CC BY
320
Journal of Stress Physiology & Biochemistry, Vol. 10 No. 2 2014, pp. 287-296 ISSN 1997-0838 Original Text Copyright © 2014 by Rathore, Singh and Singh
ORIGINAL ARTICLE
Influence of NaCl on Biochemical Parameters of Two Cultivars of Stevia rebaudiana Regenerated in vitro
Sharuti Rathore1*, Narender Singh1, S.K. Singh2
1 Department of Botany, Kurukshetra University, Kurukshetra 136119, Haryana, India
2 Division of Fruits and Horticultural Technology, Indian Agricultural Research Institute, Pusa Campus, New Delhi-110012, India
Phone: 01744-20196(501), Fax: 01744-20277 *E-Mail: shrutihp@gmail.com
Received February 21, 2014
Soil salinity occupies a prominent place among the soil problems that threaten the sustainability of agriculture over a vast area in the world. It affects plant morpho-physiology and ultimately leads to reduction in productivity. It is essential to test important medicinal plants for their salinity tolerance as research efforts aim to explore economic benefits under saline conditions. Keeping in view the importance of Stevia and salinity, present study had been designed to investigate the effect of salinity on biochemical parameters in two Stevia genotypes. Two node microcuttings were subjected to MS media supplemented with different NaCl concentrations (0, 25, 50, 75, 100, 125 mM). Chlorophyll amount was observed to be decreased as compared to sugars, proline and phenols with increased salt concentrations.
Key words: chlorophyll content, leaves, proline, phenols, sugars
ORIGINAL ARTICLE
Influence of NaCl on Biochemical Parameters of Two Cultivars of Stevia rebaudiana Regenerated in vitro
Sharuti Rathore1*, Narender Singh1, S.K. Singh2
1 Department of Botany, Kurukshetra University, Kurukshetra 136119, Haryana, India
2 Division of Fruits and Horticultural Technology, Indian Agricultural Research Institute, Pusa Campus, New Delhi-110012, India
Phone: 01744-20196(501), Fax: 01744-20277 *E-Mail: shrutihp@gmail.com
Received February 21, 2014
Soil salinity occupies a prominent place among the soil problems that threaten the sustainability of agriculture over a vast area in the world. It affects plant morpho-physiology and ultimately leads to reduction in productivity. It is essential to test important medicinal plants for their salinity tolerance as research efforts aim to explore economic benefits under saline conditions. Keeping in view the importance of Stevia and salinity, present study had been designed to investigate the effect of salinity on biochemical parameters in two Stevia genotypes. Two node microcuttings were subjected to MS media supplemented with different NaCl concentrations (0, 25, 50, 75, 100, 125 mM). Chlorophyll amount was observed to be decreased as compared to sugars, proline and phenols with increased salt concentrations.
Key words: chlorophyll content, leaves, proline, phenols, sugars
Stevia, a member of family Asteraceae, is native to Paraguay, Brazil, Venezuela and Colombia. It is an important non caloric sweetener medicinal plant, used for pharmaceutical, food and cosmetic industries. Now it is being cultivated in Japan, Taiwan, Philippines, Hawaii, Malaysia and overall South America for food and pharmaceutical products (Ahmed et al., 2007). It is commonly known as Stevia, honey leaf, ya wan, sweet grass etc. It is one of the most promising herb used in the treatment of hypoglycaemia, indigestion, skin toning, healing heart disease. It is used as a table
top sweetener, in soft drinks, baked goods, pickles, fruit juices, tobacco products, confectionery goods, jams and jellies, candies, yogurts, pastries, chewing gum and sherbets (Ali et al., 2010).
Soil salinity is one the most important stress factor which limits plant growth and productivity (Jamil et al., 2012). Salinity mainly occurs in arid and semi-arid conditions (Ehert and Ho, 1986) when the precipitation is not enough to leach the excess of soluble salts from the root zone and poor quality of water is used for irrigation (Mohammad et al., 1998). Exposure of plants to a stressful
environment during various developmental stages appears to induce various physiological and developmental changes (Islam et al., 2008). Plants cultured under salt stress show high chlorophyll degradation and high proline accumulation. The effect of salt stress on biochemical parameters on in vitro regenerated plants of Stevia has not yet been investigated. The objective of this investigation was to elucidate the effect of NaCl on biochemical parameters of in vitro regenerated plantlets of Stevia genotypes.
MATERIALS AND METHODS
Total soluble sugars
The amount of total soluble sugars was estimated using Anthrone reagents as given by Thimmaiah (2004). One hundred mg sample was taken in a boiling tube and hydrolyzed with 5 ml 2.5 N HCl in a water bath for 3 h. It was then neutralized with solid sodium carbonate until effervescence ceased. The volume was made to 100 ml followed by centrifuge at 5000 rpm for 10 min. The supernatant was collected and 1 ml sample was taken for analysis. Four ml Anthrone reagent was added to aliquot and heated for one min. in water bath (70°C). The sample was then rapidly cooled and the change of green to dark green colour was read at 630 nm against blank.
Reducing sugars
Reducing sugars were extracted from leaf samples of both the cultivars viz. CIM madhu and CIM mithi and Somogyi (1951) method was used. The reducing sugars were determined as mg/g DW. Proline content
The leaf tissue proline content was measured following the method described by Bates et al. (1973). A homogenized fresh leaf tissue (0.5 g) was added in 10 ml of 3% sulfo-salicylic acid.
Homogenates of fresh leaf samples were filtered through Whatman No. 2 filter paper. Two ml of the filterate was taken in a test tube containing 2 ml of acid ninhydrin solution (1.25 g ninhydrin in 30 ml glacial acetic acid and 20 ml of 6 M orthophosphoric acid). Then, 2 ml of glacial acetic acid was added in a test tube containing filtrate and heated for 1 h at 100°C. Test tubes were then shifted in an ice bath to terminate the reaction. Reaction mixture was then extracted with 10 ml toluene and mixed vigorously by passing a continuous air stream for 1-
2 minutes. The absorbance was noted at 520 nm. Total Phenols
The leaf tissue proline content was measured following the method described by Malik and Singh (1980). Thus, 100 mg of fresh leaf material was homogenized by adding by adding 80 per cent ethanol and then heated in a water bath at 58°C for 60 min. The absorbance was measured at 650 nm in a spectrophotometer.
Chlorophyll content
The fractions of pigments (chlorophyll a, chlorophyll b and Total chlorophyll) were estimated using the spectrophotometric method recommended by Arnon (1949). A pinch of CaCO3 was added to avoid the destruction of chlorophyll and other pigments. Extraction has to be carried out under dim light to avoid photo oxidation of the pigments. It was centrifuged in a Rim centrifuge at low speed (5000 rpm) for about 20 min. Chlorophyll contents concentrations were calculated as mg/g FW at 663 and 645 nm.
Statistical analysis
Data were analyzed for significance using oneway analysis of variance (ANOVA) and the differences contrasted using a Duncan's multiple range test (DMRT) at p < 0.05. All statistical
analyses were performed using the Statistical Package for Social Sciences (SPSS, version 11.5).
RESULTS
Soluble sugars
Addition of NaCl to proliferation medium caused an increase in total soluble sugars on in vitro raised shoots of both the cultivars (Figure 1). The lowest content (55.07 mg/g) of total soluble sugars was found in regenerants cultured on MS medium free from NaCl. The highest amount of soluble sugars (93.54 mg/g) was found in cv. CIM madhu at 75 mM NaCl. It was determined that total soluble sugars in both the cultivars increased linearly.
Reducing sugars
Changes in reducing sugars in different cultivars are more or less similar to changes in total sugars. The highest level of reducing sugars (55.78 mg/g) was recorded in media containing 75 mM NaCl whereas, minimum amount of reducing sugars (22.86 mg/g) was found on media free from NaCl. Among the two Stevia cultivars, cv. CIM madhu produced higher reducing sugars (Figure 2).
Proline
It is generally assumed that proline is acting as a compatible solute in osmotic adjustment. The highest value of (22.26 ^g/g) proline was observed
at the concentration of 75 mM of NaCl. According to results presented in Figure 3, proline content of both the cultivars increase significantly (p<0.05) with the increase of salt concentration on culture media (MS). Minimum concentration of proline was found on medium free of NaCl.
Total phenols
As the level of NaCl was increased in the medium, the total phenols were also increased concurrently (Figure 4). The highest level of phenols was recorded in cv. CIM madhu (22.42 mg/g) cultured on medium supplemented with 75 mM NaCl. Minimum concentration (16.94 mg/g) of total phenols was found on media without NaCl.
Chlorophyll contents
The Chlorophyll contents were found to be decreased in leaves produced under stress conditions (Table 1). Chlorophyll a content decreased with the increase in concentration of NaCl. The highest amount of chlorophyll b (1.23 mg/g) was found in cv. CIM madhu when cultured on medium free from NaCl. Minimum chlorophyll b (0.05 mg/g) was observed at 75 mM NaCl. The highest amount of total chlorophyll (3.27 mg/g) was recorded in cv. CIM mithi cultured on medium without NaCl.
Table 1. Effect of different sodium chloride concentrations on chlorophyll contents (mg/g FW) of S. rebaudiana cultivars.
Treatment CIM madhu CIM mithi
Chlorophyll a Chlorophyll b Total chlorophyll Chlorophyll a Chlorophyll b Total chlorophyll
Control 2.02±0.018a 1.23±0.023a 3.01±0.021a 2.05±0.006a 0.89±0.047a 3.27±0.060a
25 mM 1.64±0.184b 0.96±0.018b 2.62±0.021b 1.62±0.059b 0.87±0.054a 2.49±0.105b
50mM 1.40±0.049c 0.96±0.032b 2.36±0.020c 1.30±0.058c 1.01±0.007a 2.31±0.010b
75mM 0.24±0.053d 0.05±0.037c 0.30±0.020d 0.23±0.025d 0.04±0.009b 0.28±0.032c
100mM - - - - - -
125mM - - - - - -
LSD (p<0.05) 0.018 0.001 0.039 0.003 0.002 0.007
Values represent mean ± standard error.
Means values within a column sharing the same subscript are not significantly different at P<0.05 according to Duncan's Multiple Range Test.
£
Qj
61*
£
cd
&£■
IS
VI
i-
1
o
'S
o H
H CIM madhu -■ i CIM mithi
n-----—i--------------1------- —i---------1----------1
0 25 50 75 100 125
Treatment (mMNaCl)
Figure 1. Total soluble sugars of two S. rebaudiana cultivars cultured on MS medium fortified with NaCl (00, 25, 50, 75,100 and 125mM). Bars carrying different letters are significantly different at P< 0.05.
60
£ 50 ft
ejjj 40
G3
&£■
CO)
a
o
*0
10
0
H CIM madhu li CIM mithi
25
50
100
125
Treatment (mMNaCl)
Figure 2. Reducing sugars of two S. rebaudiana cultivars cultured on MS medium fortified with NaCl (00, 25, 50, 75,100 and 125mM). Bars carrying different letters are significantly different at P< 0.05.
20
e* 15
o>
.3 10
Рч
b b
a «CM madhu u СІМ mithi
25 50 75 100
Ti eatment (mMNaCl)
125
Figure 3. Proline of two S. rebaudiana cultivars cultured on MS medium fortified with NaCl (00, 25, 5Q, 75,100 and 125mM). Bars carrying different letters are significantly different at P< Q.Q5.
0
25
50
Я СІМ madhu -: CM mithi
75
100
125
Treatment (mMNaCl)
Figure 4. Total phenols of two S. rebaudiana cultivars cultured on MS medium fortified with NaCl (QQ, 25, 5Q, 75,100 and 125mM). Bars carrying different letters are significantly different at P< Q.Q5.
DISCUSSION
Biochemical studies were under taken when the regenerated shoots were transferred to the NaCl treated medium. The addition of sodium chloride to the culture medium resulted in marked alteration of
biochemical constituents and accordingly their levels varied with NaCl concentrations.
Sodium chloride treatment induced stimulatory effect on the accumulation of soluble sugars. The accumulation of soluble carbohydrates in plants has
been widely reported as a response to salinity or drought (Popp and Smirnoff, 1995; Murakeozy et al., 2003). Maximum amount of total soluble sugars was found at 100 mM NaCl. Significant increases in soluble sugars content of shoots of wheat cultivars were reported by Zheng et al. (2008) in response to NaCl stress. The accumulation of sucrose under salt stress supported the well-established role of the sugars as an osmoprotectant that stabilizes cellular membranes and maintains turgor (Whittaker et al., 2001; Jouve et al., 2004). In the present studies, the highest quantity of proline was observed at 75 mM NaCl in both the cultivars. Many authors have reported the increase in proline accumulation under salt stress in different plants such as pigeon pea (Waheed et al., 2006), Sesamum indicum (Koca et al., 2007), barley (Sadeghi, 2009), maize (Chaum and Kirdmanee, 2009), jojoba plant (Fayek et al., 2010). Total phenols were also found to increase with increasing concentration of NaCl. It was found to be maximum in the shoots implanted on proliferation media containing 75 mM NaCl in both the cultivars. Total phenols play a significant role in the regulation of plant metabolic processes and overall plant growth (Lewis and Yamamoto, 1990). It has been shown in some studies that polyphenols synthesis depends on abiotic factors (Ksouri et al., 2008; Megdiche et al., 2009; Naffeti et al., 2011). Salinity induced disturbances of the metabolic process leading to an increase in phenolic compounds have been reported by Ayaz et al. (2000) and Radi et al. (2013).
In the present studies, the chlorophyll contents decreased as the NaCl concentration increased in both the cultivars. Change in chlorophyll contents due to salinity is the most obvious biochemical response (Erturk et al., 2007; Sherif, 2012). Chlorophyll contents in salt stressed plants were
significantly decreased depending on NaCl concentration (Jamil et al., 2012). This trend has previously been reported by (Singh et al., 2000; Khawale et al., 2003). The depressive effect of salt stress on chlorophyll biosynthesis may be due to the formation of proteolytic enzymes such as chlorophyllase which is responsible for the chlorophyll degradation (Sabater and Rodriguez, 1978) and damaging the photosynthetic apparatus (Singh and Jain, 1981; Yasseen, 1983).
ACKNOWLEDGEMENTS
The authors are grateful to Kurukshetra University, Kurukshetra as well as Central Tissue Cultural Laboratory, NRCPB, Indian Agricultural research Institute, New Delhi for providing laboratory facilities and other institutional support.
REFERENCES
Ahmed, M.B., Salahin, M., Karim, R., Razvy, M.A., and Hannan, M.M. (2007) An efficient method for in vitro clonal propagation of a newly introduced sweetener plant (Stevia rebaudiana Bertoni.) in Bangladesh. American Eurasian Journal of Science Research, 2, 12125.
Ali, A., Gull, I., Naz, S., and Afghan, S. (2010) Biochemical investigation during different stages of in vitro propagation of Stevia rebaudiana. Pakistan Journal of Botany, 42(4), 2827-2837.
Arnon, D.I. (1949) Copper enzyme in isolated chloroplast polyphenoloxidase in Beta vulgaris L. Plant physiology., 24, 1-15.
Ayaz, F.A., Kadioglu, A., and Turgut, A. (2000) Water stress effects on the content of low molecular weight carbohydrates and phenolic acids in Ctenanthe setosa (Rosc.) Eichler. Can J Plant Sci., 80, 373-378.
Bates, L., Waldren, R.P., and Teare, I.D. (1973) Rapid determination of free proline for water-stress studies. Plant Soil, 39, 205-207.
Chaum, S., and Kirdmanee, C. (2009) Effect of salt stress on proline accumulation, photosynthetic ability and growth characters in two maize cultivars. Pakistan Journal of Botany., 41, 87-98.
Ehret, D.L. and Ho, L.C. (1986) The effects of salinity on dry matter partitioning and fruit growth in tomatoes grown in nutrient film culture. Hort. Sci., 61(3), 361-367.
Erturk, U., Sivritepe, N., Yerlikaya, C., Bor, M., Ozde Mir, F., and Turkan, I. (2007) Responses of the cherry rootstock to salinity in vitro. Biologia Plant, 51(3), 597-600.
Fayek, M.A., Shabaan, E.A., Zayed, N.S., El-Obeidy, and Taha, R.A. (2010) Effect of salt stress on chemical and physiological contents Jojoba (Simmondsia chinensis) chneider using in vitro culture. World Journal of Agricultural Science, 6(4), 446-450.
Islam, M.R., Singh, R.K., Salam, M.A., Hassan, L., and Gregorio, G.B. (2008) Molecular diversity of stress tolerant rice genotypes using SSR markers. SABRAO Journal of Plant Breeding and Genetics, 40(2), 127-139.
Jamil, M., Bashir, S., Anwar S. Bibi S., Bangash A., Ullah F., and Shikrha E. (2012) Effect of salinity on physiological and biochemical characteristics of different varieties of rice. Pakistan Journal of Botany, 44, 7-13.
Jamil, M., Bashir, S., Anwar, S. Bibi, S., Bangash, A., Ullah, F., and Shikrha, E. (2012) Effect of salinity on physiological and biochemical characteristics of different varieties of rice. Pakistan Journal of Botany., 44, 7-13.
Jouve, L., Hoffmann, L., and Hausman, J.F. (2004) Polyamine, carbohydrate, and proline content changes during salt stress exposure of Aspen (Populus tremula L.): involvement of oxidation and osmoregulation metabolism. Plant Biol., 6, 74-80.
Khawale, R.N., Singh, S.K., Patel, V.B., and Singh, S.P. (2003) Changes due to in vitro sodium chloride induced salinity in grape (Vitis vinifera L.). Indian Journal of Plant Physiology, 28, 378-382.
Koca, H., Bor, M., Ozdemir, F., and TOrkan, I. (2007) The effect of salt stress on lipid peroxidation, antioxidative enzymes and proline content of sesame cultivars. Environmental and Experimental Botany, 60, 344-351.
Ksouri, R., Megdiche, W., Falleh, H., Trabelsi, N., Boulaaba, M., Smaoui, A., and Abdelly C. (2008) Influence of biological, environmental and technical factors on phenolic content and antioxidant activities of Tunisian halophytes. Comp. Rend. Biol., 331, 865-873.
Lewis, N.G., and Yamamoto, E. (1990) Lignin: occurrence, biosynthesis and biodegradation. Annual Review on Plant Physiology, 41, 455561.
Malik, E.P., Singh, M.B. (1980) Plant enzymology and hittoenzymology (1st Edn.) Kalyani Publishers: New Delhi, 286.
Megdiche, W., Ben Amor, N., Debez, A., Hessini, K., Ksouri, R., Abdelly, C. (2009) Physiological and biochemical traits involved in the genotypic variability to salt tolerance of Tunisian Cakile maritime. African Journal of Ecology, 47, 774783.
Mohamed, A.A., and Aly, A. (2008) Alterations of some secondary metabolites and enzymes
activity by using exogenous antioxidant compound in onion plants grown under seawater salt stress. American Eurasian Journal of Science Research, 3(2), 139-146.
Mohammad, M., Shibli, R., Ajlouni, M., and Nimri, L. (1998) Tomato roots and shoots responses to salt stress under different levels of phosphorus nutrition. Journal of Plant Nutrition, 21, 1667-1680.
Murakeozy, E.P., Nagy, Z., Duhaze, C., Bouchereau, A., and Tuba Z. (2003) Seasonal changes in the levels of compatible osmolytes in three halophytic species of inland saline vegetation in Hungary. J. Plant Physiol., 160, 395-401.
Naffeti, M., Sriti, J., Hamdaoui, G., Kchouk, E.M., and Marzouk, B. (2011) Salinity impact on fruit yield, essential oil composition and antioxidant activities of Coriandrum sativum fruit extracts. Food Chemistry, 124, 221-225.
Popp, M., and Smirnoff, N. (1995) Polyol accumulation and metabolism during water deficit. In: Smirnoff N. (Ed.), environment and plant metabolism: flexibility and acclimation. Bios Scientific, Oxford. pp. 199-215.
Radi, A.A., Farghaly, F.A., and Hamada, A.M. (2013) Physiological and biochemical responses of salt-tolerant and salt-sensitive wheat and bean cultivars to salinity. Journal of Biology and Earth Science, 3(1), 72-88.
Sabater, B., and Rodriguez, M.I. (1978) Control of chlorophyll degradation in detached leaves of barley and oat through effect of kinetin on chlorophyllase levels. Physiology Plant, 43, 274-276.
Sadeghi, H. (2009) Effects of different levels of sodium chloride on yield and chemical composition in two barley cultivars. Americal-
Eurasian Journal of Sustainable Agriculture., 3(3), 314-320.
Sherif, F.El. (2012) In vitro NaCl tolerance of Artemisia dracunculus. International Journal of Medicinal and Aromatic Plants, 2249-4340.
Singh, G., and Jain, S. (1981) Effect of some growth regulators on certain biochemical parameters during seed development in chickpea under salinity. Indian Journal of Plant Physiology, 20, 167-179.
Singh, S.K., Sharma, H.C., Goswami, A.M., Dutta, S.P., and Singh, S.P. (2000) In vitro growth and leaf composition of grapevine cultivars as affected by sodium chloride. Biologia Plantarum, 6, 283-286.
Somogyi, M. (1951) Notes on sugar determination. J. Biol. Chem., 195, 19.
Thimmaiah, S.R. (2004) Standard methods of biochemical analysis. Kalyani Press, New Delhi, India.
Waheed, A., Hafiz, I.A., Qadir, G., Murtaza, G., Mahmood, T., Ashraf, M. (2006) Effect of salinity on germination, growth, yield, ionic balance and solute composition of pigeon Pea (Cajanus cajan (L.) Mill sp). Pakistan Journal of Botany, 38, 1103-1117.
Whittaker, A., Bochicchio, A., Vazzana, C., Lindsey, G., and Farrant, J. (2001) Changes in leaf hexokinase activity and metabolite levels in response to drying in the desiccation-tolerant species Sporobolus staphianus and Xerophyta viscose. J. Exper. Bot., 1, 961-969.
Yasseen, B.T. (1983) An analysis of the effects of salinity on leaf growth in Mexican wheat. Ph.D. Thesis, University of Leeds.
Zheng, Y., Jia, A., Ning, T., Xu, J., Li Z., and Jiang, G. (2008) Potassium nitrate application alleviates
sodium chloride stress in winter wheat Physiol., 165, 1455-1465.
cultivars differing in salt tolerance. J. Plant
