high throughput and low cost phenotyping options for a few ... · high throughput and low cost...
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High throughput and low cost phenotyping options for a few drought adaptive traits
Dr. Udayakumar M
Department of Crop Physiology, UAS, GKVK, Bangalore -65
18th Feb, 2014, IPPN
Drought is complex Magnitude and duration of stress is unpredictable
Plants evolved diverse adaptive strategies to sustain growth under
complex stress situations
Opuntia
ABA
A plant character/biochemical mechanisms that facilitates cell turgor, cell survival and improves adaptation under moisture stress
Drought adaptive traits - consensus
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Major emphasis has been
Identify and validate the relevant trait Is it
relevant?
This is what you have to
look for
Oh ! God NO RAIN
Capture the genetic variability
Accurate imposition of moisture stress is crucial for phenotyping drought adaptive traits
3 Managed drought environment Lysimeter
Transpiration -water uptake- drives the growth
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Plants experience stress
When Transpiration exceeds uptake
Resulting in wilting Cell Ψ/ turgor
Affects growth
Plant has to Maintain cell turgor Tolerate the stress effects
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constitutive traits
Acquired traits
Traits which are inherent to the genotype
Traits/mechanisms which are up regulated
under stress May not substantially increase under
stress
Adaptive mechanisms/traits…. Broadly classified as
Root WUE EW
Associated with water relations, postpones decrease in ψw
Phenology 5
Mainly cellular level tolerance mechanisms
Traits-water relations We developed tools / techniques to assess WUE
Stable isotope technique for WUE – 13C
For CID
Bindumadhava et al., 2005; Current Science, 89(7): 1256 - 1258 Sheshshayee et al., 2005, Journal of Experimental Botany 56: 3033–3039. Sheshshayee et al., 2011, book chapter CIMMYT Udayakumar M et al., 2013, Designer rice
Impa et al., 2005, Crop Science 45: 2517–2522.
y = -0.669x + 22.912 r = 0.831
18.50 19.00 19.50 20.00 20.50 21.00 21.50 22.00
2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 WUE (g.kg-1)
D 13
C
Gravimetry
y = -2,284x + 24,438 R² = 0,646
18,0
19,0
20,0
21,0
22,0
23,0
1,0 1,2 1,4 1,6 1,8 2,0 2,2
∆13 C
(‰)
WUE (g. kg-1) 6
Species
Range in ∆
13C (‰) Rice Germplasm (230) 18.15 – 22.79
Rice Mapping population (100) 19.21 - 22.55
Groundnut Germplasm (246) 16.19 – 22.32
Groundnut RIL (268) 16.68 – 22.96
Root structure
No
of g
enot
ypes
0
10
20
30
40
50
60
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
y = 0.0002x + 0.998r = 0.64
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
10000 15000 20000 25000 30000 35000 40000 45000
LADxd18O
Root
BM
Phenotyping for roots
Oxygen isotopes- A potential option to quantify transpiration rate and thus root traits
Root weight Sheshshayee et al., 2005 Mohankumar 2011 Udayakumar M et al., 2013
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Root traits in root structures
Mesophyll efficiency by Ci/gs ratio - 13C/18O ratio
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Mesophyll efficiency (gm) – Ci/gs Differences in Ci at given gs is reflection of gm
Bindumadhava et al., 2005; Sheshshayee et al., 2011
13C/18O ratio – a surrogate for Ci/gs
Carboxylation efficiency dA/dCi and Ci/gs
Carboxylation efficiency dA/dCi and 13C/18O
Captured the genetic variability under low cost MDE -option to simulate required precipitation levels by sprinklers
13∆C in rice genotypes Wax in rice genotypes
Semi Mechanized ROS Sprinkler system for specific water input
Prathiba, 2012
Sheela, 2012
Sheshayee et al., 2013; Mohankumar, 2011
Managed drought environment facility (MDE)
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Drought susceptibility index
When, Plant experiences stress
Cell Ψ/ turgor
Expression of stress genes
Brings in altered metabolism for
adaptation
Tolerance to oxidative stress
Protection to macro molecules
Osmotic adjustment
Protein turn over Genes expressed on exposure to stress regulate intrinsic tolerance mechanisms
involved in cell survival at low ψw
Phenomenal progress made in characterizing stress genes
Cellular level tolerance mechanisms - CLT
Acquired tolerance traits
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Two important groups of traits are emerging as relevant to achieve field level tolerance
Mechanisms regulating water relations
Mechanisms governing the cellular level tolerance
Phenotyping for acquired traits upregulated on exposure to stress is difficult to capture under field conditions and even in big containers
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Amenable to capture genetic variability even under field conditions
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Plant water status differs in the genotypes under stress
Genetic variability in water status affects the expression of acquired traits
Difference in Ψw – alters the expression of acquired traits
Genetic variability in acquired traits has to be seen -at comparable water status
Genetic variability in acquired traits
Are often assessed in
Seedlings
Excised leaves
As a compromise
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Temperature induction response (TIR)
Acquired tolerant traits
UASB
Should be assessed only on exposure to induction stress
Cotton
Sunflower
Rice
Rice
Desiccation stress response
Induced Non-Induced
Induction response seen even at plant level Induced Non-Induced
Temperature induction response (TIR)
C I NI
C I NI
Salinity induction response (SIR)
Induction response technique – an empirical assessment of survival and recovery
Babitha, 2012
Ehab et al., 2012, Journal of Cotton Science
Babitha, 2012 Sreekanthbabu et al., 2002, J. Plant Physiol
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Senthil-Kumar et al., 2003, JXB Senthil-Kumar et al., 2007, J. pl. physiol. Ganeshkumar et al., 1999, Theor Appl Gen;99:359–67.
T - Tolerant S - Susceptible
Rice
Cotton
C I NI C I NI
0
1020
30
40
5060
70
8090
100
10.0-20.0
20.0-30.0
30.0-40.0
40.0-50.0
50.0-60.0
60.0-70.0
70.0-80.0
80.0-90.0
90.0-100.0.
Range of thermotolerance
Frequ
ency Temperature
induction response in pea
T S T S
Genetic variability is seen only upon induction stress
Higher expression of Hsps in tolerant genotype
Stress transcriptome analysis should be done only after exposing to induction stress
Narendra, 2012
Ehab, 2012, Journal of Cotton Science
Senthilkumar et al., 2007, J. pl. physiol. Sreekanthbabu et al.,2002, J. Plant Physiol
Phenotype for acquired traits - only after induction UASB 15
Seeds exposed to 45 ⁰C at 100 % RH for 6/8 days
control 6 days of AA
MAS
AC35313
AC39018
IET16348
AC35310
AC39020
TELAHAMSA
KMP-175
Control
6 days of AA
wt AKR1-2
Range of root +shoot length (cm)
No.
of a
cces
sions
AKR1-2 transgenic tolerant to AA Nisarga, 2012
Seed aging is a stress – seedling vigour decreases on aging
Accelerated aging – a surrogate to phenotype for seedling vigour under stress
UASB 16
Genetic variability in Rice for oxidative stress
Untreated
Oxidative stress (Methyl viologen)
Phenotype for tolerance mechanisms – metabolic inhibitors (ROS inducers )
Frequency distribution of rice genotypes for MV induced oxidative stress
05
1015202530
Percentage reduction in growth (cm) N
umbe
r of g
enot
ypes
Class interval
UASB 17
WT M86 M97
Endoplasmic reticulum stress response
Metabolic inhibitors – to screen for specific tolerance mechanism
02468
101214
<WT 40-50 51-60 61-70 71-80 81-90 91-100
No.
of e
ntri
es
DTT induced ER stress
Survival percentage – DTT induced ER stress Rice
Groundnut
Babitha, 2012
UASB 18
Precise imposition of moisture stress is crucial
How to Phenotype acquired tolerant traits at whole plant level
By gravimetry - Capture the change in soil moisture due to ET in
mini lysimeters and replenish to maintain specified
Soil moisture status
Motorised lifting Manual lifting
Gravimetry – is an option
UASB 19
Weigh the pots to measure ET
Even in automated gravimetry systems
Water lost by ET quantified gravimetrically once a day
Limitation - frequency at which water lost by ET is replenished
Plants undergo daily cycles of stress- which again depends on leaf area
Change in the soil water status (% FC) during a 24hr cycle
Soil
moi
stur
e st
atus
(% F
C)
6.00 pm 6.00 am Percent Soil Moisture
Option to replenish water lost by ET at frequent intervals to maintain specific FC %
98 % FC 95 % FC
11.00 am 2.00 pm
93 % FC 89 % FC 85 % FC 80 % FC 78 % FC
Loadcell mini lysimeter assembly for Continuous monitoring of ET and soil moisture status (% FC)
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DATA COLLECTION & STORAGE HUB
Data + Power cable
Temperature Sensor
Relative Humidity Sensor
Data Acquisition System
Uninterrupted Power Supply
Data
Salient features of the minilysimeter assembly
Continuous measurement Fixed intervals Networked sensors Centralized Master Control Centralized data collection
Master Control Lysimeter Water Status
Load cell – minilysimeter assembly 22
Load Cell devices for continuous measurement of change in weight
in lysimeters
Load cell Lysimeter assembly
Lysimeter facility for real time monitoring of evaporation and transpiration
The mini-lysimeter facility
Open field conditions
Minilysimeters under ROS
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Cumulative transpiration of two Maize plants differing in leaf area
Soil
moi
stur
e %
FC
The system captures accurate changes in ET and soil moisture status
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65
70
75
80
85
90
95
100
0 10 20 30 40 50 60
Maize-LLA-1700cm2
Maize-HLA-3700cm2
Evap
otra
nspi
ratio
n (K
g)
12% drop
15% drop Irrigation
0
20
40
60
80
100
120
0 3 6 9 12 15 18 21 24
Diurnal changes in transpiration rate in plants differing in leaf area
Empty Pot (E)
Maize-HLA (T)
Maize-LLA (T)
12 am 3 pm 6 pm 9 pm 12 pm 9 am 6am 3 am 6 am
Time of the day
Tran
spira
tion
Rate
(g/h
r)
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Total transpiration- A function of leaf area
A significant positive correlation signifies accurate capturing of changing water loss by the lysimeter system
Leaf Area (cm2)
Tran
spira
tion
(g) /
plan
t/da
y y = 0,2764x + 125,83 R² = 0,9392
0200400600800
100012001400
1500 2500 3500 4500
Leaf Area (cm2)
Tran
spira
tion
(g) /
plan
t/da
y y = 0,2259x + 83,803 R² = 0,965
0100200300400500600700
0 500 1000 1500 2000 2500
Maize
Soybean
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Maintenance of soil moisture status by continuous replenishment of the water loss by evapotranspiration
The system provides an option to replenish within 5% change in FC
11 am 2pm 5 pm 8 pm 12 pm Overnight 8 am 5am
98 95 90 80
Continuous monitoring of ET and replenishment of water in the stationary lysimeter assembly
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Soil
moi
stur
e st
atus
(%FC
)
Can be programmed to attain required soil moisture status at desired pace
Option to program for gradual imposition of moisture stress in lysimeter grown plants
40
50
60
70
80
90
100
0 16 32 48 64 80 96 112 128 144 160 176 1 2 3 4 5 6 7 8 9 10
Reduction of 5% per day
50% FC
Soil
Moi
stur
e (%
)
Days
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Proposed lysimeter phenomics facility for growing germplasm accessions at desired soil moisture status
A pictorial outlay of the mini lysimeter facility
ROS
Each bay has 450 Mini Lysimeters
High throughput amenability
Load cell mini-lysimeter assembly
Facility under development
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Phenotyping of acquired tolerant traits at plant level
- Needs to be assessed at comparable water status
- Maintaining precise moisture status is crucial
A conceptual mini lysimeter facility developed
a) Continuous monitoring of E, T and ET in plants grown under natural conditions
b) Precise imposition of stress c) Amenable to screen large number of accessions d) Genetic variability assessment for many physiological traits
Precision high throughput phenotyping technioques for a few drought traits developed
- Water relation traits - Acquired traits at seedling level
In summary
High throughput facility is under development
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Thank you
Acknowledgement: Jalendra kumar H G Mahesh Salimath Sheshshayee M S Mohan Raju B R Arul Selvan K Bobji M
Other students and staff members
Funding from NAE, ICAR COE-program support, DBT DST-DBT National Facility
Cumulative transpiration of two Maize plants differing in leaf area Ev
apot
rans
pira
tion
(Kg)
0,0
0,5
1,0
1,5
2,0
2,5
0 10 20 30 40 50 60
Night Day
Irrigation
Time
Day-1 Day-2 Day-3
Maize-LLA-1700cm2
Maize-HLA-3700cm2
33 The system captures accurate changes in ET and soil moisture status
Emphasis We developed tools / techniques for assess
Stable isotope technique for WUE – 13C
Transpiration 18O (indirectly roots)
Mesophyll efficiency by Ci/gs ratio 13C/18O ratio
Gravimetry
Root structure
For CID
Bindumadhava et al., 1005; Sheshshayee et al., 2011
Rice
Endoplasmic reticulum stress response Genetic variability – Reactive Oxygen Scavenging
Oxidative stress
Untreated
(MV)
Rice
Groundnut