1. 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

2. 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

3. 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

4. The Ti plasmid of Agrobacterium tumafaciens and the transfer of its T-DNA to the plant nuclear genome

5. Fig The Ti plasmid of Agrobacterium tumafaciens and its T-DNA region containing eukaryotic genes for auxin, cytokinin, and opine production.

6. Fig
Crown Gall on
Tobacco
Fig Infection of a plant with A. tumefaciens and formation of crown galls

7. Fig. 17.3 Ti plasmid: structure & function
The infection process:
Wounded plant cell releases phenolics and nutrients.
Phenolics and nutrients cause chemotaxic response of A. tumefaciens
Attachment of the bacteria to the plant cell.
Certain phenolics (e.g., acetosyringone, hydroxyacetosyringone) induce vir gene transcription and allow for T-DNA transfer and integration into plant chromosomal DNA.
Transcription and translation of the T-DNA in the plant cell to produce opines (food) and tumors (housing) for the bacteria.
The opine permease/catabolism genes on the Ti plasmid allow A. tumefaciens to use opines as a C, H, O, and N source.

8. Fig 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’

9. Fig 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

10. Plant genetic engineering with the binary Ti plasmid system
(disarmed)
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.
Plant genetic engineering with the binary Ti plasmid system
Transgenic
plant

11. Table 18.1 Plant cell DNA-delivery methods
Comment
Ti plasmid-mediated gene transfer *
Excellent and highly effective, but 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 protoplasts
Only certain protoplasts can be regenerated into whole plants
Microinjetion
Tedious and slow
Electroporation *
Limited to protoplasts that can be regenerated into whole plants
Liposome fusion
* Most commonly used methods

12. Fig 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.

13. Table 18.5 Some plant cell reporter and selectable marker gene systems
Enzyme activity
Selectable marker
Reporter gene
Neomycin phosphotransferase (kanr)
Yes
Hygromycin phosphotransferase (hygr)
Nopaline synthase No
Octopine synthase
b-glucuronidase (GUS)
Firefly luciferase
b-galactosidase
Bromoxynil nitrilase
Green fluorescent protein (GFP)

14. Reporter Genes
For how reporter genes work, see:
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

15. 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 )
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)

16. Recombinase recognition
Fig Marker genes may be a safety issue, so it is best to remove them—here is one strategy
Recombinase
gene
Selectable
marker LB
Target gene RB
Recombinase recognition
sequence