Background: Members of the WUSCHEL-RELATED HOMEOBOX (WOX) gene family have important functions during all stages of plant development and have been implicated in the development of morphological novelties during evolution. Most studies have examined the function of these genes in angiosperms and very little is known from other plant species. Results: In this study we examine the presence and expression of WOX genes in the conifer Picea abies. We have cloned 11 WOX genes from both mRNA and genomic DNA and examined their phylogenetic relationship to WOX genes from other species as well as their expression during somatic embryogenesis and in adult tissues. Conclusions: Our study shows that all major radiations within the WOX gene family took place before the angiosperm-gymnosperm split and that there has been a recent expansion within the intermediate clade in the Pinaceae family. Furthermore, we show that the genes from the intermediate clade are preferentially expressed during embryo development in Picea abies. Our data also indicates that there are clear orthologs of both WUS and WOX5 present in the P. abies genome.

Proper embryo development is crucial as that is when the primary body axes are established. In Arabidopsis, AtWOX8 and AtWOX9, members of the WUSCHEL-RELATED HOMEOBOX (WOX) gene family, are critical for embryo development. In Norway spruce, PaWOX8/9, which is expressed in embryos, is the homologue of AtWOX8 and AtWOX9. In this work, it is shown that the transcript abundance of PaWOX8/9 is high during early and late embryogeny and that it decreases when the maturation phase starts. To address the function of PaWOX8/9 during embryo development, RNAi lines were established to down-regulate the transcript level of PaWOX8/9, using both constitutive and inducible promoters. Embryos in the PaWOX8/9 RNAi lines show an aberrant morphology caused by disturbed orientation of the cell division plane at the basal part of the embryonal mass during early and late embryogeny. In addition, the transcript level of several key cell-cycle-regulating genes, for example, PaE2FAB-LIKE and PaCYCLIN B-LIKE, are affected in the PaWOX8/9 RNAi lines. Taken together, our results suggest that PaWOX8/9 may perform an evolutionarily conserved function as a regulator of the establishment of the apical–basal embryo pattern.

Distinct expression domains of WUSCHEL-RELATED HOMEOBOX (WOX) gene family members are involved in patterning and morphogenesis of the early embryo in Arabidopsis. However, the role of WOX genes in other taxa, including gymnosperms, remains elusive. Here, we use somatic embryos and reverse genetics for studying expression and function of PaWOX2, the corresponding homolog of AtWOX2 in the gymnosperm Picea abies (Pa; Norway spruce). The mRNA level of PaWOX2 was transiently up-regulated during early and late embryogeny. PaWOX2 mRNA in early and early late embryos was detected both in the embryonal mass and in the upper part of the suspensor. Down-regulation of PaWOX2 during development of early embryos resulted in aberrant early embryos, which failed to form a proper protoderm. Cells on the surface layer of the embryonal mass became vacuolated, and new embryogenic tissue differentiated from the embryonal mass. In addition, the aberrant early embryos lacked a distinct border between the embryonal mass, and the suspensor and the length of the suspensor cells was reduced. Down-regulation of PaWOX2 in the beginning of embryo development, before late embryos were formed, caused a significant decrease in the yield of mature embryos. On the contrary, down-regulation of PaWOX2 after late embryos were formed had no effect on further embryo development and maturation. Our data suggest an evolutionarily conserved function of WOX2 in protoderm formation early during embryo development among seed plants. In addition, PaWOX2 might exert a unique function in suspensor expansion in gymnosperms.

Background: Catalpa bungei is an important tree species used for timber in China and widely cultivated for economic and ornamental purposes. A high-density linkage map of C. bungei would be an efficient tool not only for identifying key quantitative trait loci (QTLs) that affect important traits, such as plant growth and leaf traits, but also for other genetic studies. Results: Restriction site-associated DNA sequencing (RAD-seq) was used to identify molecular markers and construct a genetic map. Approximately 280.77 Gb of clean data were obtained after sequencing, and in total, 25,614,295 single nucleotide polymorphisms (SNPs) and 2,871,647 insertions-deletions (InDels) were initially identified in the genomes of 200 individuals of a C. bungei (7080) × Catalpa duclouxii (16-PJ-3) F₁ population and their parents. Finally, 9072 SNP and 521 InDel markers that satisfied the requirements for constructing a genetic map were obtained. The integrated genetic map contained 9593 pleomorphic markers in 20 linkage groups and spanned 3151.63 cM, with an average distance between adjacent markers of 0.32 cM. Twenty QTLs for seven leaf traits and 13 QTLs for plant height at five successive time points were identified using our genetic map by inclusive composite interval mapping (ICIM). Q16–60 was identified as a QTL for five leaf traits, and three significant QTLs (Q9–1, Q18–66 and Q18–73) associated with plant growth were detected at least twice. Genome annotation suggested that a cyclin gene participates in leaf trait development, while the growth of C. bungei may be influenced by CDC48C and genes associated with phytohormone synthesis. Conclusions: This is the first genetic map constructed in C. bungei and will be a useful tool for further genetic study, molecular marker-assisted breeding and genome assembly.

Summary: In angiosperms, WUSCHEL‐RELATED HOMEOBOX 3 (WOX3) genes are required for the recruitment of founder cells from the lateral domains of shoot meristems that form lateral regions of leaves. However, the regulation of the formation of lateral organs in gymnosperms remains unknown. By using somatic embryos of Norway spruce (Picea abies) we have studied the expression and function of PaWOX3 during embryo development. The mRNA abundance of PaWOX3 was determined by quantitative real‐time PCR, and the spatial expression of PaWOX3 was analysed by histochemical β‐glucuronidase (GUS) assays and in situ mRNA hybridization. To investigate the function of PaWOX3, we analysed how downregulation of PaWOX3 in RNA interference lines affected embryo development and morphology. PaWOX3 was highly expressed in mature embryos at the base of each cotyledon close to the junction between the cotyledons, and in the lateral margins of cotyledons and needles, separating them into an adaxial and an abaxial side. Downregulation of the expression of PaWOX3 caused defects in lateral margin outgrowth in cotyledons and needles, and reduced root elongation. Our data suggest that the WOX3 function in margin outgrowth in lateral organs is conserved among the seed plants, whereas its function in root elongation may be unique to gymnosperms.

The somatic embryogenesis (SE) process is better suited to large-scale production and automation than other clonal propagation methods such as the rooting of cuttings. SE is becoming a key technique to promote the asexual industrialization of conifers. Furthermore, somatic embryos are an ideal material to study the molecular mechanism of conifer embryo development, as the processes of somatic and zygotic embryo development are very similar. This brief review introduces the culturing techniques of the SE process in conifers and outlines the progress and deficiencies in conifer SE research. Emphasis is placed on the patterning formation of conifer somatic embryos.