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Accueil du site → Doctorat → Allemagne → 2008 → Genetic transformation of Kenyan sorghum (Sorghum bicolor (L.) Moench) with anti-fungal genes and response to Colletotrichum sublineolum infection

Universität Hamburg (2008)

Genetic transformation of Kenyan sorghum (Sorghum bicolor (L.) Moench) with anti-fungal genes and response to Colletotrichum sublineolum infection

Ayoo, Linus Moses Kosambo

Titre : Genetic transformation of Kenyan sorghum (Sorghum bicolor (L.) Moench) with anti-fungal genes and response to Colletotrichum sublineolum infection

Auteur : Ayoo, Linus Moses Kosambo

Université de soutenance : Universität Hamburg

Grade : Doctor rerum naturalium (Dr. rer. nat.) 2008

Résumé
Sorghum (Sorghum bicolor (L.) Moench) is an important food crop in Kenya as well as scores of other African and Asian countries. It ranks fifth worldwide in production among cereals and it is unique in its adaptation to adverse environmental conditions. Anthracnose, caused by Colletotrichum sublineolum, is one of the destructive fungal diseases of sorghum that cause extensive annual yield losses. Classical breeding and genetic engineering for traits conferring tolerance and resistance against fungal pathogens is one of the strategies of boosting production. Genetic engineering could be used to exploit the natural anti-fungal proteins produced by saprophytic fungi, such as Trichoderma harzianum. Lytic antifungal proteins, like the chitinases and chitosanases, degrade chitin and chitosan that are components fungal cell walls. This renders the cell walls osmotically sensitive and ultimately destroys target fungi. Constitutive expression of the chitinase (HarChit) and chitosanase (HarCho) genes from T. harzianum in transgenic plants could confer resistance to fungal diseases. Particle Bombardment and Agrobacterium tumefaciens were used to genetically transform sorghum lines sampled from Kenya with HarChit and HarCho genes from T. harzianum. Three stable transgenic lines, KOSA-1, KOSA-2 and KOSA-3, integrated the two anti-fungal genes were generated from the wild type line KAT 412 through particle bombardment of immature zygotic embryos. Quantitative RT-PCR analysis of the transgenic plants revealed that both genes were expressed in the transformants. In planta and ex planta C. sublineolum infection assays were carried out with 2 weeks old sorghum seedlings to study the level of disease tolerance by the transgenic and the parent wild type (Wt) lines. The transgenic line, KOSA-1, was found to be more tolerant to anthracnose than the parent Wt. This is the first report of successful co-transformation and genetic enhancement of sorghum after integration of HarChit and HarCho, two economically important anti-fungal genes. Response to anthracnose was also studied in six Wt sorghum sampled from Kenya : KAT L5, SDSH 513, KAT 412, KAT 487, GBK 0460812, GBK 0460844 and Serena. Qualitative and quantitative rating of the susceptibility and tolerance of different sorghum genotypes showed that the Kenyan cultivar KAT L5 was the most tolerant among the lines studied. Quantitative RT-PCR was used to study the expression of the 2 transgenes, HarChit and HarCho and 4 endogenous pathogenesis-related (PR) genes : sorghum leucine-rich repeat (SbLRR), sorghum chitinase gene (SbChit), chalcone-like synthase gene 2 (SbCHS2) and gene 8 (SbCHS8) after infection with C. sublineolum. The fold change (FC) in the expression of SbLRR, SbChit and SbCHS8 gene were found to be significantly low in the tolerant KAT L5 but high in the susceptible SDSH 513 after infection with C. sublineolum. There was a significant difference in expression pattern of the 4 PR-genes in the disease susceptible and resistant cultivars

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