Download Genetic Engineering of Plants

Genetic Engineering of Plants
BIT 220
End of Chapter 22
Why Create Transgenic Plants?
1. Improve agricultural value of plant
increase yield (herbicide-resistance, pest-resistance)
enhance nutrition
enhance taste
2. Plants can produce proteins for human needs (pharmaceutics)
3. Modified plants can be used to study effects of genes
An entire plant can be regenerated from a single cell
***TOTIPOTENCY***
No separation of germ and somatic cells
How to get a gene into a plant
TRANSFORMATION
1. Agrobacterium tumefaciens
Figure 22.15
2. Microprojectile Bombardment
(biolistics)
3. Electroporation
burst of electricity
Agrobacterium tumefaciens
Soil
Plant pathogen
Causes tumors- crown galls
Contain Ti plasmid
Only infects Dicots
1. Wounded plant
2. A. tumerfaciens attached to plant at wound site
(often at base of stem -Crown)
3. Wounded plant secretes phenolic compounds
4. These compounds activate virulence genes of A. tumefaciens
© 2003 John Wiley and Sons Publishers
Fig 22.17 Transformation of plant cells by Agrobacterium
tumefaciens harboring a wild-type Ti plasmid.
© 2003 John Wiley and Sons Publishers
Fig 22.16 Structure of the nopaline Ti plasmid pTi C58,
showing selected components.
Ti Plasmid
FIGURE 22.16
1. T-DNA (Transferred DNA)
transferred and expressed into plant causing
tumor (crown gall) formation
2. Virulence Genes
essential for the transfer and integration of the T-DNA
3. ori
4. Noc (Opine Catabolism) genes (other microbes do NOT have)
Genes within T-DNA
1. Enzymes to produce Auxin (iaa)
2. Enzyme to produce cytokinins
3. Genes for synthesis of Opines - carbon source for bacteria
no use in plants
4. Tum -genes responsible for tumor formation
Engineered Ti plasmid
1. Clone foreign gene into T-DNA
2. Delete genes responsible for tumor formation
3. Add selectable marker
Infection of Plant Cell Continued
Figure 22.17
1. Bacteria attaches to host
2. Virulence gene expression activated by compounds secreted by wounds
3. T-DNA is transferred
4. T-DNA integrates into host (plant) cell
T-DNA is cut out of plasmid at left and right border
Right border- responsible for integration
5. Other T-DNA genes are activated causing tumor formation
Biolistics Microprojectile
Bombardment
Coat gold or tungsten spheres with DNA
Shot with particle gun through cell wall of plant cells
Best to use
small plasmids (<10kb)
linear DNA (integration more efficient)
Advantage: large range of plant species can be engineered
Delivery of Gene to the
Chloroplast
Can also integrate foreign DNA into the smaller chloroplast DNA
Use Selectable marker Plus Foreign Gene on
A. Single vector
B. Dual Vector
Advantage: Two genes integrate at separate portions
of chloroplast DNA
Does NOT interrupt essential chloroplast genes
Single Vector
Double Vector
Reporter Genes/Selectable
Markers
Neomycin Phosphotransferase gene
Resistance to Kanamycin
GUS B-glucuronidase
enzyme which converts colorless substrate to blue product
Luciferase (firefly)
‘glows in dark’
Agricultural Biotechnology
• $67 billion/year
industry
•
FOOD LOSS
• One-third of world’s
crops are lost to
insects, disease and
weeds
•
• HERBICIDERESISTANT
PLANTS
• Reduce Cost, Labor,
• Environmental Stress
• 606 million pounds of
herbicide/yr in US
Herbicide-Resistance
Roundup Monsanto
Glyphosate - inhibits the enzyme necessary for amino acid
production
KILLING WEEDS
By modifying the ‘crop’ gene for amino acid production
ROUNDUP has less affinity for enzyme
Crops are resistance
Disadvantage:
Kills spiders, earthworms and fish
Other Considerations:
•Cross Pollination -Resistant weeds?
•Will herbicide application really be reduced?
Other Apps of Plant Biotech
1. Vaccinations for Fungal infections
2. Freezing Tolerance
2oC change in Canada can double output of grapes
Wild-type Bacteria
protein coat stimulates ice crystal formation on
infected plants
Ice-Minus Bacteria
NO gene for this coat protein; lowers freezing pt
Coat protein is used for snow production on ski slopes
3. Canola oil
source of hirudin- anticoagulant