Direct DNA transfer
•
•
Introduce naked DNA into cells; assay
expression immediately or select for
permanently transformed cells
DNA introduction:
1. Chemical
2. Electroporation
3. Particle bombardment (Biolistics)
Chemically-Induced Transformation
• Usually use on cells without walls
• Multiple protocols (examples):
1. Put DNA inside artificial membranes
(liposomes), they will fuse with plasma
membrane.
2. Bind DNA with polycations to neutralize
charge, some cells endocytose the
complex.
3. Combine (1) and (2)
Electroporation
•
•
•
Use on cells without walls
(plant protoplasts or animal
cells )
High-voltage pulses cause
pores to form transiently in
cell membrane; DNA pulled
in by electrophoresis or
diffusion (?)
Drawback - its more
cumbersome to regenerate
plants from single
protoplasts than from the
tissue transformations with
Agrobacterium
Particle Bombardment
•
•
•
•
Less limitations than electroporation
Can use on cells with walls, essentially any
tissue
Can transform organelles!
Method:
1. Precipitate DNA onto small tungsten or gold
particles.
2. Accelerate particles to high speeds at cells or
tissues.
3. Selective growth and regeneration of transgenic
plants as described for Agro-mediated
transformation.
Original biolistic gun. A modified 22 caliber.
DNA is bound to the microprojectiles, which impact the
tissue or immobilized cells at high speeds.
J. Sanford & T. Klein, 1988
An Air Rifle for a DNA Gun –
Circa 1990
A.Thompson, Bob ?, and D. Herrin
Repairing an organellar gene: ~ 1 x 107 cells of a mutant
of Chlamydomonas that had a deletion in the atpB gene for
photosynthesis was bombarded with the intact atpB gene. Then, the
cells were transferred to minimal medium so that only
photosynthetically competent cells could grow.
Control plate – cells were shot with tungsten
particles without DNA
The Helium Gas Gun – Circa 2000
Advantages of Chloroplast
Genetic Engineering
1. Controlled DNA integration
– goes in by homologous recombination
– Can do gene replacements (incl. gene knock-outs)
2. No gene silencing
3. High expression levels possible
4. Better containment of transgene (i.e., foreign gene)
- Chloroplasts inherited mainly from only one parent
(i.e., through the egg, not pollen)
Chloroplast Transformation by Homologous
Recombination
Transgenic Plants In Use on a Large
Scale
1. Herbicide-resistant plants
2. Pest-resistant plants
Glyphosate inhibits aromatic amino acid synthesis, et al.
The function of EPSP synthase is to
combine the substrate shikimate-3phosphate (S3P) with
phosphoenolpyruvate (PEP) to form 5enolpyruvylshikimate-3-phosphate
(EPSP).
Herbicide-resistant plants
• Resistant to herbicide “Round-up”
– Glyphosate (from Monsanto)
• Contain bacterial EPSP synthase
• Advantages: better weed control, less
tillage
• soybeans, corn, rice, wheat
Pest-resistant plants
•
Resistant to certain insects
Cry5
– Lepidopterans, Coleopterans
•
•
Carry gene(s) for Bacillus
thuringiensis (Bt) toxin
Toxin proteins produced as a
parasporal crystal
– Cry (multiple) and Cyt
genes
– encoded on a plasmid
•
Advantage: less insecticide
required, better yield
– corn, cotton, potatoes
A Transmission
Electron Micrograph
of negatively stained
spores from Bt2-56
containing a filament
(a), and a sac-like
structure containing
a spore (b) and
parasporal body (c).
Insecticide Usage on Bt and non-Bt Cotton for 1999-2001
Vaccine plants
• Pioneered by Charlie Arntzen
• Cheap vaccine-delivery system
• Plants produce a pathogen protein (or DNA) that
induces immunity (potatoes, bananas)
• Being developed for a number of diseases
– measles, cholera, foot and mouth disease, hepatitis B and C
• Four plant vaccines were successful in phase I clinical
trials:
- C.J. Arntzen et al. (2005) Plant-derived vaccines and antibodies:
Potential and limitations. Vaccine 23, 1753-1756.
Concerns that have been raised about
cultivating and consuming GM crops
1. They may be toxic or allergenic.
2. They may become established in the wild
and outcompete other plants.
3. They may negatively affect insects or other
organisms that use crops.
4. They may outcross to a nearby wild relative
spreading the transgene into a wild
population.
Hypothetical profiles of Rubisco phylogeny, the evolutionary timelines of different
photosynthetic organisms, and variation in atmospheric CO2 (thicker line) and O2 levels during
earth’s history.
Whitney S M et al. Plant Physiol. 2010;155:27-35
©2011 by American Society of Plant Biologists