1. Plant Genetic Transformation

2. In planta transformation of Arabidopsis
Vacuum infiltration method
Floral-dip method
Advantages:
Simple, fast
No somaclonal variation
Disadvantages:
Limited mostly to Arabidopsis
Will probably be useful only with those few species that produce large number of seeds per plant.

3. Several in planta methods of transformation have been described in the past 30 years. Most of them were not reproducible and the apparent positive results obtained were generally the results of artifacts or ambiguity.
Seed imbibition-germination or pollination-fertilization were the two preferred processes during which purified DNA was applied.
All attempts were futile until Agrobacterium was developed for genetic transformation.

4. Using Agrobacterium, first unambiguous but inefficient (therefore nearly irreproducible) report was published in Feldmann and Marks imbibed Arabidopsis seeds in a suspension of Agrobacterium tumefaciens bearing npt gene on T-DNA. They used MS media with 4% sucrose. Imbibition occurred for 24 h at 28oC. The imbibed seeds were grown normally and allowed to produce seeds by self-pollination. Among these seeds some gave rise to entirely transformed plants that could be selected on antibiotic selection medium. The transformation frequency averaged 1 transformant in the progeny of 100 plants derived from treated seeds.
Feldmann’s group generated more than 17,000 T-DNA lines using this method. This collection served as the first resource for forward genetics in Arabidopsis.
Feldmann KA & Marks MD (1987) Mol. Gen. Genet. 208:19

6. Vacuum infiltration method
Grow Arabidopsis to flowering stage
Uproot plants
Application of Agrobacterium in vacuum condition in sucrose containing growth media.
Re-planting
Seed collection
Bechtold, N., Ellis, J., Pelletier, G C. R. Acad. Sci. Paris Life Sciences 316:

7. Floral-dip method Procedure:
Dip a bunch of flowering plants of Arabidopsis in Agrobacterium suspension prepared by suspending fully grown culture of bacteria in 5% sucrose supplemented with surfactant (Silwet L-77).
Clough, SJ & Bent, AF (1998) The Plant Journal  16: 

8. Effect of concentration of Silwet L-77 on transformation rates following dip inoculation.

9. Effect of repetitive dip inoculations on transformation:
Plants dip-inoculated only once during the same growth period as the plants that were dipped multiple times.
(b) Plants that were dip-inoculated at the indicated day intervals during a 15 day period commencing the day after primary inflorescences were clipped.

10. Effect of various sugars on transformation
No sugar
0.04 ± 0.01
Sucrose, 0.5%
0.40 ± 0.13
Sucrose, 1.25%
0.34 ± 0.03
Sucrose, 2.5%
0.47 ± 0.13
Sucrose, 5%
0.36 ± 0.08
Sucrose, 10%
1.42 ± 0.25
Glucose, 0.5%
0.14 ± 0.08
Glucose, 1.25%
0.11 ± 0.08
Glucose, 5%
0.76 ± 0.29
Glucose, 10%
0.33 ± 0.12
Mannitol, 5% a
death
5% food-grade sucrose
0.48 ± 0.27
Plants dipped in A. tumefaciens resuspended to OD600 = 0.8 in aqueous Silwet L-77 (0.05%) with sugar as noted. Values are mean ± SE.
aSilwet L % for mannitol treatment.

11. Effect of inoculum density on rate of transformation
Inoculum OD600
% Transformation
0.15
0.50 ± 0.02
0.42
0.21 ± 0.05
0.80
0.51 ± 0.14
1.10
0.51 ± 0.09
1.75
0.57 ± 0.15
0.8 (84 h)
0.50 ± 0.05
Plants inoculated by vacuum infiltration with A. tumefaciens in MS Medium with BAP, 5% sucrose and 0.005% L-77. All bacteria resuspended from a fresh overnight liquid culture, except '84 h' from culture grown for 84 h. Values are mean ± SE.

12. Different ecotypes and Agrobacterium strains
Ecotypes Ws-O, Nd-O, No-O were transformed at rates similar to Col-O. In contrast, Ler-O, Dijon-G and Bla-2 transformed at 10- to 100-fold lower rates. In one of the experiments, zero transformants were obtained with Ler-0.
In experiments that examined the use of other Agrobacterium strains, LBA4404, GV3101, EHA105 and Chry105 were used successfully to transform ecotype Col-0 by the floral dip method.

13. Transgenic plants obtained by in planta transformation methods are hemizygous therefore transformation in flower must be occurring after the divergence of male and female germline. Only one of them gets transformed as a result generates hemizygous transgenic plants after self-fertilization.
Male or female?
These studies addressed it
Ye et al. (1999) Plant J. 19:
Bechtold et al. (2000) Genetics 155:
Desfeux et al. (2000) Plant Physiol 123:

14. Target of transformation as revealed by crosses
Ye et al. (1999)
Target of transformation as revealed by crosses
b Cross
No. attempted
No. successful
c Percentage efficiency
No. of transgenic seeds
Wt X Wt 30 13
43.3   
Inf X Wt
187 87
46.5 15
Wt X Inf
138 88
63.7
Emasc. ctrl
bCrosses: Wt X Wt where pollen donors and recipients were both wild-type plants; Inf X Wt where the infiltrated plants served as the pollen recipients and the wild-type plants served as the pollen donors; Wt X Inf where the wild-type plants served as the pollen recipients and the infiltrated plants served as the pollen donors; Emasc. Ctrl where the infiltrated plants were hand emasculated and allowed to grow to maturity.
cPercentage efficiency was expressed as the number of successful crosses divided by the number of crosses attempted (100).

15. Target of transformation revealed by crosses
Desfeux et al. (2000) Plant Physiol. 123:

16. Genetic Engineering of Plants
Must get DNA:
into the cells
integrated into the genome (unless using transient expression assays)
expressed (everywhere or controlled)
For (1) and (2), two main approaches for plants:
Agrobacterium - mediated gene transfer
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.

17. Agrobacterium tumefaciens
The species of choice for engineering dicot plants; monocots sometimes now
Some dicots more resistant than others (a genetic basis for this)
Complex bacterium – genome has been sequenced; 4 chromosomes; ~ genes

19. Infects at root crown or just below the soil line.
Can survive independent of plant host in the soil.
Infects plants through breaks or wounds.
Common disease of woody shrubs, herbaceous plants, dicots.
Galls are spherical wart-like structures similar to tumors.

21. Agrobacterium tumefaciens
Note: pili used for attachment to plant cells, and conjugation to other bacteria.

22. Infection and tumorigenesis
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 is transferred to plant cells

23. Tumor characteristics
Synthesize unique compounds, called “opines”
octopine and nopaline - derived from arginine
agropine - derived from glutamate
Opine depends on the strain of A. tumefaciens
Opines are catabolized by the bacteria, which can use only the specific opine that it causes the plant to produce.
Has obvious advantages for the bacteria, what about the plant?

24. 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.

26. Ti Plasmid

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

28. 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
Increased levels of these hormones stimulate cell division.
Explains uncontrolled growth of tumor.
Ocs – octopine synthase, produces octopine
These genes have typical eukaryotic expression signals.

29. Ti Plasmid

32. 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.

33. Vir gene functions
virA - transports AS to bacterium, activates virG post-translationally
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, to get T-DNA into the nucleus of plant cell
virB - operon of 11 proteins, gets T-DNA through bacterial membranes

34. 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

35. 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 and sometimes scorable marker gene (GUS, GFP) in T-DNA.
4. Vir genes are retained on a separate plasmid.
Put gene of interest between T-DNA borders.
6. Co-transform Agrobacterium with both plasmids.
7. Infect plant with the transformed bacteria.

36. 2 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)

37. T-DNA integration is not highly sequence-specific
Flanking sequence tags (FSTs) analysis showed no obvious site preference for integration throughout the genome.
About 40% of the integrations are in genes and more of them are in introns.