Plant transformation methods

 Agrobacterium-mediated transformation
 Biolistic transformation
 Chemical method
 Microinjection
 The pollen-tube pathway method
 Liposomes
 Shoot apex method of transformation
 Infiltration
 Silicon carbide-mediated transformation (SCMT)
 Electroporation of intact plant cells and tissues
 Electrophoresis
 Plant Transformation
• Must get DNA:
1. into the cells
2. integrated into the genome (unless using transient
expression assays)
3. expressed (everywhere or controlled)

• For (1) and (2), two main approaches for plants:

1. Agrobacterium - mediated gene transfer
2. Direct gene transfer

• For (3), use promoter that will direct expression when

and where wanted – may also require other
modifications such as removing or replacing introns.
 Agrobacterium - mediated Gene Transfer
• Most common method of engineering dicots, but also
used for monocots
• Pioneered by J. Schell (Max-Planck Inst., Cologne)

• Agrobacteria
– soil bacteria, gram-negative, related to Rhizobia
– species:
tumefaciens- causes crown galls on many dicots
rubi- causes small galls on a few dicots
rhizogenes- hairy root disease
radiobacter- avirulent
 Crown galls
caused by A.
tumefaciens on
nightshade.

More about Galls:

http://waynesword.palomar.edu/pljuly99.htm
http://kaweahoaks.com/html/galls_ofthe_voaks.
html
 Agrobacterium tumefaciens
• the species of choice for engineering dicot
plants; monocots are generally resistant
• some dicots more resistant than others (a
genetic basis for this)
• complex bacterium – genome has been
sequenced; 4 chromosomes; ~ 5500
genes
Agrobacterium tumefaciens
 Infection and tumor induction
• Infection occurs at wound sites
• Involves recognition and chemotaxis of the
bacterium toward wounded cells
• galls are “real tumors”, can be removed and
will grow indefinitely without hormones
• genetic information must be transferred to
plant cells
 Tumor characteristics

1. Synthesize a unique amino acid, called “opine”

– octopine and nopaline - derived from
arginine
– agropine - derived from glutamate
2. Opine depends on the strain of A. tumefaciens
3. Opines are catabolized by the bacteria, which
can use only the specific opine that it causes
the plant to produce.
4. Has obvious advantages for the bacteria, what
about the plant?
 Elucidation of the TIP (tumor-inducing principle)

• It was recognized early that virulent strains

could be cured of virulence, and that
cured strains could regain virulence when
exposed to virulent strains; suggested an
extra-chromosomal element.
• Large plasmids were found in A. tumefaciens
and their presence correlated with virulence:
called tumor-inducing or Ti plasmids.
 Ti Plasmid

1. Large (200-kb)
2. Conjugative
3. ~10% of plasmid transferred to plant cell
after infection
4. Transferred DNA (called T-DNA) integrates
semi-randomly into nuclear DNA
5. Ti plasmid also encodes:
– enzymes involved in opine metabolism
– proteins involved in mobilizing T-DNA (Vir
genes)
 T-DNA

LB auxA auxB cyt ocs RB

LB, RB – left and right borders (direct repeat)

auxA + auxB – enzymes that produce auxin
cyt – enzyme that produces cytokinin
Ocs – octopine synthase, produces octopine

These genes have typical eukaryotic expression signals!

 Vir (virulent) genes
1. On the Ti plasmid
2. Transfer the T-DNA to plant cell
3. Acetosyringone (AS) (a flavonoid) released by
wounded plant cells activates vir genes.
4. virA,B,C,D,E,F,G (7 complementation
groups, but some have multiple ORFs),
span about 30 kb of Ti plasmid.
 Vir gene functions (cont.)
• virA - transports AS into bacterium, activates
virG post-translationally (by phosphoryl.)
• virG - promotes transcription of other vir genes
• virD2 - endonuclease/integrase that cuts T-
DNA at the borders but only on one strand;
attaches to the 5' end of the SS
• virE2 - binds SS of T-DNA & can form channels
in artificial membranes
• virE1 - chaperone for virE2
• virD2 & virE2 also have NLSs, gets T-DNA to
the nucleus of plant cell
• virB - operon of 11 proteins, gets T-DNA
through bacterial membranes
A model for the Agrobacterium-mediated genetic transformation

Recognition and attachment

Vir genes expression by host signals
T-strand produce
T-complex export into host
Transport through cytoplasm and nuclear
T-DNA uncoating and integration.
VirE2 may get DNA-protein complex across host PM

Dumas et al., (2001), Proc. Natl. Acad. Sci. USA, 98:485

• Important: Put any DNA between the LB and RB
of T-DNA it will be transferred to plant cell!

Engineering plants with Agrobacterium:

Two problems had to be overcome:

(1) Ti plasmids large, difficult to manipulate
(2) couldn't regenerate plants from tumors
 Binary vector system
Strategy:
1. Move T-DNA onto a separate, small plasmid.
2. Remove aux and cyt genes.
3. Insert selectable marker (kanamycin resistance) gene in
T-DNA.
4. Vir genes are retained on a separate plasmid.
5. Put foreign gene between T-DNA borders.
6. Co-transform Agrobacterium with both plasmids.
7. Infect plant with the transformed bacteria.
Binary vector system
 Two Common Transformation Protocols
1. Leaf-disc transformation - after selection and
regeneration with tissue culture, get plants with
the introduced gene in every cell

2. Floral Dip – does not require tissue culture.

Reproductive tissue is transformed and the
resulting seeds are screened for drug-resistant
growth. (Clough and Bent (1998) Floral dip: a simplified method for
Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant
Journal 16, 735–743)
 Making a transgenic
plant by leaf disc
transformation with
Agrobacterium.

S.J. Clough, A.F. Bent (1998) Floral dip: a simplified method for
Agrobacterium-mediated transformation of Arabidopsis thaliana.
Plant Journal 16, 735–743.
Hand held gene gun
Sanford and colleagues at Cornell University developed the
original bombardment concept and coined the term
“biolistics” (short for “biological ballistics”) for both the
process and device.

There are several homemade “gene guns” or “particle guns,”

the process often is called by other names such as
microprojectile bombardment,
particle bombardment, particle acceleration, or ballistics.

The most widely used device for plant transformation is the

Biolistic ® PDS-1000/He Particle Delivery System
 Microinjection

Transformation via microinjection is based on introducing DNA into

the nucleus or cytoplasm by means of a glass micro capillary-injection
pipette.

This operation requires a micromanipulator.

During the introduction of DNA into the nucleus, cells are immobilized
with a holding pipette and gentle suction.

Both pipettes contain mineral oil, which works as a cylinder.

Microinjection is mainly used for the transformation of large animal

cells. Its importance for plant transformation is rather limited due to
the characteristics of plant cell walls, which contain a thick layer of
lignins and cellulose.
Vacuole hydrolases
The pollen-tube pathway method
 Silicon carbide-mediated transformation (SCMT)

SCMT is one of the least complicated methods of plant

transformation.
Silicon carbide fibers are simply added to a suspension containing
plant tissue (cell clusters, immature embryos, callus) and plasmid
DNA, and then mixed in a vortex, or in other laboratory apparatus
such as commercial shakers, blenders etc. DNA-coated fibers
penetrate the cell wall in the presence of small holes created in
collisions between the plant cells and fibers (Kaeppler et al., 1990,
1992; Wang et al., 1995).
The most often used fibers in this procedure are single crystals of
silica organic minerals like silicon carbide, which have an elongated
shape, a length of 10–80 mm, and a diameter of 0.6 mm, and which
show a high resistance to expandability. Fiber size, the parameters of
vortexing, the shape of the vessels used, the plant material and the
characteristics of the plant cells, especially the thickness of the cell
wall are the factors depending on the efficiency of SCM.
 Infiltration

Some transformation procedures do not require in

vitro culture. In the case of infiltration, the bacterial
inoculum is introduced directly into those parts of the
plant in which meiotic or mitotic divisions take place
intensively.
Infiltration has mainly been applied for the
transformation of A. thaliana for the past several last
years, and has become the main method of gene
delivery for this species.