Emir. J. Food Agric. 2011. 23 (1): 59-70 http://ejfa.info
Studies on the effects of salinity on growth, polyphenol content and photosynthetic response in Vetiveria zizanioides (L.) Nash A. V. Mane1, G. D. Saratale2,#∗, B. A. Karadge3 and J. S. Samant4 1 Department of Environmental Sciences, Fergusson College, Pune, India; 2Department of Biotechnology, Fergusson College, Pune, India; 3Department of Botany, Shivaji University, Kolhapur, India; 4Department of Environmental Sciences, Shivaji University, Kolhapur, India; # Department of Biological Environment, College of Agriculture and Life Sciences, Kangwon National University, Chuncheon, Gangwondo, 200-701, South Korea
Abstract: Salinity is one of the major abiotic stress that adversely affects on crop productivity and its quality. The present investigation was carried out to study the alterations in the growth characteristics, photosynthetic pigments and polyphenols content of Vetiveria zizanioides (L.) under the influence of sodium chloride salinity. The result shows that the shoot length of Vetiveria zizanioides was increased by 18.6% at 200 mM NaCl concentration whereas, the increase in root length about 24.8% was observed at 50 mM NaCl. Shoot to root ratio in the experimental species was reduced to 10.0% at 50 mM NaCl concentration whereas at 300 mM of salinity it was increased by 39.09%. Moreover, the average leaf area also increases under saline conditions. An increase in the photosynthetic pigments at lower salt levels indicates osmotic adjustment mechanism developed by the grass species to incumbent with salt stress. However, dry weight and fresh weight of the plant biomass was found less effective under the salinity stress. The increased levels of polyphenols at elevated salinity might be due to the accumulation of secondary metabolites. Linear increase in the electrical conductivity and total dissolved solids of the soil was observed at increasing salinity. Increase in growth and photosynthetic parameters observed at especially upto 100 mM of salinity indicate the tolerance shown by experimental species and we conclude that tolerant V. zizanioides can be grown in saline areas with salt concentrations up to 200 mM. Key words: Vetiveria zizanioides, Photosynthetic pigments, Polyphenols, Salinity, Growth characteristics, Electrical conductivity
دراﺳﺔ ﻋﻦ ﺗﺄﺛﻴﺮ اﻟﻤﻠﻮﺣﺔ ﻋﻠﻰ اﻟﻨﻤﻮ وﻣﺤﺘﻮى ﻣﺎدة اﻟﺒﻮﻟﻴﻔﻴﻨﻮل واﻻﺳﺘﺠﺎﺑﺔ اﻟﻀﻮﺋﻴﺔ ﻓﻲ Vetiveria zizanioides (L.) ﻧ ﺎش 4
ﺳﺎﻣﺎﻧﺖ. س. و ﺟﻲ3 آﺮاﺗﺶ.ا. ب،*#،2 ﺳﺎراﺗﻴﻠﻲ.د. ج،1 ﻣﺎﻧﻲ. ﻓﻲ.ا
، ﺟﺎﻣﻌﺔ ﺷﻴﻮاﺟﻲ3 ; اﻟﻬﻨﺪ، ﺑﻮﻧﻲ، آﻠﻴﺔ ﻓﺮﻗﻮس، ﻗﺴﻢ اﻟﺘﻜﻨﻮﻟﻮﺟﻴﺎ اﻟﺤﻴﻮﻳﺔ2 ; اﻟﻬﻨﺪ، ﺑﻮﻧﻲ، آﻠﻴﺔ ﻓﺮﻗﻮﺳﻮن، ﻗﺴﻢ ﻋﻠﻮم اﻟﺒﻴﺌﺔ1 ﺟﺎﻣﻌﺔ، آﻠﻴﺔ اﻟﺰراﻋﺔ وﻋﻠﻮم اﻟﺤﻴﺎة، ﻗﺴﻢ ﺑﻴﻮﻟﺠﻲ اﻟﺒﻴﺌﺔ# ; اﻟﻬﻨﺪ، آﻠﻬﺎﺑﻮر، ﺟﺎﻣﻌﺔ ﺷﻴﻮاﺟﻲ، ﻗﺴﻢ ﻋﻠﻮم اﻟﺒﻴﺌﺔ4 ; اﻟﻬﻨﺪ، آﻠﻬﺎﺑﻮر ﺟﻨﻮب آﻮرﻳﺎ،701-200 ، ﻗﺎﻧﻘﻮﻧﺪو، ﺷﻨﺸﻴﻴﻮن، آﻨﻘﻮن اﻟﻮﻃﻨﻴﺔ وﻓﻲ هﺬﻩ اﻟﺪراﺳﺔ. اﻟﻤﻠﻮﺣﺔ ﺗﻌﺘﺒﺮ واﺣﺪة ﻣﻦ اآﺒﺮ اﻟﻤﻜﻮﻧﺎت اﻟﻼ إﺣﻴﺎﺋﻴﺔ اﻟﺮﺋﻴﺴﻴﺔ اﻟﺘﻲ ﺗﺆﺛﺮ ﺳﻠﺒﺎ ﻋﻠﻰ إﻧﺘﺎﺟﻴﺔ اﻟﻤﺤﺎﺻﻴﻞ وﺟﻮدﺗﻬﺎ:اﻟﻤﻠﺨﺺ وأﻻﺻﺒﺎغ اﻟﻀﻮﺋﻴﺔ و ﻣﺤﺘﻮى اﻟﺒﻮﻟﻴﻔﻴﻨﻮل و ذﻟﻚ ﺗﺤﺖ ﺗﺄﺛﻴﺮ ﻣﻠﻮﺣﺔ آﻠﻮرﻳﺪ، اﻟﺤﺎﻟﻴﺔ و اﻟﺘﻲ أﺟﺮﻳﺖ ﻟﺪراﺳﺔ اﻟﺘﻐﻴﺮات ﻓﻲ ﺧﺼﺎﺋﺺ اﻟﻨﻤﻮ ﻣﻠﻲ ﻣﻦ ﺗﺮآﻴﺰ آﻠﻮرﻳﺪ200 ﻓﻲ٪ 18.6 ﻣﻦVetiverria zizanioides (L) ﺑﻴﻨﺖ اﻟﻨﺘﺎﺋﺞ أﻧﻪ هﻨﺎك زﻳﺎدة ﻓﻲ ﻃﻮل اﻟﺴﺎق ﻣﻦ.اﻟﺼﻮدﻳﻮم ﻧﺴﺒﺔ اﻟﻨﻤﻮ. ﻣﻞ50 آﻠﻮرﻳﺪ اﻟﺼﻮدﻳﻮم ﻓﻲ٪24.8 L) Vetiverria zizanioides) اﻟﺼﻮدﻳﻮم ﻓﻲ ﺣﻴﻦ ﻟﻮﺣﻆ زﻳﺎدة ﻓﻲ ﻃﻮل اﻟﺠﺬر ﺣﻮل ﻣﻠﻢ ﻣﻦ300 ﻣﻠﻲ ﺑﻴﻨﻤﺎ ﺗﺮآﻴﺰ آﻠﻮرﻳﺪ اﻟﺼﻮدﻳﻮم ﻓﻲ50 ﻓﻲ٪ 10،0 اﻟﺨﻀﺮي ﻟﻠﺠﺬر ﻓﻲ هﺬﻩ اﻟﺘﺠﺮﺑﺔ اﻷﻧﻮاع أوﺿﺤﺖ اﻧﺨﻔﺎض ﺑﻨﺴﺒﺔ و أوﺿﺤﺖ. ﻓﺈن ﻣﺘﻮﺳﻂ ﻣﺴﺎﺣﺔ اﻟﻮرﻗﺔ ﻳﺰداد أﻳﻀﺎ ﺗﺤﺖ ﺗﺄﺛﻴﺮ اﻟﻤﻠﻮﺣﺔ، وﻋﻼوة ﻋﻠﻰ ذﻟﻚ.٪39.09 اﻟﻤﻠﻮﺣﺔ آﺎن ﺑﻨﺴﺒﺔ اﻻﻧﺨﻔﺎض ﺣﻮاﻟﻲ اﻟﻨﺘﺎﺋﺞ إن هﻨﺎك زﻳﺎدة ﻓﻲ أﻹﺻﺒﺎغ اﻟﻀﻮﺋﻴﺔ ﻣﻊ أي اﻧﺨﻔﺎض ﻓﻲ ﻣﺴﺘﻮﻳﺎت اﻟﻤﻠﻮﺣﺔ ﻣﻤﺎ ﻳﺸﻴﺮ إﻟﻲ ﺁﻟﻴﺔ اﻟﺘﻨﺎﺿﺢ و ﻣﺪي ﻗﺪرة اﻟﻨﺒﺎﺗﺎت ﻣﺤﻞ آﺎن اﻟﻮزن اﻟﺠﺎف واﻟﻄﺎزج و وزن اﻟﻜﺘﻠﺔ اﻟﺤﻴﻮﻳﺔ اﻟﻨﺒﺎﺗﻴﺔ أﻗﻞ، وﻣﻊ ذﻟﻚ.اﻟﺘﺠﺮﺑﺔ ﻋﻠﻲ ﺗﺴﻮﻳﺔ وﺿﻌﻴﺘﻬﺎ ﻣﻤﺎ ﻳﺆآﺪ ﻗﺪرﺗﻬﺎ ﻋﻠﻲ ﻣﻘﺎوﻣﺔ اﻟﻤﻠﻮﺣﺔ وﻟﻮﺣﻆ زﻳﺎدة ﺧﻄﻴﻪ ﻟﻠﻤﻮﺻﻠﻴﺔ. ورﺑﻤﺎ زﻳﺎدة ﻣﺴﺘﻮﻳﺎت اﻟﻤﻠﻮﺣﺔ اﻟﻤﺮﺗﻔﻌﺔ ﻓﻲ اﻟﺒﻮﻟﻴﻔﻴﻨﻮل ﻳﻜﻮن ﺑﺴﺒﺐ ﺗﺮاآﻢ اﻟﻤﺮآﺒﺎت اﻟﺜﺎﻧﻮﻳﺔ.ﺗﺄﺛﺮا ﺑﺎﻟﻤﻠﻮﺣﺔ ﻣﻠﻲ100 و هﻨﺎك أﻳﻀﺎ زﻳﺎدة ﻓﻲ اﻟﻨﻤﻮ واﻟﻘﻴﺎﺳﺎت ﻟﻠﺘﻤﺜﻴﻞ اﻟﻀﻮﺋﻲ ﺧﺎﺻﺔ ﻓﻲ.اﻟﻜﻬﺮﺑﺎﺋﻴﺔ واﻟﻤﻮاد اﻟﺼﻠﺒﺔ اﻟﺬاﺋﺒﺔ ﻓﻲ اﻟﺘﺮﺑﺔ ﻣﻊ زﻳﺎدة اﻟﻤﻠﻮﺣﺔ V. وﻧﺴﺘﻨﺘﺞ ﻣﻦ هﺬا أﻧﺔ ﻳﻤﻜﻦ زراﻋﺔ اﻟﺼﻨﻒ اﻟﻤﻘﺎوم.ﻣﻦ اﻟﻤﻠﻮﺣﺔ و ﺗﺸﻴﺮ هﺬﻩ اﻟﻨﺘﺎﺋﺞ إﻟﻲ ﻣﺪي ﻣﻘﺎوﻣﺔ اﻟﺘﻲ أوﺿﺤﺘﻬﺎ اﻟﻨﺒﺎﺗﺎت ﻣﺤﻞ اﻟﺘﺠﺮﺑﺔ .ﻣﻠﻢ 200 اﻟﻰ ﻟﻴﺼﻞ اﻟﻤﻠﺢ ﺗﺮآﻴﺰ ﻣﻊ اﻟﻤﺎﻟﺤﺔ اﻟﻤﻨﺎﻃﻖ ﻓﻲ zizanioides
∗
Corresponding author, Email:
[email protected]
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basin in the USA whereas, in northeast frontier provinces of Asian continent about 300 million US $ per year and 208 million US $ per year for the Murry-Darling basin in Australia was reported (Ghassemi et al., 1995). Physiologically and genetically salt tolerance is complex among the variety of plants with a wide range of adaptations in halophytes and less tolerant plants (Flowers, 2004). Growth analysis is a fundamental characteristic to study plant’s response to an environmental stress. In addition, photosynthesis is one of the most important biochemical pathways by which plants prepare their own food material and grows; (Kojo, 2004) while levels of polyphenols acts as an indicator of stress conditions (Kate, 2008). At high sodium chloride concentration reduction in growth parameters genus Populus L. was reported earlier (Sixto et al., 2005). Moreover chlorophyll and carotenoid content are the important indicators of the healthy plant and increase in chlorophylls content under saline environment depending on salt levels has been reported (Mane et al., 2010). Some investigators reported the increase in the levels of polyphenol content under the influence of increasing levels of salinity, which indicates the induction of secondary metabolism which is one of the defence mechanism adapted by the plants to face saline environment (Kate, 2008). The present investigation was carried out to study the alterations in the growth characteristics, photosynthetic pigments and polyphenols of the Vetiveria zizanioides under the influence of sodium chloride salinity. This type of study will be useful to develop a salt tolerant species for the phytoremediation purpose.
Introduction Soil is a valuable resource with a key productive role in agriculture and forestry, since it is needed to produce crops, vegetables, fruits, timbers and other economically important items (NEP, 2006). For the welfare of the future generations, sustainable management of the soil resource, is one of the central issue worldwide (Munns, 2002). It is well established that abiotic stress including high winds, extreme temperatures, salinity, drought, flood and other natural disasters, such as tornado and wild fire have negative impact on the living organisms in a specific environment and are the most harmful factors concerning the growth and productivity of crops worldwide (Mane et al., 2010). Among which salinity is one of the major abiotic stress that adversely affect crop productivity and quality with increasing impact on the socioeconomic fabric and health, especially on the farming communities. Salinity is a general term used to describe the presence of elevated levels of different salts such as sodium chloride, magnesium and calcium sulphates and bicarbonates in soil and water (Ouda, 2008). The literature survey suggests that the statistics of the extent of salt affected areas are approximately close to one billion hectares, representing about 6% of the earth’s continental extent (FAO, 1997). In addition to these naturally salt affected areas, about 77 million hectors have been salinised by human activities such as; introduction of irrigation without proper drainage system, industrial effluents, overuse of fertilisers, removal of natural plant cover, flooding with salt rich waters, high water table and the use of poor quality groundwater (Parida and Das, 2005). Moreover Oldeman et al. (1991) estimates it to be 76 million hectors with 58% of these concentrating in irrigated areas. Current assessment made by the institute revealed that nearly 6.73 million hectare area in India is salt affected (CSSRI, 2009). When soil salinisation is assessed in economic terms, reasons to be worried about it become more apparent. For instance, the economic damage caused by secondary salinisation was estimated at 750 million US $ per year for the Colorado river
Materials and Methods Materials used in the study The seedlings of Vetiveria zizanioides (L.) Nash was collected from the government nursery, Kolhapur, India (Figure 1). The seedlings were uniformly cut to a minimum height (about 4 cm) required for their growth and then transplanted into the earthen pots (30 cm height with a narrow base). Earthen pots were used in the present investigation with the 60
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aim to see the accurate impact of the salinity doses provided to the rooting medium. To grow and establish under normal conditions regular watering was provided. After four weeks of the plants normal growth under salinity stress were studied. The plants were treated with increasing salt concentrations of sodium chloride i.e. 25, 50, 100, 200 and 300 mM concentrations. Every alternate day, they were watered with a double amount to maintain the uniform salt concentration in the pots and to cope up with the loss of water by evaporation from the soil surface and transpiration from the plant surface. The chemicals used in this study were of the highest purity available and of the analytical grade.
Estimation of photosynthetic pigments Chlorophylls content The chlorophylls of the mature leaves were estimated by following the method of Arnon (1949) with some modification. Fresh plant material (1g) was roughly homogenised in morter by keeping the temperature at 2°C in dark condition and extraction was carried out using 90% acetone, with addition of a pinch of magnesium carbonate, to protect and stabilize the chlorophylls. This extract was filtered through Whatman No.1 filter paper under suction using Buchner’s funnel. The residue was washed thoroughly 2-3 times with 90% acetone, collecting all the washings in the same filtrate and final volume of the filtrate was made to 100 ml with 90% acetone. Absorbance of chlorophyll ‘a’ and ‘b’ was recorded using double beam UV-Visible spectrophotometer (Elico SL-159, India), at 663 and 645 nm using 90 % acetone as blank. Following formulae were used to determine the chlorophylls content. Chlorophyll ‘a’ = X = 12.7 x A663 - 2.69 x A645 Chlorophyll ‘b’ = Y = 22.9 x A645 - 4.68 x A663 Total chlorophyll (a + b) =Z=8.02xA663+ 20.20 x A645
Carotenoids content The carotenoids content of the leaves was determined from the same extract used for chlorophyll estimation, by recording the absorbance at 480 nm using UV-VIS double beam spectrophotometer by putting 90% acetone as the control. The carotenoids content were calculated by using the following formula (Kirk and Allen, 1965).
Figure 1. Photograph of Vetiveria zizanioides (L.) Nash with complex root system that holds the contaminated soil.
Study of the growth characteristics Ten plants of V. zizanioides from each treatment pots were carefully uprooted and washed thoroughly with water to remove any dirt and dust particles on the surface of the plant parts and blotted to surface dry. The plant materials were analysed for root length, shoot length, shoot: root ratio, height of a plant and average leaf area. For biomass production the fresh weight, dry weight and moisture content of the randomly sampled ten leaves were recorded.
Carotenoids =
A480 x volume of extract x 10 x 100 2500x weight of plant material (g)
Polyphenols content The polyphenols content of the leaves was estimated following the method suggested by Folin and Denis (1915). Two ml of acetone extract used for chlorophyll was mixed with 10 ml of 20% sodium carbonate and adjust the volume to 35 ml with distilled water to this mixture 2 ml of Folin-Denis reagent was added, mixed thoroughly and finally adjust the volume 50 ml with distilled water. The 61
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standard tannic acid solution (0.1 mg ml-1) was used for the preparation of standard polyphenol curve by measuring the absorbance at 660 nm using double beam UV-Visible spectrophotometer.
Effects of salinity on root length of V. zizanioides Stimulation of root length with increasing salt levels has been documented in Triticum aestivum (Maghsoudi and Maghsoudi, 2008). The stimulation effect of NaCl salinity on the root length is usually more than that for the shoots at the lower level of NaCl salinity (Pessarakli et al., 2004). In the present investigation root length of V. zizanioides was observed to be increased at 50 mM NaCl concentration with an increase by 24.81% (Table 1) but at highest concentration (300 mM) it was sharply reduced by 31.77% as compared to control plants. From the present investigation, it is clear that the root growth of the selected grass species was more sensitive and severely affected at higher levels of salinity. It might be due to the toxic nature of NaCl at higher concentration. Shoot to root ratio in the experimental species was reduced to 10.04% at 50 mM NaCl concentration but later on increased with the higher doses of salinity (Table 1). It is clear that the shoot to root ratio is considerably increased in experimental grass species due to the NaCl salinity especially at higher salinity levels. It might be due to the more allocation of assimilates from the root to shoot.
Electrical conductivity and total dissolved solids Electrical conductivity (EC) and total dissolved solids (TDS) of the treated sieved soil was determined after completion of the experimental treatments (after 10th week) by preparing 1:10 soil suspension in water. This suspension was vigorously shaken, allowed to stand for 12 h and then filtered through Whatman No.1 filter paper for analysis. Both the parameters were determined by using Elico EC-TDS meter (CM 183, Make-India) where electrode was directly dipped into the well diluted and filtered solutions for the direct display of the result on a digital scale. Statistical analysis Data were analyzed by one-way analysis of variance (ANOVA) with Tukey-Kramer multiple comparisons test. Results and Discussion Effects of salinity on shoot length of V. zizanioides At higher salinity, reduction in shoot length along with a decrease in moisture content was reported (Parvaiz and Riffat, 2005). Khosravinejad et al. (2009) also noticed a significant decrease in shoot elongation in barley genotypes with increasing NaCl treatment. From the present results, it was observed that the shoot length of all V. zizanioides was stimulated at lower levels of salinity only. The shoot length of V. zizanioides was increased by 18.60% at 200 mM NaCl concentration (Table 1). However, it was adversely affected by higher 300 mM of NaCl concentration to the rooting medium. It appears that the grass species exhibit salinity tolerance upto 200 mM NaCl as far as linear growth of plant is concerned.
Effects of salinity on plant height of V. zizanioides Plant growth inhibition is one of the most important agricultural indices of salt stress tolerance as indicated by different studies (Parida and Das, 2005). The immediate effect of addition of salt to the root medium is lowering in external water potential and reduction in water uptake of plants (Munns, 2002). V. zizanioides showed the highest increase by 17.98% in the plant height at 50 mM NaCl concentration but a decreased trend was observed at higher levels and duration of NaCl treatment (Table 1).
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Emir. J. Food Agric. 2011. 23 (1): 59-70 http://ejfa.info Table 1. Effect of sodium chloride on growth characteristics of Vetiveria zizanioides. Growth parameters Shoot length (cm) Root length (cm) Shoot : Root ratio Height of the plant (cm) Average leaf area (cm2) Fresh weight of leaves plant-1 (g) Dry weight of leaves plant-1 (g) Moisture content (%)
Sodium chloride concentration (mM) Control 58.60 (±5.95) 0.0a 53.20 (±2.82) 0.0 a 1.10 (±0.05) 0.0 a 111.80 (±8.73) 0.0 a 34.93 (±3.54) 0.0 a 3.94 (±0.36) 0.0 a 2.53 (±0.38) 0.0 a 36.09 (±3.89) 0.0 a
25 60.20 (±5.25) +2.73 55.50 (±3.57) +4.32 1.08 (±0.05) -1.82 115.70 (±8.51) +3.49 35.89 (±3.13) +2.75 4.28 (±0.43) +8.63 2.69 (±0.38) +6.32 37.31 (±2.51) +3.38
a The values indicates percentage variation relative to control values
50 65.50* (±3.87) +11.77 66.40** (±2.99) +24.81 0.98** (±0.08) -10.90 131.90 (±4.51) +17.98 39.05* (±2.30) +11.80 4.48 (±0.35) +13.71 2.72 (±0.34) +7.51 39.47 (±2.98) +9.37
100 67.30*** (±4.52) +14.85 63.80*** (±2.49) +19.92 1.06 (±0.07) -3.64 131.10 (±6.01) +17.26 40.12*** (±2.70) +14.86 6.0*** (±0.35) +52.28 3.38 (±0.38) +33.60 43.78 (±3.01) +21.31
200 69.50*** (±3.10) +18.60 53.30 (±2.87) +0.19 1.31*** (±0.05) +19.09 122.80 (±5.49) +9.84 41.43*** (±1.85) +18.61 4.93 (±0.43) +25.13 3.20 (±0.37) +26.48 35.22 (±1.74) -2.41
300 55.10 (±2.28) -5.97 36.30*** (±2.11) -31.77 1.53*** (±0.15) +39.09 91.40 (±1.43) -18.25 32.85 (±1.36) -5.95 3.45 (±0.407) -12.44 2.39 (±0.37) -5.53 30.81 (±2.55) -14.63
Values are mean of ten experiments ±SD. Significantly different from the control at *P>0.05, **P
