Transgenic Development (Plant Genetic Engineering)

Genetic EngineeringGenetic 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

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

THE SCIENCE OF GENETIC THE SCIENCE OF GENETIC ENGINEERINGENGINEERING

getting DNA into a cell

getting it stably integrated

getting a plant back from the cell

Plant transformation

Requirement1.a suitable transformation method

2.2.a means of screening for transformantsa means of screening for transformants

3.an efficient regeneration system

4.genes/constructs vectors Promoter/terminator

reporter genes selectable marker genes ‘genes of interest’

Transformation techniqueTransformation technique

Biological.Biological.• Agrobacterium mediated Agrobacterium mediated

transformation.transformation. Mechanical.Mechanical.

• Particle bombardment.Particle bombardment.• Electroporation.Electroporation.• Microinjection.Microinjection.

Chemical.Chemical.• Polyethylene glycol.Polyethylene glycol.

Transformation methods

DNA must be introduced into plant cells

Indirect Agrobacterium tumefaciens

Direct 1. Microprojectile bombardment

2. Electroporation

3. Microinjection

Method depends on plant type, cost, application

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

Agrobacterium tumefaciens

Ti plasmid

AgrobacteriumGenomic DNA

Plant cellGenomic DNA

(carries the gene the gene of interestof interest)

+

Ti plasmid with the gene of interest

Gene of interest

Empty plasmid

Restriction

enzyme AA

Restriction

enzyme AA

Agrobacterium

Ti plasmid with the new gene

Plant cell

cell’s DNA

Transgenic plant Cell division

The new gene

+ Transformation

Agrobacterium tumefaciens

A. tumefaciens

binary vector

T-DNA

Success FactorSuccess Factor

Species Genotypes

Explant Agrobacterium strains

Plasmid

Direct gene transfer

Introducing gene directly to the target cell

1. Electroporation

2. Microinjection

3. Particle Bombardment

Explants: cells and protoplasts

Most direct way to introduce foreign DNA into the nucleus

Achieved by electromechanically operated devices

Transformation frequency is high

Electroporation

Duracell

DNA containingthe gene of interest

Plant cell

Protoplast

Electroporation Technique

Power supply

DNA inside the plant cell

The plant cell withthe new gene

Most direct way to introduce foreign DNA into the nucleus

Achieved by electromechanically operated devices that control the insertion of fine glass needles into the nuclei of individuals cells, culture induced embryo, protoplast

Labour intensive and slow

Transformation frequency is very high, typically up to ca. 30%

MicroinjectionMicroinjection

Microprojectile bombardment

• uses a ‘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

In the "biolistic" (a cross between biology and ballistics )or "gene gun" method, microscopic gold beads are coated with the gene of interest and shot

into the plant cell with a pulse of helium.

Once inside the cell, the gene comes off the bead and integrates into the cell's genome.

“Gene Gun” TechniqueDNA coated

golden particles

Gene gun

Cell division

A plant cell withthe new gene

Plant cell

Cell’s DNA

Transgenic plant

Model from BioRad: Biorad's Helios Gene

Gun

In Planta Transformation

♣ Meristem transformation

♣ Floral dip method♣ Pollen

transformation

Reason:There are many thousands of cells in a leaf disc or callus clump - only a proportion of these will have taken up the DNA, therefore can get hundreds of

plants back - maybe only 1% will be transformed

Screening technique

Technique which is exploited to screen the transformation product

(transformant Cell)

Screening (selection)

Select at the level of the intact plant Select in culture

• single cell is selection unit• possible to plate up to 1,000,000 cells

on a Petri-dish.• Progressive selection over a number of

phases

Selection StrategiesSelection Strategies PositivePositive Selectable marker geneSelectable marker gene NegativeNegative Selectable marker geneSelectable marker gene VisualVisual Reporter geneReporter gene

Positive selection

Add into medium a toxic compound e.g. Add into medium a toxic compound e.g. antibiotic, herbicideantibiotic, herbicide

Only those cells able to grow in the presence of Only those cells able to grow in the presence of the selective agent give coloniesthe selective agent give colonies

Plate out and pick off growing colonies.Plate out and pick off growing colonies. Possible to select one colony from millions of Possible to select one colony from millions of

plated cells in a days work.plated cells in a days work. Need a strong selection pressure - get escapesNeed a strong selection pressure - get escapes

Only individuals with characters satisfying the Only individuals with characters satisfying the breeders are selected from population to be breeders are selected from population to be used as parents of the next generationused as parents of the next generation

Seed from selected individuals are mixed, then Seed from selected individuals are mixed, then progenies are grown togetherprogenies are grown together

Negative selection

Add in an agent that kills dividing cellsAdd in an agent that kills dividing cells Plate out leave for a suitable time, wash out Plate out leave for a suitable time, wash out

agent then put on growth medium.agent then put on growth medium. All cells growing on selective agent will die All cells growing on selective agent will die

leaving only non-growing cells to now grow.leaving only non-growing cells to now grow. Useful for selecting auxotrophs.Useful for selecting auxotrophs.

The most primitive and least widely used The most primitive and least widely used method which can lead to improvement only in method which can lead to improvement only in exceptional cases exceptional cases

It implies culling out of all poorly developed It implies culling out of all poorly developed and less productive individuals in a population and less productive individuals in a population whose productivity is to be genetically whose productivity is to be genetically improvedimproved

Positive and Visual SelectionPositive and Visual Selection

How do we get plants back from cells?We use tissue culture techniques to regenerate whole plants from single cellsGetting a plant back from a single cell is important so that every cell has the new DNA

Regeneration System

Transformation series of events

Transform individual cells

Callus formation

Auxins

CytokininsRemove from sterile conditions

Gene construct

BamHI

gus-intron nptII T 35S P 35S T 35S LB RB

P SAG12 ipt P 35S T nos

Gene construct

VectorsPromoter/terminator

Reporter genesSelectable marker genes

‘Genes of interest’.

Vectors

Ti-plasmid based vectora. Co-integrative plasmidb. Binary plasmid

Coli-plasmid based vectora. Cloning vectorb. Chimeric Plasmid

Viral vectora. It is normally not stably integrated into the plant cellb. It may be intolerant of changes to the organization of its genomec. Genome may show instability

A vehicle such as plasmid or virus for carrying recombinant DNA into a living cell

Ti plasmid

The binary Ti plasmid system

Binary vector system

Binary vector system

Promoter

1.1. A nucleotide sequence within an operonA nucleotide sequence within an operon2.2. Lying in front of the structural gene or genesLying in front of the structural gene or genes3.3. Serves as a recognition site and point of Serves as a recognition site and point of

attachment for the RNA polymeraseattachment for the RNA polymerase4.4. It is starting point for transcription of the It is starting point for transcription of the

structural genesstructural genes5.5. It contains many elements which are involved It contains many elements which are involved

in producing specific pattern and level of in producing specific pattern and level of expressionexpression

6.6. It can be derived from pathogen, virus, plants It can be derived from pathogen, virus, plants themselves, artificial promoterthemselves, artificial promoter

Types of Promoter Promoter always expressed in most tissue

(constitutive)-. 35 s promoter from CaMV Virus-. Nos, Ocs and Mas Promoter from bacteria-. Actin promoter from monocot-. Ubiquitin promoter from monocot-. Adh1 promoter from monocot-. pEMU promoter from monocot

Tissue specific promoter-. Haesa promoter-. Agl12 promoter

Inducible promoter-. Aux promoter

Artificial promoter-. Mac promoter (Mas and 35 s promoter)

Easy to visualise or assay

- ß-glucuronidase (GUS) (E.coli)

-green fluorescent protein (GFP) (jellyfish)

- luciferase (firefly)

Reporter gene

GUS

Cells that are transformed with GUS will form a blue precipitate when tissue is soaked

in the GUS substrate and incubated at 37oC

this is a destructive assay (cells die)

The UidA gene encoding activity is commonly used. Gives a blue colour from a colourless

substrate (X-glu) for a qualitative assay. Also causes fluorescence from Methyl Umbelliferyl Glucuronide (MUG) for a quantitative assay.

5 -- glucuronidase Genes

very stable enzyme cleaves -D glucuronide linkage simple biochemical reaction

• It must take care to stay in linear range detection sensitivity depends on substrate

used in enzymatic assay (fast)• colorimetric and fluorescent substrates

available

5 - -glucuronidase Genes Advantages

• low background• can require little equipment (spectrophotometer)• stable enzyme at 37ºC

Disadvantages• sensitive assays require expensive substrates or

considerable equipment• stability of the enzyme makes it a poor choice for

reporter in transient transfections (high background = low dynamic range)

Primary applications• typically used in transgenic plants with X-gus

colorimetric reporter

β-Glucorodinase gene

Bombardment of GUS gene

- transient expression

Stable expression of GUS in moss Phloem-limited expression of

GUS

GFP (Green Fluorescent Protein)

GFP glows bright green when irradiated by blue or UV light

This is a non destructive assay so the same cells can be monitored all the way

through It fluoresces green under UV illumination It has been used for selection on its own

Green fluorescent protein (GFP)

Source is bioluminescent jellyfish Aequora victoriaGFP is an intermediate in the bioluminescent

reaction Absorbs UV (~360 nm) and emits visible light.

has been engineered to produce many different colors (green, blue, yellow, red)

These are useful in fluorescent resonance energy transfer experiments

Simply express in target cells and detect with fluorometer or fluorescence microscope

Sensitivity is lowSensitivity is lowGFP is non catalytic, 1 GFP is non catalytic, 1 M concentration in cells is M concentration in cells is

required to exceed auto-fluorescencerequired to exceed auto-fluorescence

Green fluorescent protein (GFP))

Advantages• can detect in living cells• inexpensive (no substrate)

Disadvantages• low sensitivity and dynamic range• equipment requirements

Primary applications• lineage tracer and reporter in transgenic

embryos

GFP

protoplast colony derived from protoplast

mass of callus

regenerated plant

Luciferase luc gene encodes an enzyme that is responsible

for bioluminescence in the firefly. This is one of the few examples of a bioluminescent reaction that only requires enzyme, substrate and ATP.

Rapid and simple biochemical assay. Read in minutes

Two phases to the reaction, flash and glow. These can be used to design different types of assays.• Addition of substrates and ATP causes a flash of light that Addition of substrates and ATP causes a flash of light that

decays after a few seconds when [ATP] dropsdecays after a few seconds when [ATP] drops• after the flash, a stable, less intense “glow” reaction continues after the flash, a stable, less intense “glow” reaction continues

for many hours - AMP is responsible for thisfor many hours - AMP is responsible for this

Luciferase flash reaction is ~20x more sensitive than flash reaction is ~20x more sensitive than

glowglow glow reaction is more stableglow reaction is more stable

• allows use of scintillation counterallows use of scintillation counter• no injection of substrates requiredno injection of substrates required• potential for simple automation in microplate potential for simple automation in microplate

formatformat

Luciferase

AdvantagesAdvantages• large dynamic range up to 7 decades, depending on

instrument and chemistry• rapid, suitable for automation• instability of luciferase at 37 °C (1/2 life of <1hr)• inexpensive • widely used

disadvantages • Equipment requirement • luminometer (very big differences between models)• liquid scintillation counter (photon counter)r)

Gene which confer tolerance to a phytotoxic substance

Most common:

1. antibiotic resistance

kanamycin (geneticin), hygromycin

Kanamycin arrest bacterial cell growth by blocking various steps in protein synthesis

2. herbicide resistance

phosphinothricin (bialapos); glyphosate

Selectable Marker Gene

Effect of Selectable Marker

Transgenic = Has Kan or Bar Gene

Plant grows in presenceof selective compound

Plant dies in presenceof selective compound

Non-transgenic = Lacks Kan or Bar Gene

X

KanamycinKanamycin

Targets 30s ribosomal subunit, causing a Targets 30s ribosomal subunit, causing a frameshift in every translationframeshift in every translation

Bacteriostatic: bacterium is unable to produce Bacteriostatic: bacterium is unable to produce any proteins correctly, leading to a halt in any proteins correctly, leading to a halt in growth and eventually cell deathgrowth and eventually cell death

Kanamycin use/resistance Over-use of kanamycin has led to many wild Over-use of kanamycin has led to many wild

bacteria possessing resistance plasmidsbacteria possessing resistance plasmids As a result of this (as well as a lot of side As a result of this (as well as a lot of side

effects in humans), kanamycin is widely effects in humans), kanamycin is widely used for genetic purposes rather than used for genetic purposes rather than medicinal purposes, especially in medicinal purposes, especially in transgenic plantstransgenic plants

Resistance is often to a family of related Resistance is often to a family of related antibiotics, and can include antibiotic-antibiotics, and can include antibiotic-degrading enzymes or proteins protecting degrading enzymes or proteins protecting the 30s subunitthe 30s subunit

G418-GentamycinG418-Gentamycin

source: aminoglycoside antibiotic related source: aminoglycoside antibiotic related to gentamycinto gentamycin

activity: broad action against prokaryotic activity: broad action against prokaryotic and eukaryotic cellsand eukaryotic cells• inhibits protein synthesis by blocking inhibits protein synthesis by blocking

initiationinitiationresistance - bacterial neo gene (neomycin resistance - bacterial neo gene (neomycin

phosphotransferase, encoded by Tn5 phosphotransferase, encoded by Tn5 encodes resistance to kanamycin, encodes resistance to kanamycin, neomycin, G418neomycin, G418•but also cross protects against bleomycin but also cross protects against bleomycin

and relatives.and relatives.

G418 - Gentamycin Stability:

• 6 months frozen selection conditions:

• E. coli: 5 g/ml• Eukaryotic cells:

300-1000 g/ml. G418 requires careful optimization for cell types and lot to lot variations Kill curves required It requires at least seven days to obtain resistant colonies, two weeks is more typicalIt requires at least seven days to obtain resistant colonies, two weeks is more typical

G418 - Gentamycin

use and availability:use and availability:• perhaps the most widely used selection in perhaps the most widely used selection in

mammalian cellsmammalian cells• vectors very widely availablevectors very widely available

Surv

ivin

g ce

lls

Increasing dose ->

Hygromycin

source: aminoglycoside antibiotic from source: aminoglycoside antibiotic from Streptomyces hygroscopicus. Streptomyces hygroscopicus.

Activity: kills bacteria, fungi and higher Activity: kills bacteria, fungi and higher eukaryotic cells by inhibiting protein eukaryotic cells by inhibiting protein synthesissynthesis• interferes with translocation causing misreading interferes with translocation causing misreading

of mRNAof mRNA resistance: conferred by the bacterial gene resistance: conferred by the bacterial gene

hphhph• no cross resistance with other selective no cross resistance with other selective

antibioticsantibiotics

Hygromycin stability:stability:

• one year at 4 ºC, 1 month at 37 ºC

selection conditions:selection conditions:• E. coli: 50 g/ml• Eukaryotic cell lines:

50 - 1000 50 - 1000 g/ml (must be optimized)g/ml (must be optimized) 10 days- 3 weeks required to generate effect10 days- 3 weeks required to generate effect

use and availability:use and availability:• vectors containing hygromycin resistance gene

are widely available• in use for many years

Glyphosate resistanceGlyphosate resistance

Glyphosate = “Roundup”, “Tumbleweed” = Systemic herbicide

Glyphosate inhibits EPSP synthase (S-enolpyruvlshikimate-3 phosphate – involved in chloroplast amino acid synthesis)

Escherichia coli EPSP synthase = mutant form less sensitive to glyphosate less sensitive to glyphosate

Cloned via Ti plasmid into soybeans, tobacco, petunias

•Increased crop yields of crops treated with herbicides

+ Glyphosate

X

RoundUp Sensitive Plants

X

X

Shikimic acid + Phosphoenol pyruvate

3-Enolpyruvyl shikimic acid-5-phosphate(EPSP)

Plant EPSP synthase

Aromaticamino acids

Without amino acids, plant dies

X

BacterialEPSP synthase

Shikimic acid + Phosphoenol pyruvate

3-enolpyruvyl shikimic acid-5-phosphate(EPSP)

Aromaticamino acids

RoundUp Resistant Plants

+ Glyphosate

With amino acids, plant lives

RoundUp has no effect;enzyme is resistant to herbicide

BialaphosBialaphos Glufosinate – active substance of a broad-

spectrum-herbicide = synthetical copy of the aminoacid phosphinothricin produced by Streptomyces viridochomogenes

Inhibit glutamine-synthetase (important enzyme in nitrogen-cycle of plants) caused plant dies

Herbicide-tolerance is reached by gene-transfer from the bacterium to the plant

The transfered gene encodes for the enzyme phophinothricin-acetyl-transferase degrade glufosinate

Bialaphos*Bialaphos (Phosphinothricin-alanyl-alanine) is an

herbicide that inhibits a key enzyme in the nitrogen assimilation pathway, glutamine

synthetase, leading to accumulation of toxic levels of ammonia in both bacteria and plant cells

Only those cells that have taken up the DNA

can grow on media containing the selection

agent