Analysis of Nitrogen Utilization Capability during the Proliferation and Maturation Phases of Norway Spruce (Picea abies (L.) H.Karst.) Somatic Embryogenesis

Abstract

Somatic embryogenesis (SE) is a laboratory-based method that allows for cost-effective production of large numbers of clonal copies of plants, of particular interest for conifers where other clonal propagation methods are mostly unavailable. In this study, the effect of L-glutamine as an organic nitrogen source was evaluated for three contrasted media (containing NH 4 + NO 3 without glutamine, or glutamine + NO 3 , or glutamine without inorganic nitrogen) during proliferation and maturation of Norway spruce somatic embryos through analyses of activities of the key enzymes of nitrogen metabolism: nitrate reductase (NR), glutamine synthetase (GS) and arginase. A major change in nitrogen metabolism was indicated by the increased activity of GS from zero in the proliferation stage through maturation to high activity in somatic embryo-derived plantlets; furthermore, NR activity increased from zero at the proliferation stage to high activity in maturing embryos and somatic-embryo derived plantlets. In contrast, arginase activity was high at all stages. In accordance with the GS and NR data, proliferation was fastest on the glutamine media, the largest number of mature embryos developed on the glutamine medium without inorganic nitrogen, and the ammonium content of proliferating cultures was much higher than that of mature somatic embryos. The best developed embryos, judged by germination, were from the glutamine + NO 3 medium. These insights into nitrogen utilization will help to improve culture medium composition.

Factors Influencing Somatic Embryo Maturation in Sugi (Japanese Cedar, Cryptomeria japonica (Thunb. ex L.f.) D. Don)

“…Similar to our experiment on C. japonica , the best results for embryo maturation in Chamaecyparis pisifera [ 29 ], Larix decidua [ 30 ], Pinus nigra [ 31 ], and Picea abies [ 32 ] were obtained in media supplemented with low IN, rather than the high concentration in the other media tested. In contrast, a survey of SE in crop plants noted that 70% of the explants were cultured on an MS medium or a modified version of it [ 33 ].…”

“…Organic nitrogen is essential for the production of healthy embryos during SE, and glutamine is one of the most common sources of organic nitrogen reported in conifers [ 32 , 61 ]. The addition of glutamine, asparagine, arginine, and other AAs to the maturation medium has been commonly used to enhance the maturation efficiency of somatic embryos [ 2 , 5 , 29 , 35 , 45 , 62 , 63 , 64 , 65 , 66 , 67 ].…”

Improved propagation efficiency in a laboratory–nursery interface for somatic embryogenesis in Norway spruce

“…Removing inorganic nitrogen from the germination media, which led to a decrease in the total amount of nitrogen in the germination media, also had a large impact on root growth during five weeks of in vitro germination (Paper I). The results herein support previous findings, in which the growth of emblings and seedlings is enhanced when less nitrogen is used (see, e.g., Brouwer 1962;Ingestad and Kähr 1985;Kaakinen et al 2004;Gruffman et al 2012;Dahrendorf et al 2018;Llébres et al 2018a, b). Due to the improved root growth, a germination medium without inorganic nitrogen was introduced in the standard protocol.…”

The Chemical Environment at Maturation Stage in Pinus spp. Somatic Embryogenesis: Implications in the Polyamine Profile of Somatic Embryos and Morphological Characteristics of the Developed Plantlets

“…The source of organic nitrogen is an important component of the culture media for the success of the SE process, and in this sense, amino acids are commonly used to improve the maturation of ses, modifying its type and concentration (Gerdakaneh et al, 2011;Satish et al, 2016;Dahrendorf et al, 2018). As Maruyama et al (2021) reported, the presence of organic nitrogen improved the production of ses.…”

“…As Maruyama et al (2021) reported, the presence of organic nitrogen improved the production of ses. Casein and glutamine are usually used in the SE of conifers (Afele and Saxena, 1995;Pullman and Bucalo, 2014;Dahrendorf et al, 2018), but, in some species, such as Pinus patula Schiede ex Schltdl. and Cham.…”