I Elements of transformation
        A. uptake of DNA into competent host cells
        B. integration vs. transient selection

II. Requirements for plant transformation
        A. cell culture and plant regeneration system
        B. cloned DNA to be introduced
                1. selectable marker gene
                        kanamycin or G148 resistance: neomycin,                                                         phosphotransferase (NPTII)
                        hygromycin B:hygromycin phosphotransferase (HygB)
                        gentamicin: gentamicin acetyltransferase
                        streptomycin: streptomycin phosphotransferase
                        Bialophos: BAR  
                2. promoter (constituitive or inducible), coding region,                                        polyadenylation site
        C. method of delivery of DNA into the cell
                1. Agrobacterium-mediated transformation
                        - cultured cells, wounded explants, vacuum infiltration
                2. direct methods
                        -protoplast polyethyleneglycol (PEG) method
                        -protoplast electroporation
                        -protoplast microinjection
                        -particle bombardment
        D. Proof of transformation of plant
III. Uses of transformation
        A. Testing function of genes or parts of genes
                -DNA complementation of mutants
                - protein dissection
                - determining place and timing of gene expression
                        reporter genes: GUS (b-glucuronidase), 
                        GFP (green fluorescent protein), LUX(luciferase),
                         CAT (chloramphenicol acetyltransferase), 
                        Barnase (RNAase- kills cells expressing it)
        B. Turning genes off
                -antisense, sense suppression, ribozymes
                -insertional mutagenesis using T-DNA
                        -see Arabidopsis sevice for doing this
                -e.g. tomato polygalacturonidase
        C. Moving genes from other organisms
                -Bt toxin for insect resistance
                -herbicide resistance -glyphosphate (Roundup™), sulfonureas                                     (Chlorosulfon), atrozine
        D. Concerns in use of transgenic plants

Links:

Plant transformation
Plant Transformation overview
On-line tour of tomato transformation- CEPRAP-UC Davis
Plant Transformation-short essay- Access Excellence
Plant transformation and use of reporter genes- PLSC731 NDSU
Vacuum infiltration of Arabidopsis
Non-vacuum protocol
Wheat trasformation
Maize biolisitic transformation
Peanut transformation
Soybean biolistic transformation
Review paper on details of Agrobacterium transformation

Genes inserted- examples
Green Fluorescent Protein -GFP
        -everything you want to know about GFP
GFP movies- click on pictures at the bottom- show optical sections through root
virus resistant squash
Flavr Savr Tomato
Engineering Bt in crops
Alternate view on herbicide-resistant crops-Greenpeace
Links to specific transgenic crop pages- Virginia
Hort 250 at Purdue-lecture notes on plant transformation, Bt, herbicide resistance, etc.
Bt toxin page-National Agricultural Library Resource Page
Essay- Ethics, law and science of genetically-engineered organisms-good overview
Report on use of Bt toxin-good summary
Corn borer resistance- Pioneer
YieldGard- Asgrow
"How we make transgenic plants"-Shockwave animation from Ag West

Wolfenbarger LL, Phifer PR .2000 The ecological risks and benefits of genetically engineered plants. Science Dec 15;290(5499):2088-93

M. Fagard and H. Vaucheret 2000. (TRANS)GENE SILENCING IN PLANTS: How Many Mechanisms?Annu. Rev. Plant Physiol. Plant Mol. Biol.51:167-194.

Last revised: March 26, 2001- Straney