Investigations on genetic transformation of tobacco and canola with potential antifungal genes
Woytowich, Adrienne Eugenie
Most plants have the ability to distinguish between pathogenic and symbiotic associations and to respond accordingly although there are similarities between pathogenic and symbiotic modes of infections. Symbiotic nodule formation is a result of nitrogen-fixing bacteroids being encapsulated in healthy leghaemoglobin-containing tissues, whereas attack by a pathogen induces the activation of a number of defense mechanisms. It is the activation of these defense mechanisms which leads to physical and biochemical changes within the plant. Originally, the main objective of this thesis was to determine if it is possible to produce transgenic tobacco and canola plants which showed increased resistance to plant pathogens by introducing the chiB gene of Serratia liquefaciens and a synthetic gene (amp2) coding for an antimicrobial peptide derived from Mirabilis jalapa, alone and in combination. However, experiments revealed that the presence of the chiB gene suppressed the oncogenic ability of A. tumefaciens, thereforepreventing regeneration of transgenic plants. As well, transgenic tobacco and canola plants carrying amp2 did not show increased resistance to plant pathogens. The chiB open reading frame (ORF), under the control of the 35S-35S promoter module, was used to transform tobacco and canola plants. However, after several transformation attempts it was found that chiB-containing tobacco or canola plants could not be regenerated using the Agrobacterium-mediated transformation procedure. It was found that in every transformation the discs and cotyledons did not swell normally, turned yellow and failed to form calli. The oncogenicity of the wild type Agrobacterium tumefaciens strain C58 was tested in the presence of the chiB construct on various plants and it was found that this strain failed to form tumors around the infected wound sites. However, tumors did form around the wound sites that were inoculated with the same strain which carried only the vector. The lack of callus and gall formation was confirmed to be due to the expression of chiB. Inhibition of its expression by the introduction of mutations led to infected discs swelling normally and forming calli within four weeks of infection. As well, the replacement of the chiB-containing plasmid with a broad host range plasmid led to the renewal of the oncogenic ability of the strain. From these results and other experiments it is being proposed that Agrobacterium-mediated transformations require a signal, similar to the Nod factors of Rhizobium, for callus and tumor formation to occur, but that the gene product of chiB somehow inactivates this signal. The antimicrobial gene amp2 was synthesized and the resulting gene product was found to display strong antimicrobial activity in vitro. The coding sequence of amp2 wastransferred to the genomes of tobacco and canola by Agrobacterium-mediated genetic transformation and protein extracts isolated from amp2--containing To tobacco and canola transgenic plants were found to lack antimicrobial activity. Assays undertaken with proteins extracted from T1 and T2 homozygous and heterozygous plants revealed that low levels of antimicrobial activity could only be observed from the progeny of the tobacco line AW708-1 when using extremely high concentrations of desalted plant protein extracts (3 mg/50 μL). However, none of the transgenic canola and tobacco seedlings tested showed any increase in tolerance towards the pathogen Rhizoctonia solani.