1. Gene Transfer in Plants

2. Gene Transfer in Plants
Two approaches of gene transfer
Chemical & Physical
Why
Biochemical and physiological characterization of gene, protein or cell function
Generate new plant strains for desired trait
Humans have been doing this by traditional crosses
This method is faster and allows for transgenic (genes from different species/organisms) plants to be created

3. Reasons for Plant Gene Transfer
Golden Rice
Grains such as rice, produce all but two of the enzymes needed to produce beta carotene (vit A precursor)
Rice feeds half the world’s population
Vit A deficiencies are associated with blindness, night blindness, diabetes, anemia and easy infections
WHO estimates 220 mil women and children affected by preventable vit A-deficiency night blindness – 400 mil world wide people are affected by beta carotene deficiency
1 million children/year dye from related diseases

4. Reasons for Plant Gene Transfer
Golden Rice
Two genes - phytoene synthase (psy) and phytoene desaturase (crt I), are transformed into rice via bacterial transmission

5. Golden Rice Synthesis
Two Daffodil genes and one bacterial gene Erwinia uredovora were cloned into agrobacterium T DNA and inserted into rice genome to generate needed enzymes
Phytoene synthase &
Lycopene-b-cyclase
Carotene desaturase
T DNA
Germ-line transformation with agrobacterium X
Cross
T-formed rice with genes
T-formed rice with gene
Progeny rice plant with complete b carotene pathway

6. Reasons for Plant Gene Transfer
Spring Canola production is limited to frost in northern climes (US and Canada)
Protoplasts were exposed to UV light to generate mutations in genomic DNA
Cells were cultured into mature plants where defense signaling for cold tolerance was elevated (salicylic acid, jasmonic acid
329 mutant embryos were screened – 74 were selected for further development with increased cold tolerance
Another approach is to cone and over express antifreeze genes

7. Reasons for Plant Gene Transfer
Drought resistant crops
Insect resistant
Increase growth rate and higher oil producing plants for biofuels
Round up ready
Production of pharmacuticals
Vaccine in bannana against diarrhea (immune stimulating disease against cholera bacteria)

8. Agrobacterium tumefaciens
Gram negative bacterium which is commonly found in soil
When plant is wounded, bacteria enter and are pathagenic causing
Crown gall disease (plant tumor)
Only infects young dicots
DNA from bacteria is transferred into plants where it binds to plant histones and is integrated into plant genome by recombination

10. Agrobacterium tumefaciens
Two DNA – genomic and extrachromasomal plasmid DNA (Ti DNA) “Tumor Inducing”
Ti plasmid has two parts
T-DNA – the portion of the plasmid inserted into the plant cells and integrated into genome (remove host cell gene and insert your gene of interest)
T-DNA also produces auxins and cytokinins (bacteria producing plant hormones?!) to induce cell proliferation (plant cancer)
And the vir region – which encodes proteins for the transfer of DNA to infected plant but proteins are not transferred

12. Agrobacterium tumefaciens

13. Agrobacterium tumefaciens
Wild type Tk plasmid = 200 kb – too large for cloning
Intermediate shuttle plasmid is used to cut in Gene of Interest
VIR genes must be removed for genetic engineering
LB and RB are required for insertion and recombination with plant genome
Insertion into plant host is random (sort of)
First cloned gene – luciferase in tobacco plant

15. Methods of Delivery Vacuum Infiltration
Plant leaf disks are placed in a suspension of bacteria and vacuum pulled
Air is release like a sponge being squeezed
Vacuum is released and solution floods tissue
Plant disk is cultured

16. Methods of Delivery Floral Dip
Simple submersion of plant into bacterium suspension
No vacuum is needed
Conducted with plants grown until just flowering
Progeny seeds are harvested and germinated using selective antibiotic

17. Methods of Delivery Cultured cells Callus or protoplasts
Easy screen for positive using selection
New plants can be cloned using plant tissue culture

18. Gene Gun “Biolistics” – shotgun cloning
Coat gold or tungsten particles with Plasmid DNA
Non-bonded DNA is preciptiated with spermidine (positive amine carbon chain)
DNA is driven by helium blast (old days gunpowder driven pistons)
Particles fired into plant tissue at 430 m/sec
Can be used against cells or whole plants (mono or dicots)
Can be used with all plants or cells or organelles
Drawbacks include tissue damage and low efficiency
Lots of rearrangement of integrated DNA – can lead to instable gene transfer

19. Other Gene Transfer Methods
PEG or Dextran Sulfate – DNA ppt and mediated transfer into cultured cells
Microinjection of naked DNA into protoplasts or cells – good for larger DNA
Transformation – similar to e.coli work – short term permiabilize cells and DNA is carried into cell