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Phenotyping
Crop Plants for
Physiological and
Biochemical Traits
Page left intentionally blank
Phenotyping
Crop Plants for
Physiological and
Biochemical Traits
P. Sudhakar
Department of Crop Physiology
S. V. Agricultural College
Acharya N. G. Ranga Agricultural University
Tirupati, A.P., India
P. Latha
Institute of Frontier Technology
Regional Agricultural Research Station
P.V. Reddy
Regional Agricultural Research Station
AMSTERDAM • BOSTON • HEIDELBERG • LONDON

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Contents
Message......................................................................................................................xi
Foreword..................................................................................................................xiii
Preface.......................................................................................................................xv
Abbreviations..........................................................................................................xvii
Introduction..............................................................................................................xix
SECTION I
CHAPTER 1 Various Methods of Conducting Crop Experiments....... 3
1.1 Field Experiments............................................................................ 3
1.2 Experiments Under Green Houses................................................... 6
1.2.1 Demerits................................................................................. 6
1.3 Experiments in Growth Chambers................................................... 6
1.3.1 Demerits................................................................................. 6
1.4 Hydroponics..................................................................................... 7
1.4.1 Precautions............................................................................. 8
1.5 Pot Culture....................................................................................... 9
SECTION II
CHAPTER 2 Seed Physiological and Biochemical Traits............... 17
2.1 Destructive Methods......................................................................17
2.1.1 Seed Viability.......................................................................17
2.1.2 Seed Vigor Tests................................................................... 18
2.2 Nondestructive Methods................................................................21
2.2.1 X-ray Analysis..................................................................... 21
2.2.2 Electrical Impedance Spectroscopy (EIS)...........................22
2.2.3 Multispectral Imaging.......................................................... 22
2.2.4 Microoptrode Technique (MOT).........................................22
2.2.5 Infrared Thermography (IRT)..............................................23
2.2.6 Seed Viability Measurement Using Resazurin
Reagent�� 23
2.2.7 Computerized Seed Imaging................................................ 23
SECTION III
CHAPTER 3 Plant Growth Measurements........................................ 27
3.1 Measurement of Growth................................................................ 27
3.2 Measurement of Below Ground Biomass...................................... 27
v
vi Contents
3.3 Growth Analysis............................................................................28

3.3.1 Growth Characteristics—Definition and Mathematical
Formulae��29
CHAPTER 4 Photosynthetic Rates................................................... 33

4.1 Net Assimilation Rate (NAR)........................................................ 33
4.2 Measuring Through Infrared Gas Analyzer (IRGA)...................... 33
4.3 Rubisco Enzyme Activity..............................................................37
4.3.1 Measurement of Rubisco Activity.......................................37
4.4 Chlorophyll Fluorescence Ratio (Fv/Fm Values)..........................39
CHAPTER 5 Drought Tolerance Traits.............................................. 41

5.1 Water Use Efficiency (WUE) Traits..............................................41
5.1.1 Carbon Isotope Discrimination............................................ 48
5.1.2 Determination of Stable Carbon Isotopes Using
Isotope Ratio Mass Spectrometer (IRMS)�� 48
5.1.3 Protocol for Carbon Isotope Discrimination
in Leaf Biomass�� 49
5.2 Root Traits.....................................................................................50
CHAPTER 6 Other Drought-Tolerant Traits...................................... 53

6.1 Relative Water Content (RWC)......................................................53
6.2 Chlorophyll Stability Index (CSI).................................................53
6.3 Specific Leaf Nitrogen (SLN)........................................................ 54
6.4 Mineral Ash Content...................................................................... 55
6.5 Leaf Anatomy................................................................................ 55
6.6 Leaf Pubescence Density............................................................... 56
6.7 Delayed Senescence or Stay-Greenness........................................ 56
6.8 Leaf Waxiness................................................................................57
6.9 Leaf Rolling................................................................................... 58
6.10 Leaf Thickness (mm).....................................................................58
6.11 Stomatal Index and Frequency......................................................58
6.12 Other Indicators for Drought Tolerance.........................................59
6.13 Phenological Traits........................................................................59
CHAPTER 7 Tissue Water Related Traits......................................... 61

7.1 Osmotic Potential........................................................................... 61
7.1.1 Determination of Osmotic Potential Using Vapor Pressure
Osmometer�� 62
7.2 Leaf Water Potential......................................................................63
7.3 Relative Water Content..................................................................64
Contents vii
7.4 Cell Membrane Injury.................................................................... 64

7.4.1 Cell Membrane Permeability Based on Leakage
of Solutes from Leaf Samples�� 64
CHAPTER 8 Heat Stress Tolerance Traits....................................... 67

8.1 Canopy Temperature......................................................................67
8.2 Chlorophyll Stability Index (CSI).................................................68
8.3 Chlorophyll Fluorescence..............................................................68
8.4 Thermo Induction Response (TIR) Technique..............................69
8.5 Membrane Stability Index.............................................................71
8.5.1 Membrane Permeability Based on Leakage of Solutes
from Leaf Samples�� 71
CHAPTER 9 Oxidative Stress Tolerance Traits................................ 73

9.1 Oxidative Damage.........................................................................73
9.1.1 Antioxidant Enzymes........................................................... 74
9.2 Superoxide Dismutase (SOD)........................................................ 74
9.3 Catalase.......................................................................................... 75
9.4 Peroxidase (POD).......................................................................... 77
9.5 Free Radicals................................................................................. 78
CHAPTER 10 Salinity Tolerance Traits.............................................. 81

10.1 Chlorophyll Stability Index...........................................................81
10.2 Proline............................................................................................ 81
10.3 Sodium (Na) and Potassium (K) Ratio.......................................... 82
10.3.1 Potassium (K).................................................................... 82
10.3.2 Sodium (Na)....................................................................... 83
10.4 Antioxidative Enzymes..................................................................84
SECTION IV
CHAPTER 11 Kernel Quality Traits.................................................... 87
11.1 Proteins.......................................................................................... 87
11.1.1 Protein Estimation by Lowry Method............................... 88
11.1.2 Protein Estimation by Bradford Method �� 89
11.2 Kernel Oil......................................................................................90
11.2.1 Oil Estimation by Soxhlet Apparatus (SOCS)................... 90
11.3 Aflatoxins....................................................................................... 91
11.3.1 Quantification of Aflatoxin Levels in Kernels...................91
viii Contents
CHAPTER 12 Carbohydrates and Related Enzymes.......................... 95

12.1 Reducing Sugars............................................................................95
12.2 Nonreducing Sugars.......................................................................96
12.3 Total Carbohydrates.......................................................................96
12.4 Estimation of Sucrose Phosphate Synthase................................... 97
12.5 Estimation of Starch Synthase....................................................... 99
12.6 Estimation of Invertases...............................................................100
CHAPTER 13 Nitrogen Compounds and Related Enzymes...............103

13.1 Total Nitrogen..............................................................................103
13.1.1 Kjeldhal Method for Quantifying Leaf
Nitrogen Content�� 103
13.1.2 Preparation of Reagents................................................... 104
13.1.3 Protein Percent can be Determined Indirectly
Using the Following Formula��104
13.2 Total Free Amino Acids...............................................................105
13.3 Nitrate Reductase......................................................................... 106
13.4 Nitrite Reductase......................................................................... 108
13.5 Leghemoglobin (Lb)....................................................................109
13.6 Glutamic Acid Dehydrogenase (GDH)........................................ 110
13.7 Glutamate Synthase (GOGAT).................................................... 111
13.8 Glutamine Synthetase (GS)......................................................... 112
13.8.1 Calculation....................................................................... 114
CHAPTER 14 Other Biochemical Traits............................................115

14.1 Total Phenols................................................................................115
14.2 Ascorbic Acid.............................................................................. 116
14.3 Alcohol Dehydrogenase (ADH).................................................. 118
14.4 Glycine Betaine........................................................................... 119
CHAPTER 15 Plant Pigments............................................................121

15.1 Chlorophylls................................................................................121
15.1.1 Estimation of Chlorophyll...............................................121
15.2 Carotenoids.................................................................................. 123
15.2.1 Quantification of Carotenoids in Green Leaves...............123
15.3 Lycopene......................................................................................126
15.4 Anthocyanin.................................................................................127
CHAPTER 16 Growth Regulators.......................................................129

16.1 Estimation of Indole Acetic Acid (IAA)...................................... 129
16.2 Estimation of Gibberellins........................................................... 130
Contents ix
16.3 Estimation of Abscisic Acid (ABA).............................................131

16.4 Estimation of Ethylene................................................................133
SECTION V
CHAPTER 17 Analytical Techniques.................................................137
17.1 Ultraviolet Visible (UV–VIS) Spectrophotometer.......................137
17.2 Thin Layer Chromatography (TLC)............................................ 138
17.3 Gas Chromatography (GC).......................................................... 140
17.3.1 Introduction...................................................................... 140
17.3.2 Principle........................................................................... 140
17.3.3 Detectors.......................................................................... 141
17.4 High-Performance Liquid Chromatography (HPLC).................. 142
17.4.1 Role of Five Major HPLC Components..........................143
17.5 Liquid Chromatography–Mass Spectrometry
(LC–MS, or Alternatively HPLC–MS)��144
17.5.1 Flow Splitting..................................................................145
17.5.2 Mass Spectrometry (MS)................................................. 145
17.5.3 Mass Analyzer................................................................. 146
17.5.4 Interface...........................................................................146
17.5.5 Applications.....................................................................146
17.6 Inductively Coupled Plasma Spectrometry (ICP)
(Soil & Plant Analysis Laboratory University of
Wisconsin–Madison http://uwlab.soils.wisc.edu)��147
17.6.1 Introduction...................................................................... 147
17.6.2 Summary of Method........................................................ 147
17.6.3 Safety............................................................................... 148
17.6.4 Interference...................................................................... 148
17.6.5 Measurement by ICP-OES.............................................. 148
17.6.6 Measurement.................................................................... 148
17.6.7 Measurement by ICP-MS................................................ 148
17.6.8 Measurement.................................................................... 149
Appendices��151
Common Buffers..........................................................................151
Appendix I: Citrate Buffer........................................................... 151
Appendix II: Sodium Phosphate Buffer....................................... 152
Appendix III: Potassium Phosphate Buffer................................. 152
Appendix IV: Sodium Acetate Buffer..........................................153
Appendix V: Tris–HCl Buffer (Tris–Hydroxymethyl
Aminomethane Hydrochloric Acid Buffer)................................. 153
x Contents
Appendix VI: 1M HEPES–NaOH pH 7.5 Buffer........................154

Appendix VII: Preparation of Stocks of Macro and
Micronutrients for Hydroponics Experiment............................... 154
Appendix VIII: Preparation of ‘Hoagland Solution’ for
Hydroponics Experiment............................................................. 155
Appendix IX: Solubility Chart of Plant Growth Regulators........156
References............................................................................................................... 157
Index.......................................................................................................................167
Message
The growing demand for food and increasing scarcity of fertile land, water, energy,
etc., present multiple challenges to crop scientists to meet the demands of future gen-
erations while protecting the environment and conserving biological diversity. The
productivity of crops greatly depends on the prevailing environmental conditions.
Although farming practices are capable of increasing crop yields through control of
pests, weeds, and application of fertilizers, the weather cannot be controlled. Occur-
rence of abiotic stress conditions such as heat, cold, drought, flooding causes huge
fluctuations in crop yields. Climatic change scenarios predict that weather extremes
are likely to become more prevalent in the future, suggesting that stress proofing our
major crops is a research priority.
Crop physiology plays a basic role in agriculture as it involves study of vital phe-
nomena in crop plants. It is the science concerned with processes and functions and
their responses toward environmental variables, which enable production potential
of crops. Many aspects of practical agriculture can be benefited from more intensive
research in crop physiology. Hence, knowledge of crop physiology is essential to all
agricultural disciplines that provide inputs to Plants Breeding, Plant Biotechnology,
Agronomy, Soil Science, and Crop Protection Sciences.
Novel directions in linking this basic science to crop and systems research are
needed to meet the growing demand for food in a sustainable way. Crop perfor-
mance can be changed by modifying genetic traits of the plant through plant breed-
ing or changing the crop environment through agronomic management practices. To
achieve that, understanding crop behavior under environmental variables plays an
important role in integrating and evaluating new findings at the gene and plant level.
Reliable crop-physiological techniques are essential to phenotype crop plants for
improved productivity through conventional and molecular breeding.
The authors of this book have been working on developing various physio-
logical and biochemical traits in different field crops for 20 years and have estab-
lished state-of-the-art laboratory and field facilities for phenotyping crop plants
at Regional Agricultural Research Station, Tirupati. I congratulate the authors
for their studious efforts in bringing out their expertise in the form of this book.
I hope this book provides an insight into several physiological and biochemical
techniques that can benefit scientists, teachers, and students of Agriculture, Plant
Biology, and Horticulture.
A. Padma Raju
xi
Foreword
The most serious challenges that societies will face over the next decades are provid-
ing food and water, in the face of mounting environmental stresses, warned by the
consequences of global climate change. There is an urgent need of developing meth-
ods to alleviate the environmental disorders to boost crop productivity especially
with existing genotypes, which are unable to meet our requirements.
The Green revolution in cereals promoted optimism about the capacity of crop
improvement in increasing yield and it drove plant physiologists to understand the
physiological basis of yield and its improvement. Although research in crop physiol-
ogy encompasses all growth phenomena of crop plants, only traits that have a likely
economic impact and show significant genetic variation can be considered in the
context of crop improvement.
The first step to be taken in this direction is to use appropriate screening tech-
niques to select germplasm adapted to various abiotic stress conditions. The im-
provement of abiotic stress tolerance relies on manipulation of traits that limit yield
in each crop and their accurate phenotyping under the prevailing field conditions in
the target population of environments.
Agricultural scientists and students often face impediments in selecting right
phenotyping method in various crop experiments. There is a dire need to bring reli-
able protocols of physiological and biochemical traits which directly or indirectly
influences final yield in a book form. I am well aware that authors of this book Dr
P. Sudhakar, Dr P. Latha, and Dr P.V. Reddy have played key role in developing
drought-tolerant peanut varieties in this University by applying various physiologi-
cal traits standardized in their laboratory. I congratulate the authors for bringing out
their expertise in the form of this book “Phenotyping crop plants for physiological
and biochemical traits.”
This publication not only is the detailed explanation of methodology of pheno-
typing but also links the physiology to a possible ideotype for its selection. Hence,
this book is highly useful to agricultural scientists, molecular biologists, and students
to select desirable ideotype for their target environment.
K. Raja Reddy
xiii
Preface
This book elaborates methods that can contribute to phenotyping of crop plants for
various physiological and biochemical traits. It contains field-based assessment of
these traits, as well as laboratory-based analysis of tissue constituents in samples
obtained from field-grown plants. Most of the phenotyping methods given in this
book are reliable, as they were validated in our research programmes.
We extend thanks to all the colleagues for their support in validating the pheno-
typing methods in several agricultural crops. We express deep sense of reverence
and indebtedness for all the team members of this crop physiology department since
1996, viz., Narsimha Reddy, D. Sujatha, Dr M. Babitha, Dr Y. Sreenivasulu, Dr K.V.
Saritha, B. Swarna, M. Balakrishna, T.M. Hemalatha, V. Raja Srilatha, C. Rajia
Begum, and K. Lakshmana Reddy. We appreciate K. Sujatha, Senior Research
Fellow of this department, for her involvement in validating phenotyping methods as
well as in preparation of this book.
We express gratitude for Dr T. Giridhara Krishna, Associate Director of Research,
Regional Agricultural Research station, Tirupati and Dr K. Veerajaneyulu, U niversity
Librarian for their constant support in accomplishing this book. We are grateful to
Acharya N G Ranga Agricultural University for facilitating the research needs and
support in bringing out this book.
We extend special thanks to our collaborate scientists Dr S.N. Nigam, ICRISAT,
Dr M. Udaya Kumar, UAS, Bangalore, Dr R.C. Nageswara Rao, ACIAR, Australia,
and Dr R.P. Vasanthi, RARS, Tirupati for their support over all these years.
Finally, we hope this book provides insightful information about various reli-
able phenotyping methods adopted in laboratory, greenhouse, and field-oriented crop
research for students and researchers of Agriculture, Horticulture, Molecular biology,
Botany, and Allied sciences.
- Authors
xv
Abbreviations
cm Centimeter
mm Millimeter
°C Degree centigrade
∆ Difference
a Alpha
b Beta
g Gamma
D.H2O Distilled water
D.D H2O Double distilled water
fr.wt Fresh weight
g Gram
GLC Gas liquid chromatography
h Hour
HPLC High-performance liquid chromatography
kg Kilogram
L Liter
mCi Micro curie
mg Microgram
mL Microliter
mmole Micromole
mg Milligram
min Minute
mL Milliliter
mmole Milli mole
M Molar
Mol Mole
N Normality
nm Nanometer
OD Optical density
rpm Resolutions per minute
s Second
TLC Thin layer chromatography
V/V Volume/volume
W/V Weight/volume
Y Year
xvii
Introduction
Agricultural crops are exposed to the ravages of abiotic stresses in various ways and
to different extents. Unfortunately, global climate change is likely to increase the
occurrence and severity of these stress episodes created by rising temperatures and
water scarcity. Therefore, food security in the 21st century will rely increasingly
on the release of cultivars with improved resistance to drought conditions and with
high-yield stability (Swaminathan, 2005; Borlaug, 2007).
We are using landraces as genetic sources for abiotic stress resistance. These are
the simple products of farmers who repeatedly selected seed that survived historical
drought for years in their fields. No science was involved, only a very long time and
a determination to provide for their own livelihood. These landraces attend to the
fact that abiotic stress resistance has been here for a very long time. We are now only
trying to improve it more effectively.
Improving the genetic potential of crops depends on introducing the right adap-
tive traits into broadly adapted, high-yielding agronomic backgrounds. The emerg-
ing concept of newly released cultivars should be genetically tailored to improve
their ability to withstand drought and other environmental constraints while optimiz-
ing the use of water and nutrients. A major recognized obstacle for more effective
translation of the results produced by stress-related studies into improved cultivars
is the difficulty in properly phenotyping relevant genetic materials to identify the
genetic factors or quantitative trait loci that govern yield and related traits across dif-
ferent environmental variables.
The Green Revolution in cereals promoted optimism about the capacity of plant
breeding to continue increasing yield and it drove plant physiologists to understand
the physiological basis of yield and its improvement. The physiological basis of the
Green Revolution in the cereals was identified very early as an increase in harvest
index from around 20–30% to about 40–50%, depending on the crop and the case.
The yield components involved in this increase were also identified, with grain
number per inflorescence as the primary one. Crop physiology then led breeders
to understand that yield formation in cereals is derived from an intricate balance
between yield components’ development, source to sink communication, crop as-
similation, and assimilate transport linked to crop phenology and plant architecture
(Tuberosa and Salvi, 2004).
Taking full advantage of germplasm resources and the opportunities offered by
genomics approaches to improve crop productivity will require a better understand-
ing of the physiology and genetic basis of yield adaptive traits. Although research in
plant physiology encompasses all growth phenomena of healthy plants, only traits
that have a likely economic impact and which show significant genetic variation can
be considered for improvement in the context of plant breeding. Many such traits are
expressed at the whole plant or organ level.
Plants exhibit a variety of responses to abiotic stresses, in other words, drought,
temperature, salt, floods, oxidative stress which are depicted by symptomatic and
xix
xx Introduction
quantitative changes in growth and morphology. The ability of the plant to cope with
or adjust to the stress varies across and within species as well as at different devel-
opmental stages. Although stress affects plant growth at all developmental stages, in
particular anthesis and grain filling are generally more susceptible. Pollen viability,
patterns of assimilates partitioning, and growth and development of seed/grain are
highly adversely affected. Other notable stress effects include structural changes in
tissues and cell organelles, disorganization of cell membranes, disturbance of leaf
water relations, and impedance of photosynthesis via effects on photochemical and
biochemical reactions and photosynthetic membranes. Lipid peroxidation via the
production of ROS and changes in antioxidant enzymes and altered pattern of syn-
thesis of primary and secondary metabolites are also of considerable importance.
Phenological traits, that is, pheno-phases of the growth and development, have
the greatest impact on the adaptation of plants to the existing environment all with
the aim of achieving a maximum productivity (Passioura, 1996). The extent by
which one mechanism affects the plant over the others depends upon many factors
including species, genotype, plant stage, composition, and intensity of stress.
Phenotype (from Greek phainein, to show) is the product of all of the possible in-
teractions between two sources of variation, the genotype, that is, the genetic blueprint
of a cultivar, and the environment, that is, the collection of biotic, abiotic, and crop
management conditions over which a given cultivar completes its life cycle. There-
fore, even discrete observations of a given phenotype can integrate many genotype
and environmental connections over time. Genotype-by-environment interactions can
play a significant role in the phenotypes collected in the field or greenhouse.
Phenotyping involves measurement of observable attributes that reflect the biologi-
cal functioning of gene variants (alleles) as affected by the environment. To date, most
phenotyping of secondary traits (ie, those traits in addition to yield, the primary trait)
has involved field assessments of easily scored morphological attributes such as plant
height, leaf number, flowering date, and leaf senescence. However, phenotyping plants
for abiotic stress tolerance involves metabolic and regulatory functions, for which mea-
surements of targeted processes are likely to provide valuable information on the un-
derlying biology and suggest approaches by which it could be modified.
Good phenotyping is a critical issue for any kind of experimental activity, but the
challenges faced by those investigating the abiotic effects on crops are particularly
daunting due to difficulties in standardizing, controlling, and monitoring the environ-
mental conditions under which plants are grown and the data are collected, especially
in the field. Phenotypic traits need to be adopted also depending on whether the
experiments are carried out in the field or in the controlled environment of a growth
chamber or greenhouse. Phenotyping means not only the collection of accurate data
to minimize the experimental error introduced by uncontrolled environmental and
experimental variability, but also the collection of data that are relevant and mean-
ingful from a biological and agronomic standpoint, under the conditions prevailing
in farmers’ fields.
Collecting accurate phenotypic data has always been a major challenge for im-
provement of quantitative traits. Success of this task is intimately connected with
Introduction xxi
heritability of the trait, namely portion of phenotypic variability accounted for by ad-
ditive genetic effects that can be inherited through sexually propagated generations
(Falconer, 1981). Trait heritability varies according to the genetic makeup of the ma-
terials under investigation, the conditions under which the materials are investigated,
the accuracy and precision of the phenotypic data. Despite this, careful evaluation
and appropriate management of the experimental factors that lower the heritability of
traits, coupled with a wise choice of the genetic material, can provide effective ways
to increase heritability and hence the response to phenotypic selection.
Moreover, excellent methods have been developed for assay of such traits and
they have been used in controlled studies to determine the mechanistic basis of stress
response. Notwithstanding their positive aspects, these methods often require highly
controlled laboratory environments and are too time consuming and expensive or
technically demanding to be used in large-scale phenotyping.
The challenge, then, is to identify those attributes that provide the most meaning-
ful phenotypic information, to design sampling methods suitable for use in the field,
and to design analytical methods that can efficiently be scaled up to the number of
samples required for phenotyping of crops in field experiments. Selection for one
trait can reduce a chance for a successful selection for some other trait, due to a com-
petitive relationship toward the same source of nutrients. However, the combination
of traits that in various ways contribute to the improvement of yields can result in a
maximum gain of each trait individually.
Although earlier studies reported several physiological and molecular traits
with the relevance field applicability, many of them are not simple, reliable, and re-
searcher friendly due to complicated protocols and high genotype and environment
interaction. This book will discuss various methods that can contribute to phenotyp-
ing of crop plants for various physiological and biochemical traits. They involve
analyzing methods for field-based assessment of these traits, as well as laboratory-
based analyses of tissue constituents in samples obtained from field-grown plants.
Researchers or students working in this direction will have several options to select
the reliable methodology according to the objective and experimenting conditions.
SECTION
1
I
Various methods of conducting crop experiments . . . . . . . . . . . . . . . . . . . . . 3
CHAPTER
Various methods
of conducting crop
experiments
1
Effective phenotyping should require a set of core setups in which plants are culti-
vated either under laboratory conditions or in experimental fields. Such experiments
enable researchers to determine the phenotypic responses of plants to defined experi-
mental treatments and evaluate the performance of different genotypes or species in
a given environment. To enable generalizations across experiments, it is necessary
that results are not only replicable, but also reproducible. Replication of results is
achieved when the same researcher finds the same results while repeating an experi-
ment in time. In plant biology, achieving a high degree of reliability and reproducibility
is a challenge. This chapter provides information on different methods of conducting
experiments for crop and data to be recorded on various abiotic environment param-
eters apart from regular plant biometric data.
1.1 FIELD EXPERIMENTS

Field experiments with rainout shelter facility are shown in Fig. 1.1. These are typi-
cally undertaken under conditions where some, but not all variables, can be controlled.
These sometimes represent a particular stress (eg, drought, nutrient, or temperature),
or under favorable conditions where the aim is to understand physiological and ag-
ronomic factors contributing to yield potential. Similarly, assessment of genotypes
under a controlled stress requires an understanding and reporting of factors contrib-
uting to their differential performance in response to stress. If some of the observed
differences in yield relate to differences in plant height, flowering, or greater leaf area,
then the cataloguing of such variation must be undertaken.
Measuring and reporting of this variation can be varied among the researchers.
This makes interpretation across multiple experiments difficult as one researcher
may view and undertake sampling differently from another. It is critical that there is
consistency in how measurements are undertaken and reported. Hence, standardizing
procedures and phenotyping among individuals will provide data that are robust,
reliable, and repeatable. This will lead to more cost-efficient research wherein high-
quality data can be produced and reused.
1. Selection of site: For critical planning and interpreting field response data,
good knowledge of the site and expected seasonal conditions based on prior
knowledge of long-term weather trends are essential. Information such as soil
Phenotyping Crop Plants for Physiological and Biochemical Traits. http://dx.doi.org/10.1016/B978-0-12-804073-7.00001-6 3
Copyright © 2016 BSP Books Pvt. Ltd. Published by Elsevier Inc. All rights reserved.
4 CHAPTER 1 Various methods of conducting crop experiments
FIGURE 1.1 Field Experimentation With Rainout Shelter Facility.
conditions, viz., soil type, soil texture, soil moisture, soil nutritional status,
soil born pest, and diseases, should be analyzed for the experiment area.
Identification of uniform blocks with perfect leveling to reduce residual (error)
variation in large size field experiments is essential. Long-term seasonal rainfall
and temperatures are to be collected and should be used in planning for the need
for sowing date, irrigation, and imposing abiotic stresses.
2. Plot type and size: Phenotyping of complex physiological traits and particularly
hose associated with canopy development, biomass, and yield is challenging
when experiments comprise diverse genotypes. This is especially so when
confounded with variation in traits such as height and maturity that are known
to affect yield.
3. Implications in row and plot experiments
a. Row plantings
Limited seed and resources may encourage field assessment in single, spaced
rows or smaller, unbordered plots. Competition for water, light, and nutrients
required for canopy growth is variable as adjacent rows are genetically
different and competition is greatest particularly under stress conditions.
Response to changes in resource availability varies among diverse genotypes,
alters genotype ranking, and thus reduces heritability. In turn, the relevance
of such growing conditions to commercial field-grown crops is unclear.
b. Plot experiments
The planting of multirow plots and the simple exclusion of plot borders
at harvest increases experimental precision and confidence in genotype
1.1 Field experiments 5
response. Well-planned field studies and particularly those focusing on the

dynamics of yield formation (canopy-related characteristics) must consider
the use of multiple-row plots and with border rows to minimize the effects
of inter-plot competition. Plots should contain two outer rows (“edge” or
“border”) and multiple inner rows to minimize inter-plot competition effect,
for example, edge effects due to shading, nutrients, water availability, or
compaction.
c. Phenotyping in the field
Assuming that both the type and the number of treatments (genotypes,
irrigation volumes, etc.) to be evaluated are adequate for the specific
objectives of each experiment, the following general factors should be
evaluated carefully to ensure the collection of meaningful phenotypic data in
field experiments conducted under water-limited conditions:
- experimental design
- heterogeneity of experimental conditions between and within
experimental units
- size of the experimental unit and number of replicates
- number of sampled plants within each experimental unit
- genotype-by-environment-by-management interaction.
4. Weather measurements
The weather has a huge impact on the crop growth and development, and the
stress that the plants will experience. Recording accurately the main weather
variable is thus crucial in success of any field experiment.
a. Stable weather station
Generally, the daily weather data, viz., solar radiation, rain, maximum and
minimum temperatures, wind speed, air humidity, pan evaporation, are
collected from research stations where experiment is conducted or nearby
organizations that have stable weather station. The demerits of such data are
- They can be far from the field trial, whereas environmental factors such
as rain can vary within short distances.
- They only deliver daily measurements that are not always accurate to
evaluate stress events.
b. Portable weather station
A better alternative is to install a portable weather station in the field trial,
to record climatic data more frequently (eg, measurements every minute).
Typically, these weather stations have a solar radiation sensor, a tipped-
bucket rainfall gauge, and an air temperature and relative humidity probe
mounted in a Stevenson screen. In addition, many other sensors can also be
included, such as:
- Thermistors to measure soil temperature
- Thermocouples to measure soil, leaf temperatures
- Infrared sensors to measure canopy temperature continuously
- Solarimeter tubes to measure light interception
- An anemometer and a wind vane to measure wind speed and direction.
6 CHAPTER 1 Various methods of conducting crop experiments
5. Merits of field experiments

a. Field conditions are relatively close to the natural environment that crops
experience in the field.
b. It provides an opportunity to compare plants under conditions in which
spatial heterogeneity is relatively small.
6. Demerits of field experiments
a. Uncontrolled variations in light, temperature, and water supply.
b. Various environmental conditions may change in concert, that is, a period of
high irradiance may come with high temperatures and low precipitations.
1.2 EXPERIMENTS UNDER GREEN HOUSES

Glasshouses and polyhouses are good alternative and provide more buffered condi-
tions for growing plants. They offer better control of water supply and protection
against too low temperatures. Additional lighting in the glasshouse may ensure a
minimal daily irradiance and a fixed photoperiod, whereas shade screens can protect
against high light intensities in summer (Max et al., 2012).
1.2.1 DEMERITS
1. In practical terms, plants grown in glasshouses will usually experience higher-
than-outdoor air temperatures during nights and winters and lower irradiance
because of shading.
2. Most glasshouses or polyhouses without humidity control have limited
possibilities of reducing temperatures during periods of strong solar irradiance
in summer.
3. In many greenhouses where there is no artificial lighting, significant spatial
heterogeneities in irradiance due to shading by the greenhouse structure itself
are observed.
1.3 EXPERIMENTS IN GROWTH CHAMBERS

Climate-controlled growth chambers (Fig. 1.2) are expensive in terms of investments
as well as running costs. They offer the most sophisticated possibilities for environ-
mental control and thereby good reliability of experiments.
1.3.1 DEMERITS
1. Conditions in growth chambers are generally the furthest away from those in
the field, not only because environmental values are often programmed within
a relatively small diurnal range, but also with regard to the absolute values
of, for example, light and temperature, at which they operate (Garnier and
Freijsen, 1994).
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“A cry of terror broke from me, as I saw in the midst of this wild
whirlwind of fire a huge black goat, loaded with glowing red chains.
The howlings grew more fearful, the flames burst into frightful
intensity, and a troop of hideous demons, also loaded with chains,
began to dance round the goat, waving their torches, and uttering
furious shouts and yells. The goat reared on to his hind legs, butted
with his horns, and appeared to be the very genius of the infernal
scene.
“‘Ah! pardieu!’ cried de Lude, ‘the comedy is well played, I own;
but I am curious to see the coulisses, and to examine the costumes
of the actors closer.’
“He grasped his pistols, and made as if he was going to step over
the circle; but at a sign from the magician, all the flames were
extinguished, the goat and the demons disappeared. We were
plunged once more into profound darkness. At the same moment
strong arms seized us, we were dragged hurriedly along the
passages, and flung outside the cavern.
“I was only too glad of this unlooked-for ending up, and did not ask
to go back and get my philtre, and I willingly left the magician in
possession of my five louis.
“The count was not at all of the same mind. He insisted on
penetrating to the solving of the enigma. We had been the victims of
a hateful and odious charlatanism. I did not feel so convinced of that
as he was, and the abominable spectacle would not quit my
imagination. For the rest of that day, and the following night, I saw
nothing but devils dancing and howling amid the flames.”
And then it was just before break of dawn, between her sleeping
and waking, came once again the Man in Black. He smilingly
asserted himself to Ninon, to be, beyond all doubt and juggling
hocus-pocus, his Satanic Majesty, the real “Simon Pure.” In calm,
grave tones he offered her the choice of the three great gifts this
world has to bestow—riches, grandeur, beauty—enduring beauty till
all-destroying Death should claim her, and with only a momentary
hesitation, Ninon chose beauty. Then in two crystal phials, like the
one the charlatans had yesterday cheated her out of in the Gentilly
cavern, he handed her the wondrous liquid—limpid, delicately rose-
tinted; enough to last the longest lifetime, since one drop only in a
wine-glass of water, to be taken after her morning bath, was all that
was needed. First, however, he produced his tablets, and writing a
few words on one of the pages, he bade her set her signature
beneath. “Very good,” he said, when she had done this. As he
placed the phials in her hands, “Now you are mine,” and he added,
as he laid his hand on her shoulder, that her health would remain
almost unbroken through all the coming years, troops of friends and
love would be ever with her, and after death the memory of her
would be unfading. Once more she would see him—years hence.
“Then beware and tremble; you will not have three more days to
live.”
And so he disappeared.[2]
In the course of their brief conversation, the Man in Black
disclosed to Ninon the manner in which his impudent imitator
produced his Mumbo-Jumbo terrors. Like the Comte de Lude, he did
not deny them effect; but he held them so essentially vulgar, that it
seemed marvellous to him how the fellow succeeded in imposing on
refined and educated clients. Moreover, they had not even the
recommendation of novelty. Perditor had, he explained, contrived
merely to get knowledge and possession of the tricks and traps of
the long since strangled César, who during his incarceration in the
Bastille had entertained his gaolers with an account of the way he
played his tricks, performed apparently at Gentilly also at that time
and therefore rendering the way the easier to his successor, since
the old quarry he had utilised and patterned about with ditches still
remained. Perditor’s ceremonial was identically the same with
César’s. The frightful cries he uttered were the signal for six men
hideously masked and garbed, he kept concealed in the cavern, to
spring forward, flinging out flashes of flame, and waving torches of
burning resin. Amid the flames was to be seen the monstrous goat,
loaded with thick iron chains painted vermilion, to give the
appearance of being red-hot. On each side, in the obscurity of the
cavern, were placed two huge mastiffs, their heads fastened into
wooden cases, wide at one end, and narrow at the other. Two men
goaded and prodded these two poor animals, which caused them to
utter the most dismal howling, filling the cavern with the appalling
noise, while the goat, a most intelligent beast, and thoroughly
understanding his part, played it to admiration, rattling his chains and
butting his huge horns.
The devil having thus shown himself, two of the men now rush
upon the unfortunate individual, and belabour him black and blue
with long bags of cloth filled full of sand, and then fling him, half-
dead, outside the cavern. “Then the parting advice is given him not
to wish to see the devil again, and he never does, concluded César.”
CHAPTER VI
Nemesis—Ninon’s Theories—Wits and Beaux of the Salons—Found at Last

—“The Smart Set”—A Domestic Ménage—Scarron—The Fatal Carnival—The
Bond of Ninon—Corneille and The Cid—The Cardinal’s Jealousy—Enlarging
the Borders—Monsieur l’Abbé and the Capon Leg—The Grey Cardinal—A
Faithful Servant.
Ninon’s intrigue with the young Marquis de Rambouillet gave great

offence to Madame de Rambouillet. It sheds a curious light on the
manner of the great world of the time, that the doors of the
marquise’s house remained still open to her, yet so they did remain.
The justly incensed lady contented herself with soliciting an order
from the Court for the young man to rejoin his regiment in Auvergne
without delay; and Ninon was left to console herself elsewhere, and
to avenge as she might her annoyance at the epigrams showered
upon her, not to speak of the severe blame cast upon women of
society who were undeterred by any sense of propriety and the
convenable—which she was well aware was mainly levelled at
herself. All moral considerations aside, the breach of good taste is
inconceivable in one who so prided herself, and generally with
justice, on the observation of the general laws governing the people
of her class. The hospitality of the famous mansion in the rue St
Thomas du Louvre, however, was still accorded her, and if it was
more chilly than formerly, Ninon consoled herself by enlisting many
who frequented the brilliant gatherings, on the side of her easy-going
philosophy, and discussing its tenets with amazing frankness.
The women were not many who upheld her arguments; but the
men vastly applauded and seconded her sallies against the theory of
Platonic love. In her opinion, it was an impossible doctrine, and on
such themes she was Madame Oracle, and her beautiful mouth
opened to expound, what dog dare bark? Unless indeed it might be
the cardinal. “Mademoiselle,” he said, one evening when he was
present, as he frequently was, in the Rambouillet salon, and Ninon
ventured an observation not quite to his taste, “I never accept
lessons, even when they issue from such pretty lips as yours.”
The stately mansion of Rambouillet, with its magnificent grand
salon, and blue chamber, the special haunt of the poets, its daintily
furnished smaller chambers, and richly-draped alcoves and cosy
corners, was only one among many houses entertaining the society
of the world which was devoted, or assumed devotion, to art and
literature. There were the Saturdays of Madame de Sablé, and
notably also the receptions of Mademoiselle Scudéri. Mademoiselle
de L’Enclos’ own apartments were thronged on her reception nights
with the company of talented and famous men and women, though
that genial admirer of hers, St Evrémond, once had the temerity to
criticise the beauty, or the lack of it, in the ladies of the côtérie. It
might, of course, as he said, arise from mere chance; but otherwise
it was a mistake; since it suggested the idea that Ninon could not
sufficiently prize her own beauty; and on the score of the hidden
compliment the audacity was condoned. After the coolness that
followed upon Ninon’s liaison with the Marquis de Rambouillet, the
society of the salon of the marquise somewhat thinned for awhile;
while the salon of the rue des Tournelles was more thronged than
ever. The cachet that admitted to all these various assemblies would
appear to have been that only of fair breeding and connexions, and
some intellectual pretension, though the supply of that was not
necessarily very great, since the leaven of would-be wits and of
absolute stupidity—the “mostly fools” Carlyle says the world is
peopled with—would seem to have been even curiously large. One
and all, however, were full of ambition to air the rhymes, and often
senseless epigrams and dreary sonnets and conceits, generated in
their miserable brains.
Perhaps the only one of this crowd of triflers who is worth
recording is the Baron de Miranges. In addition to the fact that he
was never known to sit still two consecutive minutes, he was
supremely ugly; marked with the smallpox, he squinted, his chin was
awry, his nose twisted to one side. He was the first to jest at all these
defects. One day he met a man on the Pont Neuf, an entire stranger
to him, and halting before him, Miranges, in a sort of transport of
satisfaction, gave a joyous cry and threw himself upon the
individual’s neck, saying: “Oh, sir! how charmed I am at this meeting,
and for what a number of years I have been looking for you!”
“Indeed?” said the other, in a tone of astonishment. “I do not think I
have the honour of knowing you.”
“No. Unluckily I have met you much too late; but I look at you, I
contemplate you, and I am happy.”
“But why?”
“Yes, yes, indeed,” replied Monsieur de Miranges; “let us embrace
each other again. I have always despaired of ever finding a man
uglier than myself, but now—yes, you are that man.”
Not without justice, Ninon, who about this time had in more ways
than one drawn unfavourable public criticism upon herself,
complains that she was really less culpable, infinitely more
decorously behaved in society, than many of the titled and
fashionable dames, whose behaviour, scandalous as it was, passed
unchallenged. They were constantly promenading in the Place
Royale, chattering at the top of their voices, ogling, smoking, taking
snuff, adorning their mantles and hats with knots of ribbon of various
colours, each conveying a different significance, and generally
comporting themselves after the manner of the lowest of their sex.
Ninon de L’Enclos had made a law unto herself, a law of liberty, and
she made no pretence of not abiding by it; but she rarely sinned in
outward decorum, or forgot the good breeding of her station.
In the matter of de Rambouillet, if she did not acknowledge the
false step, it was probable she was made to feel conscious of it, and
decided soon after to divert public attention to some other topics of
scandal, by absenting herself from Paris for a while and rusticating at
Loches, the estate which her aunt had left her. On reaching le Mans,
she was met by the Marquis de la Châtre—an amiable man for
whom Ninon had sufficient attachment and constancy to allow the
good provincials to imagine they were man and wife, and the two
were widely welcomed and courted.
One evening, at a supper party to which they were invited, she
met Scarron. He arrived in company with some canons from the
cathedral, and to her great surprise she learned from him that he
now held a canonry in le Mans cathedral, bestowed upon him for the
assistance of his pen, than which few were more able than his in
Lorraine, in drawing up a history of the duchy of Lorraine.
To Paul Scarron, the brilliant wit, comic poet, rhymester—so
admired of another erratic genius, Oliver Goldsmith, who translated
his Roman Comique—the sunny-natured, in earlier years
scandalously debauched, and always bon vivant—brimming with the
overflow of humour that wells from the depths of a sympathetic
temperament—generous, kind-hearted—to:
“Nothing extenuate,
Nor set down aught in malice,”
are words hardly to be more aptly applied. The sufferings of his

childhood, due to the avarice of his artful stepmother, who contrived
to separate him from his father and get possession of his fortune,
cast him nearly penniless upon the world, when scarcely more than
a child. It was one more instance of the game, ever new, which
relatives intellectually inferior, incited by envy and greed, love to play
upon the unfortunate talented one, and render life one long misery
and struggle at the best, provided sufficient bread is somehow come
upon to retain breath. So much the brave heart and exercise of his
gifts enabled the lad to acquire, and he managed to enter
ecclesiastical ranks; but only to the outermost degree—not, it may
be, aspiring to the priesthood, which hardly could have lost anything
from one whose character and mode of life were so glaringly ill
adapted for the calling. Scarron’s vocation that way was worse than
nil; nevertheless, in that lax time of ecclesiastical law and order, he
obtained the canonry of le Mans cathedral, and thus dignified,
Monsieur l’Abbé Scarron met Ninon again at the supper-table of the
local receiver-general of taxes, and was more ready than ever for
any lengths of wild uproariousness the chance brought him. It came
just then with the Carnival, and Scarron, with one or two
companions, conceived the notion of spreading a big mattress all
over with goose’s feathers and down; then, smearing themselves
from head to foot in honey, they rolled upon the mattress until they
were encased in the feathers so thickly, that the disguise was
impenetrable, and they looked like some hideous monstrosities of
the bird-tribe, face and all covered in the plumage. Passing up the
street, followed by a huge concourse, they made their way to Ninon’s
château, and forced entrance, greatly to the anger of Monsieur de la
Châtre, who quickly discovered who they were, and at once
denounced them. The mob, furious at the thought of a churchman of
their own cathedral indulging in such wild licence, set upon the
feathered monsters, and flinging them down, pommelled and beat
the unprotected bodies of the unfortunate masqueraders, and
plucked off every feather, pursuing them without mercy, until they
were compelled to jump into the rushes of the river for protection.
There they were forced to remain for hours, and two of Scarron’s
three companions died from the effects of the cold immersion, and
the violence dealt them. Scarron himself escaped with breath, but
little more. The chill and exposure brought on an illness from which
he never recovered. It crippled him in every limb, and rendered him,
as he himself says, an abridgment of human suffering—tied to his
chair by the contraction of every muscle, in never-ending pain for all
the years to come; yet never losing his gaiety, and for all the misery
he had created for himself, winning the pity and the money gifts from
the Court and from wealthy friends which enabled him to live in fair
affluence.
A short time later the domestic felicity being enjoyed at the Loches
château by Ninon and Monsieur de la Châtre was rudely broken up
by a summons from Monsieur de la Châtre’s family, at Besançon, to
repair to the deathbed of his father. The two parted with real regret,
and so much devotion on the Marquis de la Châtre’s side, that
nothing would content him short of a written and signed promise
from Ninon of eternal fidelity to him. She accordingly wrote on a leaf
of his tablets these words—
“I swear to love you always.—Ninon.”
Carefully bestowing this precious bond in black and white in an

innermost pocket of his vest, de la Châtre conducted Ninon back to
Paris. He would have preferred to leave her in Touraine, to pass the
time of his absence in the rural tranquillity of her beautiful little
domain; but if Ninon desired to ruralise, was there not her charming
country residence at Picpus?—and Picpus is much nearer Paris than
Loches; and just then the Maréchal de Sévigné had arrived in Paris,
a man of noble presence, distinguished for his recent successes in
the king’s service, and the young Vicomte de Turenne, already
entered upon the paths of his renown, by his splendid service in
Lorraine and Italy, and both, eagerly seeking introduction to Ninon,
came, saw, and were conquered by her charm.
De Sévigné’s rendered homage was, however, on somewhat
unconventional lines, the honeyed words of his admiration being
tempered with just enough fault-finding as to render it unusually
piquant; but Ninon’s favours, and just now especially, were in no
wise exclusively bestowed on the heroes of the battlefield. She was
no more précieuse than she was Platonicienne; but she was
genuinely gifted with a love of letters, which had been fostered by
the excellent education her father had given her, and she entered
ardently into the great intellectual movement of the time, in which the
drama figured so prominently. Richelieu himself was so warm a
devotee, that his ambition to excel as a dramatist equalled, if it did
not surpass, his political ambition; and while jealous to the mean
extent envy can reach, he did not withhold his patronage from the
great genius of him who has been styled the father of the French
dramatists, Pierre Corneille. Even had Richelieu not desired as he
did, to make use of the brilliant talent of Corneille for his own ends, it
would not have been possible for him to hold aloof amid the
enthusiasm of the world of letters, and of society generally, which
hailed in 1636 the production of The Cid.
As every time “doth boast itself above better gone,” so must
Corneille’s name yield place in a degree to what has since been
seen. Still, ever remembering his fathering of it—for his
predecessors in dramatic work worthy of any name were dull and
lacked artistic knowledge of their craft, and Godelet, Gamier, and
others are but names now and no more—Corneille’s masterpiece
would challenge criticism in plenty now, placed before the delicate
discrimination of the daily press of this time, or the judgment of the
gallery, alike in his native country or elsewhere. It is but recently that
the tragedy of a great French poet, not yet two generations passed
away, revived at the Comédie Française, though reverently and
finely acted, was derided and mocked at without mercy behind the
scenes by those taking part in it. Exactly what will be the opinions of
critics of future generations on the dramatic productions of the early
years of the twentieth century, fortunately the means will probably be
lacking to know; the fact remains that the fame of Pierre Corneille is
a living force and a memory for all time.
It was the fashion of that day to model plays and novels on
Spanish and Italian patterns; and advised to follow this ruling,
Corneille selected the subject of The Cid—Rodriguez—on which to
base a drama, not his first by several; but while the preceding ones
were held in great esteem, The Cid was regarded as attaining to the
highest excellence, and its fame as his crowning work has ever
remained by it. Some of his dramas of a later date were
unsuccessful; one of his comedies, Le Menteur—the only one which
had popularity—is best known in this country by Steele’s translation
of it, The Lying Lover.
Richelieu, stirred to dramatic ambition—finding probably that it
was an art less easy than it seemed—sought the assistance of five
dramatists to write up and give more effect to his tragedies; at least
any other reason for such collaboration is not easy to be imagined.
One of the five chosen was Corneille, who, naturally somewhat curt
and abrupt in speech, did not spare to find fault with some of the
details of the cardinal’s work, and the concatenation of The Cid’s
success and of Corneille’s frankness over Euterpe and Mirame,
stirred such offence in the cardinal’s jealous mind, that he
endeavoured to drive a spoke in the wheel of Corneille’s car of
triumph; and one of the earliest achievements of the recently
constituted Académie Française was a critique on The Cid
commanded of its members by its founder. It had no effect at all in
lessening the enthusiasm of the world of letters, or of the general
public for the drama. The poison did not act, in spite of the
endeavours of several of the poetasters to second the
pronouncements. One defect, that it was not original in plot and
construction, but based on a Spanish dramatic model, was to be
conceded; if defect that was which at the time was held to be almost
indispensable in a play. There is nothing new under the sun.
Shakespeare’s comedies and tragedies alike—the English historical
plays excepted—are one and all based on old legends and classic
stories which he drew from Italian, and Spanish and French, and
other sources that had, in their turn, sprung from tradition no longer
traceable, hidden in origins lost in the lapse of centuries. Richelieu’s
own dramatic effusions were reproductions of classical themes. It
was the grandeur of the verse of Corneille, its lofty thought, its
dignity and moral conception, its depicting of conflicting passions—
this it was that won the admiration, and struck home to heartfelt
sympathies, in its power of presenting character, under other names,
of living men and women, the contemporaries of Ninon’s time,
contending, suffering, striving in the stormy political atmosphere,
darkening in now with the shadows of the Thirty Years’ War.
In the delight of Corneille’s presence in Paris, Ninon sacrificed all
the ordinary routine of her life. It was in her salon, if the chronicling of
the fact is to be trusted, that Corneille read to the assembled
company his manuscript of The Cid, all the principal members being
present of the Hôtel Bourgogne, and the few other talented “rogues
and vagabonds” proscribed of the Church, though ill to be spared by
it, if the cardinal’s plays were to have any sort of success. The
Comédie Française was yet an institution of the future; and the stage
of the Hôtel Bourgogne, with the two or three other theatres were not
much more than glorified fair platforms, while the theatre in the rue
Guénégaud ordinarily confined itself to the presentment of Chinese
shadows. The drawing-room of the Louvre and of the Palais Cardinal
were utilised for masques and such plays as there were, called in
request for the Court and the more exalted circles of society.
Richelieu’s own pieces were thus performed. The drama was in
transition. It was a far cry now from Clement Marot and the antics of
the clerks of the Basoche upon the huge marble table of the Hall of
Lost Footsteps, to the Académie Française and the Hôtel de
Rambouillet; and the language of the country was undergoing
changes, even as the aspect of the city itself was no longer that of a
few years earlier, when Ninon first came to Paris. Then Notre-Dame
was nearly surrounded by green spaces of meadowland and field
and hedgerow, stretching between the streets and the grassy banks
of the Isle de la Cité. Now here, and away to the Palais de Justice;
and northwards of the Louvre, streets were gathering, and houses
began to crowd about the old towers of the Conciérgerie; while on
the banks of the Seine, right and left, the old walls of Philip Augustus
were laid low or broken up to afford room for new buildings. Behind
the Louvre, far extending to the gardens and palace of the Tuileries,
Richelieu’s magnificent residence dominated the rue de Rivoli—the
Palais Cardinal, so soon to pass as a gift to the king and take the
name of the Palais Royal, till the Revolution of 1793 changed it to
the Palais Égalité, and the lordly “pleasure-house” of the great
upholder of kingly power was cut up into gaudy shops and gaming-
houses.
After the performance of The Cid, which took place before the king
and queen, and Court, and a vast company of illustrious persons,
Corneille returned home to Rouen, to pursue the great career he
was now launched upon. The fulminations of the cardinal through the
Académie Française far from proving destructive to his fame, had
probably cast a brighter lustre on it. “I never undertake anything
without well first considering, but once I have resolved, I go straight
to my aim; I throw all down that is in my path; I mow down all, and I
cover all with my red robes,” he once said, and it was no empty
boast. Yet the ruling found its exception; his rancour and jealousy did
its worst, but it could not crush Corneille. It did not at all events do
so. Even for Richelieu it might have been dangerous and impolitic.
Gaston d’Orléans, the king’s brother, who belonged to the party of
the queen, threw in his influence to support anything he dared in
opposition to the cardinal—and at this time Gaston was a frequent
visitor at Ninon’s house. He invited himself one evening to dinner
with her, attended by several gentlemen, and Ninon, who was kept in
countenance by her friend, Marion Delorme, and another lady,
entertained her royal guest with an elegant repast of fish, flesh and
fowl, although she had ventured to remind “Monseigneur” that, being
the season of Lent, it was a questionable proceeding to have
anything but dishes of the first served up. Gaston, however, had
insisted, especially in the matter of roast capon, and good wine—
cela va sans dire. Whether the wine was partly answerable, or it was
merely the manners of the time that prompted one of the guests—
Monsieur de Boisrobert, my lord cardinal’s secretary—who was
fingering the leg-bone of a fowl, to fling it out of window at the head
of Monsieur l’Abbé Dufaure, the venerable dean of St Sulpice, that
was what he did. The abbé was a Jesuit priest, and the scandal of
insult to him was doubled by the sin of eating meat in Lent.
Monseigneur and his companions finished the evening by adjourning
to the house of Monsieur la Navarre, a neighbour of Ninon’s, and
breaking up the furniture. Then the prince himself sent for the
magistrate, and the functionary arriving, demanded to be informed
which was the culprit. The unfortunate neighbour, who did not know
who Gaston was, pointed him out, and forthwith six archers were
sent for, who laid hands on the prince, and he was threatened with
handcuffing if he did not immediately go quietly to prison. Upon this
the gentlemen in attendance, hearing the uproar, entered, and with
profoundest respect proceeded to inquire what had happened,
addressing Monseigneur by name. Terrified out of his senses at what
he had done, the magistrate besought pardon, which the prince
gravely granted, not without commanding him to make amende
honorable by holding a lighted wax taper in his hand, and, on
bended knees, confessing his crime before all and individually of the
women of the household, who were summoned to attend for the
purpose.
So much for Monseigneur’s little amusement: it was Ninon who
was the sufferer. The insulted abbé complained to his Superior, who
complained to the magistrate of the district, and from mouth to mouth
the story flew. Not one man in black, but constant contingents of the
black-soutaned fraternity haunted the rue des Tournelles, and
invaded Ninon’s apartments, subjecting her to such severe
inquisition about her affairs generally, that it became unendurable,
and she wrote to the prince in severe reproach for allowing the
blame of his folly to burden and annoy her. Whereupon Gaston sent
two of his friends to mollify the wrath of the magistrate, who tore up
the Jesuit Superior’s letter of complaint. But the scandal only
aggravated the soreness and complications of the opposing parties
of the Court, and it made an additional grievance for Richelieu
against Gaston; though, on the other hand, it was Boisrobert, his
own secretary, who was also his own jester-in-chief, who had been
at the bottom of the offence, so that the affair cut both ways, and the
cardinal may have preferred to see it hushed up.
It was about this time that Richelieu lost by death the man he
called his right hand—Père Joseph, the Capucin friar—in other
words, “The Grey Cardinal,” as he was nicknamed; but in fact and
deed the poor man never even received the bishopric long promised,
never bestowed. Richelieu himself was already in failing health, worn
by stress and anxiety for the care of the vast structure of kingly
power he had built up and sustained, as it were, by his own hand,
that was against so many, and Louis himself was almost as much a
nonentity as any of the rois fainéants of old days. It is almost
impossible to realise that he and his false-hearted, selfish brother
should have been the sons of the dauntless Henry of Navarre.
Louis was not vicious; it was his valetudinarian melancholy
temperament which appears to have rendered him indifferent to
ordinary human interests. He made less than no pretence of
affection for his Spanish wife, for whose bright glances other men
would have staked existence. For her, Buckingham forgot honour
and duty to his own royal master, and did not spare compromising
her repute. That is a page of history that remains sealed. How far it
affected Louis’s feelings towards her through the rest of his life,
remains an open question, or whether from the beginning, love and
mutual inclination were at fault. “The wind bloweth where it listeth,”
and the beauty and attractions of Anne of Austria may never have
struck a responsive chord in the king’s heart. He was not destitute of
sentiment. More than once he strove to fill the dreary void with the
sympathy of other women of repute about the Court, and, in one
instance at all events, not unsuccessfully; but he was not one to win
love and friendship generally; and the consciousness of this chilled
his manner still more, and threw him back upon himself. Gaston
d’Orléans, with all his grave faults, had at least quicker outward
intelligence and sufficient animation to win some extensive suffrages
of the gentler sex, notably of Anne herself, who tolerated his
attentions and coquetted with him up to a certain point; though how
far this was policy, or from real sentiment, Court intrigues veil too
entirely to attempt to determine, and the jealousy of Richelieu,
himself enamoured of the queen, had soon put an end to all the
aspirations of the two dukes. “There is no such word as fail,”
Richelieu was often heard to say, and he did not fail to put his foot
down very decisively when a league was formed, which the queen
herself was said to favour, whose end and aim was to depose Louis
the Just, crown Gaston, and give him Anne of Austria to wife.
“I should not have sufficiently gained by the change,” was,
however, Anne’s reply, when the accusation of her desire for this was
made against her in the course of the rigorous inquiry and treatment
to which she and her friends were subjected. If on account alone of
that time, years back now, when Gaston, to save himself, permitted
one of his noblest adherents, Chalais, to perish on the scaffold at
Richelieu’s command, Monsieur was not likely to be very favourably
regarded by her. Nearly half a score of years had passed since the
brave man had died in the flower of his life, tortured and hacked by
countless bungling strokes of a creature found at last, among the
dregs of the prison, to do the hideous task which the professional
headsman managed to evade by absenting himself and remaining
perdu. In the interval, the queen’s mother had been effectually, and
for ever, banished from France. “The Day of Dupes” had come and
gone, leaving Richelieu all-triumphant; but still the contest raged,
and the virulence of the minister against the queen broke furiously
on the pretext he found at last, of discovering that she was keeping
up a private correspondence with the King of Spain, and the cardinal
infant, her two brothers, and also with persons in Madrid and
Brussels, whose friendship she valued—the more, doubtless, for the
isolation and lack of affection and harshness surrounding her. It was
a boast of Richelieu’s, that with only two lines of an innocent man’s
writing he could ruin him. Naturally, therefore, however innocent the
correspondence, Anne was anxious to hold her letters uninspected
by the cardinal, and she kept them in her own private oratory
chamber in the Benedictine convent of the Val de Grâce, in the rue
St Jacques, which she had founded. The letters, on their arrival,
were received by one of the nuns, who placed them away in a closet
to await the queen’s coming, and her replies to them were forwarded
from thence. But Richelieu’s spies were at work; they swarmed of
course in Paris; and before long they scented out the secret
correspondence, and Richelieu informed the king of it, holding up
before His Majesty’s dreary imagination all the terrors of national
peril it signified. The alarmed king hurried the queen out of Paris to
the Château of Chantilly, where she was confined to her own rooms
and compelled to listen to a string of rigid interrogation from the
chancellor. She was in a cruelly forlorn situation; for, in fear of
Richelieu’s anger and the activity of his spies, the courtiers and
following of the royal pair did not venture so much as to lift their eyes
to her window as they passed. For her own servants, they had been
at once disposed of in various prisons; while the chancellor
proceeded to ransack the convent of Val de Grâce for more papers
and letters. But it was labour lost, which possibly was no more than
he expected; since it is believed that the queen had warning from
him of his intended visit, and the documents, for all they might be
worth, were safe in the care of Madame de Sourdis. The alarm and
suspicion intensified, when there was found upon the person of la
Porte, the queen’s confidential servant, a letter from her to the
Duchess of Chevreuse, long exiled. La Porte was thereupon, as a
man of strict honesty and fidelity to his royal mistress, locked away in
one of the towers of the Bastille, and all the efforts to draw from him
anything incriminating the queen, were absolutely abortive; though
Richelieu employed every art to shake him, from promises and
emoluments, to threats of torture, which were rendered more real to
his imagination, by his being taken to the torture-chamber for a sight
of its equipments.
Fortunately for him, a great event was at hand, which marvellously
changed the aspect of political affairs. The queen, after twenty-two
years of childlessness, was in a situation of promise to give an heir
to the throne. Then Richelieu relaxed la Porte’s durance so far as to
permit his retiring to Saumur, where he remained till the queen
recalled him, on the death of the cardinal, now shadowing in,
bringing with it the terrible tragedy which was the last act and deed
of his hand.
CHAPTER VII
Mélusine—Cinq-Mars—An Ill-advised Marriage—The Conspiracy—The Revenge

—The Scaffold—A Cry from the Bastille—The Lady’s Man—“The Cardinal’s
Hangman”—Finis—Louis’s Evensong—A Little Oversight—The King’s
Nightcap—Mazarin—Ninon’s Hero.
Some few miles from Tours, along the banks of the Loire, at one of
its most beautiful parts above Saumur, stands the little town of St
Médard, better known as Cinq-Mars. A ruined castle crowns the
heights above. It was the ancestral home of the d’Effiats, a noble
family of long lineage; and before their coming, tradition told of its
being the dwelling of Mélusine the fée, the beautiful snake-woman,
who was the wife of Raymond, Count de Lusignan, placed under the
terrible spell of transformation into a snake, from the waist
downwards, every seventh night, for having immured her father in a
rock-bound cavern, for cruelty to her mother. Disobeying Mélusine’s
command, never to intrude upon her on those fatal Saturday nights,
Raymond discovered the appalling reason for it, and in his rage cast
her forth. The despairing cry that broke from her then, is still to be
heard of stormy nights above the river; and it may be, mingles with
the lamentations of the mourners over the deed of blood which was
enacted in after centuries when Louis the Just was king.
The young lord of the castle then, was the son of the Maréchal
Cinq-Mars. He was scarcely more than a youth; for he was but
nineteen when Richelieu introduced him at Court, loading him with
favours, causing him to be made the royal master of the horse, and
otherwise specially recommending him to the notice of Louis, who
conceived so vast a liking for him, that it was even touched with
some real warmth; and Cinq-Mars, handsome, gallant, distinguished,
brave, and not a little spoiled by the splendour of his existence, but
amiable and generous-hearted, beloved by his friends—of whom a
dear one was de Thou, the son of the great historian—basked in all
the full sunshine of his young life. The pale, stern cardinal,
attenuated by bodily suffering, and more than ever soured by care,
was hardly likely to win much love from a gay butterfly of a creature
like the young marquis, and before long Cinq-Mars came to know
from Louis’s own lips, that he privately hated Richelieu, a hate
nourished by his deadly fear of him.
Meanwhile, Cinq-Mars had cast amorous eyes upon Marion
Delorme, the cardinal’s protégée. Marion, still beautiful, though no
longer young—being in fact double the age of this her latest admirer
—returned his passionate affection, and, dazzled by the prospect of
being his wife—for his infatuation impelled him to seek her as such—
she braved the consequences of her protector’s wrath, and the two
were secretly married. Richelieu, from whom nothing could long be
hidden, was furious; he had planned a brilliant alliance for the king’s
young favourite, who had shortly before leagued himself with the
queen’s party; Gaston d’Orléans, the Duc de Bouillon—burning to
supplant the cardinal-minister—and others—and they entered into
correspondence with Olivarez, the Spanish prime-minister, which
resulted in a treaty of alliance between him and the conspiring
enemies of the cardinal. Louis had for some time past treated
Richelieu with coldness; and Richelieu, suspecting the cause of it,
left Paris, and went to Tarascon, to lie in wait till his spies were able
to place him in full possession of every detail of the plot, and of a
copy of the treaty. Then, disabled by illness and infirmity, he desired
to see the king, who travelled for the interview from Perpignan,
where he was then staying, and all the thunder of the cardinal’s
reproaches and wrath was flung upon him. Apparently with justice,
Louis succeeded in justifying himself, on the plea of ignorance, and
the king departed again, enjoining everybody to obedience to
Richelieu as if he were himself.
After their marriage, Marion and Cinq-Mars went to the castle on
the Loire, where they spent a brief period of delight. Only the
servants of the household were there, and Cinq-Mars was their lord.
They showed willing, even delighted, obedience to all his behests;
but the marquise his mother returned home somewhat unexpectedly,
and her anger at the stolen marriage equalled in its way that of
Richelieu himself. Doubtless this fomented the affair to a yet
speedier issue, and Cinq-Mars was arrested, and along with him, his
friend de Thou, who was entirely innocent of complicity in the plot.
The two were taken into the presence of Richelieu at Tarascon (a
place old stories tell named after one Tarasque, “a fearful dragon
who infested the borders of the Rhone, preying upon human flesh, to
the universal terror and disturbance”), and hence his dying
Eminence—for death was very near—commanded them to be
placed, tied and bound, in a boat fastened behind his own, in which
he was returning to Paris by the waterway of the Rhone, as far as
Lyons. There, being disembarked, the two young victims were led
immediately to a hastily-erected scaffold, and there bravely they met
their fate by the headsman’s axe—de Thou guilty of refusing to
betray his friend, and Cinq-Mars’ crime not proved, suffering mainly
from the cowardly depositions laid against him by the Duke of
Orléans. Then Richelieu continued his triumphal way to Paris, where
in his magnificent palace he died; and during his last agonies, the
king was seen to smile at what he called “Death’s master-stroke of
policy.”
There was a letter, written three days before the cardinal’s death,
found among his papers. It was dated from the Bastille, and it
consisted of one bitter reproach of his injustice to the writer, in
keeping him immured in the terrible place for eleven years. It was a
letter of some length, and an eloquently written appeal for release.
“There is a time, my lord,” it began, “when man ceases to be
barbarous and unjust; it is when his approaching dissolution compels
him to descend into the gloom of his conscience, and to deplore the
cares, griefs, pains and misfortunes which he has caused to his
fellow-creatures. Had I,” the unhappy man, whose name was
Dessault, goes on to say, “performed your order, it would have
condemned my soul to eternal torment, and made me pass into

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Phenotyping crop plants for

physiological and biochemical traits 1st

Crop Production Technologies for Sustainable Use and

Sustainable Solutions for Elemental Deficiency and

Engineering Nitrogen Utilization in Crop Plants Ashok

Water Stress and Crop Plants A Sustainable Approach 2

Priming-Mediated Stress and Cross-Stress Tolerance in

Tropospheric Ozone and its Impacts on Crop Plants A

Metabolic Phenotyping in Personalized and Public

First Aid for the Surgery Clerkship 4th Edition Latha

AMSTERDAM • BOSTON • HEIDELBERG • LONDON

Academic Press is an imprint of Elsevier

For information on all Academic Press publications

Publisher: Nikki Levy

Typeset by Thomson Digital

3.3 Growth Analysis............................................................................28

CHAPTER 4 Photosynthetic Rates................................................... 33

CHAPTER 5 Drought Tolerance Traits.............................................. 41

CHAPTER 6 Other Drought-Tolerant Traits...................................... 53

CHAPTER 7 Tissue Water Related Traits......................................... 61

7.4 Cell Membrane Injury.................................................................... 64

CHAPTER 8 Heat Stress Tolerance Traits....................................... 67

CHAPTER 9 Oxidative Stress Tolerance Traits................................ 73

CHAPTER 10 Salinity Tolerance Traits.............................................. 81

CHAPTER 12 Carbohydrates and Related Enzymes.......................... 95

CHAPTER 13 Nitrogen Compounds and Related Enzymes...............103

CHAPTER 14 Other Biochemical Traits............................................115

CHAPTER 15 Plant Pigments............................................................121

CHAPTER 16 Growth Regulators.......................................................129

16.3 Estimation of Abscisic Acid (ABA).............................................131

Appendix VI: 1M HEPES–NaOH pH 7.5 Buffer........................154

1.1 FIELD EXPERIMENTS

FIGURE 1.1 Field Experimentation With Rainout Shelter Facility.

response. Well-planned field studies and particularly those focusing on the

5. Merits of field experiments

1.2 EXPERIMENTS UNDER GREEN HOUSES

1.3 EXPERIMENTS IN GROWTH CHAMBERS

Nemesis—Ninon’s Theories—Wits and Beaux of the Salons—Found at Last

Ninon’s intrigue with the young Marquis de Rambouillet gave great

are words hardly to be more aptly applied. The sufferings of his

Carefully bestowing this precious bond in black and white in an

Mélusine—Cinq-Mars—An Ill-advised Marriage—The Conspiracy—The Revenge

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