Download Plant Transformation - Lecture Notes - Bioinformatics and Genomics | BSCI 411 and more Study notes Biology in PDF only on Docsity! Lecture 11. Plant Transformation I Elements of transformation !!!!!!!1. uptake of DNA into competent host cells !!!!!!!2. integration vs. transient expression II methods of delivery 1. Agrobacterium-mediated transformation !!!!!!!!! - vacuum infiltration (floral dip) - wounded explants - Cultured cells (tobacco) can’t be applied to monocot, nor organelle 2. direct methods !!!!!!!!!!-protoplast polyethyleneglycol (PEG) method !!!!!!!!!!-protoplast electroporation !!!!!!!!!!-protoplast microinjection !!!!!!!!!!-particle bombardment Agrobacteria: -carry Ti-plasmid -induce tumor formation (crown gall tumor) in wound site of plants -produce opines (nopaline or octopine) 1. Agrobacteria tumefaciens mediated plant transformation Ti-plasmid has two parts: - T-DNA the part of DNA that is exported into the plant cell and integrated into the plant genome - vir region: encode proteins involved in this transfer, but stays within the bacteria http://www.ndsu.nodak.edu/instruct/mcclean/plsc731/transgenic/transgenic1.htm (A)Plants are grown to just flowering. (B)Plants are dipped briefly in a suspension of Agrobacterium. (C)Plants are grown until mature and then progeny seeds are harvested. (D)Seeds are germinated on selective medium (e.g. containing kanamycin) to identify successfully transformed progeny. Floral Dip (Vacuum infiltration) for Arabidopsis A. Bent (2000). Plant physiology vol 124, pp 1540-1547. Tomato transformation using wounded cotyledons
(http://www-ceprap .ucdavis.edu/Transformation/transform1. htm)
Wheat biolistic transformation (http://plantsciences,montana.edu/wheat-transformation/transformation1.htm) 2. Biolistic transformation (particle bombardment) DNA was bound to tiny metal particles, a gunpowder driven piston was fired at the target cell with a velocity of about 430 meters per second. Some of the cells that survived the bombardment incorporated the DNA into the genome. -New devices use compressed gas to accelerate the particles -tungsten or gold particles (0.5-5 mm) Advantage: universal (all plant tissues, cells, or organelles) Draw back: tissue damage, low efficiency (1-5%), size of the plasmid Summary of tools involved in transformation Name Source Substrate Visual GUS (b-glucuronidase) E.coli 5-bromo-4-chloro 3-indoyl- blue 1-glucuronide (X-gluc) LUC (Luciferase) Firefly luciferin & ATP emitting light GFP Jelly fish none emitting light (Green Flourescent Protein) breakdown Reporter genes Promoters 35S CaMV constitutive NOS constitutive Heatshock inducible CAB light inducible GAL4/GFP-mediated specific tissues, organs Gene name Resistant to Type NPT II (phosphotransferase II) Kanamycin Antibiotic Gentamicin 3-N-aceytransferase Gentamicin Antibiotic Aminoglycoside-3-adenyltransferase Streptomycin Antibiotic Phospfinothricin acetyltransferase Phosphinothricin Herbicide 5-enolpyruvylshikamate-3-phosphate synthase Glyphosate Herbicide (EPSP synthase) Acetplactate synthase (ALS) Sulfonylureas Herbicide Selectable markers
