Plant transformation

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Plant transformation
Introduction of individual gene(s) of interest
into plant genome Genetic modification with or
without integration May include regeneration
step Expected phenotypes to be analyzed
(lab/field) Stable inheritance in the offspring
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Plant transformation
Position effect from random integration of
transgene differential gene expression endogeno
us gene transgene different phenotypes from a
transformation
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Target gene
DNA insert from the same or different
species Designed to work under controlled
conditions require specific promoter for
activation of gene expression
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Gene expression control
Under the control of promoter Target gene to
express all the time / certain stage / in
response to signal whole plant / some
tissues Weak / Strong promoter determine amount
of gp
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Genetic marker
Detection / Selection of transformed plant Marker
gene bacteria or plant origin Often linked to
the transgene (same cassette) Confer a phenotype
to transformed cell/tissue Selectable /
Screenable marker
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Selectable marker
Antibiotic / Herbicide resistance
gene Transformed plants survive / grow
better under selection pressure For selection /
segregation ratio based on ability to grow on
selective agent

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Screenable marker
GUS / LUX / CAT / lacZ genes (gpenzyme) Transform
ed plants screened for phenotype
by histochemical staining fluorimetric
assay For selection / gene expression study
(level / pattern)

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Transformant selection
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Plant transformation
Transient transformation easy, rapid and
convenient approach for transient assay no
integration / similar to bacterial plasmid Stable
transformation integration of foreign DNA in
host genome stably maintained after cell division

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Transient transformation
Gene expression assay in protoplast eg. study of
functional domain of promoter deleted
promotermarker transformation with fusion
constructs phenotypic analysis

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Transient transformation
Leaf protoplast most common explant developmenta
l stages vary different response /
expression Protoplast transformation electroporat
ion polyvalent cation PEG or PLO

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Transient transformation
Electroporation electric shock to open the
membrane DNA uptake / membrane seal Polyvalent
cation activate DNA uptake by endocytosis
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Electroporation
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ElectroporatorCuvette
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Endocytosis
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Stable transformation
Stable integration of transgene into plant
chromosome Transgene / genetic marker replication
and function in plant cells Transgenic plant by
regeneration transgene inherited and active

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Stable transformation
DNA-mediated gene transfer polyvalent cation /
electroporation / microinjection Integration
during replication Homologous recombination of
plant gene/foreign DNA Protoplast as
explant Difficult for cereal / legume

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Microinjection
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Stable transformation
Particle gun mediated gene transfer to overcome
limitation of protoplast regeneration more
developed explants eg. embryogenic tissue DNA
travel through several cell layers DNA-coated
particles penetrating by force

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Stable transformation
Particle gun mediated gene transfer gold or
tungsten microparticles a.k.a. gene gun or
biolistics used with all plant species

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Stable transformation
Agrobacterium mediated gene transfer reliable
and versatile method neoplasia-causing soil
bacteria Agrobacterium natural ability to
transfer DNA into plant

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Stable transformation
Agrobacterium mediated gene transfer bacterial
plasmid as transforming vector A. tumefaciens
Ti plasmid crown gall disease A. rhizogenes
Ri plasmid hairy root disease

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Agrobacterium infection
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Agrobacterium T-DNA
Agrobacterium mediated gene transfer plasmid DNA
to be transferred T-DNA flanked by 25-bp
imperfect direct repeats left border and right
border mediating proteins Vir ABCDEG

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Agrobacterium Ti-plasmid
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Agrobacterium T-DNA
Natural T-DNA genes Changes in differentiation
and development of transformed plant
cells Neoplastic phenotype by disrupting hormone
balance Ti increase auxin / cytokinin
synthesis Ri alter hormone response induce
root differentiation

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Agrobacterium T-DNA
Natural T-DNA genes Enzymes for opines synthesis
and secretion nopaline / octopine /
agrocinopine amino acid / sugar derivatives as
a source of carbon and nitrogen

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Natural DNA transfer process
Wounded plant cells release phenolic
compounds acetosyringone to trigger following
steps chemotaxis of bacteria to plant
cells binding of bacteria to plant cell
wall induction of virulence/vir gene expression

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Natural DNA transfer process
Vir A constitutively expressed transmembrane
sensing protein In response to phenolic
compound Autophosphorylation of Vir A
Phosphorylated Vir A to activate Vir G
protein Chemotaxis of bacteria

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Natural DNA transfer process
Phosphorylated Vir G as transcriptional
activator of remaining Vir loci Vir C to bind a
sequence at right border Vir D2 strand-specific
ss endonuclease to initiate nick at bottom strand

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Natural DNA transfer process
VirE2 ssDNA-binding protein to protect T-strand
from nucleases Vir B as bacterial membrane
protein to transfer DNA from bacteria to plant
cell

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Natural DNA transfer process
Other chromosomal genes att, chvA, chvB and
pscA for tight binding of bacteria to plant
cells cel formation of cellulose fibrils for
bacterial aggregation

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Agrobacterium for Transformation
Recognition site for DNA transfer direct repeats
on T-DNA border T-DNA genes no role in DNA
transfer/integration removal with no effect Vir
genes function in trans fashion

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Agrobacterium for Transformation
Modification of Ti plasmid binary
vector cointegrative vector

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Binary vector
Split Ti plasmid to 2 molecules separate
transgene and vir genes Subclone target DNA in a
binary vector MCR / selectable marker / RB /
LB ori active both in E. coli and
Agrobacterium Transgene to replicate in E. coli
and Agrobacterium

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Binary vector
Vir genes already on a T-DNA-deleted Ti
plasmid for expression in Agrobacterium E. coli
culture to check construct and multiplication Agro
bacterium culture for plant transformation

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Cointegrative vector
Deletion derivative of Ti plasmid Replacing T-DNA
with defined DNA sequence Require helper or
intermediate vector for subcloning of DNA insert
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Cointegrative vector
Intermediate vector MCR for insert
cloning Selectable markers Sequence homologous
to defined sequence in cointegrative
vector Replication (ori) only in E. coli
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Cointegrative vector
Transform Agrobacterium with intermediate
vector DNA transfer from intermediate vector to
cointegrative vector by homologous recombination
Then Agrobacterium culture for plant
transformation

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Gene transfer method
With transgene-containing Agrobacterium Protoplas
t cocultivation Tissue explant inoculation Seed
imbibition In planta transformation
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Agrobacterium transformation
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Explant inoculation
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Regeneration of transgenics
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Application of transgenic plants
To define specific sequence (promoter)
for directing differential expression To express
foreign genes and determine or analyze
phenotypes of interest To engineer crop plants
for useful agronomic traits
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Examples of transgenic crops
Most well known transgenics Roundup Ready
glyphosate tolerant crops Bt crops Cry toxin of
Bacillus thuringiensis Other traits etc.
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Herbicide tolerant plant
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Bt BT toxin
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BT plant
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Safety assessment of transgenics
Evaluation prior to release Environmental
safety / Food safety Information on transgenic
development GM plant information (plant and
product) Safety assessment / Regulatory
decision Regulatory approval management
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Safety assessment of transgenics
Criteria
Host organism / Donor organism Modification
process Molecular characterization Genetic
stability of introduced trait Expressed material
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Safety assessment of transgenics
Criteria
Gene transfer to related plant Horizontal gene
transfer Weediness potential Secondary and
non-target traits Insect resistance
management Others