ISSN (E): 2349 – 1183

ISSN (P): 2349 – 9265

5(1): 83–87, 2018
DOI: 10.22271/tpr.2018.v5.i1.012
Research article

Estimation of the chlorophyll concentration in seven Citrus

species of Kokrajhar district, BTAD, Assam, India
Mohesh Gogoi1* and Miniswrang Basumatary2
1
Department of Botany, Science College, Kokrajhar, BTC, Assam, India
2
Institutional Biotech Hub, Science College, Kokrajhar, BTC, Assam, India
*Corresponding Author: [email protected] [Accepted: 25 April 2018]

Abstract: Green plants synthesize a wide range of primary and secondary metabolites in different
quantity. Chlorophylls belong to the primary metabolites that give the color of the leaves and
fruits, especially in the unripe stage. The spectral properties of chlorophylls are essential in
harvesting light energy and in the transduction of absorbed light energy for photosynthesis. Like
other plants, the variation of leaf colour, as well as photosynthetic activity in Citrus species, is
dependent on chlorophyll concentration. Chlorophyll content determines the photosynthetic
capacity of the plant per unit area of leaf, stress and nutritional deficiencies. The study of
chlorophyll content in seven Citrus species growing in different localities of Kokrajhar district of
Assam has shown some variability among them as an individual character.
Keywords: Citrus leaf - Pigment - Photosynthesis - Metabolite - Productivity.

[Cite as: Gogoi M & Basumatary M (2018) Estimation of the chlorophyll concentration in seven Citrus species
of Kokrajhar district, BTAD, Assam, India. Tropical Plant Research 5(1): 83–87]

INTRODUCTION
Chlorophylls are the most important green pigments in plants for the photosynthetic process (Bhatia &
Parashar 1997). Higher plants contain Chl a, Chl b, accessory pigments and several additional forms of
chlorophyll. The Chl a and Chl b are the best known among five main types of chlorophyll and are most
commonly found in all autotrophic organisms except pigment containing bacteria. Chl a has an empirical
formula of C55H72O5N4Mg and the empirical formula of Chl b is C55H70O6N4Mg. Chl a usually appears blue-
green and Chl b is yellow-green (Devlin & Witham 1997). Both Chl a and Chl b pigments are associated with
light harvesting processes (Ferus & Arkosiova 2001), which are solely responsible for photosynthesis in higher
plants.
Chlorophyll concentration in leaves is an indicator of plant health (Porra 2002). The chlorophyll a:b ratio
also indicates the developmental state of photosynthetic apparatus in plants. It has a determinative role in growth
and development of higher plants. The chlorophyll content also indicates the photosynthetic capacity per unit
area of the leaf (Kozlowski et al. 1991) that determines the rate of photosynthesis in the plant (Dickman &
Kozlowski 1968). Determination of chlorophyll content as an indirect method of estimating the productivity also
provides a good understanding of the photosynthetic regime of plants (Bojovic & Stojanovic 2005).The
chlorophyll content increases with leaf development and then decreases with the senescence phenomenon
(Pereyra et al. 2014). The rate of photosynthesis is also higher in flowering and fruiting branches of sub-tropical
fruit species in comparison to non-fruiting branches (Avery 1977). However, the pigment is a factor that might
also be responsible for the colour variation of leaf in different Citrus species. The present study was undertaken
for estimation of chlorophyll content in the mature leaf of Citrus plant growing in Kokrajhar district, Assam.

MATERIALS AND METHODS

Seven Citrus species namely C. limon Burm f, C. medica Linn., C. aurantifolia Swin., C. reticulata Blanco.,
C. nobilis Lour., C. jambhiri Lush., and C. grandis (L.) Osbeck. as reported by Gogoi & Basumatary (2017)
were selected for the present study. The matured leaves were collected from the selected Citrus spp. growing in
different areas of Kokrajhar during the month of December 2017. One gram leaf from each species is measured

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Received: 15 January 2018 Published online: 30 April 2018
https://doi.org/10.22271/tpr.2018.v5.i1.012
 Gogoi & Basumatary (2018) 5(1): 83–87

and cut into fine pieces and then grinded with mortar and pestle. Thereafter, 20 ml of 80% acetone and 0.5 g of
(MgCo3) powder was added and further grinded gently following the method of Kamble et al. (2015). The
mixture was then incubated at 4ºC for 3 hours. The mixture was centrifuged at 2500 rpm for 5 min and the
supernatant was transferred to a 100 ml volumetric flask and the volume was made up to 100ml with the
addition of 80% acetone and the solution was used for chlorophyll estimation (Fig. 1). The absorbance of the

Figure 1. Leaves and their chlorophyll extraction in acetone solution: A, Citrus medica; B, Citrus jhambiri; C, Chlorophyll
extract of C. medica & C. jhambiri; D, Citrus nobilis; E, Citrus reticulata; F, Chlorophyll extract of C. nobilis & C.
reticulata; G, Citrus aurantafolia; H, Chlorophyll extract of C. aurantafolia; I, Citrus limon; J, Chlolorophyll extract of C.
limon; K, Citrus grandis; L, Chlorophyll extract of C. grandis.
solutions was measured at 645 nm and 663 nm in LABTRONIC Spectrophotometer LT-39 taking the 80%
acetone solution as blank (Sadasivam & Manickam1996). The reading was taken in a triplicate sample and
average was considered for calculation of chlorophyll content. The chlorophyll a, b and a + b (total chlorophyll)
contents were calculated out by applying the following (Arnon 1949) formulae:-

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Where, A = absorbance at specific wavelength

V = final volume of chlorophyll extract in 80% acetone
W = fresh weight of tissue extracted

RESULTS AND DISCUSSION

Table 1. Chlorophyll concentration in seven Citrus spp. found in Kokrajhar district of Assam.
O.D. Readings Chl a Chl b Total Chl
S.No. Name of Species
645 nm 663 nm (mg g-1) (mg g-1) (mg g-1)
1 Citrus limon Burm f. 0.554±0.137 1.277±0.002 1.47 0.61 2.08
2 Citrus medica Linn. 0.404±0.003 0.904±0.002 1.03 0.50 1.53
3 Citrus aurantifolia Swin. 0.231±0.014 0.326±0.032 0.35 0.38 0.73
4 Citrus reticulata Blanco. 0.397±0.001 0.765±0.002 0.87 0.55 1.42
5 Citrus nobilis Lour. 0.354±0.001 0.699±0.002 0.79 0.48 1.27
6 Citrus jhambiri Lush. 0.269±0.004 0.456±0.004 0.51 0.41 0.92
7 Citrus grandis (L.) Osbeck. 0.339±0.010 0.583±0.010 0.65 0.49 1.14

Figure 2. The variation of chlorophyll a and b content among seven Citrus species.
The study has revealed that the Chl a ranges from 0.35 to 1.47 mg g-1 and Chl b ranges from 0.38 to 0.61 mg
g and the total chlorophyll Chl (a+b) ranges from 0.73 to 2.08 mg g-1 in seven Citrus spp. From the result it is
-1

also seen that C. limon Burm f. has the highest concentration of Chl a and Chl b whereas C. aurantifolia
(Christm.) Swin. was found to have least quantity of Chl a and Chl b among the seven species (Table 1 & Fig.
2). Among the seven citrus species the highest total chlorophyll content was found in C. limon (2.08 mg g-1)
followed by C. medica (1.53 mg g-1), C. reticulata (1.42 mg g-1), C. nobilis (1.27 mg g-1), C. grandis (1.14 mg g-
1
), C. jambhiri (0.92 mg g-1) and C. aurantifolia (0.73 mg g-1) respectively (Table 1 & Fig. 3). Similarly, Chl a
was also found to be highest in C. limon (1.47 mg g-1) followed by C. medica (1.03 mg g-1), C. reticulata (0.87
mg g-1), C. nobilis (0.79 mg g-1), C. grandis (0.65 mg g-1), C. jambhiri (0.51 mg g-1) and C. aurantifolia (0.35
mg g-1) respectively. The second highest in quantity of Chl b was found in C. reticulata (0.55 mg g-1) followed
by C. medica (0.50 mg g-1), C. grandis (0.49 mg g-1), C. nobilis (0.48 mg g-1), C. jambhiri (0.41 mg g-1), and C.
aurantifolia (0.38 mg g-1) respectively. The highest variation of chlorophyll a and b were observed in C. limon.
The quantitative variation of chlorophyll content in Citrus spp. may be due to the health condition of the plants,
habitat condition, leaf surface area and nutrients of the soil. Earlier Bojovic & Stojanovic (2005) reported that
chlorophyll and carotenoid content in wheat cultivars depends on the presence of mineral elements in the
substrate and plant physiological and environmental factors. Moreover, the application of plant growth
regulators in higher concentrations has also positive effects on leaf chlorophyll content (Sardoei 2014).
According to Pandey & Sinha (1998) Chl a and Chl b occur together in the higher plants in the ratio of 2:1.
The typical Chl a/b for shade plants is about 1.6–2.2 and daily maximum sunlight exposed plants have the
typical Chl a/b content approximately 2.6–3.4 (Anderson 1986). In Citrus species the Chl a/b was observed in
between 0.73–2.08 which is much lower than Anderson’s view. The reason of dissimilarities of Chl a/b with
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other plants may be due to the presence of inactive light harvesting complexes in Citrus resulting effect in
growth and development. Several literature and pieces of evidence suggest that the Chl a:b ratio plays an
important role to higher plants to adapt to new light regions to make optimal use of ambient light intensities and
quantities (Arnon 1949). The ratio of Chl a and b is stable in fully green leaves at nearly 3, but it can vary
depending on the physiological status of the plant (Kouril et al. 1999). An average Chl a:b ratio of 0.81:0.49
was observed in Citrus plants. Bondada & Syvertsen (2003) reported that the nitrogen-deficient leaves of Citrus
reticulata Blanco contain less chlorophyll per unit area, but a greater chlorophyll a:b ratio than N-fertilized
leaves. The Chl a/b in maximum sunlight exposure and shade plants reflect the adaptation of the chloroplast to
prevailing intensity through regulation of the amount of photosystem I (PS I) relative to photosystem II (PS II)

Figure 3. Total chlorophyll content in seven different Citrus species.

and the size and composition of the light harvesting complexes (LHCs) of each photosystem. Moreover, the leaf
colors of a plant can be used to identify stress level due to its adaptation to environmental change (Shibghatallah
2013). Therefore, from the observation it can be said that the C. limon and C. medica were well adapted under
local environmental condition. Kamble et al. (2015) opined that the leaf chlorophyll concentration plays a vital
role in maintenance of photosynthetic mechanism as well as plant metabolism. Apart from these the seasonal
variation and maturity of the leaf also affect the concentration of chlorophyll content in leaf and chlorophyll a:b
ratio remains substantially lower in plants growing in high CO 2 condition (Cave et al. 1981). Nevertheless, in
the present investigation, a variability of chlorophyll content in the Citrus species observed but it has a scope to
consider as tools for identification of Citrus species and varieties. From the (Table 1 & Fig. 2 & 3), an inference
can be made that the Chl a, Chl b and total chlorophyll content in mg/g leaf tissue of different Citrus species are
an individual character of each Citrus species studied. Remarkable variation of green colour of the leaf was
observed as the same is dependent on the variability of chlorophyll content in the species. The alteration of
Chlorophyll content may due to the factors like water, soil, temperature stress etc. which indirectly affect the
leaf area, morphology, thickness and chloroplast distribution.

CONCLUSION
The study provides a reliable data on chlorophyll content of seven citrus species growing in different
localities of Kokrajhar district. The quantitative analysis of photosynthetic pigment showed that the
photosynthetic potential and primary productivity is highest in Citrus limon followed by C. medica among the
seven Citrus spp. Further, the chlorophyll content can be used as indicators of plant health stress and nutritional
deficiencies. Our findings may be helpful in the further studies to monitor the effect of changing micro-climate
on chlorophyll content in citrus plants in terms of temperature, water, carbon dioxide concentration and soil
condition of Kokrajhar district of Assam.

ACKNOWLEDGEMENTS
Financial support from DBT, Govt. of India through the establishment of Institutional Biotech Hub is highly
acknowledged. Authors are also thankful to Principal, Science College, Kokrajhar for his encouragement and
support. Chiranjib Das, Lab Attendant is also acknowledged for his constant help during the lab work.

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