Genetic Engineering for Tolerance to Fusarium Wilt in Musa Sapientum Cv Rastali (AAB)

This study aims at developing tolerant Pisang Rastali (AAB) to Fusarium wilt via genetic engineering. Several considerations support that banana is a good candidate crop that could benefit from biotechnology applications. Single buds from multiple bud clumps (Mbcs) of Pisang Rastali (AAB) were established to be suitable target tissues for the introduction of antifungal protein and reporter genes using the Agrobacterium-mediated and particle bombardment transformation methods. Multiple bud clumps (Mbcs) were easily micropropagated in vitro that provided a reliable source of potentially regenerable single bud tissues. The effectiveness of kanamycin, neomycin, geneticin G-418, paromomycin, basta and hygromycin as selection agents to inhibit growth of Pisang Rastali (AAB) was evaluated in solid and liquid media. Of six antibiotic selection agents tested, hygromycin was the best, followed by basta and geneticin G-418. In addition, the use of liquid media has been shown to be more effective than in solid medium. Geneticin G-418 was the preferable antibiotic agent compared to kanamycin, neomycin and paromomycin since all plasmids used in this studies confers resistance to npt11 gene. Chemotaxis of Agrobacterium tumefaciens strains (EHA 101 and LBA 4404) towards wounded banana tissues has been studied using swarm agar plates. The results obtained indicate a minor role of chemotaxis in determining host specificity and suggest that it could not be responsible for the absence of tumorigenesis in banana under natural conditions. A method developed for the quantification of bacterial attachment to banana, based on the use of the gusA and gfp genes in marked Agrobacterium strains, is also described. Its application has demonstrated that attachment may also not be a main factor determining host specificity in Agrobacterium tumefaciens. In addition, optimization of several factors affecting transient gusA and gfp genes expression such as preculture period, co–cultivation period, acetosyringone concentrations, types of wounding, strains of Agrobacterium tumefactions, influence of single bud sizes and post cultivation period were evaluated to determine the efficiency of Agrobacterium-mediated transfer during the early stages of transformation in banana particularly in Pisang Rastali (AAB). Physical and biological parameters affecting DNA delivery into Pisang Rastali (AAB) single buds have been optimised. The physical parameters tested were helium pressure, distance from stopping plate to target tissues, vacuum pressure, multiple bombardment and gold microcarrier size. The optimised biological parameters were explant types, effect of various sizes of explants, effect of preculture treatment prior bombardment, effect of DNA concentrations and post bombardment incubation time. Regeneration of transgenic Pisang Rastali (AAB), molecular analyses (PCR and Southern blot), analysis of chitinase and β-1,3-glucanase protein production via Agrobacterium-mediated transformation were carried out. DNA samples from transgenic Pisang Rastali (AAB) obtained only from selection media contained geneticin G-418 as a selectable marker and were tested positive for the presence of the chitinase (RCC2) or β-1,3 glucanase (Eg) and npt11 coding sequences by PCR analyses. Genomic Southern blot hybridization confirmed the incorporation of the the chitinase (RCC2) or β-1,3-glucanase (Eg) gene into host genome. Using particle bombardment system, five different treatments using different chitinases and β-1,3-glucanase genes inserted singly or in combination were carried out. Genes encoding either chitinases (RCC2 or Chi) or β-1,3-glucanase (Eg) or both was bombarded together with gfp gene (pGEM.Ubi-SgfpS65T) for early transient expressions signal. Integration of the transgenes and stablity of the particle bombardment system were confirmed by PCR amplification of gfp, gusA, RCC2 (chitinase), Chi (chitinase), Eg (β-1, 3-glucanase) and npt11 genes. Genomic Southern blot hybridization confirmed the incorporation of the RCC2, Chi and Eg genes in host genome between one and five inserted copies in transformed plantlets. Similar to Agrobacterium-mediated transformation system, the chitinase and β-1,3-glucanase enzyme activity of transgenic plantlets obtained from particle bombardment was higher than untransformed plantlets. In addition, co-bombardment of chitinase and β-1,3 glucanase genes (Treatment 1 and 2) gave higher enzyme activities compared with single gene insertion (Treatment 3, 4 and 5). For Fusarium bioassay method, time course of Fusarium oxysporum f.sp. cubense (race 1 ; VCG 01217) spore production in vitro and the actual number of germinated spores were determined . The 28 days old Fusarium spores with 62% germinating capacity at 2x106 spores/ml were used for bioassay testing of transgenic Pisang Rastali (AAB) plantlets. Hydrogen peroxide (H2O2) and phenylalanine ammonia lyase (PAL) is the most sensitive chemical compound and enzyme to Fusarium spores inoculation. The chitinase and β-1,3-glucanase enzyme activities of transgenic plantlets was almost four folds higher than untransformed plantlets. The peroxidase (PER) and polyphenol oxidase (PPO) enzyme activities in banana roots increased markedly up to 2-3 folds higher in transformed plants compared to untransformed plantlets after 28 days of inoculation.

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