Plant Biotechnology and GMOs

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Plant Biotechnology and GMOs

• Chapter 14
• (Plus other bits and bobs)

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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.

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Figure 11.13
Increasing crop yields

• 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

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Figure 11.13
Increasing crop yields

• 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.

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Figure 11.17
Chemical pest control

• 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

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Figure 11.17
Chemical pest control

• 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

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Figure 11.18
Chemical pest control

• DDT persists in the food chain.
• It concentrates in fish and fish-eating 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.

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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

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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

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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-geneti
cally-modified-food-by-pcr/
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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.

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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

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Glyphosate - N-(phosphonomethyl) glycine

• It does this by inhibiting the enzyme 5-enolpyruvy
  lshikimate-3-phosphate synthase (EPSPS),
  which catalyzes the reaction of shikimate-3-phosph
  ate (S3P) and phosphoenol pyruvate to form
  5-enolpyruvyl-shikimate-3-phosphate (ESP).
• ESP subsequently dephosphorylated to chorismate,
  an essential precursor in plants for these
   aromatic amino acids.

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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

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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

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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.

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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)

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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.

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Genetically modified crops

• Need to build in a
• Promoter
• Stop signal

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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

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Building the Transgenes
ON/OFF Switch
Makes Protein
stop sign
Plant Transgene
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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.

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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.

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So, how do you get the DNA into the Plant?
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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!

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Particle Bombardment
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Particle Bombardment

• Particle-Gun Bombardment
• 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).

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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.
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Particle Bombardment
The DNA sometimes was incorporated into the
nuclear genome of the plant Gene has to be
incorporated into cells DNA where it will be
transcribed Also inserted gene must not break
up some other necessary gene sequence
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Agrobacterium tumefaciens
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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!!!

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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

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Overview of the Infection Process
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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.
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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.
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Agrobacterium chromosomal DNA
pscA
chvA
chvB
T-DNA-inserts into plant genome
tra
bacterial conjugation
for transfer to the plant
pTi
vir genes
opine catabolism
oriV
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Agrobacterium tumafaciens senses Acetosyringone
via a 3-component-like system
3 components ChvE, VirA, VirG
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1. ChvE

• periplasmic protein binds to sugars, arabinose,
  glucose
• binds to VirA periplasmic domain
• ? amplifies the signal

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2. VirA Receptor kinase

• 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!

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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)
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3. VirG Response Regulator

• 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)

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(No Transcript)
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Agrobacterium can be used to transfer DNA into
plants
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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
T-DNA still produced tumors.
pTi
Shuttle plasmid
conjugation
E. coli
Agrobacterium
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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 for insertion of a
foreign gene.
miniTi
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MiniTi T-DNA based vector for plants
a binary vector system
T-DNA deleted
kanr

polylinker
LB
RB
modified Ti plasmid
bom
vir
1
ori
miniTi
2
2
oriV
bom basis of mobilization
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Transfer of miniTi from E. coli to Agrobacterium
tumefaciens
miniTi kan resistance
pRK2013 kan resistance
modified pTi
E. coli
Agrobacteriumstr resistant
contains tra genes
bom site for mobilization
ColE1 ori
Ti oriV
15A ori E. coli or Agrobact.
Triparental mating
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Steps in the mating 1-2
pRK2013 kan resistance
miniTi kan resistance
E. coli
1
contains tra genes
2
ColE1 ori
Helper plasmid (pRK2013) mobilizes itself into
2nd E. coli strain containing miniTi.
Triparental mating
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Steps in the mating 2-3
E. coli
Agrobacterium
pRK2013
pRK2013
pTi
miniTi
2
miniTi kan resistance
3
pRK2013 can not replicate.
Helper plasmid mobilizes itself and the miniTi
into Agrobacterium.
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Selection of Agrobacterium containing the miniTi
on str r/kan plates
miniTi kan resistance
pRK2013 kan resistance
modified pTi
tra
bom
str r
can not replicate
miniTi
str r
pTi
pRK2013
kanr
Agrobacterium str resistant
plate on str and kan media
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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.

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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.

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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.

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Overview of the Infection Process
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And then?.......

• What is the last step?..........................
• Tissue culture
• The basics!

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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
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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

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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

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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-)

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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

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Organogenesis

• The formation of organs from a callus
• Rule of thumb Auxin/cytokinin 101-1001
  induces roots.
• 110-1100 induces shoots
• Intermediate ratios around 11 favor callus
  growth.

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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

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The End!

• Any Questions?