Simple and efficient protocols for the initiation and proliferation of embryogenic tissue of Douglas-fir

Reeves, Cathie; Hargreaves, Cathy; Trontin, Jean-François; Lelu‐Walter, Marie‐Anne

doi:10.1007/s00468-017-1622-7

Cited by 17 publications

(31 citation statements)

References 32 publications

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“…They were used as parental trees to perform the following control crosses at INRA (Orléans, France): 4455 × 4466 and 4456 × 4477. Somatic embryogenesis was induced from seed explants (isolated immature zygotic embryos at the pre-cotyledonary developmental stage) following published methodology [ 5 ]. Primary embryogenic lines SD4 (4456 × 4477) and TD17 (4455 × 4466) were initiated in 2011 and 2012, respectively [ 6 ].…”

Section: Methodsmentioning

confidence: 99%

“…Somatic embryogenesis techniques for propagating Douglas-fir ( Pseudotsuga menziesii (Mirb) Franco), a productive conifer species of the Pinaceae family with globally appreciated wood quality, have been under development for more than 30 years [ 4 ]. 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).…”

Section: Introductionmentioning

confidence: 99%

“…SE clusters of some conifer species, such as Douglas-fir, develop into polyembryogenic center of various sizes, putatively through continuous but incomplete cleavage polyembryony (somatic polyembryogenesis) and /or de novo somatic embryogenesis from proliferating early SEs [ 6 ]. In addition, some Douglas-fir lines of proliferating EMs contain both immature SEs and clusters of non-embryogenic cells (NECs) [ 5 , 16 ]. The occurrence of viable NECs interspersed with early SEs is apparently a characteristic feature of Douglas-fir EMs that has not been clearly documented in other conifer species ([ 5 ], Eliášová and Lelu-Walter, personal communication).…”

Section: Introductionmentioning

confidence: 99%

“…In addition, some Douglas-fir lines of proliferating EMs contain both immature SEs and clusters of non-embryogenic cells (NECs) [ 5 , 16 ]. The occurrence of viable NECs interspersed with early SEs is apparently a characteristic feature of Douglas-fir EMs that has not been clearly documented in other conifer species ([ 5 ], Eliášová and Lelu-Walter, personal communication). Subsequent transition from early SEs to cotyledonary SEs is stimulated when EMs are exposed to specific maturation conditions.…”

Section: Introductionmentioning

confidence: 99%

“…Such poor performance at the maturation step was shown to be associated with substantial progressive changes in cellular organization during proliferation in Pinus pinaster , resulting in reduced frequencies of immature SEs capable of completing the last stages of late embryogenesis [ 34 ]. In both maritime pine and Douglas-fir, proliferation of EMs in the presence of maltose as the main carbon source can greatly improve the key cytological features of immature SEs [ 5 , 35 ].…”

Section: Introductionmentioning

confidence: 99%

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Repetitive somatic embryogenesis induced cytological and proteomic changes in embryogenic lines of Pseudotsuga menziesii [Mirb.]

et al. 2018

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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.Electronic supplementary materialThe online version of this article (10.1186/s12870-018-1337-y) contains supplementary material, which is available to authorized users.

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Section: Methodsmentioning

confidence: 99%