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.

Coronilla viminalis Salisb. is a fodder leguminous plant from the Canary Islands and Northwestern Africa with adaptation to drought. Its conservation status is critical. Its low germination capacity and intense loss of viability of the seeds over time complicate its conservation, limiting its long-term maintenance in germplasm banks and hampering its potential use as a resilient fodder crop. Therefore, in this work, two alternative propagation methods have been addressed. The first was based on facilitating seed germination under aseptic conditions, supplementing the media with gibberellic acid (GA₃). The latter consisted of creating a micropropagation method from nodal segments and testing different media and pretreatments with indole-3-acetic acid (IAA). The quantity and quality of the roots were assessed, and the plant acclimation rate was measured. The results showed that the optimum concentration of GA₃ for germination, up to 98%, was 250 PPM. The experiments showed that using IAA in the micropropagation media is critical for in vitro rooting in this species. A hormonal pretreatment with IAA significantly improved the rooting efficiency compared to supplementing it into the culture media. One hundred percent of acclimated plants survived the process. These new protocols will help conserve the species and explore its possibilities as fodder crops.