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Journal of Stress Physiology & Biochemistry, Vol. 15, No. 3, 2019, pp. 81-91 ISSN 1997-0838 Original Text Copyright © 2019 by Reyaz Ahmad Mir, Somasundaram and Panneerselvam
ORIGINAL ARTICLE
OPEN fV\ ACCESS
3
Changes in Antioxidant Enzymes Activities Mitigates Deleterious Effects of ROS in Panicum miliaceum (L.)
under Drought Stress
Reyaz Ahmad Mir, R. Somasundaram, R. Panneerselvam
Dept. of Botany, Annamalai University, Annamalai Nagar-608 002., Tamil Nadu, India
*E-Mail: botanysundaram@gmail .com
Received August 28, 2019
In nature, plants are always subjected to various abiotic stresses such as drought, salinity, high temperatures and so on. Among these drought is a worldwide problem, responsible for limiting the growth, physiology and productivity of plants, thus has become a challenge for global food security towards growing population. Proso millet (Panicum miliaceum L.) belonging to family Poaceae grows under wide environmental conditions and different soil profile. In this view, an experiment was conducted to investigate the range of tolerance and change of metabolic activities of Proso millet under drought stress. The control plants were irrigated regularly and treated plants were irrigated at 3, 5, 7, day intervals up to 60day interval drought (DAS). The root and leaf samples were collected on 30 DAS, 45 DAS and 60 DAS respectively for morphological and biochemical analysis. It was found that with increasing duration of water deficit, tremendous increases of antioxidants activities were recorded at all growth stages compared to control on 7DID at 60DAS. Furthermore, a decreased rate of growth, biomass and chlorophyll content was recorded in treated plants than control. Therefore, it can be concluded that proso millet has affinity to survive under prolonged drought stress and can help to understand the mechanism of photosynthetic efficacy for improving crop productivity.
Key words: Abiotic stress, antioxidants, drought stress, pigments, proso millet
Drought is the most severe environmental factor which results in global warming and affects almost all living organisms in the world. Water is an important factor in agricultural and food productivity, yet a highly limited resource (Wang et al., 2012). Drought stress causes a major limitation to plant growth and development by affecting cell membrane integrity, osmotic adjustment and photosynthetic ability (Ravikumar et al., 2014). Plant experiences drought stress either when the water supply to roots becomes difficult or when the transpiration rate becomes very high (Budak et al., 2013). The climatic changes associated with global warming, drought stress may occur at any time during the growing season and thus may cause a profound decrease in biomass production and can lead to disastrous crop failure (Kabira and Muthoni, 2016).
When a plant is subjected to water deficit stress, it reacts by producing a range of reactive oxygen species (ROS) during photosynthesis, photorespiration and dark respiration. Moreover, overproduction of ROS causes damage to DNA, proteins and lipids in the plant cells that suffer from water deficit and adversely affects the crop production (Feng et al., 2015). In order to reestablish redox homeostasis, plants have an internal ROS scavenging system known as antioxidants that alleviates oxidative damages, thus ensuring normal cellular function (Abass et al., 2017). Among enzymatic antioxidants, Superoxide dismutase (SOD) acts as first defence line catalyzing superoxide radical to oxygen and hydrogen peroxide (H2O2). However, H2O2 can be further eliminated by catalase (CAT), ascorbate peroxidase (APX) and Proline oxidase (POX) present in the cell organelles (Hasheminasab et. al., 2012). On the other hand, non-enzymatic antioxidant such as ascorbic acid is the most abundant, powerful, and water-soluble antioxidant that can act to prevent or alleviate the damage caused by ROS in plant cells (Athar et al., 2008). Besides ascorbic acid, the a-tocopherol, a flavonoid compound found in chloroplasts, also helps in quenching of oxygen, alleviation of oxidative damage in chloroplast and increases membrane rigidity, thereby plays a vital role in development, signal transduction, interacting or controlling phytohormonal regulation (Arrom and Munn'e-Bosch, 2010).
Millets are small-seeded annual cereals grown for food, forage, and fuel (Kothari et al., 2005). Such crops are grown on marginal lands and under low-input agricultural conditions-situations in which major cereal crops often fail to grow or produce low yields (Amadou et. al., 2013). Proso millet (Panicum miliaceum L.) is one of the important species of the largest genus Panicum, which include more than 400 species is still widely cultivated in the arid and semi-arid areas of the world (Nazifi et al., 2009). Undoubtedly, Panicum miliaceum is considered as relief and anti-famine crop; hence, it is a very efficient user of soil water and is well adapted to different types of soils and dry weather conditions. Therefore, the present was undertaken to evaluate the growth and physiological parameters, role of antioxidants activities in Panicum miliaceum L. under drought deficit condition.
MATERIALS AND METHODS
Collection of plant materials
The seeds of Panicum miliaceum (L.) were collected from Kolimalai, of Salem district, Tamilnadu, India and were identified by Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India. The experiment was laid out in a completely randomized block design (CRBD). Quality seeds were disinfected by 0.2% HgCh solution for 2minutes with frequent shaking and rinsed with deionised water to remove traces of HgCh. Then, seeds were soaked in distilled water for 6hour and the hydrated seeds were sown in plastic pots containing sand, red soil and farmyard manure in the ratio of 1:1:1. For each treatment three replicates were maintained (each replicate is one pot containing four plants). After 15 days, drought stress treatment was given by altering the irrigation schedule at different day intervals as 3, 5 and 7 days (DID). While control (0 DID) plants were routinely irrigated daily with tap water. Samples were collected on 30, 45 and 60 DAS (days after sowing) for further analysis.
Estimation of Morphological parameters
Proso-millets were randomly uprooted carefully from each treated plant and brought to laboratory immediately, then rinsed with tap water followed by distilled water and blot dried with tissue papers for
morpho-physiological and biochemical estimation. Estimation of Root and Stem length, Fresh weight (FW) and Dry weight (DW)
The root length and the shoot length were expressed in cm plant-1. The FW and DW were taken in electronic balance and expressed in g plant-1. Estimation of Photosynthetic Pigments
Chlorophyll concentration was determined using Arnon's method (1949). Fresh leaf tissue (0.5 g) was extracted with 80 % acetone (v/v) and centrifuged at 2,500 rpm for 10 min at 40C. The extract was made up to 10 ml with 80 % acetone. The optical density (O.D.) of the extract (3ml) was measured against a blank of pure 80% aqueous acetone at wavelengths of 645 and 663nm using Hitachi U-2000 spectrophotometer and was expressed in terms of mg g-1 fresh weight. Determination of Antioxidant Enzyme Activities
For extraction of enzymes, fresh samples (0.5g) were homogenized with 1.5 cm3 of 100mM potassium phosphate buffer solution (pH 7.0) containing 2mM phenylmethylsulfonyl fluoride (PMSF), and centrifuged at 14,0009g at 40C for 20 min.
SOD (EC 1.15.1.1) activity assayed was described by Beauchamp and Fridovich (1971). The reaction mixture contained 1.17 9 10-6 M riboflavin, 0.1 M methionine, 2 x 10-5 M potassium cyanide (KCN), and 5.6 x 10-5 M nitroblue tetrazolium salt (NBT) dissolved in 3 ml of 0.05 M sodium phosphate buffer (pH 7.8) 3 ml of the reaction medium was added to 1 ml of enzyme extract. The mixtures were illuminated in glass test tubes by two sets of Philips 40 W fluorescent tubes in a single row. Illumination was started to initiate the reaction at 300C for 1 h, identical solutions that were kept under dark served as blanks. The absorbance was read at 560 nm in the spectrophotometer against the blank. SOD activity was expressed in units. One unit (U) is defined as the amount of change in the absorbance by 0.1 h-1 mg-1 protein.
CAT (EC 1.11.1.6) was determined according to the method of Chandlee and Scandalios (1984) with slight modification. The assay mixture contained 2.6 ml of 50 mM potassium phosphate buffer (pH 7.0), 0.4 ml of 15 mM H2O2 and 0.04 ml of enzyme extract. The decomposition of H2O2 is followed by the decline in
absorbance at 240 nm. The enzyme activity is expressed in U mg-1 protein (U = 1 mM of H2O2 reduction min-1 mg-1 protein).
Peroxidase (EC 1.11.1.7) activity was estimated by the method followed by Reddy et al. (1995). 20 % homogenate was prepared in 0.1 M phosphate buffer (pH 6.5). The reaction mixture contained 3.0 ml of pyrogallol solution and 0.1 ml of the enzyme extract. To the test cuvette, 0.5 ml of H2O2 was added and mixed. The change in absorbance was recorded at 430 nm every 30s up to 3min. One unit of peroxidase is defined as the change in absorbance/minute at 430 nm. Estimation of non-enzymatic antioxidants
Ascorbic acid content was estimated as described by Omay et al. (1979) and the results were expressed in mg/g DW. Alpha -tocopherol (a-toc) content was determined according to Backer et al. (1980) and result was expressed in mg/g FW.
Statistical analysis
Data were analyzed using statistical program SPSS v16.0 and presented as the means ± SD of three replicates; each replicate consisted of four plants.
RESULTS
Root and shoot length
In the present study, a significant decrease was found in the shoot length of P. miliaceum with the increase in drought period. The highest decreased rate (48.67cm) was found on 7 DID at 60 DAS as compared to control (68.67cm). However, root length of P. miliaceum was found to be increased at all growth stage with the duration of drought stress and the highest value (42.00 cm) was found on 7 DID as compared to control (29.27cm) (Table 1). Fresh weight and dry weight
Drought stress decreased the whole plant fresh weight and dry weight at all growth stages of P. miliaceum significantly than control ones. Whole plant fresh weight and dry weight at 5DID and 7DID drought treatments was more affected than at mild stress (3DID). When compared with control, fresh weight and dry weight were noted as 29.25 g plant-1 and 12.41 g plant-1 respectively, on 7DID at 60DAS (Table 2).
Chlorophyll Pigments
With increasing severity and period of drought stress there was a significant decrease in the Chlorophyll 'a' and Chlorophyll 'b' content. As compared to Chl 'a' there was a significant decrease in Chlorophyll 'b' content of P. miliaceum. The decreased rate recorded was 0.06 and 0.05mg g-1FW respectively compared to control. Moreover, total chlorophyll content (0.11 mg g-1FW) was found to be decreased with the severity of drought, compared to control (Table 3). Antioxidants
SOD activity both in the leaves and roots increased with increased drought treatment in all stages of growth. SOD activity varied in different parts of the plant. The highest activity was recorded in leaves 3.33 (U. mg-1 protein h-1) than roots 2.57 (U. mg-1 protein h-1) compared to control having less SOD content on 7DID at 60 DAS (Table 4).
Catalase activity has been increased progressively under drought period in both the root and leaves of P. miliaceum in all the treatments as compared to control.
Table 1: Effect of drought stress on shoot length and plant-1)
The highest level recorded was 0.52 (U. mg-1 protein min-1) in leaves and 0.31(U. mg-1 protein min-1) in roots at 7DID of 60 DAS respectively (Table 5).
Drought treated plants shown increased POX activity with increased duration of drought stress when compared to the control plants. Highest POX activity (2.46 U. mg-1 protein min-1) and 2.03(U. mg-1 protein min-1) was recorded in both leaves and roots of P. miliaceum at 7DID on 60DAS (Table 6).
The AsA content increased with the age of the plant in control and drought treated plants in both the leaves and root samples. Among them, the leaves recorded the highest AA content than roots and overall increase than the control. The higher AA content recorded was 3.11mg/g DW and 2.07 mg/g DW respectively, in the leaves and roots samples of P. miliaceum at 7DID on 60DAS (Table 7).
Similarly, a-Tocopherol content found in drought treated plants was higher (Table 8) than that of control on 30th, 45th and 60th DAS. The highest a-toc content recorded was 0.98 mgg-1 FW and 0.83 mgg-1FW in leaves and roots on 60th DAS in 7DID treated plants. length of P. miliaceum L. (Values are expressed in cm
Shoot Length (cm)
DAS Control 3DID 5DID 7DID
30 56.00±0.58 53.27±1.12 46.54±1.53 40.31±0.93
45 63.22±0.27 56.17±0.09 52.17±0.30 45.81±0.37
60 68.67±0.29 61.11±0.58 54.75±0.53 48.67±0.76
Root Length(cm)
DAS Control 3DID 5DID 7DID
30 19.00±0.22 23.00±0.40 25.60±0.66 28.00±0.30
45 24.21±0.25 29.00±0.34 33.34±0.30 35.24±0.20
60 29.27±0.25 33.25±0.20 38.10±0.42 42.00±0.27
Values represented above are mean of 3 replicates; '±' standard deviation.
Table 2: Effect of drought stress on fresh weight and dry weight of P. miliaceum L. (Values are expressed in g plant-1)
Fresh Weight
DAS Control 3DID 5DID 7DID
30 29.17±0.15 27.10±0.34 23.30±0.17 18.34±0.24
45 34.04±0.23 30.84±0.35 28.42±0.20 25.50±0.53
60 39.07±0.34 36.02±0.13 33.40±0.28 29.25±0.54
Dry Weight
DAS Control 3DID 5DID 7DID
30 13.02±0.61 11.12±0.25 9.10±0.23 6.70±0.44
45 17.01±0.22 15.14±0.45 11.06±0.62 8.39±0.66
60 19.30±0.16 16.00±0.60 14.60±0.50 12.41±0.30
Values represented above are mean of 3 replicates; '±' standard deviation
Table 3: Effect of drought stress on chlorophyll 'a' chlorophyll 'b' and Total Chlorophyll (a+b) of P. miliaceum L. (Values are expressed in mg g-1 FW)
Chlorophyll "a"
DAS Control 3DID 5DID 7DID
30 0.06±0.12 0.05±0.13 0.04±0.09 0.03±0.06
45 0.08±0.14 0.06±0.05 0.05±0.08 0.04±0.07
60 0.16±0.23 0.13±1.00 0.09±1.01 0.06±1.03
Chlorophyll "b"
DAS Control 3DID 5DID 7DID
30 0.05±0.03 0.04±0.03 0.03±0.01 0.02±0.05
45 0.08±0.02 0.07±0.03 0.05±0.02 0.04±0.08
60 0.11±0.12 0.09±0.04 0.08±0.03 0.05±0.10
Total Chlorophyll(a+b)
DAS Control 3DID 5DID 7DID
30 0.11±0.08 0.09±0.07 0.07±0.05 0.05±0.02
45 0.16±0.17 0.13±0.14 0.10±0.11 0.08±0.07
60 0.27±0.23 0.22±0.16 0.17±0.12 0.11±0.06
Values represented above are mean of 3 replicates; '±' standard deviation
Table 4: Effect of drought stress on superoxide dismutase activiy in shoot and root of P. miliaceum L.(Values are
expressed in U. mg-1 protein h-1)
Shoot
DAS Control 3DID 5DID 7DID
30 0.65±0.97 1.31±0.59 1.74± 1.02 2.01±1.22
45 1.54±0.82 2.01±0.91 2.12±1.19 2.53±0.83
60 2.06±0.27 2.62±1.52 3.05±1.72 3.33±1.62
Root
DAS Control 3DID 5DID 7DID
30 0.62±0.83 0.87±0.05 1.03±0.82 1.47±0.98
45 1.21±0.93 1.59±0.81 1.82±0.95 2.01±1.01
60 1.45±1.06 2.06±1.05 2.37±0.82 2.57±0.96
Values represented above are mean of 3 replicates; '±' standard deviation
Table 5: Effect of drought stress on catalase activity in shoot and root of P. miliaceum L. (Values are expressed in U. mg-1 protein min-1)
Shoot
DAS Control 3DID 5DID 7DID
30 0.23±0.06 0.28±0.06 0.33±0.11 0.40±0.12
45 0.31±0.07 0.36±0.08 0.40±0.11 0.46±0.10
60 0.39±0.10 0.43±0.11 0.47±0.13 0.52±0.13
Root
DAS Control 3DID 5DID 7DID
30 0.14±0.04 0.17±0.04 0.21±0.05 0.25±0.07
45 0.22±0.06 0.24±0.06 0.26±0.07 0.29±0.08
60 0.25±0.07 0.26±0.08 0.28±0.13 0.31±0.19
Values represented above are mean of 3 replicates; '±' standard deviation.
Table 6: Effect of drought stress on peroxidase activity in shoot and root of P.miliaceum L. (Values are expressed in U. mg-1 protein min-1).
Shoot
DAS Control 3DID 5DID 7DID
30 0.52±0.14 0.74±0.32 0.81±0.52 1.04±0.59
45 0.85±0.29 1.31±0.34 1.46±0.53 1.80±0.65
60 1.14±0.37 1.62±0.43 2.13±0.55 2.46±0.72
Root
DAS Control 3DID 5DID 7DID
30 0.32±0.23 0.57±0.24 0.66±0.35 0.81±0.45
45 0.84±0.48 1.07±0.49 1.46±0.48 1.64±0.51
60 1.03±0.81 1.43±0.74 1.68±0.70 2.03±0.71
Values represented above are mean of 3 replicates; '±' standard deviation.
Table 7: Effect of drought stress on ascorbic acid activity in shoot and root of P.miliaceum L. (Values are expressed in mg/g DW)
Shoot
DAS Control 3DID 5DID 7DID
30 1.28±0.22 1.58±0.39 1.72±0.56 2.41±0.54
45 1.90±0.26 2.18±0.49 2.46±0.59 2.73±0.58
60 2.21±0.45 2.43±0.60 2.65±0.61 3.11±0.63
Root
DAS Control 3DID 5DID 7DID
30 0.66±0.02 0.87±0.03 0.99±0.04 1.21±0.05
45 1.14±0.08 1.25±0.10 1.36±0.13 1.48±0.15
60 1.43±0.14 1.72±0.16 1.85±0.19 2.07±0.23
Values represented above are mean of 3 replicates; '±' standard deviation.
Table 8: Effect of drought stress on a-tocopherol activity in shoot and root of P. miliaceum L. (Values are expressed in mg/g FW)
Shoot
DAS Control 3DID 5DID 7DID
30 0.34±0.04 0.47±0.05 0.63±0.07 0.85±0.09
45 0.51±0.16 0.62±0.19 0.78±0.21 0.98±0.23
60 0.63±0.35 0.74±0.36 1.02±0.41 1.36±0.53
Root
DAS Control 3DID 5DID 7DID
30 0.31±0.03 0.42±0.04 0.54±0.06 0.71±0.08
45 0.48±0.12 0.57±0.16 0.68±0.19 0.82±0.22
60 0.56±0.18 0.63±0.19 0.71±0.34 0.83±0.44
Values represented above are mean of 3 replicates; '±' standard deviation.
DISCUSSION
Drought stress occurs when the plants are subjected to water shortage, which reduces photosynthesis, cell number and growth. In each of these processes, large number of genes, enzymes, hormones and metabolites are involved (Skirycz and Inze, 2010). Water deficit causes an increase in ROS production in plants, and leads to an oxidative stress. However, there is an internal ROS scavenging system in plants enzymatic and non-enzymatic antioxidants such as Superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), Ascorbic acid (AsA) and alpha-tocopherol that alleviates oxidative damages, maintains intracellular redox state homeostasis, thus ensuring normal cellular function. (Langebartels et al. 2002; Foyer and Noctor 2005, Fang and Xiong 2015, Abass et al., 2017).
The root length of P. miliaceum increased with increased drought stress treatment. Increasing root length due to water stress was reported in Pearl millet (Kusaka et al., 2005), Catharanthus roseus (Jaleel et al., 2008), Triticum aestivum (Dickin and Wright, 2008) and Panicum sumatrense (Ajithkumar and Panneerselvam, 2013). It is obvious from the results
that with increasing severity and period of drought, there was significant decrease in stem length of P. miliaceum. Similarly, significant decrease in stem length had been found in soybean (Specht et al., 2001) and stressed citrus seedlings under water deficit condition (Wu et al., 2008). Such decreased in shoot length may be due to impaired mitosis, cell elongation and expansion resulted from drought and causes reduced growth and yield traits (Hussain et al., 2008).
The whole plant fresh weight and dry weight of P. miliaceum was significantly declined with increased drought stress as compared to control. This decline was directly proportional to severity and duration of drought stress. Kobashi et al. (2000) found that the degree of drought controls the fresh weight such that it increases with moderate drought but decreases with severe drought. They attributed that decline in fresh weight to the decrease in the water content of stressed plant cells and tissues which lose their turgor and thus shrink.
Total dry weight decreased may be due to the considerable decrease in plant growth, photosynthesis and canopy structure as indicated by leaf senescence during drought stress in wheat (Gong et al., 2003).
Severe water stress may result in arrest of photosynthesis, disturbance of metabolism and finally drying (Liang et al., 2006). Mild water stress affected the shoot dry weight, while shoot dry weight was greater than root dry weight loss under severe stress in sugar beet genotypes (Mohammadian et al., 2005). Pekcan et al., (2016) also found similar results of reduction in both dry and fresh biomasses under the drought stress applications in sunflower genotypes.
The decrease in Chlorophyll content is a commonly observed phenomenon under drought stress (Mafakheri et al., 2010; Din et al., 2011). The decrease in chlorophyll content during drought stress may be due to chlorophyll degrading enzyme chlorophyllase. Increased activity of chlorophyllase and peroxidase enzymes under severe drought results decreased chlorophyll content (Abaaszsadeh et al., 2007). Moreover, decrease in net photosynthetic rate under water stress is also related to disturbances in biochemical processes of a non-stomatal nature, caused by oxidation of chloroplast lipids, changes in the structure of pigments and proteins (Marcinska et al., 2013). Similarly, Cao et al., (2015) reported that drought stress inhibited biosynthesis of the precursor of chlorophyll in tomato leaves which ultimately reduced the chlorophyll content.
A progressive increase in superoxide dismutase activity in both the shoots and roots of P. miliaceum was observed with the increasing severity of drought. Our results are in line with the results noted earlier in three cultivars of rice (Sharma et al., 2005) and Phaseolus vulgaris (Zlatev et al., 2006). Salekjalali et al. (2012) and Naderi et al., (2014) also reported that drought stress increased SOD activity in barley and wheat respectively. Increased SOD activity might be due to production of ROS under drought that has negative impacts on photosynthesis.
A positive correlation has been noticed between increased rate of catalase activity and severity of drought in both the shoots and roots of P. miliaceum L. Our results correlated with the findings of Simova-Stoilova et al. (2010), who reported increased CAT activity in sensitive varieties of wheat under drought stress. Catalase activity increased under drought stress in Zea mays (Jiang and Zhang, 2002), wheat (Dalmia
and Sawhney, 2004) and P. acutifolius (Turkan et al., 2005). The increase in catalase activity might be useful in disproportionating H2O2 that is the key product in reducing senescence under extreme moisture stress.
Similar case was found with peroxidase activity in the shoots and roots of P. miliaceum with increased drought period. Water stress could increase the accumulation of peroxidase substrate, which in turn is scavenger of activated oxygen species (Winston 1990). The important role of peroxidase in relation to oxidative tolerance has been reported in Arabidopsis thaliana (O'Kane et al., 1996) and cucumber (Lee and Lee, 2000). Increased peroxidase activity under water deficit condition was reported previously in soybean and wheat genotypes (Zhang et al., 2006; Shao et al., 2007).
Ascorbic acid can donate electrons to a wide range of enzymatic and non enzymatic reactions. Water stress resulted in significant increases in antioxidant ascorbic acid concentration in turf grass (Zhang and Schmidt, 2000) and Picea asperata seedlings (Yang et al., 2008). In Withania somnifera roots and leaves, the ascorbic acid content increased with age in drought stressed plants than normal one (Jaleel et al., 2009).
A direct proportional relationship was also observed between a-tocopherol content and duration of drought period in the shoots and roots of Panicum miliaceum i.e., with increased drought period a-tocopherol content also increased. It has been reported that water deficiency may result in an increase of tocopherol concentration in plant tissue (Wu et al., 2007; Shao et al., 2007). Synthesis of low-molecular weight antioxidants, such as, a-tocopherol has been reported in drought stressed wheat plants (Zhao et al., 2008). Some evidences implied that content of tocopherol was increased in wheat and spinach leaves as the amount of rainfall decreased or subjecting to water deficit (Shao et al., 2006, 2007).
CONCLUSION
From the above study it can be concluded that drought a kind of abiotic stress invites accelerated ROS that have deleterious effects on plant metabolism. However, P. miliaceum overcomes on decreased rate of chlorophyll contents and biomass content upto an
optimal level by increasing a series of antioxidants that possess potential of scavenging ROS with increasing duration of drought. Thus, from these observations we are able to say that P. miliaceum may be drought tolerant with adequate ROS scavenging system. Hence, in view of considerable variations in the protective mechanisms against ROS, P. miliaceum can be used to improve drought tolerance in sensitive crops by genetic engineering to get rid off from the drought and overcome food crisis.
ACKNOWLEDGEMENT
The authors are highly thankful to the Professor & Head, Department of Botany and Annamalai University for providing the facility to carry out the present work.
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