NSave Nature to Survive

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1 ISSN: : Special issue, Vol. 2; ; 2010 NSave Nature to Survive EFFECT OF ALUMINUM AND CADMIUM ON SEED GERMINATION RATE, PLANT GROWTH AND CHLOROPHYLL CONTENT IN FINGER MILLET (ELEUSINE CORACANA) S. Hemalatha et al. Aluminium and Cadmium Seed germination Chlorophyll content Paper presented in International Conference on Environment, Energy and Development (from Stockholm to Copenhagen and beyond) December 10-12, 2010, Sambalpur University 501

2 NSave Nature to Survive S. HEMALATHA, E. ANJANEYULU AND M. BALAJI* Department of Biochemistry, Sri Venkateswara University, Tirupati , A. P., INDIA E mail: bmeriga@yahoo.com ABSTRACT Seeds of four cultivars of finger millet (Sapthagiri, PBR 2700, ML 181 and ML322) were germinated in 1/10 Hoagland s nutrient solution containing different concentrations of Aluminium (Al) and cadmium (Cd) (50, 100, 200, 300, 400 and 500μM) in equal ratios. In another set of experiments, four-day old seedlings of the said cultivars of finger millet were grown for four days with different concentrations of Al and Cd under strictly controlled growth conditions. Root and shoot lengths, fresh weights and chlorophyll content of seedlings were studied. With increase in Al and Cd concentration, considerable reduction in seed germination rate, root and shoot lengths of seedlings, their fresh weights and chlorophyll content was noticed. Among the four cultivars, sapthagiri was found to be more sensitive followed by PBR 2700, ML 181 and ML 322. *Corresponding author 502

3 EFFECT OF ALUMINUM AND CADMIUM ON PLANT INTRODUCTION Heavy metals are important group of soil pollutants that limit growth and yield of several crop plants. The contamination by heavy metals causes a serious problem because they cannot be naturally degraded like organic pollutants and they accumulate in different parts of the food chain. Heavy metals make a significant contribution to environmental pollution as a result of human activities such as mining, smelting, electroplating, energy and fuel production, power transmission, intensive agriculture, sludge dumping and military operations (Nedelkoska and Doran, 2000). They present a risk for primary and secondary consumers and ultimately humans (Zeller and Feller, 1999). The unrestricted developmental activities (such as industrialization and urbanization) carried out during the past few years have given rise to serious problems of environmental contamination. A general increase in the level of heavy metals posses a pervasive threat to the natural ecosystem. Although many heavy metals when in trace amounts are essential for various metabolic processes in organisms, they create physiological stress leading to generation of free radicals and associated damage when in high concentration. Aluminum (Al) is one of the major factors limiting growth and productivity of important crops in acid soils (ph< 5.0) which occupy about 40 % of world s cultivable lands. The usage of Al has considerably increased in modern living causing its enhanced levels in the environment. Inspite of considerable research on elucidating the mechanisms of Al toxicity and tolerance in plants, the actually biochemical and molecular events of Al toxicty are still poorly understood (Delhaize and Ryan, 1995; Klein et al., 2008). One of the most important effects of Al is on the structure and function of the plasma membrane. Aluminum strongly binds to phospholipids, alters the total and relative abundance of phospholipids and other membrane lipids and changes the degree of fatty acid unsaturation (Zhang et al., 1996, 1997). There is also evidence that Al toxicity can cause excessive generation of reactive oxygen species (ROS) and an increase in peroxidation and/or breakdown of membrane lipids (Gutteridge et al., 1985; Cakmak and Horst, 1991; Ono et al., 1995). As a consequence, there is a significant modification in membrane permeability, ion transport and in the activity of a number of membrane bound enzymes, especially H±ATPase (Cooke and Burden, 1990). Cadmium is an industrial pollutant in constant rise in the environment, due to activities such as mining, smelting and refinement of zinc, manufacturing and use of fungicide and phosphorous fertilizers, metallurgy, among others Page and Bingham, (1973). Although Cd is not an essential mineral nutrient for plants, it is easily absorbed by the root system, causing a decrease in transpiration and photosynthesis (Bazzaz et al., 1974) and an increase in the respiratory rates (Lee et al., 1976; Lamoreaux and Chaney, 1978). These effects seem to be related to Cd induction of premature senescence in plants (Van Assche et al., 1988). Cadmium (Cd), being a highly toxic metal pollutant of soils, affects nutrient uptake and homeostasis, inhibits root and shoot growth and yield. It also gets accumulated in crops and then enters the food chain with a significant potential to impair animal and human health Di Toppi and Gabrielli, (1999). The application of sewage sludge, city waste, and Cd-containing fertilizers causes the increase of Cd content in soils Williams and David, (1973). MATERIALS AND METHODS Collection of seeds and their germination Seeds of finger millet viz, PBR-2700, Sapthagiri, ML-181 and ML-322 were obtained from Regional Agricultural Research Station, Tirupati and GKVK, Bangalore. Seeds were immersed in 2% HgCl 2 for five minutes followed by washing with deionized water. For evaluation germination rate, seeds were placed on double layered filter papers (3 mm, Whattman, filter papers) wetted with distilled water and test solutions (50, 100, 200, 300, 400 and 500μM of Al and Cd) in Petri dishes and incubated under white light at 22ºC. Germination tests were carried out using triplicate samples (each containing seeds). Seeds were scored as germinated when the breakage of seed coat was visible. For other experiments the seeds were germinated on distilled water for four days then, seedlings with uniform root and shoot lengths were shifted 503

4 S. HEMALATHA et al., to Petri plates containing different concentrations of Al and Cd in 1/10Hoagland s solution. Measurement of growth parameters After four days of treatment with Al and Cd in Hoaglannd s solution, seedlings were removed to measure root, shoot lengths and the fresh weight of both control and treated seedlings. Analysis of chlorophyll-a, chlorophyll-b and total chlorophyll 100 mg of fresh leaves of both control and treated seedlings of finger millet were ground in a mortar and pestle with 20 ml of 80% acetone. The homogenate was centrifuged at 3000 rpm for 15 minutes. The supernatant was saved. The pellet was reextracted with 5 ml of 80% acetone each time, until it became colorless. All the supernatants were pooled and utilized for chlorophyll determination according to the method of (Arnon, 1949). Absorbance was measured at 480nm, 645nm and 663nm in spectrophotometer. The chlorophyll content was determined by using Arnon formula and expressed as mg/g fw (fresh weight). RESULTS AND DISCUSSION Seed germination and plant growth With increase in concentration of Al and Cd in test solution, seed germination and seedlings growth was significantly reduced. At 500μM concentration of Al and Cd concentration, the inhibition of seed germination rate was 38% 42%, 40% and 36% respectively in PBR- 2700, Sapthagiri, ML-181 and ML-322 (Table 1). The per cent inhibition of root and shoot lengths was 66 and 68 in PBR-2700, 60 and 51 in Sapthagiri, 45 and 41 in ML-181 and 34 and 40 in ML-322. Similarly, the reduction in fresh weight of seedlings was 61, 59, 53 and 54 respectively in PBR- 2700, Sapthagiri, ML-181 and ML-322 (Table 1). Inhibition of plant growth and Table 1: Effect Al and Cd on seed germination rate and growth parameters in different cultivars of finger millet Cultivar *Conc. Seed germination Root Shoot Fresh in μm Rate (%) length (cm) length (cm) weight (mg) 24hrs 48hrs 72hrs 0 95% 98% 100% % 92% 96% % 84% 90% % 80% 84% % 78% 78% PBR % 68% 72% % 60% 62% % 98% 100% % 90% 92% % 80% 85% (SAPTHAGIRI) % 75% 80% % 74% 76% % 68% 70% % 58% 58% % 100% 100% % 92% 95% % 85% 90% (ML-181) % 82% 84% % 78% 80% % 68% 74% % 65% 60% % 99% 100% % 92% 94% % 87% 92% (ML-322) % 85% 85% % 78% 80% % 69% 76% % 68% 64% * Equal concentrations of Al and Cd in the test solution 504

5 Table 2: Effect of Al and Cd on chlorophyll content (chlorophyll a, chlorophyll b, total chlorophyll and carotenoids of different cultivars of finger millet Cultiver *Conc. In um Chlorophyll a Chlorophyll b Total chlorophyll Carotenoids mg/gm fw mg/gm fw mg/gmfw mg/gm fw (663nm) (645nm) (480nm) PBR (SAPTHAGIRI) (ML-181) (ML-322) * Equal concentrations of Al and Cd in the test solution. fresh weights was observed with nickel and other heavy metals on cabbage, lettuce, millet, radish, turnip, and wheat (Claire et al., 1991, Mathur et al., (1987) found that higher concentration of cadmium and chromium ( ppm) affected germination in Allium cepa. Drastic reduction in root and shoot length was observed in rice cultivars by (Balaji et al., 2004). Similar results were observed in tomato with increasing concentration of Cd and Zn in nutrient solution (Hanan and Said Deef, 2008). The probable mechanisms by which these metals inhibit plant growth are they are likely to interfere with certain enzymes and proteins involved in cell division or cytoskeleton net work. They also cause peroxidation of membrane lipids where by the dynamic properties of the membrane are disturbed ultimately affecting plant growth. The synthesis of respective phytochelatins could be attributed to the resistance exhibited by certain plants for their metal tolerance. Among the cultivars under study, Sapthagiri was found to be relatively more sensitive to Al and Cd compared to other. Chlorophyll contents in different cultivars of finger millet. Chlorophyll (chlorophyll a, chlorophyll b and total chlorophyll) and carotenoid contents were significantly reduced in treated seedlings with increasing concentrations of Al and Cd in nutrient solution. The maximum reduction in total chlorophyll content was observed at 500 μm of Al and Cd concentration. There was a 2.5 fold fall in total chlorophyll content of PBR-2700 when compared to control. Similarly, Sapthagiri, ML-181 and ML-322 witnessed a fall of 4 fold, 1 fold and 1.2 fold respectively in comparison to their respective controls. Similar tends were noted with regard to carotenoid contents in all the treated cultivars of finger millet. Once these metals are accumulated in shoots and leafs, they are supposed to intervene with chlorophyll synthesizing pathways. Similar results were observed in soybean and maize under prolonged Cd influence (Juraci Alves de Oliveira, 1994; Meeta Jain, 2007). In terms of chlorophyll content, cultivars Sapthagiri and 505 EFFECT OF ALUMINUM AND CADMIUM ON PLANT

6 S. HEMALATHA et al., PBR-2700 showed more sensitivity to Al and Cd test solution than ML-322 and ML-181. REFERENCES Arnon, D. L A copper enzyme is isolated chloroplast polyphenol oxidase in Beta vulgaries. Plant Physiol. 24: Balaji Meriga., Krishna Reddy, B. Rajender Rao, K. Ananda Reddy, L. Kavi Kishor P. B Aluminiuminduced production of oxygen radicals, lipidperoxidation and DNA damage in seedlings of rice (Oryza sativa) J. Plant Physiol. 161: Bazzaz, F.A., Rolfe, G.L. and Carlson, R.W Effect of Cd on photosynthesis and transpiration of excised leaves of corn and sunflower. Physiologia Plantarum. 32: Cakmak, I. and Horst, J. H Effects of aluminum on lipid peroxidation, superoxide dismutase, catalase, and peroxidase activities in root tips of soybean ( Glycine max). Physiologia Plantarum. 83: Claire, L. C., Adriano, D. C., Sajwan, K. S., Abel, S. L., Thoma, D. P. and Driver, J. T Effects of Selected Trace Metals on Germinatin,g Seeds of Six Plant Species. Water. Air. and Soil Pollution. 59: Cooke, D.T. and Burden, R. S Lipid modulation of plasma membranebound ATPases. Physiologia Plantarum. 78: Delhaize, E. and Ryan, P. R Aluminum toxicity and tolerance in plants. Plant Physiology. 107: Di Toppi, S. L. and Gabrielli, R Response to cadmium in higher plants. Environ. Exp. Bot. 41: Gutteridge, J. M. C., Quinlan, G. J., Clark, I. and Halliwell, B Aluminium salts accelerate peroxidation of membrane lipids stimulated by iron salts. Biochemical and Biophysical Acta. 835: Hanan, E. l., Said Deef Effect of Cadmium and Zinc on Growth Parameters of Tomato Seedlings. Academic Journal of Plant Sciences. 1(1): Hoagland, D. R., and Arnon, D. I The water culture method for growing plants without soil. Calif Agr. Expt. Sta. Circ Juraci Alves de Oliveira., Marco Antonio Oliva., and José Cambraia Effects of cadmium on chlorophyll contents and on peroxidase activity in soybean. R.Bras.Fisiol.Veg. 6(2): Kochian, L. V Cellular mechanisms of aluminum toxicity and resistance in plants.annual Review of Plant Physiology and Plant Molecular Biology. 46: Klein, M. A., Sekimoto, H., Milner, M. J. and Kochian, L. V Investigation of heavy metal hyperaccumulation at the cellular level: development and characterization of Thlaspi caerulescens suspension cell lines. Plant Physiol. 4: Lamoreaux, R. J. and Chaney, W. R The effect of cadmium on net photosynthesis transpiration, and dark respiration of excised silver maple leaves. Physiologia Plantarum. 43: Lee, K. C., Cunningham, B. A., Paulsen, B. M., Liang, B. H. and Moore, R. B Effects of cadmium on respiration rate and activities of several enzymes in soybean seedlings. Physiologia Plantarum. 36: 4-6. Mathur, K. C., Srivastava, R. K. and Chaudhary, K Effect of Cd and Cr metals on germination and early growth performance of Allium cepa seeds. Proc. Nat. Acad. Sci. India. Sect. B (Biol. Sci.). 57: Meeta Jain., Monica Pal., Priyanka gupta and Rekha Grade Effect Cadmium on chlorophyll biosynthesis and enzymes of Nitrogen assimilation in greening maize leaf segments: Role of 2-Oxoglutarate.Indian journal of experimental biology. 45: Nedelkoska, T. V. and Doran, P. M Characteristics of heavy metal uptake by plants species with potential for phytoremediation and phytomining. Minerals Engineering. 13: Ono, K., Yamamoto, Y., Hachiya, A. and Matsumoto, H Synergistic inhibition of growth by aluminum and iron of tobacco ( Nicotiana tabacum L.) cells in suspension culture. Plant and Cell Physiology. 36:

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