Plant Genetic Transformation

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-fertilizatio
n 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 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. Feldmanns
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.
20819
5
(No Transcript)
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. 1993 C. R.
Acad. Sci. Paris Life Sciences 3161194-1199.
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 
16 735-743.
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
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. 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. aSilwet L-77 0.005 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. 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. 19 249-257.
• Bechtold et al. (2000) Genetics 1551875-1887.
• Desfeux et al. (2000) Plant Physiol 123 895-904.

14
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 0
Emasc. ctrl 30 0 0 0

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. 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. 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. 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. 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). cPercentage efficiency was expressed as the number of successful crosses divided by the number of crosses attempted (100). cPercentage efficiency was expressed as the number of successful crosses divided by the number of crosses attempted (100). cPercentage efficiency was expressed as the number of successful crosses divided by the number of crosses attempted (100). 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. 123 895-904.
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 5500 genes

18
(No Transcript)
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.

20
(No Transcript)
21
Agrobacterium tumefaciens
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.

25
(No Transcript)
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
30
(No Transcript)
31
(No Transcript)
32
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.

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

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, 735743)

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.