Transgenic Plants and Plant Biotechnology

Presented by

Amith ReddyEastern 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 yieldIncrease Resistance to pests and diseasesDrought tolerance

Disadvantages of Traditional breeding:Long processLot of man powerLimited 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 cultureTissue 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 CellSmall 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 PlantsIn 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 plantsWhy 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 PlantsAgrobacterium 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 withA. 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 PlasmidsThe 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 PlantAgrobacterium 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 ChromosomesAfter 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 TissuesPlant 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 P1The 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 PathwayThe 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 PlantsThe 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 ToxinBacterial 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 PathwaysTwo 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