2. What is a transgenic plant?
โข Why are transgenic plants important?
โข What are some challenges?
โข How do we create a transgenic plant?
3. Background
โขIn nature, plant growth and development is a
product of metabolic processes such as
photosynthesis and nutrient uptake.
โขHormones regulate physiological responses
such as shoot and root growth.
โขThese cells are differentiated and are
essentially impossible to genetically modify.
4. โขUsing specific nutrient and hormone
proportions, cell differentiation can be
inhibited, resulting in undifferentiated callus
cells that are totipotent and can be genetically
modified.
callus
root
seed cap
5. โขPlant injury results in release of
acetosyringone, signaling A. tumefaciens to
infect the plant.
โขIn the lab setting, acetosyringone is contained
directly within growing medium. Cutting of
root and seed injures the callus.
6. โขAgrobacterium tumefaciens uses a Ti (tumor
inducing) plasmid to infect a plant by
integrating transfer DNA into the host plant
DNA
โขPlant expresses genes that manufacture
nutrients (opines) for the bacterium.
8. T-DNA Region
โ24-bp โ24-bp
Auxin Cytokinin Opine
T-DNA
Oncogenes (in green) promote tumor
formation due to overexpression of auxin and
cytokinin. Opines provide the bacterium with a
source of nitrogen and carbon.
LB RB
9. โขIn the lab, a designed construct replaces the
T-DNA region of the plasmid, inducing the
plant to express a desired gene rather than
tumor production.
โขAgrobacterium does most of the hard work
10. โขYoung calli are ideal for Agrobacterium
infection because they have a minor cell wall.
Older calli have thicker cell walls and generally
show recalcitrance by resisting infection.
Here, a young callus
exhibits infection by
A. tumefaciens
11. โข The T-DNA construct contains a gene that
grants the callus resistance to hygromycin
used in selection media. Transgene calli
survive while others do not.
12. โขFirst generation transgene calli are
hemizygous for the desired trait
โขSeveral strains must be produced to reach a
true-breeding result.
โขPlacement of the T-DNA in the plantโs genome
is random.
13. โขCalli are moved to a regeneration media with
higher auxin:cytokinin ratio in order to
promote root development.
โขA lower ratio would promote shoot growth.
โขPlants are eventually moved to greenhouse to
develop under typical growing conditions
15. Results
OLD calluses were darker in color and seemed
to have differentiated more. They were also
less stable upon replating.
OLD & NEW callus regeneration
OLD NEW
30. Results
Regeneration numbers were observed for both
OLD and NEW calli.
A greater proportion of the NEW calluses
regenerated with greater vigor than did the
OLD calluses.
31. Results
OLD calli exhibited latent regeneration
Agrobacterium infection of OLD calli may have
been superficial
32. Results Regeneration data for OLD and NEW calluses
0
10
20
30
40
50
60
70
80
90
Nov.16 Nov.20 Nov.30 Dec.10
PercentRegeneration
Time
Callus Regeneration v. Time
OLD
NEW