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.

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).

Protocol development for somatic embryogenesis, SSR markers and genetic modification of Stipagrostis pennata (Trin.) De Winter.

BACKGROUND: Stipagrostis pennata (Trin.) De Winter is an important species for fixing sand in shifting and semi-fixed sandy lands, for grazing, and potentially as a source of lignocellulose fibres for pulp and paper industry. The seeds have low viability, which limits uses for revegetation. Somatic embryogenesis offers an alternative method for obtaining large numbers of plants from limited seed sources. RESULTS: A protocol for plant regeneration from somatic embryos of S. pennata was developed. Somatic embryogenesis was induced on Murashige & Skoog (MS) medium supplemented with 3 mg·L-1 2,4-D subsequently shoots were induced on MS medium and supplemented with 5 mg·L-1 zeatin riboside. The highest shoots induction was obtained when embryogenic callus derived from mature embryos (96%) in combination with MS filter-sterilized medium was used from Khuzestan location. The genetic stability of regenerated plants was analysed using ten simple sequence repeats (SSR) markers from S. pennata which showed no somaclonal variation in regenerated plants from somatic embryos of S. pennata. The regenerated plants of S. pennata showed genetic stability without any somaclonal variation for the four pairs of primers that gave the expected amplicon sizes. This data seems very reliable as three of the PCR products belonged to the coding region of the genome. Furthermore, stable expression of GUS was obtained after Agrobacterium-mediated transformation using a super binary vector carried by a bacterial strain LBA4404. CONCLUSION: To our knowledge, the current work is the first attempt to develop an in vitro protocol for somatic embryogenesis including the SSR marker analyses of regenerated plants, and Agrobacterium-mediated transformation of S. pennata that can be used for its large-scale production for commercial purposes.

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.

Automation and Scale Up of Somatic Embryogenesis for Commercial Plant Production, With Emphasis on Conifers.

For large scale production of clonal plants, somatic embryogenesis (SE) has many advantages over other clonal propagation methods such as the rooting of cuttings. In particular, the SE process is more suited to scale up and automation, thereby reducing labor costs and increasing the reliability of the production process. Furthermore, the plants resulting from SE closely resemble those from seeds, as somatic embryos, like zygotic (seed) embryos, develop with good connection between root and shoot, and without the plagiotropism often associated with propagation by cuttings. For practical purposes in breeding programs and for deployment of elite clones, it is valuable that a virtually unlimited number of SE plants can be generated from one original seed embryo; and SE cultures (clones) can be cryostored for at least 20 years, allowing long-term testing of clones. To date, there has however been limited use of SE for large-scale plant production mainly because without automation it is labor-intensive. Development of automation is particularly attractive in countries with high labor costs, where conifer forestry is often of great economic importance. Various approaches for automating SE processes are under investigation and the progress is reviewed here, with emphasis on conifers. These approaches include simplification of culture routines with preference for liquid rather than solid cultures, use of robotics and automation for the harvest of selected individual mature embryos, followed by automated handling of germination and subsequent planting. Different approaches to handle the processes of somatic embryogenesis in conifers are outlined below, followed by an update on efforts to automate the different steps, which are nearing an operational stage.

Improved and synchronized maturation of Norway spruce (Picea abies (L.) H.Karst.) somatic embryos in temporary immersion bioreactors.

Somatic embryogenesis offers many benefits for clonal propagation in large-scale plant production of conifers. A key rate-limiting step is the conversion from early-stage somatic embryos in pro-embryogenic masses (PEMs) to the maturation stage. Immature embryos in PEMs are present at different developmental stages, where some are unable to respond to the maturation treatment, thus limiting yields of mature embryos. Synchronization of early somatic embryo development in PEMs could greatly improve subsequent yields of mature embryos. A temporary immersion bioreactor designed for Norway spruce (Picea abies (L.) H.Karst.) was used in this study. Through a specific system for dispersion, connected tissue of PEMs, composed of immature embryos grown in liquid medium in the temporary immersion bioreactors or on solid medium as a control, was dispersed and redistributed in a more uniform spatial arrangement. It was demonstrated that development of mature embryos could be significantly stimulated by dispersion, compared to controls, in both medium types. Synchronization of maturation was evaluated by a statistical approach. The present study shows that the yield of mature embryos from dispersed PEMs was three to five times higher than that from non-dispersed controls in three of four cell lines of Norway spruce tested, both in bioreactors and on solid medium.

Correction: Nitrogen uptake and assimilation in proliferating embryogenic cultures of Norway spruce-Investigating the specific role of glutamine.

[This corrects the article DOI: 10.1371/journal.pone.0181785.].

Metabolome and transcriptome profiling reveal new insights into somatic embryo germination in Norway spruce (Picea abies).

Transcriptome, metabolome and histological profiling were performed on normal and aberrant somatic embryo germinants of Norway spruce (Picea abies L. Karst) providing a simplistic systems biology description of conifer germination. Aberrant germinants (AGs) formed periderm-like tissue at the apical pole and lacked shoot growth above the cotyledons. Transcriptome profiling (RNA-Sequencing) revealed a total of 370 differentially expressed genes at ≥1 or ≤-1 log2-fold change, where 92% were down-regulated in AGs compared with normal germinants (NGs). Genes associated with shoot apical meristem formation were down-regulated in AGs, or not differentially expressed between AGs and NGs. Genes involved in hormone signaling and transport were also down-regulated. Metabolite profiling by gas chromatography-mass spectrometry (MS) and liquid chromatography-MS revealed biochemical difference between AGs and NGs, notably increased levels of sugars including glucose in AGs. Genes involved in glucose signaling were down-regulated and genes involved in starch biosynthesis were up-regulated, suggesting involvement of sugar signaling during late embryo development and germination. The overall results provide new data enabling further studies to confirm potential markers for a normal germination process in conifers.

Nitrogen uptake and assimilation in proliferating embryogenic cultures of Norway spruce-Investigating the specific role of glutamine.

Somatic embryogenesis is an in vitro system employed for plant propagation and the study of embryo development. Nitrogen is essential for plant growth and development and, hence, the production of healthy embryos during somatic embryogenesis. Glutamine has been shown to increase plant biomass in many in vitro applications, including somatic embryogenesis. However, several aspects of nitrogen nutrition during somatic embryogenesis remain unclear. Therefore, we investigated the uptake and assimilation of nitrogen in Norway spruce pro-embryogenic masses to elucidate some of these aspects. In our study, addition of glutamine had a more positive effect on growth than inorganic nitrogen. The nitrogen uptake appeared to be regulated, with a strong preference for glutamine; 67% of the assimilated nitrogen in the free amino acid pool originated from glutamine-nitrogen. Glutamine addition also relieved the apparently limited metabolism (as evidenced by the low concentration of free amino acids) of pro-embryogenic masses grown on inorganic nitrogen only. The unusually high alanine concentration in the presence of glutamine, suggests that alanine biosynthesis was involved in alleviating these constraints. These findings inspire further studies of nitrogen nutrition during the somatic embryogenesis process; identifying the mechanism(s) that govern glutamine enhancement of pro-embryogenic masses growth is especially important in this regard.

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.

A possible biochemical basis for fructose-induced inhibition of embryo development in Norway spruce (Picea abies).

Sugars play an important role in various physiological processes during plant growth and development; however, the developmental roles and regulatory functions of hexoses other than glucose are still largely unclear. Recent studies suggest that blocked embryo development in Norway spruce (Picea abies (L.) Karst) is associated with accumulation of fructose. In the present study, the potential biochemical regulatory mechanism of glucose and fructose was studied during development of somatic embryos of Norway spruce from pro-embryogenic masses to mature embryos. The changes in protein fluorescence, a marker of the Maillard reaction, were monitored in two cell lines of Norway spruce that were grown on media containing sucrose (control), glucose or fructose. Manual time-lapse photography showed that growth of embryogenic cultures on medium containing sucrose was characterized by normal development of mature embryos whereas the embryogenic cultures that were grown on media containing glucose or fructose did not develop mature embryos. The biochemical analyses of embryogenic samples collected during embryo development showed that: (i) the content of glucose and fructose in the embryogenic cultures increased significantly during growth on each medium, respectively; (ii) the accumulation of Maillard products in the embryogenic cultures was highly correlated with the endogenous content of fructose but not glucose; and (iii) the embryogenic cultures grown on fructose displayed the highest protein carbonyl content and DNA damage whereas the highest content of glutathione was recorded in the embryogenic cultures that had grown on sucrose. Our data suggest that blocked development of embryos in the presence of fructose may be associated with the Maillard reaction.

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.

Metabolite profiling reveals clear metabolic changes during somatic embryo development of Norway spruce (Picea abies).

Progress on industrial-scale propagation of conifers by somatic embryogenesis has been hampered by the differences in developmental capabilities between cell lines, which are limiting the capture of genetic gains from breeding programs. In this study, we investigated the metabolic events occurring during somatic embryo development in Norway spruce to establish a better understanding of the fundamental metabolic events required for somatic embryo development. Three embryogenic cell lines of Norway spruce (Picea abies (L.) Karst) with different developmental capabilities were studied during somatic embryo development from proliferation of proembryogenic masses to mature somatic embryos. The three different cell lines displayed normal, aberrant and blocked somatic embryo development. Metabolite profiles from four development stages in each of the cell lines were obtained using combined gas chromatography-mass spectrometry. Multivariate discriminant analyses of the metabolic data revealed significant metabolites (P ≤ 0.05) for each development stage and transition. The results suggest that endogenous auxin and sugar signaling affects initial stages of somatic embryo development. Furthermore, the results highlight the importance of a timed stress response and the presence of stimulatory metabolites during late stages of embryo development.

Initiation of somatic embryogenesis from immature zygotic embryos of oocarpa pine (Pinus oocarpa Schiede ex Schlectendal).

The focus of the current project was to establish somatic embryogenesis protocols for the tropical pine species Pinus oocarpa using immature zygotic embryos (ZEs) as explants. Somatic embryogenesis is best supported by mimicking natural seed-embryo developmental conditions, through a tissue culture medium formulation based on the mineral content of the seed nutritive tissue [megagametophyte (MG)]. A novel culture medium (P. oocarpa medium, PO) was tested in combination with different plant growth regulator (PGR) concentrations and compared with standard Pinus taeda media for the initiation of somatic embryogenesis from immature ZEs of P. oocarpa. Immature MGs containing immature ZEs of two mother trees were used with 12 and 8% extrusion rates for mother tree genotypes 3 and 5, respectively. In both mother trees the percentage capture was 2%. Multiplication of two captured cell lines (T5C2S01 and T5C1S12) was improved by lowering the concentrations of PGRs to 2.5 µM each 2,4-dichlorophenoxyacetic acid and abscisic acid (ABA) plus 1.0 µM each 6-benzylaminopurine and kinetin. Mature somatic embryos formed on 40 µM ABA, 6% (w/v) maltose, 12% (w/v) PEG 8000 and 0.6% (w/v) Phytagel. While PO medium appeared suboptimal for somatic embryo induction, it did exhibit potential for enhanced culture proliferation and subsequent improved maturation with cell line T5C2S01, where microscopic analysis revealed better embryo morphology on PO medium than on 1250 medium. However, this enhancement was not observed with cell line T5C1S12. Germination was preceded by partial desiccation for a period of 2-3 weeks before transferring the embryos to germination medium. Germination was observed after 7 days under low light, and apical primordia slowly expanded after transfer to ex vitro conditions. To our knowledge, this is the first report on the production of somatic seedlings in P. oocarpa.

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.

Effects from shear stress on morphology and growth of early stages of Norway spruce somatic embryos.

The shear stress effect on directional expansion of pro embryogenic masses (PEMs) and suspensor cell development of somatic embryos of Norway spruce (Picea abies) at the proliferation stage was studied by a direct and quantitative image analysis system. The experimental system allowed for detailed observations of the effect of hydrodynamic shear stress in rotating and deforming liquid cultures of proliferating Norway spruce somatic embryos. Briefly, somatic embryos at an early development stage comprised only of clusters of meristematic cells without suspensor cells were fixed on an alginate film. The alginate film was affixed on the bottom of a flow cell and the somatic embryos were subjected to laminar flow through the chamber of the flow cell. Magnified images of the cell clusters were collected every 24 h. The image data was processed based on a normalized cross-correlation method, capable of measuring morphological and size features of individual cell clusters in both temporal and spatial domains. No suspensor cells developed in the cell clusters under shear stress of 140 s(-1) for the duration of the experiments. Cell clusters in the control cultured in stationary liquid conditions developed suspensor cells after 5-9 days in culture. Furthermore, the radial growth of meristematic cell clusters was inhibited by shear rates of 86 and 140 s(-1), corresponding to shear stress of 0.086 and 0.14 N/m(2), compared to growth under stationary conditions. The shear rate showed a significant negative correlation to growth rate. Control group showed no preference for direction during growth under static conditions.

Lignin biosynthesis in transgenic Norway spruce plants harboring an antisense construct for cinnamoyl CoA reductase (CCR).

An attractive objective in tree breeding is to reduce the content of lignin or alter its composition, in order to facilitate delignification in pulping. This has been achieved in transgenic angiosperm tree species. In this study we show for the first time that changes in lignin content and composition can be achieved in a conifer by taking a transgenic approach. Lignin content and composition have been altered in five-year-old transgenic plants of Norway spruce (Picea abies [L.] Karst) expressing the Norway spruce gene encoding cinnamoyl CoA reductase (CCR) in antisense orientation. The asCCR plants had a normal phenotype but smaller stem widths compared to the transformed control plants. The transcript abundance of the sense CCR gene was reduced up to 35% relative to the transformed control. The corresponding reduction in lignin content was up to 8%, which is at the lower limit of the 90-99% confidence intervals reported for natural variation. The contribution of H-lignin to the non-condensed fraction of lignin, as judged by thioacidolysis, was reduced up to 34%. The H-lignin content was strongly correlated with the total lignin content. Furthermore, the kappa number of small-scale Kraft pulps from one of the most down-regulated lines was reduced 3.5%. The transcript abundances of the various lignin biosynthetic genes were down-regulated indicating co-regulation of the biosynthetic pathway.