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Direct organogenesis and Agrobacterium-mediated transformation of Exacum styer group

University of British Columbia

Interspecitic hybrids of Exacum, formally known as Exacum Styer Group. have significant potential as a new ornamental species. However, these hybrids are very susceptible to fungal root pathogens including Fusarium species. Therefore, the development of fungal resistant germplasm is highly desirable. In an attempt to address this goal, a genetic engineering protocol based on Agrobacterium-mediated transformation using a gene reported to confer fungal resistance was developed. A method for the regeneration of plantlets through direct organogenesis from leaf explants of Exucum, without an intervening callus phase, was developed. Four genotypes were evaluated on MS medium supplemented with TDZ (0.01, 0.05, 0.1 mg 1-¹) or combinations of BA (0, 0.1, 0.5, 1.0 or 2.0 mg 1-¹) and NAA (0, 0.01 , 0.1 or 0.5 mg 1-¹). Leaves of Exacum were very responsive to the plant growth regulators; different combinations induced different organogenesis (i.e. roots, shoots or callus). Significant genotype, media, and genotype x medium interactions were present for several variables. However, genotypes 01-09-01 and 01-37-61 had the highest number of shoots per explant across media (10.2 and 6.6, respectively), while 1.0 mg 1-¹ BA plus 0.1 mg 1-¹ NAA induced the greatest number of shoots among the genotypes evaluated. After establishing organogenesis protocols. I focused on the development of an Agrobacterium-mediated transformation protocol. Using modified thaumatin-like protein (TLP) constructs, nine transformed lines were produced using the pSM-3 TLP plasmid with the Agrobacterium strain C-58 and three transformed lines were produced using the pCambia-bar-ubi TLP plasmid with Agrohucterium strain LBA4404. Four tobacco transgenic lines also were produced and used as positive controls. The transformation percentages for Exucum were low (<I%) compared to other plants that habe been transformed with sinlilar TLP constructs and bacterial strains. Results from RT-PCR demonstrated that the expression levels of the TLP gene varied significantly among transgenic lines of both Exuczim and tobacco. Gene expression patterns showed higher transcript levels in plants transformed with the double 35s promoter/AMV leader sequence (pSM-3 plasmid) compared to plants transformed with the ubiquitin promoter (pCambia-bar-ubi). SDS-PAGE identified a small protein (~14 kDa) present in Exuct/m transgenic lines but absent from the non-transformed controls. However, it was not confirmed that this protein was TLP due to its smaller than expected size. Immunoblots displayed no signal from either Exucum or tobacco transgenic lines when probed with a rice polyclonal antibody. The lack of binding could be explained by the loss of activity of the antibody or by a post-translational modification of the protein antigens. Future studies should evaluate transgenic plants for enhanced resistance.

Text

2010-01-08

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http://hdl.handle.net/2429/17826

Plant Science

University of British Columbia

UBCV

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