EFFECT OF FLUORIDE AND SALICYLIC ACID ON SEEDLING GROWTH AND BIOCHEMICAL PARAMETERS OF WATERMELON (CITRULLUS LANATUS)

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1 49 49 EFFECT OF FLUORIDE AND SALICYLIC ACID ON SEEDLING GROWTH AND BIOCHEMICAL PARAMETERS OF WATERMELON (CITRULLUS LANATUS) Amra Ram, a Pooja Verma, a BR Gadi b Jodhpur, India SUMMARY: The effect of sodium fluoride (NaF) on seed germination and biochemical parameters were studied in in vitro grown seedlings of watermelon (Citrullus lanatus) under salicylic acid (SA) treatment. After seven days of NaF treatment, reductions were observed in percentage of seed germination, root and shoot length, vigor index, pigment content, chlorophyll stability index (CSI), and membrane stability index (MSI) with increasing concentrations of NaF (1 and 10 mm). Seedlings treated with SA, both alone and in with combination of NaF, showed an increase in seed germination as well as other growth parameters. NaF-treated seedlings were found to accumulate more soluble sugars and phenolics, which were further increased by SA treatment thereby indicating a synergistic effect of SA and NaF on the accumulation of sugars and phenols. Therefore, these parameters are not directly related to a higher vigor index, membrane stability, and seedling growth. Treatment with SA reduced the adverse effects of NaF by increasing seed germination, root and shoot length, vigor index, pigment content, CSI, and MSI by some mechanism other than the accumulation of sugars and phenols. The possible reasons for the ameliorative effects of SA in seedlings of watermelon growing under F stress are discussed. Keywords: Biochemical parameters; Chlorophyll a and b; Chlorophyll stability index. Fluoride toxicity in plants; Membrane stability index; Phenols; Pigments; Salicylic acid; Seedling growth; Sugars; Watermelon (Citrullus lanatus). INTRODUCTION Chronic fluoride (F) exposure causes diverse toxic effects in both humans 1 and animals. 2 F is not an essential element for the normal growth of plants and in higher concentration it is toxic for plants, especially for early seedling growth. 3 Seed germination and early seedling growth are important phases for the successful growth and survival of plants and these physiological parameters of plants are greatly affected by NaF at different concentrations due to its inhibitory activities. 3,4 Several physiological and biochemical processes are known to be markedly affected by F such as chlorosis and necrosis of leaf, inhibition of the nutrient uptake, reduction of plant growth, and enzyme activities. 4-7 In India, several states are F endemic 8 including Rajasthan where ground water contains a high amount F in all the 33 districts. 9 In Rajasthan, due to irregular and low rainfall and recurrent drought, ground water is the main water source for irrigation for agricultural purposes. In addition, the soil of northwest Rajasthan contains elevated F due to the excessive use of diammonium phosphate fertilizers. 10 Salicylic acid (SA) is a phenolic compound which plays an important role as a signaling molecule for the regulation of several physiobiochemical processes in plants. 11,12 Through changes in hormonal levels, it plays protective roles in plants a Department of Botany, Jai Narain Vyas University, Jodhpur , India; b For correspondence: Dr BR Gadi, Associate Professor, Department of Botany, Jai Narain Vyas University, Jodhpur , India; brgadi@rediffmail.com

2 50 50 against pathogens and many abiotic stresses including drought, salt, and heavy metals. 12,13 Watermelon (Citrullus lanatus) is a fruit crop in the arid region of Rajasthan. 14 It is used to flavor summer drinks and smoothies in the region, where the temperature may rise up to 50ºC during the summer season. Seeds of watermelon are demulcent, diuretic, tonic, and seen as helpful for lowering blood pressure and improving cardiovascular function. Immature fruits are used as vegetables and both the fresh and dry rinds are important as cattle feed. Several studies have been conducted on the effects of F stress on seed germination, early seedling growth, and biochemical content. 4-6 However, no study has been done on reducing plant F toxicity by the application of plant growth regulators such as salicylic acid. Therefore, the present investigation was undertaken to ascertain the influence of salicylic acid on NaF-induced stress on early seedling growth and certain biochemical parameters viz. pigments, sugars, phenols, chlorophyll stability index (CSI), and membrane stability index (MSI). MATERIAL AND METHODS Sterilized seeds of C. lanatus were germinated in individual petri dishes and labeled as control (distilled water) and respective treatment solutions (1 and 10 mm NaF, 1 and 10 mm NaF+0.25 mm SA, and 0.25 mm SA). Petri dishes were kept at 28±2ºC in a Biochemical Oxygen Demand (BOD) incubator. The germination percentage was recorded on the third day after seed sowing. After seven days of treatment, the root length, shoot length, and vigor index 15 were measured and whole seedlings were used for estimation of the photosynthetic pigments content, 16 chlorophyll stability index, 17 membrane stability index, 18 total soluble sugars, 19 and phenolics content. 20 For the statistical analysis, three replicates were maintained for each treatment and each experiment was repeated twice. All data were subjected to one-way ANOVA test and means were compared by Student s t-test. RESULTS AND DISCUSSION Percentage of seed germination: The present study revealed that the increasing concentration of NaF decreased the percentage of seed germination in C. lanatus (Table 1). The higher concentration of NaF (10 mm) reduced the germination percentage by 23% as compared to the control. F-induced inhibition of germination may be due to a reduction of amylase activity which is essential for seed germination. 21 SA treatment increased the seed germination, both when used alone (8% increase compared to seed germination in the control group) and in combination of NaF (22% increase when compared to seed germination with NaF 10 mm). SA stimulates the seed germination via bio-synthesis of gibberellic acid (GA) which induces the amylase synthesis. 22 Root and shoot length: As shown in Table 1, the average root and shoot length decreased with increasing NaF concentration. Shoot growth in the seedlings was more affected than root growth. F-induced reduction in root and shoot length may be due to an unbalanced nutrient uptake by seedlings. 23 SA increased the root and

3 51 51 shoot length relative to the respective control groups. In consonance to our study, SA enhances shoot and root growth in many plants possibly by regulating cell elongation and cell division and by the prevention of auxins oxidation. 13 Table 1. Effect of sodium fluoride (NaF) on seed germination, root and shoot length, and vigor index in C. lanatus seedlings under salicylic acid (SA) treatment. Values are mean±se, n=3 Group Seed Germination (% ) Root Length (cm) Shoot Length (cm) Vigor Index Control 90.81± ± ± ±25.16 NaF 1 mm 78.30±1.02* 5.83±0.18* 3.66±0.031* ±11.21* NaF 10 mm 70.40± ±0.009* 2.96±0.021* ±30.55* SA 0.25 mm 97.83±1.11* 8.66±0.003* 6.63± ±10.00 NaF 1 mm + SA 0.25mM 90.59± ± ±0.011* ±13.09 NaF 10 mm + SA 0.25mM 85.65±1.02* 5.33± ± ±6.89 CD at 5% level *p 0.05, Pesaran s cross-sectional dependence (CD) test. Vigor index: In the study, the seed vigor index was also reduced by increasing the concentration of NaF since it is determined by the multiplication of seed germination and seedling growth (root and shoot length) which were reduced by NaF. However, seedlings treated with SA alone and with combination of NaF showed the higher seed vigor index as compared to their respective controls. Pigment content: Chlorophyll-a (Chl-a), chlorophyll-b (Chl-b), total chlorophyll, and carotenoids content were found to be decreased with increasing of NaF concentration as compared to control (Figure 1). Carotenoids Figure 1. Effect of sodium fluoride (NaF) on pigment content, chlorophyll-a (Chl-a), chlorophyllb (Chl-b), total chlorophyll (Total chl), and carotenoids, in in-vitro grown seedlings of C. lanatus under salicylic acid (SA) treatment. Bar represents standard error of mean (n=3). *significantly different at p<0.05.

4 52 52 The seedlings also showed similar patterns of reduction in pigment content as shown by decrease in the Chl-a, Chl-b, and carotenoids content up to 53%, 54% and 50%, respectively under high level of NaF. The possible causes for the decreasing of the pigment content are break down of chlorophyll, inhibition of chlorophyll biosynthesis, 4 and stress-induced increase in the activity of the chlorophyll degrading enzyme chlorophyllase, 24 and a F-induced reduction in Fe +2 which is essential for chlorophyll biosynthesis. 6 Treatment with SA alone and with the combination of SA+NaF increased all the studied pigment contents as SA-treated plants synthesized more cytokinin which is implicated in higher levels of pigment by the mechanisms of chlorophyll biosynthesis and the prevention of chlorophyll degradation. 25 Percentage of chlorophyll stability index (CSI): In the present investigation, the the percentage of CSI was significantly decreased by NaF treatment and increased by SA (Figure 2). Furthermore, the CSI showed a positive correlation with pigment content as SA-treated NaF-stressed seedlings had a relatively higher CSI and pigment level. The increased CSI in the presence of SA indicates a greater stress tolerance capacity in the growing seedlings. Percentage of membrane stability index (MSI): It has also been suggested that NaF-induced stress increases membrane permeability causing higher electrolyte leakage into the external medium resulting in a decrease in the MSI. 26 It was observed that F-stress decreases the MSI with the higher concentration of NaF (10 mm) decreasing the MSI by 20% compared to the control. However, SA treatment ameliorates the adverse effect of NaF by increasing the MSI (Figure 2). Figure 2. Effect of NaF on percentage of the chlorophyll stability index (CSI) and the membrane stability index (MSI) in in-vitro grown seedlings of C. lanatus under salicylic acid (SA) treatment. Bar represents standard error of mean (n=3). *significantly different at p<0.05. Total soluble sugars content: NaF markedly increased the total soluble sugars content with the higher concentration of NaF (10 mm) increasing the soluble sugars content by 55% compared to control seedlings (Figure 3). NaF stress decreased the water potential of the plants by inhibiting root water transport 27. Water stress caused by a lower water potential has an adverse effect on plants by decreasing growth and metabolism. In the present study, the increased sugars may act as osmolytes which increase the water potential of seedlings for survival under

5 53 53 water deficit-stress and protect the membrane from damage, as reported earlier in Trifolium repens. 28 SA treated seedlings showed higher levels of sugars and these were further increased by 7% and 14% with NaF treatment in doses of 1 mm and 10 mm respectively. Consistent with our results, SA-induced accumulation of sugars under stress has also been reported in Okra seedlings. 29 The SA-induced sugar accumulation may help maintain water potential in watermelon seedlings to allow better growth in the presence of F toxicity. Figure 3. Effect of NaF on total soluble sugars content in in-vitro grown seedlings of C. lanatus under salicylic acid (SA) treatment. Bar represents standard error of mean (n=3). *significantly different at p<0.05. Phenolics content: Similar to soluble sugars, phenol levels were also enhanced by increasing the concentration of NaF (Figure 4). Figure 4. Effect of sodium fluoride (NaF) on phenolics content in in-vitro grown seedlings of C. lanatus under salicylic acid (SA) treatment. Bar represents standard error of mean (n=3). *significantly different at p<0.05. F toxicity causes oxidative stress in plants and different antioxidants play an important role in the plant defence mechanisms when under oxidative stress. 7,30 Phenols are secondary metabolites and natural antioxidants which play important roles in oxidative stress. In the present study, the higher level of phenols may be implicated in the antioxidant defence capacity of the seedlings under the influence of F toxicity. SA also increased the phenolics content in both the seedlings treated with NaF and those without NaF treatment. The NaF-treated seedlings were found to accumulate more soluble sugars and phenol content, which increased further with SA treatment thereby indicating a synergistic effect of SA and NaF. Seed

6 54 54 germination and early seedling growth is a complex process and affected by several factors. 4,7,31 The findings of the present study indicate that the high level of sugars and phenols are not directly involved in the increasing of seed germination and seedlings growth but these are adaptive responses for their survival under F toxicity. The ameliorative effect of SA on the adverse effect of NaF may be primarily through SA-induced changes in hormonal levels which regulate seed germination, seedlings growth, pigment content, and membrane stability in C. lanatus. These studies suggest that pretreatment of seeds with SA may be useful for farmers to reduce the toxic effect of F during early seedling growth. ACKNOWLEDGEMENTS One of the authors, Amra Ram is thankful to the Council of Scientific and Industrial Research, New Delhi, India for granting a Senior Research Fellowship for the present research work. The authors wish to thank Professor HS Gehlot and Dr Gyan Singh Shekhawat for their helpful critical comments. REFERENCE 1 Choubisa SL. Endemic fluorosis in southern Rajasthan, India. Fluoride 2001;34(1): Choubisa SL. Fluoride toxicosis in immature herbivorous domestic animals living in low fluoride water endemic areas of Rajasthan, India: an observational survey. Fluoride 2013;46(1): Weinstein LH, Davison AW. Fluorides in the environment: effects on plants and animals. Wallingford, Oxon, UK: CABI Publishing; Gupta S, Banerjee S, Mondal S. Phytotoxicity of fluoride in the germination of paddy (Oryza sativa) and its effect on the physiology and biochemistry of germinated seedlings. Fluoride 2009;42(2): Singh M, Verma KK. Influence of fluoride contaminated irrigation water on physiological response of poplar seedlings (Populus deltoides L, clone-s 7 C 15 ). Fluoride 2013;46(2): Elloumi N, Abdallah FB, Mezghani I, Rhouma A, Boukhris M. Effect of fluoride on almond seedling in culture solution. Fluoride 2005;38(3): Chakrabarti S, Patra PK. Effect of fluoride on superoxide dismutase activity in four common crop plants. Fluoride 2013;46(2): Choubisa SL. Status of fluorosis in animals. Proc Natl Acad Sci India Sect B Biol Sci 2012;82(3):331-9,DOI /s Choubisa SL, Choubisa L, Choubisa DK. Endemic fluorosis in Rajasthan. Indian J Environ Health 2001;43(4): Chaudhary V, Sharma M, Yadav BS. Elevated fluoride in canal catchment soils of northwest Rajasthan, India. Fluoride 2009;42(1): Gadi BR, Bohra SP. Effect of plant growth regulators on photosynthesis and some biochemical parameters in ber CV Gola. Indian J Hort 2005;62(3): Hara M, Furukawa J, Sato A, Mizoguchi T, Miura K. Abiotic stress and role of salicylic acid in plants. In: Ahmad D, Prasad MNV, editors. Abiotic stress responses in plants: metabolism, productivity and sustainability. New York: Springer; pp Miar Sadeghi S, Shekari F, Fotovat R, Zangani E. Influence of priming with salicylic acid on vigor and growth of Brassica napus L. under drought stress. Iranian J Plant Biol 2010;6: Bhandari MM. Flora of Indian Desert. Jodhpur; MPS Reports; Abdul-Baki AA, Anderson JD. Vigor determination in soybean seed by multiple criteria. Crop Sci 1973;13: Arnon DI. Copper enzymes in isolated chloroplasts: polyphenoloxidase in Beta vulgaris. Plant Physiol 1949;24:1-15.

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