Plant Genetic Engineering

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

Electroporation (electropermeabilization)

Physical Methods

1. Particle bombardment

2. Microinjection

3. Silicon Carbide whiskers

Agrobacterium tumefaciensAgrobacterium tumefaciens

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))

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 cell1. Then integrated into plant genome1. Then integrated into plant genome2. 2 x 23bp direct repeats play an important 2. 2 x 23bp direct repeats play an important

role inrole 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 Plasmid contains genes responsible for

the diseasethe disease 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 growthgrowth

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

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 and genetic engineering:Engineering the Ti plasmid

Co-integrative and binary vectorsCo-integrative and binary vectors

Binary vector

LB RB

Co-integrative

Explants: cells and protoplasts

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%

ElectroporationElectroporation

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

Pressure gauge Disk with DNA-coated particles

Stop plate

Sample goes here

Vacuum lineGas line

Rupture disk

Vacuum chamber

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

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

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

Transformation series of events

Transform individual cells

Callus formation

Auxins

CytokininsRemove from sterile conditions

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

A. tumefaciens

binary vector

T-DNA