Somatic embryogenesis and long term conservation of cocoa (Theobroma cacao L.) germplasm

Cocoa (Theobroma cacao L.) is an economically important crop for Nicaragua. It represents a secure source of income for the farmers basically because of the high demand in the international market for the chocolate industry. In Nicaragua cocoa production is carried out on small scale farms and the most critical point in the production system is the low propagation rate obtained by asexual propagation methods. The present study aimed to evaluate cocoa propagation by somatic embryogenesis following the protocol developed at the Pennsylvania State University and to validate a reliable protocol for its conservation for long term. Induction of primary somatic embryos was carried out from petals and staminodes of unopened flower buds of the genotype Scavina-6 of two physiological ages. The best frequency on primary embryogenic callus growth was obtained by 1-2 week old petals. Secondary embryo development was done from cotyledons of primary somatic embryos of the clones LCTEEN 28/S-1 and PA 169. It was determined after incubation of the embryos in four different periods in secondary callus growth (SCG) medium. The highest number of embryogenic explants was obtained for the clone PA 169 after 8 weeks in SCG medium and 6 weeks in embryo development (ED) medium whereas, the best embryo production ratio during the time in ED medium was scored by explants of LCTEEN 28/S-1 incubated twice in SCG medium. Long term conservation was evaluated by incubating immature embryos of both clones LCTEEN 28/S-1 and PA 169 in liquid nitrogen. There was not registered survival of the embryos after the freezing treatment. However, most of the non-frozen embryos could survive and regenerate from the alginate capsules suggesting that the survival was not threatened by the sucrose concentrations used to dehydrate the embryos but most likely by the intracellular ice formation from the water content that remain in the cell after the dehydration period.