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

Abstract

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.

Application of Cold Storage and Short In Vitro Germination for Somatic Embryos of Pinus radiata and P. sylvestris

“…In this work, we are unable to separate the effect of cold storage on somatic embryo germination and acclimatization from other factors (short germination/use of micro-plugs) due to the vastly different propagation protocols used in the treatments tested. Despite this, cold storage has been shown to have no adverse effects on germination in some conifers [25,41] and may, in fact, have some positive effects on preparing the embryos for germination and acclimatization, such as reduction of endogenous ABA [42] and accumulation of storage proteins [43,44]. Cold storage is also a commonly-used strategy for allowing flexibility for embling production when large numbers are required in a small window of time [39].…”

Characterization of hybrid larch somatic embryo maturation by biochemical analyses and by a novel, fast mid‐infrared approach

“…Raffinose appeared late in the maturation process of hybrid larch SE (from 8 weeks of maturation) and reached a maximum level from 11 weeks of maturation. This complex sugar always appears at the end of the maturation of somatic or zygotic embryos of conifers (Gösslová et al, 2001; Morel, Trontin, et al, 2014), or after stress treatment of SE (Eliášová et al, 2022; Konrádová et al, 2003; Välimäki et al, 2022). Raffinose, which is a part of the raffinose family oligosaccharides (RFO), is considered a storage reserve and a marker of desiccation tolerance (Konrádová et al, 2003).…”

“…After cooling to room temperature, amyloglucosidase from Aspergillus niger (SIGMA) was added to reduce starch to glucose and the samples were incubated for 1 h at 50°C. Starch content was quantified as glucose equivalents from the supernatants (Välimäki et al, 2022). Each sample was assayed in triplicate.…”

“…Soluble sugars were extracted as described by Välimäki et al (2022). SE (approximately 200 mg FW for SE at early stages and 50 mg FW for cotyledonary SE) were freeze-dried and ground in 1 mL 80% aqueous ethanol containing 0.25 mg mL À1 mannitol as an internal standard, and the extracts were heated for 30 min at 80 C. After centrifugation, the supernatants were purified on activated charcoal and polyvinylpolypyrrolidone to remove proteins and polyphenols.…”

“…After drying under vacuum, the residues were resuspended in 100 μL ultra-pure H 2 O. Samples Starch content was quantified as glucose equivalents from the supernatants (Välimäki et al, 2022). Each sample was assayed in triplicate.…”