Transcript Transformation 2015

Plant Genetic Transformation
In planta transformation of Arabidopsis
Vacuum infiltration method
Floral-dip method
Advantages:
1. Simple, fast
2. No somaclonal variation
Disadvantages:
1. Limited mostly to Arabidopsis
2. Will probably be useful only with those few species that
produce large number of seeds per plant.
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.
Using Agrobacterium, first unambiguous but inefficient (therefore nearly
irreproducible) report was published in 1987. 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
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. 1993 C. R. Acad. Sci. Paris Life Sciences 316:1194-1199.
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: 735-743.
Effect of concentration of Silwet L-77 on transformation
rates following dip inoculation.
Effect of repetitive dip inoculations on transformation:
(a) 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.
Effect of various sugars on transformation
Sugar
% 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-77 0.005% for mannitol treatment.
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.
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.
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 selffertilization.
Male or female?
These studies addressed it
1. Ye et al. (1999) Plant J. 19: 249-257.
2. Bechtold et al. (2000) Genetics 155:1875-1887.
3. Desfeux et al. (2000) Plant Physiol 123: 895-904.
Ye et al. (1999)
Target of transformation as revealed by crosses
No.
attempted
No.
successful
c
Wt X Wt
30
13
43.3
Inf X Wt
187
87
46.5
15
Wt X Inf
138
88
63.7
0
Emasc.
ctrl
30
0
0
0
b
Cross
Percentage
efficiency
No. of transgenic
seeds
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).
Target of transformation revealed by crosses
Desfeux et al. (2000) Plant Physiol. 123: 895-904.
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.
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; ~ 5500
genes
•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.
Agrobacterium tumefaciens
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
Tumor characteristics
1. Synthesize unique compounds, called “opines”
– octopine and nopaline - derived from
arginine
– agropine - derived from glutamate
2. Opine depends on the strain of A. tumefaciens
3. Opines are catabolized by the bacteria, which
can use only the specific opine that it causes
the plant to produce.
4. Has obvious advantages for the bacteria, what
about the plant?
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.
Ti Plasmid
Ti Plasmid
1. Large ( 200-kb)
2. Conjugative
3. ~10% of plasmid transferred to plant cell
after infection
4. Transferred DNA (called T-DNA) integrates
semi-randomly into nuclear DNA
5. Ti plasmid also encodes:
– enzymes involved in opine metabolism
– proteins involved in mobilizing T-DNA (Vir
genes)
T-DNA
LB
auxA auxB
cyt
ocs
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.
Ti Plasmid
Vir (virulent) genes
1. On the Ti plasmid
2. Transfer the T-DNA to plant cell
3. Acetosyringone (AS) (a flavonoid) released by
wounded plant cells activates vir genes.
4. virA,B,C,D,E,F,G (7 complementation
groups, but some have multiple ORFs),
span about 30 kb of Ti plasmid.
Vir gene functions
• virA - transports AS to bacterium, activates
virG post-translationally
• virG - promotes transcription of other vir genes
• virD2 - endonuclease/integrase that cuts TDNA 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
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
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.
5. Put gene of interest between T-DNA borders.
6. Co-transform Agrobacterium with both plasmids.
7. Infect plant with the transformed bacteria.
2 Common Transformation Protocols
1. Leaf-disc transformation - after selection and
regeneration with tissue culture, get plants with
the introduced gene in every cell
2. 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)
T-DNA integration is not highly sequencespecific
• 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.