Analysis of nitrogen utilization capability during proliferation and maturation of Norway spruce (Picea abies L. Karst) somatic embryogenesis
Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
Around 70 % of the standing trees in northern Europe are conifers, with Norway spruce being most important. To meet future wood demands, vegetative propagation methods are offering a flexible and effective way to multiply superior genotypes. The development of nitrogen metabolism during embryogenesis is not well understood and only few studies cover conifers. Norway spruce plants prefer ammonium over nitrate as an inorganic nitrogen source. However, the proliferation of somatic embryo cultures requires organic nitrogen, and ammonium nitrate as sole nitrogen source limits somatic embryo development. This raises the question how nitrogen utilization capability advances throughout the embryo development and plant formation in Norway spruce and suggests a developmental switch in nitrogen utilization capability before the plant is fully developed. Of special interest in this context is the development and activity of three key enzymes of nitrogen metabolism: nitrate reductase (NR), glutamine synthetase (GS) and arginase.
The aim of this study was to investigate the importance of L-glutamine as an organic nitrogen source and its impact on these key enzymes of nitrogen metabolism in the proliferation and maturation stage of Norway spruce somatic embryogenesis. Therefore media with modified nitrogen sources have been used to study the effects of presence and withdrawal of L-glutamine. Pro-embryogenic masses (PEMs) grown with L-glutamine (Gln) or L-glutamine and nitrate (Gln + NO3) showed a strongly improved proliferation rate in comparison to PEMs grown on ammonium nitrate (NH4NO3). Interestingly, GS and NR were inactive enzymatically in PEMs. Arginase activity was observed, and was unaffected by the presence or absence of L-glutamine. For analyzing the importance of L-glutamine as an organic nitrogen source during maturation, somatic embryos have been generated on media with modified nitrogen sources that included also autoclaved casein hydrolysate, an amino-acid mixture that lacks L-glutamine after autoclaving. Somatic embryos matured furthest regarding size and cotyledon development on Gln + NO3. Maturation on NH4NO3 resulted in well-developed cotyledonary stage somatic embryos that were smaller in size than in the presence of L-glutamine. In mature somatic embryos GS and NR were active. NR activity was highest, if embryos were matured on Gln + NO3 and notably lower if matured on Gln or NH4NO3. The tendentially highest GS activity was found if embryos were generated on NH4NO3. A striking change in nitrogen metabolism was the steady increase in GS activity from not detectable at proliferation stage, through easily detectable during maturation up to high activity in SE plantlets.
Place, publisher, year, edition, pages
Biochemistry and Molecular Biology
IdentifiersURN: urn:nbn:se:umu:diva-94896OAI: oai:DiVA.org:umu-94896DiVA: diva2:756812
SLU, Skoglig genetik och växtfysiologi
Master's Programme in Plant and Forest Biotechnology
Egertsdotter, Ulrika, ForskareBako, Laszlo, Univ.lektor
Bako, Laszlo, Univ.lektor