Chapter 18-Genetic Engineering of

Plants: Methodology
• Plant transformation with the Ti plasmid of
Agrobacterium tumefaciens
• Ti plasmid derived vector systems
• Physical methods of transferring genes to plants
• Microprojectile bombardment
• Use of reporter genes in transformed plant cells
• Manipulation of gene expression in plants
• Production of marker-free transgenic plants
 Why genetically engineer plants?

• To improve the agricultural, horticultural or

ornamental value of a crop plant
• To serve as a living bioreactor for the production of
economically important proteins or metabolites
• To provide a renewable source of energy
• To provide a powerful means for studying the action
of genes (and gene products) during development
and other biological processes
 Plant transformation with the Ti plasmid of
Agrobacterium tumefaciens

• A. tumefaciens is a gram-negative soil bacterium

which naturally transforms plant cells, resulting in
crown gall (cancer) tumors
• Tumor formation is the result of the transfer,
integration and expression of genes on a specific
segment of A. tumefaciens plasmid DNA called the
T-DNA (transferred DNA)
• The T-DNA resides on a large plasmid called the Ti
(tumor inducing) plasmid found in A. tumefaciens
The Ti plasmid of Agrobacterium tumafaciens and the
transfer of its T-DNA to the plant nuclear genome
Fig. 18.3 The Ti plasmid of Agrobacterium tumafaciens and
its T-DNA region containing eukaryotic genes for auxin,
cytokinin, and opine production.
 Fig. 28-27

Crown Gall on
Tobacco

Fig. 18.1 Infection of a

plant with A. tumefaciens and
formation of crown galls
 Fig. 17.3 Ti plasmid:
structure & function

The infection process:

1. Wounded plant cell releases phenolics and nutrients.
2. Phenolics and nutrients cause chemotaxic response of A. tumefaciens
3. Attachment of the bacteria to the plant cell.
4. Certain phenolics (e.g., acetosyringone, hydroxyacetosyringone) induce vir
gene transcription and allow for T-DNA transfer and integration into plant
chromosomal DNA.
5. Transcription and translation of the T-DNA in the plant cell to produce opines
(food) and tumors (housing) for the bacteria.
6. The opine permease/catabolism genes on the Ti plasmid allow A. tumefaciens
to use opines as a C, H, O, and N source.
Fig. 18.4 The right and left borders of the T-DNA of the Ti
plasmid are 25 bp direct “repeats” important for
mobilization of the T-DNA by vir gene products

Right 5’-TGNCAGGATATATNNNNNNGTNANN-3’
Left 5’-TGGCAGGATATATNNNNNTGTAAAN-3’
Fig. 18.7 The binary Ti plasmid system involves using a small
T-DNA plasmid (shown below) and a disarmed (i.e., no T-
DNA) Ti plasmid in A. tumefaciens
Clone YFG (your favorite gene) or
the target gene in the small T-DNA
plasmid in E. coli, isolate the plasmid
and use it to transform the disarmed
A. tumefaciens as shown.

(disarmed)

Plant genetic engineering with

the binary Ti plasmid system Transgenic
plant
 Table 18.1 Plant cell DNA-delivery methods
Method Comment
Ti plasmid-mediated gene Excellent and highly effective, but
transfer * limited to dicots
Microprojectile bombardment * Easy and effective; used with a
wide range of plants
Viral vectors Not very effective
Direct gene transfer into plant Only certain protoplasts can be
protoplasts regenerated into whole plants
Microinjetion Tedious and slow
Electroporation * Limited to protoplasts that can be
regenerated into whole plants
Liposome fusion Limited to protoplasts that can be
regenerated into whole plants

* Most commonly used methods

Fig. 18.10
Microprojectile
bombardment or
biolistic-mediated
DNA transfection
*
equipment
(a) lab version
(b) portable version

When the helium pressure

builds to a certain point, the
plastic rupture disk bursts, and
the released gas accelerates the
flying disk* with the DNA-
coated gold particles on its
lower side. The gold particles
pass the stopping screen, which
holds back the flying disk, and
penetrate the cells of the plant.
 Table 18.5 Some plant cell reporter and
selectable marker gene systems
Enzyme activity Selectable Reporter
marker gene
Neomycin phosphotransferase (kanr) Yes Yes
Hygromycin phosphotransferase (hygr) Yes Yes
Nopaline synthase No Yes
Octopine synthase No Yes
-glucuronidase (GUS) No Yes
Firefly luciferase No Yes
-galactosidase No Yes
Bromoxynil nitrilase Yes No
Green fluorescent protein (GFP) No Yes
 Reporter Genes

• For how reporter genes work, see:

http://bcs.whfreeman.com/lodish5e/pages/bcs-main.asp?v=category&s=00010&n=15000&i=
15010.01&o=|00510|00610|00520|00530|00540|00560|00570|00590|00600|00700|0071
0|00010|00020|00030|00040|00050|01000|02000|03000|04000|05000|06000|07000|08
000|09000|10000|11000|12000|13000|14000|15000|16000|17000|18000|19000|20000|
21000|22000|23000|99000|&ns=1322
• GFP Researchers Win Nobel Prize (October 8, 2008)
Osamu Shimomura, Martin Chalfie, and Roger Tsien won the Nobel Prize in chemistry
for their work on green flourescent protein, a tool that has become ubiquitous in
modern biology as a tag and molecular highlighter, vastly improving our ability to
understand what goes on inside cells.
• Perhaps you may even want to see a 10 minute YouTube video on GFP; if so please
see http://www.youtube.com/watch?v=Sl2PRHGpYuU
 Manipulation of gene expression in plants

• Strong, constitutive promoters (35S Cauliflower mosaic virus

promoter or 35S CaMV or 35S)
• Organ and tissue specific promoter (e.g., the leaf-specific
promoter for the small subunit of the photosynthetic enzyme
ribulosebisphosphate carboxylase or rbc)
• Promoterless reporter gene constructs to find new organ- and
tissue-specific promoter (see Fig. 18.15)
• Inducible promoters
• Secretion of transgene products by inclusion of a signal peptide
sequence between a root promoter and YFG and growing the
transgenic plant hydroponically (YFG product will be secreted)
Fig. 18.26 Marker genes may be a safety issue, so it is best to
remove them—here is one strategy

Recombinase Selectable
LB gene marker Target gene RB

Recombinase recognition
sequence