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.
Agrobacterium-mediated
Transformation
Only known natural example of DNA
transport between Kingdoms
1. (Virulent) strains
of A. tumefaciens
contain a 200-kb
tumor inducing (Ti)
plasmid
2. Bacteria
transfer a portion
of the plasmid DNA
into the plant host
(T-DNA).
T-DNA 
•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 T-DNA is transferred from the
Bacteria into the Nucleus of the Plant
1. Stably integrates (randomly) into the plant
genome.
2. Expression of genes in wild-type T-DNA
results in dramatic physiological changes to
the plant cell.
3.  Synthesis of plant growth hormones
(auxins and cytokinins)  neoplastic growth
(tumor formation)
Genes required to breakdown opines for use as a
nutrient source are harbored on the Ti plasmid
in addition to vir genes essential for the
excision and transport of the T-DNA to the
wounded plant cell.
T-DNA 23 kb
tra
vir genes
for transfer
to the plant
pTi
~200 kb
bacterial
conjugation
opine catabolism
Agrobacterium chromosomal DNA
pscA
chvA chvB
T-DNA-inserts into plant genome
for
transfer
to the
plant
vir genes
pTi
tra
bacterial
conjugation
opine catabolism
oriV
Ti Plasmid
Ti Plasmid
Agrobacterium
can be used to
transfer DNA
into plants
Ti plasmids and the bacterial
chromosome act in concert to transform
the plant
1. Agrobacterium tumefaciens chromosomal
genes: chvA, chvB, pscA required for initial
binding of the bacterium to the plant cell
and code for polysaccharide on bacterial
cell surface.
2. Virulence region (vir) carried on pTi, but
not in the transferred region (T-DNA).
Genes code for proteins that prepare the
T-DNA and the bacterium for transfer.
3. T-DNA encodes genes for opine synthesis and
for tumor production.
4. occ (opine catabolism) genes carried on the pTi
allow the bacterium to utilize opines as nutrient.
Generation of the T-strand
Left
Border
Right
Border
T-DNA
overdrive
5’
virD/virC
VirD nicks the lower strand (T-strand) at the
right border sequence and binds to the 5’ end.
Generation of the T-strand
Left
border
T-DNA
Right
border
gap filled in
virE
virD/virC
T-strand
D
1. Helicases unwind the T-strand which
is then coated by the virE protein.
2. ~one T-strand produced per cell.
Left
border
T-DNA
Right
border
D
T-strand coated with virE
virD nicks at Left Border sequence
1. Transfer to plant cell.
2. Second strand synthesis
3. Integration into plant chromosome
Overview of the Infection Process
pTi-based vectors for plant
transformation:
1. Shuttle vector is a small E. coli plasmid using
for cloning the foreign gene and transferring to
Agrobacterium.
2. Early shuttle vectors integrated into the TDNA; still produced tumors.
Shuttle plasmid
E. coli
conjugation
pTi
Agrobacterium
Transformation of Arabidopsis plants
Detergent added to allow
bacteria to infiltrate the
floral meristem.
Dip floral buds
in 1 ml of
Agrobacterium
culture for 5 to
15 min.
Transformation of Arabidopsis plants
700 to 900
seeds per plant.
Germinate on
kanamycin plates
to select
transformants.
10 to 20
transformed
plants per plant.
10 day old seedlings
MiniTi T-DNA based vector for plants
Disarmed vectors: do not produce tumors; can be
used to regenerate normal plants containing the
foreign gene.
1. Binary vector: the vir genes
required for mobilization and
transfer to the plant reside
on a modified pTi.
2. consists of the right and left
border sequences, a
selectable marker (kanomycin
resistance) and a polylinker
miniTi
for insertion of a foreign
gene.
MiniTi T-DNA based vector for plants
a binary vector system
T-DNA deleted
kanr
polylinker
LB
1
RB
ori
bom
modified Ti plasmid
vir
miniTi
bom = basis of mobilization
oriV
2
Alternate Methods of Transforming Plants:
Particle Bombardment
One way of physically introducing DNA into
cells is with a particlegun.
•Very tiny DNA-coated metal particles are
suspended in a drop on a macroprojectile.
•A discharge (from a gunpowder explosion or
from breakage of a membrane enclosing a
pressurized chamber) impels the
macroprojectile.
•The macroprojectile is stopped by a
stopping plate, but the microprojectiles
continue into the tissue below.
•The DNA introduced with the particles is
expressed
Particle Bombardment using the Gene Gun
1. DNA- or RNA-coated gold/tungsten particles are
loaded into the gun and you pull the trigger.