1. Agrobacterium-mediated transformation
• Transformation – the process of obtaining
transgenic plants
• Transgenic plant – a plant with a foreign gene
(or genes) from another plant/animal that is
incorporated into its chromosome
• Most common genes (and traits) in transgenic or
biotech crops
– herbicide resistance (e.g., Round-up Ready
soybeans)

2. Agrobacterium-mediated transformation
• Most common genes (and traits) in transgenic or
biotech crops
– Bt genes (European corn borer toxin) in field corn
(maize)
– virus-resistance (coat-protein) genes (papaya)
• Viewpoints of these biotech crops have stirred
debate pro and con
– growers are generally in the "pro" camp

3. Agrobacterium-mediated transformation
• Viewpoints (contin)
– environmentalists are suspicious
– organic farmers are in the "con" camp
– some consumers wonder where the benefit is to
them, others worry about potential allergens
– Europeans won't use them, but they are in a growing
minority of nations

4. Agrobacterium-mediated transformation
• Regardless of the debate over biotech crops,
they seem certain to be with us for the
foreseeable future
• We will concentrate on the way in which in vitro
methods are currently used for producing
transgenics
• Two major methods for making transgenics
– Agrobacterium-mediated transformation
– particle bombardment

5. Agrobacterium-mediated transformation
• Basic tissue-culture process for Agrobacterium-
mediated transformation
– disinfestation of an explant
– isolation and growth of the Agrobacterium vector (with
a DNA sequence that is to be transferred)
– co-culture of the explant with the Agrobacterium
– growth of the explant on a selective medium
– regeneration/rooting of the transformed shoots
– testing for the inserted DNA sequence and
propagation of the tranformed plant

6. Agrobacterium-mediated transformation
• The infection process
– wild-type Agrobacterium attaches to a plant cell, then
transfers a discrete portion of its tumor-inducing (Ti)
plasmid to the plant's chromosome
– The Ti plasmid contains 2 critical regions:
• the oncogene-containing T-DNA (the DNA that is
transferred)
• the virulence (vir) genes that encode proteins
required for T-DNA transfer

7. Agrobacterium-mediated transformation
• Binary vectors (an Agrobacterium w/2 plasmids)
– 1st plasmid contains a T-DNA region with the "trait"
gene and an antibiotic resistance gene inserted into
the T-DNA in place of the oncogenes
• removal of oncogenes, which cause crown gall
disease, make these "disarmed" vectors
• the antibiotic resistance gene can be used as a
selectable marker
– 2nd plasmid (aka "helper") contains the vir genes
necessary for infection, but w/o T-DNA

8. Agrobacterium-mediated transformation
• Binary vectors (an Agrobacterium w/2 plasmids)
– an advantage of binary vectors is that it is easier to do
the actual genetic engineering in E. coli, then transfer
that smaller, engineered plasmid into Agrobacterium
• Other features of the transformation process
– left (LB) and right (RB) border regions are ca. 23 bp
repeats and are important in the transfer process
– DNA strand synthesis starts at RB, so those genes
closest to RB are most efficiently transferred

9. Agrobacterium-mediated transformation
• Other features of the transformation process
– how T-DNA incorporates isn't known, but covalent
linkage with the plant's DNA has been proven
• Regeneration and selection
– for shoot organogenesis, cytokinin (and usu. lower
amounts of auxin) are required
– two antibiotics are required
• an antibiotic to kill the Agrobacterium, while not
affecting the plant's cell growth and division

10. Agrobacterium-mediated transformation
• Regeneration and selection
– two antibiotics are required
• a second antibiotic allows growth of transformed
shoots (w/selectable marker) but inhibits growth of
untranformed plant cells
• Detection of the "trait" gene
– PCR methods can detect the presence of the "trait"
DNA
– protein detection methods are used where a gene
product is produced that defines the trait

11. Agrobacterium-mediated transformation
• Detection of the "trait" gene
– verification of the incorporation of the trait gene into
the plant's chromosome
• by Southern hybridization
• by demonstrating transfer of the trait to the original
transformant's progeny
• Agrobacterium has a limited host range
– papaya, soybean (both dicots) are susceptible
– most monocots are not susceptible