Transcriptome profiling and gene editing for biofortification of cassava

Cassava (Manihot esculenta Crantz) is an important food crop for millions of people in sub- Saharan Africa. Cassava serves as a food security crop and a good source of energy, but it has relatively low nutritional quality, which has nutritional implications for those who rely on the crop as their main source of calorie intake. Vitamin A deficiency (VAD) is a health problem in populations whose diet constitutes mainly starchy crops like cassava. A possible solution is biofortification by conventional breeding or biotechnology to increase β-carotene content. Unfortunately, some studies report a negative correlation between β-carotene and dry matter content in certain genotypes, which could pose a challenge to these types of cassava biofortification measures. Field-grown cassava landraces were analyzed for agronomic traits and carried out specific and global transcript analyses by real-time quantitative RT-PCR and RNA-sequencing (RNA-seq). This was combined with targeted starch and carotenoids analysis by HPLC and non-targeted metabolite analysis by GC/LC-MS to understand the regulation of key enzymes and intermediate metabolites to identify genes influencing β- carotene accumulation in cassava. Also, using the CRISPR/Cas9-mediated gene editing system in the cassava cultivar TMS60444 we tried to introduce knockout mutations into cassava β-carotene hydroxylase (MeChyβ), lycopene-ε-cyclase (MeLcyε), and 9- cisepoxycarotenoid dioxygenase 1 (MeNced1) which are key genes of the carotenoid pathway. In a separate study, the biosafety regulations and policies in Kenya, Nigeria, Uganda, and the EU represented by Sweden were examined by comparing legislative texts and conducting interviews to determine if policy and regulatory frameworks present problems to perform R&D using new breeding technologies. In the cassava landraces analyzed, we found a weak negative correlation between starch and β-carotene content, whereas there was a strong positive correlation between root yield and carotenoids. Also, cassava landraces with reasonably high content of starch and β-carotene were identified that could be candidates for biofortification by further breeding or plant biotechnological means. Global gene expression profiles grouped cassava landraces into white and yellow landraces; however, at the same time there was no general correlation between the expression profiles of individual genes involved in carotenoid synthesis and accumulation with the storage root color. Gene Ontology (GO) enrichment showed over-representation of upregulated genes involved in protein-related metabolic and catabolic processes in yellow landraces while GO related to photosynthesis and light reactions were enriched in white landraces. Interestingly, we identified a previously reported amino acid change from Alanine to Aspartic acid in MePsy1 at position 191 to distinguish the yellow lines from the white lines; however, this change was absent in the paleyellow lines, confirming that the mutation in psy is not solely responsible for carotenoid accumulation in cassava. Non-targeted metabolite analysis revealed higher abundance of several amino acids in white lines, but also higher levels of a few osmolytes indicating differences in stress response. Transformation of cassava FECs with gene targets Lcyε, and Nced1 did not produce transgenic regenerated shoots, whereas MeChyβ produced in vitro plantlets still under investigation. Finally, our study showed that biosafety regulations on GMO approval in Kenya, Nigeria, and Uganda are not a major hurdle for R&D but might rather be influenced by factors outside of the regulatory framework such as perceptual and financial factors including funding opportunities

Transcriptome and metabolome profiling identify factors potentially involved in pro-vitamin A accumulation in cassava landraces

Cassava (Manihot esculenta Crantz) is a predominant food security crop in several developing countries. Its storage roots, rich in carbohydrate, are deficient in essential micronutrients, including provitamin A carotenoids.Increasing carotenoid content in cassava storage roots is important to reduce the incidence of vitamin A deficiency, a public health problem in sub-Saharan Africa. However, cassava improvement advances slowly, mainly due to limited information on the molecular factors influencing 13-carotene accumulation in cassava.To address this problem, we performed comparative transcriptomic and untargeted metabolic analyses of roots and leaves of eleven African cassava landraces ranging from white to deep yellow colour, to uncover regulators of carotenoid biosynthesis and accumulation with conserved function in yellow cassava roots.Sequence analysis confirmed the presence of a mutation, known to influence 13-carotene content, in PSY transcripts of deep yellow but not of pale yellow genotypes. We identified genes and metabolites with expression and accumulation levels significantly associated with 13-carotene content. Particularly an increased activity of the abscisic acid catabolism pathway together with a reduced amount of L-carnitine, may be related to the carot-enoid pathway flux, higher in yellow than in white storage roots. In fact, NCED_3.1 was specifically expressed at a lower level in all yellow genotypes suggesting that it could be a potential target for increasing carotenoid accumulation in cassava.These results expand the knowledge on metabolite compositions and molecular mechanisms influencing carotenoid biosynthesis and accumulation in cassava and provide novel information for biotechnological ap-plications and genetic improvement of cassava with high nutritional values

Gene Expression and Metabolite Profiling of Thirteen Nigerian Cassava Landraces to Elucidate Starch and Carotenoid Composition

The prevalence of vitamin A deficiency in sub-Saharan Africa necessitates effective approaches to improve provitamin A content of major staple crops. Cassava holds much promise for food security in sub-Saharan Africa, but a negative correlation between beta-carotene, a provitamin A carotenoid, and dry matter content has been reported, which poses a challenge to cassava biofortification by conventional breeding. To identify suitable material for genetic transformation in tissue culture with the overall aim to increase beta-carotene and maintain starch content as well as better understand carotenoid composition, root and leaf tissues from thirteen field-grown cassava landraces were analyzed for agronomic traits, carotenoid, chlorophyll, and starch content. The expression of five genes related to carotenoid biosynthesis were determined in selected landraces. Analysis revealed a weak negative correlation between starch and beta-carotene content, whereas there was a strong positive correlation between root yield and many carotenoids including beta-carotene. Carotenoid synthesis genes were expressed in both white and yellow cassava roots, but phytoene synthase 2 (PSY2), lycopene-epsilon-cyclase (LCY epsilon), and beta-carotenoid hydroxylase (CHY beta) expression were generally higher in yellow roots. This study identified lines with reasonably high content of starch and beta-carotene that could be candidates for biofortification by further breeding or plant biotechnological means

Biosafety regulatory frameworks in Kenya, Nigeria, Uganda and Sweden and their potential impact on international R&D collaborations

ABSTRACTGene technologies, such as transgenesis and new breeding techniques (NBTs), expand the toolbox for plant breeding. Many countries in Africa, however, have long been seen as “slow adopters” of gene technologies for several reasons, one being the lack of, or overly restrictive, biosafety regulatory frameworks. This is sometimes attributed to the influence of the precautionary-oriented EU biosafety policies. This study analyses and compares the biosafety regulatory frameworks and their implementation in Kenya, Nigeria and Uganda, and in the EU member state Sweden. The focus is on (1) the structure of the biosafety regulatory frameworks including the scope of the legislation, (2) the duration and cost of regulatory authorization for field trials with genetically modified (GM) plants, and (3) the regulatory approach to NBT products, i.e. to what extent NBT products are subject to the provisions of the biosafety regulatory framework. The data was collected through studying relevant legal and policy documents as well as interviewing regulatory officers and researchers in the respective countries. We found that the regulatory procedures in the selected countries are relatively straightforward, while the costs and duration may present a challenge. The regulatory approach to NBT products differ between the selected African countries and Sweden, the latter which follows EU regulations. The results are discussed in terms of the impact the regulatory developments in these four jurisdictions may have on international R&D collaborations involving the use of gene technologies and we also weigh the results against the common conception that Europe exerts a heavy influence on African countries in this technology field