Plant transformation YJ.ppt

* * * * * * * * * * * * * Direct introduction of foreign DNA Imbibition transformation Biolistics - gene gun PEG Microinjection Electroporation Liposome Silica/carbon fibers Laser microbeam Ultrasonic transformation Biolistic / Gene Gun PEG-mediated protoplast transformation Microinjection of GOI This electroporator is for low-current applications such as those using small electrodes Selectable Markers A gene encoding an enzyme Antibiotic resistance Herbicide resistance Positive selection genes genes that allow use of some necessary media component Selectable Markers NPTII - kanamycin (antibiotic) Hpt - hygromycin PMI - changes mannose to useable carbohydrate Novel Selection Genes Luciferase - gene from fireflies – substrate Green Fluorescent Protein - from jellyfish - under lights and filter the transgenic plants - GFP GUS - glucuronidase gene will convert added substrate to blue color. Production of transgenic plants Isolate and clone gene of interest Add DNA segments to initiate or enhance gene expression Add selectable markers Introduce gene construct into plant cells (transformation) Select transformed cells or tissues Regenerate whole plants 5-Bromo-4-chloro-3-indolyl β-D-glucuronide (sodium salt)? Synonym?- BC-Indicator X-GlcA/ X-Glu X-glucuronideMolecular Formula C14H12BrClNNaO7Molecular Weight 444.59 Activity - quantitative way or through visualization Beta-glucuronidase – E. Coli Richard Anthony Jefferson (1987) X-glcA (X-gluc or X-glc or X-glcU) - substrate for GUS GUS oxidative dimerization X-glu → colourless soluble → Blue precipitate of intermediate diX-indigo Selection Regeneration Cells which contain the selectable marker gene can grow Al