Crop productivity per unit of water has become an important consideration in
breeding programs aimed at developing water use efficient (WUE) varieties. WUE
or transpiration efficiency (TE, defined as the biomass produced per unit of
water transpired) has been widely perceived as a useful trait for drought tolerance
(Richards, 1996). However, it is difficult to measure
TE, a complex physiological trait, in segregating populations and breeding lines
in large-scale breeding programs. Further research has shown that TE is strongly
correlated with the easily measurable trait i.e., specific leaf area (SLA) (leaf
thickness) (Wright et al., 1993; Nageswara
Rao and Wright, 1994; Wright and Nageswara Rao, 1994).
Significant relationships between TE, SLA, chlorophyll and the photosynthetic
enzyme ribulose 1-5 bisphosphate carboxylase (Rubisco) content in peanut suggested
that photosynthetic capacity is the main cause of variation for TE in peanut
(Nageswara Rao et al., 1995).
Chlorophyll content per unit leaf area (chlorophyll density) has been used
as an index of photosynthetic capacity and growth of many crop plants and it
is most important to crop performance because chlorophyll is a major photosynthetic
pigment. The major role of this pigment is to absorb and reacts with visible
light in the photosynthesis (Bowyer and Leegood, 1997).
The ability to maintain high chlorophyll density under water deficit conditions
has been suggested as a drought tolerance mechanism in barley (This
et al., 2000) and potato (Van der Mescht et
al., 1999). In peanut, Arunyanark et al. (2008)
demonstrated that the variation in TE was closely correlated with genotypic
variation in chlorophyll density and hence with photosynthetic capacity, such
that chlorophyll density could be used as a potential indicator of TE in peanut.
Several methods with well-equipped laboratory have been available for assessing
chlorophyll density. Chlorophyll is not soluble in water and it can be extracted
in 80 % (v/v) acetone solution (Arnon, 1949) or N,N-dimethylformamide
(DMF) (Moran, 1981) and then its content is determined
by spectrophotometer. Although chlorophyll density is a drought tolerance trait,
this measurement process is laborious, time consuming, costly, destructive and
inconvenient for a large number of samples. These limitations preclude its use
for plant breeding programs in which large numbers of samples are involved.
Indirect methods which are easy and rapid to use, economical, effective and
reliable to assess chlorophyll density are required.
Minolta SPAD-502 meter has been developed to assess crop health and becomes
a useful tool for scientists conducting experiments in plant science. The SPAD
meter measures green color intensity in leaves in vivo and is, therefore,
an ideal instrument for obtaining the data without destructive sampling of a
large number of chlorophyll data in the field and the reading is performed in
very short time. Close associations between SPAD chlorophyll meter reading (SCMR)
value and chlorophyll density had been reported in leaves of maize, soybean
(Markwell et al., 1995), cotton (Wu
et al., 1998), rice (Ommen et al., 1999),
wheat and potato (Bindi et al., 2002; Uddling
et al., 2007). In peanut, the information on the relationships between
SCMR and chlorophyll density in leaves also was reported by Samdur
et al. (2000) and Arunyanark et al. (2008).
Although a close correlation between chlorophyll density and SCMR has been established,
it can therefore be inferred that chlorophyll density-SCMR relationship might
be influenced by factors such as time of observation, leaf age and water deficit
The use of SCMR to assess relative chlorophyll density as alternative to the
standard method is very attractive because it is easy to operate, low cost,
non-destructive technique and can be applied in the field conventionally. Moreover,
SCMR is easy to measure, highly correlated with SLA and TE (Nageswara
Rao et al., 2001; Sheshshayee et al.,
2006; Arunyanark et al., 2008). Thus, SCMR
could be used indirectly for evaluation of drought tolerance in peanut genotypes.
However, breeding and selection scheme for drought tolerance was limited by
other important problems. Such breeding programs have to be conducted under
dry season only or in glasshouse where drought conditions can be simulated.
In field evaluation, although the number of genotypes evaluated is not limited,
it can be grown only one crop a year. In contrast to field evaluation, although
the experiment in greenhouse can simulate drought, it has a limited space to
grow a large number of peanut genotypes. Moreover, in large-scale breeding programs,
it is difficult to complete SCMR observations within a specified time and plant
stage, or could not take sample at same leaf position for all.
Surrogate traits for drought tolerance such as chlorophyll density and
SCMR will help to increase potential of breeding scheme if they are able
to identify drought tolerance genotypes with chlorophyll density and SCMR
stability across water supply conditions. Thus, the evaluation can be
carried out for all seasons. Moreover, SCMR would be helpful to breeders
if these measurements could be observed in several times or several leaf
positions. Therefore, information on the effects of different water regimes,
plant ages and leaf positions on chlorophyll density and SCMR relationships
The experiments with a wide range of peanut germplasm and environment
conditions will help clarify the technique. The objectives of this study
were to examine the stability of chlorophyll density and SCMR and evaluate
the relationships between chlorophyll density and SCMR in different leaf
positions at different times under different drought stress conditions.
These observations can be applied for developing peanut varieties tolerant
to drought stress conditions.
MATERIALS AND METHODS
Experimental design and treatments: The field experiment was conducted
at the Field Crop Research Station, Department of Plant Science and Agricultural
Resources, Faculty of Agriculture, Khon Kaen University (latitude 16° 26
N, longitude 102° 50 E), on Yasothon soil series (loamy sand, Oxic Paleustults),
in Thailand, during the dry seasons (November 2003-May 2004). A split-plot in
a randomized complete block design with four replications was used. Three different
water regimes (field capacity; FC, 2/3 available water (AW) and 1/3 AW) were
assigned in main plots and twelve peanut cultivars in sub-plots. Twelve peanut
cultivars were chosen based on their differences in drought resistance and leaf
color. Eight of these lines (ICGV 98300, ICGV 98303, ICGV 98305, ICGV 98308,
ICGV 98324, ICGV 98330, ICGV 98348 and ICGV 98353) were drought resistant germplasm
introduced from the International Crops Research Institute for Semi-Arid Tropics
(ICRISAT). Tifton-8 is a drought resistant line introduced from the United State
Department of Agriculture (USDA). Two cultivars (KK 60-3 and Tainan 9) were
commercial varieties grown in Thailand and are known to be sensitive to drought.
A non-nodulating line (yellow- green leaf color) was used as a control for low
chlorophyll and drought susceptible genotype. The crop in each sub-plot (peanut
cultivar) was grown in eleven-row plots, 6 m long, with a spacing of 0.5 m between
rows and 0.2 m between plants within row. A subsoil-drip-irrigation system installed
at 10 cm below the soil surface and fitted with a pressure valve and water meter
ensured the uniform supply of measured amount of water across each plot. The
more detailed experiment setup, irrigation control and crop management had been
reported previously by Arunyanark et al. (2008).
Soil moisture content: Soil moisture contents were measured by the
gravimetric method at the depths of 0-5, 25-30 and 55-60 cm at planting
and harvest. Weekly changes of soil moisture were measured by the neutron
method from all sub-plots at 30, 60 and 90 cm of soil depth.
Chlorophyll density and SPAD chlorophyll meter readings: Data were recorded
for SCMR, chlorophyll density (chlorophyll content per unit leaf area) at three
times as 20, 40 and 60 (DAE). Five plants from each sub-plot were randomly sampled
and the first, second and third fully-expanded leaves from the top of the main
stems were used for SCMR assessment at 9.00-10.00 am, SCMR were recorded by
a Minolta SPAD-502 meter (Tokyo, Japan) on the four leaflets of each individual
leaf as described (Nageswara Rao et al., 2001).
An average SCMR for each plot was derived from 20 single observations (4 leafletsx5
Only one leaflet from each sample leaf in each plot was used for chlorophyll
determination. The leaflet previously used for SCMR was harvested and
kept chilled unit it was analyzed. One leaf disc was taken from each leaf
let using a cork borer with a diameter of 1 cm2. Leaf discs
were soaked in 5 mL of N,N-diamethylformamide and kept in dark for 24
h before determining chlorophyll using a light absorption with a spectrophotometer.
Chlorophyll density (per unit leaf area) was analyzed following the procedures
described by Moran (1981).
Statistical analysis: The data were pooled over times of sampling to
investigate the main effects and interaction effects. The pooled analysis of
variance for measurements over time based on a split plot design (Gomez
and Gomez, 1984) was performed for SCMR and chlorophyll density. Leaf positions
were analyzed separately.
As time of sampling is the large source of interactions, separate times
of sampling were later analyzed using a split plot design. Water regimes
were assigned in main plots and peanut genotypes in subplots. Leaf positions
were treated as sub-samples. Mean separations were conducted by using
least significant difference (LSD). All calculations were conducted by
PROC GLM in SAS (SAS Institute Inc., Cary, NC, USA).
Simple correlations among and within main effects under study were computed
for SCMR and chlorophyll density.
RESULTS AND DISCUSSION
Soil moisture: Soil moisture levels for different water regimes,
as determined by percentages of available moisture content, at different
depths of the soil profile showed a reasonable control of the water-regime
treatments. Clear differences in soil moisture percentages among water-regimes
were shown, with soil moisture levels for the individual treatments being
close to the expected percentages (10.55 , 8.48 and 6.40 % for FC, 2/3
AW and 1/3 AW) as determined by pressure plate from the soil properties.
The differences among water regimes in soil moisture contents were much
clearer at the depth of 30 cm in which soil moisture of 1/3 AW were lower
than those of FC and 2/3 AW and soil moistures of 2/3 AW were lower than
those of FC (Fig. 1a). Soil moisture at 60 cm of soil
depth has clear difference between FC and water stress levels, but soil
moisture contents at 2/3 AW and 1/3 AW levels were rather close together.
At the depth of 90 cm, the soil moisture contents among three water regimes
were overlapped (Fig. 1b, c).
||Soil moisture content (%) in three available water regimes
(field capacity (FC), 2/3 available water (2/3 AW) and 1/3 available
water (1/3 AW)) at (a) 30, (b) 60 and (c) 90 cm of the soil depth
in the field
G x E interaction effects for chlorophyll density and SCMR: Across
times of sampling, differences in SCMR were found among water regimes
in the first and second leaf positions, but the differences in chlorophyll
density were not significant for all leaf positions (Table
1). The differences were also found among peanut genotypes and times
of sampling in all leaf positions. The interaction effects associated
with time of sampling (W x T and G x T) were most significant, whereas
other interaction effects (G x W and G x W x T) were not significant for
most cases except for G x W x T for chlorophyll density in second leaf
position. Generally, G x E interactions was more pronounced for chlorophyll
density than for SCMR, suggesting the stability of SCMR trait across environments,
compared to chlorophyll density.
It is interesting to note here that, although water regimes and peanut
genotypes were significantly different, their interaction effects were
not significant for both chlorophyll density and SCMR (Table
1). This pattern was found in all leaf positions. The findings indicate
the stability of SCMR and chlorophyll density in peanut across water regimes
and peanut genotypes.
Similar to present findings, chlorophyll density and SCMR has shown to be a
stable parameter across seasons and a low genotypexseason interaction was reported
by Arunyanark et al. (2008) and Nageswara
Rao et al. (2001). The results also suggested that selection for
chlorophyll density and SCMR could be carried out under any water conditions
under investigation with similar results. Timexgenotype interaction variances
for chlorophyll density and SCMR, though significant, were much smaller than
those of their associated main effects. The variations in chlorophyll density
and SCMR across times of sampling were also affected by the differences in growth
stages in which plant development was not complete at early growth stages. The
higher G x E interaction variances in first leaf position than in second and
third leaf positions also supports these findings and also suggests to avoid
sampling at first leaf position.
Effects of drought stress on chlorophyll density and SCMR: Both chlorophyll
density and SCMR gave similar information on the effects of drought stress in
peanut. Differences in chlorophyll density and SCMR among water regimes were
found at 60 DAE only and the highest chlorophyll density and SCMR were found
at 1/3 AW (Table 2). The result indicates that more stress
higher is the density of chlorophyll. Drought stress increased chlorophyll density
as well as increasing transpiration efficiency in peanut (Arunyanark
et al., 2008). The increase in chlorophyll density is most likely
due to the thicker leaves of stressed plants (lower specific leaf area) (Nageswara
Rao and Wright, 1994), causing higher chlorophyll content per unit area.
The peanut genotypes showed significant differences in chlorophyll density
and SCMR with similar rankings at all sampling times (Table 2).
ICGV 98324, ICGV 98330 and KK 60-3 gave high chlorophyll density and SCMR, Tifton-8,
ICGV 98305, ICGV 98348 and ICGV 98353 showed intermediate, whereas non-nod was
the lowest at all sampling times. The results suggested that ICGV 98324, ICGV
98330 and KK 60-3 are promising genotypes for use as parents in drought tolerance
in breeding programs. There was an evidence that leaf chlorophyll density was
used as a criterion in selection for TE in peanut (Arunyanark
et al., 2008; Sheshshayee et al., 2006)
and wheat (Fotovat et al., 2007). Therefore, ability
to maintain chlorophyll density of water stressed plants can be a criterion
in selection for drought tolerance. This et al. (2000)
found that the measurement of the chlorophyll density could be considered as
a simple quantitative trait related to drought tolerance in barley. Moreover,
Van der Mescht et al. (1999) found that the levels
of chlorophyll density could be used as an indication of drought tolerance in
Differences in chlorophyll density and SCMR among leaf positions were
found as early as 20 DAE and were likely to rank in the order of first
< second < third, respectively. Peanut genotypes were also different
in stages of development in which the maximum chlorophyll density in leaves
was developed. Maximum chlorophyll density is dependent on plant ages
and leaf ages and both characters are under genetic and physiological
controls. This evidence indicated that Minolta SPAD-502 meter was able
to detect difference in relative chlorophyll density even among leaves
within peanut cultivars and therefore, sensitive in vivo measurement
of relative chlorophyll density in peanut leaves.
Relationship between chlorophyll density and SCMR: The correlation
coefficients between chlorophyll density and SCMR were positive and significant
across irrigation treatments, with the correlation coefficients of 0.76**,
0.94** and 0.96** at 20, 40 and 60 DAE, respectively (Fig.
2). The results indicated a close and stable relationship between
chlorophyll density and SCMR across water regimes in all plant ages.
Correlation coefficients between SCMR and chlorophyll density in peanut
leaves were calculated for each water regime, plant age and leaf position
(Table 3). Under well-watered conditions at 20 DAE,
the correlation coefficients of three leaf positions ranging from r =
0.31 to 0.75** were much lower than those at 40 (r = 0.77** to 0.99**)
and 60 (r = 0.93** to 0.97**) DAE.
|| Pooled analysis of variance for measurements over time
in split-plot design of chlorophyll density and SPAD chlorophyll meter
|Significant at *p < 0.05 and **p < 0.01 levels.
L1, L2 and L3: First, second and third leaf positions
|| Chlorophyll density and SPAD chlorophyll meter reading
(SCMR) of peanut at 20, 40 and 60 day after emergence (DAE)
|Mean in the same column within the same letters(s) with
the same letters are not significantly different by least significant
difference (LSD) at p < 0.05
|| Correlation coefficients between chlorophyll density
and SPAD chlorophyll meter reading
|*, **Significant at the 0.05 and 0.01 probability level.
DAE: Day after emergence
||Correlation between chlorophyll density and SPAD chlorophyll
meter reading (SCMR) across three water regimes (field capacity (FC),
2/3 available water (2/3 AW) and 1/3 available water (1/3 AW)) (n
= 36) at (a) 20, (b) 40 and (c) 60 days after emergence. ** Significant
at 0.01 probability level
Under 2/3 AW, the correlation coefficients (r = 0.75** to 0.80**) at
20 DAE were significant and lower than those at 40 (r = 0.92** to 0.96**)
and 60 (r = 0.87** to 0.92**) DAE. Under 1/3 AW, the correlation coefficients
(r = 0.78** to 0.91**) at 20 DAE were generally lower than those at 40
(r = 0.77** to 0.92**) and 60 (r = 0.88** to 0.94**) DAE except for the
third leaf position at 40 DAE (r = 0.77**).
The first-fully expanded leaf from the top had high correlation between chlorophyll
density and SCMR. However, the photosynthetic machinery in the leaves in this
position may not fully develop as indicated by lower chlorophyll density. Chlorophyll
density in the first fully expanded leaf was always lower than that in the second
and third fully expanded leaves. The variations in ontogenetic development of
the first-fully expanded leaves may occur in peanut population. Therefore, precaution
should be taken for determining chlorophyll density in peanut leaves. Present
results indicated that the second or third fully expanded leaf would be more
suitable for taking chlorophyll measurements or SPAD readings. Although there
was no detail on justification for leaf samplings, Nautiyal
et al. (2002) used leaflets of the second leaf from the apex of main
stem of peanut cultivars for measurements of carbon exchange, relative water
content and chlorophyll fluorescence.
When only sampling times at different water levels were considered, ignoring
the effect of leaf position, the correlation coefficients between SCMR
and chlorophyll density were highly significant (p < 0.01) at all of
three water regimes (Table 3). However, at 20 DAE, the
correlation coefficients (r = 0.65** to 0.88**) were lower than those
at 40 (r = 0.91** to 0.94**) and 60 (r = 0.88** to 0.92**) DAE.
The correlation coefficients between SCMR and chlorophyll density were
positive and highly significant, indicating closer relationship of SCMR
and chlorophyll density in the first, second and third-fully expanded
leaves and all the plant ages of sampling times, both at non-water stress
and water stress conditions, except at 20 DAE when the lowest relationships
The recent study suggested that water stress and different leaf positions
had no significant effect on the relationship between SCMR and chlorophyll
density and plant ages had a small effect on this relationship. The older
plants the stronger correlation coefficients between SCMR and chlorophyll
density would be. The results indicated that 40 and 60 DAE were the most
suitable dates for taking chlorophyll measurement by SCMR because at these
dates the correlation coefficient values were the highest. Moreover, at
40 and 60 DAE, maximum chlorophyll in leaves was developed as indicated
by the highest chlorophyll density in these date. Sampling at 20 DAE would
be less suitable due to its low correlation between chlorophyll density
and SCMR. Moreover, the lowest chlorophyll density at 20 DAE indicated
that chlorophyll was not developed completely at this age.
The SCMR is an indicator of the light-transmittance characteristics of the
leaf, which is dependent on the leaf chlorophyll density (Richardson
et al., 2002). The significant and positive correlation between SCMR
and chlorophyll density across genotypes under a range of drought stress conditions
in the present study (Table 3) demonstrated that SCMR could
be a rapid tool to assess genotypic variation for leaf chlorophyll density in
order to identify drought tolerant genotypes. Relationships between chlorophyll
density and SCMR in wheat flag leaves (Ommen et al.,
1999) and in potato leaves (Bindi et al., 2002)
were curvilinear. In this study, there was a linear relationship between chlorophyll
density and SCMR in peanut (Fig. 2 and 3c).
||Correlation between chlorophyll density (ChlD) under
non-stress (FC) and water stress (2/3 AW) conditions (a), between
SPAD chlorophyll meter reading (SCMR) under non-stress and water stress
conditions (b) and between SCMR under non-stress and ChlD under water
stress conditions (c) in the second leaf position at 40 day after
emergence. **Significant at 0.01 probability levels
Interestingly, correlation between chlorophyll density under non-stress
(FC) and water stress (2/3 AW) conditions (Fig. 3),
between SCMR under non-stress and water stress conditions and between
SCMR under non- stress and chlorophyll density under water stress conditions
were strong, positive and significant in the second leaf position at 40
DAE. As well as, high significant correlation with each other were found
for all combinations of water regimes both 40 and 60 DAE and with third
leaf position (data not shown). It implied that chlorophyll density and
SCMR, a surrogate trait of drought tolerance in peanut, were stable in
different water regime conditions. It suggested that performance of chlorophyll
density and SCMR were repeatable under both drought and non-drought stress
conditions. This gives a large flexibility to breeders who want to evaluate
genotypes for drought tolerance. Because the evaluation can be carried
out without conducting drought conditions, it can be done for several
crops a year.
However, light intensity has been shown to affect the chlorophyll meter readings
by the Minolta SPAD-502 chlorophyll meter. Irradiance during the sampling period
could also be critical. Hoel and Solhaug (1998) found
that the SPAD reading was lowest in the middle of the day and highest at dusk
and dawn. In addition, several factors affecting SPAD values consist of radiation
between seasons, plant density, varietal groups, nutrient status in soil and
biotic and abiotic stresses that induce leaf discoloration (Turner
and Jund, 1994). Users should be aware of these interfering factors and
should take adequate precaution against them while using the SCMR.
It has been concluded that plant age, drought stress and leaf positions
could affect chlorophyll density and SCMR. However, water regime x genotype
interaction was not significant for both chlorophyll density and SCMR,
indicating the stability of both chlorophyll density and SCMR across different
water regimes. The relationships between chlorophyll density and SCMR
were high, positive and significant, suggesting that SCMR is a useful
tool for assessing chlorophyll density in peanut. Plant ages had a small
effect on the relationships between chlorophyll density and SCMR, whereas
water stress and leaf positions had no significant effect on the relationship.
It is clear that using SCMR for evaluation of chlorophyll density can
be recorded at any water regime conditions in the second or third-fully
expanded leaves after 40 days of crop growth. This gives a large flexibility
to breeders who have to record observations in a large number of segregating
and breeding populations in the field. Drought simulation is not necessary
because selection under drought condition gives the results similar to
those under well watered condition.
The authors are grateful for the financial support of the Royal Golden
Jubilee Ph.D Program (Grant No. PHD/0026/2547) and the Senior Research
Scholar Project of Dr. Aran Patanothai under the Thailand Research Fund
and also supported in part by the basic research for supporting groundnut
varietals improvement for drought tolerance project under the Thailand