number: AM286747) during embryo development, were collected from nine sequential developmental stages: PEM1 (after seven days on proliferation medium), PEM2 and PEM3 (after three and seven days on pre-maturation medium respectively), EE, LE1, LE2, ME1, ME2 and ME3 (after one to eight weeks on maturation medium). The sampling was performed at mid-day and samples were frozen in liquid nitrogen and stored at −80 °C after collection.
Total RNA was isolated using the Spectrum Plant Total RNA kit (Sigma-Aldrich, USA) according to the manufacturer’s instructions. For each sample, 1 μg of total RNA was reverse transcribed with RevertAid H Minus First Strand cDNA Synthesis Kit (Fermentas, Thermo Scientific, Sweden) using an equimolar ratio of random and oligo-dT primers according to the manufacturer’s instructions.
Quantitative real-time PCR (qRT-PCR) was performed as described previously . Three reference genes, CELL DIVISION CONTROL2 (PaCDC2), ELONGATION FACTOR 1 (PaEF1) and PHOSPHOGLUCOMUTASE (PaPHOS) were used . Two to three biological replicates, each with three technical replicates were performed for each test. The primer sequences are presented in Additional file 1: Table S1. Statistical analysis was done by t-test.
RNA in situ hybridization
For RNA in situ hybridization (ISH) the following materials were used: ovules from cones collected in the end of June and somatic embryos (EEs, LE1s and ME1s). The ovules were fixed and embedded as described by Karlgren, et al. . The somatic embryos were fixed in 3.7 % formaldehyde, 5.0 % acetic acid and 50 % ethanol overnight and embedded in Technovit 8100. A gene-specific fragment was used as a probe. The probes were prepared with DIG RNA Labeling Kit (see primer sequences in Additional file 1: Table S1) (Sigma-Aldrich, USA). In situ hybridization was performed essentially as described by Karlgren et al. . Sections of 10 μm were hybridized to digoxigenin-labeled RNA probes. The pictures were processed using Adobe Photoshop CS6 13.0 software.
RNA interference vector construction
The coding sequence (CDS) of PaWOX2 was amplified from a cDNA library of early somatic embryos of Norway spruce . The full-length CDS was subcloned into the pJET1.2/blunt cloning vector using the CloneJET™ PCR Cloning Kit (Fermentas, Thermo Scientific, Sweden). To obtain RNA interference (RNAi) constructs, two overlapping fragments of PaWOX2 were amplified and fused to form a hairpin structure for PaWOX2 (Additional file 2: Figure S1). To fuse these fragments, EcoRI and BamHI digestion sites were added on forward primers as linkers. The hairpin was confirmed by sequencing. Primers are presented in Additional file 3: Table S2.
Hairpin structures were introduced into pENTR™/D-TOPO® (Invitrogen, Carlsbad, CA, USA) and then inserted by att site LR recombination into the destination vector pMDC7 [LexA-VP16-ER (XVE) β-estradiol inducible promoter, which is derived from the pER8 vector and contains the estrogen receptor-based transactivator XVE] [24, 25] or pMDC32 (35S constitutive promoter) . Hairpin structures were confirmed by sequencing. Vectors were introduced by electroporation into Agrobacterium tumefaciens strain GV3101.
Transgenic cell lines
Embryogenic cultures were transformed by co-cultivation with A. tumefaciens as described previously . Stably transformed lines were selected after four weeks. Genomic DNA was isolated from PEMs from selected lines by using the DNeasy plant mini kit (Qiagen, Germany), according to the manufacturer’s instructions. Transformed lines were confirmed by PCR.
The mRNA level of PaWOX2 in PaWOX2 RNAi lines was analyzed by qRT-PCR. In the case of the inducible XVE-WOX2i lines, cultures were induced with β-estradiol (10 μM) for 48 h before the analysis. The lines 35S:WOX2i.2, 35S:WOX2i.3, 35S:WOX2i.4 and XVE-WOX2i.12 were selected for further studies (Additional file 4: Figure S2). The untransformed 61:21 line was used as a control. In addition, in the time-laps tracking experiments we also included a transformed control line (T-control), expressing the reporter GUS (β-glucuronidase) under the 35S promoter.
To study if PaWOX2 regulates cell division, the mRNA level of nine cell-cycle-regulating genes [PaRETINOBLASTOMA-RELATED PROTEIN-LIKE (PaRBRL), PaEXTRA SPINDLE POLES (PaESP), two E2F family genes (PaE2FABL) and five CYCLIN-LIKE (PaCYCLs) genes] were analysed in EEs from the control and line 35S:WOX2i.4 by qRT-PCR as previously described .
Samples of EEs for morphological analysis were collected from the control and lines 35S:WOX2i.2, 35S:WOX2i.3, 35S:WOX2i.4 and XVE-WOX2i.12 after one week on maturation medium. The samples were embedded by mixing with 2 ml of 1.2 % (w/v) Seaplaque agarose (FMC BioProducts, USA) in 60 mm Petri dishes. The length and width of suspensor cells of about 40 EEs from the control and 35S:WOX2i lines were measured using the ImageJ software (ver. 1.48 g) .
To further study embryo morphology, 26 LE1s from the control and line 35S:WOX2i.4 were scanned with a Zeiss 780 confocal microscope (Carl Zeiss AG), using the 488 nm Argon laser line, and the 20x objective (NA = 0.80).
For histological analysis, EEs from the control and line 35S:WOX2i.4 were fixed in 3.7 % formaldehyde, 5.0 % acetic acid and 50 % ethanol overnight. Subsequently, samples were dehydrated in 50, 75, 90 and 100 % ethanol series. Finally, the samples were embedded in Technovit 8100 (Kulzer, Wehrheim, Germany). The embryos were processed for serial sectioning (10 μM) on a Zeiss HM 355 microtome.
The cuticle of untreated LE1s from the control and line 35S:WOX2i.4 was stained in freshly prepared Oil Red, 0.2 % (w/v) in water, for 5 min and then washed in water . The stained embryos were hand-sectioned and examined under a Zeiss Axioplan microscope in dark field with a 5x objective (NA = 0.12).
Time-lapse tracking analysis was performed to examine in great detail the developmental pattern from EE to ME. EEs from the controls (both untransformed and T-control) and lines 35S:WOX2i.2, 35S:WOX2i.3 and 35S:WOX2i.4 (50 embryos per line) were sampled after one week on maturation medium and transferred to fresh maturation medium. Embryo morphology was examined every second day for 15 days.
To study the effect of PaWOX2 on the maturation process, after one week on pre-maturation medium cultures from the control and lines 35S:WOX2i.2, 35S:WOX2i.3, 35S:WOX2i.4 and XVE-WOX2i.12 were re-suspended in liquid pre-maturation medium and plated out as a thin layer on filter paper placed on maturation medium. For the XVE-WOX2i line, β-estradiol (10 μM) was added to the maturation medium, either from the start or after two weeks on maturation medium, when LE2s had already developed. The development of embryos was recorded for 14 days on maturation medium. The time points examined were 1, 3, 6, 10 and 14 days. The number of ME3s developed per initial gram of tissue was estimated after seven weeks on maturation medium.