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)
• Chloroplast engineering
• 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 or horticultural value of
plants
• To serve as living bioreactors for the production of
economically important proteins or metabolites
• To provide a renewable source of energy (biofuels)
• To provide a powerful means for studying the
biological action of genes and gene products
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.
Chapter 18
Genetic Engineering of Plants: Methodology
Figure 18.3
Ti plasmid structure
Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition
Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten
Copyright © 2010 ASM Press
American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904
Chapter 18
Genetic Engineering of Plants: Methodology
Figure 18.1
Infection of a plant with A. tumefaciens
and formation of a crown gall tumor.
Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition
Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten
Copyright © 2010 ASM Press
American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904
Fig. 28-27
Crown Gall on
Tobacco
Fig. 18.1 Infection of a
plant with A. tumefaciens and
formation of crown galls
Figure 18.2 and 18.3
Ti plasmid structure and
function. Note the woundinduced plant phenolics
induce the vir genes on
the Ti plasmid.
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.
Chapter 18
Genetic Engineering of Plants: Methodology
Figure 18.4
Conserved nucleotides at the right and left borders of the Ti plasmid are imperfect
direct repeats.
Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition
Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten
Copyright © 2010 ASM Press
American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904
Chapter 18
Genetic Engineering of Plants: Methodology
Figure 18.6
Chemical structures of three
opines produced by plants.
Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition
Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten
Copyright © 2010 ASM Press
American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904
Fig. 18.7 The binary Ti plasmid system involves using a small
T-DNA plasmid (shown below) and a disarmed (i.e., no TDNA) 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)
Disarmed
Ti plasmid
Plant genetic engineering with
the binary Ti plasmid system
Transgenic
plant
Chapter 18
Genetic Engineering of Plants: Methodology
Table 18.1
Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition
Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten
Copyright © 2010 ASM Press
American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904
Chapter 18
Genetic Engineering of Plants: Methodology
Table 18.2
Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition
Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten
Copyright © 2010 ASM Press
American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904
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 DNAcoated 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.
*
Chapter 18
Genetic Engineering of Plants: Methodology
Figure 18.10
Microprojectile bombardment
(biolistics) apparatus
Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition
Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten
Copyright © 2010 ASM Press
American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904
Chapter 18
Genetic Engineering of Plants: Methodology
Chloroplasts can be genetically engineered using microparticle bombardment.
Figure 18.12
Figure 18.13
Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition
Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten
Copyright © 2010 ASM Press
American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904
Chapter 18
Genetic Engineering of Plants: Methodology
Table 18.5
Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition
Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten
Copyright © 2010 ASM Press
American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904
Table 18.5 Some plant cell reporter and
selectable marker gene systems
Enzyme activity
Selectable
marker
Reporter
gene
Neomycin phosphotransferase (kanr)
Yes
Yes
Hygromycin phosphotransferase (hygr)
Yes
Yes
Nopaline synthase
No
Yes
Octopine synthase
No
Yes
b-glucuronidase (GUS)
No
Yes
Firefly luciferase
No
Yes
b-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/lodish7e/#800911__811966__
• 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)
Chapter 18
Genetic Engineering of Plants: Methodology
Figure 18.21
Rhizosecretion using a
plant promoter active in
roots and a signal peptide
sequence.
Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition
Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten
Copyright © 2010 ASM Press
American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904
Chapter 18
Genetic Engineering of Plants: Methodology
Figure 18.26
Marker genes may be a safety issue, so it is best to remove them—here is one strategy.
Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition
Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten
Copyright © 2010 ASM Press
American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904