Direct Gene Transfer

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DIRECT GENE

TRANSFER

IN

PLANTS


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INTRODUCTION The first transgenic plant was produced via

Agrobacterium mediated-modifiedtransformation ofNicotiana tabacum

protoplasts by Horsch & co-workers in 1984.

Simultaneous development of othertechniques such as selectable markers

facilitated the development in genetic engg.

for obtaining transformed plants. But these techniques are not suitable for

monocotyledon plants as they are not natural

host ofAgrobacterium .


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Therefore, other methods of direct gene

transfer have been developed for use with

monocots & other species.

These can be categorized on the basis of

the use ofprotoplasts or cell & tissue as

the target material.


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TYPES OF DIRECT GENETRANSFER

Based on the type of target material, it can beof two types :

A. Direct Gene transfer in Protoplasts .

1) Electroporation method2) Chemically stimulated DNA uptake by

protoplasts method

3) Liposome Mediated method4) Microinjection method

5) Sonication method


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B. Direct Gene tranfer in Plant tissues.

1) Particle Bombardment method

2) Laser microbeam method

3) Silicon carbide fibres method


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DIRECT GENETRANSFER

IN

PROTOPLASTS


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1. ELECTROPORATION

It is based on the use of short electricalpulses of high field strength.

Electroporation causes the uptake of DNA

into protoplasts by temporarypermeabilization of the plasma membraneto macromolecules.

Protoplasts & foreign DNA are placed in abuffer, between two electrodes & a highintensity electric current is passed.


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Electric field damages membranes & creates

pores in membranes. DNA diffuses through

these pores immediately after the electric

field is applied, until the pores are resealed. Removal of pectin increases the amount of

DNA introduced by electroporation.

TMV was introduced in tobacco protoplastsby this method.

Plating efficiency can be as high as 0.5 %.


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CHANGES IN MEMBRANE DUE

TO ELECTROPORATION


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2. CHEMICALLY STIMULATED

DNA UPTAKE

PEG is the most commonly used chemicalfor this procedure. This process involvesmixing of freshly isolated protoplasts with

DNA & immediately adding 15-20 % PEGdissolved in a buffercontaining divalentcations.

This mixture is incubated for30 mins,protoplasts are washed & then plated inpetriplates for culture & growth .


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PEG mediated transformation is generally

preferred over eletroporation for stable

transformation of monocot protoplasts due

to relatively higher survival rates aftertreatment.

The transformation frequency of this

method is about 0.1 to 0.4 %.


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3. LIPOSOME-MEDIATED GENE

TRANSFER

Liposomes have also been used as a carrierfor the introduction of nucleic acid into plantprotoplasts.

The fusion of Liposomes with plantprotoplasts is stimulated by chemicals suchas PEG (endocytosis).

It is enhanced by including positivelycharged agents such as cations in thetransformation mixture or using the cationicliposome preparation.


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Other chemical agents like polycationPolybrene orLipofectin have also been

used for both transient & stable

transformation in maize protoplasts.

This method of direct gene transfer is

considered better than other methods of

transformation.


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4. MICROINJECTION

This method involves delivery of nucleic acid

to protoplasts using special capillary needles.

It involves skill of the worker to insert needle

into the cytoplasm or in the nucleus.

This is a labour intensive procedure that

requires special capillary needles, pumps,

micromanipulators, inverted microscope &

other equipments.


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In order to microinject protoplasts, the cells

need to be immobilized. The cells areimmobilized by:

1. The use ofholding pipette which holds the

cell by vacuum.2. Attachment of cells to poly-L-lysine coated

cover slips.

3. Embedding the cell in agarose, agar orsodium alginate.


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5. SONICATION

Ultra- sound waves of20 KHz has been used

to facilitate the uptake & transient expression

of a chloramphenicol acetyl transferase

(CAT) gene in protoplasts ofsugar beet (Betavulgaris ) & tobacco.

This method is superior than electroporation

method used for the same material.

Plating efficiency was also similar to

untreated cells.


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http://www.oardc.ohio-state.edu/plantranslab/finers_animated_gifs.htm


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DIRECT GENE

TRANSFER

IN

PLANT TISSUE


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1. PARTICLE BOMBARDMENT

This is the latest technology to transfer DNA intointact tissues.

In this method, microscopic (1-3m) tungsten orgold particles coated with genetically engineeredDNA are accelerated to high velocity, whichenables their entry into plant cells.

The particles, penetrate the cell wall & lodgethemselves within the cell & liberate the DNA,leading to the transformation of individual cellsof the explant.

It was developed by Prof. Stanford & coworkersof Cornell University(USA) in 1987.


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This process is also referred to as gene gun

method orparticle gun method orBiolisticprocess ormicroprojectile bombardment etc.

It was first used by Klien & coworkers for

transient assay in onion (Allium cepa )epidermis & was extended in 1988 to trials in

wheat, maize, rice & soybean.

Meristematic cells show higher transformationfrequency than non-dividing cells.


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Preparation of microprojectiles

The coating of DNA by precipitation is doneby: mixing 1.25-18 mg of microparticles with0.5-70 g of plasmid DNA in a CaCl (0.25-

2.5 M) & spermidine(0.1 M) solution. This solution is continuously vortexed to

ensure uniform coating .

After DNA precipitation, the microparticles aretransferred onto macrocarriermembranes,

allowed to dry & immediately used.


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2. LASER MICROBEAM

Weber & co-workers (1988) demonstrated use oflaser beam for transformation of plant cells. AnUV laser microbeam has been used to introduce

DNA into plant cells & chloroplasts. A 343 nm beam is directed into the optical path

of an inverted microscope. The focus of the laserbeam is so adjusted that it is identical with that

of the objective lens.

The laser beam is targeted by focusing on aspecimen in the microscope.


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This laser beam can then make holes in any

part of cell which is in focus.

Laser micropuncture of the cell wall & plasma

membrane allows uptake of plasmid DNA into

cells.

Brassica napus (rape seed) & microspores

have been used for transformation by this

technique.

20 % transformation efficiency has been

achieved by this method.


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3. SILICON CARBIDE FIBRES

Vortexing plasmid DNA & plant cells with

silicon carbide fibres of length 10-80m

produced tranformed cells, yet in lower

frequency.

Under vortex, silicon fibres penetrate cells and

create fine holes permitting entry of DNA into

the cells.


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CONCLUSION


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CONCLUSION

Recombinant DNA technology has made the

transfer of genesfrom any organism to any other organism a

technical feasibility.

Although, Agrobacterium mediated genetransferhas been the

most common method for gene transfer in

most of the dicotplants, its application to monocots is limited,

since this does

not infect monocots. For this reason, several


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PATIENCE

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Direct Gene Transfer