Transgenic Plants and Plant Biotechnology

Presented by

Amith Reddy
Eastern New Mexico University
Terms to know

Transgene: It is a gene or genetic material that has been transferred naturally or by
any of a number of genetic engineering techniques from one organism to another.

Transgesis : The process of introducing an exogenous gene called a transgene into
a living organisms so that the organism will exhibit a new property and transmit
that property to its offspring.

Transgenic Plants : The plants which expresses the characters coded by the
transgene are called Transgenic plants.

Selective Breeding used in the History

Genetics studies started with Mendel

Cross pollination : Pollen from one plant to stigma

of another plant.

Found dominate characteristics in plants

Uses of Traditional Breeding:
Increase crop yield
Increase Resistance to pests and diseases
Drought tolerance

Disadvantages of Traditional breeding:
Long process
Lot of man power
Limited possibility of improved traits.
The Reproductive Organs of a Typical
Plant : Pollen grains are the male
reproductive cells of the plant. They are
made in the anther (orange), the top
portion of the stamen. The female
reproductive cells, the ova, are
sequestered in the ovary. Pollen reaches
the ova via the stigma, which is attached
to the ovary by the pistil.
History of Plant Breeding
Mutation Breeding

Treat seeds with mutagens or expose to X rays or gamma rays.

Disadvantages

Less predictable results

Lot of man power

Successful in the flower world. Eg; new colours, more petals.

Seeds
UV Treatment or Mutagens
Killed Alive
Planted
Tested for Improvements
Found desirable traits
Test for Progeny
heritable Sold to Markets
Transgenic plant : Insertion of a foreign gene into a specific plant.

Difference between Trangenic Technology and traditional Breeding:

Trangenic Technology : Transform gene from any source.

Eg: animals, bacteria, virus etc

Traditional Breeding : Move genes only between members of a particular

genus of plants.
Plant Tissue Culture

Totipotency : Ability of a cell to divide into any type of cell.

Explant : Mass of tissue or cells

Solid medium Callus culture.
Tissue can be immature embryo, apical meristem, root tip

Liquid medium suspension culture
Tissue should be protoplast (cells with no cell wall), micro or macrospores.

Nutrients and hormones are used for growth and development.
Eg : 2,4 dichlorophenoxyacetic acid (analogous to auxin)

Callus : Undifferentiated cell which form a crystalline white layer on solid medium.

I. Move callus to other medium with reduced hormones which allows shoot to
develop.
II. Move the callus to other medium with no hormone which allows root hairs to
grow.
The process of regenerating a plant from a single cell may cause three types of

alterations,

1. Temporary Physiological change

2. Epigenetic change

3. True genetic changes
An Entire Plant Can Be Regenerated from a Single Cell
Small samples of tissue, or even single plant cells may
be cultured in vitro. Under appropriate conditions, these
may regenerate into complete plants.
Callus or Liquid Culture of Plant Cells Can Regenerate Entire Plants
In callus culture a mass of undifferentiated cells grows on a solid surface. In liquid culture,
separated single cells are grown. Both types of cultures can develop shoots and roots with
appropriate manipulation of plant hormone levels.
FIGURE 14.3
Plant Tissue culture
Gene transfer in plants
Why gene transfer?

Crop improvement
Disease resistance
Stress tolerance
Improved performance
Value-added traits

Basic studies

Gene expression
Reverse genetics - understanding functioning of unknown genes
Biochemistry and metabolism

Gene transfer strategies: Systems and vectors

Agro bacterium
Direct DNA uptake
Virus-based vectors
Plant transformation with the Ti plasmid of Agrobacterium tumefaciens
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 the transfer, integration and expression of
genes on a specific segment of A. tumefaciens plasmid DNA called the T-
DNA (transferred DNA)
The T-DNA resides on a large plasmid called the Ti (tumor inducing)
plasmid found in A.tumefaciens
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 be mobilized
by the vir genes
To produce and find transformed plant cells that can be regenerated into
normal, fertile plants
Requirements
A transfer cassette bounded by functioning borders
Ways to get this cassette into Agrobacterium
Disarmed Ti plasmids that retain functional vir genes
Advantages

Technically simple
Yields relatively uncomplicated insertion events (low copy number,
minimal rearrangements)
Unlimited size of foreign DNA
Efficient (for most plants)
Adaptable to different cell types, culture procedures (protoplasts, tissue
sections, non-culture methods)
Transformants are mitotically and meiotically stable

Disadvantages

Host range is limited: not all plants may be susceptible to Agrobacterium
With susceptible plants, accessible culture/regeneration systems must be
adaptable to Agrobacterium-mediated gene transfer
The Infection process
Wounded plant cell releases phenolics and nutrients.
Phenolics and nutrients cause chemotaxic response of A. tumefaciens
Attachment of the bacteria to the plant cell.
Certain phenolics (e.g., acetosyringone, hydroxyacetosyringone)
induce vir gene transcription and allow for T-DNA transfer and
integration into plant chromosomal 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 genes on the Ti plasmid allow A.
tumefaciens to use opines as a C, H, O, and N source.

Agrobacterium Transfers Plasmid DNA into Infected Plants
Agrobacterium carrying a Ti plasmid is attracted by acetosyringone to a wounded plant stem. The
Ti plasmid is cut by endonucleases to release single-stranded T-DNA, which is covered with
protective proteins, and transported into the plant cell through a conjugation-like mechanism. The
T-DNA enters the plant nucleus where it integrates into plant chromosomal DNA.
FIGURE 14.4
The Ti plasmid of Agrobacterium tumafaciens and the
transfer of its T-DNA to the plant nuclear genome
Agrobacterium tumefeciens
Infection of a plant with
A. tumefaciens and
formation of crown galls
Crown Gall on
Tobacco
Clone YFG (your favorite gene) or the
target gene in the small T-DNA plasmid
in E. coli, isolate the plasmid and use it
to transform A. tumefaciens containing
the disarmed Ti plasmid

Essential Elements for Carrying a Transgene on Ti Plasmids
The T-DNA segment contains both a transgene and a selective marker or reporter gene. These
have separate promoters and termination signals. The marker or reporter gene must be expressed
all the time, whereas the transgene is often expressed only in certain tissues or under certain
circumstances and usually has a promoter that can be induced by appropriate signals.
Ti plasmid
structure & function
22
Transfer of Modified Ti Plasmid into a Plant
Agrobacterium carrying a Ti plasmid is added to plant tissue growing in culture. The T-DNA
carries an antibiotic resistance gene (neomycin in this figure) to allow selection of successfully
transformed plant cells. Both callus cultures (A) and liquid cultures (B) may be used in this
procedure.
FIGURE 14.6
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.
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.



DNA Carried on Microscopic Gold Particles Can
Integrate into Plant Chromosomes
After penetrating the cell, the DNA unwinds from around
the gold carrier particle. Some of the DNA enters the
nucleus and is successful in integrating into the plant
chromosomes.
Particle Bombardment Technology
Detection of Inserted DNA


Use of selectable marker or reporter gene.

Widely used reporter gene is npt (neomycin phosphotransferase)

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
Include 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.

Luciferin oxidized

Use scintillation counter to view light.

Advantages :
Not stable for long.
Active protein is directly proportional to level of gene expression.
Used to test activity of a specific promoter.
Eg : cab promoter controls the expression of the luc gene, and luciferase is only
made when this promoter is turned on in the plant.
ATP + O2
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. In (A) a leaf disk is viewed by a photocell detector. In (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 deleted by Cre
recombination.
Cre gene is also included in the transgenic construct.
This approach allows selected marker genes to be removed from the plant DNA
after use.
Cre genes can be added by cross pollination.
This system has proven so easy and useful that every new variety of T.plant
released to the public contains only single trasgene of interest.
Cre/loxP System of Bacteriophage P1
The Cre protein binds to loxP recognition sites in the DNA. Two nearby loxP sites are brought
together, and recombination between them eliminates the intervening DNA. A single loxP site
remains in the target DNA molecule.
Plant Breeding and Testing


Evaluating and testing transformed Transgenic plant is most important.
Event : Each independent case of transgene integration.
The location of the integration affects the expression of transgene.
Transgene with no harmful effects must be moved from exp plant to
higher yield.
Traditional back crossing is used into high yielding varieties.
Back crossing is used for better and higher yield.
This crossing will ensure 98% of genes in the final plant are from the high
yielding variety and 2% are from the original Transgenic plant.
Herbicide Resistance

Herbicides : Chemical agents that destroy plants or inhibits their growth.

Eg : Glyphosate

Amino acid 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-phosphate syntheses)

EPSPS is product of aroA gene in chloroplast.

EPSPS is found in plants, fungi, Bacteria.

Not found in Animals and humans.

Glyphosate Inhibits EPSPS in the Aromatic Pathway
The enzyme 5-enolpyruvoylshikimate-3-phosphate synthase (EPSPS) is the product of the aroA
gene and makes 5-enolpyruvoylshikimate-3-phosphate, a precursor in the pathway to aromatic
amino acids and cofactors. Glyphosate, an analog of phosphoenolpyruvate, inhibits EPSPS.
FIGURE 14.12
Expression of the Agrobacterium aroA Gene in Plants
The bacterial aroA gene must be placed under control of a
promoter active in plants. Correct localization of the AroA
protein (EPSPS) into the chloroplast requires addition of a
chloroplast transit peptide at the N-terminus of the protein.
FIGURE 14.13
What scientists did ?
Found EPSPS resistant gene to Glyphosate in Bacteria

Bacterial terminators and sequences were replaced by plant.

Chloroplast Transit peptide was added.

Transist peptide cleaved off

Only functional enzyme enters the chloroplast.

This Glyphosate resistant gene transfer were carried out in diff crops.

Canola & cotton plants Ti plasmid method

Soybean Gene gun approach.
Insect Resistance

Insecticides : Chemicals used to kills insects

very costly, hazardous procedures.

More toxic to humans

Similar biochemical pathway in insects and humans.

Naturally occurring insecticides are only harmful to insects.

Eg : Toxin from Bacillus thuringienis (Bt toxin)

These toxin is used to prevent

1. cotton boll worm ---- destroy cotton

2. European corn borer ---- destroy corn
Bacillus thuringiensis
Insect Larvae Are Killed by Bt Toxin
Bacterial spores of Bacillus are found on food eaten by the caterpillar. The crystalline
protein is released by digestion of the spore and its breakdown produces a toxin that
kills the insect larvae.
Bacillus ------ Cry proteins ------ Insects eat ------ Cry realases delta endotoxins

(Bt toxin) ------ Toxin binds to intestinal lining ------ holes generated ------ digestive

system disturbed ------ Death of the insects.

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 Trangenic technology

Toxic gene ----- insert 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 favors diff codons of same amino acid.

Have different levels of corresponding t RNA.

Thus.

Insect toxin gene was altered by changing many bases of the third position of the

redundant codon.

20 % of its bases were altered to make it more plantlike in codon usage.
Stress Tolerance

Drought, High salinity are two major problems in growing crop plants.

In drought tolerant plants, fungi, bacteria sugar trehalose protects orgs during stress.

Trehalose

Non reducing storage carbohydrate

Absorbs and release water.
Trehalose Synthetic and Degradative Pathways
Two enzymatic reactions make trehalose. First, trehalose phosphate
synthetase converts UDP-glucose plus glucose 6-phosphate into
trehalose 6-phosphate. Next, trehalose-6-phosphate phosphatase
removes the phosphate to make trehalose. Trehalose may be broken
down into to glucose by trehalase.
Functional genomics in Plants


Functional genomics strategies are followed screening of entire genome

Mostly 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)
Insertion :

Method to find function of new genes.

Transposon or T DNA insertions are used to generate plant mutants.

T DNA includes only a reporter gene.

Clone the upstream and
downstream of the insertion

Insert into plant chromosome. Plant gene identified.



Disrupt a plant gene




Phenotype can be screened and assessed
Gene silencing:

Method to find function of new genes.

Gene silencing is done by RNAi

RNAi is triggered by double stranded RNA, which is cut into short segments (siRNA)

RISC Enzyme complex



Indentify homologous RNA



Cut



Prevents expression of mRNA to protein.
siRNA
Fast Neutron Mutagenesis


Method to generate gene knockouts.

Fast Neutrons are used to induce DNA deletions.

Fast Neutrons are created by nuclear fission.

Hence neutrons of 1 MeV kinetic energy are generated.

These neutrons cause deletions in exposed DNA.

Seeds treated with fast neutron are called M1 seeds.

M1 seeds------Grown into plants (each plant has different deletions)------

M2 seeds------Plants-----Collect DNA into pools of varying sizes.
Identifying Fast Neutron Mutants with PCR
(A) M1 seeds are mutagenized by exposure to fast neutrons. The M2 seeds are grown and DNA harvested from
each plant. The DNA is mixed to form large pools from many M2 seeds and successively smaller pools from
fewer M2 seeds. (B) The seeds are analyzed for deletions using PCR. The primers recognize specific locations
in the plant genome. If the DNA pool contains any deletions, the PCR primer will produce two bands, one from
the wild-type (full-length) gene and one from the plant with the deletion.
FIGURE 14.16
TILLING

Target induced local lesions in Genomes.

First seed are socked 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 megapool and smaller pool.

PCR primers are used to amplify the selected regions of DNA.

PCR 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 have both fluorescent labels.

Digested mutants strands can be identified by gel electrophoresis.
48
Identifying Point Mutations with TILLING

TILLING identifies point mutations in a library of plant DNA. (A) EMS, a chemical mutagen, induces point mutations in seeds.
The M1 seeds are grown into plants 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.

(B) Point mutations are identified in the DNA pools using PCR to randomly amplify different areas of the plant 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 mismatch. This leaves any mutant:
normal hybrids with a single fluorescent tag.
FIGURE 14.17
Food Safety and Starlink Corn

The term transgenic crop is more accurate than genetically modified crop.

Allergenic potential of transgenic crops has caused much controversy.

Starlink an unapproved Transgenic corn was detected in taco shell.

Transgene Cry9C is more resistant to stomach acid.

Transgenic corn was mixed with other corn and sent to markets.

Split approval given by EPA (Environmental Protection Agency)

Sent to markets and allergic reactions were reported by the public.

CDC (Center for Disease Control) examines the blood samples of public and
concluded that allergic reactions was not due to Cry9C protein.

All starlink seed and products were called back from the market.
BT Toxin and Butterflies


Journal Nature suggested that Monarch butterflies were killed by eating
pollen from corn caring Bt gene.

The result of the controversy was a surge of research on transgenic Bt plant
and their effect on the butterflies and non target organisms.

Harmful pollens were Cry1Ab, event # 176

In 2001, this transgene event was no longer approved and was no longer
grown in the United States.

Cry1Ab were only toxic to Monarch caterpillars at density greater than 1000
pollen grain per square centimeter.

Cry1 F and Cry9C showed no toxic effect.

Studies were don in labs where caterpillar had to eat only milkweed
contaminated with pollen.
Thank You