Download Plant Biotechnology and GMOs

An Ecological Perspective
(BIOL 346)
Talk Seven:
GMOs
and the Environment
Why GMOs?
• “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.”
Indeed why?
• Hunger, starvation, and malnutrition are endemic in
many parts of the world today.
• Rapid increases in the world population have
intensified these problems!
• ALL of the food we eat comes either directly or
indirectly from plants.
• Can’t just grow more plants, land for cultivation has
geographic limits
– Also, can destroy ecosystems!
Indeed
Figurewhy?
9.1
The Earth is currently experiencing
the most population increase in
Human
history.
2.5 billion in 1955 to 7 billion in 2012
At current rate, will double within
30 years!
Fastest growing nations have growth
rates at or above 4% - this will
double the countries population
every 17 years
•
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/
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
• 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
Genetically modified crops
• 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 - maize
• As Agrobacterium tumefaciens do not naturally
infect monocots
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.
Ti plasmids and the bacterial
chromosome act in concert to
transform the plant
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.
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!!!
Agrobacterium tumafaciens senses
Acetosyringone via a 3-component-like
system
3 components:
ChvE,
VirA,
VirG
The process
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.
The Process
•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.
The Process
•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
Genetically modified crops
• Issues:
• Destroying ecosystems – tomatoes are now
growing in the artic tundra with fish antifreeze
in them!
• Destroying ecosystems – will the toxin now
being produced by pest-resistance stains kill
“friendly” insects such as butterflies.
• Altering nature – should we be swapping genes
between species?
Genetically modified crops
• Issues:
• Vegetarians – what about those
tomatoes?
• Religious dietary laws – anything from a
pig?
• Cross-pollination – producing a superweed
The End!
Any Questions?