| Abstract Detail
Education & Outreach Metzler, Melissa M [1], Theis, Whitney S [1], Miles, Thomas T [1], Robinson, Dave L [1], Lau, Joann M [2]. Glowing Results: A Laboratory Exercise for Demonstrating Plant Transformation. The objective of this experiment was to develop an Agrobacterium-mediated transformation laboratory exercise for undergraduate students. The firefly luciferase gene driven by the constitutively-expressed Cauliflower Mosaic Virus (CaMV) promoter was transformed into Arabidopsis, thus allowing the plant to become bioluminescent. The plasmid construct, pLuk07 (GenBank Accession U84006), was obtained from the Arabidopsis Biological Resource Center (Ohio State University). This construct contains an intron which prevents expression in Agrobacterium thus allowing for early detection following transformation. After electroporation into A. tumefaciens, and plating on YM (Yeast Extract/Mannitol) medium containing ampicillin, wild-type Arabidopsis plants were transformed twice using the floral-dip method. After seeds matured, they were collected, sterilized, and screened on MS (Murashige and Skoog) Basal Medium containing ampicillin. Seedlings were grown to the cotyledonary stage and transplanted into soil. A subset of these seedlings were assessed for bioluminescence by soaking in either 1, 5, or 10 mM luciferin for 10 hours and transferred to x-ray film for 16 hours. Untransformed wildtype Arabidopsis seedlings served as a negative control. The intensity of illumination was greatest in transgenic seedlings incubated in 10 mM luciferin, whereas untransformed seedlings produced no detectable luminescence. Mature transformed plants (T0) were screened for transgene insertion by PCR (using luciferase-gene primers) and gel electrophoresis. This project is an effective means of demonstrating to students important molecular techniques like restriction mapping of a plasmid, floral-dip transformation using Agrobacterium, antibiotic-screening, autoradiography, DNA extraction, PCR, gel electrophoresis, and reporter analysis using a constitutively-expressed promoter. It can be accomplished in a single 4-month semester and is appropriate for courses in Plant Biology, Plant Molecular Biology, Biotechnology, or Senior Research.
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1 - Bellarmine University, Biology
2 - Bellarmine University, Biology, 2001 Newburg Road, Louisville, Kentucky, 40205, USA
Keywords:
Arabidopsis
Luciferase
Agrobacterium tumefaciens
Plant transformation
Laboratory exercise
bioluminescence.
Presentation Type: Plant Biology Abstract
Session: P
Location: Exhibit Hall (Northeast, Southwest & Southeast)/Hilton
Date: Sunday, July 8th, 2007
Time: 8:00 AM
Number: P46029
Abstract ID:2687 |
