Applications of genetic engineering techniques in agriculture

1. APPLICATIONS OF GENETIC
ENGINEERING
TECHNIQUES IN
AGRICULTURE
SUBMITTED BY
B.DEVADATHA
M.SC BMB
DEPT. OF BMB
PONDICHEERY UNIVERSITY

2. Why genetically engineer plants?
• To improve the agricultural, horticultural or ornamental value of a crop plant
• To serve as a bioreactor for the production of economically important proteins
or metabolites
• To provide a powerful means for studying the action of genes (and gene
products) during development and other biological processes

3. Genetic engineering techniques applied to plants
METHOD SALIENT FEATURES
1.VECTOR MEDIATED GENE TRANSFER
a. Agrobacterium mediated gene
transfer
b. Plant viral vectors
Very efficient but limited to a selected group of plants
Ineffective, hence not widely used
2.DIRECT OR VECTORLESS DNA
TRANSFER
a. Electroporation
b. Microprojectile
c. Liposome fusion
d. Silicon carbide fibres
Mostly confined to protoplasts that can be regenerated to viable plants
Limited use only one cell can be microinjected at a time
Confined to protoplasts that can be regenerated into viable whole
plants
Requires regenerable cell suspensions
3 CHEMICAL METHODS
a. Polyethylene glycol mediated
b.Diethylaminoethyl(DEAE)dextran-
mediated
Confined to protoplasts. Regeneration of fertile plants is frequently
problematical
Does not

4. Agrobacterium tumefaciens as a vector for
transferring foreign genes into plant
chromosome
 Characteristics
 Plant parasite that causes Crown Gall Disease
 Encodes a large (~250kbp) plasmid called Tumor-inducing (Ti) plasmid
 Portion of the Ti plasmid is transferred between bacterial cells and plant cells 
T-DNA (Tumor DNA)
 T-DNT-DNA integrates stably into plant genome
 A ss DNA fragment is converted to dsDNA fragment by plant cell
 Then integrated into plant genome
 2 x 23bp direct repeats play an important role in the excision and integration
process

5. Infection Process
 Vir genes copy T-DNA
 Open channel in bacterial cell membrane for
T-DNA to pass through
 T-DNA enters plant through wound, integrates
itself into plant chromosome

6. Applications of Plant Genetic Engineering
A.Crop Improvement
B.Genetically Engineered Traits: The Big Six
1.Herbicide Resistance
2.Insect Resistance
3.Virus Resistance
4.Altered Oil Content
5.Delayed Fruit Ripening
6.Pollen Control
C.Biotech Revolution: Cold and Drought Tolerance and
Weather-Gard Genes
D.Genetically Engineered Foods
1.Soybeans
2.Corn
3.Cotton
4.Other Crops

7. Herbicide-resistant plants:
Reducing the ability of the herbicide-sensitive target to
bind to the herbicide
• Herbicide: Glyphosate (better known as Roundup)
• Resistance to Roundup (an inhibitor of the enzyme EPSP
involved in aromatic amino acid biosynthesis) was obtained by
finding a mutant version of EPSP from E. coli that does not
bind Roundup and expressing it in plants (soybean, tobacco,
petunia, tomato, potato, and cotton)
• 5-enolpyruvylshikimate-3-phosphate synthase (EPSP) is a
chloroplast enzyme in the shikimate pathway and plays a key
role in the synthesis of aromatic amino acids such as tyrosine
and phenylalanine
• This is a big money maker for Monsanto!

8. insect-resistance plant's
• bt toxin
• cowpea trypsin inhibitor
• proteinase inhibitor ll
• alfa amylase inhibitor
• bacterial chloesterol oxidase
• combination of the above

9.  Anti-Insect Strategy – Insecticides
From Bacillus thuringensis
 Toxic crystals found during sporulation
 Alkaline protein degrades gut wall of lepidopteran
larvae
 Corn borer catepillars
 Cotton bollworm catepillars
 Tobacco hornworm catepillars
 Gypsy moth larvae
Sprayed onto plants – but will wash off

10. Transgenic plants for crop improvements
1. Virus resistant transgenic plants:
a. Pathogen-derived resistance (PDR
b. non-pathogen-derived resistance (non-PDR)
2. Stress resistant transgenic plants.

11. Anti-Viral Strategy
• TMV-coat protein inserted into tobacco and tomato
plant cells using Ti plasmid
• Viral capsids inhibit viral replication of TMV when
infected
 Cucumber mosaic virus (CMV)
• the most important viruses
• more than 800 host plants
• absent the resistance genes in the germplasm
of most crops
 Other trials using capsid proteins: potato
leaf-roll virus, cantaloupe mosaic virus, rice
strip virus

12. Development of stress- and senescence-tolerant plants:
genetic engineering of flavorful tomatoes
Fruit ripening is a natural aging or senescence process that involves two
independent pathways, flavor development and fruit softening.
Typically, tomatoes are picked when they are not very ripe (i.e., hard and
green) to allow for safe shipping of the fruit.
Polygalacturonase is a plant enzyme that degrades pectins in plant cell
walls and contribute to fruit softening.
In order to allow tomatoes to ripen on the vine and still be hard enough for
safe shipping of the fruit, polygalacturonase gene expression was
inhibited by introduction of an antisense polygalacturonase gene and
created the first commercial genetically engineered plant called the
FLAVR SAVR tomato.
Flavor development pathway
Fruit softening pathway
Green Red
Hard Soft
polygalacturonase
antisense polygalacturonase

13. Modification of plant nutritional content:
increasing the vitamin A content of plants
• 124 million children
worldwide are deficient
in vitamin A, which leads
to death and blindness
• Mammals make vitamin A
from -carotene, a
common carotenoid
pigment normally found
in plant photosynthetic
membranes
• Here, the idea was to
engineer the -carotene
pathway into rice
• The transgenic rice is
yellow or golden in color
and is called “golden
rice”
GGPP
Phytoene
Lycopene
-carotene
Vitamin A
Daffodil phytoene synthase gene
Bacterial phytoene desaturase gene
Daffodil lycopene -cyclase gene
Endogenous human gene

14. Antisense technology
• Used to produce the Flavr-Savr tomato in 1994.
• Enzyme polygalacturonase breaks down
structural polysaccharide pectin in wall of a
plant.
• This is part of the natural decay process in a
plant
• Monsanto identified the gene than encodes the
enzyme and made another gene that blocked
the production of the enzyme.

16. Edible Vaccines – Ongoing Research Areas
Hepatitis B
Dental caries - Anti-tooth decay Ab (CaroRxTM)
(anti-Streptococcus mutans)
Autoimmune diabetes
Cholera
Rabies
HIV
Rhinovirus
Foot and Mouth
Enteritis virus
Malaria
Influenza
Cancer

17. Frost Resistance
• Ice-minus bacteria
• Ice nucleation on plant surfaces caused by bacteria that aid in
protein-water coalescence  forming ice crystals @ 0oC (320F)
• Ice-minus Pseudomonas syringae
• Modified by removing genes responsible for crystal formation
• Sprayed onto plants
• Displaces wild type strains
• Protected to 23oF
• Dew freezes beyond this point
• Extends growth season
• First deliberate release experiment – Steven Lindow – 1987- sprayed
potatoes
• Frost Ban
• Different strain of bacteria – Julie Lindemann led different
project – 1987
• Strawberries in California

18. Potentially Harmful Effects
• Contamination by pesticides
• Co-purification of plant chemicals (e.g. nicotine)
• Different glycosylation in plants versus animals
• Interference with normal function of protein in animals
• Stimulation of hypersensitivity reactions in animals (allergies)
• Research is underway to engineer tobacco to synthesize “human-
compatible” glycans

19. Environmental Risks
• Pharmaceutical products may inadvertently be
introduced into the general food supply
• Cross-pollination
• Pollen from a drug-containing crop fertilizes a neighboring
related crop (or wild relatives) used for animal consumption
• Wind
• InsectsConsumption of GM plant by insects  Food
chain
• Accumulation in birds – extinction? (e.g. DDT and bald
eagle)
• Deleterious effects on non-target organisms (NTO’s)
• NTO’s = organisms in the environment that are affected by the
product unintentionally
• Insects, arthropods
• Risk to NTO’s
• Depends on recombinant protein involved
• Risk assessment carried out case-by-case