Theobroma cacao: Somatic Embryogenesis.

A two-step process combining direct and indirect somatic embryogenesis, on solid and liquid medium, respectively is described for Theobroma cacao L. Staminodes and petals from unopened bud flowers are used to induce primary direct embryos. Then, these primary embryos are cut to produce embryogenic calli which will develop secondary embryos. This step of indirect SE allows us to produce large quantities of embryos and to do mass propagation using liquid culture medium. Despite a very strong clone dependency and high batch-to-batch variability, about 80% of T. cacao cultivars respond to somatic embryogenesis and can be propagated by this method.

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

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.

Indirect somatic embryogenesis of Theobroma cacao L. in liquid medium and improvement of embryo-to-plantlet conversion rate.

The establishment of cocoa embryogenic cell lines in liquid medium starting from high frequency somatic embryogenesis (HFSE) callus is described. The growth kinetics of the cultures during the multiplication and the expression steps conducted in 250 mL Erlenmeyer flasks were described for three genotypes selected for their agronomical traits (EET95, EET96, and EET103). The glucose and dissolved oxygen concentrations and the absorption of Murashige and Skoog medium macronutrients (nitrate, ammonium, potassium, sulfate, calcium, phosphorus, and magnesium) were monitored. The multiplication of the embryogenic calluses in a medium containing 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) at 1 mg L-1, initiated with an inoculation density of 20 g L-1 of callus, was achieved. The growth rate was characterized by two phases, with the second being concomitant with a depletion of phosphorus and magnesium, and a decrease in the embryogenic potential of the callus. The expression of the callus embryogenic capacity was conducted in an auxin-free medium. The embryo production starting from 1 and 5 g L-1 inoculation densities was compared. When placed in the optimal expression conditions in flasks, 1 g of callus produced 1000 to 1500 embryos within 5 to 7 wk. Finally, two paths for improving the plantlet regenerative capacities of cocoa SE produced in liquid medium were identified. Supplementing the expression medium with myo-inositol used as an osmotic agent at a concentration of 50 g L-1 increased the embryo-to-plantlet conversion rate from 13-16% to 40-48%. A 6-wk culture of the embryos on a maturation medium in Petri dishes optimized their subsequent development into plantlets.