Abstract
Somatic embryogenesis (SE) faces many challenges in fulfilling the growing demand for elite materials. A high-throughput approach is required to accelerate the optimization of SE protocols by multiplying experimental conditions within a limited time period. For the first time in plant micropropagation, we have developed a miniaturized and automated screening system to meet high-throughput standards. Coffea arabica embryo regeneration, classically achieved in 250-ml Erlenmeyer flasks, was successfully miniaturized in 24-well plates, allowing a volume downscaling factor of 100 and a space saving of 53 cm2/well. Cell clusters were ground and filtered to fit the automated pipetting platform, leading to fast, reproducible and uniform cluster distribution (23.0 ± 5.5 cell clusters/well) and successful regeneration (6.5 ± 2.2 embryos/well). Pilot screening of active compounds on SE was carried out. Compounds belonging to the histone deacetylase inhibitor family were tested for embryo regeneration efficiency. Cells treated with 1 µM Trichostatin A showed a marked 3-fold increase in the number of regenerated embryos. When re-tested in 250-ml flasks, the same enhancement was obtained, thereby validating the miniaturized and automated screening method. These results showed that our screening system is reliable and well suited to screening hundreds of compounds, offering unprecedented perspectives in plant micropropagation.
Highlights
Somatic embryogenesis (SE) faces many challenges in fulfilling the growing demand for elite materials
We focus in this paper on one of the main three SE steps in C. arabica: embryo regeneration from embryogenic cell clusters
We proved in this study that a major step of SE processes, i.e. embryo regeneration from embryogenic cell clusters, can be successfully miniaturized and automated allowing easy, quick, cheap and reliable testing of numerous compounds at varied concentrations
Summary
Somatic embryogenesis (SE) faces many challenges in fulfilling the growing demand for elite materials. For the first time in plant micropropagation, we have developed a miniaturized and automated screening system to meet high-throughput standards. When re-tested in 250-ml flasks, the same enhancement was obtained, thereby validating the miniaturized and automated screening method. These results showed that our screening system is reliable and well suited to screening hundreds of compounds, offering unprecedented perspectives in plant micropropagation. Developing new technologies for the clonal propagation, improvement and breeding of trees can help solve these problems[2] This has been achieved in part using biotechnology methodologies, such as in vitro propagation, genetic transformation, and marker-assisted breeding for the gradual genetic improvement of woody plants[3,4]. A number of common bottlenecks have been noted throughout the main three steps of the current SE process –(1)
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