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Plant Genetic Transformation
All stable transformation methods
consist of three steps:
• Delivery of DNA into a single plant cell.
• Integration of the DNA into the plant cell
genome.
• Conversion of the transformed cell into a
whole plant.
Methods of delivering DNA into
plant cells
• Biological
– Agrobacterium
– Other bacteria
– Viruses
• Physical
– Particle bombardment
– Electroporation
– Silicon carbide whiskers
– Carbon nanofibers
Agrobacterium-mediated
Transformation
Biology of the Agrobacterium-plant
interaction
The only known natural example
of inter-kingdom DNA transfer
•Infects at root crown or just below the soil line.
•Can survive independent of plant host in the soil.
•Infects plants through breaks or wounds.
•Common disease of woody shrubs, herbaceous plants,
dicots.
•Galls are spherical wart-like structures similar to
tumors.
The genus Agrobacterium has a wide host range:
• Overall, Agrobacterium can transfer T-DNA
to a broad group of plants.
• Yet, individual Agrobacterium strains hve a
limited host range.
• The molecular basis for the strain-specific
host range is unknown.
• Many monocot plants can be transformed
(now), although they do not form crown gall
tumors.
• Under lab conditions, T-DNA can be
transferred to yeast, other fungi, and even
animal and human cells.
Why is Agrobacterium used for producing
transgenic plants?
• The T-DNA element is defined by its borders but
not the sequences within. So researchers can
substitute the T-DNA coding region with any
DNA sequence without any effect on its transfer
from Agrobacterium into the plant.
Ti Plasmid
Key steps from natural Agrobacterium
to “useful”Agrobacterium
• Some vir genes deleted—disarmed
– Opines not going to be produced
– Deleting tumorogenesis function
• Choosing strains that transfer DNA in lab
• Clone in genes of interest, antibiotic
resistance genes, etc.
• Binary system-- two plasmids are better
than one Ti plasmid
Arabidopsis Floral Dip
Biolistic Transformation
Gene Gun
Agroinfiltration
Transient method of expressing transgenes