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Plant Genetic Transformation

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Plant Genetic Transformation All stable transformation methods consist of three steps: Delivery of DNA into a single plant cell. Integration of the DNA into the plant ... – PowerPoint PPT presentation

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Title: Plant Genetic Transformation


1
Plant Genetic Transformation
2
All stable transformation methods consist of
three steps
  • Delivery of DNA into a single plant cell.
  • Integration of the DNA into the plant cell
    genome.
  • Conversion of the transformed cell into a whole
    plant.

3
Agrobacterium-mediated Transformation
4
Only known natural example of DNA transport
between Kingdoms
1. (Virulent) strains of A. tumefaciens contain a
200-kb tumor inducing (Ti) plasmid
2. Bacteria transfer a portion of the plasmid DNA
into the plant host (T-DNA).
T-DNA ?
5
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6
  • 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.

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8
The T-DNA is transferred from the Bacteria into
the Nucleus of the Plant
1. Stably integrates (randomly) into the plant
genome.
2. Expression of genes in wild-type T-DNA results
in dramatic physiological changes to the plant
cell.
3. ? Synthesis of plant growth hormones (auxins
and cytokinins) ? neoplastic growth (tumor
formation)
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11
Genes required to breakdown opines for use as a
nutrient source are harbored on the Ti plasmid in
addition to vir genes essential for the excision
and transport of the T-DNA to the wounded plant
cell.

T-DNA
23 kb
tra
bacterial conjugation
pTi 200 kb
vir genes
for transfer to the plant
opine catabolism
12
Agrobacterium chromosomal DNA
pscA
chvA
chvB
T-DNA-inserts into plant genome
tra
bacterial conjugation
for transfer to the plant
pTi
vir genes
opine catabolism
oriV
13
Ti Plasmid
14
Ti Plasmid
15
Agrobacterium can be used to transfer DNA into
plants
16
Ti plasmids and the bacterial chromosome act in
concert to transform the plant
1. Agrobacterium tumefaciens chromosomal genes
chvA, chvB, pscA required for initial binding of
the bacterium to the plant cell and code for
polysaccharide on bacterial cell surface. 2.
Virulence region (vir) carried on pTi, but not in
the transferred region (T-DNA). Genes code for
proteins that prepare the T-DNA and the bacterium
for transfer.
17
3. T-DNA encodes genes for opine synthesis and
for tumor production. 4. occ (opine catabolism)
genes carried on the pTi allow the
bacterium to utilize opines as nutrient.
18
Generation of the T-strand
Right Border
Left Border
T-DNA
overdrive
5
virD/virC
VirD nicks the lower strand (T-strand) at the
right border sequence and binds to the 5 end.
19
Generation of the T-strand
Right border
Left border
T-DNA
gap filled in
virE
T-strand
D
virD/virC
1. Helicases unwind the T-strand which is then
coated by the virE protein. 2. one T-strand
produced per cell.
20
Right border
Left border
T-DNA
D
T-strand coated with virE
virD nicks at Left Border sequence
1. Transfer to plant cell. 2. Second strand
synthesis 3. Integration into plant chromosome
21
Overview of the Infection Process
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23
pTi-based vectors for plant transformation
1. Shuttle vector is a small E. coli plasmid
using for cloning the foreign gene and
transferring to Agrobacterium.
2. Early shuttle vectors integrated into the
T-DNA still produced tumors.
pTi
Shuttle plasmid
conjugation
E. coli
Agrobacterium
24
Transformation of Arabidopsis plants
Dip floral buds in 1 ml of Agrobacterium culture
for 5 to 15 min.
Detergent added to allow bacteria to infiltrate
the floral meristem.
25
Transformation of Arabidopsis plants
700 to 900 seeds per plant. Germinate on
kanamycin plates to select transformants. 10 to
20 transformed plants per plant.
10 day old seedlings
26
MiniTi T-DNA based vector for plants
Disarmed vectors do not produce tumors can be
used to regenerate normal plants containing the
foreign gene.
1. Binary vector the vir genes required for
mobilization and transfer to the plant reside on
a modified pTi. 2. consists of the right and left
border sequences, a selectable marker (kanomycin
resistance) and a polylinker for insertion of a
foreign gene.
miniTi
27
MiniTi T-DNA based vector for plants
a binary vector system
T-DNA deleted
kanr
polylinker

LB
RB
modified Ti plasmid
bom
vir
ori
miniTi
oriV
bom basis of mobilization
28
Alternate Methods of Transforming Plants
Particle Bombardment
29
  • One way of physically introducing DNA into cells
    is with a particlegun.              
  • Very tiny DNA-coated metal particles are
    suspended in a drop on a macroprojectile.
  • A discharge (from a gunpowder explosion or from
    breakage of a membrane enclosing a pressurized
    chamber) impels the macroprojectile.
  • The macroprojectile is stopped by a stopping
    plate, but the microprojectiles continue into the
    tissue below.
  • The DNA introduced with the particles is
    expressed

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31
Particle Bombardment using the Gene Gun
  1. DNA- or RNA-coated gold/tungsten particles are
    loaded into the gun and you pull the trigger.
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