Transcript Dosya İndirin

Chapter 27 Genetic engineering
of plants
Terms to know
Transgene: It is a gene or genetic material that has been transferred naturally or by
any of a number of genetic engineering techniques from one organism to another.
Transgesis : The process of introducing an exogenous gene called a transgene into
a living organisms so that the organism will exhibit a new property and transmit
that property to its offspring.
Transgenic Plants : The plants which expresses the characters coded by the
transgene are called Transgenic plants.
History of Plant Breeding
Selective Breeding used in the History
Genetics studies started with Mendel
Cross pollination : Pollen from one plant to stigma
of another plant.
Found dominate characteristics in plants
Uses of Traditional Breeding:
Increase crop yield
Increase Resistance to pests and diseases
Drought tolerance
Disadvantages of Traditional breeding:
Long process
Lot of man power
Limited possibility of improved traits.
The Reproductive Organs of a Typical
Plant : Pollen grains are the male
reproductive cells of the plant. They are
made in the anther (orange), the top
portion of the stamen. The female
reproductive cells, the ova, are
sequestered in the ovary. Pollen reaches
the ova via the stigma, which is attached
to the ovary by the pistil.
Mutation Breeding
Treat seeds with mutagens or expose to X rays or gamma rays.
Disadvantages
Less predictable results
Lot of man power
Successful in the flower world. Eg; new colours, more petals.
UV Treatment or Mutagens
Seeds
Killed
Alive
Planted
Tested for Improvements
Found desirable traits
Test for Progeny
heritable
Sold to Markets
Transgenic plant : Insertion of a foreign gene into a specific plant.
Difference between Trangenic Technology and traditional Breeding:
Trangenic Technology : Transform gene from any source.
Eg: animals, bacteria, virus etc
Traditional Breeding : Move genes only between members of a particular
genus of plants.
Plant Tissue Culture
Totipotency : Ability of a cell to divide into any type of cell.
Explant : Mass of tissue or cells
Solid medium – Callus culture.
Tissue can be immature embryo, apical meristem, root tip
Liquid medium – suspension culture
Tissue should be protoplast (cells with no cell wall), micro or macrospores.
Nutrients and hormones are used for growth and development.
Eg : 2,4 dichlorophenoxyacetic acid (analogous to auxin)
Callus : Undifferentiated cell which form a crystalline white layer on solid medium.
I. Move callus to other medium with reduced hormones which allows shoot to
develop.
II. Move the callus to other medium with no hormone which allows root hairs to
grow.
The process of regenerating a plant from a single cell may cause three types of
alterations,
1. Temporary Physiological change
2. Epigenetic change
3. True genetic changes
An Entire Plant Can Be Regenerated from a Single Cell
Small samples of tissue, or even single plant cells may
be cultured in vitro. Under appropriate conditions, these
may regenerate into complete plants.
FIGURE 14.3
Callus or Liquid Culture of Plant Cells Can Regenerate Entire Plants
In callus culture a mass of undifferentiated cells grows on a solid surface. In liquid culture,
separated single cells are grown. Both types of cultures can develop shoots and roots with
appropriate manipulation of plant hormone levels.
Gene transfer in plants
Why gene transfer?
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Crop improvement
Disease resistance
Stress tolerance
Improved performance
Value-added traits
Basic studies
• Gene expression
• Reverse genetics - understanding functioning of unknown genes
• Biochemistry and metabolism
Gene transfer strategies: Systems and vectors
• Agro bacterium
• Direct DNA uptake
• Virus-based vectors
Plant transformatIon wIth the Ti plasmid of
AgrobacterIum tumefacIens
 A. tumefaciens is a gram-negative soil bacterium which naturally
transforms plant cells, resulting in crown gall (cancer) tumors
 Tumor formation is the result of the transfer, integration and
expression of genes on a specific segment of A. tumefaciens
plasmid DNA called the T-DNA (transferred DNA)
 The T-DNA resides on a large plasmid called the Ti (tumor
inducing) plasmid found in A.tumefaciens
Agrobacterium-mediated gene
transfer
The keys
• To make a segment of DNA that contains a selectable marker and a gene of
interest to look like a T-DNA
• To get this “T-DNA” into an Agrobacterium cell so that it can be mobilized
by the vir genes
• To produce and find transformed plant cells that can be regenerated into
normal, fertile plants
Requirements
• A transfer cassette bounded by functioning borders
• Ways to get this cassette into Agrobacterium
• Disarmed Ti plasmids that retain functional vir genes
Advantages
• Technically simple
• Yields relatively uncomplicated insertion events (low copy number,
minimal rearrangements)
• Unlimited size of foreign DNA
• Efficient (for most plants)
• Adaptable to different cell types, culture procedures (protoplasts, tissue
sections, “non-culture” methods)
• Transformants are mitotically and meiotically stable
Disadvantages
• Host range is limited: not all plants may be susceptible to Agrobacterium
• With susceptible plants, accessible culture/regeneration systems must be
adaptable to Agrobacterium-mediated gene transfer
The Infection process
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Wounded plant cell releases phenolics and nutrients.
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Phenolics and nutrients cause chemotaxic response of A. tumefaciens
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Attachment of the bacteria to the plant cell.
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Certain phenolics (e.g., acetosyringone, hydroxyacetosyringone)
induce vir gene transcription and allow for T-DNA transfer and
integration into plant chromosomal DNA.
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Transcription and translation of the T-DNA in the plant cell to
produce opines (food) and tumors (housing) for the bacteria.
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The opine permease/catabolism genes on the Ti plasmid allow A.
tumefaciens to use opines as a C, H, O, and N source.
FIGURE 14.4
Agrobacterium Transfers Plasmid DNA into Infected Plants
Agrobacterium carrying a Ti plasmid is attracted by acetosyringone to a wounded plant stem. The
Ti plasmid is cut by endonucleases to release single-stranded T-DNA, which is covered with
protective proteins, and transported into the plant cell through a conjugation-like mechanism. The
T-DNA enters the plant nucleus where it integrates into plant chromosomal DNA.
The Ti plasmıd of Agrobacterıum tumafacıens and the
transfer of ıts T-DNA to the plant nuclear genome
Crown Gall on
Tobacco
Infectıon of a plant wıth
A. tumefacıens and
formatıon of crown galls
Clone YFG (your favorite gene) or the
target gene in the small T-DNA plasmid
in E. coli, isolate the plasmid and use it
to transform A. tumefaciens containing
the disarmed Ti plasmid
Essential Elements for Carrying a Transgene on Ti Plasmids
The T-DNA segment contains both a transgene and a selective marker or reporter gene. These
have separate promoters and termination signals. The marker or reporter gene must be expressed
all the time, whereas the transgene is often expressed only in certain tissues or under certain
circumstances and usually has a promoter that can be induced by appropriate signals.
Ti plasmid
structure & function
FIGURE 14.6
Transfer of Modified Ti Plasmid into a Plant
Agrobacterium carrying a Ti plasmid is added to plant tissue growing in culture. The T-DNA
carries an antibiotic resistance gene (neomycin in this figure) to allow selection of successfully
transformed plant cells. Both callus cultures (A) and liquid cultures (B) may be used in this
procedure.
20
Gene Gun or Biolistic Method
A gene gun is used for injecting cells with
genetic information, it is also known as biolistic
particle delivery system.
Biotechnological Applications of Plant
Breeding
Genetically modified crops
• All plant characteristics, such as size, texture, and sweetness, are
determined on the genetic level.
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Also:
The hardiness of crop plants.
Their drought resistance.
Rate of growth under different soil conditions.
Dependence on fertilizers.
Resistance to various pests and diseases.
• Used to do this by selective breeding
Why would we want to modify an
organism?
• Better crop yield, especially under harsh conditions.
• Herbicide or disease resistance
• Nutrition or pharmaceuticals, vaccine delivery
• “In 2010, approximately 89% of soy and 69% of corn
grown in the U.S. were grown from Roundup Ready®
seed.”
http://www.oercommons.org/courses/detecting-genetically-modified-food-by-pcr/
Roundup Ready Gene
• The glyphosate resistance gene protects food plants against
the broad-spectrum herbicide Glyphosate - N(phosphonomethyl) glycine [Roundup®], which efficiently
kills invasive weeds in the field.
• The major advantages of the "Roundup Ready®” system
include better weed control, reduction of crop injury,
higher yield, and lower environmental impact than
traditional weed control systems.
• Notably, fields treated with Roundup® require less tilling;
this preserves soil fertility by lessening soil run-off and
oxidation.”
Glyphosate - N-(phosphonomethyl) glycine
• An aminophosphonic analogue of the
natural amino acid glycine.
• It is absorbed through foliage and
translocated to actively growing
points. (Meristems!!!)
Glyphosate
• Mode of action is to inhibit
an enzyme involved in the synthesis of
the aromatic amino acids:
• tyrosine,
• tryptophan
• phenylalanine
Glycine
Glyphosate - N-(phosphonomethyl) glycine
• It does this by inhibiting the enzyme 5enolpyruvylshikimate-3-phosphate
synthase (EPSPS), which catalyzes the
reaction of shikimate-3-phosphate (S3P)
and phosphoenol pyruvate to form 5enolpyruvyl-shikimate-3-phosphate
(ESP).
Glyphosate
• ESP subsequently dephosphorylated to
chorismate, an essential precursor in
plants for these aromatic amino acids.
Glycine
Roundup Ready Gene
• Glyphosate functions by occupying
the binding site of the phosphoenol
pyruvate, mimicking an
intermediate state of the enzyme
substrates complex.
• The "Roundup Ready®” system
introduces a stable gene alteration
which prevents Glyphosate binding
and allowing the formation of the
essential aromatic amino acids
Roundup Ready Gene
• The shikimate pathway is not present in animals, which instead
obtain aromatic amino acids from their diet.
• Glyphosate has also been shown to inhibit other plant enzymes
•Also has been found to affect animal enzymes.
•The United States Environmental Protection
Agency considers glyphosate to be relatively low in
toxicity, and without carcinogenic or teratogenic
effects
•However, some farm workers have reported
chemical burns and contact skin burns
Environmental degradation
• When glyphosate comes into contact with the soil, it can be rapidly
bound to soil particles and be inactivated.
• Unbound glyphosate can be degraded by bacteria.
– However, glyphosate has been shown to increase the infection rate of
wheat by fusarium head blight in fields that have been treated with
glyphosate.
• In soils, half-lives vary from as little as 3 days at a site in Texas to
141 days at a site in Iowa.
• In addition, the glyphosate metabolite amino methyl phosphonic
acid has been shown to persist up to 2 years in Swedish forest
soils.
• Glyphosate absorption varies depending on the kind of soil.
Insect Resistance
• B. thuringiensis (commonly known
as 'Bt') is an insecticidal bacterium,
marketed worldwide for control of
many important plant pests - mainly
caterpillars of the Lepidoptera
(butterflies and moths) but also
mosquito larvae, and simuliid
blackflies that vector river blindness
in Africa.
• Bt products represent about 1% of
the total ‘agrochemical’ market
(fungicides, herbicides and
insecticides)
Genetically modified crops
• 1992- The first commercially grown genetically
modified food crop was a tomato - was made more
resistant to rotting, by adding an anti-sense gene
which interfered with the production of the enzyme
polygalacturonase.
– The enzyme polygalacturonase breaks down
part of the plant cell wall, which is what
happens when fruit begins to rot.
Genetically modified crops
• Need to build in a:
• Promoter
• Stop signal
ON/OFF Switch
PROMOTER
INTRON
Makes Protein
CODING SEQUENCE
stop sign
poly A signal
Genetically modified crops
• So to modify a plant:
• Need to know the DNA
sequence of the gene of
interest
• Need to put an easily
identifiable maker gene
near or next to the gene of
interest
• Have to insert both of these
into the plant nuclear
genome
• Good screen process to find
successful insertion
Building the Transgenes
ON/OFF Switch
PROMOTER
INTRON
Makes Protein
CODING SEQUENCE
Plant Transgene
Plant Selectable
Marker Gene
Plasmid DNA
Construct
bacterial genes
•antibiotic marker
•replication origin
stop sign
poly A signal
Cloning into a Plasmid
• The plasmid carrying genes for
antibiotic resistance, and a DNA
strand, which contains the gene
of interest, are both cut with
the same restriction
endonuclease.
• The plasmid is opened up and
the gene is freed from its
parent DNA strand. They have
complementary "sticky ends."
The opened plasmid and the
freed gene are mixed with DNA
ligase, which reforms the two
pieces as recombinant DNA.
Cloning into a Plasmid
• Plasmids + copies of the DNA
fragment produce quantities of
recombinant DNA.
• This recombinant DNA stew is
allowed to transform a bacterial
culture, which is then exposed
to antibiotics.
• All the cells except those which
have been encoded by the
plasmid DNA recombinant are
killed, leaving a cell culture
containing the desired
recombinant DNA.
So, how do you get the DNA
into the Plant?
Meristems Injections
• REMEMBER!!!!!!!
• The tissue in most plants consisting of
undifferentiated cells (meristematic cells),
found in zones of the plant where growth can
take place.
• Meristematic cells are analogous in function
to stem cells in animals, are incompletely or
not differentiated, and are capable of
continued cellular division.
• First method of DNA transfer to a plant.
• Inject DNA into the tip containing the most
undifferentiated cells – more chance of DNA
being incorporated in plant Genome
• Worked about 1 in 10,000 times!
Tunica-Corpus model of the apical
meristem (growing tip). The epidermal
(L1) and subepidermal (L2) layers form
the outer layers called the tunica.
The inner L3 layer is called the corpus.
Cells in the L1 and L2 layers divide in
a sideways fashion which keeps these
layers distinct, while the L3 layer divides in
a more random fashion.
Particle Bombardment
Particle Bombardment
Particle-Gun Bombardment
1. DNA- or RNA-coated
gold/tungsten particles are
loaded into the gun and you
pull the trigger.
Selected DNA sticks to surface
of metal pellets in a salt
solution (CaCl2).
Particle Bombardment
2. A low pressure helium pulse delivers
the coated gold/tungsten particles
into virtually any target cell or tissue.
3. The particles carry the DNA  cells do
not have to be removed from tissue
in order to transform the cells
4. As the cells repair their injuries, they
integrate their DNA into their
genome, thus allowing for the host
cell to transcribe and translate the
transgene.
Particle Bombardment
The DNA sometimes was incorporated
into the nuclear genome of the plant
Gene has to be incorporated into
cell’s DNA where it will be
transcribed
Also inserted gene must not break
up some other necessary gene
sequence
Agrobacterium tumefaciens
Overall process
– Uses the natural infection mechanism of a
plant pathogen
– Agrobacterium tumefaciens naturally infects
the wound sites in dicotyledonous plant
causing the formation of the crown gall
tumors.
– Capable to transfer a particular DNA segment
(T-DNA) of the tumor-inducing (Ti) plasmid
into the nucleus of infected cells where it is
integrated fully into the host genome and
transcribed, causing the crown gall disease.
• So the pathogen inserts the new DNA with great
success!!!
Overall process
• The vir region on the plasmid inserts DNA between the Tregion into plant nuclear genome
• Insert gene of interest and marker in the T-region by
restriction enzymes – the pathogen will then “infect” the
plant material
• Works fantastically well with all dicot plant species
– tomatoes, potatoes, cucumbers, etc
– Does not work as well with monocot plant species - corn
• As Agrobacterium tumefaciens do not naturally infect
monocots
Overview of the Infection Process
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.
3. T-DNA encodes genes for opine synthesis and for
tumor production.
4. occ (opine catabolism) genes carried on the pTi and
allows the bacterium to utilize opines as nutrient.
Agrobacterium chromosomal DNA
pscA
chvA
chvB
T-DNA-inserts into plant genome
tra
for transfer
to the
vir genes
plant
oriV
pTi
bacterial
conjugation
opine catabolism
Agrobacterium tumafaciens senses
Acetosyringone via a 3-component-like
system
3 components:
ChvE,
VirA,
VirG