A resource of identified and annotated lincRNAs expressed during somatic embryogenesis development in Norway spruce.

Long non-coding RNAs (lncRNAs) have emerged as important regulators of many biological processes, although their regulatory roles remain poorly characterized in woody plants, especially in gymnosperms. A major challenge of working with lncRNAs is to assign functional annotations, since they have a low coding potential and low cross-species conservation. We utilised an existing RNA-Sequencing resource and performed short RNA sequencing of somatic embryogenesis developmental stages in Norway spruce (Picea abies L. Karst). We implemented a pipeline to identify lncRNAs located within the intergenic space (lincRNAs) and generated a co-expression network including protein coding, lincRNA and miRNA genes. To assign putative functional annotation, we employed a guilt-by-association approach using the co-expression network and integrated these results with annotation assigned using semantic similarity and co-expression. Moreover, we evaluated the relationship between lincRNAs and miRNAs, and identified which lincRNAs are conserved in other species. We identified lincRNAs with clear evidence of differential expression during somatic embryogenesis and used network connectivity to identify those with the greatest regulatory potential. This work provides the most comprehensive view of lincRNAs in Norway spruce and is the first study to perform global identification of lincRNAs during somatic embryogenesis in conifers. The data have been integrated into the expression visualisation tools at the PlantGenIE.org web resource to enable easy access to the community. This will facilitate the use of the data to address novel questions about the role of lincRNAs in the regulation of embryogenesis and facilitate future comparative genomics studies.

In silico characterization of putative gene homologues involved in somatic embryogenesis suggests that some conifer species may lack LEC2, one of the key regulators of initiation of the process.

BACKGROUND: Somatic embryogenesis (SE) is the process in which somatic embryos develop from somatic tissue in vitro on medium in most cases supplemented with growth regulators. Knowledge of genes involved in regulation of initiation and of development of somatic embryos is crucial for application of SE as an efficient tool to enable genetic improvement across genotypes by clonal propagation. RESULTS: Current work presents in silico identification of putative homologues of central regulators of SE initiation and development in conifers focusing mainly on key transcription factors (TFs) e.g. BBM, LEC1, LEC1-LIKE, LEC2 and FUSCA3, based on sequence similarity using BLASTP. Protein sequences of well-characterised candidates genes from Arabidopsis thaliana were used to query the databases (Gymno PLAZA, Congenie, GenBank) including whole-genome sequence data from two representative species from the genus Picea (Picea abies) and Pinus (Pinus taeda), for finding putative conifer homologues, using BLASTP. Identification of corresponding conifer proteins was further confirmed by domain search (Conserved Domain Database), alignment (MUSCLE) with respective sequences of Arabidopsis thaliana proteins and phylogenetic analysis (Phylogeny.fr). CONCLUSIONS: This in silico analysis suggests absence of LEC2 in Picea abies and Pinus taeda, the conifer species whose genomes have been sequenced. Based on available sequence data to date, LEC2 was also not detected in the other conifer species included in the study. LEC2 is one of the key TFs associated with initiation and regulation of the process of SE in angiosperms. Potential alternative mechanisms that might be functional in conifers to compensate the lack of LEC2 are discussed.

DNA methylome of the 20-gigabase Norway spruce genome.

DNA methylation plays important roles in many biological processes, such as silencing of transposable elements, imprinting, and regulating gene expression. Many studies of DNA methylation have shown its essential roles in angiosperms (flowering plants). However, few studies have examined the roles and patterns of DNA methylation in gymnosperms. Here, we present genome-wide high coverage single-base resolution methylation maps of Norway spruce (Picea abies) from both needles and somatic embryogenesis culture cells via whole genome bisulfite sequencing. On average, DNA methylation levels of CG and CHG of Norway spruce were higher than most other plants studied. CHH methylation was found at a relatively low level; however, at least one copy of most of the RNA-directed DNA methylation pathway genes was found in Norway spruce, and CHH methylation was correlated with levels of siRNAs. In comparison with needles, somatic embryogenesis culture cells that are used for clonally propagating spruce trees showed lower levels of CG and CHG methylation but higher level of CHH methylation, suggesting that like in other species, these culture cells show abnormal methylation patterns.

The effect of carbohydrates and osmoticum on storage reserve accumulation and germination of Norway spruce somatic embryos.

Somatic embryogenesis (SE) represents a useful experimental system for studying the regulatory mechanisms of embryo development. In this study, the effect of carbohydrates and osmoticum on storage reserve accumulation and germination of Norway spruce [Picea abies (L.) Karst] somatic embryos were investigated. Using time lapse photography, we monitored development from proliferation of proembryogenic masses (PEMs) to maturation of somatic embryos in two P. abies cell lines cultured on two maturation treatments. A combination of sugar assays, metabolic and proteomic analyses were used to quantify storage reserves in the mature somatic embryos. The maturation treatment containing a nonpermeating osmoticum, polyethylene glycol (PEG, 7.5%) and maltose (3%) as the carbohydrate gave significantly high maturation and low germination frequencies of somatic embryos compared to the treatment with only 3% sucrose. Somatic embryos treated with 3% sucrose contained high levels of sucrose, raffinose and late embryogenesis abundant (LEA) proteins. These compounds are known to be involved in the acquisition of desiccation tolerance during seed development and maturation. In addition the sucrose treatment significantly increased the content of starch in the somatic embryos while the maltose and PEG treatment resulted in somatic embryos with a high content of storage proteins. The high levels of sucrose, raffinose and LEA proteins in the embryos treated with 3% sucrose suggest that sucrose may improve the germination of somatic embryos by promoting the acquisition of desiccation tolerance.

In Vitro Propagation of the Blueberry 'Blue Suede™' (Vaccinium hybrid) in Semi-Solid Medium and Temporary Immersion Bioreactors.

The production of blueberries for fresh and processed consumption is increasing globally and has more than doubled in the last decade. Blueberry is grown commercially across a variety of climates in over 30 countries. The major classes of plants utilized for the planting and breeding of new cultivars are highbush, lowbush, half-high, Rabbiteye, and Southern highbush. Plants can be propagated by cuttings or in vitro micropropagation techniques. In vitro propagation offers advantages for faster generation of a large number of disease-free plants independent of season. Labor costs for in vitro propagation can be reduced using new cultivation technology and automation. Here, we test and demonstrate successful culture conditions and medium compositions for in vitro initiation, multiplication, and rooting of the Southern highbush cultivar 'Blue Suede™' (Vaccinium hybrid).

Stipagrostis pennata (Trin.) De Winter Artificial Seed Production and Seedlings Multiplication in Temporary Immersion Bioreactors

This study was conducted to develop the protocol for artificial seed production of Stipagrostis pennata (Trin.) De Winter via somatic embryo encapsulation as well as test a temporary bioreactor system for germination and seedling growth. Embryogenic calli were encapsulated using sodium alginate and calcium chloride and then sowed in the Murashige and Skoog (MS) germination medium in in vitro cultures. The experiments were conducted as a factorial based on a completely randomized design with three replications. The treatments include three concentrations of sodium alginate (1.5%, 2.5%, and 3.5%), two ion exchange times (20 and 30 min), and two artificial seed germination media (hormone-free MS and MS supplemented with zeatin riboside and L-proline). Germination percentage and number of days needed until the beginning of germination were studied. The highest percentage of artificial seed germination was obtained when 2.5% sodium alginate was used for 30 min (ion exchange time) and when the seeds were placed on the MS germination medium supplemented with zeatin riboside and L-proline. The results of the analysis of variance in the temporary immersion bioreactor system showed that the main effects observed on the seedling growth were associated with different growth hormones in culture media and the number of feeding cycles. Experimental results also indicated that the total protein analyses of zygotic seedlings and seedlings originating from the synthetic seeds showed no statistically significant differences between these samples.

Accumulated effects of factors determining plant development from somatic embryos of Abies nordmanniana and Abies bornmuelleriana.

Despite a much later inception of somatic embryogenesis (SE) propagation protocols for gymnosperms than for angiosperm species, SE is becoming increasingly important due to its applications for commercial forestry. For many conifers, there are however still major bottlenecks in the SE plant production process limiting the use of SE for forestry operations, Christmas tree production and research projects. In the present case study, the effects on plant growth from different cultural factors applied during the SE developmental process were studied in two conifer species of high value for Christmas tree production. Seven clones of Abies nordmanniana and two clones of Abies bornmuelleriana were included in the study. Accumulated effects from cultural treatments were recorded from the start of germination of mature embryos of different quality scores through development into plants in the third growing period. Experimental factors of the cultural treatments included were: germination temperature, germination time, light conditions, survival ex vitro and traits for plant growth and vitality. The results reveal that most of the studied experimental factors influenced plant growth during the first three years however their relative importance was different. Plant survival rate at end of the nursery stage was strongly impacted by germination temperature (p<0.001), initial embryo score (p=0.007), clone (p<0.001) and to a lesser extend week of germination (p=0.017). This case-study highlights and quantifies the strong interrelation between the developmental steps of somatic embryogenesis and show the importance of considering all cultural steps when optimizing SE plant production protocols.

Possible effect from shear stress on maturation of somatic embryos of Norway spruce (Picea abies).

Somatic embryogenesis is the only method with the potential for industrial scale clonal propagation of conifers. Implementation of the method has so far been hampered by the extensive manual labor required for development of the somatic embryos into plants. The utilization of bioreactors is limited since the somatic embryos will not mature and germinate under liquid culture conditions. The negative effect on mature embryo yields from liquid culture conditions has been previously described. We have described the negative effects of shear stress on the development of early stage somatic embryos (proembryogenic masses; PEMs) at shear stresses of 0.086 and 0.14 N/m(2). In the present study, additional flow rates were studied to determine the effects of shear stress at lower rates resembling shear stress in a suspension culture flask. The results showed that shear stress at 0.009, 0.014, and 0.029 N/m(2) inhibited the PEM expansions comparing with the control group without shear stress. This study also provides validation for the cross-correlation method previously developed to show the effect of shear stress on early stage embryo suspensor cell formation and polarization. Furthermore, shear stress was shown to positively affect the uptake of water into the cells. The results indicate that the plasmolyzing effect from macromolecules added to liquid culture medium to stimulate maturation of the embryos are affected by liquid culture conditions and thus can affect the conversion of PEMs into mature somatic embryos.

Temporary immersion bioreactor system for propagation by somatic embryogenesis of hybrid larch (Larix × eurolepis Henry).

Somatic embryogenesis (SE) has high potential for large-scale clonal propagation of conifers. Different types of bioreactor cultures have been tested for the conifer SE process where the temporary immersion bioreactors (TIBs) have proved to be useful across the different developmental steps of the SE process. In the present study the use of TIBs was tested for hybrid larch (Larix × eurolepis Henry). The results showed two-fold increases in both fresh weight (FW) of pro-embryogenic masses (PEMs) and yield of cotyledonary embryos in the TIBs compared to solid medium in plates. For the germination phase, the highest number of roots per plant, the root length and height of plants were also obtained in the TIBs. The results show that the TIB system can be successfully used to support scale up of plant production in all steps of the SE process from proliferation to germination of hybrid larch (Larix × eurolepis Henry).