Expression of Gus Gene in Tobacco (Nicotiana tabacum) and Corn (Zea mays) Using Dimboa-Resistant Agrobacterium Strains
This thesis evaluates the ability of Agrobacterium tumefaciens strains resistant to the compound DIMBOA [2, 4-dihydroxy-7-methoxy-2h-l,4- benzoxazin-3(4H)-one] to infect corn. DIMBOA is a secondary plant metabolite and is believed to have antimicrobial properties and provides resistance to the plant against microbial infection. Agrobacteriummediated genetic transformation technique can be employed to genetically alter most dicotyledonous plant species. However, such transformation has been limited in monocotyledonous plants, especially corn. One of the factors responsible for this recalcitrance is the presence of DIMBOA in corn shoots. In this study, we proposed the use of DIMBOA- resistant strains of Agrobacterium tumefaciens, bearing binary plasmids pGUS and pBG5. These plasmids contained reporter GUS (p-glucuronidase) gene and NPT II (Neomycin Phosphotransferase) gene conferring resistance to kanamycin. These genes were placed under the eukaryotic 35S CaMV promoter and had a NOS terminator. A plant intron was inserted into the GUS reporter gene in the pGUS plasmid. The Agrobacterium strains were subjected to treatments with virulence gene inducers like acetosyringone (plant phenolic), nopaline, glucose and arabinose at various concentrations to evaluate increase in gene transfer events. The presence of the foreign reporter gene in the plant genome was indicated by the visual GUS assay, typically by the appearance of blue color in transformed corn and tobacco tissue in the presence of substrate X-Gluc (5-bromo-4-chloro-3-indoly glucuronide). A concentration of O.lmM acetosyringone was most optimal for the gene transfer event. Nopaline, glucose and arabinose did not affect the transformation efficiency. Polymerase Chain Reaction (PCR) was employed to diagnose the presence of GUS gene in the plant genome. The GUS reporter gene is a derivative of the bacterial uidA and is about 2000bp in size. Genomic DNA extracted from putative transformants of corn and tobacco tissue was amplified using primer sequences specific to the GUS gene. The PCR products were analyzed by agarose gel-electrophoresis. The transformed tissues were represented by band size comparable to uidA of approximately 2000 base pairs. The non-transformed tissue did not indicate the presence of this gene; thus, its absence suggested transfer of the reporter gene into the host plant genome. Although the percentage of transformation in corn tissue was low, Agrobacterium-mediated genetic transformation of corn was possible. We therefore suggest the use of DIMBOA-resistant Agrobacterium strains, treated with the inducer AS at a concentration of O.lmM for genetic transformation of corn.
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