Biofortification of Cassava

Inglés. Cassava, Manihot esculenta, is the third most important staple crop in tropical re- gions. Cassava is a fibrous root that is tolerant to drought and has a flexible harvest period making it a reliable crop under poorer farming conditions (FAO, 2008). The content of provitamin A, also known as β-carotene, is unfortunately low in cassava even though there are varieties bred for a higher content. Since vitamin A deficiency, VAD, is a large problem of the regions where cassava is of major importance for calo- rie intake, an increase of the β-carotene content would be of great interest to address malnutrition. The main idea of this project is to increase the amount of β-carotene accumulated in the cassava root. This is accomplished by using the knowledge of the cassava genome and the carotenoid pathway, in order to block specific enzymes through genome editing techniques . The trials will though take many years and this Master Thesis will cover only a fragment of the entire project. Cassava needs to grow in the field for at least nine months, which will create a long time frame to consider. After creating genetic constructs and transforming the cassava plants, the carotenoid and starch content will be analysed. Methods for analysing starch and carotenoids for cassava were developed during this Master Thesis by literature research and laboratory trials.

Inglés. At the Swedish University of Agricultural Sciences, SLU, in Alnarp, Erik Alexan- dersson is leading a project that focuses on biofortification of cassava. Cassava is a staple crop grown in tropical regions and serves as the third most important source of calorie intake in these regions (FAO, 2008). Cassava is a root tolerant to drought and can be harvested when needed, which makes it a favorable crop for impover- ished families. Cassava can be cooked in several different ways, but one in particular popular method is to dry it and mill to a flour that can be stored and later used for porridge, known as garri. One major issue with cassava, however, is that it produces little β-carotene naturally. β-carotene is active as provitamin A, which is essential in our diets. If the main calorie intake consists of cassava, the lack of β-carotene in the root can cause vitamin A deficiency, leading to blindness and death. In this project, the β-carotene and starch content in cassava was evaluated in or- der to understand the genes controlling the biosynthesis. The overall project goal is to enhance the β-carotene content with retained starch levels in cassava (Alexander- sson, 2017). CRISPR/Cas9 is a powerful tool to edit plant genomes which can create new valu- able crops. The gene editing is stable and heritable. The method is also user-friendly and not hard-line considered as GMO, which would ease public acceptance. For a growing population and a harsher environment, the method holds promise to be revolutionary (Khatodia et al., 2016). In this project, CRISPR/Cas9 was used to deac- tivate enzymes in the carotenoid pathway hopefully leading to β-carotene accumu- lation in the mature root. The carotenoid content varies in plants due to for example species, genotype, ripen- ing stage and environmental conditions. Previously genetic and metabolic engineer- ing has been successful to enhance the carotenoid levels in different plants, for ex- ample in the famous so called Golden rice. The carotenoid content can be analysed using high performance liquid chromatography, HPLC, in biological samples. The long conjugated double-bonds will absorb light in at a specific wavelength and the content of specific carotenoids like β-carotene is possible to measure (Saini et al., 2015). The starch content can be analysed by using enzymatic methods that will hy- drolyse the starch to glucose, which further can be measured by using spectropho- tometry. In this thesis, the β-carotene and starch content was measured for 13 wild cassava genotypes in order to present a recommendation on which of these genotypes that produced the most favorable levels. These recommended genotypes could further be used as targets for the genetic modifications, creating a more revolutionary cas- sava variety than ever before.