Cotyledonary somatic embryos of Pinus pinaster Ait. most closely resemble fresh, maturing cotyledonary zygotic embryos: biological, carbohydrate and proteomic analyses

Abstract

Cotyledonary somatic embryos (SEs) of maritime pine are routinely matured for 12 weeks before being germinated and converted to plantlets. Although regeneration success is highly dependent on SEs quality, the date of harvesting is currently determined mainly on the basis of morphological features. This empirical method does not provide any accurate information about embryo quality with respect to storage compounds (proteins, carbohydrates). We first analyzed SEs matured for 10, 12 and 14 weeks by carrying out biological (dry weight, water content) and biochemical measurements (total protein and carbohydrate contents). No difference could be found between collection dates, suggesting that harvesting SEs after 12 weeks is appropriate. Cotyledonary SEs were then compared to various stages, from fresh to fully desiccated, in the development of cotyledonary zygotic embryos (ZEs). We identified profiles that were similar using hierarchical ascendant cluster analysis (HCA). Fresh and dehydrated ZEs could be distinguished, and SEs clustered with fresh ZEs. Both types of embryo exhibited similar carbohydrate and protein contents and signatures. This high level of similarity (94.5 %) was further supported by proteome profiling. Highly expressed proteins included storage, stress-related, late embryogenesis abundant and energy metabolism proteins. By comparing overexpressed proteins in developing and cotyledonary SEs or ZEs, some (23 proteins) could be identified as candidate biomarkers for the late, cotyledonary stage. This is the first report of useful generic protein markers for monitoring embryo development in maritime pine. Our results also suggest that improvements of SEs quality may be achieved if the current maturation conditions are refined.

Somatic Embryogenesis for More Effective Breeding and Deployment of Improved Varieties in Pinus spp.: Bottlenecks and Recent Advances

“…In P. pinaster, no differences were observed in cotyledonary se after 10-14 weeks of maturation with respect to contents of total proteins, various mono-or polysaccharide sugars, water and dry matter (Morel et al 2014b). When 12-week-old se were compared with ze at different stages of maturity (fresh cotyledonary to desiccated cotyledonary stages) using the same molecular and other biological and physiological parameters, it was clear that they corresponded the most to the fresh, maturing cotyledonary ze found in green cones that are present in France in late July/early August.…”

“…mDCR modified DCR medium from Gupta and Durzan (1985); mLV modified LV medium from Litvay et al (1985) 2006; Klimaszewska et al 2009;Humánez et al 2012). Biological parameters (dry weight, water content) and biochemical analyses (total protein and carbohydrate contents) showed that cotyledonary se harvested after 10 or 14 weeks did not differ markedly and thus confirmed that harvesting cotyledonary se after 12 weeks is appropriate (Morel et al 2014b). Cotyledonary se germinated at a high frequency (>70 %, reviewed in Trontin et al 2016a), although differences were observed among lines.…”

“…Considering protein analyses, the same storage proteins or their coding transcripts have been identified in the se of P. strobus (Klimaszewska et al 2004), P. pinaster (Morel et al 2014b), P. taeda and P. oocarpa (Lara-Chavez et al 2012), and have been shown to accumulate in different conifer species from precotyledonary to (mainly) cotyledonary stages (Lippert et al 2005;Lara-Chavez et al 2012). In P. sylvestris, it has also been reported that storage proteins were present in equivalent amounts between mature se and ze (Lelu-Walter et al 2008).…”

“…The biochemical composition of ze during late maturation has also been investigated in an attempt to define the optimal timing for transferring cotyledonary se to a germination medium (Brownfield et al 2007;Vuosku 2011;Morel et al 2014b;Pullman and Bucalo 2014). The type and contents of proteins, sugars, ethylene and polyamines were suggested to be critical for se post-maturation stages affecting ss quality.…”

Forest tree genomics: 10 achievements from the past 10 years and future prospects

“…The metabolic signature has been shown to accurately predict embryogenic potential in P. taeda (Robinson et al 2009). Transcriptomics in P. taeda (Pullman et al 2003) and proteomics in P. pinaster (Morel et al 2014b) have provided strong evidence of differences between SEs and fully mature ZEs. Transcriptomics has also proved to be of practical value in Picea glauca to check SE quality in different maturation conditions (Stasolla et al 2003).…”