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Manual on Plant Stress Physiology
Seva Nayak Dheeravathu Vikas Chandra Tyagi Chandan Kumar Gupta Edna Antony
ICAR - Indian Grassland and Fodder Research Institute, Jhansi (UP) - 284 003, India
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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 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
CHAPTERS Introduction pH and Buffer Minimum Data Set for a Abiotic Stress Experiment (MIASE) Estimations of soil particle density, soil bulk density and soil porosity Artificial saline water and saline soil preparation (Soil salinity and plant tolerance) Estimation of soil pH and soil ECe Measurement of osmotic potential using vapour pressure osmometer Determination of Sodium and Potassium in plant tissue Measurement of water content in soil and plant tissue Imposition of drought by gravimetric approach Two tier screening of germplasm under natural condition or Irrigation stops approach for stage-specific drought tolerance Determination of Water Use Efficiency (WUE) Photosynthetic pigments analysis in plants Estimation of chlorophyll stability index and carotenoid stability index in leaf tissue Cell Membrane Stability Index Estimation of abscisic acid content in leaf and root Estimation of proline content in plant tissue Photosynthesis Canopy Temperature Depression (CTD) Root aerenchyma identification under waterlogging Estimation of antioxidant enzymes Stress assessment formulas and stress related terminology Annexure-I Abbreviations
Page .No 1-2 3-4 5-9 10-11 12-20 21-28 29-30 31-33 34-39 40-42 43-49 50-54 55-57 58-60 61 62-63 64-65 66-71 72-73 74-75 76-80 81-87 88 89
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 hightemperature 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
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1
programme for crop/forage improvement.
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
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temperatures: towards genetic engineering for stress tolerance. Planta 218: 1-14.
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+ + ClStrong base NaOH→Na++OHWeak 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
acid or base are added. A buffer solution consists of a weak acid and its conjugate base.
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Buffers: Buffer solution is the one that resistant changes in pH when small amounts of
3
higher [H+] while higher pH is associated with lower [H+].
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).
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W.H. Freeman, New york. pp 65-68.
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.
adequate rainfall and drainage becomes the important problem in rainfed farming.
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more than 1150mm. There is less chances of crop failures due to dry spells. There is
5
iii. Rainfed farming Means cultivation of crops in regions where annual rainfall is
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
11. Weather data (rainfall, temp. VPD-Vapour pressure deficit).
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12. Yield and yield components.
6
by seasonal and inter annual variations in climate.
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 table5.1) 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
the maximum amount of variation/genetic makeup in smallest number of samples
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preliminary screening stage from large size population/germplasm resources for getting
7
5. Preliminary/ Rapid screening: Maximum germplasm should be discarded at
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.
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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1.
8
C) Physiological information (minimum information)
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 agrotechniques 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
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059, India
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
is calculated by formula
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decides the amount of water soil can hold. Amount of pore space or porosity of the soil
10
spaces are formed due to the movement of roots, worms and insects. The pore space
Porosity =1Where 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
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11
Sciences: Agricultural and environmental principles. pp 232.
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
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12
tolerant and tolerant (Fig-5.1 and Table-5.3).
Table-5.1 Classification of salt-affected soils Class
pH
ECe
SAR
ESP
(dS/m) Normal
6.5 -7.5
Symptom
No visible symptom and normal growth of the plant
Saline
8.5 13 >15
Sodic
Symptom
22.5)
60-120 cm (2-4 ft)
16 (> 22.5)
Crop response
(Very
Salinity effects Yield of very Yield of most crop Only tolerant crop mostly sensitive crop restricted yield satisfactorily negligible, restricted except in very sensitive plants (In parenthesis indicate irrigation water salinity: ECw) USDA classification of irrigation water salinity (adapted from Richards, 1969)
Only a few tolerant crops yield satisfactorily
13
Non-Saline/
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Soil depth
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
Moderately
Tolerant
sensitive
tolerant
Rice
Chickpea
Sorghum
Barley
Sesame
Corn and
Soybean
Canola
Peanut
Sunflower
Cotton
Pigeonpea
Sugarcane
Wheat
Guar
Walnut
Alfalfa
Barely
Oats and forage Oats
Corn (forage) Gram, Black or urd
Mango
Berseem
Guinea
Rye and forage Rye
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(forage)
14
bean
grass Banana
Cowpea
Dhaincha
Triticale
(forage) 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
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15
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) 4 8 12 16
ME for all 4 salts 45 95 150 200
ME for individual salt
MW
Salt required (g) /liter)= ME x MW
NaCl
Na2SO4
MgCl2
CaSO4
NaCl
Na2SO4
MgCl2
CaSO4
NaCl
Na2SO4
MgCl2
CaSO4
23.4 49.4 78.0 104.0
12.6 26.6 42.0 56.0
1.8 3.8 6.0 8.0
7.2 15.2 24.0 32.0
58 58 58 58
142 142 142 142
203 203 203 203
172 172 172 172
1.4 2.9 4.6 6.1
1.8 3.8 6.0 8.0
0.4 0.8 1.2 1.6
1.2 2.6 4.1 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
Table: 5.5. Electrical conductivity (EC) of pure solutions at 20°C (dS/m) equivalent with mM solution Solution 10 mM NaCl
EC (dS/m) 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).
A
B
Fig.5.2 (A and B) Concentration of saturation extraction of soil in milliequivalents per liter as
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17
related to Electrical conductivity (Conductivity v/s. concentration Source USDA Hand book No-60)
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 semiarid 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)
To calculate the salts required to prepare a soil with ECe of 4, 8, 12, 16 dSm-1
Page
Desired level of EC (dS/m): 4, 8, 12, 16
18
known weight of soil in the pots.
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 A mixture of salt ratios
Equivalent weight
ME=EC X Salt ratio
Salt required (g) /liter) = ME x MW
EC
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
Page
19
watered soil is warranted.
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.
Page
20
60. USSL, Riverside, CA, USA.
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
Application
units Conductivity (dS/m)
1 dS/m is equal
Equivalent units
to Soils
1
1 dS/m = 1 mS/cm = 1 mmho/cm
Conductivity (µS/cm)
Irrigation and river
1000 µS/cm
1 µS/cm = 1 µmho/cm
Irrigation and river
640 mg/L
1 mg/L = 1 mg/kg = 1
(mg/L)
water
(approx.)
ppm
Molarity of NaCl (mM)
Laboratory
10 mM
1 mM = 1 mmol/L
Page
Total dissolved salts
21
water
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
Page
22
salinometer methods.
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
Page
method in laboratories.
23
affect plant growth (Table-6.2). The conductivity (EC meter) method is simple and easy
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
8.6
clay, silty clay, light clay 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
6. Then filter the saturated paste with whatman paper no 42 or vacuum extractor to obtain the extract
Page
5. Remix the paste with water or soil as is needed to bring the paste saturation point
24
salts to dissolve
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
Correc
Tempera
Correc
Temperat
Correcte
Temper
Corrected
(oC)
ted
ture (oC)
ted
ure (oC)
d value
ature
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
25
(oC)
value
Page
value
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 o
C 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
cool oven/sundry 2. Take the 50 g of dried sieved soil (2mm sieve), into 500 ml beaker and
Page
1. Take a soil sample from the desired site/depth of soil surface and dried on a tray in a
26
instantaneously and easily by irrigators or farm managers in field
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) =54.48 100
=
Page
27
Formula, soil saturation %
[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
Page
28
paste extracts from 1:5 soil: water suspensions and texture. Aust. J. Soil Res. 31: 73–81.
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.
complete. Note: use only fresh or verified osmolality standards for calibration.
29
Repeat the step one to correct the offset. Calibration of the osmometer is now
Page
3.
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
Osmotic potential
concentration
Sucrose
Osmotic potential
concentration
(-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-
Page
30
pressure osmometer. Environmental and Experimental Botany. 53:77–84.
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.
standard of 5,10,15,20,30,40, and 50me Na+ L-1 concentrations
Page
solution (containing 100 mEq Na+ L-1) to 100 ml in volumetric flask to get working
31
Working standard solutions of Na+: Dilute,5,10,15,20,30,40, and 50 ml portion of stock
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
R × dilution factor
K+
=
%
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+ %
= 10000 Flame photometer reading
32
=
Page
R
R × dilution factor
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
Page
33
opportunities. Trends in Plant Science 10: 615-620.
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
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
Page
bars which are used as an approximation of soil water.
34
soil water potential at wilting ranged from -10 to -20 bars, with the average at about -15
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
Destructive sampling
2.
Labour requirement at each sampling
3.
Not applicable to field conditions
4.
More time is require
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1.
35
Disadvantages:
Table- 9. 1. Soil water content parameters for different soil textures Soil texture
Field capacity
Plant available
Permanent wilting
(in./ft)
water (in./ft)
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)
Determination of Relative Water Content (RWC) in leaf tissue
36
Fig: 9.1 Soil water parameters and classes of water (Source Juan et al E-618 08/12)
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1.
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
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37
5. Weight the samples and quickly to record the fresh weight.
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
1 2
Sample ID Control Stress
Fresh weight (g) (A) 0.95 0.90
Turgid weight (g) (B) 1 1
Dry weight (g)
RWC %= [(A-C/B-C)]x 100
55 45
89 82
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S,No
38
Observation sheet
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.
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39
3:38–42.
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)
regular intervals to monitor water status at different FCs, Replenish the water every
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irrigation (please refer the diagrammatic representation given below). Weigh the pots at
40
8. At four or six-leaf stage or at good foliage, impose drought stress by withholding
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.
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imposing precise levels of moisture stress/ drought.
41
Figure 10.1: Diagrammatic representation of gravimetric approach followed for
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.
T2 (20% ASM)
T1 (40% ASM)
Control (80% ASM)
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.
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42
6. Turner, N.C., 1997, further progress in crop water relations, Adv. Agron. 58:29-338.
Chapter 11
Two tier screening of germplasm under a natural condition or Irrigation stop approach for stagespecific 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
v. Light intensity – Cloud induced reduction in light intensity should not be > 30%.
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iv. Day length (photoperiod) – Preferably it should be in the range of 11 to 13 hours.
43
< 30% respectively
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
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Formula for available water
44
(non-stress) and 40-50% and 50-60 % in moderate and severe stress blocks respectively
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)
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45
6. Basic physiological parameters studied in selected genotypes (RBD Fig-11.2)
1.5m
Genotypes
Vegitatve Stage Stress ([50-60% or 40-50% ASM) Block-2 Block-3
1.5m
Block-1
1.5m
1.5m
Reproductive/pre mature Stage Stress ([50-60% or 40-50% ASM) Block-2 Block-3
1.5m
1.5m
46
Genotypes
Block-1
Block-3
Page
Block-1
Genotypes
Expt-1
Seedling Stage Stress ([50-60% or 40-50% ASM) Block-2
Seedling Stage Stress Block-I Moderate stress (50-60% ASM) Block-1 Block-2 Block-3
5m
Genotypes
Genotypes
1.5m
5m
Genotypes
Genotypes
1.5m
5m
1.5m
1.5m
1.5m
1.5m
1.5m
1.5m
Stress Block-II Sever stress (40-50% ASM) Block-1 Block-2 Block-3
5m
Reproductive/pre mature Stage Stress Block-I Moderate stress (50-60% ASM) Block-1 Block-2 Block-3
Non Stress Block Control (70-80 % ASM) Block-1 Block-2 Block-3
1.5m
1.5m
Vegetative stage Stress Block-I Moderate stress (50-60% ASM) Block-1 Block-2 Block-3
Non Stress Block Control (70-80 % ASM) Block-1 Block-2 Block-3
1.5m
1.5m
Genotypes
5m
Genotypes
1.5m
Stress Block-II Sever stress (40-50% ASM) Block-1 Block-2 Block-3
1.5m
1.5m
Stress Block-II Sever stress (40-50% ASM) Block-1 Block-2 Block-3
5m
Genotypes
1.5m
Genotypes
Genotypes
Non Stress Block Control (70-80 % ASM) Expt-2 Block-1 Block-2 Block-3
1.5m
1.5m
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
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47
for comparison. Note : ASM-Available soil moisture
Seedling stage R2
R3
5m
5m
5m
T2
5m
5m
5m
5m
5m
R1
Vegetatve stage R2
R3
5m
5m
5m
T2
5m
5m
5m
5m
5m
R1
Reproductive/pre mature Stage R2
R3
5m
5m
5m
T2
Genotypes
T1
T3
Genotypes
T1
T3
Genotypes
T1
R1
5m 5m
5m
T3
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
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48
% ASM), T2-Moderate stress (50-60% ASM) and T3-Sever stress (40-50% ASM)]
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.
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4. Kramer, P. J. 1983. Water deficits and plant growth. Water relation of Plants 24: 342-389.
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
water use. However, progress in breeding for improved TE has been extremely limited
50
mainly due to the lack of a suitable screening technique to determine the genetic
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volume of applied and fundamental research has been focused on optimization of crop
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
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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saturated with water and allowed to drain overnight to bring the soil to near 100% FC. On
51
should be
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
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D= A – (B + C)
52
Therefore,
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
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53
this should be treated as ASM or SMD instead of Field Capacity.
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
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tolerance. Field crops research. 120(2): 262-270.
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 sensorhold 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.
55
Close when sound is heard
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6.
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)
))
W= Weight of plant tissue (in grams)
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V=Final volume of 80% acetone (in ml)
56
Where, A = Absorbance
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
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57
improves chlorophyll meter’s estimation of rice leaf nitrogen concentration. Agron J 85:987–990.
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 o
C. 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
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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3.
58
minutes and discard water
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
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directly related with drought tolerance.
59
High CSI and CSI* corresponded with more drought tolerance. Thus, CSI, CSI* is
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,
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60
Abstract: National Seminar (ISSP), IARI, New Delhi.pp. 69.
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
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drought tolerance as affected by applied N in soybean. J. Agric. Soc. Camp 115: 63-66.
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
The amount of ascorbic acid in mg/100 g of the sample can be calculated as follows:
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9. Record the final volume of dye at the endpoint as mentioned above (V1)
62
beaker add 10ml of 3% metaphosphoric acid and titrate it against the dye.
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:
63
Albrecht, J.A. 1993. Ascorbic acid retention in lettuce. J. Food quality 16: 311-316.
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1.
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
Filter the homogenate through Whatman no. 2 filter paper and make up the volume
to 10 ml.
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2.
64
solution sulphosalicylic acid
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
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65
metabolism in drought-stressed tall fescue. Crop Sci. 49:1893–1901.
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
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wavelengths where the absorption by CO2 and water vapours coincides.
66
overcome by drying the air that is to be examined or by filtering out all the radiation at
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)
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on the real-time scale, and all such data can be logged in the experimental site itself.
67
The modern portable photosynthesis system usually measures the following processes
S.
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
o
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
no
C
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
oxygen and comparing their difference.
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rate of photorespiration by measuring the rate of CO2 consumed at 21% oxygen and 2%
68
Apart from these, the portable photosynthesis system can also be used to estimate the
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
conclusions. But mesophyll limitations were not quantified. The method proposed by
69
Farquhar and Sharkey (1982) has been used here to quantify the stomatal limitations of
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Hutmacher (1986) also adopted the same methodology and arrived at similar
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
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ML= (AT-Ao)/ATX 100
70
follows
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
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methods. Dr. WJunk NV publishers, The Hague. PP 818.
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=TaTc) 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
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
72
Adjust the emissivity knob to read accurately both the temperature at step 2(a) and
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5.
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-
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88
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
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Image J software (Fig 20.1)
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
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water-logging tolerance in tropical maize (Zea Mays L.). Maydica 55: 17–26.
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 O2radical, 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.
the volume was made up to 50 ml with doubled distilled water. 6.
Phosphate buffer (100 mM, pH 7.8)
76
Sodium carbonate (1.5 mM): Sodium carbonate 7.942 g was dissolved in water and
Page
5.
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
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
Page
3.
77
under two 15 W fluorescent lamps for 15 min.
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:
b) Guaicol (16 mM)
: 0.5 ml of 95 mM
c)
: 0.5 ml of 12 mM
Hydrogen peroxide (2 mM)
d) Enzyme
: 0.1 ml
e)
: 0. 4 ml to make a final volume of 3 ml.
Water
78
Phosphate buffer (100, mM pH 6.1) : 1 ml of 100 mM
Page
a)
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, tetraguaiacol= 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)
: 50μl
Enzyme
d) Water
: to make a final volume of 3 ml.
standard curve drawn with a known concentration of hydrogen peroxide.
Page
The initial and final content of hydrogen peroxide is calculated by comparing with a
79
Adding H2O2 started the reaction and decrease in absorbance was recorded for 1min.
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
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80
leaves.Plant physiology.74: 846-851.
Chapter 22
Stress assessment formulas and stress related terminology Index name
Outcome
Formula
Reference
Stress tolerance
The genotype with high STI values
STI =
Fernandez,
index(STI)
will be tolerant to drought
1992
Yp= irrigated condition Ys= under drought condition
Mean productivity
The genotype with high values of
Index, (MP)
this index will be more desirable
Geometric mean
The genotype with high values of
productivity
this index will be more desirable
MPI=
Hossain et al., 1990
GMP=
Fernandez, 1992
(GMP) Tolerance index
The genotype with low values of
(TOL)
this index will be more stable in
TOL = Yp – Ys
Hossain et al., 1990
two different conditions Stress
The genotype with high SSI < 1 are
susceptibility
more resistant to drought stress
Maurer,
index (SSI)
conditions
1978
Yield reduction
The genotype with high YSI values
index (YSI)/ Yield
can be regarded as stable genotype
and
stability index
under stress and non-stress
Schapaugh,
(YSI)
conditions
1984
Yield reduction
The genotype with low value of
ratio (YR)
this index will be suitable for
and anshi
drought stress conditions
and Assud
SSI =
YSI =
YR=1- (YS/YP)
Fischer and
Bouslama
Golestanni
(1998) Drought resistant
The genotype with high values of
index (DI)
this index will be more suitable for
DI = ys (ys/yp)/yp
Lan 1998
YI =
Gauzzi et
drought stress condition
this index will be more suitable for
al 1997
drought stress condition Drought
The genotype with lower DSI
sensitivity index
values can be regarded as stable
DSI= [(1- D/YP]D
Fischer and Maurer,
81
The genotype with high values of
Page
Yield index (YI)
(DSI)
genotype under stress and non-
1978
stress conditions (control)
Note: the lower the DSI the stable is the drought tolerance of the line
Salt Tolerance
The genotype with higher STI
(% STI) = (TDW
Seydi .,
Index (STI)
values can be regarded as
Value in saline
2003
tolerance genotype
environment/ TDW Value in control environment X 100) Whereas TDW: Total dry weight
Plant water
Higher the water content
PWC (g/g) =
content (PWC)
genotypes are stress tolerance
(FW-DW)/DW
(drought/salinity/water togging) Percentage of
The genotype with lower % ROC
(%ROC) = (Value
Ali Y,
reduction over the
values will be tolerant to stress
in control-value in
2004
control (% ROC)
saline environment X 100)/(Value in control)
Seed Vigour Index
Higher the seed vigour index
(SVI)
higher the rate of tolerance
Germination % Seedling length (shoot+ root length in cm)
Relative Growth
RGR= LAR
Gardner
Rate (RGR)
NAR
(1988)
LAR: Leaf area ratio, which is the amount of leaf area per unit total plant mass NAR: Net assimilation rate
mass per unit leaf area
Page
increase in plant
82
which is the rate of
Root volume
Root volume=W2W1 (cm3) W1=initial water level W2= water level after root dipped in measuring cylinder
Leaf area (LA)
Leaf area measured :
Leaf area per
Lazarove
Note: 1 Constant (Factor) 0.65
leaf=L x W x
., 1965
for cereal crops ex rice, wheat,
0.75
barley, oat (small leaf plants)
Leaf area per
Note: 2 Constant (Factor) 0.75
plant= L x W x
for cereal crops ex maize,
0.75 x Number
sorghum, pearl millets (wider
of leaves per
leaf plants) L= leaf length,
plant
W=Leaf width Allowable
The amount of plant-available water that can be removed from the soil without
Depletion Volume
seriously affecting plant growth and development. Water retained in soil pores after gravitational water has drained or is held
Capillary Water
loosely around soil particles by surface tension. Most of the soil-water available to plants is capillary water.
Crop Water Use
Maximum daily rate at which a crop can extract water from a moist soil to
Rate
satisfy PET; controlled by stage of crop development.
Crop Susceptibility Definition of salt tolerance
A measurement of crop response to a unit of stress. 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.
soil water and injury to plants.
Page
Drought
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 Absence of rainfall for a period of time long enough to result in depletion of
83
or
Drought
It is the ability of the plant to maintain water status or turgor at any given soil
avoidance
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
The upper portion of the root zone where plants get most of their water.
Depth
Effective root depth is estimated as one-half the maximum rooting depth.
Depletion Volume
evaporation from the soil surface. Water in the soil that is free to drain or move due to the forces of gravity.
Gravitational
Gravitation water is the volume of water in the soil between saturation and
Water Water
The amount of plant-available water removed from the soil by plants and
field capacity. This water is not usually used by plants. Use
Is the amount of dry matter produced per unit amount of water transired
Efficiency (WUE)
expressed as g dm g-1
Transpiration
Is the amount of water transpired per unit weight of dry matter produced
quotient
expresses as ml H2O g-1 dry weight
Cumulative water
Is the amount of water lost through transpiration. It is a reflection of the
loss (CWL)
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
In pot culture is reflection of the functional leaf area available for assimilation
(LAD)
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
The water content of soil after downward drainage of gravitational water
soil
content has become very slow and the water content has become relatively small.
Permanent wilting
Is the soil water content at which plant remain wilted unless water is added to
point
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. E= (C water –C air)/ r air Where, C- water vapour concentration of evaporating surface
84
Evaporation from a water surface can be described by following equation:
Page
Evaporation
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
Is the resistance offered by the stomata for the diffusion of water vapour into
resistance
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
Is defined as the ease with which water and CO2 diffuses across the stomata. It
conductance (gs)
is measured as the distance travelled per unit time
Mesophyll
Is the resistance offered by the mesophyll will for the diffusion of CO2 (there
conductance (gm)
are no direct methods to measure it)
Mesophyll
Is defined as the ease with which CO2diffuses through the mesophyll cells.
conductances (gm)
The rate of incorporation of CO2 into organic molecules in chloroplast al low CO2concentration is often considered as a reflection of gm
Internal
CO2
Is the CO2concentration in the intercellular spaces of the mesophyll (ppm)
CO2
The CO2concentration in the external air surrounding the canopy is termed as
concentration
Ambient CO2concentration expressed as ppm.
Vapor
Is the reduction in the partial pressure of water vapor in air compared to the
deficit
pressure
leaf of a plant (expressed pascals or bars)
Page
Ambient
85
concentration
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
Is the expression of water use efficiency at the single leaf level and is given by
efficiency
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
Term used in canopy temperature studies difference between canopy and air
temperature
temperature (Tc-Ta)
difference (CCATD) Crop water stress
Is calculated based upon CCATD and VPD
index (CWSI) Stress
stock
Certain protein induced and synthesis de novo in response to external stress
proteins
(heat, osmotic, drought). Originally termed as heat shocks proteins (HSPs)
Osmotic
Is the net increase in solutes, as distinguished from the passive increase in the
adjustment
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
Determined by energy balance approach. 1st developed by Penma, 1948,
evapotranspiration
modified by Monteith, 1965. Now referred to Penman-Monteith method E=[Ss (Rn-G) + Pa Cp gh Δ e ] / Y [ (s + y) gh/gw]
(Percolation) Temporary Wilting Permanent Wilting Point (PWP) Unavailable Water
Downward movement of gravitational water through the soil profile.
Daily cycle of plant wilting during the day followed by recovery at night. 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. Water in thin, tightly held films around soil particles; not available to plants.
86
Redistribution
Condition when all soil pores are filled with water.
Page
Saturation
Potential Rooting
The deepest rooting depth attained by crop roots depending on the type of crop
Depth
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 257270.
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.
Page
87
Nauki., 2:27-37
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
Page
88
and obtain the sum.
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
32. VPD-Vapour pressure deficit 33. WUE-Water Use Efficiency
Page
31. TDS -total dissolved salts
89
30. SPAD- Soil Plant Analysis Development chlorophyll meter
