9/30/2016 Top 4 Methods of Gene Transfer (With Diagrams)

http://www.biologydiscussion.com/gene/gene­transfer/top­4­methods­of­gene­transfer­with­diagrams/11898 1/37

Biology Discussion

Top 4 Methods ofGene Transfer (WithDiagrams)Article Shared by Parul Kumar

This article throws light upon the topfour methods of gene transfer.

The top four methods of gene transferare: (1) DNA Transfer in Protoplasts(2) Free DNA Transfer to Intact Tissue(3) Agrobacterium Mediated GeneTransfer Method and (4) Integrationand Expression.

ADVERTISEMENTS:

The first transgenic plant was produced viaAgrobacterium mediated modifiedtransformation of Nicotiana tabacumprotoplasts by Horsch and co­workers in1984. Since then several dozen plant specieshave been genetically engineered usingdifferent techniques.

Simultaneous development of othertechniques such as selectable markersfacilitated the development in geneticengineering for obtaining transformedplants. But this technique is not suitable for

9/30/2016 Top 4 Methods of Gene Transfer (With Diagrams)

http://www.biologydiscussion.com/gene/gene­transfer/top­4­methods­of­gene­transfer­with­diagrams/11898 2/37

monocotyledon plants as they are notnatural host of Agrobacterium (There isevidence that limited gene transfer ispossible in monocots by this system).

Therefore, other methods of direct genetransfer have been developed for use withmonocots and other species. These can becategorized on the basis of the use ofprotoplasts or cell and tissue as the targetmaterials. Freshly isolated protoplasts areused for genetic transformation. Protoplaststage is for a short duration, before itregenerates cell wall, and DNA transfer isperformed during this period.

I. DNA Transfer in Protoplasts:

ADVERTISEMENTS:

(i) Electroporation

(ii) Chemically stimulated DNA uptake byprotoplasts

(iii) Liposomes

(iv) Micro­injection

(v) Sonication

II. DNA Transfer in Plant Tissues:

(i) Acceleration of DNA coated micro­particles

9/30/2016 Top 4 Methods of Gene Transfer (With Diagrams)

http://www.biologydiscussion.com/gene/gene­transfer/top­4­methods­of­gene­transfer­with­diagrams/11898 3/37

(ii) Laser micro­beam

(iii) Silicon carbide fibres

Direct uptake of DNA by isolated protoplastsis a genotype dependent response.Regeneration from protoplasts is notcommon in all the species. Due to this,production of fertile transgenic plants hasremained difficult in most of the cerealspecies. However, transgenic fertile plantshave been produced in Sorghum vulgare,Oryza sativa and Hordeum vulgare. Directdelivery of free DNA molecules into plantprotoplasts by physical (electroporation andmicro injection) and chemical (polyethyleneglycol) methods have been developed tofacilitate DNA delivery across the plasmamembrane.

Method # 1. DNA Transfer inProtoplasts:

Electroporation:

This method is based on the use of the shortelectrical pulses of high field strength.Electroporation causes the uptake of DNAinto protoplasts by temporarypermeabilization of the plasma membraneto macromolecules. Protoplasts and foreignDNA are placed in a buffer between twoelectrodes and a high intensity electriccurrent is passed, the alternating current of

9/30/2016 Top 4 Methods of Gene Transfer (With Diagrams)

http://www.biologydiscussion.com/gene/gene­transfer/top­4­methods­of­gene­transfer­with­diagrams/11898 4/37

about 1 MHz is applied to align theprotoplast by di­electrophoresis.

Once aligned, fusion is induced by applyingone or more direct current pulses (1­3kV/cm, 10­100 (is), then the alternating field isreapplied briefly to maintain closemembrane contact for fusion (Fig. 16.1).Electric field damages membranes andcreates pores in membranes. DNA diffusesthrough these pores immediately after theelectric field is applied, until the pores areresealed. Technique is optimized by usingappropriate electric field strength (definedas the applied voltage divides by thedistance between two electrodes).

The optimum field strength isdependent on the followings:

1. The pulse length of electric current

2. Composition and temperature of thebuffer solution

3. Concentration of foreign DNA in thesuspension

4. Protoplasts density, and

5. Size of the protoplasts.

It has been demonstrated that the removalof pectin from the plant wall increases the

9/30/2016 Top 4 Methods of Gene Transfer (With Diagrams)

http://www.biologydiscussion.com/gene/gene­transfer/top­4­methods­of­gene­transfer­with­diagrams/11898 5/37

amount of DNA which can be introduced byelectroporation. Tobacco mosaic virus wasintroduced in tobacco protoplasts by thismethod. Electroporation has been usedsuccessfully for transient (when foreign genewhich is present in cell but not integrated inthe chromosome, shows expression incytoplasm) and stable transformation(foreign gene integration in hostchromosome and is expressed) ofprotoplasts from a wide range of species.

Plating efficiency (i.e., number of coloniesrecovered out of number of cells transferredon plates) of electroporated protoplastsgrown on selection medium (containingselective marker) can be as high as 0.5%.The highest plant transformationefficiencies have been reported for tobacco,with 0.2% of electroporated leaf mesophyllprotoplasts giving rise to transgenic calli.Low transformation efficiency is common incereals, e.g., in rice 0.002% efficiency wasrecorded.

9/30/2016 Top 4 Methods of Gene Transfer (With Diagrams)

http://www.biologydiscussion.com/gene/gene­transfer/top­4­methods­of­gene­transfer­with­diagrams/11898 6/37

Chemically Stimulated DNA Uptake:

Direct uptake of DNA by protoplasts isstimulated by polyethylene glycol (PEG) andPEG is the most widely used chemical forthis purpose. PEG mediated transformationinvolves mixing of freshly isolatedprotoplasts with DNA and immediatelyadding 15­20% PEG dissolved in a buffercontaining divalent cations. This mixture isincubated for 30 minutes; protoplasts arewashed and then plated in Petri plates forculture and growth.

The optimization of transformationfrequencies by this method includefactors that follows:

1. PEG concentration in the mixture.

2. Composition and concentration of saltsused.

3. The pH of the solution.

4. Concentration of the foreign DNA.

5. Size and form (linear, super­coiled) of theDNA molecules used.

6. Culture and selection techniques used forprotoplasts.

9/30/2016 Top 4 Methods of Gene Transfer (With Diagrams)

http://www.biologydiscussion.com/gene/gene­transfer/top­4­methods­of­gene­transfer­with­diagrams/11898 7/37

PEG mediated transformation is generallypreferred over electroporation for stabletransformation of monocot protoplasts dueto relatively higher survival rates aftertreatment.

PEG also stimulates the uptake of liposomesand improves the efficiency ofelectroporation. PEG causes precipitation ofionic macromolecules like DNA andstimulates their uptake byendocytosis. PEG mediated DNA uptaketypically transforms 0.1 to 0.4% of the totalprotoplasts treated. Production oftransgenic plants depends upon theregeneration competence of the transformedprotoplasts.

In case of Petunia, 40% transformed calliderived from mesophyll protoplasts could beinduced to form fertile plants. This is equalto about 0.1% transformation efficiency ofthe treated protoplasts. In different speciesdifferent transformation efficiency has beenobserved, e.g., in embryogenic protoplastssuspension of rice was 0.0004% and, soya­bean and tobacco was 0.7­1%.

Liposomes:

Liposomes have also been used as a carrierfor the introduction of nucleic acid intoplant protoplasts. Liposomes are small lipid

9/30/2016 Top 4 Methods of Gene Transfer (With Diagrams)

http://www.biologydiscussion.com/gene/gene­transfer/top­4­methods­of­gene­transfer­with­diagrams/11898 8/37

sacs containing plasmids and are preparedartificially. The fusion of liposomes withplant protoplasts is stimulated by chemicalssuch as PEG (endocytosis). Liposomesmediated transformation has been achievedby including positively charged agents suchas cations in the transformation mixture orusing the cationic liposome preparation(Fig. 16.2).

Other chemical agents like polycationPolybrene or lipofectin have also been usedfor both transient and stable transformationfor maize protoplast. Cationic liposome andpolycation mediated DNA delivery are newprotoplasts transformation methods and areconsidered better than other methods oftransformation. There are severaladvantages for the use of this technique.

1. Protection of DNA/RNA from nucleasedigestion.

2. They have low cell toxicity.

3. Encapsulation of nucleic acids makesthem more stable during storage.

4. High degree of reproducibility.

9/30/2016 Top 4 Methods of Gene Transfer (With Diagrams)

http://www.biologydiscussion.com/gene/gene­transfer/top­4­methods­of­gene­transfer­with­diagrams/11898 9/37

5. This method is applicable to wide range ofplant cell types.

Microinjection:

Delivery of nucleic acids to protoplasts orintact cells via microinjection is a labourintensive procedure that requires specialcapillary needles, pumps,micromanipulators, inverted microscopeand other equipment. However, injectioninto the nucleus or cytoplasm is possible andcells can be cultured individually to producecallus or plants.

In this way selection of transformants bydrug resistance or marker genes may beavoided. This method involves skill of theworker to insert needle into the cytoplasmor in the nucleus. The basic technique issimilar to that used for animal cellmicroinjection. In order to microinjectprotoplasts or other plant cells, the cellsneed to be immobilized (Fig. 16.3).

The cells are immobilized by:

1. The use of a holding pipette which holdsthe cells by vacuum.

2. Attachment of cells to poly­L­lysin coatedcover slips.

9/30/2016 Top 4 Methods of Gene Transfer (With Diagrams)

http://www.biologydiscussion.com/gene/gene­transfer/top­4­methods­of­gene­transfer­with­diagrams/11898 10/37

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

Glass micropipette are prepared to haveopenings of about 0.3 µM in diameter andare inserted into plant cell cytoplasm andnuclei with the aid of a micromanipulatorsdevice. A syringe like device is used for thecontrolled delivery of volume (10 – 10 1)into the plant cell.

Most plant cells are injected while keepinginside micro­droplets (2­50 µl) of mediumusing a chamber which is sterile, vibrationfree and permits temperature and humidityregulation. A maximum of 100­200 cells perhour can be microinjected by this method.

The recovery of trans­formants is dependentupon the regeneration ability of themicroinjected cells. Different methods havebeen used to grow injured (microinjected)single cells or protoplasts. Hanging droplets,covered under thin layer of agar or agarose,and micro­culture have been used (Fig.16.4). Attempts have been made to injectlinear or super­coiled DNA, in cytoplasm or

­11 ­4

9/30/2016 Top 4 Methods of Gene Transfer (With Diagrams)

http://www.biologydiscussion.com/gene/gene­transfer/top­4­methods­of­gene­transfer­with­diagrams/11898 11/37

in nucleus. Nuclear injections are foundbetter for transformations.

Sonication:

Mild sonication (20 KHz ultrasound) hasbeen used to facilitate the uptake andtransient expression of a chloramphenicolacetyltransferase (CAT) gene in protoplastsof sugar beet (Beta vulgaris) and tobacco.This method was found superior thanelectroporation method used for the samematerial. Plating efficiency was also similarto untreated cells. However, transgenicplant production using this technique hasnot been reported so far.

Method # 2. Free DNA Transfer toIntact Tissue:

Acceleration of DNA Loaded Micro­particles (Particle Gun or Biolistic®Method):

This is latest technology to transfer DNAinto intact tissues. Several devices aredeveloped using different methods. All toachieve the transfer of micro­sized particles(micro­projectiles) coated with DNA topenetrate the cells. In this procedure micron

9/30/2016 Top 4 Methods of Gene Transfer (With Diagrams)

http://www.biologydiscussion.com/gene/gene­transfer/top­4­methods­of­gene­transfer­with­diagrams/11898 12/37

size tungsten or gold particles areaccelerated in a gun barrel to velocitiessufficient for non­lethal penetration of cellwalls and membranes.

Klein and co­workers in 1987 developed andused for the first time the particle gun totransfer chimeric DNA and viral RNAmolecules into intact onion cells. Tungstenacted as a carrier of nucleic acids because itwas available in micro­size balls, non­toxicto cells, and dense enough (high density) forrapid penetration of target material. Micro­projectile mediated transformation is amechanical method of introducing DNA into any plant species. This method can besuccessfully used where plasmids orprotoplasts mediated transformation cannotbe used.

An acceleration device is used to propelparticles (micro projectiles) carryingplasmid DNA is called by various namesbased on machine or technique used toaccelerate the particles such as ‘particle guntechnology’, ‘biolistic method’, ‘DNAbombardment’, ‘particle acceleration ofDNA method’ and ‘electric discharge particleacceleration method’.

This is a quick method of stabletransformation and testing a gene for

9/30/2016 Top 4 Methods of Gene Transfer (With Diagrams)

http://www.biologydiscussion.com/gene/gene­transfer/top­4­methods­of­gene­transfer­with­diagrams/11898 13/37

cell and organelle specific expression,this technique has three components:

1. The basic equipment to generate particleacceleration.

2. Metal particles coated with precipitatedDNA (desired gene).

3. Plant tissues to be used for particlepenetration. The method for regenerationshould be previously standardized andproper tissues be selected for bombardment.

(a) Instrument:

The instrument is commercially available.Prototype was designed by Klein and co­workers. It uses the explosive force of gunpowder (0.22 caliber gun cartridge) toaccelerate a polypropylene cylindricalmacro­projectile. Thin piece ofpolypropylene macro­projectile is loadedwith micro­projectiles coated with DNA.Gun powder explosion forces thin macro­projectile to move with high speed towardanother end of barrel, where it is blocked bya polycarbonate disc having an aperture.

Macro­projectile is stopped but micro­projectiles move fast through the aperturetowards tissue placed in the same direction.For each transfer 50 µg tungsten is

9/30/2016 Top 4 Methods of Gene Transfer (With Diagrams)

http://www.biologydiscussion.com/gene/gene­transfer/top­4­methods­of­gene­transfer­with­diagrams/11898 14/37

accelerated up to 2000 ft per second in apartial vacuum. With this speed, particlesreach up to lower layers of cells in targettissues (Fig. 16.5).

The other devices are similar in basic designconcept but use different methods toaccelerate particles like use of compressedair or gas. Compressed air (130 kg/cmpressure) has been used to accelerate micro­projectiles at velocities (approximately 440m/sec) necessary to achieve DNA delivery toplant cells.

An electric discharge particle accelerationdevice differs in basic design from the abovedescribed devices. In this device, a highvoltage discharge (14 KV current) deliveredto a small water droplet which quicklyvaporizes and releases energy to propel DNA

2

9/30/2016 Top 4 Methods of Gene Transfer (With Diagrams)

http://www.biologydiscussion.com/gene/gene­transfer/top­4­methods­of­gene­transfer­with­diagrams/11898 15/37

coated gold spheres into target cells (Fig.16.6).

In similar way to above devices, a DNAcarrier is attracted (accelerated) due topotential differences, stopped in­between bya screen, DNA coated particles cross thescreen and fly towards target tissue anddeliver the DNA into cells.

(b) DNA coating:

This is a sophisticated technology andrequires precise preparation of DNA coatedgold or tungsten particles. The particlesshould have following properties.

1. High density (19 g/cm or greater) toensure proper acceleration and penetrationthrough cell walls.

2. Size (0.5­5 µm) should match with size ofthe cells. Large sized spheres can be usedwith large cell size.

3. Gold is costlier than tungsten, but it doesnot oxidize like tungsten. DNA isprecipitated onto the particles prior tobombardment. Most commonly, CaCl andspermidine are used to precipitate plasmidDNA (the desired gene attached to plasmidis suitable for integration into plantgenome) onto tungsten particles. Ethanol is

2

2

9/30/2016 Top 4 Methods of Gene Transfer (With Diagrams)

http://www.biologydiscussion.com/gene/gene­transfer/top­4­methods­of­gene­transfer­with­diagrams/11898 16/37

also used for precipitation of the DNA ongold particles.

(c) Plant tissue:

Plant tissue used for transformation shouldbe competent to regenerate. Mostlyembryogenic tissue is an ideal material fortransformation (e.g., in corn, cotton, soya­bean, papaya and wheat) but transformedplants have been obtained after leafbombardment in tobacco, and stembombardment in cranberry. Normally,reporter gene and selectable marker genesare used to isolate and select transformedcells/plantlets.

Laser Micro­beam:

Weber and co­workers (1988) demonstrateduse of laser beam for transformation of plantcells. An ultraviolet (UV) laser micro­beamhas been used to introduce DNA into plantcells and chloroplasts. A 343 nm beam(wavelength of UV is 200 to 400 nm) isdirected through an adjustable attenuatorinto the optical path of an invertedmicroscope.

The focus of the laser beam is adjusted sothat it is identical with that of the objectivelens. The laser beam is targeted by focusingon a specimen in the microscope. This laserbeam can then make holes in any part of cell

9/30/2016 Top 4 Methods of Gene Transfer (With Diagrams)

http://www.biologydiscussion.com/gene/gene­transfer/top­4­methods­of­gene­transfer­with­diagrams/11898 17/37

which is in focus. Laser micro­puncture ofthe cell wall and plasma membrane allowsuptake (entry) of plasmid DNA into cells.

Brassica napus (rapeseed) cells andmicrospores have been used fortransformation by this technique. Thistechnique has also been used to transfergenes into isolated chloroplasts andchloroplasts of intact protoplasts. 20%transformation was achieved by this methodbut fertile plants are yet to be produced bythis method.

Silicone Carbide Fibres:

Microinjection and electroporation methodshave also been used for transfer of DNAusing intact plant cells and tissues.Similarly, vortexing plasmid DNA and plantcells with silicon carbide fibre (0.6 µm indiameter and 10­80 µm in length) producedtransformed cells at low frequency. Undervortex (vigorous shaking by vibration),silicone fibres penetrate cells and create fineholes permitting entry of DNA.

DNA uptake by imbibition’s in driedembryos of cereals and legume species hasalso been reported. This is a very simplemethod and dried somatic embryos can alsobe used for this method. Only transient geneexpression has been observed and stable

9/30/2016 Top 4 Methods of Gene Transfer (With Diagrams)

http://www.biologydiscussion.com/gene/gene­transfer/top­4­methods­of­gene­transfer­with­diagrams/11898 18/37

transformations are yet to be achieved bythis method. Attempts have been made tointroduce gene in pollen grains bymicroinjection in the anthers. These pollengrains can be used for fertilization to obtaintransgenic plants.

Method # 3. AgrobacteriumMediated Gene Transfer Method:

Transgenic plants are produced bytwo methods:

(i) Indirect gene transfer using plasmid as avector and

(ii) Direct gene transfer. Indirect genetransfer requires construction of a vector forcarrying foreign genes based on Ti or Riplasmids.

Vectors are the carrier DNAs into which‘foreign’ DNAs or genes of interest areinserted to make a recombinant DNA(rDNA). Vectors along with this ‘foreign’DNA (i.e., rDNA) are then introduced intoappropriate host cell. Vectors are of twotypes­cloning vectors [used for obtainingmillions of copies (cloning) of DNAsegment] and expression vectors [used forexpression of cloned gene to produce theproduct (protein)].

9/30/2016 Top 4 Methods of Gene Transfer (With Diagrams)

http://www.biologydiscussion.com/gene/gene­transfer/top­4­methods­of­gene­transfer­with­diagrams/11898 19/37

Plasmids are most commonly used vectorsin gene cloning work. Isolation andpurification of plasmids is a very routineexperimental procedure. Several methodsare available for isolation and purification ofplasmid. The most critical stage in theprocess is lysis of the cell wall which is justsufficient for isolation of plasmid DNAwithout contamination by chromosomalDNA. Clear lysate contains plasmid. Alkali­SDS lysis and rapid boiling procedures arecommonly used methods for plasmidisolation.

Most vectors carry marker genes whichallow recognition e.g., antibiotic resistance –selectable markers, e.g., npt II (kanamycinresistance). Other features include (i)multiple unique restriction sites – asynthetic poly linker and (ii) bacterial originof replication e.g. (Col El). The problem isthat a vector having these properties maynot be easy to transfer to plant system.Therefore, Agrobacterium Ti plasmid ispreferred because of (1) wide host range and(2) presence of T­DNA border sequence.Plant cells do not have any endogenousplasmid. The plasmid vectors used for genetransfer in plants are based on pTi or pRi(tumour inducing or root inducing plasmid)present in Agro­bacterium tumefactions orA. rhizogenes.

9/30/2016 Top 4 Methods of Gene Transfer (With Diagrams)

http://www.biologydiscussion.com/gene/gene­transfer/top­4­methods­of­gene­transfer­with­diagrams/11898 20/37

Structure of T­DNA is described in thechapter ­14. Presence of auxin and cytokininproducing genes caused tumour formationin plants, when T­DNA present in pTi istransferred in plant cell.

Therefore, these tumour forming genes areremoved from T­DNA (called as disarmingthe plasmid) and gene of interest isintroduced in that place (between left andright border of T­DNA). Thus thisrecombinant T­DNA is ready to transfer agene by natural mechanism of gene transferin a dicot host cell.

Since pTi or pRi are large plasmids,modified plasmids (co­integrative andbinary) are prepared from Ti plasmid andused as described later on in this chapter.Thus by placing foreign genes into T­DNAregion of Ti­plasmid, it is possible to clone(make copies) the introduced genes with themultiplication of plasmid residing inside thebacteria (self replication of plasmid makesmillions of copies) which is grown on amedium and with the multiplication ofbacterial population, residing plasmid is alsomultiplied by this method. It is possible toexploit the natural ability of Agrobacteriumto transfer new DNA into the plant genome.

T­DNA: Transfer Mechanism:

9/30/2016 Top 4 Methods of Gene Transfer (With Diagrams)

http://www.biologydiscussion.com/gene/gene­transfer/top­4­methods­of­gene­transfer­with­diagrams/11898 21/37

Though exact mechanism of T­DNA transferis not clearly known, effective role of virregion is known in the process of transfer.Genes in the vir region is activated byacetosyringone, a phenolic substancesecreted by wounded cells of the host.

The phenolic signal molecules binds to thevir A gene product, the vir A proten (Fig.16.7). As consequence of this, several virrgion genes are activated and these geneproducts (proteins) help in copy of T­DNAand its insertion into host cell.

i. Nicking between 3rd and 4th base of 25 bprepeats (bottom strand). Vir­D operonencodes for an endonuclease that cause nickformation.

ii. Initiation of DNA synthesis in 5′­3′direction.

iii. Involvement of bacterial genome –synthesis and secretion of glucose, cellulose,fibrils, and cell surface proteins. This iscommon physiological response in all soilbacteria and is involved with pathogeniccharacters.

The generation of the T­strand is the firststep in the complex process of Agro­bacterium mediated plant celltransformation. Following its formation, this

9/30/2016 Top 4 Methods of Gene Transfer (With Diagrams)

http://www.biologydiscussion.com/gene/gene­transfer/top­4­methods­of­gene­transfer­with­diagrams/11898 22/37

DNA must pass through the bacterial cellmembrane, the bacterial cell wall, the plantcell wall and plant cell and nuclearmembranes. Once inside the nucleus, the T­DNA must finally integrate stably into theplant cell genome (Fig. 16.8). During thisentire transit process, the T­DNA strandalso must avoid degradation by nuclease.The T­DNA exists as a DNA proteincomplex. The T­DNA complex protects itand mediates its travel.

The final step in the genetic transformationof plant cell is integration of T­DNA copy,presumably T­strand, into plant cell DNA. Ithas been argued that the T­strand might beconverted to a double stranded (ds) DNAprior to integration.

9/30/2016 Top 4 Methods of Gene Transfer (With Diagrams)

http://www.biologydiscussion.com/gene/gene­transfer/top­4­methods­of­gene­transfer­with­diagrams/11898 23/37

Vectors Based on Ti and Ri Plasmids:

The Ti or Ri­plasmid cannot be useddirectly. There are limitations for direct useof these plasmids.

These are:

(i) Large size of vector make it difficult tomanipulate

(ii) Absence of unique restriction enzymessites and

(iii) Tumour induction.

Therefore, vectors are designed with usefulcharacteristics. This involves­removal oftumour induction property or disarming theplasmid. This is achieved by replacing oftumour induction genes in T­DNA byselectable markers such as npt­II(kanamycine). Promoters and

9/30/2016 Top 4 Methods of Gene Transfer (With Diagrams)

http://www.biologydiscussion.com/gene/gene­transfer/top­4­methods­of­gene­transfer­with­diagrams/11898 24/37

polyadenylation signal isolated fromoctopine and nopaline synthase genes wereused for expression of selectable markers.

As there is no excess production of planthormones, whole plants transformed withsuch disarmed Agrobacterium strains can beproduced and detected by the production ofopines. When a selectable marker gene(kanamycin resistant) is introduced,transformed cells can be selected by theirability to grow on media containing theselective antibiotic. Untransformed cells willnot survive on this medium. Otherpromoters – one – CaMV35S, CaMV19Sisolated from cauliflower mosaic virus havealso been used. Therefore, it is concludedthat T­DNA and Vir genes are two essentialcomponents of a vector.

Cointegrative Vectors:

Cointegrative vectors recombine. via DNAhomology, with an intermediate cloningvector, which is used for manipulation andcloning of the gene in E. coli. Agrobacteriumcontaining cointegrative vector and E. colicontaining intermediate cloning vector areallowed to undergo conjugation, but theintermediate vector cannot replicate inAgrobacterium so it has to transfer themarker genes as well as the DNA segment tothe resident Ti plasmid (cointegrative

9/30/2016 Top 4 Methods of Gene Transfer (With Diagrams)

http://www.biologydiscussion.com/gene/gene­transfer/top­4­methods­of­gene­transfer­with­diagrams/11898 25/37

vector) through recombination in the regionof DNA homology.

Example of such vector – pGV3850 fromnopaline type Ti plasmid, where almost allT­DNA has been replaced by pBR322, asmall E. coli cloning vector (Bolivar andRodriguez prepared and hence name –plasmid BR, followed by experimentnumber). The intermediate vector(pGV1103) based on pBR322 is conjugatedinto pGV3850 at the region of pBR322homology.

Binary Vector:

A significant advance that bypass theproblem of Ti­plasmid size was thediscovery that the T­DNA and the vir regioncould be separated on two differentplasmids without loss of the T­DNA transfercapacity, i.e., they worked in a trans as wellas a cis configuration. This discovery led tothe development of binary T­DNA vectorsthat involve two plasmids.

The small binary T­DNA plasmid has a widehost range that can replicate both in E. coliand Agrobacterium cells. The desiredforeign gene is inserted into the binary T­DNA plasmid between the left and rightborder sequences. A selectable plant markergene is also inserted and (along with desired

9/30/2016 Top 4 Methods of Gene Transfer (With Diagrams)

http://www.biologydiscussion.com/gene/gene­transfer/top­4­methods­of­gene­transfer­with­diagrams/11898 26/37

foreign gene) allow selection of transformedplant material. Several plant species havebeen transformed by this method.

Transformation Technique:

The critical information that made Agro­bacterium mediated gene transfer systemspossible came from the elegant work byChilton et al. (1977) who showed that incrown gall disease a region of bacterialplasmid DNA (the T­ DNA) is transferred tochromosomal DNA in the plant nucleus,stably maintained there, and expressed inthe absence of the bacterium.

The first transgenic Nicotiana tabacum plantwas produced by Horsh and co­workers in1984 using Agrobacterium. Agro­bacteriummediated gene transfer methods are beingdeveloped for a wide range ofdicotyledonous plants and gymnospermspecies. The gene tagging approach is usedto demonstrate transformation.

T­DNA containing either a reporter orstrong transcriptional enhancer transformsa large number of cells. Normally bacteriaare incubated with plant cells (few hours tofew days) during that period T­DNA transfertakes place. The cells are then washed andtreated with antibiotics to remove thebacteria. The cells are then cultured in the

9/30/2016 Top 4 Methods of Gene Transfer (With Diagrams)

http://www.biologydiscussion.com/gene/gene­transfer/top­4­methods­of­gene­transfer­with­diagrams/11898 27/37

presence of the selectable agent, andtransformed shoots are regenerated andcharacterized. (Fig. 16.9)

Plasmids of Agro­bacterium have been usedas vector for transfer of foreign DNA into anumber of dicot species. However, seedlegumes are still not amenable, exception isGlycine max. Monocotyledons (cereals)cannot be used as Agro­bacterium is hostspecific and do not infect monocots but withan exception of Asparagus (which has beentransformed with this technique). There isno cambial activity and wound healingprocess in monocots, therefore,acetosyringone is not produced; this results

9/30/2016 Top 4 Methods of Gene Transfer (With Diagrams)

http://www.biologydiscussion.com/gene/gene­transfer/top­4­methods­of­gene­transfer­with­diagrams/11898 28/37

in failure of vir genes to recognize bychemoreception.

(i) Pre­requisites for agro­infection:

1. Production of acetosyringone by the hostplant cells.

2. Bacteria have access to actively dividingcells (DNA replication) such as meri­stems,fresh protoplasts, de­differentiated tissues.

3. Regeneration in transformed tissuesshould be possible; otherwisetransformation will be of no use.

(ii) Explants for co­cultivation­following materials can be used:

(a) Protoplasts

(b) Cell suspension cultures

(c) Callus\thin cell layers – epidermis, tissueslice, organ section (leaf disc, section ofroots, stem or flower).

(d) Wounded and inoculated whole plant.

Selectable Markers:

Selectable markers are usually required forefficient recovery of transgenic cells andplants. After gene transfer, transformed cellsare few in number compared to

9/30/2016 Top 4 Methods of Gene Transfer (With Diagrams)

http://www.biologydiscussion.com/gene/gene­transfer/top­4­methods­of­gene­transfer­with­diagrams/11898 29/37

untransformed cells. It is not possible toseparate transformed and non­transformedcells by any physical method. A selectablemarker gene incorporated with the desiredgene helps the growth of transformed cellson a nutrient medium containingcorresponding selective agent.

The availability of multiple selectablemarkers is useful in developing efficienttransformation methods for diverse speciesas well as for the introduction of multiplenovel traits (characters) through successfultransformation and regeneration oftransformed plants from such cells (Table16.1).

Selective agents differ in their toxicity todifferent plant species. The differentdevelopmental states of the plant cells ortissues give different response to theselectable marker. The cells will reactdifferently than whole plant or organ.

Therefore, it is necessary to use correctconcentration of selective agent for thetransformed cells of a given species to selectthe cells and to inhibit the growth ofuntransformed cells. This can be illustratedby following example.

Transgenic plants of Lycopersiconesculentum, Brassica napus and Lactuca

9/30/2016 Top 4 Methods of Gene Transfer (With Diagrams)

http://www.biologydiscussion.com/gene/gene­transfer/top­4­methods­of­gene­transfer­with­diagrams/11898 30/37

sativa can be selected on low kanamycinconcentration (15­100 mg/1), while otherplants like Beta vulgaris requires highkanamycin concentration (400 mg/1) asselection agent.

Herbicides are generally more toxic to plantcells than antibiotics. This is due to theirspecific mode of action in plant cells as wellas their efficient uptake and translocationwithin the plant tissues. Herbicides andother highly toxic compounds may requiredelayed application in order to ensure thatthe transformed cells have producedsufficient quantities of enzyme responsiblefor the protection to such compound.

Callus and cell cultures are better systemsthan organized explants to achievetransformations and selection oftransformed cells. Explants may give rise toorgans from untransformed cells which mayescape selective agent. This is not possiblewith isolated cells and only transformedcells will be able to grow on the selectiveagent.

9/30/2016 Top 4 Methods of Gene Transfer (With Diagrams)

http://www.biologydiscussion.com/gene/gene­transfer/top­4­methods­of­gene­transfer­with­diagrams/11898 31/37

Reporter Genes:

In all transformation experiments,regeneration of non­transformed plants isalso observed along with transformedplants. If these non­transformed plants canbe detected at an early stage, a lot of time,labour and analyses can be saved. There areseveral methods to screen and identify thetransformed plants, but usually thesemethods are laborious and time consuming,e.g. southern blotting technique to compareDNA sequences requires large amount oftissue (DNA) for comparison.

The other methods use selective agent. Inboth these methods, prolonged growth andsubcultures are involved before selectioncan be made. The use of reporter geneeliminates these drawbacks andtransformed plants can be recognised easily.A reporter gene is a coding unit whoseproduct is easily assayed (such as GUSwhose product can easily be detected byhisto­chemical assay). This gene may beconnected to any promoter of interest sothat expression of the gene can be used toassay promoter function.

In fact, the reporter gene describes thetransfer and expression of other promoter.Two genes that are now widely used for thispurpose are those coding for β­

9/30/2016 Top 4 Methods of Gene Transfer (With Diagrams)

http://www.biologydiscussion.com/gene/gene­transfer/top­4­methods­of­gene­transfer­with­diagrams/11898 32/37

glucoronidase (GUS) and luciferase (LUC).The coding regions of these non­plant geneshave been fused to plant promoters andpolyadenylation sequences such that theygive high level of expression in plant cells(Table 16.2).

The anthocyanin regulatory genes can serveas a unique reporter system in maize andsome other cereals. Transformed cellsaccumulate reddish purple anthocyaninpigments. The anthocyanin genes areextremely sensitive marker used inArabidopsis and Nicotiana transgenicplants. Without selection agent, transformedplants are selected with the help of scoreablegene or reporter gene.

Examples of Transgenic Plants­ HerbaceousDicot – Tobacco, Petunia hybrida, tomato,potato, egg plant, Arabidopsis thaliana,lettuce, Apium graveolens (celary),sunflower, flax, rape oil seed, cauliflower,cabbage, cotton, soyabean, pea, chicory,carrot, licorice, sweet potato, kiwi, papaya,grape, rose, Chrysanthemum, etc.

9/30/2016 Top 4 Methods of Gene Transfer (With Diagrams)

http://www.biologydiscussion.com/gene/gene­transfer/top­4­methods­of­gene­transfer­with­diagrams/11898 33/37

Woody Dicot – Populus, Malus, Pyruscommunus, Azadiraclita indica

Monocot – Asparagus, Secale cereale, Oryzasativa, Triticum aestivum, Zea mays, Avenasativa.

Gymnosperm – Picea glauca.

Method # 4. Integration andExpression:

For expression heterologous proteins inplants, the unique capacity of a soilbacterium Agrobacterium tumefaciens tointroduce part of its resident plasmid intothe plant genomic DNA has been widelyutilized. The above bacteria causes acancerous growth in the infected plant dueto the introduction of this DNA fragmentderived from the plasmid.

The plasmid is called the Ti plasmid and theintroduced fragment of DNA is called T­DNA. The tumorigenic genes, i.e., thosewhich cause the tumour are deleted from theT­DNA and then the plasmid is incapable ofcausing tumours but is still capable oftransferring T­DNA to the plant. Anyheterologous DNA can be introduced intothe plant as part of the above plasmid.

To ensure high expression of theheterologous gene, strong promoters

9/30/2016 Top 4 Methods of Gene Transfer (With Diagrams)

http://www.biologydiscussion.com/gene/gene­transfer/top­4­methods­of­gene­transfer­with­diagrams/11898 34/37

derived from plants viruses, like thecauliflower masaic virus are generally used.Several heterologous genes have beenexpressing plants in the recent years eitherto improve the field performance of crops orto impart added qualities to the edible part,like essential vitamins or even antigenicproteins, which can serve as vaccines againstcommon disease like cholera. The low costof growing such plants expressionheterologous proteins, some of which canhave high commercial values, has madeexpression such proteins in plants a veryattractive proposition.

Several plants viruses can in fact multiply intheir plant hosts to a very high level at a veryshort time. Some of these viruses, like thetobacco mosaic virus have been used toclone heterologous genes by replacing someof the non­essential viral genes. Suchengineered viruses when allowed to infectplants, are capable of expressing theheterologous gene to very high levels in amatter of few weeks in the infected plants.

Some of the characteristic features ofDNA integrations are:

1. Integration occurs at random sites in thegenome.

9/30/2016 Top 4 Methods of Gene Transfer (With Diagrams)

http://www.biologydiscussion.com/gene/gene­transfer/top­4­methods­of­gene­transfer­with­diagrams/11898 35/37

Welcome toBiologyDiscussion! Ourmission is to provide anonline platform to helpstudents to share notesin Biology. This websiteincludes study notes,research papers, essays,articles and other alliedinformation submittedby visitors like YOU.

Before sharing yourknowledge on this site,please read thefollowing pages:

2. Linear DNA is better integrated thancircular DNA.

3. Multi­copy integration usually occurs intandem at one site (copies attached inseries).

4. Higher concentration of DNA results inhigh integration frequency with multiplecopies of gene incorporated.

5. Use of carrier DNA (plasmid) in directDNA transfer methods enhance multiplecopy integration.

Home ››

9/30/2016 Top 4 Methods of Gene Transfer (With Diagrams)

http://www.biologydiscussion.com/gene/gene­transfer/top­4­methods­of­gene­transfer­with­diagrams/11898 36/37

1. Content Guidelines2. Privacy Policy3. TOS4. Disclaimer Copyright

Share YourKnowledge

ADVERTISEMENT

ABOUTUS

Share

Your

Knowledg

e

Content

Guideline

s

Privacy

Policy

ToS

Disclaime

r

SUGGESTIONS

Report

9/30/2016 Top 4 Methods of Gene Transfer (With Diagrams)

http://www.biologydiscussion.com/gene/gene­transfer/top­4­methods­of­gene­transfer­with­diagrams/11898 37/37

Copyright © 2016 BiologyDiscussion.com, All rights reserved.

Spelling

and

Grammat

ical

Errors

Suggest

Us