1. Plant Genetic Engineering

2. 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
Genetic engineering is the process of manipulating and transferring instructions encoded by genes from one cell to another.
This process involves copying of a gene from one living organism (e.g., bacteria, plant, or animal) and adding to another living organism using biotechnology techniques.
Genetic engineering had opened up many possibilities: by adding a new gene into a living organism and thus producing new traits that may be beneficial to people. The examples of plant genetic engineering:
- increasing yields and product quality
- resistant plants to diseases and insects
- medicines and vaccines: e.g., drugs used to treat diabetes (insulin), arthritis, heart disease and others
- producing plastics or other materials traditionally synthesized artificially

3. THE SCIENCE OF GENETIC ENGINEERING
Isolate desired gene for a new trait from any organism
Isolate plasmid DNA
Gene inserted into plasmid.
Introduce modified plasmid into bacterium for replication.
Grow in culture to replicate.

4. DNA of interest is EVERYWHERE
The background is a picture showing a double stranded DNA molecule.
The DNA molecule is found in every living organism:
* ask students to come up with examples where they think they could find DNA
The universal presence of DNA (deoxyribonucleic acid) in the cells of all living organisms makes the genetic engineering possible.

5. 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

6. Requirement Plant transformation getting DNA into a cell
getting it stably integrated
getting a plant back from the cell
Requirement
a suitable transformation method
a means of screening for transformants
an efficient regeneration system
genes/constructs
Vectors Promoter/terminator
reporter genes selectable marker genes
‘genes of interest’

7. Plant Transformation DNA Delivery to Target Cells
Selection and Regeneration
Event Selection

8. Transformation methods
DNA must be introduced into plant cells
Technique
Methods
Indirect
Agrobacterium mediated gene transfer
Viral vector
Direct
Lipid-mediated method,
Calcium-phosphate mediated,
Dextran-mediated,
Electroporation,
Biolistics,
Polybrene,
Laser transfection,
Gene transfection enhanced by elevated temperature.
Method depends on plant type, cost, application

9. 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

10. Agrobacterium tumefaciens10

11. Ti plasmid 23 kb tra pTi ~200 kb vir genes T-DNA bacterial conjugation
for transfer to the plant
opine catabolism

12. 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
Cloning
vector
YFG
T-DNA
Ti plasmid
Disarm
Disarmed
Ti plasmid
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

13. The binary Ti plasmid system

14. Binary vector system

15. Binary vector system

16. 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

17. Agrobacterium tumefaciens
Ti plasmid
Agrobacterium
Genomic DNA
Plant cell
Genomic DNA (carries the gene of interest)
Gene of interest
Empty plasmid
Restriction enzyme A +
Ti plasmid with the gene of interest
Agrobaterium tumefaciens is a bacterium capable of inserting new DNA into the host plant cell upon infection. This ability makes Agrobacterium a natural genetic engineer. It contains a plasmid called the Ti plasmid that carries one or more genes. Once in the host cell, the gene(s) gets integrated into the plant cell’s DNA.
Scientists alter the Ti plasmid to contain the gene they whish to incorporate into the plant cell. Next, the plasmid is reintroduced into the bacterial cell. When the bacterium with the altered Ti plasmid infects a plant cell, the new gene will be incorporated into the plant cell’s genome.
Steps of the process:
the Ti plasmid is removed from the bacterium
using a restriction enzyme (biological scissors) the plasmid is cut at a specific manner
2) a gene of interest is cut out of the DNA of another organism (e.g. a plant cell) using the same restriction enzyme that will cut the DNA at the same manner as the Ti plasmid in the previous step
3) the gene is inserted into the plasmid (producing a recombinant DNA)

18. Agrobacterium tumefaciens
Ti plasmid with the new gene +
Transformation
Plant cell
cell’s DNA
The new gene
Transgenic plant
4) the plasmid is reinserted into Agrobacterium and incubated with plant cells
5) Agrobacterium transfers the new gene into the plant cell’s nucleus and the gene gets incorporated into the plant cell’s genome (DNA)
7) as the plant cell divides subsequent cells receive the new gene
8) a transgenic plant is produced where every cell contains the new gene
A. tumefaciens infects broadleaf plants like tomatoes and soybean.
Cell division

19. 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

20. DNA coated golden particles
“Gene Gun” Technique
DNA coated golden particles
Plant cell
Cell’s DNA
Gene gun
A plant cell with
the new gene
Transgenic plant
The other methods have been developed for the plant species that Agrobacterium does not infect (e.g., corn, rice, wheat, etc.).
The gene gun techniques is known as the biolistic particle delivery system. Gold particles (1mm in diameter) are coated with DNA containing the gene of interest, and are propelled using the gene gun into the host plant tissue.
The particles are able to penetrate the plant cell walls and deliver the DNA inside the cell. Only those cells that incorporate the new gene into their genomic DNA are selected and grown into the full transgenic plants.
Cell division

21. Tearless Onion

22. Colorful Cauliflowers

23. Purple tomatoes

24. Blue Roses

25. 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.

26. 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)

27. 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

28. δ -endotoxin gene (Cry gene) of Bacillus thuriengenesis
GENE FOR Bt TOXIN WAS TRANSFERRED
TO OBTAIN BT TRANSGENIC PLANTS
PLANT SYNTHESIZES INACTIVE PROTOXIN
PROTEINASE
DIGESTION IN
INSECT GUT
MAKES THE
ACTIVE TOXIN
INSECT FEEDS ON
TRANSGENIC PLANT
Toxin binds a receptor on the gut epithelial cells, forms a channel on the membrane. This causes electrolyte leakage and insect death

29. Papaya infected with the papaya ringspot virus
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

30. 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.

31. Flav’r Sav’r tomato “Rot-Resistant Tomato”
Anti-sense gene  complementary to polygalacturonase (PG)
PG = pectinase  accelerates plant decay/rotting

32. 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

33. Gernayl Gernayl diphosphate (GGPP)
Phytoene synthase
Phytoene
Phytoene desaturase
Lycopene
Lycopene cyclase
Beta carotene
Complete biochemical pathway in the rice for production of beta-carotene, a precursor for vitamin A.

34. 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 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.

35. Approved Transgenic plants
Soybean
Corn
Cotton
Oil Seed rape
Sugarbeet
Squash
Tomato
Tobacco
Carnations
Potato
Flax
Papaya
Chicory
Rice
Melon