Inhibition of cell death as an approach for development of transgenic resistance against Fusarium wilt disease

Fusarium oxysporum f. sp. cubense (Foc) is one of the major threats to dessert banana (Musa spp.) production. In Uganda, ‘Sukali Ndiizi’ is one of the most popular dessert banana cultivars and it is highly susceptible to Fusarium wilt. Development of resistant cultivars through transgenic approaches has shown to offer one of the most effective control options for most diseases. The transgenic approaches for providing plant disease resistance have mainly been through either enzymatic destruction of pathogen structures, neutralization of pathogen and its products or production of metabolites that eventually kill the pathogen. However in recent years, methods that prevent cell death of host plant after infection especially for necrotrophic pathogens like F. oxysporum have registered success in providing resistance in several crops. We investigated whether the transgenic expression of a programmed cell death inhibition gene in Sukali Ndiizi could be used to confer Fusarium resistance to Foc race 1. Embryogenic cell suspensions of cv. ‘Sukali Ndiizi, were stably transformed with a synthetic, plant-codon optimise mCed-9 gene. Twenty-eight independently transformed plant lines were regenerated. The lines were inoculated with Foc race 1 and observed for 13 weeks in small-plant glasshouse. Three transgenic lines showed significantly lower internal and external disease symptoms than the wild-type susceptible ‘Sukali Ndiizi’ banana plants used as controls. This is the first report from Africa on the generation of Fusarium wilt tolerant transgenic ‘Sukali Ndiizi’, a very popular but rapidly diminishing African dessert banana.Key words: Fusarium wilt, banana, Sukali Ndiizi, Fusarium oxysporum f. sp. Cubense race 1, programmed cell death, disease resistance

Generating transgenic banana (cv. Sukali Ndizi) resistant to Fusarium Wilt

Banana is one of the world’s most popular fruit crops and Sukali Ndizi is the most popular dessert banana in the East African region. Like other banana cultivars, Sukali Ndizi is threatened by several constraints, of which the Fusarium wilt disease is the most destructive. Fusarium wilt is caused by a soil-borne fungus, Fusarium oxysporum f.sp. cubense (Foc). No effective control strategy currently exists for this disease and although disease resistance exists in some banana cultivars, introducing resistance into commercial cultivars by conventional breeding is difficult because of low fertility. Considering that conventional breeding generates hybrids with additional undesirable traits, transformation is the most suitable way of introducing resistance in the banana genome. The success of this strategy depends on the availability of genes for genetic transformation. Recently, a novel strategy involving the expression of anti-apoptosis genes in plants was shown to result in resistance against several necrotrophic fungi, including Foc race 1 in banana cultivar Lady Finger. This thesis explores the potential of a plant-codon optimised nematode anti-apoptosis gene (Mced9) to provide resistance against Foc race 1 in dessert banana cultivar Sukali Ndizi. Agrobacterium-mediated transformation was used to transform embryogenic cell suspension of Sukali Ndizi with plant expression vector pYC11, harbouring maize ubiquitin promoter driven Mced9 gene and nptII as a plant selection marker. A total of 42 independently transformed lines were regenerated and characterized. The transgenic lines were multiplied, infected and evaluated for resistance to Foc race 1 in a small pot bioassay. The pathogenicity of the Ugandan Foc race 1 isolate used for infection was pre-determined and the spore concentration was standardised for consistent infection and symptom development. This process involved challenging tissue culture plants of Sukali Ndizi, a Foc race 1 susceptible cultivar and Nakinyika, an East African Highland cultivar known to be resistant to Foc race 1, with Fusarium inoculum and observing external and internal disease symptom development. Rhizome discolouration symptoms were the best indicators of Fusarium wilt with yellowing being an early sign of disease. Three transgenic lines were found to show significantly less disease severities compared to the wild-type control plants after 13 weeks of infection, indicating that Mced9 has the potential to provide tolerance to Fusarium wilt in Sukali Ndizi