Lecture 26

Gene Delivery methods –Agrobacterium and particle gun

Requirements for plant genetic transformation

Source Tissues for Plant Gene

Transfer

Gene to be

transferred

Gene Transfer Methods

Regeneration of whole

Transformed Plant

Plant Transformation Vector

Effecting plant gene transfer

Gene transfer methods

DirectIndirect

Indirect Gene Transfer Methods

Whole Plant Infection

In vitro infection of tissues

Co-cultivation of protoplasts and A.tumifaciens

Leaf disc transformation

Agrobacterium mediated gene transfer

Agrobacterium tumifacienswith vector DNA having the gene of interest

Plant cell

T DNA getting into the cell

Agrobacterium tumifaciens: A natural genetic engineer

This opportunistic soil phytopathogen

causes crown gall tumours in wounded

gymnosperms and dicotyledonous

angiosperms (dicots).

Oncongenic strains contain a single

copy of a large (150-250 kb) tumour-

inducing (Ti) plasmid.

Part of this plasmid DNA (the ‘transfer’

or T-DNA) is transferred to the

wounded plant cell and stably

integrated into the genome.

Agrobacterium mediated gene transfer

Direct Gene Transfer Methods

Electroporation

Liposome mediated gene transfer

Microinjection

Electroinjection

Sonication method

Microprojectile bombardment

Gene transfer can be effected by certain means that do not usevectors (Direct gene transfer). These include

a. Electroporation: Temporary holes are formed in the plasmamembrane of the host cell by applying a high voltage. Thesepores permit entry of foreign DNA.

b. Chemical mediated genetic transformation some chemicals,such as polyethylene glycol, help foreign DNA to enter thehost cell.

c. Micro-injection: The host cell is immobilized by applying amild suction with a blunt pipette. The foreign gene is theninjected with a micro-injection needle.

When the electric field is applied, the ions move accordingto their charge.

Pathways are formed across the cell membrane allowingDNA to enter.

When the electric field is deactivated, the membrane heals.

ElectroporationTransformation is stimulated by physically disrupting the cell membranes sothat the genetic material may enter.

This method was introduced by Fromm and his coworkers in 1986

The voltage range varies from 250-2000V.

This method was reported by Krens and his colleagues in l982

Non-biological methodsPolyethylene Glycol (PEG) method:

In fusion conditions, PEG is usually present at a concentration of40 percent for several seconds whereas for DNA transformation the optimalPEG concentration is 13.3 percent for an incubation of 30 mins.

Microinjection method:Crossway and Reich in l986

Using the holding pipette, the protoplast has to be held and the DNA to beinjected into the nucleus using the injection pipette. After the micro injectionthe injected cells are cultured by hanging droplet culture method.

Micro projectile bombardment method:In 1987 Klein and his colleagues evolved a method by which the deliveryof DNA into cells of intact plant organs or cultured cells is done by aprocess called Projectile Bombardment

These particles with high kinetic energy penetrate the cells and membranesand carry foreign DNA inside of the bombarded cells. This method isotherwise called as "Biolistics Method”

Biolistic Transformation

Some of the particles will pass through the cell wall and enter individual cells.

Sonication method:

• This is a simple technique formulated by Xu and his co-workers.

• In this method the explants (especially leaves) are excised and cutinto segments, immmersed in sonication buffer containing plasmidDNA and carrier DNA in a sterile glass Petri dish. Then the sampleswere sonicated with an ultrasonic pulse generator at 0.5 c/cm2

acoustic intensity for 30 mins.• After 30 mins., the explants were rinsed in buffer solution without

DMSO and transferred to the culture medium.