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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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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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THANKSFOR
YOUR
PATIENCE
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