Study of Post-Harvest Physiological Deterioration in Transgenic Cassava

Abstract

Cassava (Manihot esculenta Crantz) was domesticated approximately 8,000 years ago and is a staple food for over 500 million people in about 105 tropical and subtropical countries. Vegetatively propagated for its starch-rich storage roots, cassava has an exceptional capacity to grow on marginally fertile soils and in regions with low annual rainfall. However, production in Africa - the largest producer of cassava - is constrained by numerous biotic and abiotic factors, including viral infection (e.g. cassava mosaic viruses and cassava brown streak viruses), pests and post-harvest physiological deterioration (PPD). PPD is an endogenous process that renders the roots unmarketable and unpalatable within approximately 24-48 hours after harvest. Although harvesting triggers a wound response, cassava is unable to modulate the accumulation of reactive oxygen species (ROS), resulting in oxidative damage and the development of symptoms referred to as vascular streaking. Over-expression constructs containing selected genes involved in ROS detoxification (ASCORBATE PEROXIDASE (APX), CATALASE, GALACTURONIC ACID REDUCTASE, γ-GLUTAMYLCYSTEINE SYNTHETASE (GSH1) and SUPEROXIDE DISMUTASE) and driven by the root-specific PATATIN promoter (StPAT) were successfully crafted. The protocol for Agrobacterium-mediated transformation of friable embryogenic callus (cultivar TMS60444) was extensively modified to guarantee production of transgenic cassava and progress was monitored using constructs harbouring the GUSPlus reporter gene. PCR-based analyses and Southern blot hybridisation revealed successful and stable integration of the transgenes with >85% of lines having T-DNA inserted into a single genomic fragment. The APX transgene and peroxidase activity were successfully up-regulated in transgenic cassava storage roots. Additionally, enhanced accumulation of the antioxidant thiol, glutathione, was measured in GSH1 transformed plants. Unique data elucidating suitable reference genes to study transgene expression profiles using real-time PCR is provided. And experiments to develop an assay to measure PPD in glasshouse-cultivated storage roots were performed. The data presented in this thesis aims to expand our knowledge of cassava tissue culture, transformation, PPD and prolong the shelf-life of cassava storage roots via enhancement of ROS-detoxifying pathways.

Date of Award	1 Jul 2011
Original language	English
Awarding Institution	University of Bath
Supervisor	John Beeching (Supervisor)