An innovative automated active compound screening system allows high-throughput optimization of somatic embryogenesis in Coffea arabica

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 cm 2 /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.

Histone Deacetylase Inhibitors Increase the Embryogenic Potential and Alter the Expression of Embryogenesis-Related and HDAC-Encoding Genes in Grapevine (Vitis vinifera L., cv. Mencía)

“…However, treatment with 0.5 mM NaB resulted in some of the HDAC-encoding genes becoming overexpressed, even significantly, with respect to the control without NaB. These differences tended to decrease after 48 h, indicating that the gene response to NaB treatment occurs during the first hours after its application, as previously suggested [32,40,41].…”

“…With that goal, we analyzed the expression of several embryogenesis-related and HDACencoding grapevine genes. Since it has been suggested that the gene response to HDAC inhibitors occurs during the first hours of their application [32,40,41], we studied the expression of those genes in cotyledonary somatic embryos that were cultured for 24 h and 48 h in the presence of NaB.…”

miRNAs as key regulators via targeting the phytohormone signaling pathways during somatic embryogenesis of plants

“…However, few bottlenecks including poor responsiveness and somatic mutations during the SE process hampered the propagation of many plant species. (Awada et al 2020). Though some woody plants are recalcitrant to SE, still it's a promising technology for large scale propagation of important woody plants (Awada et al 2020;Guan et al 2016;Isah 2016).…”

Osmotic stress-induced somatic embryo maturation of coffee Coffea arabica L., shoot and root apical meristems development and robustness

“…It is important to notice that shoot and root apical meristems were developed compared to untreated SE. Failure in forming proper SAM and RAM in SEs was published elsewhere 1 , 3 , 7 , 9 , 13 , 50 . The importance of the apical meristems in plant growth and development has long been recognized 51 , but in woody plants, little is known about meristems formation during SE 11 .…”

“…The complete plant regeneration of C. arabica derived from indirect SE was first achieved by Yasuda et al 2 using the cytokinin (BAP) as plant growth regulator. After that report, many approaches and protocols using the synthetic auxin (2,4-D) as an inducer of indirect SE have been tested with different rates of efficiency (For reviews, see: 3 – 9 ).…”