Regeneration and RNAi-Mediated Downregulation of Cyano-Glycoside Biosynthesis in Cassava (Manihot esculenta, Crantz)
Ngugi, Mathew Piero
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Cassava (Manihot esculenta, Crantz) is an important perennial tropical crop for millions of people around the world particularly in Sub-Saharan Africa. It is preferred due to its agronomical attributes such as ability to grow in poor soils and drought resistance. Cassava storage roots are also a good source of starch. In addition, cassava leaves and tender shoots are eaten as vegetables in many parts of Africa, Kenya included and are an excellent source of vitamins, minerals and protein. Although most cassava is used for food, it is also used in the production of ethanol for fuel, for animal feed, and as a raw material for the starch industry. Cassava has high photosynthetic rates and its roots can persist in the soil for 8-24 months without decaying, thereby making it an ideal food security crop. In Kenya cassava is a major source of subsistence and cash income to farmers in agroclimatically- disadvantaged regions and high potential areas of Coast, Central and Western regions of Kenya. Sadly, cassava leaves and roots contain potentially toxic levels of cyanogenic glycosides, a demerit that generated interest in this study. Cassava is largely propagated clonally making it an ideal plant for improvement through genetic engineering. The objective of this study was to develop and optimize regeneration protocol for Kenyan cassava varieties and produce transgenic acyanogenic cassava plants in which the expression of the cytochrome P450 genes (CYP79D1/D2) is knocked down through RNAi. Three Kenyan cassava genotypes; Adhiambo lera, Kibanda meno and Serere along with an exotic model cultivar TMS 60444 were used. As a prerequisite for transformation, a reproducible in vitro regeneration protocol was optimized for Kenyan cassava lines using immature leaf lobes as explants. The transformable lines were then taken through Agrobacterium-mediated transformation with an RNAi cassette harbouring cytochrome P450 genes (CYP92D1) to down regulate production of cyanoglycosides. Molecular analysis by PCR and RT-PCR confirmed transformation of the putative transformants. Analysis of cyanide content of the transgenic Kenyan cassava lines corroborated with the molecular analysis data that transformation had indeed occurred. From this study, an optimized and reproducible transformation protocol for Kenyan cassava varieties has been developed. In addition, transgenic cassava lines with cyanide content three folds less than the cyanide content of the wild type relatives were produced. The results of this study disapproves the view that African cassava genotypes are recalcitrant to in vitro manipulations. Production of transgenic lines with greatly reduced cyanide contents will further add value to cassava utilization. This, therefore, is an impetus for further genetic manipulations on Kenyan cassava cultivars to mitigate the various genetic demerits associated with cassava in order to ultimately maximise on the numerous benefits of cassava.