Plant Biotechnology and GMOs

workkinkajouBiotechnology

Dec 5, 2012 (4 years and 7 months ago)

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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.”

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


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.



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

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

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.

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
-
enolpyruvylshikimate
-
3
-
phosphate synthase

(EPSPS),
which

catalyzes

the reaction
of

shikimate
-
3
-
phosphate

(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.

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

CODING SEQUENCE

INTRON

poly A signal

PROMOTER

ON/OFF Switch

Makes Protein

stop sign

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

CODING SEQUENCE

INTRON

poly A signal

PROMOTER

Building the Transgenes

Plant Transgene

bacterial genes


antibiotic marker


replication origin

Plant Selectable


Marker Gene

Plasmid DNA


Construct

ON/OFF Switch

Makes Protein

stop sign

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
-
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 (CaCl
2
).


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

Particle Bombardment

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

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.

vir

genes

opine catabolism

pTi

tra

for
transfer
to the
plant

bacterial
conjugation

Agrobacterium chromosomal DNA

chvA

chvB

pscA

oriV

T
-
DNA
-
inserts into plant genome

Agrobacterium tumafaciens

senses
Acetosyringone

via a 3
-
component
-
like
system


3 components:

ChvE
,


VirA
,


VirG

Periplasmic domain

acetosyringone

ChvE

VirA

VirG

sugars

Transmitter

Inhibitory domain

receiver

DNA
-
binding

1. ChvE

periplasmic protein binds to sugars, arabinose,
glucose


binds to VirA periplasmic domain



amplifies the signal

Periplasmic domain

acetosyringone

ChvE

VirA

VirG

sugars

Transmitter

Inhibitory domain

receiver

DNA
-
binding

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!


Periplasmic domain

acetosyringone

ChvE

VirA

VirG

sugars

Transmitter

Inhibitory domain

receiver

DNA
-
binding

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)

Periplasmic domain

acetosyringone

ChvE

VirA

VirG

sugars

Transmitter

Inhibitory domain

receiver

DNA
-
binding

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)


Periplasmic domain

acetosyringone

ChvE

VirA

VirG

sugars

Transmitter

Inhibitory domain

receiver

DNA
-
binding

Agrobacterium

can be used to
transfer DNA
into plants

pTi
-
based vectors for plant
transformation:

2. Early shuttle vectors integrated into the T
-
DNA;

still produced tumors.

1.

Shuttle vector is a small E. coli plasmid using
for cloning the foreign gene and transferring to
Agrobacterium.

E. coli

Agrobacterium

pTi

Shuttle plasmid

conjugation

MiniTi T
-
DNA based vector for plants

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.

Disarmed vectors: do not produce tumors; can
be used to regenerate normal plants containing
the foreign gene.

miniTi

MiniTi T
-
DNA based vector for plants



modified Ti plasmid

a binary vector system

oriV

vir

T
-
DNA deleted

LB

RB

ori

kan
r

polylinker

miniTi

bom

bom

= basis of mobilization

2

1

1

2

Transfer of miniTi from
E. coli
to
Agrobacterium tumefaciens

Triparental mating:

bom

site

for
mobilization

miniTi;

kan resistance

E. coli

Agrobacterium
str resistant

pRK2013;

kan resistance

contains

tra

genes

modified pTi

15A ori;

E. coli or Agrobact
.

ColE1

ori

tra

bom

Ti

oriV

Steps in the mating 1
-
2:

Triparental mating:

pRK2013;

kan resistance

contains

tra

genes

tra

ColE1

ori

bom

tra

1

2

E. coli

Helper plasmid
(pRK2013) mobilizes
itself into 2
nd

E. coli

strain containing miniTi
.

miniTi;

kan resistance

1

2

Steps in the mating 2
-
3:

E. coli

miniTi;

kan resistance

Agrobacterium

Helper plasmid mobilizes itself and the miniTi
into
Agrobacterium.

2

miniTi

3

pTi

pRK2013

miniTi

pRK2013
can not
replicate.

pRK2013

2

3

Selection of
Agrobacterium

containing
the miniTi on str r/kan plates

miniTi;

kan resistance

pRK2013;

kan resistance

modified

pTi

Agrobacterium

str resistant

plate on str and kan media

tra

str r

bom

can

not

replicate

pTi

miniTi

pRK2013

kanr

str r

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
.

Summary

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
-
)


cell division factor


stimulates adventitious bud
formation


delays senescence


promotes some stages of root
development

Cytokinin
(zeatin, ZR,
IPA)

Produced in root meristems, young leaves,
fruits, seeds

Organogenesis

The formation of organs
from a callus



Rule of thumb
:
Auxin/cytokinin 10:1
-
100:1

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?