Transcript Plant Genetic Engineering Genetic Engineering

Plant Genetic Engineering
Genetic Engineering
The process of manipulating and transferring instructions
carried by genes from one cell to another
Why do scientists want to change gene
instructions?
 to produce needed chemicals
 to carry out useful processes
 to give an organism desired characteristics
THE SCIENCE OF GENETIC
ENGINEERING
Isolate desired gene
for a new trait from
any organism
Gene inserted into
plasmid.
Isolate plasmid DNA
Introduce modified plasmid into
bacterium for replication.
Grow in culture to
replicate.
DNA of interest is
EVERYWHERE
Plant Genetic Engineering
Product Concepts and Technical Feasibility
Building the Transformant
Plant Transformation
Selection
Plant Breeding
Seed Production and Marketing
Detection of GMO Crops in the Commodity Chain
Plant transformation
getting DNA into a cell
getting it stably integrated
getting a plant back from the cell
Requirement
1. a suitable transformation method
2. a means of screening for transformants
3. an efficient regeneration system
4. genes/constructs
Vectors
reporter genes
‘genes of interest’
Promoter/terminator
selectable marker genes
Plant Transformation
DNA Delivery to Target Cells
Selection and Regeneration
Event Selection
Transformation methods
DNA must be introduced into plant cells
Technique
Methods
Indirect
1. Agrobacterium mediated gene transfer
2. Viral vector
Direct
1. Lipid-mediated method,
2. Calcium-phosphate mediated,
3. Dextran-mediated,
4. Electroporation,
5. Biolistics,
6.
Polybrene,
7.
Laser transfection,
8.
Gene transfection enhanced by elevated temperature.
Method depends on plant type, cost, application
Agrobacterium tumefaciens
plasmid
• A large (~250kbp) plasmid
called Tumor-inducing (Ti)
plasmid)
• Plasmid contains genes
responsible for the crown
gall disease
• Portion of the Ti plasmid is
transferred between
bacterial cells and plant
cells  T-DNA (Transfer
DNA)
• There are 2 x 23bp direct
repeat border
Agrobacterium tumefaciens
Ti plasmid
T-DNA 23 kb
tra
vir genes
for transfer to the
plant
bacterial
conjugation
pTi
~200 kb
opine catabolism
Genetic engineering of the Ti plasmid
Some considerations:
 Ti plasmid is 200 kb - too big for easy cloning
 need to remove tumor-inducing genes from T-DNA
T-DNA
Disarm
Ti plasmid
YFG
Disarmed
Ti plasmid
Cloning
vector
VIR
genes
Agrobacterium
Remember:
– Only VIR genes are required for T-DNA
transfer
– Only LB and RB are required for T-DNA
insertion into genome
The binary Ti plasmid system
Binary vector system
Binary vector system
Agrobacterium-mediated
transformation
Transformation by the help of agrobacterium
Agrobacterium is a ‘natural genetic engineer’
i.e. it transfers some of its DNA to plants
Agrobacterium tumefaciens
Agrobacterium
Plant cell
Genomic DNA
Genomic DNA
Ti plasmid
(carries the gene of
interest)
Restriction
enzyme A
Restriction
enzyme A
+
Empty
plasmid
Gene of
interest
Ti plasmid with the gene of interest
Agrobacterium tumefaciens
Ti plasmid with the new gene
cell’s
DNA
+
Agrobacterium
Transformation
Plant cell
The new
gene
Transgenic plant
Cell division
Biolistics
 ‘gene gun’
 DNA is coated onto
gold (or tungsten)
particles (inert)
 Gold is propelled by
helium into plant cells
 if DNA goes into the
nucleus it can be
integrated into the plant
chromosomes
 Cells can be
regenerated to whole
plants
“Gene Gun” Technique
DNA coated
golden particles
Gene gun
Cell’s DNA
Plant cell
A plant cell with
the new gene
Transgenic plant
Cell division
Tearless Onion
Colorful Cauliflowers
Purple tomatoes
Blue Roses
Herbicide Resistance
A problem in agriculture is the reduced growth of crops imposed by
the presence of unwanted weeds. Herbicides such as RoundupTM and
Liberty LinkTM are able to kill a wide range of weeds and have the
advantage of breaking down easily. Development of herbicide
resistant crops allows the elimination of surrounding weeds without
harm to the crops.
ROUNDUP (Glyphosate) TOLERANCE
( HERBICIDE TOLERANCE IN CROPS)
Glucose
ROUNDUP
(Glyphosate )
3 phosphoglycerate
Tryptophan
Glycolysis
EPSP SYNTHASE
Phosphoenol
pyruvate
EPSP SYNTHASE
Tyrosine
Phenylalaline
ROUNDUP
(Glyphosate )
In transgenic plant, herbicide can not bind the mutant of EPSP synthase
(Example: RR-Cotton, RR-Soybean)
Insect Resistance
Various insect resistant crops have been produced. Most of
these make use of the Cry gene in the bacteria Bacillus
thuringiensis (Bt); this gene directs the production of a protein
that causes paralysis and death to many insects.
Corn hybrid with a Bt gene
Corn hybrid susceptible to European
corn borer
δ -endotoxin gene (Cry gene) of Bacillus thuriengenesis
GENE FOR Bt TOXIN WAS TRANSFERRED
TO OBTAIN BT TRANSGENIC PLANTS
PLANT SYNTHESIZES INACTIVE PROTOXIN
INSECT FEEDS ON
TRANSGENIC PLANT
PROTEINASE
DIGESTION IN
INSECT GUT
MAKES THE
ACTIVE TOXIN
Toxin binds a receptor on the gut epithelial cells, forms a channel on the
membrane. This causes electrolyte leakage and insect death
Virus Resistant Crops
Papaya infected with the papaya
ringspot virus
Virus resistance gene
introduced
The Freedom II squash has a
modified coat protein that confer
resistance to zucchini yellows
mosaic virus and watermelon
mosaic virus II.
Scientists are now trying to
develop crops with as many as
five virus resistance genes
Delayed Fruit Ripening
 Tomatoes are usually picked and sprayed with the plant
hormone ethylene to induce ripening, although this does
not improve taste
 Tomatoes have been engineered to produce less ethylene
so they can develop more taste before ripening, and
shipment to markets.
 It is produced by blocking the polygalacturonase (PG)
gene, which is involved in spoilage. PG is an enzyme that
breaks down pectin, which is found in plant cell walls.
 Plants were transformed with the anti-sense PG gene,
which is mRNA that base pair with mRNA that the plant
produces, essentially blocking the gene from translation.
Flav’r Sav’r tomato
“Rot-Resistant Tomato”
Anti-sense gene  complementary to polygalacturonase (PG)
PG = pectinase  accelerates plant decay/rotting
Golden Rice
Normal rice
Transgenic technology produced a type of rice that
accumulates beta-carotene in rice grains. Once inside the body,
beta-carotene is converted to vitamin A.
“Normal” rice
“Golden” rice
Gernayl Gernayl diphosphate (GGPP)
Phytoene synthase
Phytoene
Phytoene desaturase
Lycopene
Lycopene cyclase
Beta carotene
Complete biochemical pathway in the rice for production of betacarotene, a precursor for vitamin A.
Edible Vaccines
Edible vaccines are vaccines
produced in plants that can be
administered directly through the
ingestion of plant materials
containing the vaccine. Eating
the plant would then confer
immunity against diseases.
The first human clinical trial took
place in 1997. Vaccine against the
toxin from the bacteria E.coli was
produced in potato. Ingestion of
this transgenic potato resulted in
satisfactory vaccinations and no
adverse effects.
Approved Transgenic
plants








Soybean
Corn
Cotton
Oil Seed rape
Sugarbeet
Squash
Tomato
Tobacco







Carnations
Potato
Flax
Papaya
Chicory
Rice
Melon