An efficient system for or 'Agrobacterium'-mediated transformation of <i>Brassica carinata</i> was used together with a promoterless <i> gus</i>::<i>nptII</i> gene fusion to isolate putative promoter sequences. Cotyledonary petioles were transformed using the promoterless gene fusion construct. Only transformation events in which the promoterless gene fusion had integrated downstream from plant regulatory sequences were expected to produce viable tissue under kanamycin selection. Forty-two transgenic plants were recovered. Transformation efficiency was approximately 0.6%. Regenerated plants were screened for GUS expression in different tissues and organs by histological and fluorometric assays. Tissue-specific GUS expression was detected (stigmas, seed coat, leaf edges and vascular tissue) in some plants, while strong constitutive GUS expression was detected in others (based on GUS histological assays). Using subgenomic libraries, putative promoter fragments were isolated from the plants which exhibited GUS expression in stigmas, leaf edges and constitutively. A putative promoter fragment from a plant which exhibited GUS expression only in the stigma was fused with the gus gene and reintroduced by <i>Agrobacterium </i> -mediated transformation into <i>B. napus, B. carinata, Arabidopsis' and tobacco </i>. GUS expression was observed in the stigma of <i>B. napus </i> but not in ' B. carinata'. In <i>Arabidopsis </i> and tobacco GUS expression. was not tissue specific (weakly constitutive or restricted to two or more tissues). The 3' DNA sequence (15 kb) flanking the <i> gus</i>::<i>nptII </i> insert in the plant with GUS expression in the stigma was also isolated using a subgenomic library. A gene for a cytochrome P450 like protein was discovered on the minus DNA strand of the 3' sequence with a start codon approximately 6.5 kb from the T-DNA left border.
| Identifer | oai:union.ndltd.org:USASK/oai:usask.ca:etd-10212004-002246 |
| Date | 01 January 1998 |
| Creators | Babic, Vivijan |
| Contributors | Keller, Wilfred |
| Publisher | University of Saskatchewan |
| Source Sets | University of Saskatchewan Library |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://library.usask.ca/theses/available/etd-10212004-002246 |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Saskatchewan or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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