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Assessment of promoter function and enhancing disease resistance in transgenic carrot (Daucus carota L.)

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(Thesis) Ph.D.
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Genetic engineering is a promising strategy for creating agriculturally significant resistance in carrot. Several different transgenic strategies, in addition to conducting a detailed quantification of commonly used promoters to achieve disease resistance were investigated. Tissue-specific patterns and levels of protein expression were characterized in transgenic carrot plants transformed with the ?-glucuronidase gene driven by five promoters: Cauliflower moasaic virus 35S, double 35S (D35S), Arabidopsis ubiquitin (UBQ3), mannopine synthase (mas2) or rooting loci promoter (rolD). UBQ3 promoter provided the highest levels of expression in roots, while D35S and 35S promoters had high expression in leaves. rolD and Mas2 promoters had enhanced root expression; however levels were much lower compared to the constitutive promoters, which were subsequently used in this research. Genes encoding a rice peroxidase (POC1), wheat chitinase (383) and ?1,3- glucanase (638) were introduced into carrot. Lines expressing 638 alone had no enhanced resistance to B. cinerea or S. scleorotiorum, while 383 lines reduced disease symptoms by up to 40%. When 638 and 383 were co-expressed resistance levels were similar to 383 alone. High levels of disease resistance were seen in lines expressing POC1 with 70-90% reduction in symptoms to B. cinerea and S. sclerotiorum. POC1 lines had 20-30% increases in lignin levels in petioles and roots, which was enhanced with pathogen challenge. Several defence genes exhibited strong induction in POC1 expressing plants when induced with fungal cell wall elicitor. Additionally, POC1 lines had reduced H2O2 accumulation during oxidative burst response. POC1 derived resistance was effective towards necrotrophic pathogens. Over-expression of Arabidopsis Nonexpressor of Pathogenesis Related protein 1 was studied in carrot, with two independent lines successfully generated and analyzed. There was no detectable activation of the systemic acquire resistance (SAR) pathways in the absence of pathogens, however, the lines exhibited more intense and longer lasting activation of SAR when elicited with fungal cell walls or Salicylic acid. Both lines were highly resistant against biotrophic and necrotrophic foliar pathogens and the roots were resistant towards A. radicina, indicating broad-spectrum disease resistance. The results from this study demonstrate the feasibility of engineering disease resistance in carrot using several different approaches.
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