Biotechnological studies in Theobroma cacao L: Micropropagation, characterization of polysaccharides, and estimation of genetic diversity using molecular markers

A rapid system of micropropagation for Theobroma cacao (cacao), a species recalcitrant under conventional protocols, was developed based on exposure to high levels of CO$\sb2$. Increasing CO$\sb2$ concentration from ambient (470 ppm) up to chamber levels of 30,200 ppm significantly increased total axillary shoot elongation, number of leaves, and leaf area per explant, but decreased budbreak. The presence of light was necessary for the CO$\sb2$ effect, but estimation of net photosynthesis indicated that photosynthesis stimulation alone did not account for all the improved growth responses. Elevated CO$\sb2$ was associated with increased ethylene production in vitro and increased nutrient uptake. Nucellar embryony was induced by exposing nucellar tissue to 2,4 dichlorophenoxyacetic acid, 6-($\gamma,\gamma$-dimethylallylamino) purine (2iP), and coconut water. A protocol for conversion of nucellar somatic embryos into seedlings was developed involving preculture of somatic embryos in liquid medium and transfer to semi-solid medium in chambers receiving 20,000 ppm CO$\sb2$. A polysaccharide present in large amounts in all tissues of cacao, was hypothesized to be responsible for the recalcitrance to in vitro culture. Histochemical studies located lysigenous cavities throughout the pith and cortex of stems and pericarp of pod husks. Stem and pod husk gums contained the same monosaccharides as gum karaya (Sterculia spp), with higher proportion of rhamnose and acidic sugars (glucuronic and galacturonic). Cacao pod and stem gums have a higher viscosity at concentrations below 1% than gum karaya, but a lower viscosity on higher concentrations. The size of the haploid genome of cacao was estimated using laser flow cytometry as 0.43 pg. An improved DNA extraction procedure was developed based on isolation of a crude nuclei preparation from leaf tissue that effectively eliminated contamination of the DNA by polysaccharides and produced DNA that was on average greater than 50 kb in length. Differences in random amplified polymorphic DNA (RAPD) was used to evaluate the conventional classification of cacao into three major horticultural races (Criollo, Forastero, and Trinitario). A phenogram based on molecular markers (RAPD) indicated essentially continuous variation among different genotypes, with only a superficial rationale for the conventional classification, but a clear separation between wild and cultivated genotypes. DNA blot hybridization experiments using a flax ribosomal DNA probe revealed restriction fragment length polymorphisms (RFLP), and was used to establish phylogeny of Theobroma and Herrania species. A phenogram based on the rDNA polymorphism separated a T. cacao cluster and a Theobroma-Herrania cluster