DIRECT TRANSFORMATION METHODS

Submitted by Submitted by

Suvendhu S. DuttaSuvendhu S. Dutta

MBB/11-09, IBT, MBB/11-09, IBT, ANGRAUANGRAU

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

TRANSFORMATIONTRANSFORMATION

Introduction of DNA into the cells of an Introduction of DNA into the cells of an organism by a method other than conventional organism by a method other than conventional crossing/the introduction of a gene of choice into crossing/the introduction of a gene of choice into the genome of an organism. the genome of an organism.

Transformant is a cell that has been genetically Transformant is a cell that has been genetically altered through the uptake of foreign DNA .altered through the uptake of foreign DNA .

Requirement

1.a suitable transformation method

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

3.an efficient regeneration system

4. vectors, Promoter/terminator, reporter genes, selectable marker

genes, ‘genes of interest’

Transformation methods

Indirect Agrobacterium tumefaciens

Direct

Method depends on plant type, cost, application

Particle bombardment

Polyethylene glycol (PEG)-mediated protoplast transformation.

Electroporation

Microinjection

Ultrasound mediated

Pollen-mediated

limitation of Agrobacteriumlimitation of Agrobacterium

• Host range is limited: not all plants may be Host range is limited: not all plants may be susceptible to Agrobacterium.susceptible to Agrobacterium.

• With susceptible plants, accessible With susceptible plants, accessible culture/regeneration systems must be adaptable culture/regeneration systems must be adaptable to Agrobacterium-mediated gene transferto Agrobacterium-mediated gene transfer

1.Electroporation

Explants: cells and protoplasts

Most direct way to introduce foreign DNA into the nucleus

Achieved by electromechanically operated devices

Transformation frequency is high

Direct Transformation methods

http://horticulture.unh.edu/gen_tech.html

2. Microinjection2. Microinjection

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%

3. PEG-mediated protoplast transformation

It is the oldest (direct DNA) reliable method for plant transformation.

The first report of generating transgenic plants using this method was provided by Paszkowski et al. (1984). They regenerated transformed protoplasts into plants that were kanamycin (drug) resistant.

This method has been very useful and applied to several plant species.

But it is a tedious procedure!

4. Pollen mediated transformation

In 1976, attempts were made to use pollen to take up foreign DNA and then cross fertilize a related species. glauca pollen were incubated in DNA isolated from langsdorfii. The DNA treated pollen were used to pollinate emasculated glauca plants. One group claimed that the sexual progeny thus obtained formed tumors on the stem.

However, these experiments were not reproduced in other labs.

5.Ultrasound-induced (sonication) DNA uptake

Ultrasound treatment causes the formation of bubbles with generation of high pressure and temperature and violent-flow or streaming of fluids. This method has been used to introduce DNA into plant protoplasts. First report described the introduction of DNA into tobacco protoplasts but no transgenic plant was obtained. Subsequent reports described generation of transgenic tobacco and wheat but insufficient molecular evidence was provided.

It appears that this method may be effective in introducing DNA into cell but not to the nucleus. Further, it does not present much advantage over other methods and therefore it hasn’t been explored much.

6. Gene Delivery Biolistic-mediated 6. Gene Delivery Biolistic-mediated transformationtransformation

• Known as:Known as:

• Particle BombardmentParticle Bombardment

• BiolisticsBiolistics

• Microprojectile bombardmentMicroprojectile bombardment

• Particle accelerationParticle acceleration

• Particle inflow gunParticle inflow gun

• Gene gunGene gun

• The gene gun was originally a Crosman air The gene gun was originally a Crosman air pistol modified to fire dense tungsten particles. pistol modified to fire dense tungsten particles.

• It was invented by It was invented by John C SanfordJohn C Sanford, Ed Wolf and , Ed Wolf and Nelson Allen at Cornell in 1987.Nelson Allen at Cornell in 1987.

• The original target was onions (chosen for their large The original target was onions (chosen for their large cell size) and it was used to deliver particles coated with cell size) and it was used to deliver particles coated with a marker gene.a marker gene.

• Bt-Maize is the first commercial GM crop produced by Bt-Maize is the first commercial GM crop produced by this method.this method.

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

the cell's genome.the cell's genome.

• Preparation of micro carriers:Preparation of micro carriers:• Use 50mg of tungsten power for 100 bombardments.Use 50mg of tungsten power for 100 bombardments.• Add 1ml of absolute ethanol.Add 1ml of absolute ethanol.• Centrifuge for 10 sec.(2X).Centrifuge for 10 sec.(2X).• Remove ethanol and add 1ml SDW, repeat 3x.Remove ethanol and add 1ml SDW, repeat 3x.• Sample preparation for Sample preparation for 6 bombardments6 bombardments, ,

• 50ul tungsten suspension50ul tungsten suspension,10ul DNA in TE, 50ul 2.5M cacl,10ul DNA in TE, 50ul 2.5M cacl2,2,

20ul 0.1 M spermidine (20ul 0.1 M spermidine (SpermidineSpermidine is a polyamine involved  is a polyamine involved in cellular metabolism) Binds and precipitates DNA.in cellular metabolism) Binds and precipitates DNA.

• Add 200ul ethanol.Add 200ul ethanol.• Centrifuge for 10sec. And resuspend in 30ul.Centrifuge for 10sec. And resuspend in 30ul.

PSD-1000/He Particle Delivery System

Particle Gun components

� Target embryos

� Rupture disk

� Stopping screen

� Carrier disk with gene-coated particles

• Helium is released into the barrel

When the gun is fired:Biolistic Method Mechanism:

When the gun is fired:

• Pressure breaks the rupture disk

When the gun is fired:

• The carrier disk and the DNA-coated particles strike the stopping screen

When the gun is fired:

• The particles pass through the screen.

When the gun is fired:

• Particles enter the petri dish. Some penetrate the walls of the cells in it.

• Cell wall

• Nucleus

• DNA-coated particle

• Cell wall

• Nucleus

• DNA-coated particle

Cell after particle gun useCell after particle gun useCell after particle gun useCell after particle gun use

Biolistic Method Advantage and DisadvantageBiolistic Method Advantage and Disadvantage

Advantages:Advantages:

• This method can be use to transform all plant speciesThis method can be use to transform all plant species

• No binary vector is required. No binary vector is required.

• Transformation protocol is relatively simple.Transformation protocol is relatively simple.

Disadvantage:Disadvantage:

• Difficulty in obtaining single copy transgenic events. Difficulty in obtaining single copy transgenic events.

• High cost of the equipment and microcarriers. High cost of the equipment and microcarriers.

• Intracellular target is random (cytoplasm, nucleus, vacuole, Intracellular target is random (cytoplasm, nucleus, vacuole,

plastid, etc.).plastid, etc.).

• Transfer DNA is not protectedTransfer DNA is not protected

Parameters Affecting Gene Gun MethodParameters Affecting Gene Gun Method

• A number of parameters has been identified A number of parameters has been identified and need to be considered carefully in and need to be considered carefully in experiments involving particle bombardmentexperiments involving particle bombardment

Parameter categories:Parameter categories:

• Physical –ParametersPhysical –Parameters

• Biological –ParametersBiological –Parameters

• Environmental – ParametersEnvironmental – Parameters

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

Regeneration of shoots from leaf protoplasts in Arabidopsis thaliana

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

Not all cells take up DNA & not all cells can regenerate so

Need an efficient regeneration system and transformation system i.e. lots of cells take up DNA and lots of cells regenerate into a plant

to maximize chance of both happening

Transformed cellsregenerable cells

Cells containing new DNA that are able to regenerate into a new plant

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, costlyOr use reporter genes & selectable marker genes

Screening TechniqueScreening Technique

SelectionSelection• Transformation frequency is low (Max 3% of Transformation frequency is low (Max 3% of

all cells) and unless there is a selective all cells) and unless there is a selective advantage for transformed cells, these will be advantage for transformed cells, these will be overgrown by non-transformed.overgrown by non-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 Neomycin phospho-transferase II phosphorylates kanamycin group antibiotics phosphorylates kanamycin group antibiotics and is commonly usedand is commonly used. .

Reporter genes -It is the coding unit of a test gene whose product is easily assayed during genetic transformation

most common - ß-glucuronidase (GUS) (E.coli)

- green fluorescent protein (GFP) (jellyfish)

- luciferase (firefly, bacterial, jellyfish etc)

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)

GUS

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

GFP

protoplast colony derived from protoplast

mass of callus

regenerated plant

Selectable marker genes -A gene whose expression allows the identification of a specific trait or gene in an organism or cells that have been transformed.

Most common:

antibiotic resistance - e.g. kanamycin, hygromycin [ only phytotoxic antibiotics can be used]

herbicide resistance - e.g. phosphinothricin (PPT); glyphosate

APPLICATIONS

transfer of exogenous genes

manipulation of endogenous genes Gene silencing/

downregulation

Pathogen resistance

Herbicide resistance

Bioreactors/molecular farming

Delivery systems

Plant improvement

Reference :

‘Plant Biotechnology’ by Adrian Slater et al.

Wikipedia.org.com

Google.com

Klein et al 2003