STRESS
When some factors
of the environment
interfere with the
expression of
genotypic potential
• ABIOTIC
• BIOTIC
Stresses are abiotic or biotic
ABIOTIC STRESSES
Environmental, nonbiological
•Temperature (high /
low)
•Water (high / low)
•Salt
•Radiation
•Chemical
BIOTIC STRESSES
Caused by living
organisms
•Fungi
•Bacteria
•Viruses
•Insects
•Herbivores
•Other
plants/competition
 Stresses induce metabolic
developmental responses
and
 Injuries occuring in susceptible
plants, can lead to impeding
flowering, death
Preferable!
Productivity losses due to stress
 Loss due to diseases range from 20 to 30 %, in case of severe
infection, total crop may be lost
 Estimated global loss due to insect pests in potential yields of
all crops is ~14%.
 In India losses due to insect pests ranges from 10 to 20 %
 Abiotic stresses reduce average yield of crops by upto 50%
(Bray EA 1997)
 In India also 67% of the area is rain-fed and crops in these
areas invariably experience droughts of different magnitudes
 Annually about 42% of the crop productivity is lost owing to
various abiotic stress factors ( Oerke et.al., 1994).
Stress resistant crops are a dire need
• Human population continues to increase:
Nine billion expected by 2050.
• Global warming and climate perturbations
are likely to accentuate biotic as well as
abiotic stresses
Climate Change analysis in Punjab
Since 1970
Average minimum temperature has increased by about 1oC
Relative humidity increased at :
Ludhiana,
Ballowal Saunkhri,
Jalandhar
Bathinda
Sunshine hours have decreased at :
Ludhiana,
Ballowal Saunkhri,
Jalandhar
Pan Evaporation has decreased at :
Ludhiana,
Ballowal Saunkhri,
Bathinda
Anticipated abiotic stress induced crisis in
Indo-Gangetic plains
• Conventional rice cultivation may become unsustainable in the
Indo-Gangetic plains due to ground water exploitation greatly in
excess of recharge. Recharging of aquifers to be hit further by less
rain and snow and shrinking of himalayan snow cover
• Temperature effects predicted to be more pronounced in this
region. Wheat with already strained adaptation in the region is
likely to be hit hard. For every 1 C increase in mean temperature
above normal, grain yield is reduced by 12-23 per cent.
Climate change induced pest problems in the region
• Increasing incidence of several species of cereal
aphid in wheat, barley and oats
• Attack of mealy bug (Phenococcus solenopsis), white
fly (Bemisia tabaci) and Spodoptera litura on cotton
• Leaf folder (Cnaphalocrocis medinalis) and plant
hoppers (Nilaparvata lugens and Sogatella furcifera)
have emerged as major pests of rice
• Shoot fly (Atherigona spp.) and pyrilla are emerging
as important pests of maize and sorghum crops
Contd.
Climate change induced pest problems in the region
• Stemfly (Ophiomyia phaseoli) and blister beetle
Mylabris spp. have emerged as major pests of pulse
crops
• Cabbage caterpillar (Piersis brassicae), tobacco
caterpillar (Spodoptera litura), American bollworm
(Helicoverpa armigera), several species of aphids,
whitefly, leafminer, spider mites and blister beetle
are causing increasing damage in different
vegetable crops
• Fruit piercing moth (Eudocoma spp.), mealy bugs
and fruit flies are causing increasing damage to fruit
crops
Emerging disease problems
CROP
Wheat
Rice
DISEASE
Powdery mildew, foliar blight and black tip of grain
Sheath blight, neck blast, false smut, foot rot, brown leaf
spot, kernel bunt, grain discoloration
Cotton
Cotton leaf curl, foliar leaf spot
Maize
Bacterial stalk rot
Sugarcane
Oilseeds
Red stripe
Sclerotinia stem rot
Potato
Scab scurf, fusarial dry rot
Tomato
Early blight
Cucurbits
Cucumber mosaic
Chillies
Leaf curl
Citrus
Fruit drop, gummosis, sooty mold
Papaya
Papaya leaf curl
Increasing
disease
problems
Soil health
problems
Shift in
status of
insect pests
Thermal
stress
Less water/
flooding
Climate
changes
By 2025, India will need to produce:
•125 million tonnes (mt) of rice (current: ~104 mt)
•115 mt of wheat (current: ~93 mt)
Responding to challenges posed by biotic and
abiotic stresses through crop improvement
• Every new objective added to a breeding
programme almost doubles the magnitude of work
• Unlike past successes (e.g., dwarf varieties), future
increases in productivity potential are not likely to
be accompanied by enhanced inputs
• Genetic improvements need to be accomplished
under demanding time frames
Can routine breeding programmes meet
these challenges ?
Elements of an enhanced breeding strategy
• Wide
hybridization
• Marker assisted
selection
• Transgenics
Integration with conventional
breeding for biotic and abiotic stress tolerance
Illustrating the integrated approach: some
initiatives at PAU
• Heat tolerance in wheat using wide hybridization and
molecular markers
• Wide hybridization for disease resistance in wheat
• Marker assisted selection for rust resistance in wheat
• Marker assisted selection for bacterial blight in rice
• Water use efficiency in rice using transgenic and molecular
marker strategies
• Molecular markers for drought and flooding tolerance in
maize
• Rootstock transformation for salinity tolerance in citrus
• Cloning genes for disease resistance
• DH facilitated accelerated breeding for resistance to biotic
and abiotic stresses
Developing a complete panel of T. durum-Ae. speltoides
chromosome segment substitution lines as a source of heat
tolerance in wheat
Strategy used for developing T. durum – Ae. speltoides
introgression lines
Triticum durum
Ae. speltoides
X
(PDW233/PDW
274)
X
(pau3809)
T. durum
F1
BC1F1
X
T. durum
BC2F1
selfing
BC2F6
T. durum – Ae. speltoides introgression lines
800 introgression lines developed
384 taken up for molecular marker analysis
and phenotyping
100 randomly selected ILs phenotyped and
genotyped for developing introgression
profiles and association analysis as a pilot
study
Molecular
marker profile
of 90
introgression
lines from
Triticum durum
x
Aegilops
speltoides
Ae. speltoides chromosome segments
associated with heat stress tolerance
Trait
linked markers Chromosome
LOD Score
PVE(%)
Plant yield
Xcfa2278
2B
3.3225
10.9
Chlorophyll content
Xgwm148
2B
2.7567
14.7
Chlorophyll content
Xgwm566
3B
2.7386
12.9
Xcfd60
5B
3.1114
16.1
TTC cell viability
Wide hybridization for disease resistance
in wheat
• More than a 1000 accessions of wild and related
species germplasm maintained as active collection
• More than 20 disease resistance genes being
introgressed from alien species T. monococcum, T.
boeoticum, Ae. tauschii, Ae. ovata, Ae. triuncialis,
Ae. umbellulata, Ae. caudata and Ae. variabilis to
cultivated wheats
• PAU has the distinction of designating the first three
alien rust resistance genes from India: Yr40 (yellow
rust), Lr57 (brown rust) and Lr58 (brown rust) using
molecular marker technology
Transfer and mapping of leaf rust and stripe rust
resistance genes from Aegilops geniculata and Ae.
triuncialis in wheat.
Alien genes introgressed into cultivated wheat varieties
from wild Aegilops and Triticum species
Donor
Trait
Gene(s)
Ae. geniculata
Leaf rust, Stripe rust
Lr5-Yr40 (5DS)
Ae. triuncialis
Lr58 (2BL),
LrT, PmT, CreT (5U)
Ae. umbellulata
Leaf rust
Powdery mildew, CCN,
Karnal bunt
Leaf rust, Stripe rust
Ae. caudata
Leaf rust, Stripe rust
LrC (5DS)
Ae. variabilis
Leaf rust
Stripe rust
Leaf rust
Stripe rust
CCN
Powdery mildew
Leaf rust
Karnal bunt
Powdery mildew
Leaf rust
Stripe rust
LrV (2AL)
YrV
LrTm (6A); QYrtm.pau-2A(2A)
QYrtb.pau-5A(5A); Qcretm.pau1A (1A) Qcretm.pau-2A (2A);
PmTb7A.1 & PmTb7A.2(7A)
LrAt (2D)
KbAt (1D, 2D, 6D)
PmTa
LrTa
YrTa
T. monococcum
&
T. boeoticum)
Ae. tauschii
T. araraticum
LrU-YrU (5DS)
WORKING WITH MARKER TAGGED RUST RESISTANCE GENES IN
THE WHEAT BREEDING PROGRAMME
Sr2, Sr 22,
Sr26,Sr 39,
Year
Crosses (F1s) with a
Yr 5,
known gene parent
Yr 10, Yr 15,
2007-08
3 out of 592
Yr 24, Yr 36
Yr C591,
2011-12
455 out of 764
Yr40/Lr57
Lr19/Sr25
Lr24/Sr25
Lr 28,
Lr28,
Lr 24/Sr24
Lr34/Yr18,
Lr 58,
Yr/Lr Ae. Umb
Yr/Lr T.m.-T.b.
2007-08
2011-12
PBW343 +
Lr24 + Lr28
Avocet
6*/Yr10
X
PBW343 +
Lr24 + r28
Avocet
6*/Yr15
X
MAS
MAS
BC2F1
BC2F1
MAS
MAS
Yr10
positive
Yr15
positive
X
Crossed seed
F1- complex
Sowing at Off season location
(Keylong-2009)
Yr
PYRAMIDING OF
FOUR GENES : Lr
24, Lr 28, Yr 10
and Yr 15 IN PBW
343
Six hundred and fifty one (651) single
plants sown at Ludhiana (2009-10) : MAS
MAS
2850 F4 COMP PROGENIES (2010-11)
Positive plants advanced to
offseason location
(Keylong 2010)
MAS
Bulking of selected single plant
progenies
(Keylong 2011)
MAS
Yield trials:
688 entries (2011-12)
Seed multiplication at
Keylong
Recreating mega varieties:
Marker assisted background selection (MABS)
MABS
•
•
•
•
Phenotypic selection
PBW621*3/(AVOCET+Yr5 )
PBW621*2/(PBW 568+Yr36)
PBW343*2//PBW621/(AVOCET+Yr5)
PBW343*2//PBW621/(PBW
568+Yr36)
High throughput
genotyping system
sanctioned under
FIST project (DST)
Initiative of
PAU &
WSU
• Varietal background
recovered in just 2
backcrosses
• Minimum linkage drag
• Limited testing needed
in the field
Molecular marker interventions in wheat:
Towards commercialization in North Western Plains Zone
Entry
PBW
697
PBW
698
PBW
702
PBW
703
PBW
722
PBW
723
Stage
(2013-14)
Conditions
AVT 1st yr
AVT 1st yr
Timely
sown,
Irrigated
AVT 1st yr
AVT 1st yr
AVT 1st yr
Special
trial for
MAS
products
Late sown,
Irrigated
Timely
sown,
Irrigated
Genetic background +
genes
% yield increase
over check
DBW 18+ Lr57/Yr40
9.30
PBW 343
+Lr24+Lr28+Yr10
+Yr15
7.94
PBW 533+Yr15
13.94
PBW 343
+Lr24+Lr28+Yr10
+Yr15
7.09
PBW 343
+Lr37/Yr17+Lr57/Yr40
PBW 343 +Lr57/Yr40
+Yr15
--
--
Wide hybridization for bacterial blight and
brown plant hopper resistance in rice
• Four bacterial blight resistance genes Xa38, xa-g(t), xab(t) and Xa-r(t) has been transferred to cultivated rice
background from Oryza nivara, O. glaberrima, O. barthii
and O. rufipogon, respectively
• Candidate genes for Xa38 has been identified and work
for cloning this gene is in progress
• Genes for brown plant hopper has also been identified
in wild rice germplasm and are being mobilized to
cultivated rice.
Identification, transfer and mapping of BB
resistance gene Xa38 from Oryza nivara
Development of
improved Basmati386
through MAS by
pyramiding of bacterial
blight resistance genes
xa13 and Xa21, semi
dwarfing gene sd1 and
monitoring the retention
of genes conferring
aroma, amylose content
and grain elongation
genes and release of
Punjab Basmati 3.
Improving water use
efficiency in rice
Genetic engineering
approach
Molecular marker based
interventions
Genetic transformation of rice variety PR 121 using gly I and gly II genes through
particle bombardment
Genetic transformation of PR 121
Genes used
Calli bombarded
Calli selected on hygromycin (30
mg/l)
Calli transferred to regeneration
medium
No. of putative transgenic
plantlets regenerated
gly I and gly II
1440
432 (30.0%)
415
251 (17.43%)
Genetic transformation of PR 122
Genes used
gly I and gly II
Calli bombarded
195
Calli transferred to
regeneration medium
Calli selected on hygromycin
(30 mg/l)
No. of putative transgenic
plantlets regenerated
117
118 (60.51%)
65 (33.33%)
Genetic transformation of PR 118
Gene used
Calli bombarded
Calli selected on hygromycin
(30 mg/l)
No. of calli regenerated
No. of putative transgenic
plantlets regenerated
Plants analyzed for PCR
No. of PCR-positive plants
gly I
2600
322 (13.39%)
91
249 (9.57%)
155
13 (0.5%)
558 bp
PCR-positive
samples of PR 118
(T0)
[L= Ladder,
PC = Positive control,
NT = Non-transgenic
control]
PCR-positive
plants of PR 118
(gly I gene)
Preliminary evaluation of transgenic lines for salt stress tolerance
Material screened for salinity tolerance at 0, 8 and 12 dS/m
salinity levels under transgenic glasshouse: T2 generation of 1 line of PR118 (DREB1A gene)
 T1 generation of 2 lines of PR 118 (gly I gene)
0 dS/m
8 dS/m
12 dS/m
Generation of three RIL populations for investigating
adaptation to aerobic conditions
S.
No.
Cross
Generation
Population
size
1
PAU3116/CT 6510-24-1-2*
F10
228
2
PR120/UPLRi7**
F10
193
F9
379
3
PR115/CRR 615-PR-27699-D808-4-4***
*CT 6510-24-1-2 & **UPLRi7- are upland rice and
***CRR615-PR- drought tolerant line from CRRI, Cuttack
Traits studied:
• Iron deficiency induced chlorosis
(IDIS)
• Plant Height
• Days to 50% flowering
• Number of tillers
• Number of grains /panicle
• Spikelet fertility
• Plot yield
Twenty high yielding entries from each RIL
population were taken up for testing as part of
the breeding programme for aerobic rice
A
B
Variation of chlorosis (IDIC) in RILs at early growth stage under
aerobic conditions. (A)- Chlorotic (B)- Non chlorotic
Molecular profile of PAU3116 x CT6510-24-2-1 RIL population using
SSR markers
Recombinant inbred lines
P1 P2 C
RM566
P1 P2 C
Recombinant inbred lines
RM302
P1 P2 C
Recombinant inbred lines
RM212
Molecular data is being generated on PAU 3116/Ct651024-2-1 population for tagging QTL’s related to yield
components under aerobic conditions
Markers associated with important traits
under water logging stress in maize
TRAIT
ASSOCIATED
MARKERS
CHROMOSOME
NUMBER
Plant Height
umc1701
1
Plant mortality
umc1701
umc1117
umc1029
phi027
1
4
7
9
Leaf senescence
umc1636
9
Leaf rolling
bnlg1496
phi076
umc1577
3
4
7
Total no. of ears/line
umc1701
1
No. of grains/line
umc1035
3
Grain weight
bnlg1601
umc1035
3
3
Anthesis silking interval
umc1053
10
I 110 (S) x I 172 (T) mapping population
Chromosome 1
umc1685 (0.0)
Chromosome 3
Chromosome 4
phi104127 (0.0)
umc1117 (0.0)
umc1577 (0.0)
umc1793 (27.8)
bnlg1189
(26.1)
umc2061 (0.0)
umc1808 (35.2)
umc1701 (22.9)
bnlg1144 (62.3)
umc2002 (0.0)
Leaf senescence
Leaf rolling
Total no. of ears/line
No. of grains/line
Grain weight
ASI
SPAD1 (C)
SPAD2 (C)
umc1015 (61.5)
umc1667
(31.1)
SPAD 1
SPAD 2
Plant Height
Plant mortality
Chromosome 7
umc1399
(35.9)
bnlg1601
(57.7)
umc1539
(75.3)
umc1035
(108.3)
umc2000
(140.6)
umc2152
(169.8)
phi034 (85.5)
umc2291
(60.7)
umc1339
(119.2)
umc2325 (0.0)
phi076 (92.0)
Both
stress
and
non
stress
umc1029 (34.3)
phi328175 (54.0)
bnlg1070 (83.8)
Plant height (C)
No. of grains/line (C)
Grain weight (C)
ASI (C)
bnlg1496
(199.4)
umc1409 (111.4)
Chromosome 8
bnlg1194 (0.0)
phi119 (25.5)
Chromosome 9
umc1583
(0.0)
bnlg1401
(23.5)
phi027 (0.0)
phi115 (0.0)
umc1636 (28.1)
Chromosome 10
bnlg2190 (0.0)
umc1962 (29.5)
umc1318 (54.6)
bnlg2046 (32.5)
SPAD 1
SPAD 2
umc1470 (64.5)
Plant Height
Plant mortality
umc1471 (93.9)
Leaf senescence
Leaf rolling
Total no. of ears/line
No. of grains/line
Grain weight
ASI
SPAD1 (C)
SPAD2 (C)
Plant height (C)
No. of grains/line (C)
Grain weight (C)
ASI (C)
umc 2371-1 (61.0)
umc1084 (85.2)
umc2338 (81.5)
bnlg1012 (108.4)
umc1053 (0.0)
umc2067 (16.9)
umc1657 (126.7)
umc 2371-3
(149.3)
umc 2371-4 (179.1)
umc 2371-2 (0.0)
umc1366 (15.1)
umc1077 (47.9)
GENETIC
TRANSFORMATION
OF CITRUS ROOT
STOCK FOR
TOLERANCE TO
SALINITY
Epicotyls bombarded with gly 1
Proliferation of shoot buds
Subculture on MS + BAP (0.5 mg/L)
Doubled haploidy as a breeding tool
• Cuts short the breeding cycle by 3-4 years
• Freezes every recombination event for evaluation,
selection and use in further crosses
• Genetic advance higher than any other method
• Strengthens the ‘breeding option’ in face of sudden
biotic and abiotic challenges
ACCELERATED BREEDING:
WHEAT OFFSEASON OPERATIONS AT KEYLONG
Doubled haploid facilitated accelerated
breeding initiated in major crops
• Wheat: wheat x maize crosses-3000
lines/year
• Maize: haploid inducer lines from
CIMMYT
• Rice: anther/microspore culture
Is our knowledge of
molecular-genetic basis
of plant resistance to
stress adequate to allow us
to step beyond natural variation
and design transgenics that
combine stress tolerance
and high productivity?
Abiotic and biotic stress tolerance:
a basic unity
Cascade is strikingly similar
signal perception, signal transduction,
activation of transcription factors,
activation of cis-acting response
elements and finally a well coordinated
expression of a large set of genes
Abiotic and biotic stress tolerance:
a basic unity
• The difference: Biotic stress tolerance in
most cases based on a strong central
switch (the R gene) while a network of
switches (transcription factors) governs
reponse to abiotic stresses
A unifying pattern across species
• Molecular and genetic studies show
Arabidopsis and rice share common
mechanisms regulating response to
abiotic stress
• Transcription factors (TFs) which play an
important role in regulating gene
expression are common between
grasses and Arabidopsis
The current reach of transgenic
manipulations
• Transcription factors (TFs) such as DREB1/CBF,
DREB2, AREB/ABF, NAC are powerful targets for
genetic engineering of stress tolerance
• Constitutive expression of TFs is, however, counter
productive-stunting, dwarfing, developmental
abnormalities
• Effective inducible expression systems (including
promoters) need to be established
• Cloning of new genes for resistance to biotic and
abiotic stresses remains as a major need
Cloning of antifungal genes from local antagonistics and
generation of gene cassettes for genetic transformation
GENE NAME
GENE
SOURCE
ANTAGONISTIC
TARGET
CROP
TRAIT
Glucanase &
Chitinase
Trichoderma
viride
Phytophthora
parasitica
Roughlemon Foot rot
root stock
Glucanase &
Chitinase
Trichoderma
viride
Phytophthora
parasitica
Sugarcane
(CoJ 83)
Red rot
Glucanase
Trichoderma
viride
Phytophthora
parasitica
Cotton
(RG8)
Fusarium
wilt
Glucanase
Trichoderma
viride
Rhizoctonia
solani
Rice
(Kitaake)
Sheath
blight
Future Outlook
• Scientists are able to visualize in
individual experiments
hundreds of genes involved in
the complex phenomenon of
plant response to stress
• Made possible by advent of new
powerful technologies:
» High throughput or
next generation
DNA sequencing
» Genome wide gene
expression profiling
» Advances in
bioinformatics
Strengthening
precision screening
high throughput
phenotyping:
Phenomics
THANKS