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