The document discusses Agrobacterium-mediated transformation, which is a common method for inserting foreign genes into plant chromosomes to create transgenic plants. It involves using Agrobacterium containing binary vectors with genes of interest and antibiotic resistance markers. The Agrobacterium infects plant cells and transfers T-DNA containing the genes into the plant genome. Transformed plants are selected and tested to verify insertion of the new genes.

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