Download Plant Biotechnology and GMOs

Plant Biotechnology and
GMOs
Chapter 14
(Plus other bits and bobs)
Plant Biotechnology
• “For centuries, humankind has made improvements
to crop plants through selective breeding and
hybridization — the controlled pollination of plants.
• Plant biotechnology is an extension of this
traditional plant breeding with one very important
difference —
– plant biotechnology allows for the transfer of a
greater variety of genetic information in a more
precise, controlled manner.”
•
Increasing
crop
yields
Figure
11.13
To feed the increasing
population we have to increase
crop yields.
• Fertilizers - are compounds to
promote growth; usually applied
either via the soil, for uptake
by plant roots, or by uptake
through leaves. Can be organic
or inorganic
• Have caused many problems!!
• Algal blooms pollute lakes near
areas of agriculture
Increasing
crop
yields
Figure 11.13
• Algal blooms - a relatively rapid
increase in the population of
(usually) phytoplankton algae in
an aquatic system.
• Causes the death of fish and
disruption to the whole
ecosystem of the lake.
• International regulations has led
to a reduction in the
occurrences of these blooms.
Chemical
pest
control
Figure 11.17
• Each year, 30% of crops are lost to insects and other crop
pests.
• The insects leave larva, which damage the plants further.
• Fungi damage or kill a further 25% of crop plants each year.
• Any substance that kills organisms that we consider
undesirable are known as a pesticide.
• An ideal pesticide would:–
–
–
–
Kill only the target species
Have no effect on the non-target species
Avoid the development of resistance
Breakdown to harmless compounds after a short time
Chemical
pest
control
Figure 11.17
• DDT was first developed in the 1930s
• Very expensive, toxic to both harmful and
beneficial species alike.
• Over 400 insect species are now DDT
resistant.
• As with fertilizers, there are run-off
problems.
• Affects the food pyramid.
– Persist in the environment
•
Chemical
pest
control
Figure
11.18
DDT persists in the food chain.
• It concentrates in fish and fisheating birds.
• Interfere with calcium
metabolism, causing a thinning in
the eggs laid by the birds –
break before incubation is
finished – decrease in population.
• Although DDT is now banned, it
is still used in some parts of the
world.
Plant Biotechnology
• The use of living cells to make products
such as pharmaceuticals, foods, and
beverages
• The use of organisms such as bacteria to
protect the environment
• The use of DNA science for the production
of products, diagnostics, and research
Genetically modified crops
• All plant characteristics, such as size, texture, and
sweetness, are determined on the genetic level.
•
•
•
•
•
•
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!!!)
• Mode of action is to inhibit
an enzyme involved in the
synthesis of the aromatic amino
acids:
• tyrosine,
• tryptophan
• phenylalanine
Glyphosate
Glycine
Glyphosate - N-(phosphonomethyl) glycine
• It does this by inhibiting
the enzyme 5-enolpyruvylshikimate3-phosphate synthase (EPSPS),
which catalyzes the reaction
of shikimate-3-phosphate (S3P)
and phosphoenol pyruvate to form
5-enolpyruvyl-shikimate-3phosphate (ESP).
• ESP
subsequently dephosphorylated to
chorismate, an essential precursor
in plants for these aromatic amino
acids.
Glyphosate
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
Makes Protein
PROMOTER INTRON 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
Makes Protein
PROMOTER INTRON CODING SEQUENCE
Plant Transgene
Plant Selectable
Marker Gene
bacterial genes
•antibiotic marker
•replication origin
Plasmid DNA
Construct
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 T-region 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
for
transfer
to the
plant
vir genes
pTi
tra
bacterial
conjugation
opine catabolism
oriV
Agrobacterium tumafaciens senses
Acetosyringone via a 3-component-like
system
3 components:
ChvE,
VirA,
VirG
1. ChvE
periplasmic protein binds to sugars, arabinose,
glucose
binds to VirA periplasmic domain
 amplifies the signal
VirA
Periplasmic domain
acetosyringone
Transmitter
Inhibitory domain
sugars
ChvE
receiver
VirG
DNAbinding
2. VirA : Receptor kinase
1. Membrane protein five functional domains:
a) Periplasmic binds ChvE-sugar complex does NOT bind
acetosyringone
b) Transmembrane domain
c) Linker region BINDS acetosyringone NOTE this is on the
cytoplasmic side!
VirA
Periplasmic domain
acetosyringone
Transmitter
Inhibitory domain
sugars
ChvE
receiver
VirG
DNAbinding
2. VirA : Receptor kinase
d) Transmitter domain (His) auto- phosphorylates and then
transfers to the response regulator protein VirG
e) Inhibitory domain  will bleed off the phosphate from the
His in the transmitter domain (to an Asp)
VirA
Periplasmic domain
acetosyringone
Transmitter
Inhibitory domain
sugars
ChvE
receiver
VirG
DNAbinding
3. VirG : Response Regulator
a) Receiver domain that is phosphorylated on an Asp residue by
the His on the transmitter domain of VirA
b) Activates the DNA binding domain to promote transcription
from Vir-box continaing promoter sequences (on the Ti
plasmid)
VirA
Periplasmic domain
acetosyringone
Transmitter
Inhibitory domain
sugars
ChvE
receiver
VirG
DNAbinding
sugars
VirA
Periplasmic domain
ChvE
receiver
acetosyringone
Transmitter
Inhibitory domain
VirG
DNAbinding
Agrobacterium
can be used to
transfer DNA
into plants
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 TDNA; still produced tumors.
Shuttle plasmid
E. coli
conjugation
pTi
Agrobacterium
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
miniTi
for insertion of a foreign
gene.
MiniTi T-DNA based vector for plants
a binary vector system
kanr
polylinker
LB
11
RB
ori
T-DNA deleted
bom
modified Ti plasmid
vir
miniTi
bom = basis of mobilization
oriV
2
Transfer of miniTi from E. coli to
Agrobacterium tumefaciens
pRK2013;
kan resistance
tra
modified pTi
bom
E. coli
contains tra genes
ColE1 ori
miniTi;
kan resistance
bom site for
mobilization
15A ori;
E. coli or Agrobact.
Triparental mating:
Agrobacterium
str resistant
Ti oriV
Steps in the mating 1-2:
pRK2013;
miniTi;
E. coli
kan resistance
kan resistance
11
tra
contains tra genes
ColE1 ori
Triparental mating:
tra
bom
22
Helper plasmid
(pRK2013) mobilizes
itself into 2nd E. coli
strain containing miniTi.
Steps in the mating 2-3:
E. coli
Agrobacterium
pRK2013
pRK2013
pTi
miniTi
miniTi;
kan resistance
miniTi
2
pRK2013
can not
replicate.
33
Helper plasmid mobilizes itself and the miniTi
into Agrobacterium.
Selection of Agrobacterium containing
the miniTi on str r/kan plates
miniTi;
pRK2013;
modified pTi
kan resistance kan resistance
tra
can not replicate
bom
str r
miniTi
pRK2013
kanr
plate on str and kan media
pTi
str r
Agrobacterium
str resistant
Summary
Agrobacteria are biological vectors for
introduction of genes into plants.
•Agrobacteria attach to plant cell
surfaces at wound sites.
•The plant releases wound signal
compounds, such as acetosyringone.
•The signal binds to virA on the
Agrobacterium membrane.
•VirA with signal bound activates virG.
Summary
•Activated virG turns on other vir genes,
including vir D and E.
•vir D cuts at a specific site in the Ti
plasmid (tumor-inducing), the left border.
The left border and a similar sequence, the
right border, delineate the T-DNA, the DNA
that will be transferred from the bacterium
to the plant cell
•Single stranded T-DNA is bound by vir E
product as the DNA unwinds from the vir D
cut site. Binding and unwinding stop at the
right border.
Summary
•The T-DNA is transferred to the
plant cell, where it integrates in
nuclear DNA.
•T-DNA codes for proteins that
produce hormones and opines.
Hormones encourage growth of the
transformed plant tissue. Opines feed
bacteria a carbon and nitrogen source.
Overview of the Infection Process
And then?.......
• What is the last step?..........................
Tissue culture
The basics!
What is Plant Tissue Culture?
Of all the terms which have been applied to the process,
"micropropagation" is the term which best conveys the
message of the tissue culture technique most widely in use
today.
The prefix "micro" generally refers to the small size of the
tissue taken for propagation, but could equally refer to the
size of the plants which are produced as a result.
Relies on two plant hormones
Auxin
Cytokinin
Protoplast to Plant
• Callus: Induced by
• 2, 4 dichlorphenoxyacetic
acid (2,4D)
• Unorganized, growing mass
of cells
• Dedifferentiation of explant
– Loosely arranged thinned
walled, outgrowths
– No predictable site of
organization or
differentiation
Protoplast to Plant
• 2, 4 dichlorphenoxyacetic acid
(2,4D)
• Stops synthesis of cellulose
• Knocks out every other rosette
• Makes b 1,3 linked glucose
– Callose
• Temporarily alters the cell wall
Auxin (indoleacetic acid)
Produced in apical and root meristems, young
leaves, seeds in developing fruits
• cell elongation and expansion
• suppression of lateral bud growth
• initiation of adventitious roots
• stimulation of abscission (young fruits) or
delay of abscission
• hormone implicated in tropisms (photo-, gravi, thigmo-)
Cytokinin (zeatin, ZR,
IPA)
Produced in root meristems, young leaves,
fruits, seeds
• cell division factor
• stimulates adventitious bud
formation
• delays senescence
• promotes some stages of root
development
Organogenesis
The formation of organs
from a callus
• Rule of thumb:
Auxin/cytokinin 10:1100:1 induces roots.
• 1:10-1:100 induces
shoots
• Intermediate ratios
around 1:1 favor
callus growth.
Edible Vaccines
Transgenic Plants Serving Human Health Needs
• Works like any vaccine
• A transgenic plant with a pathogen protein gene is
developed
• Potato, banana, and tomato are targets
• Humans eat the plant
• The body produces antibodies against pathogen protein
• Humans are “immunized” against the pathogen
• Examples:
Diarrhea
Hepatitis B
Measles
The End!
Any Questions?