DOI: 10.52228/NBW-JAAB.2022-4-1-2

Introduction

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 0^th and 30^th 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 100^oC 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.

Proline content

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 4^oC. 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 0^oC, 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:

where, €= Extinction coefficient; d= Size of cuvette side, t= 1 or 3 min, c= Protein content in test sample in mg.

Protein content

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.

Chlorophyll content

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

                                    1000

Chl b = 20.97 × A645− 3.94 × A663+ 3.66 × A630

                                     1000

Chl a + b = 20.2 × A645 + 8.02 × A663

                                     1000

Statistical analysis of data

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 30^th DAI in the leaves of C. caesia, whereas the total sugar content did not increase at 30^th DAI in the leaves grown on medium with 3% sucrose (Fig. 2A) when compared to control (cultures on both the medium at 0^th day). Similarly, a significant increase (3.17 fold) observed in the proline content at 30^th 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 30^th 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 30^th DAI whereas H₂O₂ content did not increase in the leaves grown on 3% sucrose at 30^th 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 30^th 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 30^th 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 30^th 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. 2002).

Conclusion

The 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

     Figure: 2 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 ±standard error.

Conflict of interest

Authors had no conflict of interest.

Acknowledgement

Whole hearted thanks to School of Studies in Biotechnology, Pandit Ravishankar Shukla University, Raipur (C.G.)

References

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, 39(1):205-207.

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, 72:248-254.

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.

Dubois M, 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.

Haida Z, 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. Horticulturae, 8(8):740.

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, 8:100-127.

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, 4(3):36-40.

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.

Sarma I, 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 Physiology, 41:1585-1590.

Swedlund B, Locy RD (1993) Sorbitol as the primary carbon source for the growth of embryogenic callus of maize. Plant Physiology, 103:1339-1346.

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 Physiology, 162:650-661.

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, 30:841-847.

Winicov I, Button JD (1991) Accumulation of photosynthesis gene transcripts in response to sodium chloride by salt-tolerant alfalfa cells. Planta, 183:478-483.

Zhang J, 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.