Norway spruce somatic embryogenesis benefits from proliferation of embryogenic tissues on filter discs and cold storage of cotyledonary embryos.

Vegetative propagation opens opportunities for the multiplication of elite tree progeny for forest regeneration material. For conifers such as Norway spruce (Picea abies) the most efficient vegetative propagation method is seed multiplication through somatic embryogenesis. Efficient culture methods are needed for somatic embryogenesis to be commercially viable. Compared to culturing as clumps, filter disc cultures can improve the proliferation of embryogenic tissue (ET) due to more even spread and better developmental synchronization. In this study, ET proliferation on filter discs was compared to proliferation as clumps. The study comprised 28 genotypes in four trials. The benefits of adding a pre-maturation step and the selection of fresh ET for the subculture were evaluated. Pre-maturation on hormone-free media before maturation did not significantly improve embryo yield but improved greenhouse survival from 69% to 80%, although there was high variation between lines. Filter disc cultivation of ET did result in better growth than in clumps but was more dependent on ET selection and the amount of ET than the clump cultivation method. Filter proliferation also favors certain lines. Post-maturation storage can be used to change the storage compound composition of the produced mature embryos. The embryo storage compound profile was analyzed after post-maturation cold storage treatments of 0, 4, 8, 31, and 61 weeks and compared to that of the zygotic embryos. Cold storage made the storage compound profile of somatic embryos closer to that of zygotic embryos, especially regarding the raffinose family oligosaccharides and storage proteins. Sucrose, hexose, and starch content remained higher in somatic embryos even through cold storage. Prolonged storage appeared less beneficial for embryos, some of which then seemed to spontaneously enter the germination process.

Somatic Embryo Yield and Quality From Norway Spruce Embryogenic Tissue Proliferated in Suspension Culture.

Somatic embryogenesis is being piloted for the commercial production of genetically improved Norway spruce (Picea abies L. Karst) forest regeneration material in Finland. The main challenge to making the process commercially relevant is the dependence on time-consuming and highly skilled manual labor. Automation and scaling up are needed to improve cost-effectiveness. Moving from the proliferation of embryogenic tissue on semisolid media to suspension cultures could improve process scalability. In a series of four experiments (overall, with 20 cell lines, 4-9 per experiment), the suitability of proliferation in suspension culture for Norway spruce somatic embryogenesis was evaluated based on the growth rate, indicators of stress conditions, good-quality cotyledonary embryo yield, and embling survival in a greenhouse. The proliferation rate in suspension was found equal to on semisolid media, but with a remarkable genotypic variation. Embryogenic tissue matured directly without pre-treatments from suspension onto semisolid media produced lower numbers of good-quality embryos than tissue matured from semisolid media. Rinsing the suspension-grown tissue with hormone-free liquid media before maturation improved embryo yield, bringing it closer to that of semisolid-grown tissue. Decreasing 6-benzylaminopurine and 2,4-dichlorophenoxyacetic acid concentrations in suspension proliferation media to 0.5 or 0.1 times those in semisolid media did not affect tissue growth and did not improve embryo production. The hydrogen peroxide (H2O2) content and guaiacol peroxidase activity were elevated in suspension cultures compared with semisolid medium, which had the same plant growth regulator content. In one experiment out of four, the greenhouse survival of germinants was lower when proliferation was carried out in full strength suspension than on semisolid media; in other experiments the survival rates were equal.

Correction: Adipocytes as a Link Between Gut Microbiota-Derived Flagellin and Hepatocyte Fat Accumulation.

[This corrects the article DOI: 10.1371/journal.pone.0152786.].

Adipocytes as a Link Between Gut Microbiota-Derived Flagellin and Hepatocyte Fat Accumulation.

While the role of both elevated levels of circulating bacterial cell wall components and adipose tissue in hepatic fat accumulation has been recognized, it has not been considered that the bacterial components-recognizing adipose tissue receptors contribute to the hepatic fat content. In this study we found that the expression of adipose tissue bacterial flagellin (FLG)-recognizing Toll-like receptor (TLR) 5 associated with liver fat content (r = 0.699, p = 0.003) and insulin sensitivity (r = -0.529, p = 0.016) in humans (n = 23). No such associations were found for lipopolysaccharides (LPS)-recognizing TLR4. To study the underlying molecular mechanisms of these associations, human HepG2 hepatoma cells were exposed in vitro to the conditioned culture media derived from FLG or LPS-challenged human adipocytes. The adipocyte-mediated effects were also compared to the effects of direct HepG2 exposure to FLG and LPS. We found that the media derived from FLG-treated adipocytes stimulated fat accumulation in HepG2 cells, whereas either media derived from LPS-treated adipocytes or direct FLG or LPS exposure did not. This is likely due to that FLG-treatment of adipocytes increased lipolysis and secretion of glycerol, which is known to serve a substrate for triglyceride synthesis in hepatocytes. Similarly, only FLG-media significantly decreased insulin signaling-related Akt phosphorylation, IRS1 expression and mitochondrial respiratory chain ATP5A. In conclusion, our results suggest that the FLG-induced TLR5 activation in adipocytes increases glycerol secretion from adipocytes and decreases insulin signaling and mitochondrial functions, and increases fat accumulation in hepatocytes. These mechanisms could, at least partly, explain the adipose tissue TLR5 expression associated with liver fat content in humans.