TRANSGENIC PLANTS

Methods used in Transgenic Plants

Agrobacterium-mediated Transformation
Microinjection
Direct Transformation
Application of Genetic Engineering in Agriculture
Engineering Herbicide Resistance
Engineering Stress Resistance
Engineering Nutritional Quality
Conventional Breeding vs Transgenic Plants
TRANSGENIC PLANTS -INTRODUCTION
Terminology
Transgene: a gene or genetic material that has been
transformed naturally or by any number of genetic
engineering techniques from one organism to another

Transgesis: the process of introducing an exogenous gene

called transgene into a living organisms so that the
organism will exhibit a new property and transmit that
property to its offspring

Transgenic Plants: Plants which expresses the characters

coded by the transgene as a result of insertion of a foreign
gene into a specific plant
TRANSGENIC PLANTS -INTRODUCTION
Difference between Transgenic Technology and Traditional
Breeding

Transgenic Technology Traditional Breeding

Transform gene from any Move genes only between
source e.g. animals, members of a particular
bacteria, virus etc genus of plants
METHODS USED IN TRANSGENIC PLANTS
The process of regenerating a plant from a single
cell may cause three types of alterations:

1. Temporary Physiological Change

2. Epigenentic Change
3. True Genetic Change

An entire plant can be regenerated from a single cell,

small samples of tissue or even a single plant cells may be
cultured in vitro. Under appropriate conditions, these
may regenerate into complete plants
METHODS USED IN TRANSGENIC PLANTS
Gene Transfer in Plants
 Why?

1. Crop Improvement
2. Disease Resistance
3. Stress Tolerance
4. Improved Performance
5. Value-added traits

Basic Studies
1. Gene Expression
2. Reverse Genetics – understanding functioning
of unknown genes
3. Biochemistry and metabolism
METHODS USED IN TRANSGENIC PLANTS
 Gene transfer strategies: Systems and Vectors

1. Agro bacterium
2. Direct DNA uptake
3. Virus-based vectors
METHODS USED IN TRANSGENIC PLANTS
 Plant Transformation with Ti Plasmid of
Agrobacterium tumesfaciens

 A. tumefaciens is a gram-negative soil bacterium

which naturally transforms plant cells, resulting
in crown gall (cancer). tumors.
 Tumor formation is the result of transfer ,
integration and expression of genes on a specific
segment of A. tumefaciens plasmid DNA called T-
DNA (transferred DNA).
 The T-DNA resides on a large plasmic called the
Ti (tumor including) plasmid found in A.
tumefaciens
METHODS USED IN TRANSGENIC PLANTS

 Agrobacterium-mediated gene transfer

 The keys
 To make a segment of DNA that contains a selectable marker and
a gene of interest to look like a T-DNA.
 To get this “T-DNA” into an Agrobacterium cell so that it can
mobilized by the ir genes.
 To produce and find transformed plant cells that can be
regenerated into normal, fertile plants.
 Requirements
 A transfer cassette bounded by functioning border
 Ways to get this cassette into Agrobacterium
 Disbarment of plasmids that retain the retain functional genes
METHODS USED IN TRANSGENIC PLANTS
 METHODS USED IN TRANSGENIC PLANTS
 The Infection Process
Wounded plant cells releases
phenolics and nutrients

Phenolics and nutrients

causes chemotaxis
response of A. tumefaciens
Certain phenolics e.g. acetosyringone, Attachment of the
hydroxyacetosyringone induce virus bacteria to plant cell
gene transcription and allow for T-DNA
transfer and integration into chrosomal DNA.

Transcription and translation of the T-DNA in the plant cell to produce opines (food) and tumors
(housing) for the bacteria

The opine permease/

catabolism gene on the
Ti plasmid allow A. tumefaciens use
opines as a C, H, O and N source.
METHODS USED IN TRANSGENIC PLANTS
 The Ti plasmid of A. tumafaciens and the transfer
of its T-DNA to the plant nucleus genome
METHODS USED IN TRANSGENIC PLANTS
METHODS USED IN TRANSGENIC PLANTS
METHODS USED IN TRANSGENIC PLANTS
 Plant Tissue Culture
METHODS USED IN TRANSGENIC PLANTS
 Transfer of Modified Ti Plasmid into a Plant

Agrobacterium carrying Ti plasmid is added to plant tissue growing in

culture. The T-DNA carries an antibiotic resistance gene (necomycin) to
allow the selection of successfully transformed plant cells. Both callus
cultures (A) and liquid cultures (B) may be used in this procedure.
METHODS USED IN TRANSGENIC PLANTS
 Plant Cell DNA-delivery Methods
Method Comment
Ti Plasmid-mediated gene Excellent and highly effective, but
transfer limited to dicots
Microprojectile Bombardment Easy and Effective, used with a wide
range of plants
Viral Vectors Not very effective

Direct Gene transfer into Only certain protoplasts can be

plant protoplast regenerated into whole plants
Microinjection Tedious and Slow

Electroporation Limited to protoplasts that can be

regenerated into whole plants
Liposome Fusion Limited to protoplasts that can be
regenerated into whole plants
PARTICLE BOMBARDMENT TECHNOLOGY
 Works with all types of plants.
 DNA is carried on microscopic metal particle.
 Fired by a gun into plant tissue.

Method
 DNA coated on microscopic gold beeds.
 Beeds are placed at the end of a plastic bullet.
 Blast of helium used to project them.
 Plastic meshwork stop is used to stop the bullet.
 Alternative method is by strong electrical discharge.
 Amount of penetration into tissue can be changed.
PARTICLE BOMBARDMENT TECHNOLOGY

 Beeds enter the cytoplasm or nucleus of the cell.

 DNA is free and recombine with chromosomal DNA.
 Leaf transferred to selection media for cell to grow.
 Only cells with selectable marker grow other die.
 Transformed plants are regenerated using tissue
culture techniques.
 Screened for gene of interest.
DETECTION OF INSERTED DNA

 Use of selectable marker or reporter gene.

 Widely used reported gene is not (neomycin
phosphotranserse.
 In activates Ab neomycin by attaching a
phosphate group.
 Cells with Ab are not killed but other cells which
do not integrate with DNA.
LUCIFERASE
 Includes a reporter gene coding for luciferase.
 Luciferase provides light with its substrate luciferin.
 Found in luminous creatures.
 Gene coding Eukaryotes is luc and in Prokaryotes is lux
 Different chemical nature

ATP + O2
Luciferin oxidized
 Use scintillation counter to view light.

Advantages
1. Not stable for long.
2. Active protein is directly proportional to level of gene expression.
3. Used to test activity of a specific promoter.
4. Used to test activity of a specific promoter.
e.g. cab promoter controls the expression of the luc gene, and
luciferase is only made when this promoter is turned on in the
plants.
 LUCIFERASE AS A REPORTER IN PLANT
TISSUES

Plant tissue carrying the luc gene for firefly luciferase emits blue light when
provided with the substrate luciferin.
A – a leaf disk is viewed by a photocell detector.
B – the luc gene in a seedling is expressed under control of an inducible
promoter.
CRE/LOXP SYSTEM
 C re: Cause recombination
 Found in Bacteriophage
 Cre protein is a recombinase enzyme
 Recognizes 34 base pair DNA seq
(loxP site)
 Catalyses recombination between
two loxP sites.
 Placing loxP on either side of DNA,
the enclosed region may be detected by Cre recombination.
 Cre gene is also included in the transgenic construct.
 This approach allows selected market genes to be removed fro
plant DNA after use
 Cre genes can be added by cross pollination.
PLANT BREEDING AND TESTING
 HERBICIDE RESISTANCE
Herbicides: Chemical agents that destroys
plants or inhibits their growth e.g.
Glyphosate
 Amino aid phosphate derivative of
Glysine
 Environmental friendly
 Breaks down into non toxic compounds
 Kills plants by blocking the synthetic
pathway of Aromatic amino acid
 Inhibits the enzyme EPSPS (5-
enolpyruvoylshikimate-3-phosphaate
syntheses
 EPSPS ia product of aroA gene in
chloroplast.
 EPSPS is found in plants, fungi,
bacteria.
 Not found in animals and humans.
WHAT SCIENTISTS DID?
 INSECT RESISTANCE
Insecticides: Chemicals used to kill insects
 Very costly, hazardous procedures

 More toxic to humans

 Similar biochemical pathway in insects and humans

 Naturally occurring insecticides are only harmful to

insects.
e.g. Toxin from Bacillus thurgienis (Bt toxin)
 This toxin is used to prevent

1. Cotton boll worm – destroy cotton

2. European corn borer – destroy corn
DIFFERENT CRY PROTEINS PRODUCED BY
BACILLUS

Cry I : kills Butterflies and Moths

Cry II : kills Butterflies and Flies
Cry III : kills Beetles
Cry IV : kills only flies
USE OF TRANSGENIC TECHNOLOGY
Toxic gene ----- insect into tomato plant ----- showed partial
protection
Plant made only low levels of toxin. Why???
The toxin gene is from bacterium and is designed to express well
in bacteria and not in plants
So here genes expressed in different host cell.
 Use of codon is a problem.
 Several different codons encode the same amino acid.
 Different orgs favour codons of same amino acid.
 Have different levels of corresponding tRNA.
Thus …..
 Insect toxin gene was altered by changing many bases of third
position of the redundant codons.
 20% of its bases were altered to make it more plantlike in codon
usage.
STRESS TOLERANCE
FUNCTIONAL GENOMICS IN PLANTS

Strategies are following screening of entire genome.

Most rely on removal and blockage of gene expression.
Methods to know the function of plant genes are:
1. Insertions
2. Gene silencing
3. Fast Neutron Mutagenesis
4. TILLING (Target Induced Local Lesions in Genomes)
INSERTIONS
GENE SILENCING
 Method to find function of new genes.
 Done by RNAi
 RNAi is triggered by doubled stranded RNA, which is cut into
short segments (siRNA)
RISC Enzyme Complex

Indentify homologous RNA

Cut

Prevents expression of mRNA to protein

FAST NEUTRON MUTAGENESIS
METHODS USED IN TRANSGENIC PLANTS
 Plant Tissue Culture
METHODS USED IN TRANSGENIC PLANTS
 Plant Tissue Culture (Tilling)
 Target induced local lesions in Genomes.
 First seed are soaked in chemical mutagens (EMS) ----induce G/C
and A/T transitions in DNA
 M1 seeds  grown into plants M2 seeds  Plants  DNA is
harvested and pooled into large mega pool and smaller pool.
 PCR primers used to amplify the selected regions f DNA
 PRC products are labeled with two different labels.
 Heteroduplexex of mutants and wild type DNA are created.
 If PCR product is cleaved by CEL-1, it will have one fluorescent
label.
 Uncleaved DNA will both fluorescent labels.
 Digested mutant gene strands can be identified by gel
electrophoresis.
IDENTIFYING POINT MUTATIONS WITH
TILLINGS

TILLING identifies point mutations in a library of plant DNA.

EMS, a chemical mutagens, induces point mutations in seeds.
The M1 seeds are grown into plant and the M2 seeds are
harvested. Most M2 seeds are stored as a stock, while the
remaining M2 seeds are grown into plants. DNA is harvested
from each plant and pooled. The larger pools contain DNAs
from all the M2 plants, and the smaller pools contain DNA
from one or two different M2 plants.
IDENTIFYING POINT MUTATIONS WITH
TILLINGS

Point mutations are identified in the DNA pools using PCR to

randomly amplify different areas of the plants genomes. Some
PCR products will contain point mutations and others will be
normal. These are denatured and annealed so that some of the
normal and mutant strands form hybrids. The reannealed PCR
products are labeled at each end with a different fluorescent tag.
The PCR products are then digested with the enzyme CEL-1,
which cuts only where the helix has a mismatched. This leaves
any mutants normal hybrids with a single fluorescent tag.
FOOD SAFETY AND STARLINK CORN
BT TOXIN AND BUTTERFLIES
WHY ALTER PLANTS?
CONVENTIONAL BREEDING VS TRANSGENIC
PLANTS
Conventional Breeding
1. Limited to exchanges between
the same or very closely related
species
2. Little or no guarantee of any
particular gene combination
from the million of crosses
generated
3. Undesirable genes can be
transferred along with
desirable genes
4. Takes a long time to achieve
desired results
Transgenic Plants
1. Allows the direct transfer of
one or just a few genes,
between either closely or
distantly related organisms
2. Crop improvement can be
achieved in a shorter time
compared to conventional
breeding
3. Allows plants to be modified by
removing or switching off
particular genes