TRANSGENIC TRANSGENIC PLANTSPLANTS

IntroductionIntroduction

These are plants whose DNA is modified using These are plants whose DNA is modified using genetic engineering techniques.genetic engineering techniques.

In most cases the aim is to introduce a In most cases the aim is to introduce a new trait to the plant which does not occur new trait to the plant which does not occur naturally in this species. naturally in this species.

Examples include resistance to certain pests, Examples include resistance to certain pests, diseases or environmental conditions, or the diseases or environmental conditions, or the production of a certain nutrient or production of a certain nutrient or pharmaceutical agent.pharmaceutical agent.

IntroductionIntroduction

During the last couple of decades, considerable During the last couple of decades, considerable progress has been made to understand the progress has been made to understand the function of genes, isolation of novel genes and function of genes, isolation of novel genes and promoters as well as the utilization of these genes promoters as well as the utilization of these genes for the development of transgenic crops with for the development of transgenic crops with improved and new characters.improved and new characters.

In fact, in 2002, more than 5.5million farmers In fact, in 2002, more than 5.5million farmers worldwide cultivated about 58.7million hectares worldwide cultivated about 58.7million hectares the crops that were genetically manipulated for the crops that were genetically manipulated for herbicide tolerance, insect resistance, delayed herbicide tolerance, insect resistance, delayed fruit ripening and improved oil quality.fruit ripening and improved oil quality.

Stress ToleranceStress Tolerance

Crop plants are very productive under ideal culture Crop plants are very productive under ideal culture conditions, but ideal growing conditions rarely occur.conditions, but ideal growing conditions rarely occur.

They face so many strategies like soil nutrient They face so many strategies like soil nutrient depletion, water scarcity, increase in salt level, effect of depletion, water scarcity, increase in salt level, effect of so many microorganisms and other biological factors, so many microorganisms and other biological factors, etc..etc..

Based on this stress tolerant transgenic plants are of Based on this stress tolerant transgenic plants are of two types:two types:

-- Biotic (viral, bacterial, fungal, pathogens, nematodes, Biotic (viral, bacterial, fungal, pathogens, nematodes, and insect pests resistance)and insect pests resistance) -- Abiotic (salinity, drought, extreme temperatures, Abiotic (salinity, drought, extreme temperatures, nutrient deficiency resistance)nutrient deficiency resistance)

Stress tolerance- BioticStress tolerance- Biotic1.1. Insect Resistance:Insect Resistance: All crop plants are affected by a variety of insects, All crop plants are affected by a variety of insects,

mites and nematodes, that significantly reduce their mites and nematodes, that significantly reduce their yield and quality.yield and quality.

To minimize these losses, farmers use synthetic To minimize these losses, farmers use synthetic pesticides extensively which cause severe effects on pesticides extensively which cause severe effects on human health & environment.human health & environment.

The transgenic technology provides an alternative & The transgenic technology provides an alternative & innovative method pest control management which innovative method pest control management which are eco friendly, effective, sustainable & beneficial in are eco friendly, effective, sustainable & beneficial in terms of yields.terms of yields.

The 1The 1stst gene available for GE of crop plants for pest gene available for GE of crop plants for pest resistance were resistance were CryCry gene (Bt gene) from gene (Bt gene) from Bacillus Bacillus thuringiensisthuringiensis..

Clone gene coding for BT toxin - pesticide

Protein toxin from Bacillus thuringiensisKills larvae of

Lepidopterans (butterflies, moths, boll worms)

Dipterans (2 winged flies (gnats, mosquitos))

Coleopterans (beetles) orthoptera (grass hoppers) Homoptera (aphids)

Agricultural importance - Kills corn borer, corn root worm and cotton bollworm larvae

Insect resistant plants

Corn borer Corn root worm

By using insect control protein genes Insect resistant transgenic plants – contain

either a gene from bacterium B.thuringiensis or some other gene

Insect resistance - First reported in Tobacco(Vaeck 1987) & Tomato (fischhoff 1987)

Insect resistance transgenes – of plant, bacterial or other origin – can be introduced in to plants to increase level of insect resistance

Insect-resistant plants

Bt toxin – Bt gene from B.thuringiensis Ipt(Isopentyl transferase) from Agrobacterium

tumefaciens cholesterol oxidase from streptomyces fungus Pht gene from photorhabdus luminescens Cowpea trypsin inhibitor Combinations of the above (e.g., Bt toxin and

proteinase inhibitor II)

RESISTANCE GENES FROM HIGHER LANTS

2 categories

1) Proteinase inhibitor II & -amylase inhibitor

2) Lectins: - Snow drop lectin (Pea lectin, rice lectin)

Resistance genes of animal origin – Serine proteinase inhibitors from mammals & tobacco hornworm (Manduca sexta)

Bacillus thuringiensis

Discovered by Ishiwaki (1901)

Gram –ve soil bacterium

Produces a parasporal crystalline proteinous toxin with insecticidal activity

Proteins produced are referred to as ICP – Insecticidal crystalline protein

B.thuringiensis – used since world war I – in europe to control insect pests

Classified based on serological tests 30 different types

The The Bt geneBt gene of of B.thuringiensis - encode the - encode the toxins called toxins called EndotoxinsEndotoxins,, such as beta- such as beta-endotoxin & delta-endotoxin - which pose endotoxin & delta-endotoxin - which pose cidal effectcidal effect on certain insect pests. on certain insect pests.

Cry gene of B.thuringiensis – produces a protein

Protein forms crystalline inclusions in the bacterial spores during sporulation

These crystal proteins – responsible for insecticidal activities of the bacterial strains

CRY GENE

They are converted to active form upon infection by susceptible insect

They kill the insect by disruption of ion transport across the brush border/membranes of susceptible insect

Cry genes – grouped in to 18 groups – which either code for a 130 kDa or a 70 kDa protein

Available as Available as Bt gene powderBt gene powder

Different Bt Crys Cry 1s—kills

caterpillars (lepidoptera)

Cry 2s—kills caterpillars (lepidoptera)

Cry 3s—kills beetles (coleoptera)

Canola plant expresses a Bt cry1Ac gene

Bt COTTON – CRY PROTEINBt COTTON – CRY PROTEIN

The insecticidal toxin of Bt cry proteins has The insecticidal toxin of Bt cry proteins has been classified 4 main groups as: Crystal been classified 4 main groups as: Crystal proteins - proteins - cry I, cry II, cry III & cry IVcry I, cry II, cry III & cry IV

based on insecticidal activitiesbased on insecticidal activities cry V, cry VI groups were also addedcry V, cry VI groups were also added These proteins – nematicidal in actionThese proteins – nematicidal in action They do not harm beneficial insects.They do not harm beneficial insects.

Bt Cry structure

IIII

II

BT ACTION

Cry proteins – are solubilized in alkaline environment of insect midgut

Then proteolytically processed to yield a 60kDa toxic core fragment (except cry IVD)

The toxin function – is localised in N terminal half of the 130 kDa proteins

C terminal half of these proteins – highly conserved – involved in crystal formation

None of the truncated proteins – crystallizes in the typical bipyramidal shape of most of the 130kDa proteins

12c terminal aminoacid residues – of toxic fragment – are highly conserved among cry proteins

Cry IA(c) – are packaged in vivo in association with 20 kb non specific chromosomal DNA

DNA protein complex forms virus –like structure in which central DNA core is surrounded by N terminal half of the cry protein

C terminal half of protein extends outward

Insect midgut cells that have bound Bt toxin.

Same gut cells a few hours later– note the damage and leakage.

Bt toxin

Insect midgut cells that have bound Bt toxin.

Bt

Mutated receptors cannot bind Bt toxin.

Receptors are not present– cells cannot bind Bt Stewart, 2004. Genetically Modified Planet 2004

When cry proteins are ingested by insects – they are dissolved in alkaline juices present in the midgut lumen

The gut proteases process them hydrolytically to release the core toxic fragments

The toxic fragments – believed to bind to specific high affinity receptors present in the brush border of midgut epithelial cells

TOXIC ACTION OF CRY PROTEINS

As a result – brush border membranes develop pores – nonspecific in nature –

Permitting influx in to the epithelial cells of ions & water – causes their swelling & eventual lysis

Toxic Action of Cry Proteins:Toxic Action of Cry Proteins:

Expression of cry Genes in Plants:Expression of cry Genes in Plants:

Isolation of Bt toxin gene( Bt 2 from ) Isolation of Bt toxin gene( Bt 2 from ) B.thuringiensis strain Berlkiner 1715

It produces a 1155 aa Bt2 proteinIt produces a 1155 aa Bt2 protein Inserting into plant genome, here its the cotton plant, Inserting into plant genome, here its the cotton plant,

to produce insect resistant plants.to produce insect resistant plants. This was done using Ti-plasmid transfer method – This was done using Ti-plasmid transfer method –

introduction of toxin gene in to Ti DNA plasmid of introduction of toxin gene in to Ti DNA plasmid of Agrobacterium tumefaciensAgrobacterium tumefaciens

The genetically modified A.tumefaciens was allowed The genetically modified A.tumefaciens was allowed to infect the desired plantto infect the desired plant

Ti plasmid mediated transformation of several plants Ti plasmid mediated transformation of several plants have been done – Tobacco, corn, Tomato, Cotton have been done – Tobacco, corn, Tomato, Cotton brinjal, cauliflower, cabbage, canolabrinjal, cauliflower, cabbage, canola

Approach 1Approach 1

Field experiments with Manducta sexta – Field experiments with Manducta sexta – pest of tobaccopest of tobacco

75-100% larvae of M.sexta – Died75-100% larvae of M.sexta – Died The insects die when they chew the leaves The insects die when they chew the leaves

of transgenic tobacco.of transgenic tobacco. Control plants (not transgenic) – severly Control plants (not transgenic) – severly

damageddamaged Tobacco plants crossed with normal control Tobacco plants crossed with normal control

plant – resistance gene was inherited as plant – resistance gene was inherited as per mendelian principleper mendelian principle

Segregates as a single dominant geneSegregates as a single dominant gene

A binary T-DNA plasmid for delivering the Bt gene to plants (not a cointegrate vector)

(NPT or kanr)(35S-Bt gene-tNOS)

(Spcr)

Several genes – encoding lepidopteran type toxins have been isolated

Fischhoff 1987 One gene from B.thuringneisis subsp –

Kurstaki HD-1 – contains an open reading frame of 3468bp encoding a protein of 1156 aa

Chimeric B.thuringneisis Kurstaki genes – containing Camv 35S promoter & a sequence coding for an active truncated variant as well as full length

Constructued & expressed in tomato plants

2. KURSTAKI HD-1

Level of insecticidal protein – was sufficient to kill larvae of Manduca sexta, Heliothis virescenes & heliothis zea

Analysis of progeny plants showed B.thuringneisis Kurstaki gene – segregated as a single dominant mendelian marker

Bt corn‘Plant cells are totipotent’

Bt Corn from Phillipines

Mechanism of toxin action:Binds to receptors in insect gutIonophore- ion channel that allows ions to flow across plasma membrane

Note: organic farmers spray crops with intact Bt bacterium

Strategies to avoid Bt resistant insects

Use of inducible promoters (that can be turned on only when there is an insect problem)

Construction of hybrid Bt toxins Introduction of the Bt gene in combination with

another insecticidal gene Spraying low levels of insecticide on Bt plants Use of spatial refuge strategies

Genetically engineered Bt-plants in the field

Product Institution(s) Engineered Trait(s) Sources of New Genes

Name

Corn Bayer Resist glufosinate herbicide to control weeds/Bt toxin to control insect pests (European corn borer)

Bacteria, virus StarLink-1998 (animals only)

Corn Dow/Mycogen Bt toxin to control insect pests (European corn borer) Corn, bacteria, virus NatureGard-1995

Corn Dow/Mycogen Resist glufosinate herbicide to control weeds/Bt toxin to control insect pests (Lepidopteran)

Corn, bacteria, virus Herculex I-2001

DuPont/Pioneer  

Corn Monsanto/DeKalb

Bt toxin to control insect pests (European corn borer) Bacteria Bt-Xtra-1997

Corn Monsanto Bt toxin to control insect pests (European corn borer) Bacteria YieldGard-1996

Corn Monsanto Resist glyphosate herbicide to control weeds/Bt toxin to control insect pests (European corn borer)

Arabidopsis, bacteria, virus

?-1998

Corn  Syngenta Bt toxin to control insect pests (European corn borer) Bacteria Bt11-1996

Corn Syngenta Bt toxin to control insect pests (European corn borer) Corn, bacteria, virus Knock Out-1995

Corn (pop)

Syngenta Bt toxin to control insect pests (European corn borer) Corn, bacteria, virus Knock Out-1998

Corn (sweet)

Syngenta Bt toxin to control insect pests (European corn borer) Bacteria Bt11-1998

Cotton Monsanto/Bayer

Resist bromoxynil herbicide to control weeds/Bt toxin to control insect pests (cotton bollworms

Bacteria ?-1998

    and tobacco budworm)    

Cotton Monsanto Bt toxin to control insect pests (cotton bollworms and tobacco budworm) Bacteria Bollgard-1995

Potato Monsanto Bt toxin to control insect pests (Colorado potato beetle) Bacteria NewLeaf-1995

Potato Monsanto Bt toxin to control insect pests (Colorado potato beetle)/resist potato virus Y Bacteria, virus NewLeaf Y-1999

Potato Monsanto Bt toxin to control insect pests (Colorado potato beetle)/resist potato leafroll virus Bacteria, virus NewLeaf Plus-1998

 

Mode of treatment Resistance developed inB. thuringensis formulations

Laboratory selection(Cry protein resistance)

Diamonback moth (Plutella xylostella) population in several countries

LepidopteraHelicoverpa armigera, spodoptera exigua, S. Littorallis, P. Xylosetella, Ephestia kuehniella, Cadra cautella, Indian meal moth(Plodia interpunctella), Christoneura fumiferanaColeopteraChrysomella scripta, Leptinotarsa decemlineataDipteraAedes aegypti, Culex quniquefasciatus, Drosophila melanogester, Musca domestica

Agriculture Transgenics On the Market

Insect resistant cotton – Bt toxin kills the cotton boll worm• transgene = Bt protein

Insect resistant corn – Bt toxin kills the European corn borer• transgene = Bt protein

Normal Transgenic

Cry gene designationToxic to these insect

orders

CryIA(a), CryIA(b), CryIA(c) Lepidoptera

Cry1B, Cry1C, Cry1D Lepidoptera

CryII Lepidoptera, Diptera

CryIII Coleoptera

CryIV Diptera

CryV Lepidoptera, Coleoptera

RESISTANCE GENES RESISTANCE GENES FROM HIGHER PLANTSFROM HIGHER PLANTS

Discovery of non bt toxin genes having Discovery of non bt toxin genes having insecticidal activityinsecticidal activity

Non bt insecticidal proteins interfere with Non bt insecticidal proteins interfere with nutritional needs of the insectnutritional needs of the insect

Retarding insect growth & development Retarding insect growth & development

PROTEINASEPROTEINASE

Plants contain peptides acting as Plants contain peptides acting as protease inactivating proteins(PIPs)protease inactivating proteins(PIPs)

Different proteinases – serine, cysteine, Different proteinases – serine, cysteine, aspartic, metallo proteinasesaspartic, metallo proteinases

They catalyze the release of aa from They catalyze the release of aa from dietary protein – thereby providing the dietary protein – thereby providing the nutrients crucial for normal growth & nutrients crucial for normal growth & development of insectsdevelopment of insects

PROTEINASE (PROTEASE) PROTEINASE (PROTEASE) INHIBITORSINHIBITORS

PROTEINASE (PROTEASE) PROTEINASE (PROTEASE) INHIBITORSINHIBITORS

Proteinase inhibitors – proteins that inhibit Proteinase inhibitors – proteins that inhibit the activity of proteinase enzymesthe activity of proteinase enzymes

Certain plants naturally produce Proteinase Certain plants naturally produce Proteinase inhibitors to provide defence against inhibitors to provide defence against herbivorous insectsherbivorous insects

Possible – since inhibitors when ingested by Possible – since inhibitors when ingested by insects interfere with digestive enzymes of insects interfere with digestive enzymes of the insectthe insect

This results in nutrient deprivation causing This results in nutrient deprivation causing death of insectsdeath of insects

Proteinase inhibitors – deprive the insect Proteinase inhibitors – deprive the insect of nutrients by interfering with digestive of nutrients by interfering with digestive enzymes of the insectenzymes of the insect

2 types2 types

Possible to control insects by introducing Possible to control insects by introducing proteinase inhibitor genes in to crop plants proteinase inhibitor genes in to crop plants that normally do not produce these proteinsthat normally do not produce these proteins

1.cowpea trypsin inhibitor (CpTI) gene

cowpea trypsin inhibitor (CpTI) gene

Wild species of cowpea plants growing in Wild species of cowpea plants growing in africa – were resistant to attack by a wide africa – were resistant to attack by a wide range of insectsrange of insects

Insecticidal protein was a trypsin inhibitor Insecticidal protein was a trypsin inhibitor – capable of destroying insects belonging – capable of destroying insects belonging to orders of lepidopters & coleopterato orders of lepidopters & coleoptera

It has no effect on mammalian trypsins – It has no effect on mammalian trypsins – non toxic to mammalsnon toxic to mammals

CpTI – found in cowpea (vigna unguiculata) – active CpTI – found in cowpea (vigna unguiculata) – active inhibitorinhibitor

Inhibitor gene produces antimetabolite substances – Inhibitor gene produces antimetabolite substances – which provide major protection against the major which provide major protection against the major storage pest Bruchid beetel (callosobruchus storage pest Bruchid beetel (callosobruchus maculatus)maculatus)

Also harmful to various lepidopteran insects Also harmful to various lepidopteran insects (Heliothis virescens), spodopteran insects (Manduca (Heliothis virescens), spodopteran insects (Manduca sexta), Coleopteran insects (Callosobruchus, sexta), Coleopteran insects (Callosobruchus, Anthonomus grandis), orthopteran insects (locusta Anthonomus grandis), orthopteran insects (locusta migratoria) migratoria)

CpTI has been cloned & constructs CpTI has been cloned & constructs containing CaMV 35S promoter & a full containing CaMV 35S promoter & a full length cDNA clone 550bp long were used to length cDNA clone 550bp long were used to transform leaf discs of tobaccotransform leaf discs of tobacco

Bioassay for insecticidal activity – of Bioassay for insecticidal activity – of transgenic tobacco plants was done with transgenic tobacco plants was done with cotton bollworm (helicoverpa zea)cotton bollworm (helicoverpa zea)

Insect survival & plant damage – decreased Insect survival & plant damage – decreased in transgenic plants compared with controlin transgenic plants compared with control

CpTI gene – also introduced in to potato & CpTI gene – also introduced in to potato & oil seed rape oil seed rape

Binary cloning vector carrying a cowpea trypsin inhibitor (CTI) gene

(pNOS-NPT-tNOS)(35S-CTI-tNOS)

(Kanr)

ADVANTAGESADVANTAGES

Many insects not controlled by Bt – can Many insects not controlled by Bt – can be effectively controlledbe effectively controlled

Use of proteinase gene along with Bt Use of proteinase gene along with Bt gene – help to overcome Bt resistance gene – help to overcome Bt resistance development in plantsdevelopment in plants

LIMITATIONSLIMITATIONS High levels of proteinase inhibitors are High levels of proteinase inhibitors are

required to kill insectsrequired to kill insects Necessary that expression of proteinase Necessary that expression of proteinase

inhibitors – should be low in plant parts inhibitors – should be low in plant parts consumed by humansconsumed by humans

High in parts of plants utilised by insectsHigh in parts of plants utilised by insects

2. ALPHA AMYLASE INHIBITOR

ALPHA AMYLASE ALPHA AMYLASE INHIBITORINHIBITOR

Genes for three alpha amylase inhibitors Genes for three alpha amylase inhibitors expressed in tobaccoexpressed in tobacco

Transferring the gene of alpha amylase Transferring the gene of alpha amylase inhibitor (alpha AI-Pv) isolated from Adzuki inhibitor (alpha AI-Pv) isolated from Adzuki bean (phaseolus vulgaris) – transferred & bean (phaseolus vulgaris) – transferred & expressed in tobaccoexpressed in tobacco

Works & provides resistance against Works & provides resistance against coleoptra - Zabrotes subfasciatus & coleoptra - Zabrotes subfasciatus & callosobruchus chinensiscallosobruchus chinensis

Inhibitor – blocks larval feeding in the midgutInhibitor – blocks larval feeding in the midgut The insect larvae secrete a gut enzyme – The insect larvae secrete a gut enzyme –

called alpha amylase that digests starchcalled alpha amylase that digests starch By adding a protein that inhibits insect gut By adding a protein that inhibits insect gut

alpha amylase & blocking the activity of this alpha amylase & blocking the activity of this enzyme by alpha amylase inhibitor – weevil enzyme by alpha amylase inhibitor – weevil undergoes starvation & dies undergoes starvation & dies

LECTINSLECTINS LECTINS – are plant glycoproteinsLECTINS – are plant glycoproteins They provide resistance to insects by They provide resistance to insects by

acting as toxinsacting as toxins Lectin gene (GNA) from snow drop Lectin gene (GNA) from snow drop

(Galanthus nivalis) has been transferred (Galanthus nivalis) has been transferred & expressed in potato & tomato, oil seed & expressed in potato & tomato, oil seed raperape

Activity against aphidsActivity against aphids

Also act against piercing & sucking insectsAlso act against piercing & sucking insects LIMITATIONLIMITATION Works well only when ingested in large Works well only when ingested in large

quantities quantities

Stress tolerance- BioticStress tolerance- Biotic2.2. Virus Resistance:Virus Resistance: Several approaches have been used to engineer plants for Several approaches have been used to engineer plants for

virus resistance, which are as follows: virus resistance, which are as follows: (1) coat protein gene, (1) coat protein gene, (2) cDNA of satellite RNA, (2) cDNA of satellite RNA, (3) defective viral genome,(3) defective viral genome,(4) antisense RNA approach, and (4) antisense RNA approach, and (5) ribozyme  mediated protection. (5) ribozyme  mediated protection.

Of these strategies, use of coat protein gene has been the Of these strategies, use of coat protein gene has been the most successful. Transgenic plants having virus coat protein most successful. Transgenic plants having virus coat protein gene linked to a strong promoter have been produced m gene linked to a strong promoter have been produced m many crop plants, tobacco, tomato, alfalfa, sugarbeet, many crop plants, tobacco, tomato, alfalfa, sugarbeet, potato, etc. potato, etc.

The disease rasistance generated by employing pathogen The disease rasistance generated by employing pathogen genes is called pathogen derived resistance(PDR).genes is called pathogen derived resistance(PDR).

VIRUS RESISTANCEVIRUS RESISTANCE Virus infections of crops – result in retarded cell Virus infections of crops – result in retarded cell

division (hypoplasia), excessive cell division division (hypoplasia), excessive cell division (hyperplasia) & cell death (necrosis)(hyperplasia) & cell death (necrosis)

Strategy to integrate or create new resistance factors Strategy to integrate or create new resistance factors in plant virus systemsin plant virus systems

Approach – to identify those viral genes or gene Approach – to identify those viral genes or gene products – when present at an improper time or in products – when present at an improper time or in wrong amount – will interfere with normal functions of wrong amount – will interfere with normal functions of the infection process & prevent disease development the infection process & prevent disease development

Possible to immunize plants against viral Possible to immunize plants against viral damages by expressing viral proteins in damages by expressing viral proteins in plant cellsplant cells

COAT PROTEIN MEDIATED COAT PROTEIN MEDIATED CROSS PROTECTIONCROSS PROTECTION

Cross protection – the ability of one virus to Cross protection – the ability of one virus to prevent or inhibit the effect of a second prevent or inhibit the effect of a second challenge viruschallenge virus

If susceptible strain of a crop is inoculated with If susceptible strain of a crop is inoculated with mild strain of a virus – then the susceptible strain mild strain of a virus – then the susceptible strain develops resistance against more virulent strainsdevelops resistance against more virulent strains

Transgenic plants having virus coat protein – Transgenic plants having virus coat protein – gene linked to promoter – produced in many gene linked to promoter – produced in many cropscrops

The first transgenic plant of this type was tobacco The first transgenic plant of this type was tobacco produced in 1986; it contained & expressed the produced in 1986; it contained & expressed the coat protein gene of tobacco mosaic virus (TMV) coat protein gene of tobacco mosaic virus (TMV) strain U I. strain U I.

Exhibited high levels of resistance to TMVExhibited high levels of resistance to TMV When these plants were inoculated with TMV U I, When these plants were inoculated with TMV U I,

symptoms symptoms either failed to develop or were either failed to develop or were considerably delayed.  considerably delayed. 

Further, there was a much less accumulation of Further, there was a much less accumulation of virus than in the control plants in both inoculated virus than in the control plants in both inoculated and systemically infected leaves.and systemically infected leaves.

In addition, these plants In addition, these plants showed delayed showed delayed expression of disease symptomsexpression of disease symptoms when inoculated when inoculated with the related tomato mosaic virus (ToMV) and with the related tomato mosaic virus (ToMV) and with tobacco mild green mosaic virus (TMGMV)with tobacco mild green mosaic virus (TMGMV)

Inhibition of viral replication at initial point of Inhibition of viral replication at initial point of infectioninfection

Initial step in viruslife cycle – is disruptedInitial step in viruslife cycle – is disrupted Most likely the resistance generated by CP is due to Most likely the resistance generated by CP is due to

the blocking of the process of uncoating of virus the blocking of the process of uncoating of virus particles, which is necessary for viral genome particles, which is necessary for viral genome replication as well as expressionreplication as well as expression

It appears to be a common feature that expression It appears to be a common feature that expression of a virus coat protein gene not only confers of a virus coat protein gene not only confers resistance to the concerned virus but also gives a resistance to the concerned virus but also gives a measure of resistance to related viruses. measure of resistance to related viruses.

The effectiveness of coat protein (CP) gene in The effectiveness of coat protein (CP) gene in conferring virus resistance can be affected by both conferring virus resistance can be affected by both the amount of the amount of coat protein produced in coat protein produced in transgenic plants and by the concentration of transgenic plants and by the concentration of virus inoculumvirus inoculum. .

In most cases – virus resistance is produced by the In most cases – virus resistance is produced by the virus coat proteins & not by the mRNA transcript of virus coat proteins & not by the mRNA transcript of the genesthe genes

However, other effects seem to be involved However, other effects seem to be involved in producing coat protein mediated virus in producing coat protein mediated virus resistance; one such mechanism appears to resistance; one such mechanism appears to be the prevention or delay of systemic be the prevention or delay of systemic spread of the viruses.  spread of the viruses. 

But at least in some cases, the resistance But at least in some cases, the resistance mechanism does not involve the coat mechanism does not involve the coat protein itself since CP genes even in protein itself since CP genes even in antisense orientation produce resistance to antisense orientation produce resistance to the virus.the virus.  

Transgenic papaya plants show resistance (right) while non-transgenic plants (left) are susceptible to papaya ringspot virus under field conditions. Photo courtesy of Dennis Gonsalves, Cornell University.

Transgenic plant providing coat protein Transgenic plant providing coat protein mediated resistance to virus – are rice, mediated resistance to virus – are rice, potato, wheat, tobacco, peanut, sugar beet, potato, wheat, tobacco, peanut, sugar beet, alfalfa, tomato.alfalfa, tomato.

Viruses include – Alfalfa mosaic Viruses include – Alfalfa mosaic virus(AIMV), cucumber mosaic virus virus(AIMV), cucumber mosaic virus (CMV), Potato virus X (PVY), Citrus tristeza (CMV), Potato virus X (PVY), Citrus tristeza virus (CTV) and R rice stripe virus (RSV)virus (CTV) and R rice stripe virus (RSV)

MECHANISM OF ACTIONMECHANISM OF ACTION As transgenic plant – expresses the gene As transgenic plant – expresses the gene

for coat protein of a given virus – the for coat protein of a given virus – the ability of the same virus to infect the ability of the same virus to infect the plants again is drastically reducedplants again is drastically reduced

Molecular mechanism – not unknownMolecular mechanism – not unknown

OTHER VIRAL GENESOTHER VIRAL GENES Viral genes other than that for coat proteins Viral genes other than that for coat proteins

used to generate virus resistance in plantsused to generate virus resistance in plants Poty viruses & genes that were used for Poty viruses & genes that were used for

encoding these poty virus genome linked encoding these poty virus genome linked proteins (VPg), VPg proteinases (Nla), RNA proteins (VPg), VPg proteinases (Nla), RNA dependent RNA polymerase(Nlb), cylindrical dependent RNA polymerase(Nlb), cylindrical inclusion proteins (CI)inclusion proteins (CI)

cDNA OF SATELLITE RNAcDNA OF SATELLITE RNA Some RNA viruses have small RNA Some RNA viruses have small RNA

molecule s called satellitesmolecule s called satellites They depend on viral genomes for their They depend on viral genomes for their

replication – not necessary for viral replication – not necessary for viral functionsfunctions

In many cases – satellite may increase or In many cases – satellite may increase or decrease the severity of disease produced decrease the severity of disease produced by the virus carrying itby the virus carrying it

cDNA copies of the satellites that reduce cDNA copies of the satellites that reduce disease severity – have been integrated in to disease severity – have been integrated in to host genomeshost genomes

Expression of the satellites – shown to reduce Expression of the satellites – shown to reduce disease symptoms as well as virus disease symptoms as well as virus accumulation under both green house & firld accumulation under both green house & firld conditionsconditions

Tobacco plants – expressing the satellites of Tobacco plants – expressing the satellites of cucumber mosaic virus showed reduced cucumber mosaic virus showed reduced disease symptoms when infected with CMV or disease symptoms when infected with CMV or with the related tomato aspermy virus (TAV)with the related tomato aspermy virus (TAV)

Transgenic pepepr, tomato, tobacco plants Transgenic pepepr, tomato, tobacco plants – expressing CMV satellite – grown in field – expressing CMV satellite – grown in field showed reduced disease symptoms & less showed reduced disease symptoms & less virus accumulation than the control plants virus accumulation than the control plants inoculated with CMVinoculated with CMV

DEFECTIVE VIRAL DEFECTIVE VIRAL GENOMESGENOMES

Defective or deleted genomes of some Defective or deleted genomes of some RNA & DNA viruses disrupt the RNA & DNA viruses disrupt the replication of complete genomes of those replication of complete genomes of those viruses with which they are associatedviruses with which they are associated

Eg African cassava mosaic virus (ACMV) Eg African cassava mosaic virus (ACMV) genome consists of two ss DNA genome consists of two ss DNA molecules – designated as A & B DNAsmolecules – designated as A & B DNAs

In addition – a 50% deleted B DNA – is also In addition – a 50% deleted B DNA – is also found associated with ACMV particlesfound associated with ACMV particles

Tobacco plants containing this deleted B DNA Tobacco plants containing this deleted B DNA integrated in their genomes showed reduced integrated in their genomes showed reduced systemic spread when they were infected with systemic spread when they were infected with ACMVACMV

Disease severity - reducedDisease severity - reduced

ANTISENSE RNA APPROACHANTISENSE RNA APPROACH RIBOZYME MEIDATED PROTECTIONRIBOZYME MEIDATED PROTECTION

ANTISENSE RNA ANTISENSE RNA APPROACHAPPROACH

Stress tolerance- BioticStress tolerance- Biotic3. Fungi & Bacteria Resistance:3. Fungi & Bacteria Resistance: In case of bacterial and fungal pathogens, resistance has been sought In case of bacterial and fungal pathogens, resistance has been sought

to be generated by expression of the following transgenes:  to be generated by expression of the following transgenes:  1) genes encoding insensitive target enzymes, 1) genes encoding insensitive target enzymes, 

(2) genes specifying toxin inactivation, (2) genes specifying toxin inactivation, (3) expression of antibacterial peptides,(3) expression of antibacterial peptides,(4) expression of bacterial lysozymes, (4) expression of bacterial lysozymes, (5) genes specifying artificially programmed cell death (in items 1-5, (5) genes specifying artificially programmed cell death (in items 1-5,

transgenes are from non plant sources),transgenes are from non plant sources),(6) expression of heterologous phytoalexins, (6) expression of heterologous phytoalexins,

(7) genes encoding ribosome inactivating proteins,(7) genes encoding ribosome inactivating proteins,(8) expression of heterologous thionins, (8) expression of heterologous thionins, (9) ectopic (out of the natural place) expression of pathogenesis related (9) ectopic (out of the natural place) expression of pathogenesis related

proteins, and proteins, and (10) ectopic expression of chitinases (items 6-10 use plant genes). In (10) ectopic expression of chitinases (items 6-10 use plant genes). In

almost all the approaches, transgenic plants showed increased almost all the approaches, transgenic plants showed increased

resistance to the concerned diseases. resistance to the concerned diseases.

The strategy of artificially programmed cell death has been The strategy of artificially programmed cell death has been designed to mimick hypersensitive response. designed to mimick hypersensitive response.

A programmed cell death is brought about by endogenous A programmed cell death is brought about by endogenous gene action, particularly in response to some specific gene action, particularly in response to some specific stimulus, e.g., the elicitor specified by (avirulence) avr stimulus, e.g., the elicitor specified by (avirulence) avr genes of the pathogen in the case of hypersensitive genes of the pathogen in the case of hypersensitive response. response.

However, hypersensitive response depends on specific However, hypersensitive response depends on specific pairs of avr genes of pathogens and R (resistance) genes pairs of avr genes of pathogens and R (resistance) genes of the host. Therefore, each such pair specifies resistance of the host. Therefore, each such pair specifies resistance to a single race of a pathogen and is not of general to a single race of a pathogen and is not of general applicability.applicability.

In contrast, the artificially programmed cell death is so In contrast, the artificially programmed cell death is so designed as to cover all the races of a pathogen and designed as to cover all the races of a pathogen and possibly, more than one pathogen as well. There are two possibly, more than one pathogen as well. There are two schemes for artificial cell death, viz., schemes for artificial cell death, viz.,

1) two-component and 1) two-component and (2) single-component systems. (2) single-component systems.

The single-component system is based on the expression of The single-component system is based on the expression of a toxic polypeptide in response to pathogen infection. a toxic polypeptide in response to pathogen infection.

The transgenes usable in this scheme may be those that The transgenes usable in this scheme may be those that encode toxins, ribonucleases, or other enzymes, whose encode toxins, ribonucleases, or other enzymes, whose products are toxic to plant cells.products are toxic to plant cells.

The barnase gene from Bacillus amyloliquefaciens was The barnase gene from Bacillus amyloliquefaciens was placed under the control of infection  specific promoter prp1-placed under the control of infection  specific promoter prp1-1and was transferred into potato. 1and was transferred into potato.

Transgenic potatoes showed effective control of Transgenic potatoes showed effective control of Phytophthora infestans. Phytophthora infestans.

Promoter prp1-1j ensures the expression of barnase gene in Promoter prp1-1j ensures the expression of barnase gene in such cells that are infected by a fungal pathogen. such cells that are infected by a fungal pathogen. 

Synthesis of Barnase protein, an RNase, in such cells leads Synthesis of Barnase protein, an RNase, in such cells leads to their death; the pathogen would also die along with the to their death; the pathogen would also die along with the dying host cells. dying host cells.

Obviously, the strategy of artificially programme cell death Obviously, the strategy of artificially programme cell death will be effective against obligate parasites, but not against will be effective against obligate parasites, but not against facultative parasites; in fact, facultative parasites may be facultative parasites; in fact, facultative parasites may be pleased to use it to their own advantage.pleased to use it to their own advantage.

TWO COMPONENT TWO COMPONENT SYSTEMSYSTEM

2 component system : 2 precisely matched 2 component system : 2 precisely matched transgenes are expressed in the same celltransgenes are expressed in the same cell

1 transgene – avr gene – avr 9 driven by a 1 transgene – avr gene – avr 9 driven by a promoter inducible by a nonspecific elicitor promoter inducible by a nonspecific elicitor produced during by pathogen invasionproduced during by pathogen invasion

Other transgene R Gene – corresponding to Other transgene R Gene – corresponding to the avr gene used – cf9the avr gene used – cf9

This transgene - driven by a constitutive This transgene - driven by a constitutive promoterpromoter

Expression of avr gene – precisly regulatedExpression of avr gene – precisly regulated It must be expressed immediately following It must be expressed immediately following

pathogen attack but only in the infected cellspathogen attack but only in the infected cells Expression of avr gene – would produce Expression of avr gene – would produce

elicitor – that would be recognised by the elicitor – that would be recognised by the coressponding R gene productcoressponding R gene product

This recognition – initiate & culmiante hyper This recognition – initiate & culmiante hyper sensitive response sensitive response

Stress tolerance-Abiotic Stress tolerance-Abiotic

1.1. Herbicide tolerance:Herbicide tolerance: Development of transgenic plants resistant to certain Development of transgenic plants resistant to certain

biodegradable herbicides was the 1biodegradable herbicides was the 1stst major major achievement from genetic eng’g in plants.achievement from genetic eng’g in plants.

Infact this activity generated the basic tools and Infact this activity generated the basic tools and techniques for gene transfer in plants, and several techniques for gene transfer in plants, and several gene that confer herbicide resistant serve as useful gene that confer herbicide resistant serve as useful selectable marker.selectable marker.

Transgenic plants resistant to several Transgenic plants resistant to several herbicides,eg.,Glyphosate,glufosinate,sulfonylurease, herbicides,eg.,Glyphosate,glufosinate,sulfonylurease, etc., have been successfully developed and are etc., have been successfully developed and are commercial cultivation in U.S.commercial cultivation in U.S.

Strategies for Herbicide Resistance:Strategies for Herbicide Resistance: Herbicide resistance in plants can be generated by expressing Herbicide resistance in plants can be generated by expressing

in them transgenes that serve one of the following purposes.in them transgenes that serve one of the following purposes.

1.Overproduction of EPSPS enzyme1.Overproduction of EPSPS enzyme This strategy effectively involves titrating the herbicide out by This strategy effectively involves titrating the herbicide out by

overproduction of the target protein.overproduction of the target protein. For eg, if the herbicide is a specific inhibitor of one particular For eg, if the herbicide is a specific inhibitor of one particular

enzyme, production of sufficient excess enzyme will partially enzyme, production of sufficient excess enzyme will partially overcome the inhibition.overcome the inhibition.

Over expression can be achieved by the integration of multiple Over expression can be achieved by the integration of multiple copies of the genee and/or the use of a strong promoter plus copies of the genee and/or the use of a strong promoter plus translational enhancer to drive expression of the gene.translational enhancer to drive expression of the gene.

2. Mutation of the target protein:2. Mutation of the target protein: The logic behind this approach is to find a modified target The logic behind this approach is to find a modified target

protein that substitutes functionally for the native protein protein that substitutes functionally for the native protein and which is resistant to inhibition by herbicide, and to and which is resistant to inhibition by herbicide, and to incorporate the resistant target protein gene into the plant incorporate the resistant target protein gene into the plant genome. genome.

Several sources of resistant proteins can be exploited.Several sources of resistant proteins can be exploited.

3. Detoxification of the herbicide, using a single gene from a 3. Detoxification of the herbicide, using a single gene from a foreign source:foreign source:

Detoxification is a means of converting the herbicide to a less toxic form and/or removing it from the system.

This strategy can be contrasted with the previous two b’cos it does not require a detailed knowledge of the site of action.

4. Enhanced plant detoxification: The aim here is to improve the natural

plant defences toxic compounds. This requires detailed information about

endogenous plant detoxification pathways and the mechanism by which compounds are recognized and targeted for detoxification by the plant.

HERBICIDE RESISTANT HERBICIDE RESISTANT PLANTSPLANTS

Glyphosate Resistance:Glyphosate Resistance: Glyphosate is a broad spectrum herGlyphosate is a broad spectrum herbicide that bicide that

inhibits the enzyme inhibits the enzyme 5-enolpyruvylshikimate-3-5-enolpyruvylshikimate-3-phosphate synthase (EPSPSphosphate synthase (EPSPS).).

Enzyme EPSPS is involved in aromatic amino Enzyme EPSPS is involved in aromatic amino acid biosynthesis in plants. acid biosynthesis in plants.

Thus the killing action of glyphosate results Thus the killing action of glyphosate results primarily from primarily from starvingstarving the cells of aromatic the cells of aromatic amino acid, which disrupts their amino acid, which disrupts their protein protein synthesissynthesis..

This is reputedly effective against 76 of the This is reputedly effective against 76 of the world’s worst 78 weeds and is marketed as world’s worst 78 weeds and is marketed as “Roundup” by American chemical company “Roundup” by American chemical company Monsanto. Monsanto.

Glyphosate binds more tightly to the EPSPS-shikimate-3-phosphate complex than does PEP-its dissociation rate from the complex is 2300 times slower than PEP.

EPSPS is inactivated once the glyphosate binds to the enzyme-substrate complex.

EPSPS is a key enzyme in the biosynthetic pathway of the aromatic amino acids Phe, Tyr, Trp.

Thus, the herbicidal activity of the glyphosate result from inhibition of the biosynthesis of aromatic amino acids and other products of the shikimate pathway.

Stress tolerance-Abiotic Stress tolerance-Abiotic

2. Drought Resistance:2. Drought Resistance: A number of such genes have been identified; isolated, cloned and A number of such genes have been identified; isolated, cloned and

expressed in plants, which are potential sources of resistance to expressed in plants, which are potential sources of resistance to abiotic stresses.abiotic stresses.

These genes include Rab (responsive to abscisic acid) and SalT (induced in response to salt stress) genes of rice; genes for enzymes involved in proline biosynthesis in bacteria (proBA and proC in E. coli) and plants, spinach genes involved in betaine synthesis, etc.

In plants, proline is preferentially produced from ornithine under normal conditions. However, under stress it is made directly from glutamate, the first two reactions of the pathway being catalyzed by a single enzyme ∆1-pyrroline  5-carboxylate synthetase (P5CS).

The gene encoding P5CS has been isolated from soybean and moth bean, and cloned. 

The moth bean P5CS gene has been transferred and over expressed in tobacco.

The transgenic plants produced 10- to 18-fold more proline than the control plants.

The leaves of transgenic plants retained a higher osmotic potential and showed a greater root biomass under water stress than did the control plants. 

These findings indicates that over expression of P5CS in plants enhances their tolerance to osmotic stress.

The primary function of accumulation of proline and other solutes, e.g., glycine betaine appears to be the regulation of intracellular water activity; under water stress, they may induce the formation of strong H-bonded water around proteins, thereby preserving the native state of cell biopolymers.

But it should be kept in mind that accumulation of proline is only one of the factors, which enable plants to sustain growth under water stress. Other factors also allow plants to overcome osmotic stress. 

Stress tolerance-Abiotic Stress tolerance-Abiotic 3. Salt Resistance:3. Salt Resistance: Plant species vary in how well they tolerate salt-Plant species vary in how well they tolerate salt-

affected soils. Some plants will tolerate high levels of affected soils. Some plants will tolerate high levels of salinity while others can tolerate little or no salinity. The salinity while others can tolerate little or no salinity. The relative growth of plants in the presence of salinity is relative growth of plants in the presence of salinity is termed their salt tolerance.termed their salt tolerance.

Salt tolerances are usually given in terms of the stage of plant growth over a range of electrical conductivity

(EC) levels.  Electrical conductivity is the ability of a solution to

transmit an electrical current. To determine soil salinity EC, an electrical current is imposed in a glass cell using two electrodes in a soil extract solution taken from the soil being measured (soil salinity). The units are usually given in deciSiemens per metre (dS/m).

Table 1 categorizes salinity into general ranges from non- saline to very strongly saline. These values are used for plant selection for saline soils.

Salinity levels vary widely across a saline seep. Salinity also varies from spring to fall. Salinity usually appears on the soil surface just after spring thaw.

ds/m = decisiemens per metre.

Table 1. Salinity rating and electrical conductivity value

Soil DepthNon-Saline

Weakly

Saline

Moderately

Saline

StronglySalin

e

Very Strongly

Saline

0-60 cm (0-2 ft) <2 ds/m*2-4

ds/m

4-8 ds/m 8-16 ds/m >16 ds/m

60-120 cm(2-4 ft)

<4 ds/m4-8

ds/m

8-16 ds/m16-24

ds/m>24 ds/m

The dominant salts in prairie saline seeps are calcium (Ca), magnesium (Mg), sodium (Na) cations and sulfate (SO4) anions.

If Na levels are high or not balanced with the Ca and Mg, soil tilth can also be effected. The positively charged Na cations attach to the negatively charged clay particles in the soil, causing the soil to be sticky when wet, and hard and impermeable when dry.

Table 2 gives salinity tolerance ratings for a range of plant species and a range of salinity levels.

New research underway may modify the rating of some plant types.

As a general rule, plants that have low drought tolerance will have low salinity tolerance.

Table 2. Salt tolerance of various types of plants

Salt ToleranceEC (ds/m)

Field Crops Forages Vegetables Trees, Shrubs

Very High20

  beardless wildryefulks altai grasslevonns alkaligrassalkali sucatan

   

High16

kochiasugar beets

altai wildryetall wheatgrassRussian wildryeslender wheat grass

  Siberian salt treesea buckthornsilver buffaloberry

8 6-row barleysafflowersunflower2-row barleyfall ryewinter wheatspring wheat

birdsfoot trefoilsweetcloveralfalfabromegrass

garden beetsasparagusspinach

hawthornRussian oliveAmerican elmSiberian elmvillosa lilaclaurel leaf willow

Moderate oatsyellow mustard

crested wheatgrassintermediate wheatgrass

tomatoesbroccoli

spreading juniperpoplar

  meadow fescueflaxcanola

reed canary grass cabbage ponderosa pineapplemountain ash

4 corn   sweet cornpotatoes

common lilacSiberian crab appleManitoba mapleViburnum

Low timothypeasfield beans

white dutch cloveralsike cloverred clover

carrotsonionsstrawberriespeasbeans

Colorado blue spruceroseDouglas firbalsam fircottonwoodaspen, birchraspberry

0       black walnutdogwoodlittle-leaved lindenwinged euonymusspirealarch

Delayed Fruit RipeneingDelayed Fruit Ripeneing

Ripening is a normal phase in the maturation process Ripening is a normal phase in the maturation process of fruits and vegetables. of fruits and vegetables.

Upon its onset, it only takes about a few days before Upon its onset, it only takes about a few days before the fruit or vegetable is considered inedible. the fruit or vegetable is considered inedible.

This unavoidable process brings significant losses to This unavoidable process brings significant losses to both farmers and consumers alike.both farmers and consumers alike.

Scientists have been working to delay fruit ripening so Scientists have been working to delay fruit ripening so that farmers will have the flexibility in marketing their that farmers will have the flexibility in marketing their goods and ensure consumers of “fresh-from-the-goods and ensure consumers of “fresh-from-the-garden” produce.garden” produce.

A notable example of this kind is the “Flavr Savr” A notable example of this kind is the “Flavr Savr” transgenic tomatoes, which are commercialized in U.S. transgenic tomatoes, which are commercialized in U.S. about 6 years ago. about 6 years ago.

The Fruit Ripening Process Ethylene is a natural plant hormone associated

with the growth, development, ripening and aging of many plants. 

This phytohormone is said to promote ripening in a variety of fruits including bananas, pineapples, tomatoes, mangoes, melons, and papayas. 

When the concentration of ethylene reaches 0.1-1.0 ppm (parts per million), the ripening process in climacteric fruits is considered irreversible.

In tomatoes, it takes about 45-55 days for the fruit to reach full maturity. After which, it starts to undergo the ripening process

Controlling the Ripening Process There are several ways by which scientists can control

the ripening process by genetic modification. 1. Regulation of Ethylene Production The amount of ethylene produced can be controlled

primarily by “switching off” or decreasing the production of ethylene in the fruit and there are several ways to do this. They include:

Suppression of ACC synthase gene expression. ACC (1-aminocyclopropane-1-carboxylic acid) synthase is the enzyme responsible for the conversion of S-adenosylmethionine (SAM) to ACC; the second to the last step in ethylene biosynthesis. Enzyme expression is hindered when an antisense (“mirror-image”) or truncated copy of the synthase gene is inserted into the plant’s genome.

Insertion of the ACC deaminase gene. The gene coding for the enzyme is obtained from Pseudomonas chlororaphis, a common nonpathogenic soil bacterium. It converts ACC to a different compound thereby reducing the amount of ACC available for ethylene production.

Insertion of the SAM hydrolase gene. This approach is similar to ACC deaminase wherein ethylene production is hindered when the amount of its precursor metabolite is reduced; in this case SAM is converted to homoserine. The gene coding for the enzyme is obtained from E. coli T3 bacteriophage.

Suppression of ACC oxidase gene expression. ACC oxidase is the enzyme which catalyzes the oxidation of ACC to ethylene, the last step in the ethylene biosynthetic pathway. Through anti-sense technology, down regulation of the ACC oxidase gene results in the suppression of ethylene production, thereby delaying fruit ripening.

2. Control of Ethylene Perception Since ethylene signals the onset of fruit

ripening, delayed ripening on some plants can be achieved by modifying their ethylene receptors.

The gene ETR1 is one example, and it has been shown to encode an ethylene binding protein.

Plants with modified ETR1 lack the ability to respond to ethylene.

3. Suppression of Polygalacturonase Activity

Polygalacturonase (PG) is the enzyme responsible for the breakdown of pectin, the substance that maintains the integrity of plant cell walls.

Pectin breakdown occurs at the start of the ripening process resulting in the softening of the fruit.

To produce a fruit with DR trait using this method, scientists insert an anti-sense or a truncated copy of the PG gene into the plant’s genome resulting in a dramatic reduction of the amount of PG enzyme produced thereby delaying pectin degradation.

Male SterilityMale Sterility

Male sterility can be produced by transferring Male sterility can be produced by transferring certain genes from other species against certain genes from other species against endogenous genes,e.g., endogenous genes,e.g., rolBrolB and and rolCrolC genes genes from from Agrobacterium rhizogenesAgrobacterium rhizogenes, , barnasebarnase gene gene from from Bacillus amyloliquefaciensBacillus amyloliquefaciens, etc.,., etc.,.

Gene barnase is the 1st transgene that was used to produce male sterility by Mariani & coworkers in 1990.

Its an effective fertility restoration system in barstar.

Barnase-barstar system: In 1990, C. Mariani and others from Belgium,

successfully used a gene construct having an anther specific promoter (from TA29 gene of tobacco) and bacterial coding sequence for a ribonuclease (barnase gene from Bacillus amyloliquefaciens) for production of transgenic plants in B. napus. 

The results were spectacular in the sense that the transferred gene prevented normal pollen development leading to male sterility.

The product of barnase gene is cytotoxic, killing the tapetal cells, thus preventing pollen development.

Utilizing this male sterility barnase gene construct (TA-29- RNase), it was possible to introduce male sterility in other crops also.

These crops include tobacco, lettuce, cauliflower, cotton, tomato, corn, etc.

The same group of workers (Mariani el al., 1992) used another gene construct later involving the same anther specific promoter i.e. T A 29 and the barstar gene from B. amyloliquefaciens, for production of transgenic plants in B. napus.

The product of barstar gene is a ribonuclease inhibitor.

It forms a complex with ribonuclease and neutralizes its cytotoxic properties. In Bacillus, the ribonuclease is active extracellularly and the bacterium itself is protected by a ribonuclease inhibitor protein coded by barstar gene.

When transgenic male sterile plants (with barnase gene) were crossed with transgenic male fertile plants (with barstar gene), the F1 plants expressed both genes so that male fertility was restored due to suppression of cytotoxic ribonuclease activity in the anther by the formation of cell specific RNase/RNase inhibitor complexes.

This system of transgenic plants should facilitate hybrid seed production in crop plants in general.

Methods of Gene Methods of Gene TransferTransfer

These include:1. Electroporation2. Particle Gun3. Microinjection4. Agrobacterium-mediated transfer,5. Co-cultivation method6. Leaf disc transformation method7. Virus-mediated transformation8. Pollen-mediated transformation9. Liposome-mediated transformation, etc..

Applications1. They have proved to be extremely valuable tools in studies on

plant molecular biology, regulation of gene action, identification of regulatory/promotary sequences, etc.

2. Specific genes have been transferred into plants to improve their agronomic and other features.

3. Genes for resistance to various biotic stresses have been engineered to generate transgenic plants resistant to insects, viruses, etc.

4. Several gene transfers have been aimed at improving the produce quality.

5. Transgenic plants are being used to produce novel biochemicals like hirudin, etc. which are not produced by normal plants.

6. Transgenic plants can be used vaccines for immunization against pathogens; this is fast emerging as an important objective.