This document discusses molecular breeding methods for developing plant resistance. It outlines several approaches including using transgenics with antimicrobial molecules like pathogenesis related (PR) proteins from microbes, manipulating disease resistance genes, and using genes from Bacillus thuringiensis (Bt) to develop insect resistance. The molecular approaches described include using coat protein-mediated resistance, movement protein-mediated resistance, and ribozymes to develop virus resistance in transgenic plants. Examples are provided of transgenic crops developed with resistance to various fungal, bacterial, viral, and insect pests.

1. Molecular Breeding
methods for Plant
Resistance
Siddhartha Swarup Jena
RAD/10-30
Ph.D. Mol. Bio & Biotechnology
Presented by:

2. OUTLINE
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3. Need for development of Plant Resistance
25%
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4. Disease developmentDisease development
EnvironmentEnvironmentPathogenPathogen
HostHost
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5.  In-vitro selection of disease resistant mutants:
Somaclonal variation
 Recombinant DNA technology
Through plant transformation techniques
Molecular approaches for development ofMolecular approaches for development of
plant resistanceplant resistance
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6. I. DEVELOPMENT OF PLANTI. DEVELOPMENT OF PLANT
RESISTANCE TO BACTERIARESISTANCE TO BACTERIA
AND FUNGIAND FUNGI
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7. Generally host and pathogen show ‘gene for gene
relationship’ for disease reaction.
Gene for gene relationship is of two types
i) Incompatible reaction:
Found in biotrophic pathogens viz. rusts, smuts etc.
The alleles for resistance in host (R) and those for avirulence in the
pathogen (A) produce specific compounds which recognize each other
and produce resistant response in the host.
The other 3 combinations of alleles produce susceptible reaction
ii) Compatible reaction:
Found in heterotrophic pathogens
The alleles for susceptibility in host (r) and those for virulence in the
pathogen (a) produce specific compounds which interact with each
other to produce susceptible response in the host.
Gene for geneGene for gene
relationshiprelationship
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8. Approaches for developing disease resistance is
broadly classified into 2 categories
I. Transgenics with antimicrobial molecules like
proteins and toxins
II. Transgenics which generate a hypersensitive
response through R genes or by manipulating
genes of the SAR pathway
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9. 1. Pathogenesis related proteins:
Low molecular weight proteins accumulate to significant levels in
infected plant tissues
Major classes of pathogenesis related proteins
PR-1, PR-2 (ß 1-3 glucanase)
PR-3 ( Chitinases)
PR-4 (Hevein like)
PR-5 (Thaumatin like & Osmotin)
The ability of hydrolytic enzymes to break chitin and Glucan in the cell
walls of fungal pathogen has been exploited to develop crop
resistance to pathogens.
Ex: Tobacco – chitinase gene obtained from soil bacteria Serratia
marcescens – post emergent damping off.
I. Transgenics with antimicrobial moleculesI. Transgenics with antimicrobial moleculesI. Transgenics with antimicrobial moleculesI. Transgenics with antimicrobial molecules
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10. Transgenic plants generated for resistance to fungal and bacterial
diseases using PR protein
CropsCrops Gene transferredGene transferred Controlled pathogenControlled pathogen
TobaccoTobacco
Chitinase fromChitinase from SerratiaSerratia
marcescensmarcescens
Bean chitinase geneBean chitinase gene
PR-1-a genePR-1-a gene
ChitinaseChitinase
ChitinaseChitinase
Chitinase and 1,3-Chitinase and 1,3-ββ glucanaseglucanase
Alternaria longipesAlternaria longipes
Rhizoctonia solaniRhizoctonia solani
Peronospora tabacina PhytophthoraPeronospora tabacina Phytophthora
Parasitica var. nicotianaeParasitica var. nicotianae
Sclerotinia sclerotiorumSclerotinia sclerotiorum
Rhizoctonia solaniRhizoctonia solani
Cercospora nicotinaeCercospora nicotinae
TomatoTomato Chitinase and 1,3-Chitinase and 1,3-ββ glucanaseglucanase Fusarium oxysporum lycopersiciFusarium oxysporum lycopersici
Brassica napusBrassica napus ChitinaseChitinase Rhizoctonia solaniRhizoctonia solani
Brassica napusBrassica napus
var. oleiferavar. oleifera
ChitinaseChitinase Cylindrosporium concentricum;Cylindrosporium concentricum;
Phoma lingam; SclerotiniaPhoma lingam; Sclerotinia
sclerotiorumsclerotiorum
RiceRice ChitinaseChitinase Rhizoctonia solaniRhizoctonia solani
CarrotCarrot Chitinase and 1,3-Chitinase and 1,3-ββ glucanaseglucanase Alternaria dauci, Alternaria radicina,Alternaria dauci, Alternaria radicina,
Cercospora carotae, ErysipheCercospora carotae, Erysiphe
heracleiheraclei
PotatoPotato PR5PR5 Phytopthora infestansPhytopthora infestans
PotatoPotato 1,3-1,3-ββ glucanaseglucanase Phytopthora infestansPhytopthora infestans
Kiwi fruitKiwi fruit 1,3-1,3-ββ glucanaseglucanase Botrytis cinereaBotrytis cinerea S S Jena

11. 2. Ribosome inactivating proteins (RIPS)2. Ribosome inactivating proteins (RIPS)
 Cysteine rich proteins such as lectins, defensins,
thionins, lysozyme, polygalacturonase inhibitors etc.
 RIPs have N-glycosidase activity which cleaves a
specific ‘A’ residue from the large subunit rRNA.
 There are more than 40 RIPs from range of plant
families. RIPs do not inhibit ribosomes from the plant of
origin, but active against fungal ribosomes.
 e.g. Barley α-thionin tobacco
Pseudomonas syringae pv tabaci &
Pseudomonas syringae pv syringae
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12. 3. Phytoalexins3. Phytoalexins
 Low molecular weight anti-microbially active secondary
metabolites synthesized by the plant in response to an infection
 Identified in many different plant families like Leguminosae,
Solanaceae, Vitaceae, Gramineae & some gymnosperm
 Structurally diverse, synthesized from a wide range of
precursors.
 Ex: terpenes, acetylenes, isoflavonoids, stibenes.
 Resveratol is one of the commonest stilbene. The key enzyme
of synthesis is resveretol synthase also known as stilbene
synthase (STS).
 During infection the stored phytoalexins are mobilized & quickly
accumulate to very high levels around the site of attack, while
genes for biosynthetic pathways are induced.
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13. 4. Hydrogen peroxide
Transgenic potato expressing a H2O2 generating fungal gene
for glucose oxidase was found to have high levels of H2O2
and enhanced levels of resistance both to fungal and
bacterial pathogens
5. Engineering toxin insensitivity
• Bacterial haloblight pathogen of bean Pseudomonas
phaseolicola produces a tripeptide toxin, Phaseolotoxin,
which causes chlorotic halos
• Phaseolotoxin inhibits the enzyme Ornithine
transcarbamylase (OC)
• The gene encoding the enzyme was cloned and transferred
in tobacco, where its expression has shown to prevent the
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14. II. Manipulation of disease resistance genesII. Manipulation of disease resistance genes
 Introduction of resistance (R) gene from a plant
variety resistant to a certain pathogen into
susceptible varieties
 In rice Xa21 gene confers resistance to 30 distinct
strains of bacterium Xanthomonas oryzae pv oryzae
that causes leaf blight in rice.
 HM1 gene from maize which confers resistance to
Cochliobolus carbonum has been cloned by
transposon tagging.
 Bacterial resistant genes like Arabidopsis Rps2 and
tomato Pto have been cloned.
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15. II. DEVELOPMENT OF PLANTII. DEVELOPMENT OF PLANT
RESISTANCE TO VIRUSRESISTANCE TO VIRUS
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16. Virus resistance development is broadly
classified into two categories, based on the
source of gene used.
A. Pathogen derived resistance
B. Non-pathogen derived resistance
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17. A. Pathogen derived resistanceA. Pathogen derived resistance
Plant pathogen’s genome itself is the source of
resistance genes.
1. Virus Coat protein mediated resistance :
 Cross protection: Ability of one virus to prevent or
inhibit the effect of a second challenge virus.
 Untimely presence of coat protein of the virus responsible
for the first infection prevents unpacking or replication of
the newly arrived genetic material of the second virus.
 e.g. Papaya ring spot virus resistance
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18. Papaya ring spot virus
Virus resistant Papaya Construct
35Sp nos!PRSV coat
protein
nptII GUS
Selectable Markers S S Jena

19. 2. Non coat protein mediated resistance
Viral genes other than coat protein gene
a) Replicase mediated resistance:
Non-structural proteins necessary for replication
b) Movement protein mediated resistance
 Involved in Cell to cell movement of plant viruses
 Resistance is based on competition between wild type
virus encoded movement protein and the defective viral
movement protein to bind to the plasmodesmatal sites.
 Ex: Transgenic tobacco, Poty virus PVX in potato
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20. c) Satellite RNA
 RNA viruses have small RNA molecules called satellites.
 These depend on viral molecules for their replication but are not
necessary for viral functions
 Satellites either increase or decrease the severity of disease
produced by the virus carrying it.
 The satellites that reduce disease severity are used for integration
into host genome.
 CMV when inoculated with a symptom attenuating satellite RNA
successfully protected tobacco, pepper, tomato and cucumber
plants from a virulent strain of CMV and reduced yeild losses
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21. d) Defective viral genomes
• Defective or deleted genomes of some RNA and DNA
viruses disrupt the replication of complete genome of
those viruses with which they are associated
• African Cassava Mosaic virus consists of two single
stranded DNA molecules A & B DNA
• In addition a 50% deleted B DNA is also found associated
with ACMV
• Tobacco plants containing this deleted B DNA integrated
in their genomes showed reduced systemic spread when
they were infected with ACMV
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22. e) Antisense RNA approach
 Antisense RNA can be produced by inverting a cDNA copy of
an mRNA with respect to the promoter in an expression vector
 Interact with mRNA molecules by base pairing to form ds RNA
 Transgenic tobacco expressing antisense RNA of the CMV coat
protein gene showed reduced virus accumulation
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23. 1. Ribozyme mediated resistance
Ribozymes are RNA based RNA restriction enzymes
capable of cleaving RNA molecules at specific sites
Strategies:
 Producing a ribozyme specific to a part of the target
virus genome.
 To produce cDNA of this ribozyme
 To integrate it into the host plant genome
Example:
Transgenic Tomato resistant to Citrus exocortis viroid
(CEVD)
B. Non-Pathogen derived resistance
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24. 2. Anti viral proteins/Ribose inactivating proteins (RIPs)
o Inhibit ribosome function by modifying ribosomal RNA and there by
interfere with polypeptide translation
o Identified in Poke weed (Phytolacca americana)
o Tobacco plants with PAPs (Poke weed anti viral proteins) are
resistant to PVX, PVY, TMV and CMV
3. Mammalian Oligo adenylate synthase gene
 Higher vertebrates resist virus infection by using interferon system
 Interferons induce the synthesis of additional proteins that lead to the
inhibition of virus multiplication. e.g. 2’ 5’ Oligoadenylate synthase
 Transgenic tobacco expressing the mammalian
2’, 5’-Oligoadenylate synthase was generated with resistance to CMV
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25. TransgeneTransgene Transgene mode of actionTransgene mode of action CropCrop VirusVirus
controlledcontrolled
Replicase proteinReplicase protein Competition for enzymeCompetition for enzyme
PotatoPotato PVYPVY
PeaPea PSbMVPSbMV
RiceRice RYMVRYMV
Movement proteinMovement protein Interference with transportInterference with transport
TobaccoTobacco TMVTMV
PotatoPotato PVX, PVYPVX, PVY
TobaccoTobacco TEVTEV
Viral proteaseViral protease Polyprotein processingPolyprotein processing PotatoPotato PVYPVY
Antisense RNAAntisense RNA Blocks viral RNA andBlocks viral RNA and
prevents translationprevents translation
PotatoPotato PLRVPLRV
RibozymeRibozyme Cleaves viral RNACleaves viral RNA TomatoTomato CEVdCEVd
Satellite RNASatellite RNA Competes for capsidsCompetes for capsids TobaccoTobacco CMVCMV
Antiviral proteinAntiviral protein
ribonucleaseribonuclease
Degrades ds-RNA (viroids)Degrades ds-RNA (viroids) PotatoPotato PSTVPSTV
Pokeweed antiviral proteinPokeweed antiviral protein Inhibits rRNA of 60S subunitInhibits rRNA of 60S subunit TobaccoTobacco TMV, PVXTMV, PVX
2’, 5’ oligoadenylate antiviral2’, 5’ oligoadenylate antiviral
proteinprotein
Degrades ds-RNADegrades ds-RNA TobaccoTobacco CMVCMV
Few examples of Virus resistant transgenic plants
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26. III. DEVELOPMENT OF PLANTIII. DEVELOPMENT OF PLANT
RESISTANCE TO INSECTSRESISTANCE TO INSECTS
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27. 1. Insect1. Insect ResistantResistant genes from micro-organismsgenes from micro-organisms
GenesGenes SourceSource
BtBt Bacillus thuringiensisBacillus thuringiensis
VIP (vegetative insecticidalVIP (vegetative insecticidal
protein)protein)
Bacillus thuringiensisBacillus thuringiensis
iptipt (isopentenyl transferase)isopentenyl transferase) Agrobacterium tumifaciens
Cholesterol oxidase geneCholesterol oxidase gene Streptomyces sp.Streptomyces sp.
Pht genePht gene Photorhabtitus luminescensPhotorhabtitus luminescens
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28. BtBt
 Bt: Bacillus thuringiensis
 Gram positive, spore forming, soil
Bacterium
 cry gene of B. thuringiensis
produces CRY proteins which
forms crystalline inclusions in
bacterial spores.
 Otherwise called ‘insecticidal
crystal proteins’ (ICP).
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29. Classification of CRY protein
CRY proteins are classified into 6 main groups or first ranks viz.
CryI, CryII, CryIII, CryIV, CryV & CryVI on the basis of their
insecticidal activities.
Cry I : Lepidoptera specific
Cry II : Lepidoptera and Diptera specific
Cry III : Coleoptera specific
Cry IV : Dipteran specific
Cry V, Cry VI : Nematodes
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30. 1. Ingestion and
solubilisation of
pro-toxin.
2. Proteolytic
activation at N- and
C-termini.
3. Interaction with cell
surface binding
protein.
4. Conformational
change exposing α-
4,5 helical hairpin.
Mode of action of Cry toxin
3
5. Oligomerisation and insertion in membrane to form pore.
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31. • Bt cotton: Commercialized in
India
• Bt corn: Commercially planted in the
Philippines.
• Bt Brinjal: moratorium in India
• Bt Rice: Bt rice is “extensively field
tested in China and awaiting approval
by the Chinese regulatory authorities”
for commercialization.
Achievements
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32. CropsCrops GenesGenes Target insectTarget insect
Cotton
Cry 1Ab
Heliothis zea,
H. armigera,
Cry 1Ac Pectinophora gossipiella
Maize Cry 1Ab Ostrinia nubilalis (1993)
Rice (japonica) Cry 1Ab Chilo suppressalis
Rice (indica) Cry 1Ac Scirpophaga incertulas
Tomato Cry 1Ab Heliothis armigera
Tobacco Cry 1Ab Heliothis virescens
Eggplant Cry 1Ab Leucinodes orbonalis (1998)
Tomato Cry 1Ac H. armigera
Canola Cry 1Ac Plutella xylostella
Alfalfa Cry 1C Spodoptera littoralis
Potato Cry 13a Leptinotarsa decemlineata
Transgenic crop plants with synthetic Bt genes
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33. 2. Resistance genes from higher plants2. Resistance genes from higher plants
i. Proteinase
inhibitor
a) Cow pea trypsin inhibitor gene ( CpTI)
b) Alpha amylase Inhibitor ((ααAl-Pv)Al-Pv)
ii. LectinsLectinsi) Proteinase Inhibitors
Interfere with digestive enzymes of the insect depriving
the insect of nutrients.
a) Cowpea trypsin inhibitor gene (CpTI)
 Cowpea (Vigna unguiculata)
 The inhibitor gene produces antimetabolite substances
that provide protection against the major storage pest
Bruchid beetle (Callosobruchus maculatus).
 e.g. Tobacco
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34. b) α-Amylase inhibitor
 α-amylase inhibitor protein inhibits insect gut α-amylase resulting
the weevil starved to death
 α-amylase inhibitor (αAl-Pv) isolated from common bean
(Phaseolus vulgaris) is commonly used
 e.g. Potato, Oilseed rape, Pea and Tomato against Bruchus beetle
& Pea weevil
ii) Lectins:
 Plant glycoproteins which act against piercing & sucking insects
like aphids.
 Source: Snowdrop (Galanthus nivalis)
 e.g. Potato
Contd…
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35.  Serine protease inhibitors:
From mammals and the tobacco hornworm (Manduca
sexta).
 Bovine pancreatic trypsin inhibitor (BPTI):
α-antitrypsin(α1 AT) and spleen inhibitor (SI) from
mammals
3. Resistance genes from animals3. Resistance genes from animals
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36. Few transgenic plants conferring resistanceFew transgenic plants conferring resistance
against insectsagainst insects
CropsCrops Gene transferredGene transferred Insect (s) controlledInsect (s) controlled
TobaccoTobacco CpTICpTI
CpTICpTI
Sweet potato trypsin inhibitorSweet potato trypsin inhibitor
genegene
Heliothis armigeraHeliothis armigera
M. SextaM. Sexta
Spodoptera lituraSpodoptera litura
PotatoPotato Snowdrop lectin (GNA)Snowdrop lectin (GNA) Tomato moth (Tomato moth (Lacanobia oleraceaLacanobia oleracea))
PeaPea ααAI from beanAI from bean
ααAI from beanAI from bean
Bruchus beetleBruchus beetle
Pea weevilPea weevil
RiceRice Corn cysteine geneCorn cysteine gene Coleopteran (Coleopteran (Sitophilus zeamisSitophilus zeamis))
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