International Research Journal of Environment Sciences________________________________ ISSN 2319–1414Vol. 3(11), 20-28, November (2014) Int. Res. J. Environment Sci. International Science Congress Association 20 An in vitro hydroponic study on Physiological and Biochemical responses of Indian wild Rice to varying doses of Hexavalent Chromium Nayak J., Mathan J., Mohanty M. and Pradhan C.* Laboratory of Plant Biochemistry and Environmental Biotechnology, Post Graduate Department of Botany. Utkal University, Bhubaneswar-751004, Odisha, INDIA Available online at: www.isca.in, www.isca.me Received 29th August 2014, revised 17th October2014, accepted 16st November 2014 AbstractIndian wild rice (Oryza nivara) grown as a weed in most of the cultivated paddy fields is used as an experimental tool in current investigation and studied for its various physiological and biochemical alterations in response to hexavalent chromium. After exposure to varying concentrations of toxic hexavalent chromium, the two cultivars of Oryza nivara i.e. IC-283169 and IC-336684 showed significant changes in its morphobiometric and physiological parameters. The present hydroponic study exhibited deleterious effects on germination, plant height and biomass of 14 days treated seedlings. IC-336684 cultivar of wild rice showed significant reduction in root-shoot length as well as dry and fresh biomass of shoot beyond a treatment dose of 50 M Cr+6 as compared to IC-283169 cultivar. Growth parameters, chlorophyll and carotenoid content showed 50% reduction with treatment dose of 25 M Cr+6. More than 80% reduction in all the above parameters were observed at 100 M Cr+6 treatments. Present preliminary study screens the tolerance and sensitivity of wild rice cultivars to toxic doses of Cr+6. The aim of this article is to give an overview of the impact of varying doses of hexavalent chromium on two cultivars of Indian wild rice and removal of these toxic contaminants from soil by potent application of weeds like Indian wild rice. Keywords: Chromium, chlorophyll, antioxidative enzymes, protein, hydroponically. Introduction Plants are exposed to various types of biotic and abiotic stresses. Heavy metal stress is considered as one of the emerging abiotic environmental stress issues now-a-days. Various natural and manmade activities are the causative factors for increased heavy metal content in the environment. Discharge of heavy metals from various anthropogenic activities leads to pollution in soil and water and pose threat to mankind. Irrigation through wastewater, applications of sludge, unsafe disposal of solid wastes, exhaust from automobiles and effluent discharge from industrial activities are listed as some common manmade sources of heavy metals in the environment. Extensive use of chromium (Cr) in various activities like electroplating, tanning, textile dyeing and as a biocide in power plant cooling water create environmental disturbances due to its toxic potentiality and high persistence in nature. Cr has an electronic configuration of [Ar] 3d4s and exist in two most common and stable forms i.e, the trivalent Cr(III) and the hexavalent Cr(VI) form. The toxic effect of Cr (VI) on various plants has been reported2, 3. Presence of excess amount of chromium in soil and irrigated water beyond the tolerance limit will cause harm to crop growth and yield. Some of the phytotoxic effects induced by Cr(VI) in plants are delayed seed germination, reduced seedling growth, less pigment content, nutrient content and enzyme activities of various5-10. Toxicity of Cr also affects human being and cattles when it entered through food chain, causing bronchitis and cancer 11-13. Oryza nivara Sharma et Shastry, an annual short (usually 2 m) seasonal grass found growing in swampy areas, at edge of pond and tanks, beside streams, in ditches, in or around rice fields is a wild progenitor of the cultivated rice Oryza sativa L. This research program was envisaged to assess the phytotoxic effects and accumulation of Cr6+ in different plant parts that may be transferred to humans through food chain, particularly in the contaminated areas. Material and Methods Plant Material: Dry seeds of two varieties of Indian wild rice Oryza nivara Sharma et Shastry; Accesion No. IC-283169 and IC-336684) were collected from Central Rice Research Institute (CRRI), Cuttack. Germination study: Pre-treated uniform sized seeds were placed in sterilized petriplates over saturated tissue paper for germination under varying concentrations (0 M as Control, 10M, 25 M, 50M, 75M and 100M) of Cr [source: Cr] in different petriplates [Pre-treatment of seeds includes surface sterilization with 0.1% mercuric chloride (HgCl)] at 25º C in darkness for two days. Germinated seeds with 2 mm radicle in different treatment petripalted were International Research Journal of Environment Vol. 3(11), 20-28, November (2014) International Science Congress Association analysed for calculating Germination percentage using following formula14-15. % of Germination = No. of seeds ger minated/ Total no. of seeds taken X 100 Seedling growth and growth parameter study: germinated seeds were grown in well aerated hydroponic culture vessels containing Hoagland’s nutrient solution (half strength) as control and Hoagland’s solution supplemented with varying concentrations of Cr for seedling growth. The seedlings were grown under white light provided by white fluorescent tubes (36 W Philips TLD) with a photon flux density of 52 with a 12h photo period inside the growth chamber for. 7 sand 14 days. After 7 and 14 days growth the Indian wild rice seedlings were analysed for various growth parameters like root length, shoot length, fresh matter and dry matter and a comparison was made between different Cr treatment vessels along with control vessels. For study of dry biomass the seedlings were kept in an oven at 80º C for a period of 3 days or more (till constant weight was attained). Analysis of Chlorophyll Content: Fresh leaves of 7 and 14 days treated seedlings were grin ded in 10 ml of 80% cold acetone and kept in dark in refrigerator for 48 hours at 4 chlorophyll extraction study with further spectrophotometric analysis of chlorophyll content using the method of Porra (2002). The leaf samples from different cultur absorbance value of extracted liquid was recorded at 663.6 nm, 646.6 nm and 470 nm for Chlorophyll- a, Chlorophyll carotenoid respectively15. Estimation of Proline: Plant material (0.5) was grinded in 10ml of 3% sulfo- salicylic acid then the homogenized mixture was centrifuged at 3000 rpm for 10 minutes. Then to the 2ml of supernatant 2ml of acid Ninhydrin reagent and 2ml of Glacial acid was added and boiled in water bath at 100 toluene was added to it and allowed to cyclom toluene layer containing chromophore was collected carefully with help of micropipette and the absorbance was measured at 520nm15. Estimation of catalase activity: For catalase assay 2 ml of sodium phosphate buffer and 0.5 ml of 12 mM of H added to 0.5 ml of the plant leaf extract and the O.D was taken at 240 nm. Activity of Catalase enzymes of seedlings grown under different Cr treatments were assayed with measurement of the initial rate of disappearance of H . [Extinction coeffic for H is 40mM1 cm-1 at 240nm]15-16. Estimation of peroxidase activity: For assay of peroxidase activity, 2.8 ml of potassium phosphate buffer (pH = 7.0), 50 of 10 mM H and 50l of guaiacol was added to diluted leaf extract. The mixture was mixed properly and absorbance value Environment Sciences_______________ _________________________ International Science Congress Association analysed for calculating Germination percentage using minated/ Total no. of seeds Seedling growth and growth parameter study: Two-days- germinated seeds were grown in well aerated hydroponic culture vessels containing Hoagland’s nutrient solution (half strength) supplemented with varying concentrations of Cr for seedling growth. The seedlings were grown under white light provided by white fluorescent tubes (36 W Philips TLD) with a photon flux density of 52 /m2s (PAR) chamber for. 7 sand 14 days. After 7 and 14 days growth the Indian wild rice seedlings were analysed for various growth parameters like root length, shoot length, fresh matter and dry matter and a comparison was made between different Cr treatment vessels along with control vessels. For study of dry biomass the seedlings were kept in an oven at 80º C for a period of 3 days or Fresh leaves of 7 and 14 ded in 10 ml of 80% cold acetone and kept in dark in refrigerator for 48 hours at 4 C for chlorophyll extraction study with further spectrophotometric analysis of chlorophyll content using the method of Porra (2002). The leaf samples from different cultur e pots were. The absorbance value of extracted liquid was recorded at 663.6 nm, a, Chlorophyll -b and Plant material (0.5) was grinded in 10ml then the homogenized mixture was centrifuged at 3000 rpm for 10 minutes. Then to the 2ml of supernatant 2ml of acid Ninhydrin reagent and 2ml of Glacial acid was added and boiled in water bath at 100 \rC. 4ml of toluene was added to it and allowed to cyclom ixer. The upper toluene layer containing chromophore was collected carefully with help of micropipette and the absorbance was measured at For catalase assay 2 ml of sodium phosphate buffer and 0.5 ml of 12 mM of H was added to 0.5 ml of the plant leaf extract and the O.D was taken at 240 nm. Activity of Catalase enzymes of seedlings grown under different Cr treatments were assayed with measurement of . [Extinction coeffic ient For assay of peroxidase activity, 2.8 ml of potassium phosphate buffer (pH = 7.0), 50l l of guaiacol was added to diluted leaf extract. The mixture was mixed properly and absorbance value was recorded at 436 nm. Activity was calculated using the extinction coefficient (25.5 mM tetraguaiacol15-16. Results and Discussion Maximum inhibition of seed germination was observed at 100 M Cr6+ treatments for both the varieties of 283169 and IC- 336684). The inhibition in seed germination was recorded as 62 % and 58% in IC cultivars respectively (figure- 1a and 1b) Figure - Bar graph showing the effects of Hexavalent Chromium on seed germination of two varieties of seven days and fourteen days grown Oryza nivara Changes in growth parameters recorded in different growth parameters of 7 and 14 days grown cultivars of Indian wild rice seedlings ( figure- 3a and 3b) grown under different treatment concentrations of Cr6+. Shoot length decreased markedly with increased dose of Cr6+ treatment from 10 decreasing trend in growth parameters corroborates the findings of other researchers in different plants stress . Root length and shoot length of Oryza nivara seedlings showed high sensitivity to Cr toxicity in comparison to IC- 283169 cultivar of evident from their reduced values at Root and shoot length values of IC nivara in response to varying concen of that of IC-283169 cultivar. Fresh weight values of roots and shoots were gradually decreased with increased in concentrations of Cr+6 treatments in nutrient solution. Parallel growth trend values were found for fresh biomass and figure- 5a and 5b) and dry biomass production ( and 6b and figure-7a and 7b). The two cultivars of Indian wild rice seedlings were examined for their tolerance and sensitivity towards different concentrations of _________________________ ______ ISSN 2319–1414 Int. Res. J. Environment Sci. 21 was recorded at 436 nm. Activity was calculated using the extinction coefficient (25.5 mM -1 cm-1 at 436 nm) for Maximum inhibition of seed germination was observed at 100 treatments for both the varieties of Oryza nivara (IC- 336684). The inhibition in seed germination was recorded as 62 % and 58% in IC -283169 and IC-336684 1a and 1b) - 1 Bar graph showing the effects of Hexavalent Chromium on seed germination of two varieties of seven days and fourteen Oryza nivara Changes in growth parameters : Notable changes have different growth parameters of 7 and 14 days grown cultivars of Indian wild rice seedlings ( figure-2a and 2b and 3a and 3b) grown under different treatment Shoot length decreased markedly with treatment from 10M to 100M. The decreasing trend in growth parameters corroborates the findings of other researchers in different plants 16-17 with response to Cr . Root length and shoot length of IC-336684 cultivar of high sensitivity to Cr toxicity in 283169 cultivar of Oryza nivara seedlings as reduced values at 100M treatment of Cr6+. Root and shoot length values of IC -336684 cultivar of Oryza in response to varying concen tration of Cr showed 50 % Fresh weight values of roots and shoots were gradually decreased with increased in treatments in nutrient solution. Parallel growth trend values were found for fresh biomass (figure-4a, 4b 5a and 5b) and dry biomass production ( figure-6a The two cultivars of Indian wild rice seedlings were examined for their tolerance and sensitivity towards different concentrations of Cr+6. International Research Journal of Environment Sciences______________________________________________ ISSN 2319–1414 Vol. 3(11), 20-28, November (2014) Int. Res. J. Environment Sci. International Science Congress Association 22 Figure-2a and b Effect of different concentrations of Cr6+ on shoot and root length of 7 and 14 days old IC-283169 variety of Oryza nivaraseedlings. [NB: A-7 days grown; A’- 14 days grown] Figure-3a and b Effect of different concentrations of Cr6+ on shoot and root length of 7 and 14 days old IC-336684 variety of Oryza nivaraseedlings. [NB: A-7 days grown; A’- 14 days grown] A'-IC336101214161820Control10255075100Cr treatments (M)Length (cm) RL SL b A-IC336Control10255075100Cr treatments (M)Length (cm) RL SL a A'1012141618Control10255075100Cr treatments (M)Length (cm) RL SL b Control10255075100Cr treatments (M)Length (cm) RL SL a International Research Journal of Environment Sciences______________________________________________ ISSN 2319–1414 Vol. 3(11), 20-28, November (2014) Int. Res. J. Environment Sci. International Science Congress Association 23 Figure-4a and b Effect of different concentrations of Cr6+ on root fresh weight and shoot fresh weight of 7 and 14 days old IC-283169 variety of Oryza nivara plant. [NB: B-7 days grown; B’- 14 days grown] Figure-5a and b Effect of different concentrations of Cr6+ on root fresh weight and shoot fresh weight of 7 and 14 days old IC-336684 variety of Oryza nivara plant. [NB: B-7 days grown; B’- 14 days grown] B'-IC3360.10.20.30.40.50.60.70.8Control10255075100Cr treatments (M)Fresh weight (gm) RFW SFW b B-IC3360.050.10.150.20.250.3Control10255075100Cr treatments (M)Fresh weight (gm) RFW SFW a B'0.10.20.30.40.50.60.70.8Control10255075100Cr treatments (M)Fresh weight (gm) RFW SFW b 0.050.10.150.20.250.30.350.40.45Control10255075100Cr treatments (M)Fresh weight (cm) RFW SFW a International Research Journal of Environment Sciences______________________________________________ ISSN 2319–1414 Vol. 3(11), 20-28, November (2014) Int. Res. J. Environment Sci. International Science Congress Association 24 Figure-6a and b Effect of different concentrations of Cr6+ on root dry weight and shoot dry weight of 7 and 14 days old IC-283169 variety of Oryza nivara plant. [NB: C-7 days grown; C’- 14 days grown] Figure-7a and b Effect of different concentrations of Cr6+ on root dry weight and shoot dry weight of 7 and 14days old IC-336684 variety of Oryza nivara plant. [NB: C-7 days grown; C’- 14 days grown] Effects on Chlorophyll and Carotenoid Content: By comparing the total chlorophyll content of two cultivates of O. nivara, it was noted that IC-336684 showed high Chlorophyll content in comparison to IC-283169 cultivar. Gradual decrease in chlorophyll content was found with increased treatment concentrations of Cr6+ except for treatment of 10 M Cr6+. Maximum chlorophyll content was recorded in 10M-Cr+6 and minimum in 100M-Cr+6 treated seedlings. Analogous trend of increase/decrease in carotenoid content was observed (figure-8a and 8b). C'-IC3360.010.020.030.040.050.06Control10255075100Cr treatments (M)Dry weight (gm) RDW SDW b C-IC3366840.0050.010.0150.020.025Control10255075100Cr treatments (M)Dry weight (gm) RDW SDW a C'0.010.020.030.040.050.060.070.08Control10255075100Cr treatments (M)Dry weight (gm) RDW SDW b 0.0050.010.0150.020.0250.03Control10255075100Cr treatments (M)Dry weight (gm) RDW SDW a International Research Journal of Environment Vol. 3(11), 20-28, November (2014) International Science Congress Association Effect of different concentrations of chromium on chlorophyll and carotenoid content (mg / gm fresh wt.) of two cultivars of Or yza nivara Accumulation of proline: Higher proline content was found with increased concentration of Cr6+ . Elevated proline accumulation was noted in seedlings treated with 100 treatments for IC-283169 cultivar and 75M- Cr IC- 336684 cultivar for both 7 and 14 days old seedling. A comparative proline accumulation in two cultivars of Indian wild rice shows that IC- 283169 cultivar has higher C omparative proline accumulation in two cultivars of 0.51.52.5Control (0)10255075100Control (0)1025 Total Chlorophyll Carotenoid Treatments of Cr (M) Bar graph with standard error bars showing a comparison of the effects of Chromium in two cultivated varieties of fourteen days grown respect to total chlorophyll and carotenoid content. Total Chlorophyll and Carotenoid content (mg/gm fr. wt.) IC283 b Environment Sciences_______________ _________________________ International Science Congress Association Figure-8a and b Effect of different concentrations of chromium on chlorophyll and carotenoid content (mg / gm fresh wt.) of two cultivars of yza nivara seedlings after7 and 14 days exposure Higher proline content was found . Elevated proline accumulation was noted in seedlings treated with 100 -Cr+6 Cr +6 treatments for 336684 cultivar for both 7 and 14 days old O. nivara seedling. A comparative proline accumulation in two cultivars 283169 cultivar has higher proline accumulation (figure- 9a and 9b) Alterations in antioxidative enzyme activity responses were exhibited by antioxidants like catalase, peroxidase activities and carotenoids to different chromium treatments (figure-10a, 10b, figure - The activity of peroxidase (POX) increased at high chromium stress of 100 . Enhanced peroxidase activity was observed in Figure-9 omparative proline accumulation in two cultivars of Oryza nivara seedlings after 7 and 14 days exposure 0.51.52.5Control (0)10255075100Control (0) Total Chlorophyll Treatments of Cr (M) Bar graph with standard error bars showing a comparison of the effects of Chromium in two cultivated varieties of seven days grown respect to total chlorophyll and carotenoid content. Total Chlorophyll and Carotenoid content (mg/gm fr. wt.) a 5075100 Carotenoid Bar graph with standard error bars showing a comparison of the effects of Chromium in two cultivated varieties of fourteen days grown Oryza nivara with respect to total chlorophyll and carotenoid content. IC283 IC336 _________________________ ______ ISSN 2319–1414 Int. Res. J. Environment Sci. 25 Effect of different concentrations of chromium on chlorophyll and carotenoid content (mg / gm fresh wt.) of two cultivars of 9a and 9b) . Alterations in antioxidative enzyme activity : Variable responses were exhibited by antioxidants like catalase, peroxidase activities and carotenoids to different chromium - 11a, 11b, and figure-8a, 8b). activity of peroxidase (POX) increased at high chromium . Enhanced peroxidase activity was observed in 7 and 14 days exposure 10255075100 Carotenoid Treatments of Cr (M) Bar graph with standard error bars showing a comparison of the effects of Chromium in two cultivated varieties of seven days grown Oryza nivara with respect to total chlorophyll and carotenoid content. IC283 IC336 International Research Journal of Environment Sciences______________________________________________ ISSN 2319–1414 Vol. 3(11), 20-28, November (2014) Int. Res. J. Environment Sci. International Science Congress Association 26 IC-336684 cultivar than other cultivar. Catalase (CAT) activity showed decreasing trend with increased chromium concentrations for IC-283169 cultivar whereas in IC-336684 cultivar the activity was significantly enhanced at 50 M treatment of Cr6+ than other treatments after 14 days exposure. An imbalance arises between generation and removal of ROS in plant tissues subjected to excess chromium or any other heavy metal exposure18. Discussion:Though chromium induced phytotoxic effects (reduced rate of growth, damage to cell wall, cell membranes and changes in the metabolic status of plants) was reported long time back, still there is a huge dearth of information regarding Figure-10a and b Effect of different concentrations of Cr6+ on catalase activity in two cultivars of Oryza nivara after7 and 14 days exposure Figure-11a and b Effect of different concentrations of Cr6+ on peroxidase activity in two cultivars of Oryza nivara after7 and 14 days exposure its impact in wild cultivars of Indian wild rice. In view of the seriousness if Cr pollution, the present study has been undertaken with an effort to assess the phytotoxic impacts with special reference to biochemical lesions in 7days and 14 days -0.010.010.020.030.040.050.060.07CONTROL10255075100Cr treatments(M)Change in Peroxidase activity(A)/min after 7 Days Exposure IC283 IC336 a 0.020.040.060.080.10.12CONTROL10255075100Cr treatments(M)Change in Peroxidase activity(A)/min after 14 Days Exposure (IC283)0.0050.010.0150.020.025Change in Peroxidase activity(A)/min after 14 Days Exposure (IC336) IC283169 IC336684 b 0.0050.010.0150.020.0250.030.035CONTROL10255075100Cr treatments(M)Change in Catalase activity(A)/min after 14 Days Exposure IC283 IC336 b -0.050.050.10.150.20.25CONTROL10255075100Cr treatments(M)Change in Catalase activity(A)/min after 7 Days Exposure-0.04-0.020.020.040.060.080.10.120.14Change in Catalase activity(A)/min after 7 Days Exposure IC283 IC336 a International Research Journal of Environment Sciences______________________________________________ ISSN 2319–1414 Vol. 3(11), 20-28, November (2014) Int. Res. J. Environment Sci. International Science Congress Association 27 grown Oryza nivara seedlings. The part of the present hydroponics study provides a promising start for revising and comparing the level of chromium toxicity in two varieties of Oryza nivara with a potentiality of their tolerance after exposure to varying concentrations of Cr. The work signifies the potential of Oryza nivara plants towards Cr phytotoxicity and tolerance. Study of proline accumulation in response to heavy metal stress is one of the important indicators in plants under stress. Increased proline provide osmoprotection, regulates the redox potential, scavenges hydroxyl radicals which will denature of various macromolecules and thus protects the macromolecules from damage. Non-enzymic free radical detoxifications are often associated with proline accumulation16. The increased proline biosynthesis in the Cr6+ treated seedlings signifies a stress regulating factor that gives protection in plants under heavy metal toxicity environment17. Essential components of plant antioxidant defence system are associated with activities of POX and CAT. Earlier studies on elevated POX activity in nickel treated O. sativa, suggests its role in the detoxification of H19. Plants with enhanced POX activity in response to excess chromium and cadmium supply might suffer from peroxidative damage of the thylakoid membrane or lower auxin and protein contents in tissues20. Though CAT and POX activities protect the plant metabolic system21 still it has negative impacts with its excess. Considering the above phytotoxic effects, Cr removal from pollutes sites becomes very much essential for which different biosorbents are effectively used22-24. Conclusion Plant tolerance to heavy metals is the ability to survive in contaminated soil which is manifested by an interaction between a genotype and its environment. Above studies reveal some interrelationship between different metabolic effects induced by chromium in a wild plant like Oryza nivara. Intensive future research on the effects of accumulation of Cr on plant metabolism is essential. Further the ability of different cultivars of this plant for increasing phytoaccumulation potential needs to be tried alongwith various biosorption models. Suitable post harvest bioremediation techniques should be adopted for disposal of plants and plant parts containing accumulated toxic chromium from mining environment. The overall view of the impact of varying doses of hexavalent chromium on tolerance and sensitivity ability of two cultivars of Indian wild rice suggest removal of these toxic contaminants from soil by potent application of weeds like Indian wild rice. 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