Wamalwa, Lydia Nanjala2015-09-082015-09-082015http://ir-library.ku.ac.ke/handle/123456789/13524A Thesis Submitted in Fulfillment of the Requirements for the Award of the Degree of Doctor of Philosophy (plant Biotechnology and Molecular Biology) in the School of Pure and Applied Sciences of Kenyatta University. QK 495 .C78W3Abstract Sweetpotato is an important root and tuber crop in Sub-Saharan Africa (SSA) used as food and feed but weevil infestation remains a major concern. Weevils account for production losses between 28 and 100% but no resistance genes are available in the natural gene pool. When weevils attack, they create avenues for secondary infection, which in turn elicit phytoalexin production, toxic to farm animals. Since genetic transformation has been widely used to introduce genes into germplasm that may not be available in the natural gene pool, it was used in this study. Objectives of this study included (i) to develop a regeneration protocol of 32 SSA cultivars through indirect organogenesis, (ii) to develop a transformation protocol for the best cultivars using uidA gene and the double gene construct (cry7AaJ and ET33-34), and (iii) to isolate a fungus that infects sweetpotato after weevil infestation and subsequent analysis of the furanoterpenoid elicited from infected roots. Regeneration and transformation were conducted via indirect organogenesis. Four gene constructs were used in this study: pCIPlOO, pCIP87, pCIP88 and pCIP85. Phytoalexin levels were evaluated on four consumer-preferred sweetpotato cultivars using coupled gas chromatography mass spectrophotometry (GC-MS), thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and Nuclear Magnetic Resonance (NMR). Results showed 6 SSA cultivars with RE above 40% while TE of 5 SSA cultivars revealed GUS-stained calli between 6 and 98%. Six transgenic events were produced using the double gene construct as confirmed by southern blot but they had low protein levels. For phytoalexins, levels between 0.3 and 2,900 mg/kg were recorded. The results suggested that pre-screening for high RE and TE was important in identification of the best cultivars. Low protein concentration levels obtained could be due to post transcription or/and post-translational factors. Such high variations in furanoterpenoid levels have previously been reported, and could be an indicator of a potential health concern to both animals and humans on consumption.en-USThesis