Since the 1990s, somatic embryogenesis (SE) has enabled the propagation of selected varieties, Arabica F1 hybrid and Robusta clones, originating from the two cultivated coffee species, Coffea arabica and Coffea canephora, respectively. This paper shows how mostly empirical research has led to successful industrial transfers launched in the 2000s in Latin America, Africa, and Asia. Coffee SE can be considered as a model for other woody perennial crops for the following reasons: (i) a high biological efficiency has been demonstrated for propagated varieties at all developmental stages, and (ii) somaclonal variation is understood and mastered thanks to intensive research combining molecular markers and field observations. Coffee SE is also a useful model given the strong economic constraints that are specific to this species. In brief, SE faced four difficulties: (i) the high cost of SE derived plants compared to the cost of seedlings of conventional varieties, (ii) the logistic problems involved in reaching small-scale coffee growers, (iii) the need for certification, and (iv) the lack of solvency among small-scale producers. Nursery activities were professionalized by introducing varietal certification, quality control with regard to horticultural problems and somaclonal variation, and sanitary control for Xylella fastidiosa. In addition, different technology transfers were made to ensure worldwide dissemination of improved F1 Arabica hybrids and Robusta clones. Innovations have been decisive for successful scaling-up and reduction of production costs, such as the development of temporary immersion bioreactors for the mass production of pre-germinated embryos, their direct sowing on horticultural soil, and the propagation of rejuvenated SE plants by rooted mini-cuttings. Today, SE is a powerful tool that is widely used in coffee for biotechnological applications including propagation and genetic transformation. Basic research has recently started taking advantage of optimized SE protocols. Based on -omics methodologies, research aims to decipher the molecular events involved in the key developmental switches of coffee SE. In parallel, a high-throughput screening of active molecules on SE appears to be a promising tool to speed-up the optimization of SE protocols.

Somatic embryogenesis is affected by highly variable maturation yields in Pinus pinaster. Origins of this variability were investigated by testing effects of spatial and temporal division of initial embryogenic tissue into independently cultivated pieces. One embryogenic cell-line was proliferated to obtain six embryonal-suspensor masses (ESM) treated as six sub-lines within a single dish. After proliferation to reach 2, 4 and 8 dishes (12, 24, 48 ESM), the 48 ESM (six sub-lines) were maintained by weekly subculture (34 weeks). ESM observations and maturation tests were regularly performed during the amplification and maintenance periods. Relations between maturation yields as well as cotylfedonary embryo length and immature embryo morphology were analyzed. ESM yielded variable results, independently from culture spatial (dish) and temporal (sub-line) subdivision. Maturation yields and length of regenerated embryos globally decreased as a function of subculture number. Concomitantly, morphological degradation of immature embryos occurred, indicating a global loss of embryogenic ability. Maturation variability probably results from immature embryos heterogeneity, which could be increased by manipulations during subculture. This is the first time that this evolution along time in culture is precisely described in conifers somatic embryos. Keywords Maturation AE Pinus pinaster AE Somatic embryogenesis AE Subculture AE Culture ageing Abbreviations ESM embryonal-suspensor masses f.wt fresh weight CE cotyledonary somatic embryos AE abnormally developed embryos PC precotyledonary and early cotyledonary embryos Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at http://dx.

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Somatic embryogenesis (SE) is one of the most promising processes for large-scale dissemination of elite varieties. However, for many plant species, optimizing SE protocols still relies on a trial-and-error approach. Using coffee as a model plant, we report here the first global analysis of metabolome and hormone dynamics aiming to unravel mechanisms regulating cell fate and totipotency. Sampling from leaf explant dedifferentiation until embryo development covered 15 key stages. An in-depth statistical analysis performed on 104 metabolites revealed that massive re-configuration of metabolic pathways induced SE. During initial dedifferentiation, a sharp decrease in phenolic compounds and caffeine levels was also observed while auxins, cytokinins and ethylene levels were at their highest. Totipotency reached its highest expression during the callus stages when a shut-off in hormonal and metabolic pathways related to sugar and energetic substance hydrolysis was evidenced. Abscisic acid, leucine, maltotriose, myo-inositol, proline, tricarboxylic acid cycle metabolites and zeatin appeared as key metabolic markers of the embryogenic capacity. Combining metabolomics with multiphoton microscopy led to the identification of chlorogenic acids as markers of embryo redifferentiation. The present analysis shows that metabolite fingerprints are signatures of cell fate and represent a starting point for optimizing SE protocols in a rational way.