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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
Plant transformation
Position effect from random integration of transgene
differential gene expression
endogenous gene
transgene
different phenotypes from a transformation
Target gene
DNA insert from the same or different species
Designed to work under controlled conditions
require specific promoter
for activation of gene expression
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
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
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
Screenable marker
GUS / LUX / CAT / lacZ genes (gp=enzyme)
Transformed plants screened for phenotype by
histochemical staining
fluorimetric assay
For selection / gene expression study (level / pattern)
Transformant selection
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
Transient transformation
Gene expression assay in protoplast
eg. study of functional domain of promoter
deleted promoter:marker
transformation with fusion constructs
phenotypic analysis
Transient transformation
Leaf protoplast: most common explant
developmental stages vary
different response / expression
Protoplast transformation
electroporation
polyvalent cation: PEG or PLO
Transient transformation
Electroporation
electric shock to open the membrane
DNA uptake / membrane seal
Polyvalent cation
activate DNA uptake by endocytosis
Electroporation
Electroporator&Cuvette
Endocytosis
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
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
Microinjection
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
Stable transformation
Particle gun mediated gene transfer
gold or tungsten microparticles
a.k.a. gene gun or biolistics
used with all plant species
Stable transformation
Agrobacterium mediated gene transfer
reliable and versatile method
neoplasia-causing soil bacteria: Agrobacterium
natural ability to transfer DNA into plant
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
Agrobacterium infection
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
Agrobacterium Ti-plasmid
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
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
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
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
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
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
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
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
Agrobacterium for Transformation
Modification of Ti plasmid
binary vector
cointegrative vector
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
Binary vector
Vir genes already on a T-DNA-deleted Ti plasmid
for expression in Agrobacterium
E. coli culture to check construct and multiplication
Agrobacterium culture for plant transformation
Cointegrative vector
Deletion derivative of Ti plasmid
Replacing T-DNA with defined DNA sequence
Require helper or intermediate vector
for subcloning of DNA insert
Cointegrative vector
Intermediate vector
MCR for insert cloning
Selectable markers
Sequence homologous to defined sequence
in cointegrative vector
Replication (ori) only in E. coli
Cointegrative vector
Transform Agrobacterium with intermediate vector
DNA transfer from intermediate vector
to cointegrative vector
by homologous recombination
Then Agrobacterium culture for plant transformation
Gene transfer method
With transgene-containing Agrobacterium
Protoplast cocultivation
Tissue explant inoculation
Seed imbibition
In planta transformation
Agrobacterium transformation
Explant inoculation
Regeneration of transgenics
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
Examples of transgenic crops
Most well known transgenics
Roundup Ready: glyphosate tolerant crops
Bt crops: Cry toxin of Bacillus thuringiensis
Other traits: etc.
Herbicide tolerant plant
Bt & BT toxin
BT plant
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
Safety assessment of transgenics
Criteria
Host organism / Donor organism
Modification process
Molecular characterization
Genetic stability of introduced trait
Expressed material
Safety assessment of transgenics
Criteria
Gene transfer to related plant
Horizontal gene transfer
Weediness potential
Secondary and non-target traits
Insect resistance management
Others