NewBioWorld (2022) 4(1):1-6
Investigation of biochemical changes in the leaves of Curcuma caesia Roxb. under
sucrose-induced osmotic stress environment
Afreen Anjum and Afaque Quraishi*
School of Studies in Biotechnology, Pt.
Ravishankar Shukla University, Raipur, Chhattisgarh, INDIA
*Corresponding Author Email-
30 March 2022
Received in revised form
28 June 2022
Curcuma caesia Roxb., also known as black turmeric,
is a rare rhizomatous herb in the Zingiberaceae family. In the present study,
we used the two different sucrose concentrations (3% and 9% sucrose) in
Murashige and Skoog medium supplemented with 8 mg/l benzyladenine, 8 mg/l kinetin,
100 mg/l citric acid, 200 mg/l adenine sulfate and 2 mg/l indole-3-acetic
acid to investigate the biochemical differences in the leaves of C. caesia. Biochemical parameters such
as total sugar, proline, protein, chlorophyll, and hydrogen peroxide (H2O2)
content, as well as superoxide dismutase (SOD) and catalase (CAT) activity,
were analyzed to study the differences. All the parameters were performed on
the 0th and 30th day after inoculation (DAI). It has
been found that as sucrose concentration raised from 3% to 9% sucrose, the
SOD activity, as well as the total sugar, proline, protein, chlorophyll, and
H2O2 content increases significantly at 30th
DAI. These increased enzymatic and non-enzymatic defense systems, thus
helping the plant to maintain its growth under a stressed environment.
Reactive oxygen species,
Curcuma caesia (C.
caesia) is an endangered rhizomatous herb with high medicinal and economic
value (Sharma et al. 2017). Volatile essential oil of rhizomes of this species
consists of about 30 components representing 97.48% of oil with camphor (28.3%)
and others as their major constituent (Verma et al. 2010). The rhizome of C .caesia is widely used in making
Ayurvedic drugs in India (Mishra 2013). It possesses anti-neurodegenerative,
antidiabetic, antiulcer, smooth muscle relaxant, anticonvulsant, and anxiolytic
effects and cures metabolic disorders like leukoderma, asthma, piles, tumor, bronchitis,
etc. (Devi et al. 2015). Tribes of Chhattisgarh use a paste of fresh rhizomes
in curing skin diseases (Mahato and Sharma 2017).
Presently, C. caesia is considered to be an
endangered species by the Central Forest department of India (Sharma et al.
2017). Plant density is declining due to poor regeneration and wild harvesting
in Madhya Pradesh and Chhattisgarh states and if this condition continues this
species will soon get completely vanished from the natural forest of Central
India (Mishra 2013). In vitro propagation
and conservation methods can salvage the C.
caesia from extinction (Krishnan et al. 2011). Pharmaceutically important
secondary metabolites of medicinal plants can be enhanced by in vitro elicitation for their higher
exploration (Chauhan et al. 2018). The most popular carbon source utilized in
plant tissue culture media, sucrose, is also known to cause abiotic osmotic
stress when applied in excess (Javed
2002c; Ahmad et al. 2007). The plant underwent certain biochemical or molecular
modifications to maintain osmotic balance in the stressful environment, which
resulted in a shift in the solute content in the apoplast of the plant (Munnik
and Meije 2001). Under stressful conditions, it is also known that plants
activate many signaling molecules that aid in maintaining their metabolism and
allowing them to live (Zhang et al. 2006). Literature is available on in vitro propagation studies (Haida et al. 2022), antioxidant
properties of rhizomes of C. caesia
(Karmakar et al. 2011), and in vitro
microrhizome induction (Sarma et al. 2021). Not many reports are available on
biochemical studies of sucrose-induced osmotic stress on C. caesia. Therefore in the present study, biochemical changes were
used to investigate the sucrose-induced osmotic stress.
Materials and Method
Three-month-old cultures which were grown on Murashige and Skoog (MS) medium
(Murashige and Skoog 1962), supplemented with 8
mg/l benzyladenine, 8 mg/l kinetin, 100 mg/l citric acid, 200 mg/l adenine
sulfate, and 2 mg/l indole-3-acetic acid (standard medium) (Anjum et al. 2022)
(Fig. 1), they were inoculated on the standard medium along with 3% and 9%
sucrose. Biochemical examination of both of the aforesaid mediums was performed
at 0th and 30th DAI (the day after inoculation). Three
replicates were used in each test, which was repeated twice. The following
experiments were carried out:
Total sugar content
Dubois et al. (1956) described the method of detecting the total
sugar concentration in a sample. 0.025 gm of leaf sample were crushed in 5 ml
of 80% ethanol and centrifuged at 4000g for 10 minutes. The supernatant was
made up to 10 ml. The reaction is then finished by mixing 0.1 ml extract with 4
ml anthrone reagent diluted in sulfuric acid and placing it in a bath at 100oC
for 10 minutes. The absorbance is measured at 625 nm after the solution has
cooled. The total sugar content was measured in mg g-1 of fresh
weight. Pure glucose was used as the standard.
Bates et al. (1973) described a method to calculate proline
content. According to this, 5 ml of 3% aqueous sulfosalicylic acid was used to
crush 0.1 gm of leaf sample. It is then centrifuged for 10 minutes at 5000g. To
the 1 ml of filtrate, 2 ml glacial acetic acid and 2 ml acid-ninhydrin solution
are added. The mixture was heated to 100°C for 1 hour and after that cooled in
an ice bath. At last, 4 ml toluene was added and mixed thoroughly. The lower
aqueous phase was removed and absorbance was taken at 520 nm. The proline
content was expressed in mg g-1 fresh weight.
Superoxide dismutase (SOD) activity
Enzyme extracts were prepared using 0.2 gm of leaf sample that was completely pulverized using
a cold mortar and pestle in an ice bath in 0.1 M potassium phosphate buffer
with 0.5 mM EDTA (ethylene-diamine tetra-acetic acid) (pH 7.5). The sample is
then centrifuged at 15000g for 10 minutes at 4oC. The filtrate is
used for enzymatic assay. For determining SOD activity, Dhindsa et al. (1981)
were used. The reaction mixture consist of 50 mM potassium phosphate buffer (pH
7.8), 13 mM methionine, 75 µM nitroblue tetrazolium chloride (NBT), 0.1 mM EDTA
and 50 mM sodium carbonate. The reaction was begun by adding 2 µM riboflavin to
the mixture, which was then incubated for 10 minutes under fluorescent lights
before being left in the dark to cease the reaction. The mixture's absorbance
was measured at 560 nm. Due to the highest rate of NBT reduction, the reaction
mixture without enzyme acquired the most color. The amount of enzyme that
inhibits 50% NBT photo-reduction was determined to be one unit of SOD. Enzyme
units are measured in micrograms of protein as well as per gram of fresh
weight. The Enzyme unit was calculated using the formula:
Hydrogen peroxide (H2O2) content
Velikova et al. (2000) described a method for determining H2O2
content. At 0oC, 0.25 gm of leaf tissue was crushed in 3 ml of 0.1 %
TCA (tricarboxylic acid) with 0.1 g of activated charcoal. After that, it is
centrifuged for 15 minutes at 1200g. To the 0.5 ml of supernatant, 0.5 ml of 10
mM potassium phosphate buffer (pH 7.0) and 1 ml of 1M potassium iodide were
added. Absorbance was taken at 390 nm. H2O2 content was
expressed in µM H2O2 g-1 FW.
Catalase (CAT) activity
The activity of CAT was assessed using the procedures described
by (Mahely and Chance 1959). The reaction mixture consist of 2.5 ml potassium
phosphate buffer (pH 7.4), 0.1 ml 1% H2O2, and 50 µl
enzyme extract. The activity was determined by measuring the decrease of H2O2
absorbance at 240 nm, which was calculated using the extinction coefficient of
H2O2, which is 36 mM-1 cm-1. The
reaction is started by adding the H2O2. For 0, 1, and 3
minutes, absorbance (A) was recorded. The enzyme activity was measured in min-1
mg-1 of protein. CAT activity was calculated using the formula:
€= Extinction coefficient; d= Size of cuvette side, t= 1 or 3 min, c= Protein
content in test sample in mg.
Bradford method (Bradford 1976) is used to determine protein
content. To 0.1 ml of extract, 3 ml Bradford reagent was added and absorbance
was measured at 595 nm. Protein concentrations were measured in mg gm-1
of fresh weight. Using bovine serum albumin as a standard curve, the protein
content was measured.
Hiscox and Israelstam (1979) method for determining chlorophyll
(Chl) content was used. 1 gm of a fresh leaf was crushed in 5 ml of 80% acetone
that had been pre-chilled. The material was then centrifuged at 4°C for 20
minutes at 5000g. The supernatant was then collected and absorbance were made
at three different wavelengths: 630, 645, and 663 nm. Furthermore, the Chl
content was determined using the following equation:
Chl a = 11.64 ×
A663− 2.16 × A645+ 0.01 × A630
Chl b = 20.97 ×
A645− 3.94 × A663+ 3.66 × A630
a + b = 20.2 × A645 + 8.02 × A663
Statistical analysis of
The data obtained were analyzed using analysis of variance
(ANOVA) using SPSS software version 10 (SPSS Inc 1999) and the mean differences
were calculated by Duncan's multiple range test (DMRT) at a significance level
of p = 0.05
Results and Discussion
Sucrose is the most often utilized carbon source in plant tissue
culture media because it promotes optimal development when supplied at a
specific dosage (Swedlund and Locy 1993). It acts as an osmotic agent when
administered at a specific concentration, but when present over a certain level,
it can cause osmotic stress in the in
vitro condition (Mehta et al. 2000; Kim and Kim 2002). In this experiment,
we employed a higher sucrose concentration (9%) in the medium. The exogenous
addition of a higher concentration of sucrose induces a significant increase in
the total sugar content (4.05 fold) at 30th DAI in the leaves of C. caesia, whereas the total sugar
content did not increase at 30th DAI in the leaves grown on medium
with 3% sucrose (Fig. 2A) when compared to control (cultures on both the medium
at 0th day). Similarly, a significant increase (3.17 fold) observed
in the proline content at 30th DAI in the leaves of C. caesia grown on 9% sucrose, whereas
leaves grown on medium containing 3% sucrose did not show any differences in
the proline content at the same 30th DAI (Fig. 2B). This finding
supports Javed and Ikram's (2008) findings that free proline and total soluble
carbohydrate increased in two wheat genotypes, S-24 and MH-97, along with
increased sucrose concentration in Linsmaier and Skoog medium (Linsmaier and Skoog 1965). The addition of exogenous sugar boosted proline
concentration because a-ketoglutarate, a five-carbon precursor necessary for
proline accumulation, is produced by sugar oxidation (Stewart et al. 1966).
Sucrose when added beyond the normal limit, changed the osmotic potential,
causing carbohydrates and proline to accumulate in greater amounts, allowing
the plant to take up more water and develop faster (Javed 2002c; Ahmad et al.
2007). SOD is a key intracellular antioxidant enzyme that protects cells from
oxidative stress caused by reactive oxygen species. SOD is well-known for
acting as the first line of defense against the stress caused by reactive
oxygen species (Gill and Tuteja 2010). The SOD activity was observed to
increase significantly by 21.6 fold with increased sucrose concentration (Fig.
2C), which agrees with Wang and Li (2008), who discovered higher SOD activity
in white clover (Trifolium repens)
leaves under water stress conditions, also Moharramnejad et al. (2016)
discovered the similar pattern in SOD activity of maize seedling shoots under
stress condition. In the present study, H2O2 content also
increased significantly by 2.38 fold in the leaves grown on 9% sucrose at 30th
DAI whereas H2O2 content did not increase in the leaves
grown on 3% sucrose at 30th DAI (Fig. 2D). In the case of CAT
activity, the non-significant difference observed between the leaves grown on
medium with 3% and 9% sucrose (Fig. 2E). Kolarovic et al. (2009) found an increase
in CAT activity in maize under stressful conditions. However, some studies have
found that under water stress, such as in sunflowers, CAT activity remains
similar or even decreases (Luna et al. 2004). Total chlorophyll content also
increased significantly by 4.8 fold in the leaves of 9% sucrose and also showed
a significant increase (2.25 fold) in the leaves grown on 3% sucrose at 30th
DAI as compared to control (Fig. 2G) and previous studies (Winicov and Button
1991; Chang et al. 1997) also reported the same, i.e., increased chlorophyll
production in dicot chlorophyllic cells under saline stress, while Valenzuelaa
et al. (2005) found the same trend in graminaceous chlorophyllic cells under
osmotic stress. Like the other defensive biomolecules, the protein was also
found to be significantly elevated (6.46 fold) at 30th DAI in the
leaves grown on a medium with 9% sucrose, whereas almost the same protein
content as that of control was maintained in the cultures grown on 3% sucrose
at 30th DAI (Fig. 2F). Noman et al. (2018) showed a significant
increase in shoot proteins in water-stressed wheat (Triticum aestivum). Under the osmotic stress environment, these
enzymatic and non-enzymatic scavenging systems accumulate thereby showing their
increased amount at high sucrose concentration, which helps the plant in
lowering the osmotic potential and thus maintain its growth (Ikeda et al.
present study shows that high sucrose concentration induces osmotic stress in C. caesia which
causes a rise in the enzymatic and non-enzymatic scavenging components that
helps in avoiding or lowering stress. This study thus helps in understanding
the physiological and biochemical changes in C. caesia during osmotic
stress and how they adapt themselves to that environment.
Figure: 1 Three-month-old cultures on standard medium
Effect of sucrose on: A total sugar content, B proline content, C superoxide
dismutase activity, D hydrogen peroxide content, E catalase activity, F protein
content, and G total chlorophyll content. Values are represented as mean
Conflict of interest
Authors had no conflict of interest.
thanks to School of Studies in Biotechnology, Pandit Ravishankar Shukla
University, Raipur (C.G.)
Ahmad MSA, Javed F, Ashraf M (2007)
Iso-osmotic effect of NaCl and PEG on growth, cations and free proline
accumulation in callus tissue of two indica rice (Oryza sativa L.) genotypes. Plant Growth Regulation, 53:53-63.
Anjum A, Singh V, Adil S, Quraishi A
(2022) In Vitro Propagation of Curcuma caesia Roxb. via Bud Culture
Technique and ISSR Profiling of the Plantlets for Genetic
Homogeneity. Research Journal of Biotechnology (Accepted).
Bates LS, Waldren RP, Teare ID (1973)
Rapid determination of free proline for water-stress studies. Plant and Soil,
Bradford MM (1976) A rapid and
sensitive method for the quantitation of microgram quantities of protein
utilizing the principle of protein-dye binding. Analytical biochemistry,
Chance B, Maehly AC (1959) The assay of
catalase and peroxidase. In: Click D (ed) Methods of Biochemical Analysis, Vol
1 Interscience Publishers, New York.
Chang CC, Locy RD, Smeda R, Sahi SV,
Singh NK (1997) Photoautotrophic tobacco cells adapted to grow at high
salinity. Plant Cell Reports, 16:495-502.
Chauhan R, Keshavkant S, Quraishi A
(2018) Enhanced production of diosgenin through elicitation in micro-tubers of Chlorophytum borivilianum Sant et
Fernand. Industrial Crops and Products, 113:234-239.
Devi HP, Mazumder PB, Devi LP (2015)
Antioxidant and antimutagenic activity of Curcuma
caesia Roxb. rhizome extracts. Toxicology report, 2:423-428.
Dhindsa RS, Dhindsa PP, Thorpe TA
(1981) Leaf senescence: Correlated with increased levels of membrane
permeability and lipid peroxidation, and decreased levels of superoxide
dismutase and catalase. Journal of Experimental Botany, 32:93-101.
Gilles KA, Hamilton JK, Roberts PA, Smith F (1956) Colorimetric method for
determination of sugars and related substances. Analytical chemistry, 28(3):350-356.
Gill SS, Tuteja N (2010) Reactive
oxygen species and antioxidant machinery in abiotic stress tolerance in crop
plants. Plant Physiology and Biochemistry, 48:909-930.
Sinniah UR, Nakasha JJ, Hakiman M (2022) Shoot Induction, Multiplication,
Rooting and Acclimatization of Black Turmeric (Curcuma caesia Roxb.): An Important and Endangered Curcuma Species.
Hiscox JDT, Israelstam GF (1979) A
method for the extraction of chlorophyll from leaf tissue without maceration. Canadian
Journal of Botany, 57(12):1332-1334.
Ikeda T, Fujime Y, Terabayashi S, Date
S (2002) Water status of garlic callus under various salt and osmotic stress
conditions. Horticultural Science, 37:404-405.
Javed F (2002c) In vitro salt tolerance in wheat. III: Water relations in callus.
International Journal of Agriculture and Biology, 4:465-467.
Javed F, Ikram S (2008) Effect of
sucrose induced osmotic stress on callus growth and biochemical aspects of two
wheat genotypes. Pakistan Journal of Botany, 40(4):1487-1495.
Karmakar I, Dolai N, Saha P, Sarkar N,
Bala A, Haldar P K (2011) Scavenging activity of Curcuma caesia rhizome against reactive oxygen and nitrogen
species. Oriental Pharmacy and Experimental Medicine, 11:221-228.
Kim SH, Kim SK (2002) Effect of sucrose
level and nitrogen source on fresh weight and anthocyanin production in cell
suspention culture of ‘Sheridan’ Grape (Vitis
spp). Journal of Plant Biotechnology, 4:2327-2330.
Kolarovic L, Valentovic P, Luxova M, Gasparıkova O
(2009) Changes in antioxidants and cell damage in heterotrophic maize seedlings
differing in drought sensitivity after exposure to short-term osmotic stress.
Plant Growth Reregulation, 59:21-26.
Krishnan PN, Decruse SW, Radha RK
(2011) Conservation of medicinal plants of Western Ghats, India and its
sustainable utilization through in vitro
technology. In Vitro Cellular and Developmental Biology-Plant, 47(1):110-122.
Linsmaier EM, Skoog F (1965) Organic
growth factor requirements of tobacco tissue cultures. Physiologia Plantarum,
Luna CM, Pastori GM, Driscoll S, Groten
K, Bernard S, Foyer CH (2004) Drought controls on H2O2
accumulation, catalase (CAT) activity and CAT gene expression in wheat. Journal
of Experimental Botany, 56:417-23.
Mahato D, Sharma HP (2017) Kali Haldi,
an ethnomedicinal plant of Jharkhand state- A Review. Indian Journal of
Traditional Knowledge, 17(2):322-326.
Mehta UJ, Krishnamurthy VK, Hazra S
(2000) Regeneration of plant via adventitious bud formation from zygotic embryo
axis of tamarind (Tamarindus indica).
Current Science, 78: 1231-12.
Mishra M (2013) Conservation of
critically endangered medicinal plant Curcuma
caesia in the natural forests of Mandla district, Madhya Pradesh. A Journal of Environment and Biodiversity,
Moharramnejad S, Sofalian O, Valizadeh
M, Agari A, Shiri M (2016) Response of antioxidant defense system to osmotic
stress in maize seedlings. Fresenius Environmental Bulletin, 25:805-811.
Munnik T, Meijer HJ (2001)
Osmotic stress activates distinct lipid and MAPK signalling pathways in plants.
FEBS Letters, 498:172-178.
Murashige T, Skoog F (1962) A revised
medium for rapid growth and bioassays with tobacco tissue culture. Physiologia Plantarum, 15:473-497.
Noman A, Ali Q, Naseem J, Javed MT, Kanwal
H, Islam W, Aqeel M, Khalid N, Zafar S, Tayyeb M, Iqbal N,
Buriro M, Maqsood J, Shahid S (2018) Sugar beet extract acts
as a natural bio-stimulant for physio-biochemical attributes
in water stressed wheat (Triticum
aestivum L.) Acta Physiologiae Plantarum, 40(6):1-7.
Deka AC, Sarma TC (2021) A Protocol for Rapid Clonal Propagation and
Microrhizome Production of Curcuma caesia Roxb. (Zingiberaceae): A
Critically Endangerd Medicinal Plant of North East India. Indian Journal of
Agricultural Research, 55(1):13-22.
Sharma N, Verma PP, Murthy SN (2017)
Pharmacognostical evaluation and conservation of threatened species Curcuma
caesia Roxb. International Journal of Ayurvedic Medicine, 8(2):68-72.
Stewart CR, Morris CJ, Thompson JF
(1966) Changes in Amino Acid Content of Excised Leaves During Incubation II.
Role of Sugar in the Accumulation of Proline in Wilted Leaves. Plant
Swedlund B, Locy RD (1993) Sorbitol as
the primary carbon source for the growth of embryogenic callus of maize. Plant
Valenzuelaa XG, Moyab EG, Cruza QR,
Estrellac LH, Santacruza GAA (2005) Chlorophyll accumulation is enhanced by
osmotic stress in graminaceous chlorophyllic cells. Journal of Plant
Velikova V, Yordanov I, Edreva A (2000)
Oxidative stress and some antioxidant system in acid rain-treated bean plants,
protect role of exogenous polyamines. Plant Sciience, 151:59-66.
Verma D, Srivastava S, Singh V, Rawat
AKS (2010) Pharmacognostic evaluation of Curcuma
caesia Roxb. rhizome. Natural Product Sciences, 16(2):107-110.
Wang CQ, Li RC (2008) Enhancement of
superoxide dismutase activity in the leaves of white clover (Trifolium repens L.) in response to
polyethylene glycol-induced water stress. Acta Physiological Plantarum,
Winicov I, Button JD (1991)
Accumulation of photosynthesis gene transcripts in response to sodium chloride
by salt-tolerant alfalfa cells. Planta, 183:478-483.
Jia W, Yang J, Ismail AM (2006) Role of ABA in integrating plant responses to
drought and salt stresses. Field Crops Research, 97:111-119.