Agrobacterium-mediated transformation

polarbearfellowshipBiotechnology

Dec 12, 2012 (4 years and 6 months ago)

212 views

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)

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

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

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

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

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

Agrobacterium
-
mediated transformation


Binary vectors (an Agrobacterium w/2 plasmids)


1
st

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


2
nd

plasmid (aka "helper") contains the vir genes
necessary for infection, but w/o T
-
DNA


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

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

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

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