Abstract
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.
Highlights
Theobroma cacao L. trees are grown in the humid tropics to produce cocoa beans
The differences observed between the occurrence of cocoa secondary somatic embryogenesis (SE) and embryogenic callus are very similar to the differences reported in Arabica coffee, between the LFSE and high frequency somatic embryogenesis (HFSE) processes (Söndahl et al 1985; Etienne 2005)
A confined environment induced a larger number of well-formed mature embryos and a lower rate of precocious germination than a ventilated environment, and the addition of 5% CO2 or 10% O2 in a vented environment promoted SE (El Meskaoui and Tremblay 1999; El Meskaoui et al 2006). This is the first detailed report describing the establishment of cocoa embryogenic cell lines and their growth kinetics in liquid medium
Summary
Theobroma cacao L. trees are grown in the humid tropics to produce cocoa beans. Cocoa is considered as a cash crop, but this is a crucial crop for the confectionary business, as it provides the key raw material for chocolate-based products. Cocoa production from T. cacao trees is vulnerable in most producing countries due to the low income of farmers, diseases, pests, and adverse environmental conditions. To face these challenges, integrated propagation approaches based on somatic embryogenesis (SE), combined with traditional propagation techniques, have been described for the rapid dissemination of selected clones (Maximova et al 2005; Miller 2009; Guillou et al 2014). Due to SE, disease-free materials can be produced with an orthotropic growth habit, which the farmers are familiar with in some countries
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