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ICAR - Indian Grassland and Fodder Research Institute,
Jhansi (UP) - 284 003, India
Man
ual
on
Pla
nt
Str
ess
Ph
ysi
olo
gy
Seva Nayak Dheeravathu
Vikas Chandra Tyagi
Chandan Kumar Gupta
Edna Antony
This page is left blank intentionally.
Manual on Plant Stress Physiology
Seva Nayak Dheeravathu
Scientist (Plant Physiology)
Division of Crop Improvement, IGFRI, Jhansi (UP), India
Vikas Chandra Tyagi
Scientist (Economic Botany & PGR)
Division of Grassland and Silvipasture Management, IGFRI, Jhansi (UP), India
Chandan Kumar Gupta
Scientist (Plant Physiology)
Division of Seed Technology, IGFRI, Jhansi (UP), India
Edna Antony
Sr. Scientist (Plant Physiology)
IGFRI-Regional Research Station, Dharwad (Karnataka), India
ICAR-Indian Grassland and Fodder Research
Institute, Jhansi (UP), India
Manual on Plant Stress Physiology. No. /2017
November, 2018
Citation:
Seva Nayak Dheeravathu, Vikas Chandra Tyagi, Chandan Kumar Gupta, Edna Antony.
(2018), Manual on Plant Stress Physiology. ICAR-Indian Grassland and Fodder Research
Institute, Jhansi.
Published on:
November, 2018
Published by:
Director
ICAR-Indian Grassland and Fodder Research Institute
Jhansi- 284003, Uttar Pradesh, India.
© 2018 All right reserved. No part of this publication may be reproduced or transmitted in
any form by any means, electronic or mechanical photocopy, recording or any information
storage and retrieval system without the permission in writing from the copyright owners.
Cover page design:
Vikas C Tyagi & Seva Nayak D
ACKNOWLEDGEMENTS
The authors express their profound gratitude towards Dr Khem Chand, Director and Dr
R.V. Kumar, Ex-Director, ICAR-Indian Grassland and Fodder Research Institute, Jhansi for his
ever moral boosting encouragement and also for providing the necessary facilities in coming out
with the present endeavor. Authors also thank Dr Shahid Ahmed Pr. Scientist (I/C-Head), Dr
Geetanjali Sahay, Pr. Scientist, Dr Nilamani Dikshit, Pr. Scientist., Dr Manoj Srivastava, Pr.
Scientist, Dr AK Singh, Sr. Scientist, Dr K K Dwivedi, Sr. Scientist, Dr Tejveer Singh, Scientist,
Dr. A Radhakrishnan Scientist, Dr Maneet Rana, Scientist, Mr Rahul Gajghate, Scientist, Dr.
Reetu, Scientist, Mr Neeraj Kumar, Scientist., Mr. Maharishi Tomar, Scientist, Dr. Hanamant
M. Hali Scientist and Dr. Mahendra Prasad Scientist, IGFRI, Jhansi. Sincere thanks to Dr P
Saxena and Dr P Kaushal, former, Head, Division of Crop Improvement, ICAR-IGFRI, Jhansi
for his keen interest, guidance and for providing essential Facilities for manual preparation.
Authors also thank Dr. Rodelio Carating, Supervising Science, Research Specialist, Bureau of
Soils and Water Management (Philippines), Dr. Bhupinder Singh, Principal Scientist, Centre for
Environment Science and Climate Resilient Agriculture (CESCRA), IARI, New Delhi, Dr P S
Deshmukh and Dr R K Sairam former Heads, Division of Plant Physiology IARI, New Delhi,
Dr Asit Mandal, Scientist, Indian Institute of Soil Science, ICAR-IISS, Bhopal, Prof. RV Koti,
Dr. B C Patil, Head, UAS, Department of Crop Physiology, College of Agriculture, Bijapur,
Karnataka and Dr B Mohan Raju, Associate professor, UAS, Department of Crop Physiology,
College of Agriculture, Bangalore for their critical input in preparation of this manual. Authors
also acknowledge suggestions and critical review of the manuscript made by the publication
committee viz., Dr V K Yadav, Chairman, and publication committee members Dr Manoj
Choudhary, Dr.V K Wasnik and Sri. P K Tyagi.
AUTHORS
Date: 30/10/2018
Place: Jhansi
S.No CHAPTERS Page .No
1. Introduction 1-2
2. pH and Buffer 3-4
3. Minimum Data Set for a Abiotic Stress Experiment (MIASE) 5-9
4. Estimations of soil particle density, soil bulk density and soil porosity 10-11
5. Artificial saline water and saline soil preparation (Soil salinity and plant
tolerance)
12-20
6. Estimation of soil pH and soil ECe 21-28
7. Measurement of osmotic potential using vapour pressure osmometer 29-30
8. Determination of Sodium and Potassium in plant tissue 31-33
9. Measurement of water content in soil and plant tissue 34-39
10. Imposition of drought by gravimetric approach 40-42
11. Two tier screening of germplasm under natural condition or Irrigation stops
approach for stage-specific drought tolerance
43-49
12. Determination of Water Use Efficiency (WUE) 50-54
13. Photosynthetic pigments analysis in plants 55-57
14. Estimation of chlorophyll stability index and carotenoid stability index in
leaf tissue
58-60
15. Cell Membrane Stability Index 61
16. Estimation of abscisic acid content in leaf and root 62-63
17. Estimation of proline content in plant tissue 64-65
18. Photosynthesis 66-71
19. Canopy Temperature Depression (CTD) 72-73
20. Root aerenchyma identification under waterlogging 74-75
21. Estimation of antioxidant enzymes 76-80
22. Stress assessment formulas and stress related terminology 81-87
23. Annexure-I 88
24. Abbreviations 89
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e1
Chapter 1
Introduction
Drought, flooding, high temperature, cold, salinity, and nutrient availability are abiotic
factors that have a significant impact on world agriculture and account for more than
50% reduction in average potential yields for most major food and fodder crops (Wang
et al., 2003). These comprise mostly of high temperature (40%), salinity (20%), drought
(17%), low temperature (15%) and other forms of stresses (Ashraf, 2008). Climate
prediction models show increased occurrences of drought, flooding, salinity and high-
temperature spells during the crop growing periods (IPCC, 2008; Mittler and
Blumwald, 2010). Plant genetic resources for food and agriculture comprises of a
diversity of genetic materials in the form of traditional varieties, modern cultivars, crop
wild relatives and other native species that are the basis of global food security. Genetic
diversity provided farmers, plant physiologists, plant breeders and biotechnologists
with options to develop, through the natural selection, breeding and genetic
manipulation, new crops, that are resistant to pests, diseases and adapted to changing
environments (abiotic stress). Human population is increasing and is expected to grow
from 6.9 billion to 9 billion by 2050. To feed the increasing population, we need to
improve the food production by 60% up to 2050 with the limited land and water
resources (FAO, 2012b). The demand for food and livestock production will continue
to rise with the increase in global population; therefore improving production and
productivity to ensure sustainable yields under changing environmental conditions is
essential. To achieve this predicted global food security, we need to increase our
understanding of plant responses to abiotic stress. Knowledge of natural selection,
stress breeding and genetic manipulation of plants that can maintain higher
photosynthetic rates, better foliage growth and improved yield under stress conditions
(Condon et al., 2004; Morison et al., 2008) are must for achieving this goal.
Agronomists, soil scientists, plant genetic resource (PGR) scientists, plant
physiologists and plant geneticists and breeders can play an essential role in boosting
crop production by collection, evaluation, documentation, identification,
characterisation of stress adaptive traits and utilisation of these traits into the breeding
programme for crop/forage improvement.
Pag
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References:
1. Ashraf, M., H.R. Athar, P.J.C. Harris and T.R Kwon. 2008. Some prospective strategies for
improving crop salt tolerance. Adv Agron 97: 45-110.
2. Condon, A.G., R.A. Richards, G.J. Rebetzke and G.D. Farquhar. 2004. Breeding for high
water-use efficiency. J. Exp. Bot. 55: 2447-2460.
3. FAO (Food and Agriculture Organization of the United Nations), 2012 b. World Agriculture
towards 2030/2050: the 2012 Revision. ESA Working Paper No. 12-03. Food and Agriculture
Organization of the United Nations, Rome, Italy.
4. IPCC, 2008. Climate change and water. In: Bates, B.C., Kundzewicz, Z.W., Palutikof, J., Wu,
S. (Eds.), Technical Paper of the Intergovernmental Panel for Climate Change. Secretariat, Geneva,
pp. 210.
5. Mittler, R. and E Blumwald. 2010. Genetic engineering for modern agriculture: challenges and
perspectives. Annu. Rev. Plant Biol. 61:443-462.
6. Morison, J.I.L., N.R. Baker, P.M. Mullineaux and W.J Davies. 2008. Improving water use in
crop production. Philos. Trans. R. Soc. Biol. Sci. 363: 639-658.
7. Wang W., B. Vinocur, A. Altman. 2003. Plant responses to drought, salinity and extreme
temperatures: towards genetic engineering for stress tolerance. Planta 218: 1-14.
Pag
e3
Chapter 2
pH and Buffer
Acid and base: According to Bronsted concept a proton donor is denoted as an acid
and proton acceptor as a base
Strong acids or bases: These compounds are completely ionised in solution. So that
the concentration of free H+ or OH- is the same as the concentration of the acid or base
Strong acid, HCl→H+ + Cl-
Strong base NaOH→Na++OH-
Weak acids or bases: The dissociation of this compound is incomplete. The
concentration of free of H+ or OH- depends on the value of their dissociation constant:
Ionization of water: Water molecules tend to undergo reversible ionisation to yield a
hydrogen ion (H+) and a hydroxyl ion (OH-)
The concept of pH: In 1909 Sorenson introduced the term pH as a convenient way of
expressing hydrogen ion by mean of a logarithmic function and is defined as the
negative of logarithmic hydrogen ion concentration pH = -log [H+] concentration
Hydroxyl ion may be defined as pOH = -log [OH-]
The equation for Kw can be written as -log Kw= pH + pOH=14
Thus the sum of pH and pOH is 14, and the two components are related reciprocally.
Neutrality prevail at pH pOH =7. The pH of material ranges on a logarithmic scale
from 1-14, where
pH 1-6 is acidic, pH 7 is neutral, and pH 8-14 is basic. Lower pH corresponds with
higher [H+] while higher pH is associated with lower [H+].
Buffers: Buffer solution is the one that resistant changes in pH when small amounts of
acid or base are added. A buffer solution consists of a weak acid and its conjugate base.
Pag
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References:
1. Conn, Eric E. and Stumpf, P. K. 1977. Outlines of biochemistry (4th Edition). John wiley and
sons, London. pp 3-23.
2. David, L. N. and Michael M. Cox Lehninger. 2004. Principles of Biochemistry (4th Edition).
W.H. Freeman, New york. pp 65-68.
Pag
e5
Chapter 3
Minimum Data set for Abiotic Stress Experiment
(MIASE)
Before conduct of the experiment, minimum information should be known, i.e. about
agronomical, physical properties of soils and physiological and molecular responses of
plants to abiotic stresses for varietal development to abiotic stress tolerance.
A) Agronomic/soil information:
1. Agronomic conditions of crop growth: Seed rate, spacing, number (quantity and
interval) of irrigations, fertiliser application schedule, irrigation schedule (irrigation
should be started when about 50 percent of the available moisture (%) in the soil root
zone is depleted, the available water is the soil moisture, which lies between field
capacity and wilting point), IW/CPE ratio (Irrigation water /cumulative pan
evaporation).
2. Physical properties of soils and types: texture, structure, colour, soil particle
density, soil bulk density, soil porosity and pH and EC of soil.
3. Soil moisture data: At different depth at least two points preferably in root zone; at
least two time points one each at start and end of drought stress.
4. Defining dry land agriculture scientifically based on Reddy and Reddis
definition, Dryland Agriculture may be classified into three groups on the basis of
annual rainfall.
i. Dry Farming Cultivation of crops in areas where annual rainfall is less than
750mm and crop failures due to prolonged dry spells during crop period are most
common. Dry farming is practiced in arid regions with the help of moisture
conservation practices.
ii. Dry land farming Cultivation of crops in areas where annual rainfall is more than
750 mm but less than 1150mm is called Dry land farming. Dry spells may occur, but
crop failures are less frequent. Higher Evapotranpiration (ET) than the total
precipitation is the main reason for moisture deficit in these areas .The soil and
moisture conservation measures is the key for dryland farming practices in semi-arid
.regions. Drainage facility may be required especially in black soils.
iii. Rainfed farming Means cultivation of crops in regions where annual rainfall is
more than 1150mm. There is less chances of crop failures due to dry spells. There is
adequate rainfall and drainage becomes the important problem in rainfed farming.
Pag
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5. Classification of drought on water deficit at the following five levels:
i. Severe water deficit—Available soil moisture (ASM) between 40 -50% or Soil
Moisture depletion (SMD) between 50-60% during the plant growth period
ii. Moderate water deficit— Available soil moisture (ASM) between 50 - 60 % or Soil
Moisture depletion (SMD) between 40-50% during the plant growth period
iii. Mild water deficit— Available soil moisture (ASM) between 60 - 70 % or Soil
Moisture depletion (SMD) between 30-40% during the plant growth period
iv. No deficit or full irrigation— Available soil moisture (ASM) between 70-80 % or
Soil Moisture depletion (SMD) between 20-30% during the plant growth period
v. Over-irrigation—the amount of water irrigated may be more than plants requirement
for optimal growth
6. Relative water content (RWC): Normal values of RWC range between 98% in fully
turgid transpiring leaves to about 30-40 in severely desiccated and drying leaves,
depending on plant species. In most species, the typical leaf RWC at around initial
wilting is about 60 to 70% with exceptions.
7. Crop growth stage at which the stress was imposed: At three stages (seedling,
vegetative and reproductive or premature stage).
8. Duration of stress: At least 7-15, 20-30, 15-20, days for seedling, vegetative and
reproductive or premature stages respectively (less duration in case of premature
because natural senescence occurs) but in case of range grasses 30-45 days for
vegetative/reproductive (crop to crop vary).
9. Type of design: For Rapid screening- augmented design, for basic/confirmative
study- RBD and CRD field and laboratory respectively.
10. Minimum number of plant sample should be taken from segregation populations
is 30-35 plants from augmented design [(at least BC1 to BC5 (Back crosses) or more for
getting stable trait)]
11. Phenology: Phenology is the study about event in the lifecycle of a plant influenced
by seasonal and inter annual variations in climate.
12. Yield and yield components.
11. Weather data (rainfall, temp. VPD-Vapour pressure deficit).
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12. National/regional check genotype can be used as control for comparison.
13. Problematic soils /salinity: The soil sample should be collected from different soil
layers to different depths based on the plant species preferably in the root zone. For
deep-rooted plants, sample soil layers from 0-5, 5-10, 10-20, 20-40, and 40 -60 cm and
so on to at least 1m deep. The samples from different layers should be mixed
uniformly.
14. Problematic soil classifications, saline soil types and plant and crop plant tolerance
ratings:
i) Classification of salt-affected soils based the on pH, ECe, SAR; ESPs (see the table-
5.1)
ii) Classification of saline soils based the on soil pH and soil ECe ranges (see the table
No.5.2)
iii) Ratings of plants and crop plants, tolerance to salt stress based on pH and soil ECe
ranges verses to relative crop yield or yield potential reductions (see the fig No-5.1
and table No-5.3)
15. Ayers and Westcot (1985) reported that in irrigation water 0.7 EC (dS/m) would
not affect plant growth or slightly affect plant growth in the field with increasing
number of irrigations, because salts may go down or leach out may occur. In pot
condition, salt concentration may increase with increasing number of irrigations and
affect plant growth and development.
B) Exploration/Rapid Screening Techniques
1. Exploration: Capture maximum amount of variation in smallest number of samples
(allelic richness for given locus)
2. Handling and maintenance: Handling, maintaining, conducting the experiment and
screening of large size population/ germplasm is difficult, so maximum germplasm
should be discarded at ground level/ preliminary screening
3. Critical level of stress: To find out the critical stress level where we can discard the
maximum of germplasm /population
4. Rapid screening techniques/ methodology/protocol: Find out the Rapid screening
techniques/ methodology/protocol for rapid screening / Preliminary screening
5. Preliminary/ Rapid screening: Maximum germplasm should be discarded at
preliminary screening stage from large size population/germplasm resources for getting
the maximum amount of variation/genetic makeup in smallest number of samples
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6.Screening criteria: Agronomic characters such as survival, biomass accumulation
(multi-cut forage crops 2nd cut is preparable for biomass), GFY and DMY and HI and
physiological parameters, seedling vigour index, Relative growth rate, chlorophyll
content (SPAD reading) and Relative water content (RWC), Membrane Stability Index
(MSI), Root/shoot ratio, K+/ Na+ ratio in the plant are the most commonly used criteria
for identifying the adaptive traits among the genotypes or germplasm to abiotic stress
tolerance.
7. Techniques/ methodologies/ tools: Hydroponics, petri dishes, in vitro test tube
method, germination paper method, cup method/ pot/field methods, tools- SPAD meter,
Leaf ara meter IRGA, CTD.
General points:
1. Passport data: Collect the germplasm/genotype passport data from the passport
data also, we can minimise sample
2. Grouping: Grouping the germplasm on morphology, phenology/phenotypic or
genotypic (seed vigour index and flowering and maturity)
3. Take the diverse genetic group of germplasm for experiment
4. Multiply the germplasm/seed for sufficient material for experiment
5. Collect weather data from meteorological department and find out/known for
target environment
6. Find out /known for target trait for crop improvement
7. Use check lines/genotype/variety for trait comparison and variety development
8. Experimental design: Augmented design/ germination paper method/ in vitro test
tube methods are easy for rapid/ preliminary screening.
C) Physiological information (minimum information)
1. Chlorophyll Stability Index (CSI) and Carotenoids Stability Index (CSI)
2. Relative Water Content (RWC)
3. Membrane Stability Index (MSI)
4. Water Use Efficiency (WUE)
5. Abscisic acid and proline content
6. Photosynthesis (stomatal conductance)
7. Canopy Temperature Depression (CTD)
8. High K+ / Na+ ratio or low Na+/ K+ ratio -for tolerant genotypes
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9. Root aerenchyma formation (RAF), Root volume and Root length
10. Antioxidant enzymes
11. Seedling Vigour Index (SVI)
12. Relative growth rate (RGR)
13. Root to shoot ratio
14. Leaf area ratio (LAR), Flag leaf area, Leaf area per plant, leaf Area Index
15. Net assimilation rate (NAR)
16. Plant water content
Note: These definitions provide a “standardised” approach with which water deficit
treatments and the responses reported in various published studies can be assessed
using a similar scale.
References:
1.T .Yellamanda Reddy and G.H. Sankara Reddy. 2016. Principles of Agronomy. New
Delhi, Kalyani publishers.
2. M. Mudasir Magray, Nayeema. Jabeen, M.A. Chattoo, F.A. Parray1 Alima. Shabir and
S.N. Kirmani.2014.Various problems of dryland agriculture and suggested agro-
techniques suitable for dryland vegetable production, Int. Jour. of App. Sc. and Eng. 2(2) :
45-57.
3.Reddy, N.N., Reddy, M.J.C., Reddy, M.V., Reddy, Y.V.R., and Singh, H.P. 2002. Role
of Horticultural Crops in Watershed Development Programmes Under Semi-Arid Sub
Tropical Dryland Conditions of Western India. 12th ISCO Conference Beijing.Central
Research Institute for Dryland Agriculture Santoshnagar, Saidabad (P.O.), Hyderabad-500
059, India
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Chapter 4
Estimations of soil particle density, soil bulk
density and soil porosity
Particle density: The particle density of soil is the mass of a soil sample in a given
volume of particles (mass divided by volume).
Purpose: To measure the soil particle density of each horizon in a soil profile
Procedure:
1. Take the 50 ml measuring cylinder
2. Add 25ml DH2O and warm it then cool at room temperature
3. Add 10g of dried and sieved soil (2mm sieve)
4. Note the changes in water level this gives volume of 10g soil repeat it for 3-4 times
and take the average
Formula: PD=Mass/Volume
Bulk density:
Soil bulk density is the weight of soil that is dry per unit volume. This volume includes
the volume of soil particles and volume of the pores present in the soil. Bulk density or
BD is expressed in g/ cm3. Bulk density is an important soil parameter used to convert
the weight and volume of the soil Soil bulk density can vary among different soil types
and is affected by management practices. Organic matter incorporation into the soil will
lower the bulk density, while any processes that compact the soil will increase bulk
density. The bulk density of mineral soils ranges from 1.0 to 1.8 g/cm3.
Procedure:
1. Soil core sampler is inserted into undisturbed soil without compressing the soil
2. Remove the excess soil from both ends with the help of knife
3. Now dry this core in oven at 105 o C records the dry weight and measure the radius
of the core and core height and calculate the volume of the core
Soil porosity is the amount of pore space occurring in between soil particles. Pore
spaces are formed due to the movement of roots, worms and insects. The pore space
decides the amount of water soil can hold. Amount of pore space or porosity of the soil
is calculated by formula
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Porosity =1-
Where in BD is Bulk density and PD is particle density
References:
1. Steven, Thien and Graveel John. 2002. (8th edition) Adapted from Laboratory manual for Soil
Sciences: Agricultural and environmental principles. pp 232.
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Chapter 5
Artificial saline water and saline soil preparation
(Soil salinity and plant tolerance)
Soils containing an excess concentration of soluble salts or exchangeable sodium in the
root zone, it is called as salt-affected soils (Conway 2001; Denise 2003; Jim 2002).
Salt-affected soils (Usara/ Kalar) can be broadly categorised into three types based on
their salinity and sodicity (Gonzalez et al., 2004) Table-5.1. When soils contain
excessive concentration of water-soluble salts containing positive charge cations such
as sodium (Na+), potassium (K+), calcium (Ca2+) and magnesium (Mg2+) along with
negative charge anions chloride (Cl-), sulphate (SO42-), nitrate (NO3
-), bicarbonate
(HCO3-) and carbonate (CO3
2-), these are called saline (Rhoades and Miyamoto, 1990).
These dissolved salts cause the harmful effect on seed germination, plant growth and
yield when the concentration in the root zone exceeds critical level (Conway 2001;
Denise 2003).The more soluble salts such as sodium chloride (NaCl), sodium sulfate
(NaSO4), sodium bicarbonate (NaHCO3), and magnesium chloride (MgCl2) cause more
plant stress than less soluble salts such as calcium sulfate (CaSO4), magnesium sulfate
(MgSO4), and calcium carbonate (CaCO3). Irrigation water and saline soils were
classified into four and five major groups respectively, depending on salinity levels
(Table-5.2). The electrical conductivity (EC) or EC of the saturated soil paste (ECe) is
an important parameter because this value is used to characterise crop salt tolerance.
Salt susceptible (glycophytes /sweet plants) and tolerant plants (halophytes/ salt tolerant
plants) are classified into four groups viz, sensitive, moderately sensitive, moderately
tolerant and tolerant (Fig-5.1 and Table-5.3).
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Table-5.1 Classification of salt-affected soils
Class pH ECe
(dS/m)
SAR ESP
Normal 6.5 -7.5 <4 <13 <15
Symptom No visible symptom and normal growth of the plant
Saline <8.5 >4 <13 <15
Symptom White crust on the soil surface. Water-stressed plants. Leaf tip burn/ non-sodic soil
with sufficient soluble salts to interfere with the growth of most crops
Sodic >8.5 <4 >13 >15
Symptom Poor drainage. Black powdery residue on soil surface. Soils with sufficient
exchangeable sodium to interfere with the growth of most plants, but without
appreciable quantities of soluble salts
Saline-
Sodic
<8.5 >4 >13 >15
Symptom Grey-colored soil. Plants showing water stress. Soils with sufficient exchangeable
sodium to interfere with the growth of most plants and containing appreciable
quantities of soluble salt
(Source: Horneck et al. 2007)
Table-5.2. Crop response to salinity, measured as the electrical conductivity of the soil
saturation extract (ECe)
(In parenthesis indicate irrigation water salinity: ECw)
USDA classification of irrigation water salinity (adapted from Richards, 1969)
Soil
depth
Saline Soil Classes/ Interpretation (Classification of irrigation water salinity)
Non-Saline/
salt-free
Weakly Saline/
Slightly
saline(Low
salinity water)
Moderately
Saline(Medium
salinity water)
Strongly
Saline (High
salinity water)
Very Strongly
Saline (Very
high salinity
water)
ECe (dS/m) at 25 oC [(ECw (dS/m)]
0-60 cm
(0-2 ft)
0-2
(up to 0.7 )
2-4
(0.7- 2.5)
4-8
(2.5-7.5)
8-16
(7.5-22.5)
>16
(> 22.5)
60-120
cm
(2-4 ft)
<4 4-8 8-16 8-16
(7.5-22.5)
>16
(> 22.5)
Crop
response
Salinity effects
mostly
negligible,
except in very
sensitive plants
Yield of very
sensitive crop
restricted
Yield of most crop
restricted
Only tolerant crop
yield satisfactorily
Only a few tolerant
crops yield
satisfactorily
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Fig: 5. 1 Relative crop yield (or yield potential) as a function of average root zone salinity (dS/m)
grouped according to relative tolerance or sensitive to salinity. Source: Adapted from Maas and
Grattan 1999; Grieve et al .2012)
Table- 5.3 Salt tolerance ratings of various crops
Sensitive
Moderately
sensitive
Moderately
tolerant
Tolerant
Rice Chickpea Sorghum Barley
Sesame Corn and
Corn
(forage)
Soybean Canola
Gram, Black or urd
bean
Peanut Sunflower Cotton
Pigeonpea Sugarcane Wheat Guar
Walnut Alfalfa Barely
(forage)
Oats and forage Oats
Mango Berseem Guinea Rye and forage Rye
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grass
Banana Cowpea
(forage)
Dhaincha Triticale
Apple Buffel grass Wheat (semidwarf)
Wheat (durum)
Kallar grass
Date palm
Source: Adapted from Maas and Grattan 1999; Grieve et al. (2012)
A) Preparation of saline water (Source: USDA Hand book No-60)
Known standard mixtures of salt ratios are used for conducting the experiment under
(specify your actual experiment-test tube, hydroponics, pot, and field) for screening the
salt tolerant/transgenic cultivars based on Table 5.4, Fig.5.2 (A and B) and Table 5.5
Fig-5.3 using the following formula:
Desired EC = mEq or ME x MW
Where,
mEq or ME = milli equivalent for desired EC
MW = molecular weight of the salt
Desired mixture of salts and its ratios: NaCl, Na2SO4, MgCl2, and CaSO4, 13:7:1:4
respectively
Level of desired saline EC (dS/m): 4, 8, 12, 16
Ex: NaCl at 4 EC at 4 EC = 45meq L-1 (Fig.5.2 (A and B)
= Concentrations of salt (me L-1)
Total salt ratio
ME =
Test the EC of the water before using it to saturate the soil, germination paper (Test the
EC of the water before using it to saturate the soil, germination paper (salinity levels
raised on germination paper)
Table: 5.4.Computed salt requirements for desired saline water levels given for various types of experiment (Test tube, hydroponics, pot,
and field soils)
EC
(dS/m)
ME for all
4 salts
ME for individual salt MW Salt required (g) /liter)= ME x MW
NaCl
Na2SO4
MgCl2
CaSO4
NaCl
Na2SO4
MgCl2
CaSO4
NaCl
Na2SO4
MgCl2
CaSO4
4 45 23.4 12.6 1.8 7.2 58 142 203 172 1.4 1.8 0.4 1.2
8 95 49.4 26.6 3.8 15.2 58 142 203 172 2.9 3.8 0.8 2.6
12 150 78.0 42.0 6.0 24.0 58 142 203 172 4.6 6.0 1.2 4.1
16 200 104.0 56.0 8.0 32.0 58 142 203 172 6.1 8.0 1.6 5.5
Note: This prepared saline solution/or saline water directly used for germination study in petri dish/germination paper study/ in vitro test tube
method or hydroponic study (Hoagland solution) or saline irrigation method- mostly useful/preferable to laboratory conditions, but not good for
pot/field conditions. This is why because soil ECe generally comes down into lower than desired or targeted saline soil ECe
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Table: 5.5. Electrical conductivity (EC) of pure solutions at 20°C (dS/m)
equivalent with mM solution
Solution EC
(dS/m)
10 mM NaCl 1.0
100 mM NaCl 9.8
500 mM NaCl 42.2
10 mM KCl 1.2
10 mM CaCl2 1.8
10 mM MgCl2 1.6
50 mM MgCl2 8.1
The solutions represent those of salts found in soils or in seawater. Data from the Handbook of
Physics and Chemistry (CRC Press, 55th edition, 1975).
Fig.5.2 (A and B) Concentration of saturation extraction of soil in milliequivalents per liter as
related to Electrical conductivity (Conductivity v/s. concentration Source USDA Hand book No-60)
A
B
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Fig-5.3 Concentration of single-salt solutions in mill equivalents per liter as related to
electrical conductivity
B. Preparation of artificial saline soil (I.C. Gupta et al 2012)
Artificial saline soils are usually used in pots and micro plot experiments. To develop a
given salinity level, application of salts like NaCl, CaCl2 and Na2SO4 dissolved in the
ratio of 7:2:1, gives good results as it is the ratio in which these salts are found in semi-
arid areas. Other composition of salts could be used depending upon the kind of [(Ex.
NaCl, Na2SO4, MgCl2, and CaSO4, (13:7:1:4 ratio) for petri dish, test tube, hydroponic,
pot/pit experiments] experiments. In this case, take dry, grounded and sieved (2mm)
known weight of soil in the pots.
Desired level of EC (dS/m): 4, 8, 12, 16
To calculate the salts required to prepare a soil with ECe of 4, 8, 12, 16 dSm-1
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Calculate the salt required for1 litre of 4, 8, 12, 16 (each) EC water (Table-5.6) that
would be able to saturate about 2.5 kg of the soil if the porosity of the soil is taken as
0.4 by weight (for semi-arid soils). The calculation of salinity depends on the
percentage saturation of the soil which needs to be estimated individually for the type
of soil used for the experiment.
Table: 5.6.Computed salt requirements for desired salinity levels given various types of soils
EC
A mixture of salt ratios Equivalent weight ME=EC X Salt ratio
Salt required (g) /liter) =
ME x MW
NaCl CaCl2 Na2SO4 NaCl CaCl2 Na2SO4 NaCl CaCl2 Na2SO4 NaCl CaCl2 Na2SO4
4 7 2 1 59 56 71 28 8 4 1.6 0.4 0.3
8 7 2 1 59 56 71 56 16 8 3.3 0.9 0.6
12 7 2 1 59 56 71 84 24 12 4.9 1.3 0.9
16 7 2 1 59 56 71 112 32 16 6.6 1.8 1.1
Dissolved NaCl and CaCl2 in approximately half of the total water and Na2SO4 in the
remaining half of the water.
Test the EC of the water before using it to saturate the soil.
[Note- 1: Equivalent weight of salt = ,
Note-2: Na2SO4 = = 71]
Note: Initial checking of ECe is required to know the salt concentration already present
Note: This prepared saline soil, directly used for sowing/transplanting in pot conditions.
The soil containing salts should is irrigated with ordinary water. The drain holes in the
pot should be plugged or seald with M-seal. An equal volume of water should be added
to the pots having different ECe (dS/m) soils. Before planting seedlings /root slips, the
pot should be watered for two weeks, and salts should be allowed to distribute within
the pot uniformly. Check the EC of irrigation water. If the water is saline, then the salts
will get added to the soil salinity. So before planting/sowing, measuring the EC of
watered soil is warranted.
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References:
1. Ayers, R.S., and D.W. Westcot. 1985. Water Quality for Agriculture, FAO Irrigation and
Drainage Paper 29 rev 1.
2. Conway, T. 2001. Plant materials and techniques for brine site reclamation. Plant materials
technical note degraded Soils: Origin, Types and Management.
3. Denise, M. W. 2003. Soil salinity and sodicity limits efficient plant growth and water use.
Rio grande regional soil and water series guide A-140, New Mexico State University, New
Mexico.
4. Grattan, S.R. and C.M. Grieve. 1992. Mineral element acquisition and growth response of
plants grown in saline environments. Agric. Ecosyst. Environ. 38: 275–300.
5. Grieve C.M., S.R. Grattan and E.V. Maas. 2012. Plant Salt Tolerance. In: Wallender,
W.W., Tanji, K.K. (eds), Agricultural Salinity Assessment and Management. American
Society of Civil Engineers, Reston, Virginia. pp. 405-459.
6. Horneck, D.S., J.W. Ellsworth, B.G. Hopkins, D.M. Sullivan and R.G. Stevens. 2007.
Managing Salt-Affected Soils for Crop Production. PNW 601-E. Oregon State University,
University of Idaho, Washington State University.
7. I.C.Gupta, N.P.S.Yadavashi, S.K gupta 2012. Standard methods for Analysis of soil plant
and water. Scientific Publishers, India .pp 50
8. Jim, M. 2002. Managing salt affected soils. NRCS, South Dakota
9. Mass, E.V. and S.R. Grattan.1999. Crop yields as affected by salinity. In Agricultural
Drainage; Skaggs, R.W., van Schilfgaarde, J., Eds.; American Society of Agronomy, Crop
Science Society of America, Soil Science Society of America, Madison, WI, USA. pp. 55–
108.
10. Richards, L.A. 1969. Diagnosis and improvement of saline and alkali soils. United States
Department Of Agriculture (USDA); Washington.
11. USDA. 1954. Diagnoses and improvement of saline and alkali soils. Agric. Handbook No.
60. USSL, Riverside, CA, USA.
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Chapter 6
Estimation of soil pH and soil ECe
Soil pH is a measure of the acidity or basicity of a soil, the plant optimum pH range for
most plants is between 5.5-7.5. However, many plants have adapted to thrive at pH
value outside this range. Because pH level controls many chemical processes that take
place in the soil, soils maintain the proper pH levels to plant nutrient availability. Soil
pH does not directly measure soil salinity. Irrigation water and soil salinity are
measured by passing an electrical current between the two electrodes of a salinity miter
in a sample of soil solution or irrigation water. EC of a soil or water sample is
influenced by the concentration and composition of soluble salts. There are two
common methods are available for measuring salt concentration in soil and water i.e.
EC meter and TDS (total dissolved salts), units and conversion factor mentioned in
table 6.1.and Annexure-I. The salt concentration in the soil solution and irrigation
water determined by the electrical conductivity (EC) meter method is very rapid a more
and accurate method than the TDS method.
Principle:
The soil pH reflects whether a soil is acidic, basic (alkaline) or neutral. The acidity,
basicity (alkalinity) or neutrality of the soil is measured in terms of hydrogen or
hydroxyl ion activity of the soil -water system. It indicates whether the soil is acid (pH
1-6), neutral (pH) or alkaline in reaction (pH 8).The pH range normally found in soils
varies from 3 to 9. The presence of neutral soluble salts as in saline soils is not
normally reflected in its pH, but when their content is excessively high it reduces
hydrogen (H+) activity. Crop growth and yield may reduce under both very low (acidic
soils) as well as very high pH (alkaline soils) conditions.
Table 6.1: Units for measuring salinity, and conversion factors.
Measurement and
units
Application 1 dS/m is equal
to
Equivalent units
Conductivity (dS/m) Soils 1 1 dS/m = 1 mS/cm = 1
mmho/cm
Conductivity (µS/cm) Irrigation and river
water
1000 µS/cm 1 µS/cm = 1 µmho/cm
Total dissolved salts
(mg/L)
Irrigation and river
water
640 mg/L
(approx.)
1 mg/L = 1 mg/kg = 1
ppm
Molarity of NaCl (mM) Laboratory 10 mM 1 mM = 1 mmol/L
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The most convenient method for measuring pH is by the use of glass electrode pH
meter
Some general guidance on the use of pH meter:
1. New glass electrodes should be soaked in 0.1 M HCl for a minimum of 6-8 hours
before use
2. The solution should be thoroughly mixed before measuring the pH
3. The temperature should be maintained constantly as it affects the pH of the
solution
4. The electrodes should be rinsed with distilled water before and after use and must
not be touched
5. Calibrate the pH meter before use using a standard buffer solution (pH 9.2, 7.0 and
4.0).
6. The calibration should be done with buffer solution whose pH is close to that under
test
Testing of sample:
1. In a clean, dry 100 ml beaker take the sample and place in a magnetic stirrer
2. Stir well with the Teflon coated stirring bar
3. Place the electrode in the beaker containing a sample (soil solution/ water/
chemical solution) and note the pH reading with pH Meter.
4. Wait until a stable reading is displayed.
Soil salinity measures
Water soluble salts in the soil and irrigation water are strong electrolytes and as such
soil solution and irrigation water has conductivity. The electrical conductivity reflects
the conductivity capacity of the soil solution and irrigation water within a certain range
of salt concentration. The salt content in the soil solution and irrigation water is
positively related to the electrical conductivity. The electrical conductivity of the soil
extract can reflect the soil salt content, but it cannot reflect the component of the mixed
salt. The main methods of measuring the total water-soluble salt in the soil and
irrigation water are weight method (gravimetric), electrical conductivity and
salinometer methods.
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Procedures for gravimetric method
Total salts in a soil sample can be measured by dissolving them in water and
evaporating the water by heat and estimating them by their weight (also applies to
water samples)
Procedure
1. Take 50 ml sample solution (A) in a evaporating tin box record the weight (B0)
and evaporate it in water both, followed by oven drying at 105-110 oC for over night
2. Take the constant weight (C1) ( weight difference two times is not more than
1mg)
3. Add 15% H2O2 in drops to wet the residue then evaporate to dryness in the water
both (until the entire residue turn white) then take the weight (D2)
4. Calculate the content of total water-soluble salt in the soil.
Formula
Total dried residue (%) =
Where A, is the weight of the sample (g) that the drawn extract is equivalent to
The result of the weight method is reliable but the operation is tedious and time
consuming.
Electrical Conductivity:
ECe estimation can be determined by two ways first soil saturated paste extracts
(saturation-extract) and soil-water ratio extracts method. SP method is time consuming
and need more skills are needed for determining the correct saturation point and it is an
uneasy and costly method to determine soil salinity for high sampling frequency
(Aboukila and Norton, 2017). Soil-water extract (different soil and water ratios 1:1,
1:2, 1:2.5, 1:5, and 1:10 commonly utilized in soil laboratories) method is simple and
easy than soil saturated paste extracts method (Aboukila and Norton, 2017). Among the
1:1, 1:2, 1:2.5, 1:5, and 1:10 soil-water ratio extractions, 1:5 ratio extraction is
preferably used as a method for calculating soil salinity (Shirokova et al., 2000; Wang
et al., 2011) or EC (1:5) test value can be converted to an estimated electrical
conductivity of a saturation paste (ECe) by multiplying with a texture factor, because
soil texture influences the degree to which the amount of salt present in the soil will
affect plant growth (Table-6.2). The conductivity (EC meter) method is simple and easy
method in laboratories.
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Formula:
ECe estimated = EC 1:5 x texture conversion factor
Note: 1 Note that ECe is the term used to indicate actual soil salinity, so then convert
your salinity EC meter readings to soil salinity (ECe), by multiplying the value by the
conversion factor based on the texture of the soil sample (Table : 6.2)
Note : 2 Sonmez et al. (2008) observed high correlations between ECe and the EC
values of 1:1, 1:2.5, and 1:5 soil-to-water suspensions for soils in Turkey with a slightly
better correlation using the 1:2.5 suspensions
Table: 6.2 EC 1.5 to ECe conversion factors
S. no Soil texture Multiplication factor
1 Sand, loamy sand, clayey sand 23
2 Sandy loam, fine sandy loam, light sandy clay loam 14
3 Loam, fine sandy loam, silty loam, sandy clay loam 9.5
4 Clay loam, silty clay loam, fine sandy clay loam, sandy
clay, silty clay, light clay
8.6
5 Light medium clay 8.6
6 Medium clay 7.5
7 Heavy Clay 5.8
8 Peat 4.9
Source: Slavich and Petterson (1993)
Procedure for saturated soil paste preparation:
1. Take 200-400 g of sieved 2mm air dried soil into plastic beaker (500ml capacities)
2. Add DDH2O or deionized water into soil and mix with spatula until all the soil
become moist and soil become smooth paste with adding water or soil as necessary/no
free water on soil surface
3. The paste should the be glistened as it reflects light, flows slightly when the
container is tipped, slides freely and cleanly off a spatula
4. Keep the saturated paste for overnight with lid for soil to fully imbibe water and the
salts to dissolve
5. Remix the paste with water or soil as is needed to bring the paste saturation point
6. Then filter the saturated paste with whatman paper no 42 or vacuum extractor to
obtain the extract
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7. If the filtrate is not clear, the procedure must be repeated. Transfer the clear filtrate
into a 50-ml bottle. Switch on the conductivity meter and immerse the electrode in the
saturation extract and record the reading at a standard temperature of 25°C. (Some
instrument automatically has preset reading at 25oC)
8. If temperature adjustment is not available in the same instrument, then correct with
correction factor (Table-6.3)
Calculation: The EC of the soil extract at 25 oC (EC25) is used to reflect the soil salt
content.
It is calculated as follows: EC25= ECt x ft
Where,
EC25: EC of the soil extract at 25oC, ECt: measured EC of the soil extract at t oC,
ft : the corrected value of EC at t oC (Table : 6.3)
Table-6.3. The corrected values of electrical conductivity rate under different temperatures
Temperature
(oC)
Correc
ted
value
Tempera
ture (oC)
Correc
ted
value
Temperat
ure (oC)
Correcte
d value
Temper
ature
(oC)
Corrected
value
3.00 1.709 20.00 1.112 25.00 1.000 30 0.907
4.00 1.66 20.20 1.107 25.20 0.996 30.2 0.904
5.00 1.613 20.40 1.102 25.40 0.992 30.4 0.901
6.00 1.569 20.60 1.097 25.60 0.988 30.6 0.897
7.00 1.528 20.80 1.092 25.80 0.983 30.8 0.894
8.00 1.488 21.00 1.087 26.00 0.979 31 0.890
9.00 1.448 21.20 1.082 26.20 0.975 31.2 0.887
10.00 1.411 21.40 1.078 26.40 0.971 31.4 0.884
11.00 1.375 21.60 1.073 26.60 0.967 31.6 0.880
12.00 1.341 21.80 1.068 26.80 0.964 31.8 0.877
13.00 1.309 22.00 1.064 27.00 0.960 32 0.873
14.00 1.277 22.20 1.06 27.20 0.956 32.2 0.870
15.00 1.247 22.40 1.055 27.40 0.953 32.4 0.867
16.00 1.218 22.60 1.051 27.60 0.950 32.6 0.864
17.00 1.189 22.80 1.047 27.80 0.947 32.8 0.861
18.00 1.163 23.00 1.043 28.00 0.943 33 0.858
18.20 1.157 23.20 1.038 28.20 0.940 34 0.843
18.40 1.152 23.40 1.034 28.40 0.936 35 0.829
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18.60 1.147 23.60 1.029 28.60 0.932 36 0.815
18.80 1.142 23.80 1.025 28.80 0.929 37 0.801
19.00 1.136 24.00 1.02 29.00 0.925 38 0.788
19.20 1.131 24.20 1.016 29.20 0.921 39 0.775
19.40 1.127 24.40 1.012 29.40 0.918 40 0.763
19.60 1.22 24.60 1.008 29.60 0.914 41 0.750
19.80 1.117 24.80 1.004 29.80 0.911
Source Bado S et al (2008)
In addition, when the temperature of the soil extract is 17-35, the electrical conductivity
of the oC soil extract increases about 2% for every 1in the differences of the soil extract
oC temperature and the standard temperature at (25oC). So the oC EC of the soil extract
at 25 can also be calculated according to the fallowing the formula when the soil extract
is 17-35 oC (Bado S et al (2008))
EC25 = ECt x [1 – (t – 25) x 2%]
Where: EC25: electrical conductivity of the soil extract at 25℃, ECt: measured electrical
conductivity of the soil extract at t oC, t: the temperature of the soil extract (oC).
Note: 1 Check accuracy of the EC meter using a 0.01 NKCI solution, which should
give a reading of 1.413 dS/m at 25°C.
Note: 2 Electrolytic conductivity (unlike metallic conductivity) increases at a rate of
approximately 1.9% per degree Centigrade increase in temperature. Therefore, EC
needs to be expressed at a reference temperature for purposes of comparison and
accurate salinity expression; 25°C is most commonly used in this regard. The best way
to correct for the temperature effect on conductivity is to maintain the temperature of
the sample and cell at 25° ± 0.5°C while EC is being measured.
3. The salinometer / Salinity sensors is mostly used in agricultural research, where
continuous monitoring of soil salinity in soil columns, lysimeters, and field experiments
is required
Procedure for soil pH and soil EC 1.5 estimation (1:5 soil and water ratio)
EC is a much more useful measurement than TDS, because it can be made
instantaneously and easily by irrigators or farm managers in field
1. Take a soil sample from the desired site/depth of soil surface and dried on a tray in a
cool oven/sundry
2. Take the 50 g of dried sieved soil (2mm sieve), into 500 ml beaker and
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3. Then add 250ml DDH2O, stirred and intermittently for 1hr
5. Allow the solution to settle for minute before testing
6. On the EC meter and adjust temperature at 25 oC then wait for 30 min
7. Place the EC meter electrode in the solution (not to be touching the bottom of soil)
and read display once it has stabilized it is test value EC (dS/m) ex = 0.366
8. Place pH meter electrode in the same soil solution (not to be touch the bottom of
soil) and read display once it has stabilized.
Note: ECe is the term used to indicate actual soil salinity, so then convert your salinity
EC meter readings to soil salinity (ECe), by the formula
Test value EC (dS/m) converted into ECe (dS/m) formula:
ECe=EC Constant
Formula, Constant
Estimation of soil saturation percentage:
Procedure:
1. Take 20 g of dried, sieved soil (2mm sieve) and add some water to make it into a
paste
Note: Paste should glossy and it should drop freely from a spatula with a small jerk
2. Record the weight of the paste
3. Keep the sample in hot air oven at 108 oC for overnight and record dry weight of
paste
Ex: Initial dry soil weight (g) = A= 19.58gm
Tin weight (g) = B= 34.9 gm
Wet soil wt (g) = A+B+C (weight of water is C) =65.28 gm
Final soil dry wt (g) =54.48
Formula, soil saturation % 100
=
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[1:5 (soil and water ratio) =50gm=250ml dilution likewise calculate the other soil
water ratios]
Constant=
EC Test value (dS/m) ex: 0.366
Therefore,
ECe (dS/m) =EC Constant = 0.366 4.5= 1.65
Or
8. Multiplying the value by the conversion factor based on the texture of the soil sample
(table-3)
Ex: EC 1.5 (soil: water ratio) test value (dS/m): 0.366
Suppose soil type: Medium and high clay, Multiplication factor= 7
Therefore= EC 1.5 test value Multiplication factor= 0.366 7=2.56
Actual medium and high clay soil salinity is = 2.56 (dS/m)
Note: In general studies on dynamic changes of water and salt contents in the soil, the
water/soil ratio of 5:1 is usually used, whereas the water/soil ratio of 1:1 is suitable for
the analysis of alkaline soil.
References:
1. Aboukila, E. F. and J. B. Norton. 2017. Estimation of saturated soil paste salinity
2. Souleymane, B., B. P. Forster, Abdelbagi M. A. Ghanim, Joanna Jankowicz-Cieslak, Günter
Berthold, Liu Luxiang. Protocol for measuring soil salinity. In: Protocols for Pre-Field
Screening of Mutants for Salt Tolerance in Rice, Wheat and Barley. Springer, Switzerland. pp.
13-20.
3. Shirokova, Y., I. Forkutsa and N. Sharafutdinova. 2000. Use of electrical conductivity instead
of soluble salts for soil salinity monitoring in Central Asia. Irrig. Drain. Syst. 14:199-205.
4. Slavich, P. G. and G. H. Petterson. 1993. Estimating the electrical-conductivity of saturated
paste extracts from 1:5 soil: water suspensions and texture. Aust. J. Soil Res. 31: 73–81.
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Chapter 7
Measurement of osmotic potential using vapour
pressure osmometer
Solute potential (Ψs), also called osmotic potential, osmotic potential created due to the
addition of salts or solutes. Solutes reduce the free energy of the water by diluting the
water. Its value is negative or maximum zero. The minus sign indicates that dissolved
solutes reduce the water potential of a solution relative to the reference state of the pure
water. The potential (Ψs) is negative in a plant cell and zero in distilled water. Typical
values for cell cytoplasm are –0.5 to –1.0 MPa. The osmotic potential in plants can be
measured by the following methods.
1. Vapour pressure method, Plasmolytic method, Cryoscopic method
Aim: to measure osmotic potential in plant by using vapour pressure osmometer
Materials required: vapour pressure osmometer, plant sample, sucrose solution
Principle: Properties of solution which are functions of mole fraction are called
colligative properties, which include boiling point, melting point, vapour pressure and
osmotic potential. Therefore, the measurement of total solution concentration, or
osmolality, can be indirectly assessed by comparing one of the colligative properties of
the solution with the corresponding cardinal property of the solvent. The Wescor
vapour pressure osmometer measures the osmotic potential by measuring vapour
pressure depression by thermocouple hygrometer.
Procedure:
1. Place the filter paper disc in the sample well and load 10 µl of 290 mmol/kg
standard provided along with the instrument. Set the display to read 290 using calibrate
290 controls.
2. Next, calibrate with 1000 mol/kg standard. Locate the instrument reading on the
left hand (READ) side of the calibration nomograph. Then using the calibrate 1000
control, adjust the display to read corresponding SET value found on the right-hand
side of the calibration Nomograph.
3. Repeat the step one to correct the offset. Calibration of the osmometer is now
complete. Note: use only fresh or verified osmolality standards for calibration.
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Measurement of osmotic potential of sample:
1. Keep the sample in 70 oC for the required duration and then take the sap out from
the tissue.
2. Open the sample chamber and withdraw sample slide.
3. Place the filter paper disc in sample holders.
4. Load about 10 µl of sap on the paper disc or directly use the leaf sample (cut the
leaf sample holder sample size)
5. Gently push the sample slide entirely into the instrument until it stops.
6. In this process, the indicator will go out, and an audible tone will sound when the
measurement is completed. The number displayed represents the osmolality of the
specimen.
7. Rotate the chamber looking level to the open (vertical) position, and then withdraw
the sample slide. Clean the sample holder.
Table: 7.1 Osmotic potential (Ψs) of sucrose solutions of various molar
concentrations at 20 of (m moles per 1 litre of the solution)
Sucrose
concentration
Osmotic potential Sucrose
concentration
Osmotic potential
(-bars) MPa
(-bars) MPa
0.00 0.00 0.00 0.80 -25.9 -2.59
0.10 2.7 -0.27 0.83 -27.2 -2.72
0.20 5.4 -0.54 0.90 -30.1 -3.01
0.30 8.2 -0.82 1.00 -35.1 -3.51
0.40 11.3 -1.13 1.05 -35.4 -3.54
0.50 14.5 -1.45 1.10 -40.3 -4.03
0.60 18.0 -1.80 1.20 -45.3 -4.53
0.70 21.8 -2.18 1.30 -52.3 -5.23
1bar [bar] =0.1 mega Pascal [MPa]
References:
R. A. B Oosterhuis D.M. 2005. Measurement of root and leaf osmotic potential using the vapor-
pressure osmometer. Environmental and Experimental Botany. 53:77–84.
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Chapter 8
Determination of Sodium and Potassium in plant
tissue
The concentrations of sodium (Na+) and potassium (K+) in plant tissues are important
determinants of salt stress tolerance. A high leaf Na+concentration inhibits
photosynthetic enzymes and carbohydrate metabolism, and induce oxidative damage
leading to cell death (Chaves et al., 2009; Wang et al., 2003). Leaf Na+concentrations
also correlate with pollen sterility (Pushpavalli et al., 2016). Plants have developed salt
tolerance mechanisms that reduce uptake and exclude Na+ from roots as well as
sequester Na+ into vacuoles to protect the cytosolic enzymes (Munns & Tester, 2008).
Inclusion mechanisms also control Na+ concentrations in the cytosol and maintain a
high cytosolic K+/Na+ ratio, indicating that the maintenance of a high cytosolic
K+/Na+ratio is important for plant growth under salt stress (Yamaguchi & Blumwald,
2005). The capacity of plant to maintain a high cytosolic K+/Na+ ratio is one of the key
determinants of plant salt tolerance (Serrano et al., 1999; Frans and Amtmann, 1999).
Under typical physiological conditions, plants contain about 100 mM K+ and maintain a
high K+/Na+ ratio in their cytosol cells, rarely tolerating cytosolic Na+ levels above 20
mM (Blumwald., 2000).
Potassium (K) and Sodium estimation:
Instruments: Flame photometer
Reagents for K+: 1N ammonium acetate: Dissolve 77.08 g of ammonium acetate in
500ml of distilled water and make the volume to 1L. Adjust the pH to 7.0 with glacial
acetic acid
Standard K+ solution for K+: Prepare 1000mg L-1 K+ solution by dissolving 1.908 g of
KCl salt per litre solution. Dilute suitable volumes of this solution to get 100 ml of
working standards containing 5, 10, 15, 20, 25 30 and 40 mg KCl L-1.
Reagents for Na+: Standard stock solution (100 mEq Na+ L-1): Dissolve 5.845 g of
NaCl in distilled water and make the volume to 1L.
Working standard solutions of Na+: Dilute,5,10,15,20,30,40, and 50 ml portion of stock
solution (containing 100 mEq Na+ L-1) to 100 ml in volumetric flask to get working
standard of 5,10,15,20,30,40, and 50me Na+ L-1 concentrations
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Digestion for K+ and Na+
One gram dried and powdered plant sample (20 mesh) was taken in a 50 ml digestion
tube and 10 ml di-acid mixture (4:1 v/v HNO3: HClO4) was added to it and was kept
overnight. It was then digested on a block digester till a colourless solution was
obtained. The volume of acid was reduced till the flask contained only moist residue.
The flask was cooled, and 25 ml of distilled water was added. The solution was filtered
into a 50 ml volumetric flask and diluted up to mark.
Estimation of potassium in leaf
Potassium content of leaf sample was determined by Flame Photometer method
(Jackson, 1973). The digested extract was used directly for flame photometer
determination of potassium. K+ content was calculated using the standard curve and
expressed as total K+ (%).
Total K+
% =
R × dilution factor
10000
R =Flame photometer reading
Estimation of sodium in leaf:
The sodium content of leaf sample was determined by Flame Photometer method
(Jackson, 1973). The digested extract was used directly for flame photometer
determination of potassium. K+ content was calculated using the standard curve and
expressed as total K+ (%).
Total Na+
% =
R × dilution factor
10000
R = Flame photometer reading
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References:
1. Blumwald, E. 2000. Sodium transport and salt tolerance in plants. Current opinion in cell
biology 12: 431-434.
2. Chaves, M.M., J. Flexas, C. Pinheiro. 2009. Photosynthesis under drought and salt stress:
regulation mechanisms from whole plant to cell. Ann. Bot. 103: 551-560.
3. Frans, J. M. M. and A. amtmann. 1999. K+ Nutrition and Na+ Toxicity: The Basis of Cellular
K+/Na+ Ratios. Annals of Botany 84: 123–133.
4. Jackson, M.L, (1973). Soil chemical analysis, prentice hall of India Pvt. Ltd, New Delhi, Pp
498
5. Munns, R. and Tester M. 2008. Mechanisms of salinity tolerance. Ann. Rev. Plant Biol. 59:
651–681.
6. Pushpavalli, R., J. Quealy, T.D .Colmer, N.C. Turner, K.H.M. Siddique, M.V. Rao and V.
Vadez. 2016. Salt stress delayed flowering and reduced reproductive success of chickpea (Cicer
arietinum L.), a response associated with Na+ accumulation in leaves. Journal of Agronomy and
Crop Science 202: 125–138.
7. Serrano, R., F.A.Culianz-Macia and V Moreno. 1999. Genetic engineering of salt and drought
tolerance with yeast regulatory genes. Sci Hortic 78:261–269.
8. Wang, D., S.M. King, T.A. Quill, L.K. Doolittle and D.L. Garbers. 2003. A new spermspecific
NaC/HC exchanger required for sperm motility and fertility. Nature Cell Biology 5:1117–1122.
9. Yamaguchi, T. and E. Blumwald. 2005. Developing salt-tolerant crop plants: challenges and
opportunities. Trends in Plant Science 10: 615-620.
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Chapter 9
Measurement of water content in soil and plant
tissue
A) Measurement of water content in soil
Quantification of available water in the soil is mandatory in the studies related to water
management, irrigation scheduling, development of drought-tolerant varieties and
studies concerned with stress physiology. Usually, the moisture content at field capacity
and the wilting point is -0.3 bar and -15.0 bar respectively. The soil moisture held
between field capacity and the permanent wilting point is called available water; called
available water should not be less than 50% for healthy plant growth. There are several
methods of determining the soil moisture content. Field capacity plant available water
and the permanent wilting point (Fig-9.1). These levels of soil water content can be
expressed in inches of water per foot of soil (Table-9.1) as well as in bars.
Following methods are commonly employed ones:
1. Gravimetric method
2. Time domain reflectometry
3. By Neutron probe
The energy regarding either soil matric potential or soil moisture potential can be
measured by the following method also
1. Resistance block
2. Tensiometer
3. Psychrometer
Field capacity (FC): the field capacity of the soil is described as the water content of
the downward flow of gravitational water has become very slow, and water content has
become relatively stable. This situation exists several days (1-3) after the soil has been
wetted by rain or irrigation.
Permanent wilting point (PWP): this is the soil water content at which plants remain
wilted unless water is added to the soil. Richards and Wadleigh (1952) found that the
soil water potential at wilting ranged from -10 to -20 bars, with the average at about -15
bars which are used as an approximation of soil water.
Plant-Available Water (PAW): The amount of water held in the soil that is available
to plants; the difference between field capacity and permanent wilting point. Since field
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capacity and PWP represent the upper and lower limit of soil water availability, this
range has considerable significance in determining the agricultural values of soils. The
following methods can measure the quantity or content of water in the soil. As a
general rule, plant available water is considered to be 50 percent of the water holding
capacity.
A). Estimation of soil moisture by gravimetric method
Aim: to determine the moisture content of the soil by gravimetric method
Materials: Screw augar, aluminium tins (moisture tins), oven, balance
Procedure:
1. Take Soil samples with the help of a screw type auger at 0-15, 15, 30 and 50 and
75cm depths from the control and stress plot
2. After determining the wet soil weight, the soil samples were dried in a hot air oven at
80 oC for 72hours, and the dry weight recorded. The soil moisture content expressed in
percent soil moisture availability.
Percept moisture content=
Advantages:
1. Cheap method
2. Accurate method than other methods
3. Used for calibration of other instruments
Disadvantages:
1. Destructive sampling
2. Labour requirement at each sampling
3. Not applicable to field conditions
4. More time is require
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Table- 9. 1. Soil water content parameters for different soil textures
Soil texture Field capacity
(in./ft)
Plant available
water (in./ft)
Permanent wilting
point (in./ft)
Sand 1.2 (0.10)* 0.7 (0.06) 0.5 (0.04)
Loamy sand 1.9 (0.16) 1.1 (0.09) 0.8 (0.07)
Sandy loam 2.5 (0.21) 1.4 (0.12) 1.1 (0.09)
Loam 3.2 (0.27) 1.8 (0.15) 1.4 (0.12)
Silt loam 3.6 (0.30) 1.8 (0.15) 1.8 (0.15)
Sandy clay loam 4.3 (0.36) 1.9 (0.16) 2.4 (0.20)
Sandy clay 3.8 (0.32) 1.7 (0.14) 2.2 (0.18)
Clay loam 3.5 (0.29) 1.3 (0.11) 2.2 (0.18)
Silty clay loam 3.4 (0.28) 1.6 (0.13) 1.8 (0.15)
Silty clay 4.8 (0.40) 2.4 (0.20) 2.4 (0.20)
clay 4.8 (0.40) 2.2 (0.18) 2.6 (0.22)
Numbers in parenthesis are volumetric water content expressed as foot of water per foot of soil.
(Source: Hanson 2000)
1. Fig: 9.1 Soil water parameters and classes of water (Source Juan et al E-618 08/12)
Determination of Relative Water Content (RWC) in leaf tissue
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The relative water content (RWC) is one of the reliable parameters to know the water
status in plants and it decreases gradually with increases in severity of drought stress.
Decline of RWC as response of stress were reported by several investigators under
different stress conditions (Barr and Weatherley, 1962). Further it has been suggested
that the plants to retain a high RWC during stress period are conspired as tolerant once
(Barr and Weatherley, 1962). The relative water content (RWC; or ‘relative turgidity)
of a leaf is a measurement of its hydration status (actual water content) relative to its
maximal water holding capacity at full turgidity. RWC provides a measurement of the
‘water deficit’ of the leaf and may indicate a degree of stress expressed under drought
and heat stress. A genotype with the ability to minimise stress by maintaining turgid
leaves in stressed environments will have physiological advantages (e.g., this allows
turgor dependent processes such as growth and stomatal activity, and to protect and
maintain the photosystem complex). The term was introduced by Weatherly in 1962, is
a modification of an older term, water saturation deficit (WSD). This term expresses the
leaf water content as a percentage of turgid water content and is calculated by the
following equation.
RWC (%) =
WSD and RWC are related; RWC = 100-WSD or RWC+WSD=100%. Barrs and
Weatherly (1962) have found 4 hours to be the optimum time for floating leaf discs or
whole leaves in water to determine turgid weight. Hewlett and Kramer (1963) found
entire leaves are more satisfactory than discs for some species.
Procedure:
1. Collect the leaf sample; usually fully expanded topmost leaf is preferable. Time
of sampling 11-12noon is desirable.
2. Immediately after sampling place the sample in a polythene bag and seal properly
to minimizing water loss from the leaf.
3. Samples should reach the lab as soon as possible and place these sample in picnic
cooler (temperature around10-15 °C)
4. Cute 5-10 cm length mid-leaf sections or 5-10 cm leaf discs of around 1.5cm in
diameter or take the several leaf lets depending upon the plant species (in smaller
composite leaves). Avoid the midribs and veins
5. Weight the samples and quickly to record the fresh weight.
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6. Hydrate the samples to full turgidity by floating on DDH2O or de-ionized water or
normal tap water in a closed petri-dish for 4hrs at normal room temperature and
light
7. Add 0.01% Tween 20 in case the leaf sample surface is waxy and not getting wet
by water.
8. After 4hrs take out the samples; remove the surface moisture quickly and lightly
with filter paper or blotting paper and immediately weigh to obtain fully turgid weight
9. Keep the sample in an hot air oven for 48 hours at 75-80 oC and record the oven
drying weight of the sample
Advantages:
1. Simple and needs no sophisticated equipment
Disadvantages:
1. Unfortunately, a given water deficit or RWC does not represent the same level of
water potential in leaves of different species or ages or from different environments.
Leaf and cell characteristics (thickness, elasticity) can cause changes in RWC although
water potential may be unaltered, particularly as the leaf matures
2. Time consuming
Note:
1. With good and careful work the method should normally result in about 2% to 3% of
RWC being a statistically significant difference between treatments.
2. Estimation of relative water content (RWC) in large size of population/genotypes is not
possible, so first short out the germplam by Plant Water Content [(PWC) whole plant]
or Leaf Water Content (only leaf):
Formula
PWC (g/g) = (FW-DW)/DW
Whereas FW-Fresh weight, DW-Dry weight
Observation sheet
S,No
Sample
ID
Fresh weight (g)
(A)
Turgid weight
(g) (B)
Dry weight (g)
RWC %= [(A-C/B-C)]x
100
1 Control 0.95 1 55 89
2 Stress 0.90 1 45 82
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References:
1. Barr, H.D. and Weatherley, P.E. 1962. A re-examination of the relative turgidity technique for
estimating water deficit in leaves. Aust. J. Biol. Sci. 15:413-428.
10. Juan, M. E., P. Dana, R. E. Steven, P. Xavier and P. Troy. Irrigation Monitoring with Soil
Water Sensors. E-618 08/12.
11. Hanson, B.,Orloff S., P. Douglas. 2000. California Agriculture, Volume 54, No.
3:38–42.
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Chapter 10
Imposition of moisture stress by gravimetric
approach
Objective: To generate drought/moisture stress induced plant tissues for assessing
various physiological and molecular assays.
Materials: Pots or battery containers, garden soil, sand and manure, mobile weighing
devices, seed/plant material, rain-out-shelter (ROS) or polythene sheet covered on net
house
Procedure:
1. Weigh the empty pots and record the accurate weights for each pot (A)
2. Fill the pots with soil: sand: farmyard manure mixture in the ratio of 2:1:1. or 2:1
ratio of soil: farmyard manure mixture. While filling the pots, makes sure that the soil
mixture is not compacted
3. Weigh the pot along with soil (B) and deduct the empty pot weight to obtain the dry
soil weight (C).
C= B-A
4. Carefully flood the pot with water (not splashing the soil from the pot). Allow it for
overnight to drain excess water and attain field capacity (FC).
5. Take the pot weight after saturation (D) and deduct empty pot weight (A) to get full
soil weight (E) at field capacity.
E=D-A
6. Subtract the dry soil weight from the full soil weight to get the amount of water
required to attain 100% FC (E-C).
7. Sow seeds of the crop under investigation in the pots. Maintain two to four seedlings
in each pot and water regularly to maintain moisture level at desired level of FC viz
100% FC, 75% FC, 60% FC etc., Ensure to protect the pots from rains or any other
source of water by keeping them under rain out shelter (ROS)
8. At four or six-leaf stage or at good foliage, impose drought stress by withholding
irrigation (please refer the diagrammatic representation given below). Weigh the pots at
regular intervals to monitor water status at different FCs, Replenish the water every
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time by adding the required amount of water depending on the loss of water occurred
previously and also based on the set FC value. The amount of water to be replenished to
maintain the required FC in the containers can be arrived at based on the formula given
below.
To maintain 100% FC, X ml of water is required. Therefore, to maintain Y%
FC, it is
Y% FC = Y% x X ml of water
100%
For example, the amount of water required to maintain 100% FC = 200ml
Therefore, the amount of water required to maintain 80% FC = 80 x 200ml = 160ml
100
The plants under different treatments are to be grown for a week or longer depending
on the crops. During this period, soil water potential (Mpa) and osmotic potential (Mpa)
are measured with Dew Point Potentiometer and Osmometer respectively. Similarly,
Relative water content (RWC %) is quantified according to Barrs and Weatherly (1962)
to assess the tissue water status and Electrical conductivity (EC %) is quantified to
assess the stress-induced cell damage.
Figure 10.1: Diagrammatic representation of gravimetric approach followed for
imposing precise levels of moisture stress/ drought.
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Note: Better terms are Available soil moisture (ASM) or Soil Moisture depletion (SMD),
instead of Field Capacity (FC)
Ex: In the literature, Available Soil Moisture (ASM) between 40 -50% or Soil Moisture
depletion (SMD) between 50-60%, 40-50% has been used as Field Capacity (FC) whereas
this should be treated as ASM or SMD instead of Field Capacity.
Figure.10.2. Diagrammatic representation of gravimetric approach followed for
imposing precise levels of drought (Berseem crop). ASM- Available Soil Moisture
References:
1. Allen, L.H., J.R.R. Valle, J.W. Mishoe, and J.W. Jones. 1994. Soybean leaf gas exchange
responses to carbon dioxide and water stress. Agron. J. 86: 625-636.
2. Barrs, HD and PE. Weatherley. 1962. A re-examination of the relative turgidity technique for
estimating water deficits in leaves. Aust. J. Biol. Sci. 24: 519-570.
3. Nissanka, S.P., M.A. Dixon and M. Tollenaar. 1997. Canopy gas exchange response to moisture
stress in old and new maize hybrid. Crop Sci. 37:172-181.
4. Pennypacker, B.W., K.T. Leath, W.L. Stout, and R.R. Hill. 1990. Technique for simulating field
drought stress in the greenhouse. Agron. J. 82:951-957.
5. Ray, JD., and Sinclair, TR.,1998, The effect of pot size on growth and transpiration of maize and
soybean during water deficit stress, J. Exp. Bot. 49:1381-1386.
6. Turner, N.C., 1997, further progress in crop water relations, Adv. Agron. 58:29-338.
Control (80% ASM) T1 (40% ASM) T2 (20% ASM)
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Chapter 11
Two tier screening of germplasm under a natural
condition or Irrigation stop approach for stage-
specific drought tolerance
Objective
To evaluate and identify germplasms/breeding elite lines for drought tolerance/generate
plant tissues exposed to drought stress at whole plant level for various physiological,
molecular assays.
Principle
Rain-free conditions permit to impose variable stress treatments to evaluate the genetic
variability of crop/forage plants to drought tolerance. Screening in rain-free conditions
is reliable as it allows variable stress imposition with the definite advantage of avoiding
genotype x season interactions which can affect genotype response to stress. Rain-free
screening condition has the benefits of scale, reliability and economy but the choice of
rain-free location is crucial for screening.
Location/Site:
Drought stress tolerance of genotypes can be efficiently screened in field conditions
during rain-free periods provided the selected site fulfils the desirable meteorological
conditions. It includes consideration of the rainfall distribution, temperature regimes,
day length, wind velocity and relative humidity. Further, these parameters must meet
the screening criteria as identified below;
i. Rainfall distribution – Rain free period of 120-150 days depending on the target
crop species.
ii. Temperature – mean maximum temperature must not exceed 35-38 oC. Mean
minimum temperature should be more than 5oC above the base temperature of the crop
species.
iii. Relative humidity (RH) – Mean maximum and minimum should not be < 60% and
< 30% respectively
iv. Day length (photoperiod) – Preferably it should be in the range of 11 to 13 hours.
v. Light intensity – Cloud induced reduction in light intensity should not be > 30%.
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vi. Soil characteristics – Soil texture, soil depth and water holding capacity amenable to
impose variable stress treatments. It may vary depending on plant species
Experimental design and layout
The experimental design for screening in rain-free conditions has two methods; i)
Augmented randomized block design ii) Randomized field block method
Experimental design requires that each block is randomized with adequate replicates
(minimum 3) to allow effective statistical data analysis in RBD. The stress treatments
indicated in the layout are indicative and vary depending on the objective of the
screening and genotypes. In Augmented randomized block design treatment blocks
must be separated by 1.5 meters and 3 to 4 meters long and 2 to 3 rows for each
line/genotypes planting/sowing (spacing depend upon the crop). Introduction of
background checks in each of the block after ten genotypes/lines or ever 5 to 10 metres
will help account for the heterogeneity and soil parameters (Fig-11. 1).
The non-stress and stress treatment blocks must be separated by 5 meters to overcome
the seepage of moisture (Fig-11.2).
1. Crop raised and irrigated in the respective crop seasons, i.e. kharif, rabi, summer
2. Irrigation can be scheduled when soil water content drops below 70 percent of the
total available soil moisture for non-stress treatment
3. Soil moisture will be recorded 2 to 3 days after irrigation by gravimetric method;
subsequently soil moisture content (gravimetric method) will be recorded for getting
the desired stress (at 5-10 days interval) during crop growth stages at seedling,
vegetative and reproductive stages
a) Under natural condition record the soil moisture, when the available soil moisture
(%) 70-80%, 50-60% and at 40-50 % for control (non-stress), moderate and severe
stress respectively
Or
b) Stress was imposed by irrigation stop approach in control (non-stress) and stress
treatment blocks when the soil moisture content depleted by 20-30 % in control blocks
(non-stress) and 40-50% and 50-60 % in moderate and severe stress blocks respectively
Formula for available water
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Available soil moisture (%) = Soil Moisture in (SM %) or Field capacity- Soil moisture
in PWP (%)
SM: Soil Moisture (%)
FC: Field capacity, PWP: Permanent Wilting Point.
i) Non stress (T1)= ASM
SM-PWP=20-8=12%
=75% (70-80%=AVG=75) of the 12% is 9%
ii) Moderate stress (T2)=ASM
SM -PWP=20-8=12%
=55% (50-60%=AVG=55) of the 12% is 6.6%
iii) Severe stress(T3) =ASM
SM -PWP=20-8=12%
=55% (40-50%=AVG=45) of the 12% is 5.4%
4. Shortlist the genotypes/line from the large size population/lines based on GFY and
DMY data (Augmented design Fig-11.1)
6. Basic physiological parameters studied in selected genotypes (RBD Fig-11.2)
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Expt-1 Block-1 Block-2 Block-3
Block-1 Block-2 Block-3
Block-1 Block-2 Block-3
Geno
type
sGe
noty
pes
Geno
type
s
Seedling Stage
1.5m 1.5m
1.5m 1.5m
Stress ([50-60% or 40-50% ASM)
Vegitatve Stage
Stress ([50-60% or 40-50% ASM)
Reproductive/pre mature Stage
Stress ([50-60% or 40-50% ASM)
1.5m 1.5m
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Expt-2 Block-1 Block-2 Block-3 Block-1 Block-2 Block-3 Block-1 Block-2 Block-3
Block-1 Block-2 Block-3 Block-1 Block-2 Block-3 Block-1 Block-2 Block-3
Block-1 Block-2 Block-3 Block-1 Block-2 Block-3 Block-1 Block-2 Block-3
1.5m 5m
Ge
no
typ
es
1.5m 1.5m
Stress Block-I
Ge
no
typ
es
1.5m 1.5m 5m
Ge
no
typ
es
1.5m
Non Stress Block Stress Block-I Stress Block-II
Control (70-80 % ASM) Moderate stress (50-60% ASM) Sever stress (40-50% ASM)
1.5m 5m
Ge
no
typ
es
1.5m 1.5m
Reproductive/pre mature Stage
Ge
no
typ
es
1.5m 1.5m 5m
Ge
no
typ
es
1.5m
Non Stress Block Stress Block-I Stress Block-II
Control (70-80 % ASM) Moderate stress (50-60% ASM) Sever stress (40-50% ASM)
1.5m 5m
Ge
no
typ
es
1.5m 1.5m
Vegetative stage
Ge
no
typ
es
1.5m 1.5m 5m
Ge
no
typ
es
1.5m
Seedling Stage
Non Stress Block Stress Block-II
Control (70-80 % ASM) Moderate stress (50-60% ASM) Sever stress (40-50% ASM)
Figure -11.1: Layout of plot design with some genotypes and augmented randomization
of blocks. In expt-1 augmented with check line and in expt-2 augmented with control
for comparison. Note : ASM-Available soil moisture
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R1 R2 R3
5m 5m 5m
5m 5m 5m
R1 R2 R3
5m 5m 5m
5m 5m 5m
R1 R2 R3
5m 5m 5m
5m 5m 5m
T3T2
T2
Gen
oty
pes
G
eno
typ
es
Gen
oty
pes
T1T2
T3T1
T1T3
Reproductive/pre mature Stage
Vegetatve stage
Seedling stage
5m 5m
5m 5m
5m 5m
Figure-11.2: Plots (size 4 x 3 m2 area) in each block must have minimum three
replicates. Experimental design requires that each block is randomized with adequate
replicates (minimum 3) to allow effective statistical data analysis [(T1-Control (70-80
% ASM), T2-Moderate stress (50-60% ASM) and T3-Sever stress (40-50% ASM)]
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References:
1. Blum, A. and A. Ebercon. 1981. Cell membrane stability as a measure of drought and heat
tolerance in wheat. Crop. Sci. 21: 43-47.
2. Fisher, R.A. and R. Maurer. 1978. Drought resistance in spring wheat cultivars. I.Grain yield
responses in spring wheat. Australian J. Agric. Sci. 29: 892-912.
3. Fischer, K.S. and G. Wood. 1981. Breeding and selection for drought tolerance in tropical
maize. In: Proc. Symposium on Principles and Methods in Crop Improvement for Drought
Resistance with Emphasis on Rice, (23-25th May 1981) IRRI, Philippines.
4. Kramer, P. J. 1983. Water deficits and plant growth. Water relation of Plants 24: 342-389.
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Chapter 12
Determination of Water Use Efficiency (WUE)
Water plays a crucial role in the life of a plant. Plants use water in vast amounts, but
only a small part of that remains in the plant. Up to 97% of water taken up by plants is
lost to the atmosphere, where the remaining 2% is used for volume increase or cell
expansion, and 1% goes to metabolic processes, predominantly photosynthesis. The
uptake of CO2 is coupled to the loss of water, because the driving gradient for water
loss from leaves is much larger than that for CO2 uptake, as many as 400 water
molecules are lost for every CO2 molecule gained. Water-use efficiency (WUE- also
called transpiration efficiency (TE)) is broadly defined as the ratio of water used by the
plant for metabolism to the water lost through transpiration or amount of water
transpired per unit biomass produced by the plant. Physiologically or at a single leaf
level, WUE is defined as amount of CO2 fixed (assimilation rate) to the amount of
water transpired (transpiration rate) (WUE=A/E).
The physiological yield model proposed by Passioura (1986), says that, Yield = T x TE
x HI, where T is transpiration, TE is transpiration efficiency and HI is harvest index,
which clearly implicates the physiological basis that determine yield. TE is defined as
the ratio of total biomass produced over a period of time to the total transpiration during
the same period, expressed as g kg-1. TE is an important physiological trait for drought
tolerance and genotypic variation in TE was identified by Briggs and Shantz as early as
1914. In fact with two-fold variability in TE among C3 and C4 crop species, a low
genotype x environment interaction and high broad-sense heritability for TE renders
this trait a potential one for crop improvement programs.
With diminishing water resources for agriculture, it is imperative to grow the crops
with less water. Moreover, climate change predictions show clear increases in
temperatures (and concomitant increase in potential evapotranspiration) and more
frequent episodes of climatic anomalies, such as droughts and heat waves. All of these
climate change phenomena are prevalent in most semiarid areas. Consequently, the
optimization of water use for crops by improvement of WUE is a challenge for securing
agricultural sustainability in semiarid areas. In response to this challenge, a large
volume of applied and fundamental research has been focused on optimization of crop
water use. However, progress in breeding for improved TE has been extremely limited
mainly due to the lack of a suitable screening technique to determine the genetic
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variability for WUE among germplasm lines as well as in segregating populations and
inbreed lines. WUE can be measured following various approaches which include
Carbon Isotopic discrimination approach, Gravimetric method, Minilysimeter/lysimeter
based approach and based on SLA and SCMR readings. Each of these methods have
their own inherent disadvantages. However, although the gravimetric approach is
cumbersome, time consuming and labour intensive, it is still considered to be an
efficient and effective and most accurate method of determining water use efficiency of
crop plants.
Gravimetric determination of Water Use Efficiency (WUE)
The gravimetric determination in transpiration efficiency (TE) and the associated
physiological traits involve the frequent weighing of the pots to determine the daily
evapotranspiration
Objective
To estimate WUE by gravimetric approach (Gravimetric approach is the most accurate
and reliable approach to determine WUE)
Materials: Pots, field soil, sand and manure, weighing balance, seed/plant material,
rain-out-shelter (ROS) or polythene sheet covered on net house
Procedure:
1. Take the empty plastic pots and fill the pots with dry soil (Soil: sand: farmyard
manure mixture in the ratio of 2:1:1).while filling the pots, make sure that the soil
mixture is not compacted and close the hole with M-Seal
2. Carefully flood the pot with water (not splashing the soil from the pot). Allow it for
overnight to drain excess water and attain field capacity (FC).
3. Sow the seeds of the crop under investigation in the pots. Maintain two to four
seedlings in each pot and water regularly to maintain moisture level at desired level of
FC viz 100% FC,75% FC, 60% FC.....etc.,
4. Raise the crop unto 30 to 55 DAS.
5. On a specific day, designated as the START of the experiment, all the containers
should be
saturated with water and allowed to drain overnight to bring the soil to near 100% FC. On
the same day, the initial biomass of the plant has to be determined by culling out plants
from a couple of pots.
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Note: For each genotype, plants are to be raised at least in 8-10 pots of which plants from
2-3 pots should be up rooted at the beginning of the experiment to determine the initial
biomass of that particular genotype. The remaining plants from 5-6 pots should be
harvested at the end of gravimetry experiment to determine the final biomass. The
difference between final biomass and initial biomass is actually the biomass accrued
during the experimental period which we it as delta biomass.
6. Spread small plastic pieces or polythene sheet or small foam sheet pieces on the soil
surface as mulch to minimize direct soil evaporation
7. High-density polythene feeder pipe, of 50 cm length, 50 mm inner diameter, with
perforations of 7.5 cm intervals and one end sealed, can be buried to a depth of 30cm.
8. Ensure to protect the pots from rains or any other source of water by keeping them
under rain out shelter (ROS)
9. The weight of individual container with soil at 100% field capacity or desired level of
FC viz 75% FC, 60% FC.....etc., or read as 75%, 60% available soil moisture…, etc
plastic pieces and plant must be recorded on the day of starting the experiment.
10. The required amount of water to reach the desired level of FC can be added manually
through the feeder pipe after weighing. This will ensures water availability at the root
zone.
11.The containers should be weighed along with feeder pipe once daily between 9 to 11
am to record the amount of transpirational losses.
The difference in the weight between subsequent weighing is replaced to bring the soil
back to 100% FC or desired FC levels viz 75% FC, 60% FC or read as 75%, 60%
available soil moisture etc.,
The detailed procedure adopted is as follows:
A =B+ C + D
Where, A is the container weight at 100 % FC or desired FC levels viz 75% FC,
60% FC.....etc
B is dry soil weight
C is the weight of plastic pieces spread on the soil surface and feeder tubes,
D is the quantity of water present at 100 % FC or at desired FC levels viz 75% FC,
60% FC.....etc
Therefore,
D= A – (B + C)
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The amount of water (E) to be added every day/ every time should be matched with
amount of water lost over the last observations which can be determined by weighing the
pots every day and noting down how much water is has lost in comparison to the weight
of previous day (basically at the set FC level)
For example, Pot A has a total weight of 20 kgs at 100% FC which includes, empty pot,
soil, water, mulch and plant. If this plant weighs 19.5 kgs next day, it infers that the plant
has lost 0.5 kg of water. Therefore, to bring the pot again to 100% FC, 0.5 kg of water
has to be replenished.
The amount of water added should be noted down and likewise, on a daily basis how
much water was added should be noted down which will be called as cumulative water
added (CWA).
Though necessary care is taken to reduce the direct surface evaporation losses, some
amount of water would still be lost from the soil surface. To give a correction to this, a
set of empty containers without plants (with the same amount of soil and plastic pieces as
that of planted pots) should be maintained and weighed to measure daily evaporation
loss. The total water evaporated during the experimental period known as cumulative
evaporative loss (CEL) has to be summed up.
Therefore to arrive at cumulative water transpired (CWT), CEL has to be subtracted
from CWA.
With this WUE is calculated by taking into account Delta biomass and CWT
WUE= Delta biomass/ CWT
Note: Better terms are Available soil moisture (ASM) or Soil Moisture depletion (SMD),
instead of Field Capacity (FC)
Ex: In the literature, Available Soil Moisture (ASM) between 40 -50% or Soil Moisture
depletion (SMD) between 50-60%, 40-50% has been used as Field Capacity (FC) whereas
this should be treated as ASM or SMD instead of Field Capacity.
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Fig: 10.2. Diagrammatic sequence/ events to determine Water Use Efficiency by gravimetric
approach (Berseem crop).
References:
1. Jongrungklang, T.B., N. Vorasoot, S. Jogloy, K.J. Boot, G. Hoogenboom and A. Patanothan.
2011. Rooting traits of peanut traits with different yield responses to pre-flowering drought
tolerance. Field crops research. 120(2): 262-270.
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Chapter 13
Photosynthetic pigments analysis in plants
Methods: Two types
1. Non- Destructive method 2. Destructive method (Acetone and DMSO methods)
Non- destructive method:
Estimation of Chlorophyll by SPAD or Chlorophyll meter
The SPAD (Soil Plant Analysis Development) chlorophyll meter is a simple, rapid, and
non-destructive method for evaluation of chlorophyll contents in leaves and can be used
in the field and laboratory. Chlorophyll meters are widely used to guide nitrogen (N)
management by monitoring leaf N status in agricultural systems.These instruments
determine the light attenuation at 430 nm and 750 nm. SPAD it is useful for rapid
screening for crop improvement.
Procedure:
1. The SPAD readings are more stable under the standard light between 10 AM to 4
PM.
2. Switch on the instrument and let it warm up for about 10-20 min.
3. Calibrate the device for accuracy checking using a particular disc provided with
the apparatus.
4. As soon as the beep sound is over, put (Normally the 2nd or 3rd) wholly expanded
leaf from apex is chosen and clamped after avoiding the mid-rib portion into the sensor-
hold of the SPAD meter.
5. A gentle stroke should be given to record the SCMR (SPAD chlorophyll meter
reading)/ SPAD value, and the average of several measurements can be considered.
6. Close when sound is heard
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Figure- 13.1. Recording the SCMR using the SPAD meter
Destructive method:
Estimation of carotenoid and chlorophyll content in leaf tissue: (Acetone method)
Materials required:
1. Falcon Tubes (15ml)
2. Acetone (80%)
3. Microbalance
4. Scissor
5. Spectrophotometer
6. Plant Material: leaf
Procedure:
1. Take the 100 mg leaf sample into Falcon Tubes (15ml) (ovoid the midribs)
2. Add the 10 ml acetone (80%) then close Falcon Tubes with cap then keep in dark for
overnight
3. Take the 1ml sample and add the 2ml acetone (80%) (1:2 ratio)
4. Read the absorbance of the extract at 645, and 663 and 470 nm using acetone (80%)
blank.
5. The amount of chlorophyll ‘a’ and ‘b’ are determined using the formula given by
Arnon (1949)
Chl ‘a’= ((12.7 A663)-(2.69 A645) ))
Chl ‘b’=((22.9 A645)-(4.68 A643) ))
Total chlorophyll (a+b) = ((20.2 (A645) +8.02(A 663) ))
Where, A = Absorbance
V=Final volume of 80% acetone (in ml)
W= Weight of plant tissue (in grams)
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The Chlorophyll content is expressed as mg/g fw
Carotenoid content use the formula by Method by Lichtenthaler (1987)
Total carotenoids 1000 A470- (1.82 Chl a)-(85.02 Chl b
198
Where Chl a and Ch b are
Chl ‘a’ (µg/ml) (12.25 A663.2)- (2.79 A646.3) and
Chl ‘b’ (µg/ml) (21.50 A646.3) - (5.10 A663.2)
µg g-1 fresh weight (µg/ml final volume)/leaf weight (g)
DMSO method:
1. Take the 100 mg of freshly cut fine pieces of leaf sample is placed in the into test
tubes to which 20 ml DMSO is added (avoid the midribs)
2. The tubes are covered with aluminium foil and kept in an oven or water both at 65 oC
for 4-5 hrs
3. Cool the sample at room temperature, record absorbance at 645, 663nm using DMSO
as a blank
Calculate chlorophyll ‘a’ and ‘b’ using Arnon (1949) formulas
References:
1. Arnon, D.I. (1949). Copper enzymes in isolated chloroplast, polyphenol oxidase in Beta
vulgaris.L. Plant physiology. 24:1-15
2. Hiscox, J.D.and Israelstam,G.F.(1979).A method for extraxtion of chlorophyll from leaf tissue
without maceration.Can.J.Bot.57:1332-1334.
3. Lichtenthaler, H.K., 1987. Chlorophylls and carotenoids, the pigments of photosynthetic
biomembranes. In: Douce, R., Packer, L. (ed.), Methods in Enzymology 350–382, Academic Press
Inc., New York.
4. Peng S, R.C. Laza, F.C. Garcia and K.G. Cassman.1995b. Chlorophyll meter estimates leaf
area-based N concentration of rice. Commun Soil Sci Plant Anal 26:927–935.
5. Peng, S., F.C. Garcia, R.C, Laza and K.G. Cassman. 1993. Adjustment for specific leaf weight
improves chlorophyll meter’s estimation of rice leaf nitrogen concentration. Agron J 85:987–990.
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Chapter 14
Estimation of chlorophyll stability index and
carotenoid stability index in leaf tissue
Carotenoid and chlorophyll pigment content provides valuable information about the
physiological status of plants. Chlorophylls a and b are essential pigments to absorb the
energy of light and convert it to store chemical energy. Carotenoids have several
physiological functions associated with photosynthesis, including a structural role in the
organisation of photosynthetic membranes, participation in light harvesting and energy
transfer, as well as quenching of Ca + b excited state and photoprotection. Carotenoid
content is known to be correlated with plant stress and photosynthetic capacity. Green
plant pigments are thermosensitive, and degradation occurs when they are subjected to
a higher temperature. This method is based on pigment changes induced by heating.
The chlorophyll destruction commences rapidly at a critical temperature of 55 oC to 56
oC. Thus, chlorophyll stability is a function of temperature. This base has been formerly
used in pine needles immersed in water and heated gradually in a temperature regulated
water bath at 58 oC. Thus, chlorophyll stability is a function of temperature. This
property of chlorophyll stability was found to correlate well with drought resistance.
Aim: To estimate carotenoid content and chlorophyll stability index in leaf sample
Materials required:
7. Glass test tube of 2.5 cm in diameter
8. Acetone (80%)
9. Balance
10. Water bath with thermostatic control
11. Spectrophotometer
Procedure:
1. Two clean glass tubes are taken and add 100 mg of representative leaf sample is
placed in them with 10 ml of distilled water.
2. One tube is then subjected to heat on water bath at 56 oC ± 1 oC for precisely 30
minutes and discard water
3. Add the 10 ml acetone (80%) in both the sample and keep in dark for overnight
4. Take the 1ml sample and add the 2ml acetone (80%) (1:2 ratio)
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5. Read the absorbance of the extract at 645, and 663 and 470 nm using acetone
(80%) s blank.
Formula:
Total chlorophyll content = 20.2 (A 645) + 8.02 (A 663) x V/ (1000 x W x a) (mg/g fr.
Wt.)
Carotenoid (mg/g): 46.95 (A 470- 0.268 x Chl a + b)
Where, A = Absorbance
a= path length of light (3 cm)
V= final volume made (ml)
W= fresh weight of sample (g)
Calculations: CSI = Cs/Cc X 100
Where, CSI = chlorophyll stability index
Cs = Chlorophyll content of stressed plant (mg/g)
Cc = Chlorophyll content of control plant mg/g)
Calculations: CSI = Cs/Cc X 100
Where, CSI* =Carotenoid stability index
Cs = Carotenoid content of stressed plant (mg/g)
Cc = Carotenoid content of control plant mg/g)
* Carotenoid OR
The chlorophyll stability index (CSI) was determined according to Sairam et al. (1997)
and calculated as follows:
CSI = (total chlorophyll under stress/total chlorophyll under control) × 100
CSI* = (total carotenoid under stress/total carotenoid under control) × 100
*= Carotenoid
Note: Here control and treatment plot is needed
High CSI and CSI* corresponded with more drought tolerance. Thus, CSI, CSI* is
directly related with drought tolerance.
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Note: Here take the leaf sample 100 to 500 mg or more depend upon the degree of
stress
Reference:
1. Hiscox, JD and Isrealstam GF. 1979. A method of extraction of chlorophyll from leaf tissue
without maceration. Can J. Bot. 57: 1332-1334.
2. Sairam, R.K., P.S. Deshmukh and D.S. Shukla. 1997. Increased antioxidant enzyme activity in
response to drought and temperature stress related with stress tolerance in wheat genotypes,
Abstract: National Seminar (ISSP), IARI, New Delhi.pp. 69.
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Chapter 15
Cell Membrane Stability Index
A significant impact of plant environmental stress is cellular membrane modification,
which results in its total dysfunction of the plant. The cellular membrane dysfunction
due to stress is well studied. The dysfunction of membranes is expressed as increased
permeability and leakage of ions, the efflux of electrolytes is used to calculate this
Index. Hence cellular electrolyte leakage is used to screen for stress resistance. The
method was initially developed by the late C.Y. Sullivan (University of Nebraska) in
the late 1960's for assessing sorghum and maize heat tolerance. Variations of this
methods were developed for cold and desiccation (drought) tolerance. This assay is
found in many reports to be associated across diverse genetic materials with yield under
stress.
Aim: To estimate the salinity, heat and drought stress tolerance of plant tissue by
Sairam
Materials required: leaf sample, beakers, test tubes, water bath, and EC meter
Leaf MSI was determined according to the method of Premchandra et al. (1990), as
modified by Sairam (1994). Leaf discs (100 mg) were thoroughly washed in running
tap water followed by washing with double distilled water after that the discs were
heated in 10 mL of double distilled water at 40 °C for 30 min. Then EC (C1) was
recorded by EC meter. Subsequently, the same samples were placed in a boiling water
bath (100 °C) for 10 min, and their EC was also recorded (C2) in a conductivity meter
MSI= [1- (C1/C2)] x100
High CMSI corresponded with more stress tolerance
Reference:
1. Sairam, R.K., P.S. Deshmukh and D.S. Shukla. 1997. Increased antioxidant enzyme activity in
response to drought and temperature stress related with stress tolerance in wheat genotypes,
Abstract: National Seminar (ISSP), IARI, New Delhi. p. 69
2. Premachandra, G.S., H. Saneoka and Ogata. 1990. Cell membrane stability an indicator of
drought tolerance as affected by applied N in soybean. J. Agric. Soc. Camp 115: 63-66.
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Chapter 16
Estimation of Abscisic acid content in leaf and
root
Abscisic acid (ABA) is a plant stress hormone that is observed to accumulate under
drought stress and mediates many stress responses, like heavy metal stress, drought,
thermal or heat stress, high level of salinity, low temperature, and radiation stress.
Abscisic acid regulates drought stress responses by mediating stomatal closure, thereby
reducing transpiration water loss.
Aim: To determine Abscisic acid content in leaf and root by Titration Method
Materials required: Centrifuge
Reagents: 3% dichlorophenol indophenol
Principle:
2,6 dichlorophenol indophenol (2,6-DCPIP) is a blue coloured dye but turns pink when
reduced by ascorbic acid. Oxalic acid or metaphosphoric acid may be used titrating
medium because it increases the stability of ascorbic acid in the medium
Procedure:
1. Take 0.5 to 5 g of plant sample
2. Add 10-20ml of 3% metaphosphoric acid
3. Centrifuge at 1000xg for 10min
4. Take the supernatant and make the volume upto 100ml
5. Take the 5ml supernatant and add 10 ml of 3% metaphosphoric acid
6. Titrate it against standard 2, 6 dichlorophenol indophenol solution of concentration
0.5mg/ml until the pink colour develops completely
7. Note down the difference between final and initial volume of the dye (V2)
8. Take 5ml of the working standard of ascorbic acid (0.1mg/ml concentration) in
beaker add 10ml of 3% metaphosphoric acid and titrate it against the dye.
9. Record the final volume of dye at the endpoint as mentioned above (V1)
The amount of ascorbic acid in mg/100 g of the sample can be calculated as follows:
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Where,
A = 0.5 mg (the concentration of working standard of ascorbic acid=0.5mg in 5ml
taken for titration.
B = 5 ml (volume of sample taken for titration)
V1 = Volume of dye in case of titration with standard solution
V2 = volume of dye in case of titration with the sample solution.
References:
1. Albrecht, J.A. 1993. Ascorbic acid retention in lettuce. J. Food quality 16: 311-316.
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Chapter 17
Estimation of proline content in plant tissue
Proline a compatible solute and an amino acid, is involved in osmotic adjustment (OA)
and protection of cells during dehydration (Zhang et al., 2009). Cell turgor is
maintained due to Osmotic Adjustments which allow cell enlargement and plant growth
during water stress. It also enables stomata to remain partially open and
CO2 assimilation to continue at water potentials that would be otherwise inhibitory for
CO2 assimilation. (Alves and stter, 2004). Proline can scavenge free radicals and reduce
damage due to free radicles during drought stress. Growing body of evidence indicated
that proline content increases during drought stress and proline accumulation is
associated with improvement in drought tolerance in plants (Seki et al., 2007; Zhang et
al., 2009).
Aim: To determine the free proline content of plant tissue following Bates et al., (1973)
method.
Materials required: test tubes, pestle and mortar, pipettes, funnels, Whatman no. 1
filter paper, water bath, heater, ice bath, separating funnel
Reagents:
3% aqueous sulphosalicylic acid, Glacial acetic acid, Orthophosphoric acid (6M),
Toluene, Proline
Acid ninhydrin, warm 1.25 g ninhydrin in 30 ml glacial acetic acid and 20 ml 6M
phosphoric acid with agitation until dissolved. Store at 4 oC and use within 24 hours.
Principle:
During selective extraction with aqueous sulphosalicylic acid, proline is precipitated as
a complex. Other interfering materials are removed by absorption to the protein
Sulphosalicylic acid complex. The extracted protein is made with ninhydrin in acidic
conditions (pH = 1.0) to form the chromophore (red colour) to read at 520 nm.
Procedure:
1. Extract 0.5 g of plant material fresh by homogenising in 3-5 ml of 3% aqueous
solution sulphosalicylic acid
2. Filter the homogenate through Whatman no. 2 filter paper and make up the volume
to 10 ml.
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3. Take 2 ml of filtrate in a test tube and add 2 ml of glacial acetic acid and 2 ml acid-
ninhydrin
4. Heat the test tube in boiling water bath for one hr.
5. Terminate the reaction by placing the tube in ice bath
6. After attaining room temperature transfer the contents to a separate funnel
7. Add 4 ml toluene to the reaction mixture and stir well for 22-30 sec
8. Take out the lower coloured layer and discard the upper toluene layer
9. Measure the red colour intensity at 520 nm
10. Simultaneously run a blank with 2 ml distilled water instead aliquot.
Calculations:
Express the proline content on fresh-weight basis as follows:
"µmoles per gram tissue = [(µg proline/ml) x ml toluene)/115.5 µg/µmole] / [(g
sample)/5]
Or
"µmoles per gram tissue = [(µg proline/ml) x ml toluene x ml salicylic acid]/(115.5 µg
µmole x sample (g))
Notes:
1. The colour intensity is stable for at least one hr.
2. The relationship between the amino acid concentration and absorbance is linear in
the range of 0.02 to 0.1 µ M per ml of proline.
References:
1. Alves, A.A.G. and T.L. Setter. 2004. Abscisic acid accumulation and osmotic adjustment in
cassava under water deficit. Environ. Exp. Bot. 51: 259–279.
2. Bates, LS., R.P. Waldren and I.D. Tear. 1973. Rapid determination of free proline water stress
studies. Plant and Soil. 39: 205-208.
3. Seki, M., T. Umezawa, K. Urano, and K. Shinozaki. 2007. Regulatory metabolic networks in
drought stress responses. Curr. Opin. Plant Biol. 10: 296–302.
4. Zhang, X., E.H. Ervin, G.K. Evanylo and K.C. Haering. 2009. Impact of biosolids on hormone
metabolism in drought-stressed tall fescue. Crop Sci. 49:1893–1901.
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Chapter 18
Photosynthesis
A. Effect of water stress on photosynthesis and associated leaf characters in crop
Plants
Photosynthesis is fundamental parameter in plant physiological studies. Photosynthesis
is the process in which the green plants’ chlorophyll pigments produce organic matter
by utilising CO2 and water. There are several methods of measuring CO2 fixation or
exchange in the plant but, the modern techniques of determining CO2 fixation using
infrared gas analysis (IRGA) of CO2 is widely used to the precision of detecting
minimal changes in CO2 concentrations. This method is sensitive for CO2 uptake by
tiny leaves or even leaf segments.
Aim: To measure the effect of water stress on the rate of photosynthesis, conductance,
transpiration and leaf temperature
Materials required: portable photosynthesis system with accessories
Principle:
Heteroatomic gas molecules like CO2, H2O, NH3, N2, NO absorbs radiation at a specific
wavelength. The major absorption band of CO2 is at 425 nm with secondary peaks at
266, 277 and 1499 nm. The rate of CO2 uptake is measured by enclosing leaf in an
airtight leaf chamber, passing air over the leaf for a specific period and measuring the
changes in CO2 concentration with an infrared gas analyser (IRGA). The IRGA will
have an infra-red source which emits IR rays continuously and this IR being absorbed
by the CO2 and IRGA measures the difference in the CO2 concentration of the air
before and after it passes through the leaf chamber. The change in the amplitude of
vibration of the membrane, produced by the CO2 concentration difference between the
analysis and reference tubes of IRGA is inversely proportional to the voltage change
which is measured by the output meter.
The only heteroatomic gas molecule which interferes with CO2 is H2O vapour, whose
absorption spectrum overlaps with that of CO2. The interference of water vapour is
overcome by drying the air that is to be examined or by filtering out all the radiation at
wavelengths where the absorption by CO2 and water vapours coincides.
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Construction:
IRGA consists of 3 parts viz. IR source, the sample chamber and the detector. The IR
source is a nichrome spiral which is heated at 600-800oC and produces a beam of IR
light which is being passed through the reference and analysis tube of ‘Sample
chamber’. The CO2 concentration difference (as a result of CO2 fixation by the leaves)
between the analysis and reference tube create, voltage change across the condenser.
This change is amplified and measured by the detector.
Calibration:
For calibration, a source of CO2 free air and a source of air containing a precisely
known concentration of CO2 is required. There are two ways of calibration.
Absolute calibration: Analyzer will be used to determine the exact CO2 of an air sample
by comparing with CO2 free air.
Open system: Analyzer will be used to determine a change in CO2 concentration, i.e.
the difference in CO2 concentration in an air stream before and after it has passed over
a leaf. In this mode, it is possible to detect tiny changes in CO2 concentration down to
100 mg m-3
Open system: In open system, IRGA is calibrated in differential mode, and air of a
known and controlled CO2 and water vapour concentration from outside the system
through a leaf chamber is drawn. A sample of the incoming air stream is passed through
the reference tube, and the air is leaving the chamber is passed through the analysis
tubes. Thus, the IRGA measures the difference in the CO2 content of the air before and
after it passes through the leaf chamber.
FCO2= f Ca/A
Where,
f= the flow rate of air through the leaf chamber
Ca= the difference in CO2 concentration before and after passing through the leaf
chamber
A= leaf area (m2)
The modern portable photosynthesis system usually measures the following processes
on the real-time scale, and all such data can be logged in the experimental site itself.
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S.
no
Process determined/some measured parameters Units
1 Change in CO2 concentration (sample-reference) µmol CO2 mol-1
2 Change in H2O concentration (sample-reference) mmol H2O mol-1
3 Photosynthetic rate (A) µmol CO2m-2 s-1
4 Stomatal conductance µmol m-2 s-1
5 Conductance to H2O mol H2O m-2 s-1
6 Rate of respiration µ mol m-2 S-1
7 Photosynthetically active radiations (PAR) µ mol m-2 S-1
8 Transpiration rate mmol H2O m-2 s-1
9 Temperature of leaf thermocouple oC
10 Temperature in sample cell oC
11 Initial CO2 concentration ppm
12 Ambient CO2 concentration ppm
13 Water Use efficiency, WUE (ΔA/ΔT) mg/g
14 Light Use Efficiency, LUE (ΔA/PAR) µmol
15 Carboxylation efficiency (Ci/Ca) -
16 Output of quantum sensor µmol m-2 s-1
17 Vapor pressure deficient based on air tem kPa
18 Vapor pressure deficient based on leaf tem kPa
19 Flow rate to the sample cell µmol s-1
20 Intercellular CO2 concentration µmol CO2mol-1
The IRGA chamber should be covered with black cloth to cut off the light completely and continuing
measurements in which case CO2 will be released instead of consuming.
These equipment have automatic control of climatic parameters (like CO2, temperature,
light and humidity) which help in determining the gas exchange parameters
(photosynthesis and associated parameters) at desired levels through following studies
S. no Parameters controllable Studies that can be made
1 CO2 concentration A/Ci curves, CO2 compensation point
2 Light intensity A/PAR or light response curve
3 Temperature Temperature response
4 Relative humidity Response to gas exchange parameter for
change in RH
Apart from these, the portable photosynthesis system can also be used to estimate the
rate of photorespiration by measuring the rate of CO2 consumed at 21% oxygen and 2%
oxygen and comparing their difference.
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Procedure:
1. On the instrument, after the opening menu comes on the console press F1
(measurement mode)
2. Latch the leaf in the chamber of LI-6400P
3. On the console press 5, F1 and move the cursor to auto log mode
4. Give the file name for the treatment/plant/leaf
5. Go on answering for the default settings.
6. After the measurements are logged press F3 ( to close the files)
7. Go to next treatment/plant/leaf
8. Measure comparable leaf in both control and water-stressed plants
9. At the end of the measurements dump the data into a computer for further
processing the data (refer sample output of the logged file)
B. Estimation of stomatal and mesophyll limitations of photosynthesis during
water stress
Photosynthesis in a water stressed leaf is limited by stomatal and non-stomatal factors.
Stomatal limitation can account for only 25% reduction in net photosynthesis rate due
to water deficit. The rest of the limitation is contributed by non-stomatal or mesophyll
factors. Though essentially a biochemical process, photosynthesis can also be
considered as a diffusive process; stomatal (gs) and mesophyll resistance (gm) being
the two major resistances for gas exchange. Broadly the difference in assimilation rate
between species or amongst genotype is predominantly due to these two factors.
Depending upon the abiotic stress and its magnitude the ‘gm’ and ‘gs’ is affected
differentially, ultimately affecting (observed photosynthetic rate) ‘A’. To optimize ‘A’
under abiotic stresses it is, therefore, essential to quantify the relative stomatal and
mesophyll limitations of photosynthesis under a given abiotic stress. This approach
assumes importance even under non stress conditions also when we try to assess the
reason for differences in photosynthetic rate between the varieties.
Farquhar and Sharkey (1982) proposed a simple method to estimate stomatal
limitations of ‘A’. They observed that the ‘gs’ induced limitations of ‘A’ did not
increase with stress though there was reduction in the absolute values of ‘gs’. Hence,
they concluded that the mesophyll limitations of ‘A’ were more under stress. Kreig and
Hutmacher (1986) also adopted the same methodology and arrived at similar
conclusions. But mesophyll limitations were not quantified. The method proposed by
Farquhar and Sharkey (1982) has been used here to quantify the stomatal limitations of
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photosynthesis. Their method has been further modified to estimate mesophyll
limitations of photosynthesis also. The modified method is described here.
This analysis involves the development of A/Ci curves. One of the approaches for
developing A/Ci curves is by measuring the gas exchange traits in plant or leaves
exposed to different ambient CO2 concentrations. The first step therefore to make A/Ci
curves in plants experiencing different degree of moisture, light or temperature or
salinity or nutrient stress.
On the A/Ci curves the following points were marked and from these measured and
observed points, the stomatal and mesophyll limitations are computed.
i. A’- Observed photosynthetic rate at any given time
ii. Ao- potential photosynthetic rate when stomatal factors are not limiting and
mesophyll factors are limiting
iii. Ag- Potential photosynthetic rate when mesophyll factors are not limiting and
stomatal factors are limiting
iv. AT- Potential photosynthetic rate when neither mesophyll factors are nor stomatal
factors are limiting
v. A’- Observed photosynthetic rate under stress
vi. A’o-potential photosynthetic rate when stomatal factors are not limiting and
mesophyll factors are limiting under stress
vii. Is-Im- Stomatal and mesophyll limitations.
Control Is= (Ao’-A)/Ao X 100 Stress Is= (Ao- A’)/ Ao X 100
Control Im=(Ag- A)/ Ag X 100 Stress Im=( Ag - A’)/ Ag X 100
Farquhar and Sharkey (1982) gave the following formula to estimate the relative
stomatal limitations (Is)
Is= (Ao- A)/ Ao X 100
We further define the mesophyll limitation of the observed photosynthesis (Im) as
follows
Im= (Ag- A)/ Ag X 100
We further define the mesophyll limitation to the potential photosynthesis (AT) as
follows
ML= (AT-Ao)/ATX 100
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These limitations are estimated for selected crops under control and moisture stress
conditions.
To arrive at the extent of stomatal and mesophyll limitations in stress the A/Ci
curves developed from normal and stressed plants, the following points are marked in
addition to the above described points.
A’- observed photosynthetic rate under stress
A’ó= potential photosynthetic rate when stomatal factors are not limiting under stress.
Relative stomatal and mesophyll limitations (Is, Im) under stress are calculated as
follows
Stress Is= (A’o- A’)/ A’oX 100
Stress Im=(A’g- A’)/ A’gX 100
Fig-18.1. Estimation of stomatal and mesophyll limitations in control and stress
References:
1. Farquhar, G.D. and T.D. Sharkey. 1982. Stomatal cqnductance and photosynthesis. Ann. Rev.
Plant Physiol. 33: 317-345.
2. Hall, D.O., H.R. Securlock, H.R. Bolhar- Nordenkamp, R.C. Leegood, S.P. Long. 1993.
Photosytheis and production in a changing environment. Chamman and Hall, UK. pp464
3. Sestak, Z., J. Katsky and P.G. Jarvis. 1971. Plant photosynthesis production, manual of
methods. Dr. WJunk NV publishers, The Hague. PP 818.
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Chapter 19
Canopy temperature depression (CTD)
Canopy air temperature is a direct measure of energy which is directly released by
plant. Canopy temperature depression (CTD) the difference between air temperature
(Ta) and canopy temperature (Tc). It is trait which is being used successfully as
selection criteria for tolerance to drought in breeding programme. Canopy temperature
depression played an important role for identification drought adaptive traits on
physiological and biochemical basis of abiotic stress tolerance. High CTD (CTD=Ta-
Tc) value indicates the leaf canopy temperature is cool. It has been used in various
practical applications of plant responses to environmental stress to the drought. Leaf
temperature is found to be a valuable indicator of plant water stress. Canopy leaf
temperature at a given situation depends on transpiration rate, leaf temperature. Leaf
water status directly affects the stomatal conductance, which regulates transpiration rate
at a given VPD. Therefore, leaf water status, transpiration rate and leaf/canopy
temperature are interrelated.
Aim: To measure canopy temperature using an infrared thermometer.
Materials required: Infrared thermometer
Principle:
This instrument works on a principle that all objects which has temperature emit
infrared wave radiation. The intensity of infrared radiation emitted is directly
proportional to its body temperature. Infrared gun detects the intensity of temperature
via LCD regarding degree Celsius (oC) directly.
Procedure:
1. Charge the battery of IR gun to full
2. Check the instrument reading
3. By focusing on ice cube
4. By focussing on objects whose temperature can be accurately measured using
conventional thermometer
5. Adjust the emissivity knob to read accurately both the temperature at step 2(a) and
2(b)
6. While measuring the canopy temperature in field avoid overheating the IR gun. For
this purpose cover the IR gun with thermocouple sheets
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7. Focus the gun on the canopy target by holding gun pistol-grip at an angle of 45 oC
and a distance 0.5 to 1 m above the canopy
8. The instrument records the air temperature constantly
9. To record the differences in the canopy and air temperature press the trigger
(differential mode). Before pressing the trigger wave the gun back and forth above the
canopy to avoid stagnation of air around the thermistor located in the nose
10. Repeat the operation 4-7.
References:
1. Morgan, JM. 1980. Osmotic adjustment in the spikelet and leaves of wheat. Journal of
Experimental biology 31: 665-666.
2. Turner, N.C. and M.M. Jones. 1980. Turgor maintenance by osmotic adjustment. A review and
evaluation. In “Adaptation of plants to water and high-temperature stress” (NC Turner and PJ
Kramer, eds. Wiley, New York, 87-103
3. Wilson, R., M.J. Fisher, E.D. Schulze, G.R. Dovler and M.M. Ludlow. 1979. Ecologia 41: 77-
88
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Chapter 20
Root aerenchyma identification under water
logging
More than one-third of the world’s irrigated areas suffer occasional or more frequent
waterlogging (Donmann and Houston, 1967). In Southeast Asia, 18% of total maize
growing areas are significantly affected by waterlogging, causing 25-30% losses in
maize production every year (Rathore et al. 1998 and Zaidi et al. 2010). Systematic
information on the cascade of events conferring the stress tolerance in maize is not yet
established which is necessarily required for genetic enhancement of tropical maize
germplasm for improved tolerance to extreme moisture situation. A large volume of
information is available on the responses of excessive moisture/waterlogging stress on
maize. However, the primary challenge is to identify the stress-adaptive traits in maize
and teosinte essential for abiotic stress crop improvement. In maize plants, to escape
the water logging, several strategies like the development of adventitious roots near to
the surface and formation of internal gas space are present. Internal gas space
(aerenchyma) provides a conduit for the transport of oxygen, this structural
modification in roots is significant for the survival of the plants under low oxygen
availability
Procedure:
1. Three days old aerobically grown (African tall and teosinte and maize lines)
seedlings were further grown for 12,24,36 48 hr under waterlogged conditions
2. Isolated segments of primary roots at 0.05cm,0.5-1cm,1.5 -2.0cm and 2.5-3cm
from the root-shoot junctions for observation of aerenchyma formation
3. Transverse section of primary roots was used to determine the extent of
aerenchyma formation (defined as the area of the aerenchyma per area of the whole
root-on the section)
4. Each section was photographed using a light microscope (LEICA MD 2500 LESD)
with a LEICA MC 170 HD camera (digital light DS-LI, Nikon) area was measured with
Image J software (Fig 20.1)
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Fig-20.1 Root aerenchyma identification using a light microscope
References:
1. Campbell, R., M.C. Drew. 1983. Electron-microscopy of gas space (aerenchyma) formation in
adventitious roots of Zea mays L. subjected to oxygen shortage. Planta 157: 350-357.
2. Donmann, W.W. and Houston C.E. 1967. Drainage related to irrigation management. In:
Drainage of Agricultural Lands. R.W. Hagan, H.R. Haise, and T.W. Ediminster (eds.). Am Soc
Agronomy pp. 974-987.
3. Mano, Y., F. Omori, T. Takamizo, B. Kindiger, R.M. Bird and C.H. Loaisiga. 2006. Variation
for root aerenchyma formation in flooded and non-flooded maize and teosinte seedlings. Plant Soil
281(1-2): 269–279.
4. Rathore, T.R., Warsi M.Z.K, N.N. Singh, S.K. Vasal. 1998. Production of Maize under excess
soil moisture (Waterlogging) conditions. 2nd Asian Regional Maize Workshop PACARD, Laos
Banos, Phillipines, (Feb 23-27, 1998) pp 23.
5. Lenochová Z., A. Soukup and O. Votrubová. 2009. Aerenchyma formation in maize roots.
Biologia Plantarum 53 (2): 263-270.
6. Zaidi, P. H., P. Maniselvan, A. Srivastava, P. Yadav and R.P. Singh. 2010. Genetic analysis of
water-logging tolerance in tropical maize (Zea Mays L.). Maydica 55: 17–26.
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Chapter 21
Estimation of antioxidant enzymes
Oxidative stress results from conditions are promoting the formation of Reactive
Oxygen Species (ROS: Molecular oxygen, singlet oxygen, superoxide anion, hydrogen
peroxide, hydroxyl radical, per hydroxyl radical and ozone) that damage or kill cells.
Environmental factors that cause oxidative stress includes air pollution (ozone and
sulphur dioxide), herbicides (Paraquat) drought, heat, cold, wounding, UV light, intense
light, pathogen infection and during senescence. Plant scavenges and disposes of the
reactive molecules by use of anti-oxidative defence systems present in several
subcellular compartments. The antioxidant defence systems include non-enzymatic and
enzymatic antioxidants. Some major antioxidant enzymes Superoxide dismutase
(SOD), Peroxidase (PX), Catalase (CAT).
A) Estimation of Super Oxide Dismutase enzyme
Principle:
The assay is based on the formation of blue colour by nitro-blue tetrazolium and O2-
radical, which absorbs at 560 nm and the enzymes (SOD) decreases this absorbance
due to a reduction in the formation of O2- radical by the enzyme (Dhindsa et al. 1981).
Requirements:
Reagents:
1. Methionine (200 mM): L-methionine 0.298 g was dissolved in water and the
volume was made up to 10 ml with doubled distilled water.
2. Nitroblue tetrazolium chloride (NBT) (2.25mM): NBT 0.0184 g was dissolved in
doubled distilled water, and the volume was made up to 10 ml with d doubled distilled
water.
3. EDTA (3mM: EDTA 0.0558 g was dissolved in water and volume was made up to
50 ml with d doubled distilled water.
4. Riboflavin (60µM): Riboflavin 0.0023 g was dissolved in water, and the volume
was made up to 100 ml with doubled distilled water.
5. Sodium carbonate (1.5 mM): Sodium carbonate 7.942 g was dissolved in water and
the volume was made up to 50 ml with doubled distilled water.
6. Phosphate buffer (100 mM, pH 7.8)
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Solution A: Potassium dihydrogen phosphate 6.80 g was dissolved in water, and the
volume was made up to500 ml with doubled distilled water.
Solution B: Di- potassium hydrogen phosphate 8.71 g was dissolved in water and the
volume was made up to500 ml with doubled distilled water.
Mix 8.5 ml of Sol.A and 91.5ml of Sol.B and final pH 7.8 was adjusted with the help of
PH meter
7. Grinding media: (0.1M phosphate buffer, pH7.5., containing 0.5 mM EDTA in
case of SOD, CAT, and POX and 1mM ascorbic acid In case of APOX
8. (EDTA 0.0186 g is dissolved in phosphate buffer 0.1M, pH 7.5 (made by mixing
16 ml of Sol A and 84 ml Sol B and final pH is adjust with the help of pH meter) and
volume is made to up to 100 ml with the buffer)
Preparation of enzyme extract:
Enzyme extract for SOD, peroxidase, and catalase was prepared by first freezing the
weighed amount of sample (1g) in liquid nitrogen to prevent proteolytic activity
followed by grinding with 10 ml extraction buffer. Ground plant material was passed
through 4 layers of cheesecloth and filtrate was centrifuged for 20 min at 15000 g and
the supernatant was used as enzyme
Enzyme assay:
SOD activity was estimated by recording the decreases in optical density of formazone
made by superoxide radical and nitro-blue tetrazolium dye by the enzyme (Dhandsa et
al. 1981).
1. Three ml of reaction mixture contained
a) 13.33 mM methionine (0.2 ml of 200 mM)
b) 75µM Nitro blue tetrazolium chloride (0.1ml of 2.25 mM)
c) 0.1mM EDTA (0.1 ml of 3 mM)
d) 50 mM Phosphate buffer (pH 7.8 ) (1.5 ml of 100 mM)
e) 50 mM sodium carbonate (0.1 ml of 1.5M)
f) 0.05 to 0.1 ml enzyme
g) 0.9 to 0.95 of water (to make final volume of 3 ml)
2. Reaction was started by adding 2 µM riboflavin (0.1 ml) and placing the test tubes
under two 15 W fluorescent lamps for 15 min.
3. A complete reaction mixture without enzyme, which gave the maximal colour,
served as control
4. To stop the reaction, turn off the lights and keep the tubes in darkness
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5. A non –irradiated complete reaction mixture served as a blank
6. The absorbency was recorded at 560 nm, and 1 unit of enzyme activity was taken
as that amount of enzyme, which reduced the absorbency reading to 50 % in
comparison with tubes lacking enzyme.
1uinit (of enzyme) Control-Sample
Control/2
B) Estimation of Peroxidase enzyme
Principle:
The enzyme peroxidase catalyses the oxidation of the substrate by oxygen generated
from the decomposition of hydrogen peroxide:
2H2O2 → 2H2O + O2
Substrate + O2 → Oxidized substrate.
Reagents:
1. Phosphate buffer (100, mM pH 6.1)
Solution A: Potassium dihydrogen phosphate 6.80g was dissolved in water, and the
volume was made up to 500 ml with doubled distilled water.
Solution B: Dipotassium hydrogen phosphate 8.71g was dissolved in doubled distilled
water, and the volume was made up to 500 ml with doubled distilled water.
Mix 15 ml of sol. A and 85ml of sol. B and final pH 6.1 was adjusted with the help of
pH meter
1. Hydrogen peroxide (12 mM): Dissolve 124 µl of 30% H2O2 in doubled distilled
water and the volume was made up to100 ml
2. Guaicol (96 mM): Dissolve 1075 µl of analytical grade guiacol in doubled
distilled water and the volume was made up to100 ml
The reaction mixture contained:
a) Phosphate buffer (100, mM pH 6.1) : 1 ml of 100 mM
b) Guaicol (16 mM) : 0.5 ml of 95 mM
c) Hydrogen peroxide (2 mM) : 0.5 ml of 12 mM
d) Enzyme : 0.1 ml
e) Water : 0. 4 ml to make a final volume of 3 ml.
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Absorbance due to the formation of tetra-guaiacol was recorded at 470 nm and enzyme
activity was calculated as per extinction coefficient of its oxidation product, tetra-
guaiacol= 26.6 mM-1 cm-1
Enzyme activity is expressed as μm tetra-guaiacol formed per min per fresh weight or
per mg protein
C) Estimation of Catalase enzyme
Principle
The enzyme catalase mediates the breakdown of hydrogen peroxide into oxygen and
water.
Reagents:
1. Hydrogen peroxide: 77754 µl of 30% H2O2 is dissolved in doubled distilled water
and make up the volume was made to100 ml to get 75 mM Hydrogen peroxide
2. Phosphate buffer (100 mM, pH 7.0)
Solution A: Potassium dihydrogen phosphate 6.80g was dissolved in water and the
volume was made up to 500 ml with doubled distilled water.
Solution B: Di- potassium hydrogen phosphate 8.71 g was dissolved in doubled
distilled water and the volume was made up to500 ml with doubled distilled water.
Mix 39 ml of sol. A and 61 ml of sol. B and final pH 6.1 was adjusted with the help of
pH meter
Enzyme Assay:
The reaction mixture contained:
a) Phosphate buffer 50 mM :1.5 ml of 100 mM buffer, pH 7.0
b) Hydrogen peroxide 12.5 mM :0.5 ml of 75 mM Hydrogen peroxide
c) Enzyme : 50μl
d) Water : to make a final volume of 3 ml.
Adding H2O2 started the reaction and decrease in absorbance was recorded for 1min.
The initial and final content of hydrogen peroxide is calculated by comparing with a
standard curve drawn with a known concentration of hydrogen peroxide.
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Enzyme activity is calculated as the concentration of hydrogen peroxide (initial
reading- and final reading = quantity of hydrogen peroxide) per min per mg protein.
References:
1. Aebi, H. 1984. Catalase in vitro. Meth Enzymology 105:121-126.
2. Sairam, R.K., P.S. Deshmukh and D.S. Shukla. 1997. Tolerance of drought and temperature
stress in relation to increased antioxidant enzyme activity in wheat. Journal of Agronomy and Crop
Science 178: 171–178.
3. Dhindsa, R.A., P.P. Dhindsa and T.A. Thorpe. 1981. Leaf senescence: Correlated with
increased permeability and peroxidation, and decreased the level of SOD and CAT. J. Exp .Bot.
126: 93-101.
4. Yu, Q R.Z. 1999. Drought and salinity differentially influence activities od SOD in narrow-
leafed lupins. Plant Sci. 142: 1-11.
5. Catillo FI, Penel I and Greppin H (1984). Peroxidase release induced by ozone in sedum album
leaves.Plant physiology.74: 846-851.
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Chapter 22
Stress assessment formulas and stress related
terminology
Index name Outcome Formula Reference
Stress tolerance
index(STI)
The genotype with high STI values
will be tolerant to drought
Yp= irrigated condition
Ys= under drought condition
STI = Fernandez,
1992
Mean productivity
Index, (MP)
The genotype with high values of
this index will be more desirable
MPI= Hossain et
al., 1990
Geometric mean
productivity
(GMP)
The genotype with high values of
this index will be more desirable
GMP=
Fernandez,
1992
Tolerance index
(TOL)
The genotype with low values of
this index will be more stable in
two different conditions
TOL = Yp – Ys
Hossain et
al., 1990
Stress
susceptibility
index (SSI)
The genotype with high SSI < 1 are
more resistant to drought stress
conditions
SSI = Fischer and
Maurer,
1978
Yield reduction
index (YSI)/ Yield
stability index
(YSI)
The genotype with high YSI values
can be regarded as stable genotype
under stress and non-stress
conditions
YSI =
Bouslama
and
Schapaugh,
1984
Yield reduction
ratio (YR)
The genotype with low value of
this index will be suitable for
drought stress conditions
YR=1- (YS/YP)
Golestanni
and anshi
and Assud
(1998)
Drought resistant
index (DI)
The genotype with high values of
this index will be more suitable for
drought stress condition
DI = ys (ys/yp)/yp
Lan 1998
Yield index (YI) The genotype with high values of
this index will be more suitable for
drought stress condition
YI =
Gauzzi et
al 1997
Drought
sensitivity index
The genotype with lower DSI
values can be regarded as stable
DSI= [(1- D/YP]D Fischer and
Maurer,
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(DSI) genotype under stress and non-
stress conditions (control)
Note: the lower the DSI the stable
is the drought tolerance of the line
1978
Salt Tolerance
Index (STI)
The genotype with higher STI
values can be regarded as
tolerance genotype
(% STI) = (TDW
Value in saline
environment/ TDW
Value in control
environment X
100)
Whereas TDW:
Total dry weight
Seydi .,
2003
Plant water
content (PWC)
Higher the water content
genotypes are stress tolerance
(drought/salinity/water togging)
PWC (g/g) =
(FW-DW)/DW
Percentage of
reduction over the
control (% ROC)
The genotype with lower % ROC
values will be tolerant to stress
(%ROC) = (Value
in control-value in
saline environment
X 100)/(Value in
control)
Ali Y,
2004
Seed Vigour Index
(SVI)
Higher the seed vigour index
higher the rate of tolerance
Germination %
Seedling length
(shoot+ root length
in cm)
Relative Growth
Rate (RGR)
RGR= LAR
NAR
LAR: Leaf area
ratio, which is the
amount of leaf area
per unit total plant
mass
NAR: Net
assimilation rate
which is the rate of
increase in plant
mass per unit leaf
area
Gardner
(1988)
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Root volume Root volume=W2-
W1 (cm3)
W1=initial water
level
W2= water level
after root dipped
in measuring
cylinder
Leaf area (LA) Leaf area measured :
Note: 1 Constant (Factor) 0.65
for cereal crops ex rice, wheat,
barley, oat (small leaf plants)
Note: 2 Constant (Factor) 0.75
for cereal crops ex maize,
sorghum, pearl millets (wider
leaf plants) L= leaf length,
W=Leaf width
Leaf area per
leaf=L x W x
0.75
Leaf area per
plant= L x W x
0.75 x Number
of leaves per
plant
Lazarove
., 1965
Allowable
Depletion Volume
The amount of plant-available water that can be removed from the soil without
seriously affecting plant growth and development.
Capillary Water
Water retained in soil pores after gravitational water has drained or is held
loosely around soil particles by surface tension. Most of the soil-water
available to plants is capillary water.
Crop Water Use
Rate
Maximum daily rate at which a crop can extract water from a moist soil to
satisfy PET; controlled by stage of crop development.
Crop
Susceptibility A measurement of crop response to a unit of stress.
Definition of salt
tolerance
Definition of salt tolerance is the ability of plants to survive and produce
harvestable yields under salt stress is called salt resistance or Plant salt
tolerance or resistance is generally thought of in terms of the inherent ability
of the plant to withstand the effects of high salts in the root zone or on the
plant's leaves without a significant adverse effect called salt tolerance.
or
Salt resistance is a complex phenomenon, and plants manifest a variety of
adaptations at subcellular, cellular, and organ levels such as stomatal
regulation, ion homeostasis, hormonal balance, activation of the antioxidant
defense system, osmotic adjustment, and maintenance of tissue water status to
grow successfully under salinity
Drought Absence of rainfall for a period of time long enough to result in depletion of
soil water and injury to plants.
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Drought
avoidance
It is the ability of the plant to maintain water status or turgor at any given soil
water deficit
Drought tolerance Is the ability to maintain life functions under decreasing tissue water potential
Drought escape The ability of plant to complete its life cycle before serious soil and plant
water deficit develop
Effective Root
Depth
The upper portion of the root zone where plants get most of their water.
Effective root depth is estimated as one-half the maximum rooting depth.
Depletion Volume The amount of plant-available water removed from the soil by plants and
evaporation from the soil surface.
Gravitational
Water
Water in the soil that is free to drain or move due to the forces of gravity.
Gravitation water is the volume of water in the soil between saturation and
field capacity. This water is not usually used by plants.
Water Use
Efficiency (WUE)
Is the amount of dry matter produced per unit amount of water transired
expressed as g dm g-1
Transpiration
quotient
Is the amount of water transpired per unit weight of dry matter produced
expresses as ml H2O g-1 dry weight
Cumulative water
loss (CWL)
Is the amount of water lost through transpiration. It is a reflection of the
amount of water used by plant for transpiration and also includes evaporation
and also includes evaporation losses, expressed as ml water per unit land area
(per plot)
Leaf area duration
(LAD)
In pot culture is reflection of the functional leaf area available for assimilation
on during the active growth period calculated by the following formula:
LAD= (L1+L2)/2 X (t2-t21)
Where, L1= leaf area dm2 at time t1
L2= leaf area dm2 at time t2
T2-t1= duration in days between initial and final samples
LAD- expressed as dm2 days
Rate of water loss Mean transpiration rate-is a product of the cumulative water transpired (CWT)
divided by the leaf area duration (LAD) expressed as ml H2O dm2 days-1
Field capacity of
soil
The water content of soil after downward drainage of gravitational water
content has become very slow and the water content has become relatively
small.
Permanent wilting
point
Is the soil water content at which plant remain wilted unless water is added to
the soil- Richard and Wadleish (1952) found that the soil water potential at
wilting ranged from -1.0 to 2.0 M Pa. the volume of -1.5 MPa (15 bars) is
generally used as an appropriation of soil water at permanent wilting.
Evaporation Evaporation from a water surface can be described by following equation:
E= (C water –C air)/ r air
Where, C- water vapour concentration of evaporating surface
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C air= water vapour concentration in the bulk air
Transpiration The amount of water lost from the plant surface during the growth and
development expressed in m mol H2O m-2 s-1
Evapotranspiration The amount of water lost both through transpiration and evaporation
Bars Is the unit of expression of the stress level in the water relations of plant soil
Pascals 10 bars= 1 mega pascal
Relative water
content (% RWC)
Is the expression of leaf water content as percentage of turgid water content
given by the following formula
[(Fresh Weight-Dry weight)/( turgid weight- Dry weight)] X 100
Water potential In thermodynamic terminology, it is the free energy status of water in a system
compared to that of pure water at atmospheric pressure and temperature under
isothermal conditions. The various factors involved in cell all water relations
at equilibrium can be summarized by U= Us+Up+Um
Solute potential The contribution of solute to the total U. it is a negative term because solutes
in water decrease the chemical potential and follows Raoults law.
Pressure potential The contribution of the pressure potential, to total U. also called turgor
pressure important for cell enlargement, guard cell movement. Usually
positive in leaves.
Soil metric
potential
It is a component of total water potential contributed by metric forces in the
soil
Available soil
moisture
Is the amount of water retained in the soil between field capacity and
permanent wilting point i.e. -0..3 M Pa and -1.5 M Pa. expresses the effect of
water binding colloids and surfaces and capillary effect in cells and cell walls
Stomatal
resistance
Is the resistance offered by the stomata for the diffusion of water vapour into
the atmosphere or for the CO2 entry. It is measured as the time taken by tha
gases to diffuse through a unit distance across the stomata (sec cm -1)
Stomatal
conductance (gs)
Is defined as the ease with which water and CO2 diffuses across the stomata. It
is measured as the distance travelled per unit time
Mesophyll
conductance (gm)
Is the resistance offered by the mesophyll will for the diffusion of CO2 (there
are no direct methods to measure it)
Mesophyll
conductances (gm)
Is defined as the ease with which CO2diffuses through the mesophyll cells.
The rate of incorporation of CO2 into organic molecules in chloroplast al low
CO2concentration is often considered as a reflection of gm
Internal CO2
concentration
Is the CO2concentration in the intercellular spaces of the mesophyll (ppm)
Ambient CO2
concentration
The CO2concentration in the external air surrounding the canopy is termed as
Ambient CO2concentration expressed as ppm.
Vapor pressure
deficit
Is the reduction in the partial pressure of water vapor in air compared to the
leaf of a plant (expressed pascals or bars)
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Partial pressure Is the partial free energy associated with the gases such as CO2and O2 or water
vapor
Osmoregulation Osmoregulation as distinguished from osmotic adjustment has been defined
recently as regulation of osmotic pressure resulting in a constant internal
osmotic pressure when the external osmotic pressure varies. This delineation
has been given to describe regulation of tissues or volume occurring in some
fresh water algae
Transpiration
efficiency
Is the expression of water use efficiency at the single leaf level and is given by
the rates of the two gas exchange process in molar units.
T efficiency = m moles CO2/mol H2O used in transpiration
Osmoprotectants Some osmotic solutes like proline, glycine, betaine or known to be protectants
of certain enzymes
Crop canopy air
temperature
difference
(CCATD)
Term used in canopy temperature studies difference between canopy and air
temperature (Tc-Ta)
Crop water stress
index (CWSI)
Is calculated based upon CCATD and VPD
Stress stock
proteins
Certain protein induced and synthesis de novo in response to external stress
(heat, osmotic, drought). Originally termed as heat shocks proteins (HSPs)
Osmotic
adjustment
Is the net increase in solutes, as distinguished from the passive increase in the
concentration caused by loss of water. It results in maintenance of turgor at a
lower water potential than would otherwise be possible
Pan evaporation Standard measurements of evaporation for weather bureau purposes, are
generally made within evaporation. A standard pan measuring 25.4 cm deep X
120.6 cm inside diameter. The coefficient from such pan to the free water
surface of a shallow lake is approximately 0.7.
Potential
evapotranspiration
Determined by energy balance approach. 1st developed by Penma, 1948,
modified by Monteith, 1965. Now referred to Penman-Monteith method
E=[Ss (Rn-G) + Pa Cp gh Δ e ] / Y [ (s + y) gh/gw]
Saturation Condition when all soil pores are filled with water.
Redistribution
(Percolation) Downward movement of gravitational water through the soil profile.
Temporary
Wilting Daily cycle of plant wilting during the day followed by recovery at night.
Permanent Wilting
Point (PWP)
The soil-water content of which healthy plants can no longer extract water
from the soil at a rate fast enough to recover from wilting. The permanent
wilting point is considered the lower limit of plant-available water.
Unavailable Water Water in thin, tightly held films around soil particles; not available to plants.
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Potential Rooting
Depth
The deepest rooting depth attained by crop roots depending on the type of crop
and independent of soil conditions.
Maximum
Rooting Depth
Deepest rooting depth attained by a crop under specific soil conditions.
Physical and chemical barriers in the soil often limit actual rooting depths to
less than potential rooting depth.
Refernces:
1. Bouslama, M. and, W.T. Schapaugh. 1984. Stress tolerance in soybean. Part 1: evaluation of
three screening techniques for heat and drought tolerance. Crop Science 24: 933-937.
2. Fernandez, G.C.J. 1992. Effective selection criteria for assessing plant stress tolerance. In: Kus
EG (ed) Adaptation of Food Crop Temperature and Water Stress. Proceeding of 4th International
Symposium, Asian Vegetable and Research and Development Center, Shantana, Taiwan, pp 257-
270.
3. Fischer, R.A. and R. Maurer. 1978. Drought resistance in spring wheat cultivars. I. Grain yield
responses. Australian Journal of Agricultural Research 29: 892-912.
4. Hossain, A.B.S., A.G. Sears, T.S. Cox and G.M. Paulsen. 1990. Desiccation tolerance and its
relationship to assimilate partitioning in winter wheat. Crop Science 30: 622-627.
5. Rosielle, A.A. and J. Hamblin. 1981. Theoretical aspects of selection for yield in stress and
non-stress environment. Crop Science 21: 943-946.
6. Seydi AB. 2003. Determination of the salt tolerance of some barley genotypes and the
characteristics affecting tolerance. Turkish Journal of Agriculture and Forestry, 27:253-260.
7. Lazarove R (1965).Coefficient for determining the leaf area in certain agricultural crops. Rast.
Nauki., 2:27-37
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ANNEXURE-I Here's a quick guide to converting units:
1 part per million (ppm) = 1 milligram per liter (mg/L)
1 milligram per liter (mg/L) = milliequivalents per liter (meq/L) x the element's
equivalent weight (e.g., 23 for sodium, 35 for chloride)
1 millimho per centimeter (mmho/cm) = 1 decisiemen per meter (dS/m) = 1,000
micromhos per centimeter (µmhos/cm) = 0.1 siemen per meter (S/m)
Electrical conductivity of irrigation water (ECiw) approximately equals the total
dissolved solids in parts per million or milligrams per liter divided by 640. That number
640 is an average conversion factor applicable under most circumstances (consult your
testing laboratory if you're not sure about this).
Stated another way, and using symbols:
TDS in mg/L or ppm = 640 × ECiw in dS/m
Note: for EC value less than 5dS/m, 1dS/m=640 mg/L TDS
And 1dS/m=about 800 mg/L for EC values above 8dS/m
Note: The SI unit of conductivity is ‘Siemens’ symbol ‘S’ per metre. The equivalent
non-SI unit is mho’ and 1 mho = 1 Siemens. Thus for those unused to the SI system
mmhos/cm can be read for dS/m without any numerical change.
Conductivity 1 S cm-1 (1 mho/cm) = 1000 mS/cm (1000 mmhos/cm)
1 mS/cm-1 (1 mmho/cm) = 1 dS/m = 1000 mS/cm (1000 micromhos/cm)
Conductivity to mmol (+) per litre: mmol (+)/1 = 10 × EC (EC in dS/m)
For irrigation water and soil extracts in the range 0.1-5 dS/m.
Conductivity to osmotic pressure in bars:
OP = 0.36 × EC (EC in dS/m)
For soil extracts in the range of 3-30 dS/m.
Conductivity to mg/l: mg/l = 0.64 × EC x 103, or (EC in dS/m) mg/l = 640 × EC
For waters and soil extracts having conductivity up to 5 dS/m.
nmol/l (chemical analysis) to mg/l: Multiply mmol/l for each ion by its molar weight
and obtain the sum.
Pag
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Abbreviations
1. AW-Available water
2. ASM-Available soil moisture
3. BC- Back cross
4. BD-Bulk density
5. CAT-Catalase
6. CRD- Completely Randomized Design
7. CTD- Canopy Temperature Depression
8. DMSO- Dimethylsulphoxide
9. DMY- Dry matter yields
10. EAR- Exchangeable sodium ratio
11. EC- Electrical conductivity
12. ECe- Electrical conductivity of the saturated soil paste
13. ECw- irrigation water salinity
14. ESP- Exchangeable sodium percentage
15. GFY - Green fodder yield
16. HI- Harvest index
17. IRGA- Infrared Gas Analyzer
18. ME or mEq = mill equivalent
19. MSI-Membrane Stability Index ,
20. MW = molecular weight
21. oC-Temparature
22. PD- Particle density
23. PX- Peroxidase
24. RBD- Randomized Block Design
25. R-Flame photometer reading
26. RWC- Relative water content
27. SAR- sodium adsorption ratio
28. SCMR-SPAD chlorophyll meter readings
29. SOD-Superoxide dismutase
30. SPAD- Soil Plant Analysis Development chlorophyll meter
31. TDS -total dissolved salts
32. VPD-Vapour pressure deficit
33. WUE-Water Use Efficiency