Abstract
BackgroundTo explore poorly understood differences between primary and subsequent somatic embryogenic lines of plants, we induced secondary (2ry) and tertiary (3ry) lines from cotyledonary somatic embryos (SEs) of two Douglas-fir genotypes: SD4 and TD17. The 2ry lines exhibited significantly higher embryogenic potential (SE yields) than the 1ry lines initiated from zygotic embryos (SD4, 2155 vs 477; TD17, 240 vs 29 g− 1 f.w.). Moreover, we observed similar differences in yield between 2ry and 3ry lines of SD4 (2400 vs 3921 g− 1 f.w.). To elucidate reasons for differences in embryogenic potential induced by repetitive somatic embryogenesis we then compared 2ry vs 1ry and 2ry vs 3ry lines at histo-cytological (using LC-MS/MS) and proteomic levels.ResultsRepetitive somatic embryogenesis dramatically improved the proliferating lines’ cellular organization (genotype SD4’s most strongly). Frequencies of singulated, bipolar SEs and compact polyembryogenic centers with elongated suspensors and apparently cleavable embryonal heads increased in 2ry and (even more) 3ry lines. Among 2300–2500 identified proteins, 162 and 228 were classified significantly differentially expressed between 2ry vs 1ry and 3ry vs 2ry lines, respectively, with special emphasis on “Proteolysis” and “Catabolic process” Gene Ontology categories. Strikingly, most of the significant proteins (> 70%) were down-regulated in 2ry relative to 1ry lines, but up-regulated in 3ry relative to 2ry lines, revealing a down-up pattern of expression. GO category enrichment analyses highlighted the opposite adjustments of global protein patterns, particularly for processes involved in chitin catabolism, lignin and L-phenylalanine metabolism, phenylpropanoid biosynthesis, oxidation-reduction, and response to karrikin. Sub-Network Enrichment Analyses highlighted interactions between significant proteins and both plant growth regulators and secondary metabolites after first (especially jasmonic acid, flavonoids) and second (especially salicylic acid, abscisic acid, lignin) embryogenesis cycles. Protein networks established after each induction affected the same “Plant development” and “Defense response” biological processes, but most strongly after the third cycle, which could explain the top embryogenic performance of 3ry lines.ConclusionsThis first report of cellular and molecular changes after repetitive somatic embryogenesis in conifers shows that each cycle enhanced the structure and singularization of EMs through modulation of growth regulator pathways, thereby improving the lines’ embryogenic status.
Highlights
To explore poorly understood differences between primary and subsequent somatic embryogenic lines of plants, we induced secondary (2ry) and tertiary (3ry) lines from cotyledonary somatic embryos (SEs) of two Douglas-fir genotypes: SD4 and TD17
Many Gene Ontology (GO) terms are enriched in significant proteins associated with both cycles of somatic embryogenesis (Table 3), including processes involved in chitin and polysaccharide catabolism, lignin and L-phenylalanine metabolism, phenylpropanoid biosynthesis, oxidation-reduction, and response to karrikin
The expression of proteins assigned to these GO term categories declined following the first cycle but increased after the second cycle, corroborating the finding that different protein profiles were established after each cycle of somatic embryogenesis
Summary
To explore poorly understood differences between primary and subsequent somatic embryogenic lines of plants, we induced secondary (2ry) and tertiary (3ry) lines from cotyledonary somatic embryos (SEs) of two Douglas-fir genotypes: SD4 and TD17. Plant somatic embryogenesis is the generation of embryos from vegetative cells, usually in vitro. Significant advances have been made in the development of techniques to improve somatic embryogenesis of increasing numbers of tree species, from initiation of embryogenic cultures to maturation of high-quality somatic embryos (SEs). Such progress towards large-scale production of vigorous somatic seedlings has been reported for both hardwood [1, 2] and softwood (mostly coniferous) species (reviewed in [3]). There is a wealth of patented methods, but some recent improvements for steps from initiation to efficient production of somatic seedlings have just been made publicly available ([5, 6], and references therein)
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