Reducing Pre- and Post-Treatments in Cryopreservation Protocol and Testing Storage at -80 °C for Norway Spruce Embryogenic Cultures.

Somatic embryogenesis (SE) is considered the most effective method for vegetative propagation of Norway spruce (Picea abies L. Karst). For mass propagation, a storage method that is able to handle large quantities of embryogenic tissues (ETs) reliably and at a low cost is required. The aim of the present study was to compare freezing at -80 °C in a freezer to cryopreservation using liquid nitrogen (LN) as a method for storing Norway spruce ETs. The possibility of simplifying both the pre-treatment and thawing processes in cryopreservation was also studied. The addition of abscisic acid (ABA) to the pre-treatment media and using polyethylene glycol PEG4000 instead of PEG6000 in a cryoprotectant solution were tested. Both the pre-and post-treatments on semi-solid media could be simplified by reducing the number of media, without any loss of genotype or embryo production capacity of ETs. On the contrary, the storage of ETs in a freezer at -80 °C instead of using LN was not possible, and the addition of ABA to the pre-treatment media did not provide benefits but increased costs. The lower regeneration rate after using PEG4000 instead of PEG6000 in a cryoprotectant solution in cryovials was unexpected and unwanted. The simplified pre-and post-treatment protocol will remarkably reduce the workload and costs in the mass-cryopreservation of future forest regeneration materials and in thawing the samples for mass propagations, respectively.

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.

Telomere Length in Norway Spruce during Somatic Embryogenesis and Cryopreservation.

Telomeres i.e., termini of the eukaryotic chromosomes protect chromosomes during DNA replication. Shortening of telomeres, either due to stress or ageing is related to replicative cellular senescence. There is little information on the effect of biotechnological methods, such as tissue culture via somatic embryogenesis (SE) or cryopreservation on plant telomeres, even if these techniques are widely applied. The aim of the present study was to examine telomeres of Norway spruce (Picea abies (L.) Karst.) during SE initiation, proliferation, embryo maturation, and cryopreservation to reveal potential ageing or stress-related effects that could explain variation observed at SE process. Altogether, 33 genotypes from 25 families were studied. SE initiation containing several stress factors cause telomere shortening in Norway spruce. Following initiation, the telomere length of the embryogenic tissues (ETs) and embryos produced remains unchanged up to one year of culture, with remarkable genotypic variation. Being prolonged in vitro culture can, however, shorten the telomeres and should be avoided. This is achieved by successful cryopreservation treatment preserving telomere length. Somatic embryo production capacity of the ETs was observed to vary a lot not only among the genotypes, but also from one timepoint to another. No connection between embryo production and telomere length was found, so this variation remains unexplained.

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.

Somatic Embryogenesis and Plant Regeneration From Primordial Shoot Explants of Picea abies (L.) H. Karst. Somatic Trees.

The recalcitrance of adult conifer tissues has prevented vegetative propagation of trees with known and desired characteristics. Somatic embryogenesis (SE) initiation protocol, recently developed for white spruce (Picea glauca, Klimaszewska et al., 2011), was applied in order to examine the feasibility, frequency and timing of SE induction from primordial shoots (PS) of Norway spruce (P. abies). In total, 39 genotypes were screened from 2015 to 2017 using 4-6 years old trees of SE origin as explant donors. Two genotypes responded: 11Pa3794 produced six proliferating embryonal mass (EM) sublines and 11Pa4066 produced 23 EM sublines. SE initiations occurred at the beginning of April, when the temperature sum (d.d.) started to accumulate, and at the end of October or beginning of November when the chilling unit (ch.u.) sum was over 500. EM sublines from both genotypes contained numerous early somatic embryos as detected by acetocarmine staining. The sublines of 11Pa4066 produced the mean of 78.6 ± 12.8 cotyledonary somatic embryos /g FW, but 11Pa3794 produced only a few cotyledonary somatic embryos that were able to germinate. The original EM lines (from which the trees were regenerated) had produced the same number of somatic embryos in 2011 maturations, which was approximately 120 somatic embryos /g FW. Microsatellite analyses conducted with both responsive genotypes confirmed the genetic stability of the EM sublines compared with the donor trees growing in the field. SE protocol developed for white spruce PS explants was also suitable for PS of Norway spruce if the explants were in the responsive developmental stage.

Reliable and practical methods for cryopreservation of embryogenic cultures and cold storage of somatic embryos of Norway spruce.

Somatic embryogenesis (SE) is considered as the most-effective method for vegetative propagation of Norway spruce (Picea abies L. Karst). For mass propagation, a cryopreservation method able to handle large numbers of embryogenic tissues (ETs) reliably and at low costs is needed. The aim of the present study was to compare pretreatments, cryoprotectants and slow-cooling devices for cryopreservation of Norway spruce ETs, with 12 variations of methods and a total of 136 spruce genotypes. Secondly, possible applications for cold storage of mature somatic embryos were studied with the aim of developing a flexible time window for embling production. At best, 100% of the embryogenic lines were recovered following cryopreservation, but the results varied among the sets of lines. Also physiological condition of the tissues, pre-treatment and cryoprotectant applied, as well as the slow-cooling device used were found to affect the recovery. The best option for cryopreservation of Norway spruce is to select fresh growth from young ETs as samples, pretreat them on semi-solid medium with increasing sucrose concentration (0.1 M for 24 h; 0.2 M for another 24 h), apply a mixture of polyethylene glycol 6000, glucose, and dimethylsulfoxide, 10% w/v each, as cryoprotectant and use a programmable freezer with a slow cooling rate (0.17 °C/min). On average, 87% of the genotypes can be recovered, without any effect on their genetic fidelity, as shown by microsatellite markers and embryo production capacity. Mature somatic embryos of Norway spruce can also be safely cold-stored at +4 °C, without adverse effects on their germination ability.

Molecular phylogeny and systematics of anoplocephaline cestodes in rodents and lagomorphs.

A molecular phylogenetic hypothesis is presented for the anoplocephaline cestodes of placental mammals based on sequence data from the mitochondrial cytochrome c oxidase I (COI) gene, the nuclear-encoded 28S rRNA gene and the internal transcribed spacer region I of rRNA (ITS1). The material consists of 35 species representing nine genera of cestodes, with emphasis on taxa parasitising rodents and lagomorphs in the Holarctic region. The resulting phylogenies show considerable disagreement with earlier systematic and phylogenetic hypotheses derived from morphology. Specifically, the results contradict the view of uterine morphology being the primary determinant of deeper phylogenetic splits within Anoplocephalinae. Also, the role of genital duplication as a means of generic divergence was not found to follow consistently the pattern suggested by earlier hypotheses. Colonisation of novel host lineages has evidently been the predominant mode of diversification in anoplocephaline cestodes of placental mammals; evidence for phyletic co-evolution was obscure. The phylogenies consistently distinguished a large monophyletic group including all species from arvicoline rodents (voles and lemmings), primarily representing the genera Anoplocephaloides Baer, 1923 and Paranoplocephala Lühe, 1910. Phylogenetic relationships within the "arvicoline clade" of cestodes were generally poorly resolved. Consistent support for nodes above and below the unresolved polytomy indicates a rapid radiation involving a nearly simultaneous diversification of many lineages, a scenario also proposed for the arvicoline hosts.