TRANSGENIC TECHNOLOGY

getting DNA into a cell

getting it stably integrated

getting a plant back from the cell

Plant transformation

1. 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’

Requirement

Transformation methods

DNA must be introduced into plant cells

Indirect - Agrobacterium tumefaciens

Direct - Chemical method

- Electrical method

- Physical methods

Chemical Method

1. Use of PEG (Polyethylene glycol (PEG)-mediated )

2. Protoplasts are incubated with a solution of DNA and PEG

Electrical method

1. Electroporation (electropermeabilization)

2. Cells or protoplast are subjected to short electrical pulse

Physical Methods

1. Particle bombardment

2. Microinjection

3. Silicon Carbide whiskers

AgrobacteriumAgrobacterium-mediated -mediated transformationtransformation

A natural genetic A natural genetic engineerengineer

2 species2 species• A.tumefaciensA.tumefaciens

(produces a gall)(produces a gall)• A. rhizogenesA. rhizogenes

(produces roots)(produces roots) OncOncoogenesgenes (for (for

auxin and cytokinin auxin and cytokinin synthesis) + Opinessynthesis) + Opines

In the presence of In the presence of exudates (e.g. exudates (e.g. acetosyringone) from acetosyringone) from wounded plants, wounded plants, VirVirulence (Vir) genes ulence (Vir) genes are activated and are activated and cause the t-DNA to be cause the t-DNA to be transferred to plants. transferred to plants. Everything between Everything between the left and right the left and right border is transferred.border is transferred.

BACTERIAL GALL DISEASESBACTERIAL GALL DISEASES

Galls:Galls: overgrowth or proliferation of tissue, primarily overgrowth or proliferation of tissue, primarily due to increased cell division (hyperplasia) and due to increased cell division (hyperplasia) and increased cell size (hypertrophy).increased cell size (hypertrophy).

Bacterial Galls:Bacterial Galls:

induced by bacteria in 3 different genera. induced by bacteria in 3 different genera. • AgrobacteriumAgrobacterium• PseudomonasPseudomonas• ClavibacterClavibacter

Genes for plant hormone production found Genes for plant hormone production found on bacterial plasmids!on bacterial plasmids!

Crown Gall Disease: Crown Gall Disease: Agrobacterium tumefaciensAgrobacterium tumefaciens

Gram -Gram - DicotsDicots WorldwideWorldwide

Disease Cycle

Agrobacterium tumefaciensAgrobacterium tumefaciens

CharacteristicsCharacteristics• Plant parasite that causes Crown Gall Plant parasite that causes Crown Gall

DiseaseDisease• Encodes a large (~250kbp) plasmid called Encodes a large (~250kbp) plasmid called

Tumor-inducing (Ti) plasmidTumor-inducing (Ti) plasmid Portion of the Ti plasmid is transferred between Portion of the Ti plasmid is transferred between

bacterial cells and plant cells bacterial cells and plant cells T-DNA (Tumor T-DNA (Tumor DNADNA))

Agrobacterium tumefaciensAgrobacterium tumefaciens

T-DNA integrates stably into plant genomeT-DNA integrates stably into plant genomeSingle stranded T-DNA fragment is Single stranded T-DNA fragment is converted to dsDNA fragment by plant cellconverted to dsDNA fragment by plant cell Then integrated into plant genomeThen integrated into plant genome 2 x 23bp direct repeats play an important role in 2 x 23bp direct repeats play an important role in

the excision and integration processthe excision and integration process

Agrobacterium tumefaciensAgrobacterium tumefaciens

Tumor formation = hyperplasiaTumor formation = hyperplasia Hormone imbalanceHormone imbalance Caused by Caused by A. tumefaciensA. tumefaciens

• Lives in intercellular spaces of the plantLives in intercellular spaces of the plant• Plasmid contains genes responsible for the Plasmid contains genes responsible for the

diseasedisease Part of plasmid is inserted into plant DNAPart of plasmid is inserted into plant DNA Wound = entry point Wound = entry point 10-14 days later, 10-14 days later,

tumor formstumor forms

Agrobacterium tumefaciensAgrobacterium tumefaciens

What is naturally encoded in T-DNA?What is naturally encoded in T-DNA?• Enzymes for auxin and cytokinin synthesisEnzymes for auxin and cytokinin synthesis

Causing hormone imbalance Causing hormone imbalance tumor tumor formation/undifferentiated callusformation/undifferentiated callus

Mutants in enzymes have been characterizedMutants in enzymes have been characterized

• Opine synthesis genes (e.g. octopine or Opine synthesis genes (e.g. octopine or nopaline)nopaline)

Carbon and nitrogen source for Carbon and nitrogen source for A. tumefaciensA. tumefaciens growth growth Insertion genes Insertion genes

• Virulence (vir) genesVirulence (vir) genes• Allow excision and integration into plant genomeAllow excision and integration into plant genome

Ti plasmid of Ti plasmid of A. tumefaciensA. tumefaciens

1. Auxin, cytokinin, opine synthetic genes transferred to plant

2. Plant makes all 3 compounds

3. Auxins and cytokines cause gall formation

4. Opines provide unique carbon/nitrogen source only A. tumefaciens can use!

Agrobacterium tumefaciensAgrobacterium tumefaciens How is T-DNA modified to allow genes of How is T-DNA modified to allow genes of

interest to be inserted?interest to be inserted?• In vitroIn vitro modification of Ti plasmid modification of Ti plasmid

T-DNA tumor causing genes are deleted and replaced T-DNA tumor causing genes are deleted and replaced with desirable genes (under proper regulatory control)with desirable genes (under proper regulatory control)

Insertion genes are retained (vir genes)Insertion genes are retained (vir genes) Selectable marker gene added to track plant cells Selectable marker gene added to track plant cells

successfully rendered transgenic [antibiotic resistance successfully rendered transgenic [antibiotic resistance gene gene geneticin (G418) or hygromycin] geneticin (G418) or hygromycin]

Ti plasmid is reintroduced into Ti plasmid is reintroduced into A. tumefaciensA. tumefaciens A. tumefaciensA. tumefaciens is co-cultured with plant leaf disks is co-cultured with plant leaf disks

under hormone conditions favoring callus under hormone conditions favoring callus development (undifferentiated)development (undifferentiated)

Antibacterial agents (e.g. chloramphenicol) added to Antibacterial agents (e.g. chloramphenicol) added to kill kill A. tumefaciensA. tumefaciens

G418 or hygromycin added to kill non-transgenic plant G418 or hygromycin added to kill non-transgenic plant cellscells

Surviving cells = transgenic plant cellsSurviving cells = transgenic plant cells

Agrobacterium and genetic engineering:Engineering the Ti plasmid

Co-integrative and binary vectorsCo-integrative and binary vectors

Binary vector

LB RB

Co-integrative

cause ‘Crown gall’ disease

Agrobacterium tumefaciens

Agrobacterium-mediated transformation

Agrobacterium is a ‘natural genetic engineer’

i.e. it transfers some of its DNA to plants

Electroporate T-DNA vector into Agrobacterium and select for tetr

Expose wounded plant cells to transformed agro strain

Induce plant regeneration and select for Kanr cell growth

Explants: cells and protoplasts

Most direct way to introduce foreign DNA into the nucleus

ElectroporationElectroporation

Diagram of one techniqueDiagram of one technique

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

Once inside the cell, the gene comes off the bead and Once inside the cell, the gene comes off the bead and

integrates into the cell's genome.integrates into the cell's genome.

Model from BioRad: Model from BioRad: Biorad's Helios Gene Biorad's Helios Gene GunGun

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

Silicon carbide forms long, needle like crystals

Cells are vortex mixed in the present of whiskers and DNA

DNA can be introduced in the cells following penetration by the whiskers

Silicon Carbide WhiskersSilicon Carbide Whiskers

Gene constructGene construct

BamHI

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

P SAG12 ipt P 35S T nos

Gene constructGene construct

VectorsPromoter/terminator

reporter genesselectable marker genes

‘genes of interest’.

VectorsVectors

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

PromoterPromoter

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 in It contains many elements which are involved in

producing specific pattern and level of expressionproducing specific pattern and level of expression6.6. It can be derived from pathogen, virus, plants It can be derived from pathogen, virus, plants

themselves, artificial promoterthemselves, artificial promoter

Types of PromoterTypes of Promoter Promoter always expressed in most tissue Promoter always expressed in most tissue

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

Tissue specific promoterTissue specific promoter-. Haesa promoter-. Haesa promoter-. Agl12 promoter-. Agl12 promoter

Inducible promoterInducible promoter-. -. Aux promoterAux promoter

Artificial promoterArtificial promoter-. Mac promoter (Mas and 35 s 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 The UidA gene encoding activity is commonly used. Gives a blue colour from a colourless used. Gives a blue colour from a colourless substrate (substrate (X-gluX-glu) for a qualitative assay. Also ) for a qualitative assay. Also causes fluorescence from causes fluorescence from MMethyl ethyl UUmbelliferyl mbelliferyl GGlucuronide (lucuronide (MUGMUG) for a quantitative assay.) for a quantitative assay.

GUS

Bombardment of GUS gene

- transient expression

Stable expression of GUS in moss Phloem-limited expression of

GUS

HAESA gene encodes a receptor protein kinase that controls floral organ abscission. (A) transgenic plant expressing a HAESA::GUS fusion. It is expressed in the floral abscission zone at the base of an Arabidopsis flower.

Transgenic plants that harbor the AGL12::GUS fusions show root-specific expression.

Inducible expressionInducible expression

GFP (Green Fluorescent Protein)

GFP glows bright green when irradiated by blue or UV light

This is a nondestructive assay so the same cells can be monitored all the way through

Fluoresces green under UV illuminationFluoresces green under UV illumination Problems with a cryptic intron now resolved.Problems with a cryptic intron now resolved. Has been used for selection on its own.Has been used for selection on its own.

GFP

protoplast colony derived from protoplast

mass of callus

regenerated plant

let you kill cells that haven’t taken up DNA- usually genes that confer resistance to a phytotoxic substance

Most common:

1. antibiotic resistance

kanamycin, hygromycin

2. herbicide resistance

phosphinothricin (bialapos); glyphosate

Selectable Marker Gene

Only those cells that have taken up the DNA can grow on media containing the selection agent

Gene of interestGene of interest

Sequence of DNA which will be inserted Sequence of DNA which will be inserted to the host cell and its product will be to the host cell and its product will be

studied or beneficial for mankindstudied or beneficial for mankind

Origin of gene interest:

1. Non plant genes

2. Plant genes

Exogenous genes (non-plant

genes)

pathogen-derived genes

bacterial genes

any other organism

Endogenous genes (Plant

genes)

Enzymes in biochemical pathway

Natural resistance genes

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

How do we know which plants have taken up the DNA?

Could test each plant - slow, costly

Or use reporter genes & selectable marker genes

Screening technique

ScreeningScreening

Transformation frequency is low (Max 3% of all Transformation frequency is low (Max 3% of all cells) and unless there is a selective advantage for cells) and unless there is a selective advantage for transformed cells, these will be overgrown by non-transformed cells, these will be overgrown by non-transformed.transformed.

Usual to use a positive selective agent like Usual to use a positive selective agent like antibiotic resistance. The NptII gene encoding antibiotic resistance. The NptII gene encoding Neomycin phospho-transferase II phosphorylates Neomycin phospho-transferase II phosphorylates kanamycin group antibiotics and is commonly kanamycin group antibiotics and is commonly

usedused. .

Screening (selection)Screening (selection)

Select at the level of the intact plantSelect at the level of the intact plant Select in cultureSelect in culture

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

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

phasesphases

Selection StrategiesSelection Strategies

PositivePositive NegativeNegative

VisualVisual

Positive selectionPositive 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 Only those cells able to grow in the presence of the selective agent give presence of the selective agent give coloniescolonies

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

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

escapesescapes

Negative selectionNegative selection

Add in an agent that kills dividing cells Add in an agent that kills dividing cells e.g. chlorate / BUdR.e.g. chlorate / BUdR.

Plate out leave for a suitable time, Plate out leave for a suitable time, wash out agent then put on growth wash out agent then put on growth medium.medium.

All cells growing on selective agent All cells growing on selective agent will die leaving only non-growing cells will die leaving only non-growing cells to now grow.to now grow.

Useful for selecting auxotrophsUseful for selecting auxotrophs..

Visual selectionVisual selection

Only useful for coloured or Only useful for coloured or fluorescent compounds fluorescent compounds

Plate out at about 50,000 cells per Plate out at about 50,000 cells per plate.plate.

Pick off coloured / fluorescent Pick off coloured / fluorescent compoundscompounds

Possible to screen about 1,000,000 Possible to screen about 1,000,000 cells in a days work.cells in a days work.

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 cells

getting a plant back from a single cell is important so that every cell has the new DNA

Regeneration System

Regeneration

Regeneration of shoots from leaf protoplasts in Arabidopsis thaliana

Plant tissue culture uses growth regulators and nutrients to regenerate plants in vitro

Somatic embryogenesis in peanut