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Quality of cocoa (Theobroma cacaoL AbstractTheobroma cacao L. and its products are consumed worldwide. Those products are of great research interest due to antioxidant properties of some of their polyphenolic constituents. The amount of these polyphenols and polysaccharides has shown that can interfere with the high quality and quantity of nucleic acids for molecular research. Therefore, cocoa DNA extraction protocols can require a large amount of plant material and optimization time according with plant material source. The aim of this study was to evaluate the quality and quantity of DNA isolated from field plant leaves at different developmental stages from TSH565 genotype using different DNA extraction protocols. In addition, DNA extraction protocol was evaluated for small amount of young leaf tissue collected from in vitro plantlets from CCN51 and TSH565 genotype. Subsequently, the selectivity of different polymerase enzymes for PCR amplification using the obtained DNA was evaluated. This study revealed that D stage of development in field leaves was efficient for extraction of high-quality genomic DNA using the PowerPlant® Pro Kit modified (183.80 ng.µL -1 (1.98 A260/A280-1.98 A260/A230)). Highest DNA concentrations were obtained for FPL with 128.68 ng.µL -1 and 114.42 ng.µL -1 for CCN51 and TSH565 respectively and with IVL, which was obtained 54.24 ng.µL -1 for CCN51 and 56.52 ng.µL -1 for TSH565 per 0.1 g of leaf tissue. Taq DNA Polymerase recombinant of Thermo Scientific® showed the highest performance specifically for this study, contributing to the undoubted amplification of molecular markers like microsatellites (SSRs). The results obtained have allowed improvements in genetic analyses and molecular studies using a reduced amount of plant tissue.

In order to understand the causes of lack of regeneration in cacao somatic embryos, two cacao varieties with different responses to regeneration potential were described based on their capacity to store different compounds. It is well known that seed reserves play a central role in the regenerative capability of somatic embryos; thus, we followed histochemical changes and reserve fluctuations of proteins, polysaccharides and polyphenols during somatic embryogenesis (SE) in the two cacao varieties. The study showed that, in somatic embryos of the regenerating variety, polyphenols were localized mainly in the periphery of the embryo (epidermal cells) and proteins were the main storage substance in the embryo expression medium, while the non-regenerating variety had a high presence of polysaccharides with random distribution of polyphenols at the end of the embryo induction step.Keywords: antioxidants, histology, reserve accumulation, recalcitrance, somatic embryogenesis. RESUMENDos variedades de cacao con diferentes respuestas a la regeneración fueron descritas en función de su capacidad para almacenar diferentes compuestos, con el fin de aproximarse al entendimiento de las causas de la falta de regeneración en embriones somáticos de cacao. Es bien sabido que las reservas de semillas desempeñan un papel central en la capacidad de regeneración de embriones somáticos; por tanto, se realizó un seguimiento de cambios histoquímicos y fluctuaciones de reserva de proteínas, polisacáridos y polifenoles durante la embriogénesis somática (SE) en dos variedades de cacao. El estudio mostró que, en los embriones somáticos de la variedad regenerante, los polifenoles se localizaron principalmente en la periferia del embrión (células de la epidermis) y las proteínas fueron el componente principal de almacenamiento en el medio de expresión de embriones, mientras que la variedad no regenerante tenía una alta presencia de polisacáridos y una distribución aleatoria de los polifenoles en el final de la etapa de inducción de embriones. Palabras clave: acumulación de reservas, antioxidantes, embriogénesis somática, histología, recalcitrancia.

In vitro cacao (Theobroma cacao L.) production via somatic embryogenesis (SE) is being implemented to mass propagate clonal plant material with the donor material’s prominent characteristics. Though it is an advanced technology, it is con-sidered expensive compared to other propagation techniques. This work focused on identifying the critical financial feasi-bility factors for the SE productive process. The process's costs were estimated, identifying factors influencing each la-boratory's standardized ES process. A Monte Carlo Simulation (MCS) was performed to evaluate different variables upon increasing productive scale in a biofactory (commercial-scale production). The projected lot volume was 100,000 plantlets in solid media, considering the process flow from in vitro introduction to acclimation. A biofactory operational model was projected, establishing time and operator movements and identifying direct and indirect costs. Costs were defined by the standardized or integral method, with estimated and budgeted calculations to set the cost per plantlet. The identified cost components were culture medium (CM), indirect manufacturing costs (IMC), labor (direct and indirect) and operating expenses. Labor had the most significant share of the costs, at 53%, followed by operating expenses, at 30%, CM, at 12%, and IMC, at 5%. The MCS helped define that the variables with the highest impact on unit price were the embryos’ response in the germination-acclimation stage and the proliferation coefficient during the maturation stage. This projection yielded a figure of US $0.73 per plantlet. However, strategies to reduce this cost have been proposed. These strategies are mainly conducive to optimizing labor and implementing practices that increase multiplication. Keywords. Plant Tissue culture, Cost analysis, Large-scale production, Biofactory, Monte Carlo Simulation (MCS)

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