Research Journal of Recent Sciences ______ _______________ _______________ ______ ____ ___ ISSN 2277 - 2502 Vol. 2 ( ISC - 2012 ), 24 - 28 (201 3 ) Res. J. Recent . Sci. International Science Congress Association 24 Effects of heavy metal stress on callus induction and regeneration of Finger millet ( Eleusine coracana ) (L.) Gaertn Krishania Suman and Agarwal Kalpana Department of Life Sciences,The IIS University, SFS Mansarovar, Jaipur - 20 (Raj) INDIA Available onli ne at: www.isca.in Received 20 th October 2012, revised 31 st December 2012, accepted 30 th January 201 3 Abstract Today Abiotic stress is a major global problem limiting crop productivity and Stress factors are a serious problem limiting the yield potential of modern cultivars faced by mankind now a days. Stress causes nutritional imbalances in the plant causing reduction in water uptake and toxicity, decreasing the production and Seeds of Eleusine coracana (L.) Gaertn va riety PR202 were taken as explants here and inoculated on callus induction medium with varied ( Cd(NO 3 ) 2 levels (100 然 , 300 然 and 500 然 ) Calli were formed in treatments with 100 然 concentration of Cd. Induced Callus were sub cultured on maintenance media and then on regeneration medium (MS + 1mg/l NAA) supplemented with toxic level of heavy metals. Cd above 100 然 concentration was inhibitory for callus induction as well as for plantlet regeneration . Keywords : Heavy metal, MS media, Cd (NO 3 ) 2 . Introdu ction Stresses are increasing drastically today because of pollution, declining availability of quality water and land degradation. Ragi is used as cereal, which is also called finger millet and scientific name is Eleusine coracana. Eleusine coracana belong ing to family Poaceae Figure - 2 . Cereals constitute a major source of food for the human population of the world. Important cereals in day to day use are rice, wheat and millets Figure1 . Of all the millets Eleusine contains more percentage of different nutr ients as compared to rice and wheat. It is a rich source of calcium, magnesium and potassium. Eleusine is nutritionally very rich cereal and is a staple food for poor and invalids 1 . Due its nutritional superiority and requirement by poor people production needs to be improved. Eleusine coracana mainly growing in dry condition faces heat, salinity and heavy metal stress which may cause negative effect on its yield. Stress tolerant plants can also be developed by breeding and by transgenesis which are complex processes 2 . Tissue culture techniques offer an easy and important tool in developing stress tolerant variants 3 . Studies were conducted on diabetics (male and female) living in different rural and urban locations in 2008 in different countries 4 . Finger mil let is especially valuable as it contains amino acid methionine , which is lacking in the diets of millions of the po or people. Abiotic stresses like salinity, heavy metals and pesticides are the primary cause of crop failures in India. It has been estimated that about 8.6 million ha of land is affected by salinity in India 5 . 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 6 . They present a risk for primary and seconda ry consumers and ultimately humans 7 . Figure1 Figure2 Figure1 - Seeds of Eleusine coracana Figure2 - Mature Plant of Eleusine coracana Cadmium is an industrial pollutant in constant rise in the environment, due to activiti es such as mining, smelting and refinement of zinc, manufacturing and use of fungicide and phosphorous fertilizers, metallurgy, among others 8 . 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 and an increase in the respiratory rates 9,10,11 . These effects seem to be related to Cd induction of premature senescence in plants 12 . Cadmium, being a highly toxic metal pollutant of soils, affects nutrient upt ake and homeostasis, inhibits root and shoot growth and yield. Zn supplementation efficiently reduced lipid peroxidation, electrical conductivity and lipoxygenase activity induced by Cd 13 . Stress - induced enzyme, peroxidase, in the regenerating tissues of L inum usitatissimum showed less activity in cadmium and zinc combinations 14 . Looking to the scope and realizing its importance, the present study was undertaken to investigate the effects of different concentrations of Cd elements on callus induction and pl ant regeneration with the following Objectives - To standardize a medium with growth regulators for tissue Research Journal of Recent Sciences ______ _ _ _______________ ________________ ______________ _ ________ ISSN 2277 - 2502 Vol. 2 ( ISC - 2012 ), 24 - 28 (201 3 ) Res. J. Recent. Sci . International Science Congress Association 25 culture of Eleusine coracana and to study the influence of Cd in the morphogenesis of Eleusine coracana tissue culture and Implantation in field to in crease the productivity of cereals. Levels of different heavy metals were varied taking MS as the standard medium. Materials and Methods Important plant Eleusine corcana (L.) Gaertn of family Poaceae was taken as the model plant for the study. Seeds of ag ronomically superior and released variety (PR - 202) of Ragi were procured from Agricultural University, Bangalore and this study was conducted in 201 - 12 at International College for girls, Department of Life Sciences, Jaipur. Common Name: Finger millet - Ver nacular Name: Ragi, Explants Taken: - Explants taken for the experiment - Seeds. Sterilization and Preparation of Explants - Explants taken were aseptically sterilized. Seeds were surface sterilized in 0.1 percent mercuric chloride solution for 3 minutes. These explants (seeds) were inoculated on MS medium supplemented with auxin 2, 4 - D (2 mg/l) and cytokinin Kn (0.5 mg/l).Basal Medium Preparation - Basal medium used in the present study was MS (Murashige and Skoog, 1962) medium.(Sucrose: 3% (w/v), Agar: 0.8 - 1% ( w/v) and pH: 5.8). A septic Manipulations - Aseptic culture was carried out in laminar air flow chamber. Incubation - Cultures were incubated in growth chamber equipped with two air conditioners and temperature controlled at 261 軏. A photoperiod of 16 hours a lternating with 8 hours of darkness was maintained. Statistical Analysis - The observations recorded for the various experiments were subjected to following. Average: The average (mean) was calculated by dividing the sum of values of observations for a part icular treatment by the total number of observations for that treatment. Standard Deviation This is a measure of dispersion which was calculated by squaring the deviation of each observation from the mean. Preparation of Medium with Varied Concentration of Heavy Metals (Cd) : Callus Induction, Embryogenic callus and Plant Regeneration A) Seeds of Eleusine coracana were inoculated on callus induction medium with varied Cd levels (100 然 , 300 然 and 500 然 ) . Seeds was inoculated on MS Medium supplemented wit h 2mg/l 2,4 - D+0.5mg/l Kinetin (control) .In addition to this Cd concentration were varied in callus induction medium and the amount of callus formed in each case was record and after 3 - 4 weeks all the calli were transferred to MS medium with lower concent ration on (2,4 - D(0.2mg/l)+Heavy metal) maintenance media and then on regeneration media with MS+1mg/l NAA + heavy metal. In this experiment heavy metal was added in each step of subculturing in compare to control. B). Seeds was inoculated on MS Medium supp lemented with 2mg/l 2,4 - D+0.5mg/l Kinetin (control) the amount of callus formed in each case was record and after 3 - 4 weeks all the calli were transferred to MS medium with lower concentration (2,4 - D(0.2mg/l) maintenance media without heavy metal and then on regeneration media (MS+1mg/l NAA) with heavy metal. In control there is no heavy metal in any stage of subculturing. Approximately 4 - 5 seeds were cultured in each flask and approx 5 replica of each concentration were made. Approx 250mg of callus was tra nsferred to each flask for regeneration. In this experiment heavy metal were added only in regeneration media in compare to control. Results and Discussion Seed Culture Table - 1 Seeds of Eleusine coracana were inoculated on callus induction medium with va ried Cd levels (100 然 , 300 然 and 500 然 ) After 2 - 3 weeks of inoculation of seeds, it was observed that the callus was healthy, compact, nodulated, dark green embryogenic sectors along with watery translucent non embryogenic sectors Figure 3a . Amount of ca llus formed was recorded. Only the embryogenic sectors were transferred to maintenance medium, the non embryogenic portion of the callus was excluded from being subculture and then obtained callus were transferred on Regeneration media Figure - 3b . Callus Cu lture and Plant Regeneration Table - 2 Approx 250mg of maintenance callus was transferred to each flask for regeneration. Experiments for evaluating the effects of heavy metal (Cd) were conducted and regenerated shoots were transferred on soil pots Figure - 3 c . T able - 1 Callus induction medium for E.coracan with various concentrations of Cadmium Medium Concentration of Cd(然 ) MS + 2,4 - D + Kn (Control) Nil MS + 2,4 - D + Kn+Cd 100 然 MS + 2,4 - D + Kn +Cd 300 然 Growth regulators added: Auxin (2, 4 - D), Cytoki ne (Kinetin/Kn), Agar (8 g/l) Table2 Callus formation from seeds of Eleusine coracana L . cultured on MS medium supplemented with 2, 4 - D and Kn. and on Maintenance Medium. (Control) Media Amount of callus formed per seed (mg) Callus Induction Medium(CIM) MS +2,4 - D (2mg/l)+Kn (0.5mg/l 200 Maintenance Medium(MM) MS+2,4 - D (0.2mg/l) 250 C.I.M.=Callus Induction Medium, C=c ontrol, S.D= Standard deviation, R.M.=Regeneration Medium and M.M=M aintenance Medium and Number of explants =5 seeds/flask Research Journal of Recent Sciences ______ _ _ _______________ ________________ ______________ _ ________ ISSN 2277 - 2502 Vol. 2 ( ISC - 2012 ), 24 - 28 (201 3 ) Res. J. Recent. Sci . International Science Congress Association 26 Figure 3a Figure3b Figure3c Figure - 3 a - b Response of seeds on Callus Induction Medium and Response after Primary callus transferred on Regeneration media. Figure 3 - a Callus induced from seeds of on MS + 2, 4 - D (2 mg/l) +Kn (0.5 mg/l), Figure 3 - b Embryogenic callus after subculture on regeneration media, Figure3 - c Regenerates were transfer to pot from regeneration media Effects of Heavy Metal on Callus Induction and Plant Regeneration (Cd) , Effects of Cd on primary callus induction and plant reg eneration t able - 3 . Table - 3 Response on callus and regeneration with continues vari ed concentration of Cd in media Concentrations of Cd in Callus Induction Medium(然) Amount of callus formed / explant(mg) Number of shoots per seed callus on plant regenerati on medium S.D. 0 然 250 12.4 6.4 100然 150 3.30.4 300然 0 0 500然 0 0 Table - 4 Response on plant regeneration with varied concentration of Cad mium only in regeneration media Concentrations of Cd in plant Regeneration Medium (然) Mean number of shoot formed / Callus Piece S.D. 0 然 13.4 1.45 100然 6.8 2.2 300然 0 500然 0 Initial weight of callus=200mg Seeds of Eleusine coracana were inoculated on callus induction medium with varied Cd levels (100 然 , 300 然 and 500 然 ) Calli were for med in treatments with 100 然 concentration of Cd Figure - 4b .While there was no callus formation in treatments with 300 and 500 然 concentrations of Cadmium Figure4c - d.Calli were nodulated, compact, embryogenic, soft and watery. Control callus was more com pact in compare to heavy metal callus Figure - 4a. Cd containing media callus was worse than control, only the embryogenic sectors of calli were transferred to maintenance medium with heavy metal, the non embryogenic portion of the callus was excluded from b eing subculture and then obtained callus were transferred on regeneration media with heavy metal. After 2 - 3 weeks deep green and well developed regenerates appeared. Less shoots are developed when heavy metal was added continue in media in compare to Heavy metal added only in regeneration media. Effects of Cd on Plant Regeneration Table - 4 Embryogenic callus from maintenance medium (MS + 0. 2 mg/l 2, 4 - D + without heavy metal) were further transferred on regeneration medium (MS + 1mg/l NAA) supplemented with varied concentrations of Cd (100 然 , 300 然 and 500 然) to see the effects of Cd concentrations on regeneration of Eleusine coracana . After 2 - 3 weeks, differentiation of shoot buds took place in medium containing 100 然 concentration of Cd Figure - 4f. Number of shoots per callus piece was counted. Regeneration was worse than control Figure - 4e. Shoots are better than continue containing heavy metals media. In this experiment heavy metal were added only in regeneration . medium Figure - 4g Eleusine Coracana (L) Ga ertn which mainly grows in dry condition faces the toxicity of cadmium which may have negative effect on its yield and these heavy metals decrease the growth rate of explants in in vitro conditions. Holarrhena antidysenterica L nodal part when treated with ZnSO 4 proved to be less inhibitory in comparison to CuSO 4 , Pb(NO 3 ) 2 and CdCl 2 . There is no response on CdCl 2 containing medium 15 . Cu and Zn significantly reduced seed germination and root growth in compare to Cd, Pb in Pimpinella anisum L. (anise), Carum carvi L. (caraway), and Foeniculum vulgare L. (fennel) 16 . In Cabbage when Zn, Cu, Cd, Pb and Hg presence in excess reduced glutathione (GSH) greatly and increases the soluble and immobilized peroxidase activity 17 . And also observed effects of different he avy metals Ni,Cd,Cu,Zn on different variety of Eleusine coracana . 18,19, 20 . If low concentration of auxin along with cytokinin increases the rate of shoot multiplication and found that 2iP was the best for shoot elongation 21 . An appropriate concentration of IBA and 2ip was effective on traits of micropropagation of Cymbidium orchid 22 . The value of SSI increases with increasing salinity levels for thirteen sorghum genotypes 23 . In Barley Results showed that paclobutrazol (40 mg l - 1) minimizes the negative ef fects of water stress (60% ETc) with evidence of enhancing leaf water potential by up regulating the endogenous production of proline and antioxidant enzymes like SOD and CAT leading to Research Journal of Recent Sciences ______ _ _ _______________ ________________ ______________ _ ________ ISSN 2277 - 2502 Vol. 2 ( ISC - 2012 ), 24 - 28 (201 3 ) Res. J. Recent. Sci . International Science Congress Association 27 maximization of fruit yield accompanied with higher WUE 24 . Experiment for evaluating the effects of heavy metal (Cd) was conducted. Callus induction was obtained only in the medium containing 100 然 concentrations of Cd .These results show that Cd above 100 然 concentration were toxic for callus induction in Eleusine coraca na . Embryogenic callus obtained from 100 然 concentration of Cd was sub cultured in the regeneration medium (MS + 1 mg/l NAA) for shoot regeneration. Sub cultured regenerates appear deep green and well developed. These results show that there is no shoot r egeneration in Eleusine coracana above 100 然 concentration of heavy metal . Shoots of heavy metals containing media were worst then control. In first Experiment Heavy metal are added in each step of experiment like initiation of callus and then in subcultu ring on maintenance media and in last in regeneration media to observe the heavy metal stress in all stages of subculturing and survivability of plant in every stage with heavy metals. In experiment second heavy metal was added only in regeneration media t o observe the stress only in regeneration media. Figure 4a - f Effects of varied concentrations of Cd on primary callus induction and plant Regeneration as compare to control Figure 4a MS+2, 4 - D and kn(control), Figure 4b MS+2, 4 - D and kn +Cd(100然) Figu re 4c MS+2, 4 - D and kn+Cd (300然)Figure 4d MS+ 2, 4 - D and kn +Cd (500) Figure 4e Primary callus sub cultured on regeneration media (control) Figure 4f Primary callus obtained from (100然) Cd concentration media sub cultured on Regeneration medium with hea vy metal. Figure 4g No heavy metals containing Primary callus subcultured on regeneration media with heavy metals Conclusion These experiments led to a conclusion that Cadmium above 100 然 concentration is inhibitory for callus induction as well as for pl antlet regeneration in Eleusine coracana but callus and regeneration was worse than control. Acknowledgments This work was supported by International College for girls, Jaipur, the IIS University Jaipur - 20 References 1. Panda P.R., Incorporating millets in h igh intensity cropping., Yojana (1999) 2. Babu S., Sheeba A., Yogameenakshi J. and Rangaswamy P., Effect of salt stress in the selection of salt tolerant hybrids in rice (Oryza sativa L.) under in vivo and in vitro conditions . Asn J Plt Sci., 6, 137 - 142 (2007 ) 3. Nabors M.W. and Dykes T.A., Tissue culture of cereal cultivars with increased salt, drought and acid tolerance. Biotech in international agri res, IRRI Phillipines, 121 - 138 (1985) 4. Pradhan Adikant, Nag S.K. and Pati, Dietary management of finger millet ( E leusine coracana L. Gaerth) controls diabetes,Current Science . 98 , 6 - 25 (2010) 5. Pathak, A tool for arresting land degradation - Indian Farming. Agro forestry. 49 , 15 - 19 (2000) 6. Nedelkoska T.V. and Doran. P.M ., Characteristics of heavy metal uptake by plant spe cies with potential for phytoremediation and phytomining , Journal on Minerals Engineering, 13 , 549 - 561 (2000) 7. Zeller S. and Feller U., Long - distance transport of cobalt and nickel in maturing wheat , European Journal of Agronomy. 10 , 91 98 (1999) 8. Page.A.L., and Bingham, F. T.Cadmium residues in the environment. Residue Reviews. 48 , 1 - 44 ( 1973) 9. 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 , 373 - 376 (1 974) 10. 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 (1976) 11. Lamoreaux R.J and Chaney W.R. The effect of cadmi um on net photosynthesis transpiration, and dark respiration of excised silver maple leaves, Physiologia Plantarum, 43 , 231 - 236 (1978) 12. Van assche F., Cardinales C. and Clijsters J. , Induction of enzyme capacity in plants as a result of heavy Dose - response relations in Phaseolus vulgaris L., treated with zinc and cadmium . Environmental Pollution, 52, 103 - 115 (1988) 13. Aravind P., Narasimha M. and Prasad V., Zinc alleviate cadmium - induced oxidative stress in Ceratophyllum demersum L.: A free floating freshwater macrophyte, Journal of Plant Physiology and Biochemistry , 41, 391 - 397 (2003) 14. Chakravarty B. and Srivastava S., Effect of cadmium and zinc interaction on metal uptake and regeneration of Research Journal of Recent Sciences ______ _ _ _______________ ________________ ______________ _ ________ ISSN 2277 - 2502 Vol. 2 ( ISC - 2012 ), 24 - 28 (201 3 ) Res. J. Recent. Sci . International Science Congress Association 28 tolerant plants in linseed, Journal of Agriculture, Ecosystems & Envir onment , 61 , 45 - 50 (1999) 15. Agrawal V. and Sharma K. Phytotoxic effects of Cu, Zn, Cd and Pb on in vitro regeneration and concomitant protein changes in Holarrhena antidysenterica, Journal of Biologia Plantarum, 50 , 307 - 310 (2006) 16. Jeliazkova E.A. and Craker L .E. Seed Germination of Some Medicinal and Aromatic Plants in a Heavy Metal Environment, Journal of Herbs, Spices & Medicinal Plants, 10 , 105 (2002) 17. Brzyska M., Grzywnowicz K., Obarzewski J. and Greppin H. Influence of reduced glutathione (GSH) and metal ions on soluble and immobilized cabbage peroxidase , Journal of Molecular Catalysis , 78 ,113 - 120. (2001) 18. Krishania.S and Agarwal K,. Effects of heavy metals on productivity of cereals Lambert Academy Publication House, 1 - 76 (2010) 19. Krishania S. and Agarwal K. , Effect of Cu and Zn concentrations with nutrient doses on callus induction and regeneration of finger millet ( Eleusine coracana ), World Journal of Science and Technology, 2(7), 118 - 123 ( 2012) 20. Krishania.S and Agarwal. K., Effects of heavy metals on Eleusi ne coracana (L.) Gaertn Research in Plant Biology, 2(4), 43 - 54 (2012) 21. Eman A.A., Initiation of Pharmaceutical Factories depending on more Application of Biotechnology on some Medicinal Plants , Research Journal of Recent Sciences, 1 , 398 - 404 (2012) 22. Chikwe T .N., Osuji L.C., Ogali R.E. and Okoye I.P. , The effects of IBA and 2ip on callogenesis and shoot formatting of Cymbidium orchid var Red Tiffani 2(1) , 1 - 8 (2013) 23. Chauhan R.R., Chaudhary R., Singh A. and Singh P.K., Salt Tolerance of Sorghum bicolor Cultiv ars during Germination and Seedling Growth, 1(3) , 1 - 10 (2012) 24. Mostafa M. Rady and Maybelle S. Gaballah, Improving Barley Yield Grown Under Water Stress Conditions, 1(6) , 1 - 6 (2012)