Methods of plant transformation and direct gene transfer

Khushpreet Singh B tech Biotech ‘09 Thapar University Patiala INDIA

Plant Transformation
 The genetic manipulation of plants has been an ongoing

science since prehistoric times, when early farmers along the Euphrates began carefully selecting and maintaining seed from their best crops to plant for the next season. Early Americans also bred plants, and modern corn is a result of thousands of years of genetic manipulation.  With the advent of recombinant DNA technology in the 1970s, the genetic manipulation of plants entered a new age. Genes and traits previously unavailable through traditional breeding became available through DNA recombination, and with greater specificity than ever before.  Genes from sexually incompatible plants, or from animals, bacteria or insects can now be introduced into plants. Products of modern plant genetic engineering are already on the market in various regions of the U.S. Examples include a slow-softening tomato and cotton plants resistant to herbicides and insects. With many more products in the pipeline, the genetic engineering of plants will have a profound impact on the future

Keys
• • • • DNA must be moved into nucleus DNA must be integrated into the genome DNA must be stable Tissue culture techniques are needed for – Preparation of cells amenable to receive DNA – Selecting transgenic cells and tissues – Regenerating transgenic plants – Transgene(s) inherited in Mendelian fashion

Formula Tissue culture + DNA delivery and

Plant Transformation using Agarobacterium tumefaciens
 Modern plant genetic engineering involves the transfer of the

desired genes into the plant genome, and then regeneration of a whole plant from the transformed tissue.  Currently, the most widely used method for transferring genes into plants is Agrobacterium-mediated transformation.  Agrobacterium is a naturally occurring pathogenic bacteria in the soil that has the ability to transfer its DNA into a plant's genome.  Agrobacterium infection and gene transfer normally occurs at the site of a wound in the plant, and causes a characteristic growth referred to as a crown gall tumor.  Scientists have taken advantage of this naturally occurring transfer mechanism, and have designed DNA vectors from the tumorinducing plasmid DNA found in the bacteria that are capable of carrying desired genes into the plant.  The engineered or constructed genes are inserted into the Agrobacterium vectors and enter the plant by the bacteria's own internal transfer mechanisms.  Transformation is typically done on a small excised portion of a plant known as an explant. This small piece of transformed plant tissue is then regenerated into a mature plant through tissue culture techniques.

Agrobacterium tumefaciens
Agrobacterium tumefaciens is the causal agent of Crown Gall disease (the formation of tumours) in over 140 species of dicot. It is a rod shaped, Gram negative soil bacterium. Symptoms are caused by the insertion of a small segment of DNA (known as the T-DNA,

Causative agent of crown gall disease

Transformation by A. tumefaciens

If a plant is wounded or damaged, A.

tumefaciens can infect the at the wound site. Crown gall formation depends on the presence of a plasmid in A. tumefaciens known as Ti plasmid. Part of this plasmid is actually transferred from the bacterium into the plant cell, where it integrates into the host genome. The T-DNA is involved in the biosynthesis ofPlant hormones (auxins and cytokinins). Novel plant metabolites (opines and

agropines).

The Ti plasmid
Ti plasmid has a central role in crown gall

formation and it is the portion of Ti plasmid that is integrated into the host genome. This Ti plasmid is responsible for the tumorous phenotype. Ti plasmid featuresThey They They They

contain one or more T-DNA regions. contain a vir region. contain an origin of replication. contain a region enabling conjugative transfer. They contain genes for the catabolism of opines(a class of amino acid conjugates).

The T-DNA
 The T-DNA region of any Ti plasmid is defined by the

presence of the right and the left border sequences.  These border sequences are 24 bp imperfect repeats.  Any DNA between the borders will be transferred into the genome of the plant.  The oncogenes: Two genes auxA and auxB encode proteins involved in

the production of auxins, similarly gene cyt for cytokinin production which are the prime determinants of the tumour phenotype.

 Other genes :-

 The tml gene which is involved in determining tumor

size is found in some species, is also found in the TDNA.  Genes responsible for the T-DNA transfer are also situated on the Ti plasmid.

Key steps from natural Agrobacterium to “useful
• Some vir genes deleted--disarmed – Opines not going to be produced – Deleting tumorogenesis function • Choosing strains that transfer DNA in lab • Clone in genes of interest, antibiotic resistance genes, etc. • Binary system-- two plasmids are better than one Ti plasmid

The process of T-DNA transfer and integration
Agrobacteriumcontains a tumour-inducing (Ti)

plasmid, which includes virulence (vir) genes and a transferred-DNA(T-DNA) region. Genes of interest can be inserted into the T-DNA. Wounded plant cells produce phenolic defence compounds, which can trigger the expression of the Agrobacterium virgenes. The encoded virulence (Vir) proteins process the T-DNA region from the Ti-plasmid, producing a 'T-strand'. After the bacterium attaches to a plant cell, the Tstrand and several types of Vir proteins are transferred to the plant through a transport channel. Inside the plant cell, the Vir proteins interact with the T-strand, forming a T-complex.

The process of T-DNA transfer and integration
 Step 1. Signal recognition by Agrobacterium.  Step 2. attachment to plant cells.
 Two step process, attachment via polysaccharide and

subsequently by a mesh of cellulose fibers produced by bacterium. phosphorylates Vir G which induces the induction of all the vir genes.many sugars enhance the vir genes induction. and Vir D2 becomes covalently attached to 5’ end and displaces single stranded T-DNA strand .

 Step3. induction of vir genes.

 Vir A senses phenolics, autophosphorylates and subsequently

 Step 4. T-strand production.

 the left and right borders are recognized by VirD1-VirD2 complex

 Step 5. transfer of T-DNA out of the bacterial cell.

 Done by a T-pilus(membrane channel secretory system) composed

of proteins encoded by virB operon and Vir D4. Vir E2 and Vir F are exported from bacterial cell.

 Step 6. transfer of T-DNA and Vir proteins into the

plant cell and nuclear localization.

 TDNA-VirD2 complex and other vir proteins cross the plant plasma

Desirable features of any plasmid vector
Be of a small size. Small plasmids are easy

to handle invitro as they are less liable to damage by shearing. There is also less chance of the vector having other sites for restriction enzymesmaking the design and integrationof a MCS simple. Confer a selectable phenotype on the hist cell so that they can be selected for like antibiotic resistant gene. Contain single site for a large number of restriction enzymes to enable the

DIRECT GENE
BIOLISTIC GUN ELECTROPORATION TRANSFORMATION USING SILICON CARBIDE

FIBERS PEG MEDIATED TRANSFER

Direct gene transfer
Advantage:
 This method can be use to transform all plant

species.  No binary vector is required.  Transformation protocol is relatively simple.

Disadvantage:
 Difficulty in obtaining single copy transgenic events.  High cost of the equipment and microcarriers.  Intracellular target is random (cytoplasm, nucleus,

vacuole, plastid, etc.).  Transfer DNA is not protected.

Gene Delivery System Biolistic-mediated transformation
Also known as:  Particle Bombardment  Biolistics  Microprojectile bombardment  Particle acceleration  Particle inflow gun  Gene gun  Using a gene gun directly shoots a piece of DNA into the recipient plant tissue.
 Tungsten or gold beads are coated in

the gene of interest and fired through a stopping screen, accelerated by Helium, into the plant tissue. The particles pass through the plant cells, leaving the DNA

Overview of the process

Mechanism

Equipment

DNA-coated microcarriers are

loaded on microcarrier. Micro-carriers are shot towards target tissues during helium gas decompression. A stopping screen placed allowing the coated microprojectiles to pass through and reach the

Particle bombardment of Vicia faba cotyledons

Biolistic Transformation parameters
A number of parameters has been

identified and need to be considered carefully in experiments involving particle bombardment Parameter categories: - Physical parameters - Biological parameters - Environmental parameters

Physical parameters
 Nature, chemical and physical properties of the metal particles used as

a macrocarrier for the foreign DNA

 Particles should be high enough mass in order to possess adequate

momentum to penetrate into appropriate tissue. rhodium,platinum and iridium. and cell components.

 Suitable metal particles include gold, tungsten, palladium,  Metals should be chemically inert to prevent adverse reaction with DNA

 Nature, preparation and binding of DNA onto the particles

 The nature of DNA (single vs double stranded, circular vs linerized DNA).

Optimal: double stranded circular DNA molecules (e.g. plasmid)  In the process of coating the metal particls with DNA certain additives such as spermididne and CaCl 2 appear to be useful.
 Target tissue

 It is important to target the appropriate cells that are competent for both

transformation and regeneration.  Depth of penetration is one of the most important variables in order to achieve particle delivery to particular cells.

Biological parameters
-Biological parameters
Temperature, photoperiod and humidity These parameters have a direct effect on the

physiology of tissues. Such factors will influence receptiveness of target tissue to foreign DNA delivery and also affect its susceptibility to damage and injury that may adversely affect the outcome of transformation process. Some explants may require a “healing” period after bombardment under special regiments of light, temperature, and humidity.

PROTOPLAST ELECTROPORATION
 Protoplasts are cells

stripped of their cell walls and maintained in culture  Electroporation, or electropermeabilizati on, is a significant increase in the electrical conductivity and permeability of the cell plasma membrane caused by an externally applied electrical field.  The electroporation of cells can be used to

 The vectors used

can be simple plasmids.  Material is incubated in buffer solution containing DNA & subjected to high voltage electrical pulse.  DNA migrates through high voltage induced pores in plasma membrane and integrates into the genome.  Plant material used for electroporation require specific treatment such as pre and post

The vectors used can be simple plasmids. Material is incubated in buffer solution

containing DNA & subjected to high voltage electrical pulse. DNA migrates through high voltage induced pores in plasma membrane and integrates into the genome. Plant material used for electroporation require specific treatment such as pre and post electroporation incubation in buffers of high osmotic pressure.

Advantages of protoplast Electroporation
Amount of DNA deliverd to each cell is low

which has a benefit of producing transformants with a low transgene copy number.
All the cells are in the same physiological

state after transformation unlike the situation with particle bombardment where damage can occur from transformation.

Transformation by Silicon carbide fibres : WHISKERS™

Transformation by Silicon carbide fibres : WHISKERS™(contd.)
This technique requires no special

equipment. Plant material is introduces into a buffer containing DNA and silicon carbide fibres, which is then vortexed. The fibre penetrates the cell wall and plasma membrane allowing DNA to gain access to the inside of the cell.

Drawbacks
Related to availability of suitable plant

materials. Fibres require careful handling. This procedure has been utilized for friable callus for maize and is limited only to few genotypes of maize and oats. Many cereals ,the natural targets for the procedure produce embryogenic callus that is hard and compact and is not easily transformed at present.

Microinjection
• The microinjection method uses a fine needle to inject a solution of DNA into a cell.

Transformation with Protoplasts
Microinjection
 Protoplasts attached to

slide by embedding in agarose using a holding pipet injection pipet

 DNA injected using

PEG mediated transformation
Polyethylene Glycol Transformation of naked

DNA done by treatment with PEG in presence of divalent cations. PEG and divalent cationsdestabilize the plasma membrane of plant protoplast and render it permeable to naked DNA. DNA then enters the

Figure: GUS staining of PEG-transformed protoplasts derived from roots of Arabidopsis ecotype Columbia after

Advantages
The PEG-mediated transformation is

simple and efficient, allowing a simultaneous processing of many samples. Yields a transformed cell population with high survival and division rates Method utilizes inexpensive supplies and equipments, Helps to overcome a hurdle of host range limitations of Agrobacterium-mediated transformation. The PEG-mediated DNA transfer can be readily adapted to a wide range of plant

Disadvantages
Plant protoplasts are not easy to work with,

and the regeneration of fertile plants from protoplasts is problematic for some species. The DNA used is also susceptible to degradation and rearrangement

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