An in vitro hydroponic study on Physiological and Biochemical responses of Indian wild Rice to varying doses of Hexavalent Chromium

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1 Interntionl Reserch Journl of Environment Sciences ISSN Vol. 3(11), -8, Novemer (14) An in vitro hydroponic study on Physiologicl nd Biochemicl responses of Indin wild Rice to vrying doses of Hexvlent Chromium Nyk J., Mthn J., Mohnty M. nd Prdhn C.* Lortory of Plnt Biochemistry nd Environmentl Biotechnology, Post Grdute Deprtment of Botny. Utkl University, Bhuneswr- 7514, Odish, INDIA Astrct Aville online t: Received 9 th August 14, revised 17 th Octoer 14, ccepted 16 st Novemer 14 Indin wild rice (Oryz nivr) grown s weed in most of the cultivted pddy fields is used s n experimentl tool in current investigtion nd studied for its vrious physiologicl nd iochemicl ltertions in response to hexvlent chromium. After exposure to vrying concentrtions of toxic hexvlent chromium, the two cultivrs of Oryz nivr i.e. IC nd IC showed significnt chnges in its morphoiometric nd physiologicl prmeters. The present hydroponic study exhiited deleterious effects on germintion, plnt height nd iomss of 14 dys treted seedlings. IC cultivr of wild rice showed significnt reduction in root-shoot length s well s dry nd fresh iomss of shoot eyond tretment dose of 5 µm Cr +6 s compred to IC cultivr. Growth prmeters, chlorophyll nd crotenoid content showed 5% reduction with tretment dose of 5 µm Cr +6. More thn 8% reduction in ll the ove prmeters were oserved t 1 µm Cr +6 tretments. Present preliminry study screens the tolernce nd sensitivity of wild rice cultivrs to toxic doses of Cr +6. The im of this rticle is to give n overview of the impct of vrying doses of hexvlent chromium on two cultivrs of Indin wild rice nd removl of these toxic contminnts from soil y potent ppliction of weeds like Indin wild rice. Keywords: Chromium, chlorophyll, ntioxidtive enzymes, protein, hydroponiclly. Introduction Plnts re exposed to vrious types of iotic nd iotic stresses. Hevy metl stress is considered s one of the emerging iotic environmentl stress issues now--dys. Vrious nturl nd mnmde ctivities re the custive fctors for incresed hevy metl content in the environment. Dischrge of hevy metls from vrious nthropogenic ctivities leds to pollution in soil nd wter nd pose thret to mnkind 1. Irrigtion through wstewter, pplictions of sludge, unsfe disposl of solid wstes, exhust from utomoiles nd effluent dischrge from industril ctivities re listed s some common mnmde sources of hevy metls in the environment. Extensive use of chromium (Cr) in vrious ctivities like electroplting, tnning, textile dyeing nd s iocide in power plnt cooling wter crete environmentl disturnces due to its toxic potentility nd high persistence in nture. Cr hs n electronic configurtion of [Ar] 3d 5 4s 1 nd exist in two most common nd stle forms i.e, the trivlent Cr(III) nd the hexvlent Cr(VI) form. The toxic effect of Cr (VI) on vrious plnts hs een reported, 3. Presence of excess mount of chromium in soil nd irrigted wter eyond the tolernce limit will cuse hrm to crop growth nd yield 4. Some of the phytotoxic effects induced y Cr(VI) in plnts re delyed seed germintion, reduced seedling growth, less pigment content, nutrient content nd enzyme ctivities of vrious 5-1. Toxicity of Cr lso ffects humn eing nd cttles when it entered through food chin, cusing ronchitis nd cncer Oryz nivr Shrm et Shstry, n nnul short (usully < m) sesonl grss found growing in swmpy res, t edge of pond nd tnks, eside strems, in ditches, in or round rice fields is wild progenitor of the cultivted rice Oryz stiv L. This reserch progrm ws envisged to ssess the phytotoxic effects nd ccumultion of Cr 6+ in different plnt prts tht my e trnsferred to humns through food chin, prticulrly in the contminted res. Mteril nd Methods Plnt Mteril: Dry seeds of two vrieties of Indin wild rice (Oryz nivr Shrm et Shstry; Accesion No. IC nd IC ) were collected from Centrl Rice Reserch Institute (CRRI), Cuttck. Germintion study: Pre-treted uniform sized seeds were plced in sterilized petripltes over sturted tissue pper for germintion under vrying concentrtions ( µm s Control, 1µM, 5 µm, 5µM, 75µM nd 1µM) of Cr [source: K Cr O 7 ] in different petripltes [Pre-tretment of seeds includes surfce steriliztion with.1% mercuric chloride (HgCl )] t 5º C in drkness for two dys. Germinted seeds with mm rdicle in different tretment petriplted were Interntionl Science Congress Assocition

2 Interntionl Reserch Journl of Environment Sciences ISSN Vol. 3(11), -8, Novemer (14) nlysed for clculting Germintion percentge using following formul % of Germintion = No. of seeds germinted/ Totl no. of seeds tken X 1 Seedling growth nd growth prmeter study: Two-dyshydroponic culture germinted seeds were grown in well erted vessels contining Hoglnd s nutrient solution (hlf strength) s control nd Hoglnd s solution supplemented with vrying concentrtions of Cr for seedling growth. The seedlings were grown under white light provided y white fluorescent tues (36 W Philips TLD) with photon flux density of 5 µ /ms (PAR) with 1h photo period inside the growth chmer for. 7 snd 14 dys. After 7 nd 14 dys growth the Indin wild rice seedlings were nlysed for vrious growth prmeters like root length, shoot length, fresh mtter nd dry mtter nd comprison ws mde etween different Cr tretment vessels long with control vessels. For study of dry iomss the seedlings were kept in n oven t 8º C for period of 3 dys or more (till constnt weight ws ttined). Anlysis of Chlorophyll Content: Fresh leves of 7 nd 14 dys treted seedlings were grinded in 1 ml of 8% cold cetone nd kept in drk in refrigertor for 48 hours t 4 C for chlorophyll extrction study with further spectrophotometric nlysis of chlorophyll content using the method of Porr (). The lef smples from different culture pots were. The sornce vlue of extrcted liquid ws recorded t nm, nm nd 47 nm for Chlorophyll-, Chlorophyll- nd crotenoid respectively 15. Estimtion of Proline: Plnt mteril (.5) ws grinded in 1ml of 3% sulfo- slicylic cid then the homogenized mixture ws centrifuged t 3 rpm for 1 minutes. Then to the ml of superntnt ml of cid Ninhydrin regent nd ml of Glcil cid ws dded nd oiled in wter th t 1 C. 4ml of toluene ws dded to it nd llowed to cyclomixer. The upper toluene lyer contining chromophore ws collected crefully with help of micropipette nd the sornce ws mesured t 5nm 15. Estimtion of ctlse ctivity: For ctlse ssy ml of sodium phosphte uffer nd.5 ml of 1 mm of H O ws dded to.5 ml of the plnt lef extrct nd the O.D ws tken t 4 nm. Activity of Ctlse enzymes of seedlings grown under different Cr tretments were ssyed with mesurement of the initil rte of disppernce of H O. [Extinction coefficient for H O is 4mM - 1 cm -1 t 4nm] Estimtion of peroxidse ctivity: For ssy of peroxidse ctivity,.8 ml of potssium phosphte uffer (ph = 7.), 5µl of 1 mm H O nd 5µl of guicol ws dded to diluted lef extrct. The mixture ws mixed properly nd sornce vlue ws recorded t 436 nm. Activity ws clculted using the extinction coefficient (5.5 mm tetrguicol cm -1 t 436 nm) for Results nd Discussion Mximum inhiition of seed germintion ws oserved t 1 µm Cr 6+ tretments for oth the vrieties of Oryz nivr (IC- in seed germintion nd IC ). The inhiition ws recorded s 6 % nd 58% in IC nd IC cultivrs respectively (figure-1 nd 1) Figure-1 Br grph showing the effects of Hexvlent Chromium on seed germintion of two vrieties of seven dys nd fourteen dys grown Oryz nivr Chnges in growth prmeters: Notle chnges hve recorded in different growth prmeters of 7 nd 14 dys grown cultivrs of Indin wild rice seedlings (figure- nd nd figure-3 nd 3) grown under different tretment concentrtions of Cr 6+. Shoot length decresed mrkedly with incresed dose of Cr 6+ tretment from 1µM to 1µM. The decresing trend in growth prmeters corroortes the findings of other reserchers in different plnts with response to Cr stress. Root length nd shoot length of IC cultivr of Oryz nivr seedlings showed high sensitivity to Cr toxicity in comprison to IC cultivr of Oryz nivr seedlings s evident from their reduced vlues t 1µM tretment of Cr 6+. Root nd shoot length vlues of IC cultivr of Oryz nivr in response to vrying concentrtion of Cr showed 5 % of tht of IC cultivr. Fresh weight vlues of roots nd shoots were grdully decresed with incresed in concentrtions of Cr +6 tretments in nutrient solution. Prllel growth trend vlues were found for fresh iomss (figure-4, 4 nd figure-5 nd 5) nd dry iomss production (figure-6 nd 6 nd figure-7 nd 7). The two cultivrs of Indin wild rice seedlings were exmined for their tolernce nd sensitivity towrds different concentrtions of Cr +6. Interntionl Science Congress Assocition 1

3 Interntionl Reserch Journl of Environment Sciences ISSN Vol. 3(11), -8, Novemer (14) A RL SL A' RL SL Length (cm) 5 4 Length (cm) Control Figure- nd Effect of different concentrtions of Cr 6+ on shoot nd root length of 7 nd 14 dys old IC vriety of Oryz nivr seedlings. [NB: A-7 dys grown; A - 14 dys grown] Control A- 8 RL SL A'- 18 RL SL 7 16 Length (cm) Length (cm) Control Figure-3 nd Effect of different concentrtions of Cr 6+ on shoot nd root length of 7 nd 14 dys old IC vriety of Oryz nivr seedlings. [NB: A-7 dys grown; A - 14 dys grown] Control Interntionl Science Congress Assocition

4 Interntionl Reserch Journl of Environment Sciences ISSN Vol. 3(11), -8, Novemer (14) B RFW SFW B' RFW SFW.45.8 Fresh weight (cm) Fresh weight (gm) Control Figure-4 nd Effect of different concentrtions of Cr 6+ on root fresh weight nd shoot fresh weight of 7 nd 14 dys old IC vriety of Oryz nivr plnt. [NB: B-7 dys grown; B - 14 dys grown] Control B- RFW SFW B'- RFW SFW Fresh weight (gm) Fresh weight (gm) Control Figure-5 nd Effect of different concentrtions of Cr 6+ on root fresh weight nd shoot fresh weight of 7 nd 14 dys old IC vriety of Oryz nivr plnt. [NB: B-7 dys grown; B - 14 dys grown] Control Interntionl Science Congress Assocition 3

5 Interntionl Reserch Journl of Environment Sciences ISSN Vol. 3(11), -8, Novemer (14) C.3 RDW SDW C'.8 RDW SDW Dry weight (gm) Dry weight (gm) Control Figure-6 nd Effect of different concentrtions of Cr 6+ on root dry weight nd shoot dry weight of 7 nd 14 dys old IC vriety of Oryz nivr plnt. [NB: C-7 dys grown; C - 14 dys grown] Control C RDW SDW C'-.6 RDW SDW..5.4 Dry weight (gm).15.1 Dry weight (gm) Control Figure-7 nd Effect of different concentrtions of Cr 6+ on root dry weight nd shoot dry weight of 7 nd 14dys old IC vriety of Oryz nivr plnt. [NB: C-7 dys grown; C - 14 dys grown] Control Effects on Chlorophyll nd Crotenoid Content: By compring the totl chlorophyll content of two cultivtes of O. nivr, it ws noted tht IC showed high Chlorophyll content in comprison to IC cultivr. Grdul decrese in chlorophyll content ws found with incresed tretment concentrtions of Cr 6+ except for tretment of 1 µm Cr 6+. Mximum chlorophyll content ws recorded in 1µM-Cr +6 nd minimum in 1µM-Cr +6 treted seedlings. Anlogous trend of increse/decrese in crotenoid content ws oserved (figure-8 nd 8). Interntionl Science Congress Assocition 4

6 Interntionl Reserch Journl of Environment Sciences ISSN Vol. 3(11), -8, Novemer (14) Totl Chlorophyll nd Crotenoid content (mg/gm fr. wt.) Control () Control () 1 IC Totl Chlorophyll nd Crotenoid content (mg/gm fr. wt.) Control () Control () 1 5 IC Totl Chlorophyll Crotenoid Totl Chlorophyll Crotenoid Tretments of Cr (µm) Tretments of Cr (µm) Br grph with stndrd error rs showing comprison of the effects of Br grph with stndrd error rs showing comprison of the effects of Chromium in two cultivted vrieties of fourteen dys grown Oryz nivr with Chromium in two cultivted vrieties of seven dys grown Oryz nivr with respect to totl chlorophyll nd crotenoid content. respect to totl chlorophyll nd crotenoid content. Figure-8 nd Effect of different concentrtions of chromium on chlorophyll nd crotenoid content (mg / gm fresh wt.) of two cultivrs of Oryz nivr seedlings fter 7 nd 14 dys exposure Accumultion of proline: Higher proline content ws found with incresed concentrtion of Cr 6+. Elevted proline ccumultion ws noted in seedlings treted with 1µM-Cr +6 tretments for IC cultivr nd 75µM-Cr +6 tretments for IC cultivr for oth 7 nd 14 dys old O. nivr seedling. A comprtive proline ccumultion in two cultivrs of Indin wild rice shows tht IC cultivr hs higher proline ccumultion (figure-9 nd 9). Altertions in ntioxidtive enzyme ctivity: Vrile responses were exhiited y ntioxidnts like ctlse, peroxidse ctivities nd crotenoids to different chromium tretments (figure-1, 1, figure-11, 11, nd figure-8, 8). The ctivity of peroxidse (POX) incresed t high chromium stress of 1µM. Enhnced peroxidse ctivity ws oserved in Figure-9 Comprtive proline ccumultion in two cultivrs of Oryz nivr seedlings fter 7 nd 14 dys exposure Interntionl Science Congress Assocition 5

7 Interntionl Reserch Journl of Environment Sciences ISSN Vol. 3(11), -8, Novemer (14) IC cultivr thn other cultivr. Ctlse (CAT) ctivity showed decresing trend with incresed chromium concentrtions for IC cultivr wheres in IC cultivr the ctivity ws significntly enhnced t 5 µm tretment of Cr 6+ thn other tretments fter 14 dys exposure. An imlnce rises etween genertion nd removl of ROS in plnt tissues sujected to excess chromium or ny other hevy metl exposure 18. Discussion:Though chromium induced phytotoxic effects (reduced rte of growth, dmge to cell wll, cell memrnes nd chnges in the metolic sttus of plnts) ws reported long time ck, still there is huge derth of informtion regrding Chnge in Ctlse ctivity(a)/m in fter 7 Dys Exposure Figure-1 nd Effect of different concentrtions of Cr 6+ on ctlse ctivity in two cultivrs of Oryz nivr fter 7 nd 14 dys exposure Chnge in Peroxidse ctivity(a)/min fter 14 Dys Exposure (IC83) IC83 CONTROL Cr tretments(µm) CONTROL Cr tretments(µm) Chnge in Ctlse ctivity(a)/m in fter 7 Dys Exposure IC Chnge in Peroxidse ctivity(a)/min fter 14 Dys Exposure () Chnge in Ctlse ctivity(a)/m in fter 14 Dys Exposure Figure-11 nd Effect of different concentrtions of Cr 6+ on peroxidse ctivity in two cultivrs of Oryz nivr fter 7 nd 14 dys exposure Chnge in P eroxidse ctivity(a)/m in fter 7 Dys E xposure CONTROL Cr tretments(µm) CONTROL Cr tretments(µm) IC83 IC83 its impct in wild cultivrs of Indin wild rice. In view of the seriousness if Cr pollution, the present study hs een undertken with n effort to ssess the phytotoxic impcts with specil reference to iochemicl lesions in 7dys nd 14 dys Interntionl Science Congress Assocition 6

8 Interntionl Reserch Journl of Environment Sciences ISSN Vol. 3(11), -8, Novemer (14) grown Oryz nivr seedlings. The prt of the present hydroponics study provides promising strt for revising nd compring the level of chromium toxicity in two vrieties of Oryz nivr with potentility of their tolernce fter exposure to vrying concentrtions of Cr. The work signifies the potentil of Oryz nivr plnts towrds Cr phytotoxicity nd tolernce. Study of proline ccumultion in response to hevy metl stress is one of the importnt indictors in plnts under stress. Incresed proline provide osmoprotection, regultes the redox potentil, scvenges hydroxyl rdicls which will denture of vrious mcromolecules nd thus protects the mcromolecules from dmge. Non-enzymic free rdicl detoxifictions re often ssocited with proline ccumultion 16. The incresed proline iosynthesis in the Cr 6+ treted seedlings signifies stress regulting fctor tht gives protection in plnts under hevy metl toxicity environment 17. Essentil components of plnt ntioxidnt defence system re ssocited with ctivities of POX nd CAT. Erlier studies on elevted POX ctivity in nickel treted O. stiv, suggests its role in the detoxifiction of H O 19. Plnts with enhnced POX ctivity in response to excess chromium nd cdmium supply might suffer from peroxidtive dmge of the thylkoid memrne or lower uxin nd protein contents in tissues. Though CAT nd POX ctivities protect the plnt metolic system 1 still it hs negtive impcts with its excess. Considering the ove phytotoxic effects, Cr removl from pollutes sites ecomes very much essentil for which different iosorents re effectively used -4. Conclusion Plnt tolernce to hevy metls is the ility to survive in contminted soil which is mnifested y n interction etween genotype nd its environment. Aove studies revel some interreltionship etween different metolic effects induced y chromium in wild plnt like Oryz nivr. Intensive future reserch on the effects of ccumultion of Cr on plnt metolism is essentil. Further the ility of different cultivrs of this plnt for incresing phytoccumultion potentil needs to e tried longwith vrious iosorption models. Suitle post hrvest ioremedition techniques should e dopted for disposl of plnts nd plnt prts contining ccumulted toxic chromium from mining environment. The overll view of the impct of vrying doses of hexvlent chromium on tolernce nd sensitivity ility of two cultivrs of Indin wild rice suggest removl of these toxic contminnts from soil y potent ppliction of weeds like Indin wild rice. Acknowledgement The uthors cknowledge the finncil support provided y UGC-DRS-SAP scheme to Deprtment of Botny, Utkl University Bhuneswr, Indi. References 1. Rouphel Y., Crdrelli M., Re E. nd Coll G., Grfting of cucumer s mens to minimize copper toxicity, Env. Exp. Bot., 63, (8). Cervntes C., Grci J.C., Devrs S., Coron F.G. nd Tver H.L., Crlos Torres-guzmn J, Snchez RM. Interctions of chromium with micro-orgnisms nd plnts, FEMS Micro. Rev., 5, (1) 3. McGrth S.P., The uptke nd trnsloction of tri- nd hexvlent chromium nd effects on the growth of ot in flowing nutrient solution nd in soil, New Phytologist, 9, (198) 4. Shrm R.M.S. nd Rju N.S., Correltion of hevy metl contmintion with soil properties of industril res of mysore, krntk, Indi y cluster nlysis, I. Res. J. Environment Sci., (1), -7, (13) 5. Poschenrieder C., Vzquez M.D., Bonet A. nd Brcelo J., Chromium III iron interction in iron sufficient en plnts. II Ultrsructurl spects, J. Plnt Nutr., 14, (1991) 6. Brcelo J., Poschenrieder C., Vzquez M.D. nd Gunese B., Aluminium phytotoxicity. A chllenger for plnt scientists, Fertile. Res., 43, 17-3 (1996) 7. Brcelo J. nd Poschenrieder C., Chromium in plnts. In: Crti, S., tottrelli, F., Seqmi, P. (ed): Chromium Environment Issues, Frncotngti Press, Miln, (1997) 8. Pnd S.K. nd Ptr HK., Physiology of chromium toxicity. A review, Plnt Physiol. Biochem., 4,1-17 (1997) 9. Pnd S.K. nd Ptr H.K., Alterntion of nitrte reductse ctivity y chromium ions in excised whet leves, Indin J. Agr. Biochem., 11(), (1998) 1. Pnd S.K nd Ptr H.K., Does Chromium (III) produce oxidtive dmge in excised whet leves?, J.Plnt Biol., 7, () 11. Sudermn F.W., Crcinogenic effect of metls, Fed. Proc., 37(1), 4 46 (1978) 1. Khsim I. nd Nnd Kumr N.V., Environmentl contmintion of chromium in griculturl nd niml products ner chromte industry, Bull. Environ. Contm. Toxicol., 43, (1989) 13. Hq1 B.I.U., Nursfur E. B. nd Zki K., Adsorption Studies of Cr (VI) nd Fe (II) Aqu Solutions Using Ruer Tree Leves s n Adsorent, I. Res. J. Environment Sci., (1), 5-56, (13) 14. Pttnik N., Mohnty M. nd Ptr H.K., Effect of chelting gents nd metl ions on nickel iovilility nd chlorophyll fluorescence response in whet- An Interntionl Science Congress Assocition 7

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