1. Gene delivery techniques

2. Genetic Engineering of Plants
Must get DNA:
into the cells
integrated into the genome
Expressed
For (1) and (2), two main approaches for plants:
Direct gene transfer
Agrobacterium - mediated gene transfer
For (3), use promoter that will direct expression when and where wanted.

3. Commonly used gene delivery techniques
Microinjection
Electroporation
Microprojectiles
Agrobacterium tumifaciens mediated transfer

4. Microinjection Mechanical method of gene insertion
Very fine micropipette used to inject DNA molecule directly into the nucleus
Most commonly used for animal embryos
Not very successful in case of plants

5. Microinjection
Source:

6. Electroporation Electric current is provided to plant cells
Tiny holes are created
DNA molecules present in the liquid medium
Diffusion into the cells
Cells allowed to recover
Regeneration into whole plant

7. Microprojectile/Gene Gun
Most commonly used for monocots
Tungsten or gold particles coated with DNA
Shot into the cells

8. Agrobacterium tumefaciens
Mainly for engineering dicot plants; monocots generally
Some dicots more resistant than others (a genetic basis for this)
Complex bacterium – genome has been sequenced; 4 chromosomes; ~ 5500 genes
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
Agrobacterium tumefaciens: The Gram-negative soil bacterium as pathogen results in crown gall tumors in plants. T-DNA, a part of the bacterial tumor-inducing (Ti) plasmid, is transferred from the bacterium to the plant cell with the aid of a number of Virulence (Vir) proteins encoded by the Ti-plasmid.
Agrobacterium is the only known prokaryote to transfer genes to a eukaryote.
Agrobacterium is used throughout microbiology to transfer DNA into plant cells

10. A. tumefaciens Ti (Tumor inducing) Plasmid
A plasmid is a small DNA molecule that is physically separate from, and can replicate independently of, chromosomal DNA within a cell. Most commonly found as small circular, double-stranded DNA molecules in bacteria

11. Structure of Ti Plasmid

12. Ti Plasmid containing T-DNA region

13. T-DNA auxA auxB cyt ocs LB 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

14. Agrobacterium mechanism of plant infection
The transferred genes form the T-DNA (transferred DNA) region of the Ti plasmid (tumour inducing).
T-DNA is less than 10% of the whole plasmid, encodes only 3 or so genes and enters a plant that has at least genes
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)
Agrobacteria usually infects plants from their wounds, which occurred quite frequent after frost.
In practice, protection from subfreezing winter temperatures and control of chewing insects and nematodes can prevent infection by agrobacteria

15. Effect of T-DNA genes on plant cells
Changes the plant’s metabolism to produce food materials (opines) that only the bacterium can use
Produces the plant hormones auxin and cytokinin that remove the controls that normally limit cell division and cell expansion.
Result - cells showing altered metabolism multiply uncontrollably

16. RESULT
Even if the T-DNA originally change only one cell in the plant, because that cell divides uncontrollably and passes on those bacterial genes to all the new cells, there are soon millions of cells feeding the bacterium at the expense of the plant.

17. Infection of a plant with A
Infection of a plant with A. tumefaciens and formation of a crown gall tumor.
Agrobacterium tumefaciens and A. rhizogenes infect wounded plants and transfer plasmid DNA (T-DNA) and virulence (Vir) proteins into plant cells.
The Plasmid shown here in the plant cell codes for T-DNA and Virulence proteins, this T-DNA is then transferred to the host
with the aid of the virulence proteins.
You can see a cellular view in which a portion of the plasmid DNA and the Vir proteins are transferred here
This dna then encodes for oncogenes and enzymes that aid in the synthesis of opines, which is an amino acid and sugar complex that the bacteria can metabolize and use as an energy source. Thus the bacterium has the plant make food for it.

18. EM of A. tumefaciens attached to plant cell

19. Vir (virulent) genes On the Ti plasmid
Transfer the T-DNA to plant cell
Acetosyringone (AS) (a flavonoid) released by wounded plant cells activates vir genes
virA,B,C,D,E,F,G (7 complementation groups, but some have multiple ORFs), span about 30 kb of Ti plasmid.

20. 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 get T-DNA to the nucleus of plant cell
virB - operon of 11 proteins, gets T-DNA through bacterial membranes

21. A. tumifaciens mediated gene transfer
Removal & Replacement of T-DNA region

22. Engineered A. Tumefaciens Ti Plasmid vector
Use engineered instead of wild-type A. tumefasciens Ti plasmid
Still possesses virulence genes (allow transfer of T-DNA to target cell) but lacks opine and PGR synthesis genes

23. Common Transformation Protocols
Leaf-disc transformation - after selection and regeneration with tissue culture, get plants with the introduced gene in every cell
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)

24. T-DNA transfer mechanism
ANIMATION