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9/30/2016 Methods of Gene Transfer in Plants: 2 Methods

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Biology Discussion

Methods of GeneTransfer in Plants: 2MethodsArticle Shared by Nandkishor Jha

This article throws light upon the twomethods used for gene transfer in plants.

The two methods are: (1) VectorMediatedGene Transfer and (2) Direct or VectorlessDNA Transfer.

Gene Transfer Methods:

The gene transfer techniques in plantgenetic transformation are broadlygrouped into two categories:

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I. Vectormediated gene transfer

II. Direct or vector less DNA transfer

The salient features of the commonly usedgene (DNA) transfer methods are given inTable 49.1.

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Method # I. VectorMediated GeneTransfer:

Vectormediated gene transfer is carried outeither by Agrobacteriummediatedtransformation or by use of plant viruses asvectors.

AgrobacteriumMediated GeneTransfer:

Agrobacterium tumefaciens is a soilborne,Gramnegative bacterium. It is rod shapedand motile, and belongs to the bacterialfamily of Rhizobiaceae. A. tumefaciens is aphytopathogen, and is treated as thenature’s most effective plant geneticengineer.

Some workers consider this bacterium as thenatural expert of interkingdom genetransfer. In fact, the major credit for thedevelopment of plant transformation

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techniques goes to the natural uniquecapability of A. tumefaciens. Thus, thisbacterium is the most beloved by plantbiotechnologists.

There are mainly two species ofAgrobacterium:

i. A. tumefaciens that induces crown galldisease.

ii. A. rhizogenes that induces hairy rootdisease.

Crown Gall Disease and Ti Plasmid:

Almost 100 years ago (1907), Smith andTownsend postulated that a bacterium wasthe causative agent of crown gall tumors,although its importance was recognizedmuch later. As A. tumefaciens infectswounded or damaged plant tissues, ininduces the formation of a plant tumorcalled crown gall (Fig. 49.1). The entry of thebacterium into the plant tissues is facilitatedby the release of certain phenoliccompounds (acetosyringone,hydroxyacetosyringone) by the woundedsites.



Crown gall formation occurs when thebacterium releases its Ti plasmid (tumorinducing plasmid) into the plant cellcytoplasm. A fragment (segment) of Tiplasmid, referred to as TDNA, is actuallytransferred from the bacterium into the hostwhere it gets integrated into the plant cellchromosome (i.e. host genome). Thus,crown gall disease is a naturally evolvedgenetic engineering process.

The TDNA carries genes that code forproteins involved in the biosynthesis ofgrowth hormones (auxin and cytokinin) andnovel plant metabolites namely opines —amino acid derivatives and agropines —sugar derivatives (Fig. 49.2).

The growth hormones cause plant cells toproliferate and form the gall while opinesand agropines are utilized by A. tumefaciensas sources of carbon and energy. As such,opines and agropines are not normally partof the plant metabolism (neither producednor metabolised). Thus, A. tumefaciens




genetically transforms plant cells andcreates a biosynthetic machinery to producenutrients for its own use.

As the bacteria multiply and continueinfection, grown gall develops which is avisible mass of the accumulated bacteria andplant material. Crown gall formation is theconsequence of the transfer, integration andexpression of genes of TDNA (or Tiplasmid) of A. tumefaciens in the infectedplant.

The genetic transformation leads to theformation of crown gall tumors, which




interfere with the normal growth of theplant. Several dicotyledonous plants (dicots)are affected by crown gall disease e.g.grapes, roses, stonefruit trees.

Organization of Ti plasmid:

The Ti plasmids (approximate size 200 kbeach) exist as independent replicatingcircular DNA molecules within theAgrobacterium cells. The TDNA(transferred DNA) is variable in length inthe range of 12 to 24 kb, which depends onthe bacterial strain from which Ti plasmidscome. Nopaline strains of Ti plasmid haveone TDNA with length of 20 kb whileoctopine strains have two TDNA regionsreferred to as T and T that are respectively14 kb and 7 kb in length.

A diagrammatic representation of a Tiplasmid is depicted in Fig. 49.3. The Tiplasmid has three important regions.

L R




1. TDNA region:

This region has the genes for thebiosynthesis of auxin (aux), cytokinin (cyt)and opine (ocs), and is flanked by left andright borders. These three genesaux, cytoand ocs are referred to as oncogenes, as theyare the determinants of the tumorphenotype.

TDNA borders — A set of 24 kb sequencespresent on either side (right and left) of TDNA are also transferred to the plant cells.It is now clearly established that the rightborder is more critical for TDNA transferand tumorigenesis.

2. Virulence region:

The genes responsible for the transfer of TDNA into the host plant are located outsideTDNA and the region is referred to as vir orvirulence region. Vir region codes forproteins involved in TDNA transfer. Atleast nine virgene operons have beenidentified. These include vir A, vir G, vir B ,vir C , vir D , D and D , and vir E , and E .

3. Opine catabolism region:

This region codes for proteins involved inthe uptake and metabolisms of opines.Besides the above three, there is ori region

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1 1 2 4 1 2



that is responsible for the origin of DNAreplication which permits the Ti plasmid tobe stably maintained in A. tumefaciens.

TDNA transfer and integration:

The process of TDNA transfer and itintegration into the host plant genome isdepicted in Fig. 49.4, and is brieflydescribed.

1. Signal induction to Agrobacterium:

The wounded plant cells release certainchemicals phenolic compounds and sugarswhich are recognized as signals byAgrobacterium. The signals induced resultin a sequence of biochemical events inAgrobacterium that ultimately helps in thetransfer of TDNA of Tplasmid.

2. Attachment of Agrobacterium toplant cells:




The Agrobacterium attaches to plant cellsthrough polysaccharides, particularlycellulose fibres produced by the bacterium.Several chromosomal virulence (chv) genesresponsible for the attachment of bacterialcells to plant cells have been identified.

3. Production of virulence proteins:

As the signal induction occurs in theAgrobacterium cells attached to plant cells, aseries of events take place that result in theproduction of virulence proteins. To startwith, signal induction by phenolicsstimulates vir A which in turn activates (byphosphorylation) vir C. This inducesexpression of virulence genes of Ti plasmidto produce the corresponding virulenceproteins (D1, D2, E , B etc.). Certain sugars(e.g. glucose, galactose, xylose) that inducevirulence genes have been identified.

4. Production of TDNA strand:

The right and left borders of TDNA arerecognized by vir D /vir D proteins. Theseproteins are involved in the productionsinglestranded TDNA (ss DNA), itsprotection and export to plant cells. The ssTDNA gets attached to vir D .

5. Transfer of TDNA out ofAgrobacterium:

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1 2

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The ss TDNA — vir D complex inassociation with vir G is exported from thebacterial cell. Vir B products form thetransport apparatus.

6. Transfer of TDNA into plant cellsand integration:

The TDNAvir D complex crosses the plantplasma membrane. In the plant cells, TDNA gets covered with vir E . This coveringprotects the TDNA from degradation bynucleases; vir D and vir E interact with avariety of plant proteins which influences TDNA transport and integration.

The TDNAvir D vir E — plant proteincomplex enters the nucleus through nuclearpore complex. Within the nucleus, the TDNA gets integrated into the plantchromosome through a process referred toillegitimate recombination. This is differentfrom the homologous recombination, as itdoes not depend on the sequence similarity.

Hairy Root Disease of A. Rhizogenes— R Plasmids:

Agrobacterium rhizogenes can also infectplants. But this results in hairy roots and notcrown galls as is the case with A.tumefaciens. The plasmids, of A. rhizogeneshave been isolated and characterized. These

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2 2

2 2

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plasmids, referred to as Ri plasmids, (Ristands for Root inducing) are of differenttypes. Some of the Ri plasmid strainspossess genes that are homologous to Tiplasmid e.g. auxin biosynthetic genes.

Instead of virulence genes, Ri plasmidscontain a series of open reading frames onthe TDNA. The products of these genes areinvolved in the metabolism of plant growthregulators which gets sensitized to auxinand leads to root formation.

Vectors of A. rhizogenes:

As it is done with A tumefaciens, vectors canbe constructed by using A. rhizogenes. Thesevectors are alternate strategies for genetransfer. However, employment of A.rhizogenebased vectors for planttransformation is not common since moreefficient systems of A. tumefaciens havebeen developed.

Importance of hairy roots:

Hairy roots can be cultured in vitro, andthus are important in plant biotechnology.Hairy root systems are useful for theproduction of secondary metabolites,particularly pharmaceutical proteins.

Ti PlasmidDerived Vector Systems:



The ability of Ti plasmid of Agrobacteriumto genetically transform plants has beendescribed. It is possible to insert a desiredDNA sequence (gene) into the TDNA region(of Ti plasmid), and then use A. tumefaciensto deliver this gene(s) into the genome ofplant cell.

In this process, Ti plasmids serve asnatural vectors. However, there areseveral limitations to use Ti plasmidsdirectly as cloning vectors:

i. Ti plasmids are large in size (200800 kb).Smaller vectors are preferred forrecombinant experiments. For this reason,large segments of DNA of Ti plasmid, notessential for cloning, must be removed.

ii. Absence of unique restriction enzymesites on Ti plasmids.

iii. The phytohormones (auxin, cytokinin)produced prevent the plant cells beingregenerated into plants. Therefore auxin andcytokinin genes must be removed.

iv. Opine production in transformed plantcells lowers the plant yield. Therefore opinesynthesizing genes which are of no use toplants should be removed.



v. Ti plasmids cannot replicate in E. coli.This limits their utility as E. coli is widelyused in recombinant experiments. Analternate arrangement is to add an origin ofreplication to Ti plasmid that allows theplasmid to replicate in E. coli.

Considering the above limitations, Tiplasmid based vectors with suitablemodifications have been constructed.

These vectors are mainly composed ofthe following components:

1. The right border sequence of TDNAwhich is absolutely required for TDNAintegration into plant cell DNA.

2. A multiple cloning site (polylinker DNA)that promotes the insertion of cloned geneinto the region between TDNA borders.

3. An origin of DNA replication that allowsthe plasmids to multiply in E. coli.

4. A selectable marker gene (e.g. neomycinphosphotransferase) for appropriateselection of the transformed cells.

Two types of Ti plasmidderived vectors areused for genetic transformation of plants—cointegrate vectors and binary vectors.

Cointegrate vector:



In the cointegrate vector system, thedisarmed and modified Ti plasmid combineswith an intermediate cloning vector toproduce a recombinant Ti plasmid (Fig.49.5).

Production of disarmed Ti plasmid:

The TDNA genes for hormone biosynthesisare removed (disarmed). In place of thedeleted DNA, a bacterial plasmid (pBR322)DNA sequence is incorporated. Thisdisarmed plasmid, also referred to asreceptor plasmid, has the basic structure ofTDNA (right and left borders, virulencegenes etc.) necessary to transfer the plantcells.

Construction of intermediate vector:




The intermediate vector isconstructed with the followingcomponents:

i. A pBR322 sequence DNA homologous tothat found in the receptor Ti plasmid.

ii. A plant transformation marker (PTM) e.g.a gene coding for neomycinphosphotransferase II (npt II). This geneconfers resistance to kanamycin in the plantcells and thus permits their isolation.

iii. A bacterial resistance marker e.g. a genecoding for spectinomycin resistance. Thisgene confers spectinomycin resistance torecipient bacterial cells and thus permitstheir selective isolation.

iv. A multiple cloning site (MCS) whereforeign genes can be inserted.

v. A Co/E origin of replication which allowsthe replication of plasmid in E. coli but notin Agrobacterium.

vi. An oriT sequence with basis ofmobilization (bom) site for the transfer ofintermediate vector from E. coli toAgrobacterium.

Production and use of cointegratevectors:

1



The desired foreign gene (targetgene) isfirst cloned in the multiple cloning site ofthe intermediate vector. The cloning processis carried out in E. coli, the bacterium wherethe cloning is most efficient. Theintermediate vector is mated withAgrobacterium so that the foreign gene ismobilised into the latter.

The transformed Agrobacterium cells withreceptor Ti plasmid and intermediate vectorare selectively isolated when grown on aminimal medium containing spectinomycin.The selection process becomes easy since E.coli does not grow on a minimal medium inwhich Agrobacterium grows.

Within the Agrobacterium cells,intermediate plasmid gets integrated intothe receptor Ti plasmid to produce cointegrate plasmid. This plasmid containingplant transformation marker (e.g. npt II)gene and cloned target gene between TDNAborders is transferred to plant cells. Thetransformed plant cells can be selected on amedium containing kanamycin when theplant and Agrobacterium cells are incubatedtogether.

Advantages of cointegrate vector:

i. Target genes can be easily cloned



ii. The plasmid is relatively small with anumber of restriction sites.

iii. Intermediate plasmid is convenientlycloned in E. coli and transferred toAgrobacterium.

Binary vector:

The binary vector system consists of anAgrobacterium strain along with a disarmedTi plasmid called vir helper plasmid (theentire TDNA region including bordersdeleted while vir gene is retained). It may benoted that both of them are not physicallylinked (or integrated). A binary vector withTDNA can replicate in E. coli andAgrobacterium.

A diagrammatic representation of a typicalbinary vector system is depicted in Fig. 49.6.The binary vector has the followingcomponents.

1. Left and right borders that delimit the TDNA region.




2. A plant transformation marker (PTM) e.g.npt II that confers kanamycin resistance inplant transformed cells.

3. A multiple cloning site (MCS) forintroducing target/foreign genes.

4. A bacterial resistance marker e.g.tetracycline resistance gene for selectingbinary vector colonies in E. coli andAgrobacterium.

5. oriT sequence for conjugal mobilization ofthe binary vector from E. coli toAgrobacterium.

6. A broad hostrange origin of replicationsuch as RK that allows the replication ofbinary vector in Agrobacterium.

Production and use of binary vector:

The target (foreign) gene of interest isinserted into the multiple cloning site of thebinary vector. In this way, the target gene isplaced between the right and left borderrepeats and cloned in E. coli. By a matingprocess, the binary vector is mobilised fromE. coli to Agrobacterium. Now, the virulencegene proteins of TDNA facilitate thetransfer of TDNA of the vector into plantcells.

Advantages of binary vectors:

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i. The binary vector system involves only thetransfer of a binary plasmid toAgrobacterium without any integration. Thisis in contrast to cointegrate vector systemwherein the intermediate vector istransferred and integrated with disarmed Tiplasmid.

ii. Due to convenience, binary vectors aremore frequently used than cointegratevectors.

Plant Transformation TechniqueUsing Agrobacterium:

Agrobacteriummediated technique is themost widely used for the transformation ofplants and generation of transgenic plants.The important requirements for genetransfer in higher plants throughAgrobacterium mediation are listed.

i. The explants of the plant must producephenolic compounds (e.g. autosyringone)for activation of virulence genes.

ii. Transformed cells/tissues should becapable to regenerate into whole plants.

In general, most of the Agrobacteriummediated plant transformations have thefollowing basic protocol (Fig. 49.7)



1. Development of Agrobacterium carryingthe cointegrate or binary vector with thedesired gene.

2. Identification of a suitable explant e.g.cells, protoplasts, tissues, calluses, organs.




3. Coculture of explants withAgrobacterium.

4. Killing of Agrobacterium with a suitableantibiotic without harming the plant tissue.

5. Selection of transformed plant cells.

6. Regeneration of whole plants.

Advantages of Agrobacteriummediated transformation:

i. This is a natural method of gene transfer.

ii. Agrobacterium can conveniently infectany explant (cells/tissues/organs).

iii. Even large fragments of DNA can beefficiently transferred.

iv. Stability of transferred DNA is reasonablygood.

v. Transformed plants can be regeneratedeffectively.

Limitations of Agrobacteriummediated transformation:

i. There is a limitation of host plants forAgrobacterium, since many crop plants(monocotyledons e.g. cereals) are notinfected by it. In recent years, virulent



strains of Agrobacterium that can infect awide range of plants have been developed.

ii. The cells that regenerate more efficientlyare often difficult to transform, e.g.embryonic cells lie in deep layers which arenot easy targets for Agrobacterium.

VirusMediated Gene Transfer (PlantViruses as Vectors):

Plant viruses are considered as efficient genetransfer agents as they can infect the intactplants and amplify the transferred genesthrough viral genome replication. Virusesare natural vectors for genetic engineering.They can introduce the desirable gene(s)into almost all the plant cells since the viralinfections are mostly systemic.

Plant viruses are nonintegrativevectors:

The plant viruses do not integrate into thehost genome in contrast to the vectors basedon TDNA of A. tumefaciens which areintegrative. The viral genomes are suitablymodified by introducing desired foreigngenes. These recombinant viruses aretransferred, multiplied and expressed inplant cells. They spread systemically withinthe host plant where the new geneticmaterial is expressed.



Criteria for a plant virus vector:

An ideal plant virus for its effectiveuse in gene transfer is expected toposses the following characteristics:

i. The virus must be capable of spreadingfrom cell to cell through plasmodesmata.

ii. The viral genome should be able toreplicate in the absence of viral coat proteinand spread from cell to cell. This is desirablesince the insertion of foreign DNA will makethe viral genome too big to be packed.

iii. The recombinant viral genome mustelicit little or no disease symptoms in theinfected plants.

iv. The virus should have a broad host range.

v. The virus with DNA genome is preferredsince the genetic manipulations involveplant DNA.

The three groups of viruses —caulimoviruses, Gemini viruses and RNAviruses that are used as vectors for genetransfer in plants are briefly described.

Caulimoviruses as Vectors:

The caulimoviruses contain circular doublestranded DNA, and are spherical in shape.



Caulimoviruses are widely distributed andare responsible for a number ofeconomically important diseases in variouscrops. The caulimovirus group has around15 viruses and among these cauliflowermosaic virus (CaMV) is the most importantfor gene transfer. The other caulimovirusesinclude carnation etched virus, dahliamosaic virus, mirabilis mosaic virus andstrawberry vein banding virus.

Cauliflower mosaic virus (CaMV):

CaMV infects many plants (e.g. members ofCruciferae, Datura) and can be easilytransmitted, even mechanically. Anotherattractive feature of CaMV is that theinfection is systemic, and large quantities ofviruses are found in infected cells.

A diagrammatic view of the CaMV geneticmap is depicted in Fig. 49.8. The genome ofCaMV consists of a 8 kb (8024 bp) relaxedbut tightly packed circular DNA with sixmajor and two minor coding regions. Thegenes II and VII are not essential for viralinfection.



Use of CaMV in gene transfer:

For appropriate transmission of CaMV, theforeign DNA must be encapsulated in viralprotein. Further, the newly inserted foreignDNA must not interfere with the nativeassembly of the virus. CaMV genome doesnot contain any noncoding regions whereinforeign DNA can be inserted. It is fortunatethat two genes namely gene II and gene VIIhave no essential functions for the virus. It istherefore possible to replace one of themand insert the desired foreign gene.

Gene II of CaMV has been successfullyreplaced with a bacterial gene encodingdihydrofolate reductase that providesresistance to methotrexate. When thechimeric CaMV was transmitted to turnip




plants, they were systemically infected andthe plants developed resistance tomethotrexate.

Limitations of CaMV as a vector:

i. CaMV vector has a limited capacity forinsertion of foreign genes.

ii. Infective capacity of CaMV is lost if morethan a few hundred nucleotides areintroduced.

iii. Helper viruses cannot be used since theforeign DNA gets expelled and wildtypeviruses are produced.

Gemini Viruses as Vectors:

The Gemini viruses are so named becausethey have geminate (Gemini literally meansheavenly twins) morphological particles i.e.twin and paired capsid structures. Theseviruses are characterized by possessing oneor two singlestranded circular DNAs (ssDNA). On replications, ss DNA forms anintermediate doublestranded DNA.

The Gemini viruses can infect a wide rangeof crop plants (monocotyledons anddicotyledons) which attract plantbiotechnologists to employ these viruses forgene transfer. Curly top virus (CTV) andmaize streak virus (MSV) and bean golden



mosaic virus (BGMV) are among theimportant Gemini viruses.

It has been observed that a large number ofreplicative forms of a Gemini virus genomeaccumulate inside the nuclei of infectedcells. The singlestranded genomic DNAreplicates in the nucleus to form a doublestranded intermediate.

Gemini virus vectors can be used to deliver,amplify and express foreign genes in severalplants/ explants (protoplasts, culturedcells). However, the serious drawback inemploying Gemini viruses as vectors is thatit is very difficult to introduce purified viralDNA into the plants. An alternatearrangement is to take the help ofAgrobacterium and carry out gene transfer.

RNA Plant Viruses as Vectors:

There are mainly two type’s singlestranded RNA viruses:

1. Monopartite viruses:

These viruses are usually large and containundivided genomes for all the geneticinformation e.g. tobacco mosaic virus(TMV).

2. Multipartite viruses:



The genome in these viruses is divided intosmall RNAs which may be in the sameparticle or different particles, e.g. bromemosaic virus (BMV). HMV contains fourRNAs divided between three particles. PlantRNA viruses, in general, are characterizedby high level of gene expression, goodefficiency to infect cells and spread todifferent tissues. But the major limitation touse them as vectors is the difficulty ofjoining RNA molecules in vitro.

Use of cDNA for gene transfer:

Complementary DNA (cDNA) copies of RNAviruses are prepared in vitro. The cDNA sogenerated can be used as a vector for genetransfer in plants. This approach is tediousand cumbersome. However, some successhas been reported. A gene sequenceencoding chloramphenicol resistance(enzyme chloramphenicolacetyltransferase) has been inserted intobrome mosaic virus genome. This geneexpression, however, has been confined toprotoplasts.

Limitations of Viral Vectors in GeneTransfer:

The ultimate objective of gene transfer is totransmit the desired genes to subsequentgenerations. With virus vectors, this is not



possible unless the virus is seedtransmitted. However, in case ofvegetatively propagated plants, transmissionof desired traits can be done e.g. potatoes.Even in these plants, there is always a riskfor the transferred gene to be lost anytime.For the reasons referred above, plantbiotechnologists prefer to insert the desiredgenes of interest into a plant chromosome.

Method # II. Direct or VectorlessDNA Transfer:

The term direct or vector less transfer ofDNA is used when the foreign DNA isdirectly introduced into the plant genome.Direct DNA transfer methods rely on thedelivery of naked DNA into the plant cells.This is in contrast to the Agrobacterium orvectormediated DNA transfer which may beregarded as indirect methods. Majority ofthe direct DNA transfer methods are simpleand effective. And in fact, several transgenicplants have been developed by thisapproach.

Limitations of direct DNA transfer:

The major disadvantage of direct genetransfer is that the frequency of transgenerearrangements is high. This results inhigher transgene copy number, and highfrequencies of gene silencing.



Types of direct DNA transfer:

The direct DNA transfer can be broadlydivided into three categories.

1. Physical gene transfer methods—electroportion, particle bombardment,microinjection, liposome fusion, siliconcarbide fibres.

2. Chemical gene transfer methods—Poly ethylene glycol (PEG)mediated, diethylamino ethyl (DEAE) dextranmediated,calcium phosphate precipitation.

3. DNA imbibition by cells/tissues/organs.

The salient features of the different methodsfor direct DNA transfer are given in Table49.1 .

(A) Physical Gene Transfer Methods:

An overview of the general scheme for theproduction of transgenic plants byemploying physical transfer methods isdepicted in Fig. 49.9. Some details of thedifferent techniques are described.



1. Electroporation:

Electroporation basically involves the use ofhigh field strength electrical impulses toreversibly permeabilize the cell membranesfor the uptake of DNA. This technique canbe used for the delivery of DNA into intactplant cells and protoplasts.

The plant material is incubated in a buffersolution containing the desiredforeign/target DNA, and subjected to highvoltage electrical impulses. This results inthe formation of pores in the plasma




membrane through which DNA enters andgets integrated into the host cell genome.

In the early years, only protoplasts wereused for gene transfer by electroporation.Now a days, intact cells, callus cultures andimmature embryos can be used with suitablepre and postelectroporation treatments.Electroporation has been successfully usedfor the production of transgenic plants ofmany cereals e.g. rice, wheat, maize.

Advantages of electroporation:

i. This technique is simple, convenient andrapid, besides being costeffective.

ii. The transformed cells are at the samephysiological state after electroporation.

iii. Efficiency of transformation can beimproved by optimising the electrical fieldstrength, and addition of spermidine.

Limitations of electroporation:

i. Under normal conditions, the amount ofDNA delivered into plant cells is very low.

ii. Efficiency of electroporation is highlyvariable depending on the plant materialand the treatment conditions.



iii. Regeneration of plants is not very easy,particularly when protoplasts are used.

2. Particle Bombardment (Biolistics):

Particle (or micro projectile) bombardmentis the most effective method for genetransfer, and creation of transgenic plants.This method is versatile due to the fact thatit can be successfully used for the DNAtransfer in mammalian cells andmicroorganisms.

The micro projectile bombardment methodwas initially named as biolistics by itsinventor Sanford (1988). Biolistics is acombination of biological and ballistics.There are other names for this techniqueparticle gun, gene gun, bio blaster. Adiagrammatic representation of microprojectile bombardment system for thetransfer of genes in plants is depicted in Fig.49.10, and briefly described below.




Micro carriers (micro projectiles), thetungsten or gold particles coated with DNA,are carried by macro carriers (macroprojectiles). These macrocarriers areinserted into the apparatus and pusheddownward by rupturing the disc.

The stopping plate does not permit themovement of macro carrier while the microcarriers (with DNA) are propelled at a highspeed into the plant material. Here the DNAsegments are released which enter the plantcells and integrate with the genome.

Plant material used in bombardment:

Two types of plant tissue arecommonly used for particlebombardment:

1. Primary explants which can be subjectedto bombardment that are subsequentlyinduced to become embryo genic andregenerate.

2. Proliferating embryonic tissues that canbe bombarded in cultures and then allowedto proliferate and regenerate.

In order to protect plant tissues from beingdamaged by bombardment, cultures aremaintained on high osmoticum media orsubjected to limited plasmolysis.



Transgene integration inbombardment:

It is believed (based on the gene transfer inrice by biolistics) that the gene transfer inparticle bombardment is a two stageprocess.

1. In the preintegration phase, the vectorDNA molecules are spliced together. Thisresults in fragments carrying multiple genecopies.

2. Integrative phase is characterized by theinsertion of gene copies into the host plantgenome.

The integrative phase facilitates furthertransgene integration which may occur atthe same point or a point close to it. The netresult is that particle bombardment isfrequently associated with high copynumber at a single locus. This type of singlelocus may be beneficial for regeneration ofplants.

The success of bombardment:

The particle bombardment technique wasfirst introduced in 1987. It has beensuccessfully used for the transformation ofmany cereals, e.g. rice, wheat, maize. In fact,the first commercial genetically modified



(CM) crops such as maize containing Bttoxin gene were developed by this approach.

A selected list of the transgenic plants(developed by biolistics) along with thesources of the plant materials used is givenin Table 49.2.

Factors affecting bombardment:

Several attempts are made to study thevarious factors, and optimize the system ofparticle bombardment for its most efficientuse. Some of the important parameters aredescribed.

Nature of micro particles:

Inert metals such as tungsten, gold andplatinum are used as micro particles to carry




DNA. These particles with relatively highermass will have a better chance to move fastwhen bombarded and penetrate the tissues.

Nature of tissues/cells:

The target cells that are capable ofundergoing division are suitable fortransformation. Some more details on thechoice of plant material used inbombardment are already given.

Amount of DNA:

The transformation may be low when toolittle DNA is used. On the other hand, toomuch DNA may result is high copy numberand rearrangement of transgenes.Therefore, the quantity of DNA used shouldbe balanced. Recently, some workers havestarted using the chemical aminosiloxane tocoat the micro particles with low quantitiesof DNA adequate enough to achieve highefficiency of transformation.

Environmental parameters:

Many environ mental variables are known toinfluence particle bombardment. Thesefactors (temperature, humidity, photoperiodetc.) influence the physiology of the plantmaterial, and consequently the genetransfer. It is also observed that some



explants, after bombardment may requirespecial regimes of light, humidity,temperature etc.

The technology of particle bombardmenthas been improved in recent years,particularly with regard to the use ofequipment. A commercially producedparticle bombardment apparatus namelyPDS1000/HC is widely used these days.

Advantages of particle bombardment:

i. Gene transfer can be efficiently done inorganized tissues.

ii. Different species of plants can be used todevelop transgenic plants.

Limitations of particle bombardment:

i. The major complication is the productionof high transgene copy number. This mayresult in instability of transgene expressiondue to gene silencing.

ii. The target tissue may often get damageddue to lack of control of bombardmentvelocity.

iii. Sometimes, undesirable chimeric plantsmay be regenerated.

3. Microinjection:



Microinjection is a direct physical methodinvolving the mechanical insertion of thedesirable DNA into a target cell. The targetcell may be the one identified from intactcells, protoplasts, callus, embryos,meristems etc. Microinjection is used for thetransfer of cellular organelles and for themanipulation of chromosomes.

The technique of microinjection involves thetransfer of the gene through a micropipette(0.510.0 pm tip) into thecytoplasm/nucleus of a plant cell orprotoplast. While the gene transfer is done,the recipient cells are kept immobilized inagarose embedding, and held by a suctionholding pipette (Fig. 49.11).

As the process of microinjection is complete,the transformed cell is cultured and grownto develop into a transgenic plant. In fact,transgenic tobacco and Brassica napus havebeen developed by this approach. The majorlimitations of microinjection are that it isslow, expensive, and has to be performed bytrained and skilled personnel.

4. LiposomeMediatedTransformation:




Liposomes are artificially created lipidvesicles containing a phospholipidmembrane. They are successfully used inmammalian cells for the delivery of proteins,drugs etc. Liposomes carrying genes can beemployed to fuse with protoplasts andtransfer the genes.

The efficiency of transformation increaseswhen the process is carried out inconjunction with polyethylene glycol (PEG).Liposomemediated transformation involvesadhesion of liposomes to the protoplastsurface, its fusion at the site of attachmentand release of plasmids inside the cell (Fig.49.12).

Advantages of liposome fusion:

i. Being present in an encapsulated form ofliposomes, DNA is protected fromenvironmental insults and damage.

ii. DNA is stable and can be stored for sometime in liposomes prior to transfer.

iii. Applicable to a wide range of plant cells.




iv. There is good reproducibility in thetechnique.

Limitations of liposome fusion:

The major problem with liposomemediatedtransformation is the difficulty associatedwith the regeneration of plants fromtransformed protoplasts.

5. Silicon Carbide FibreMediatedTransformation:

The silicon carbide fibres (SCF) are about0.30.6 pm in diameter and 10100 pm inlength. These fibres are capable ofpenetrating the cell wall and plasmamembrane, and thus can deliver DNA intothe cells. The DNA coated silicon carbidefibres are vortexed with ‘plant material(suspension culture, calluses). During themixing, DNA adhering to the fibres entersthe cells and gets stably integrated with thehost genome. The silicon carbide fibres withthe trade name Whiskers are available in themarket.

Advantages of SCFmediatedtransformation:

i. Direct delivery of DNA into intact walledcells. This avoids the protoplast isolation.



ii. Procedure is simple and does not involvecostly equipment.

Disadvantages of SCFmediatedtransformation:

i. Silicon carbide fibres are carcinogenic andtherefore have to be carefully handled.

ii. The embryonic plant cells are hard andcompact and are resistant to SCFpenetration.

In recent years, some improvements havebeen made in SCFmediated transformation.This has helped in the transformation ofrice, wheat, maize and barley by using thistechnique.

(B) Chemical Gene Transfer Methods:

1. Polyethylene glycol (PEG)mediatedtransfer:

Polyethylene glycol (PEG), in the presenceof divalent cations (using Ca ), destabilizesthe plasma membrane of protoplasts andrenders it permeable to naked DNA. In thisway, the DNA enters nucleus of theprotoplasts and gets integrated with thegenome.

The procedure involves the isolation ofprotoplasts and their suspension, addition

2+



of plasmid DNA, followed by a slow additionof 40% PEG4000 (w/v) dissolved inmannitol and calcium nitrate solution. Asthis mixture is incubated, protoplasts gettransformed.

Advantages of PEGmediatedtransformation:

i. A large number of protoplasts can besimultaneously transformed.

ii. This technique can be successfully usedfor a wide range of plant species.

Limitations of PEGmediatedtransformation:

i. The DNA is susceptible for degradationand rearrangement.

ii. Random integration of foreign DNA intogenome may result in undesirable traits.

iii. Regeneration of plants from transformedprotoplasts is a difficult task.

2. Deae DextranMediated transfer:

The desirable DNA can be complexed with ahigh molecular weight polymer diethylamino ethyl (DEAE) dextran andtransferred. The major limitation of this



approach is that it does not yield stabletransformants.

Calcium Phosphate Co PrecipitationMediated Transfer:

The DNA is allowed to mix with calciumchloride solution and isotonic phosphatebuffer to form DNAcalcium phosphateprecipitate. When the actively dividing cellsin culture are exposed to this precipitate forseveral hours, the cells get transformed. Thesuccess of this method is dependent on thehigh concentration of DNA and theprotection of the complex precipitate.Addition of dimethyl sulfoxide (DMSO)increases the efficiency of transformation.

Dna Imbibition By Cells/Tissues:

Some workers have seriously tried totransform cells by incubating cellsuspensions, tissues, embryos and evenseeds with DNA. The belief is that the DNAgets imbibed, and the cells get transformed.DNA imbibition approach has met with littleor no success.

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methods of gene transfer in plants: 2 methods · the gene transfer techniques in plant genetic...

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