Methods of producing
transgenic plants
Part II
Plant Biotechnology
?Traditional
crossbreeding
Recombinant
DNA techniques
http://www.insp.mx/xcongreso/ponencias/5
The results of traditional Agbiotech
are not that bad,
but they are time-consuming
teosinte
cob
corn
John Doebley photo
Wild tomato
(Lycopersicon pimpinellifolium )
D= 1 cm
www.quirkyworks.com/design/ Samples/plant-breeding.jpg
Hybrid plants
(heterosis)
Agricultural Biotechnology
Crossbreeding Recombinant DNA
Species Related Related/unrelated
Efficiency
Susceptibility to
external gene
influences
Genes shuffled in one
exepriment
10s of 1000s 1 – few
Random insertion + +
Insecti and herbicides
Fertilizers
Pollution/run off
Potential toxins/risk + +
http://www.insp.mx/xcongreso/ponencias/5
Introducing genes into plants
1) infecting plant cells with plasmids
as vectors carrying the desired gene;
2) shooting microscopic pellets
containing the gene directly into the cell.
Genetic engineering of plants with
Agrobacterium tumefaciens
? A,tumefaciens,used
extensively for genetic
engineering of plants,
? Contains T-DNA
(bacterial plasmid)
? Genes colud be
integrated into the plant
chromosomes when
the T-DNA is
transferred,
http://courses.washington.edu/z490/gmo/natural.html
Tumor induced by
A,tumefaciens
A.Tumefaciens gall is not a tiny thing
Biology of A,tumefaciens
www-genvagar.slu.se/teknik/ djup/plasm.htm
Well known to induce crown gall tumor
A.tumefaciens lives around
root surfaces (in rhizosphere)
where it using nutrients
that leak from the root tissues
infects only through wound sites
and actively chemotactic to them
Plant wound produces
acetosyringone
Bacterial T-plasmid produces
receptors for acetosyringone
The basis of Agrobacterium-
mediated genetic engineering
? T-DNA of A,tumefaciens is excised and integrates into
the plant genome as part of the natural infection
process,
? Any foreign DNA inserted into the T-DNA will also be
integrated,
1,Cytokinins
(plant hormone for cell plant division and tumorous growth)
2,Enzymes for indoleacetic acid (auxin) synthesis
Another plant hormone (inducing stem and leaf elongation,inducing parthenocarpy and
preventing aging)
3,Enzymes for synthesis and release of novel plant
metabolites,
the opines (uniques amino acid derivatives)
the agrocinopines (phosphorylated sugar derivatives),
Opines and agrocinopines are NUTRIENTS for A.tumefacies,
They can not be used by other bacterial species
It provides unique niche for A.tumefaciens
Important genes encoded by
Ti plasmid
Nopaline
Cytokinins are plant hormones that are
derivatives of the purine adenine.
Cell specific expression of cytokinin
in the A.tumefaciens infected cell
Zeatin is one of cytokinines which synthesis
may be encoded by Ti plamid
isolated from corn (Zea mays),
Opines are nutrients that are also
for quorum sensing
The plant cells start to secrete
the opines
from transferred bacterial T DNA
opine diffuses
into the surrounding cells
and serves as a signal molecules
for the conjugation
of the agrobacterium
(Quorum sensing)
Ti Plasmid
Tumor-
producing
genes
Virulence region
Opine catabolism
ORI
T-DNA
region
DNA between
L and R borders is
transferred to plant
as ssDNA;
T-DNA encoded
genes can be
substituted by
target genes
Ti-plasmid based vectors
Binary systems Co-integrated vectors
Needs 2 vectors,Needs 3 vectors
Disarmed Ti plasmid
with gene of interest
(no vir genes)
Helper vector
for infection
(with vir genes)
Disarmed Ti plasmid
capable for infection
Intermediate vector
with T-region
and gene of interest
(transferred by conjugation)
Form
co-integrated plasmid
after homologous
recombination on T-DNA
Helper vector
for transfer of
intermediate plasmid into A.tum
Co-integrated vectors
(hybrid ti-plasmids)
DISADVANTAGES:
1) Long homologies required between the Ti plasmid
and the E,coli plasmids (pBR322 based Intermediate vectors)
making them difficult to engineer and use
2) Relatively inefficient gene transfer compared to the binary vecto
Right now
rarely used
Ti plasmid vector systems
are often working as binary vectors
Virulence
region
T DNA region removed
ori for A,tum
Gene of interest
Plant selectable marker
Bacterial selectable
marker
ori for A,tumefaciensori for E.coli
HELPER
plasmidDisarmed Ti
plasmid
DISADVANTAGE,Depending on the orientation,
plasmids with two different origins of replication may be unstable in E,coli
ADVANTAGE,small vectors are used,which increases transfer efficiency
from E,coli to Agrobacterium,
No intermolecular recombination is needed
Promoters used for expression in
transgenic plants
35S,cauliflower mosaic virus 35S promoter
CaMV 35S is a strong promoter
that is active
in essentially all dicot plant tissues.
CaMV is a circular dsDNA genome virus
Procedure for creation a transgenic
plant
1,Both plasmids are transfected into A.tumefaciens
2,Plant cell culture is infected with A.tumefaciens
3,Products of Vir genes excised gene of interest within T-DNA
and transfer it to plant chromosome
Polylinker Kan-resistance geneT-DNA Repeat T-DNA Repeat
Gene of interest
4,Plant cells are selected on kanamycine
5,Presence of transgene confirmed by PCR
6,Whole plant could be grown from transformed cells !!!
Callus
specialized plant tissues that form
over a wound;
cork cambium may form
and the cells produced
will gradually seal the wound
Callous cells
are easiest cells to transform
(as their cell wall is very thin)
Protoplasts (no cells wall)
is even easier to transfrom,
but it is very difficult
to grow something viable from it,
Callus nay be produced by manipulation
with external concentration of plant hormones
Major Plant Hormones
Auxins Cytokinins
Structurally related to adenine
auxin < CYTOKININ? shoots
AUXIN > cytokinin ? roots
auxin = cytokinin ?undifferentiated callus
Produced by actively growing tissues
particularly roots,embryos,and fruits
Natural auxin is indoleacetic acid (IAA)
Apical meristem is the major site
of auxin synthesis
Produce callus ? transform callus ?
stimulate shooting by cytokinin addition
Biology of Plants,Raven et,al.,Freeman Worth Publishing,1999
+ cytokinin
This procedure is easy
for dicotyledone plants
(legumes etc)
Monocotyledones are not easy to handle –
callus is very difficult to be initiated,and
A.tumefaciens is not pathogenic for them
1,Pericarp sholud be pulled back and
the immature embryos (0.5 - 1.0 mm) are removed,
2,The immature embryos
are placed on
a callus induction medium
high osmotic media
prepare calli
for transfomation
plantsciences.montana.edu/,../transform1.htm
Transformation
is performed
by gene gun method
DNA with desired gene and antibiotic resistance
is coated onto the surface of gold particles.
plantsciences.montana.edu/,../transform1.htm
vacuum chamber
Calli are placed
in vacuum chamber,
Helium pressure
shot DNA into cells
Gene gun Coating gold
particles with DNA
Calli remain
on the high osmotic media
for 20 hours
following shooting,
Closer look on (“gene gun”)
After shooting calli are placed on a selective
media containing a herbicide for three weeks.
Then calli are transferred to a media
to induce the production of shoots,
After they form small shoots,
they are transferred to
DARKER containers on a root induction media,
plantsciences.montana.edu/,../transform1.htm
The small plantlets are transplanted into soil
and acclimated under high humidity conditions
With current procedures only 10-20% of the plants are actually transgenic,
so they should be tested on transgene expression
plantsciences.montana.edu/,../transform1.htm
transgenic plants
Part II
Plant Biotechnology
?Traditional
crossbreeding
Recombinant
DNA techniques
http://www.insp.mx/xcongreso/ponencias/5
The results of traditional Agbiotech
are not that bad,
but they are time-consuming
teosinte
cob
corn
John Doebley photo
Wild tomato
(Lycopersicon pimpinellifolium )
D= 1 cm
www.quirkyworks.com/design/ Samples/plant-breeding.jpg
Hybrid plants
(heterosis)
Agricultural Biotechnology
Crossbreeding Recombinant DNA
Species Related Related/unrelated
Efficiency
Susceptibility to
external gene
influences
Genes shuffled in one
exepriment
10s of 1000s 1 – few
Random insertion + +
Insecti and herbicides
Fertilizers
Pollution/run off
Potential toxins/risk + +
http://www.insp.mx/xcongreso/ponencias/5
Introducing genes into plants
1) infecting plant cells with plasmids
as vectors carrying the desired gene;
2) shooting microscopic pellets
containing the gene directly into the cell.
Genetic engineering of plants with
Agrobacterium tumefaciens
? A,tumefaciens,used
extensively for genetic
engineering of plants,
? Contains T-DNA
(bacterial plasmid)
? Genes colud be
integrated into the plant
chromosomes when
the T-DNA is
transferred,
http://courses.washington.edu/z490/gmo/natural.html
Tumor induced by
A,tumefaciens
A.Tumefaciens gall is not a tiny thing
Biology of A,tumefaciens
www-genvagar.slu.se/teknik/ djup/plasm.htm
Well known to induce crown gall tumor
A.tumefaciens lives around
root surfaces (in rhizosphere)
where it using nutrients
that leak from the root tissues
infects only through wound sites
and actively chemotactic to them
Plant wound produces
acetosyringone
Bacterial T-plasmid produces
receptors for acetosyringone
The basis of Agrobacterium-
mediated genetic engineering
? T-DNA of A,tumefaciens is excised and integrates into
the plant genome as part of the natural infection
process,
? Any foreign DNA inserted into the T-DNA will also be
integrated,
1,Cytokinins
(plant hormone for cell plant division and tumorous growth)
2,Enzymes for indoleacetic acid (auxin) synthesis
Another plant hormone (inducing stem and leaf elongation,inducing parthenocarpy and
preventing aging)
3,Enzymes for synthesis and release of novel plant
metabolites,
the opines (uniques amino acid derivatives)
the agrocinopines (phosphorylated sugar derivatives),
Opines and agrocinopines are NUTRIENTS for A.tumefacies,
They can not be used by other bacterial species
It provides unique niche for A.tumefaciens
Important genes encoded by
Ti plasmid
Nopaline
Cytokinins are plant hormones that are
derivatives of the purine adenine.
Cell specific expression of cytokinin
in the A.tumefaciens infected cell
Zeatin is one of cytokinines which synthesis
may be encoded by Ti plamid
isolated from corn (Zea mays),
Opines are nutrients that are also
for quorum sensing
The plant cells start to secrete
the opines
from transferred bacterial T DNA
opine diffuses
into the surrounding cells
and serves as a signal molecules
for the conjugation
of the agrobacterium
(Quorum sensing)
Ti Plasmid
Tumor-
producing
genes
Virulence region
Opine catabolism
ORI
T-DNA
region
DNA between
L and R borders is
transferred to plant
as ssDNA;
T-DNA encoded
genes can be
substituted by
target genes
Ti-plasmid based vectors
Binary systems Co-integrated vectors
Needs 2 vectors,Needs 3 vectors
Disarmed Ti plasmid
with gene of interest
(no vir genes)
Helper vector
for infection
(with vir genes)
Disarmed Ti plasmid
capable for infection
Intermediate vector
with T-region
and gene of interest
(transferred by conjugation)
Form
co-integrated plasmid
after homologous
recombination on T-DNA
Helper vector
for transfer of
intermediate plasmid into A.tum
Co-integrated vectors
(hybrid ti-plasmids)
DISADVANTAGES:
1) Long homologies required between the Ti plasmid
and the E,coli plasmids (pBR322 based Intermediate vectors)
making them difficult to engineer and use
2) Relatively inefficient gene transfer compared to the binary vecto
Right now
rarely used
Ti plasmid vector systems
are often working as binary vectors
Virulence
region
T DNA region removed
ori for A,tum
Gene of interest
Plant selectable marker
Bacterial selectable
marker
ori for A,tumefaciensori for E.coli
HELPER
plasmidDisarmed Ti
plasmid
DISADVANTAGE,Depending on the orientation,
plasmids with two different origins of replication may be unstable in E,coli
ADVANTAGE,small vectors are used,which increases transfer efficiency
from E,coli to Agrobacterium,
No intermolecular recombination is needed
Promoters used for expression in
transgenic plants
35S,cauliflower mosaic virus 35S promoter
CaMV 35S is a strong promoter
that is active
in essentially all dicot plant tissues.
CaMV is a circular dsDNA genome virus
Procedure for creation a transgenic
plant
1,Both plasmids are transfected into A.tumefaciens
2,Plant cell culture is infected with A.tumefaciens
3,Products of Vir genes excised gene of interest within T-DNA
and transfer it to plant chromosome
Polylinker Kan-resistance geneT-DNA Repeat T-DNA Repeat
Gene of interest
4,Plant cells are selected on kanamycine
5,Presence of transgene confirmed by PCR
6,Whole plant could be grown from transformed cells !!!
Callus
specialized plant tissues that form
over a wound;
cork cambium may form
and the cells produced
will gradually seal the wound
Callous cells
are easiest cells to transform
(as their cell wall is very thin)
Protoplasts (no cells wall)
is even easier to transfrom,
but it is very difficult
to grow something viable from it,
Callus nay be produced by manipulation
with external concentration of plant hormones
Major Plant Hormones
Auxins Cytokinins
Structurally related to adenine
auxin < CYTOKININ? shoots
AUXIN > cytokinin ? roots
auxin = cytokinin ?undifferentiated callus
Produced by actively growing tissues
particularly roots,embryos,and fruits
Natural auxin is indoleacetic acid (IAA)
Apical meristem is the major site
of auxin synthesis
Produce callus ? transform callus ?
stimulate shooting by cytokinin addition
Biology of Plants,Raven et,al.,Freeman Worth Publishing,1999
+ cytokinin
This procedure is easy
for dicotyledone plants
(legumes etc)
Monocotyledones are not easy to handle –
callus is very difficult to be initiated,and
A.tumefaciens is not pathogenic for them
1,Pericarp sholud be pulled back and
the immature embryos (0.5 - 1.0 mm) are removed,
2,The immature embryos
are placed on
a callus induction medium
high osmotic media
prepare calli
for transfomation
plantsciences.montana.edu/,../transform1.htm
Transformation
is performed
by gene gun method
DNA with desired gene and antibiotic resistance
is coated onto the surface of gold particles.
plantsciences.montana.edu/,../transform1.htm
vacuum chamber
Calli are placed
in vacuum chamber,
Helium pressure
shot DNA into cells
Gene gun Coating gold
particles with DNA
Calli remain
on the high osmotic media
for 20 hours
following shooting,
Closer look on (“gene gun”)
After shooting calli are placed on a selective
media containing a herbicide for three weeks.
Then calli are transferred to a media
to induce the production of shoots,
After they form small shoots,
they are transferred to
DARKER containers on a root induction media,
plantsciences.montana.edu/,../transform1.htm
The small plantlets are transplanted into soil
and acclimated under high humidity conditions
With current procedures only 10-20% of the plants are actually transgenic,
so they should be tested on transgene expression
plantsciences.montana.edu/,../transform1.htm
