Cassava (Manihot esculenta Crantz) biofortification with provitamin A carotenoids is an ongoing process that aims to alleviate vitamin A deficiency. The moderate content of provitamin A carotenoids achieved so far limits the contribution to providing adequate dietary vitamin A levels. Strategies to increase carotenoid content focused on genes from the carotenoids biosynthesis pathway. In recent years, special emphasis was given to ORANGE protein (OR), which promotes the accumulation of carotenoids and their stability in several plants. The aim of this work was to identify, characterize and investigate the role of OR in the biosynthesis and stabilization of carotenoids in cassava and its relationship with phytoene synthase (PSY), the rate-limiting enzyme of the carotenoids biosynthesis pathway. Gene and protein characterization of OR, expression levels, protein amounts and carotenoids levels were evaluated in roots of one white (60444) and two yellow cassava cultivars (GM5309-57 and GM3736-37). Four OR variants were found in yellow cassava roots. Although comparable expression was found for three variants, significantly higher OR protein amounts were observed in the yellow varieties. In contrast, cassava PSY1 expression was significantly higher in the yellow cultivars, but PSY protein amount did not vary. Furthermore, we evaluated whether expression of one of the variants, MeOR_X1, affected carotenoid accumulation in cassava Friable Embryogenic Callus (FEC). Overexpression of maize PSY1 alone resulted in carotenoids accumulation and induced crystal formation. Co-expression with MeOR_X1 led to greatly increase of carotenoids although PSY1 expression was high in the co-expressed FEC. Our data suggest that posttranslational mechanisms controlling OR and PSY protein stability contribute to higher carotenoid levels in yellow cassava. Moreover, we showed that cassava FEC can be used to study the efficiency of single and combinatorial gene expression in increasing the carotenoid content prior to its application for the generation of biofortified cassava with enhanced carotenoids levels.

An optimized high-quality DNA isolation protocol was developed using body segment tissue from the Fall Armyworm (Spodoptera frugiperda), that will allow documenting genetic variability based on biotypes, facilitating studies on the appearance, distribution and population dynamics of the fall armyworm at the molecular level. The resulting protocol is an easy-to-use, timesaving method that can rapidly achieve high quality, high-yielding total genomic DNA, using chemicals and everyday consumables available in a molecular laboratory. This new method of DNA extraction avoids the contamination of polysaccharides, salts, phenols, proteins and other cellular by-products that can interfere with subsequent reactions. DNA purity estimates reveal A260: A280 ratios greater than 1.9, which were evidenced by quality test on agarose gel, observing complete integrity and high purity of the resulting samples, and yielded 30–99 µg/g of total DNA. Therefore, the quality of the DNA produced from this extraction is suitable for subsequent molecular applications: (i) next generation whole genome sequencing, (ii) conventional polymerase chain reaction for genotyping, (iii) barcodes and (iv) gene cloning. In addition, to become an anticipating diagnostic tool for invasive lepidopteran larval stages: The resulting protocol is an easy-to-use time-saving method. This new extraction method prevents contamination from polysaccharides, salts, phenols, proteins, and other cellular sub-products. DNA purity estimations reveal A260:A280 ratios above 1.9.