Genetic transformation of cassava (manihot esculenta crantz) for abiotic stress tolerance

Abstract

Cassava (Manihot esculenta Crantz) is a major source of food for over 800 million people worldwide. Although cassava is relatively easy to grow, even in poor soils and under erratic rainfall conditions, its roots have a short shelf-life of only 24-72 hours due to post-harvest physiological deterioration (PPD). PPD is a process initiated on harvesting and mediated by reactive oxygen species (ROS) that ultimately renders storage roots unpalatable and unmarketable. Control of PPD through conventional breeding is difficult and do not appear to offer a solution due to lack of resistance genes in existing germ plasm, high heterozygosity, poor flowering and low pollen fertility. As an alternative, a transgenic approach focusing on preventing ROS accumulation post-harvest has the potential to extend the shelf-life of cassava storage roots. However, an essential pre-requisite for genetic transformation of cassava is the availability of an efficient and reproducible transformation and regeneration system. Therefore, the first objective of this study was to optimize a transformation protocol for African farmer-preferred cassava cultivars using p-glucuronidase (GUS) reporter gene. To establish an efficient transformation system, the conditions for production of friable embryogenic calli (FEC) and Agrobacterium-mediated transformation were optimized for a selection of African farmer-preferred cultivars (i.e. Albert, Ebwanatereka, Kibaha, Kibandameno, Mkombozi, Serere and TME 14). FECs from Ebwanatereka, Serere, Kibandameno and 60444 cultivars were transformed with Agrobacterium strain LBA4404 harboring a binary vector pCAMBIA 130 1 and 17-28 transgenic plants per 100 mg of FEC were regenerated. Histochemical GUS assay demonstrated transient and stable expression of uidA "gene in both calli and regenerated plants. The presence, integratlbij'~and expression of the trsnsgenes were confirmed by PCR, Southern blot and RT-PCR analysis, respectively. An efficient transformation and regeneration protocol has been established in this study and will provide a useful platform to transfer novel traits to farmer-preferred cassava cultivars in Africa. The second objective was to modulate PPD through overexpression of glutathione peroxidase (GPX) gene in cassava storage roots. Transgenic plants of cultivar 60444 were generated expressing Ar~bid.opsis GPX (AtGPX) gene, driven by a root-specific patatin promoter. Molecular analysis indicated that AtGPX had been integrated in the genome of transgenic plants and functionally expressed. Transgenic cassava storage roots showed a reduction in ROS accumulation and malondialdehyde (MDA) content compared to non-transgenic plants, with an extension in shelf-life. The third objective was to develop transgenic cassava expressing dehydroascorbate reductase (DHAR) gene and evaluate the level of tolerance to oxidative stress. DHAR is an important enzyme functioning in the regeneration of ascorbate (AsA), and since AsA blocks ROS accumulation, the hypothesis that DHAR overexpression protects cassava plants from abiotic stress was tested. Transgenic cassava plants of cultivar 60444 were generated expressing Arabidopsis DHARI (AtDHARl) gene driven by CaMV35S promoter. Molecular analyses of transgenic lines revealed stable integration and expression of AtDHARI gene. DHAR activity and AsA content were significantly (p<0.05) increased in transgenic lines compajedto non-transgenic plants. Overexpression of AtDHARl in cassava led to enhanced oxidative stress tolerance measured by enhanced root elongation, reduced ROS actumulat-ion and reduced lipid peroxidation products. This study has developed an effective transgenic approach for delaying PPD and enhancing oxidative stress tolerance in cassava.