International Research Journal of Biological Sciences ___________________________________ ISSN 2278-3202Vol. 4(5), 80-86, May (2015) Int. Res. J. Biological Sci. International Science Congress Association 80 Phytotoxic Effects of Cadmium on Seed Germination and Seedling Growth of Brassica juncea L.Czern Coss) cv.Bohra Alfiya and Sanadhya Dheera School of Life Sciences, Jaipur National University, Jaipur, Rajasthan, INDIAAvailable online at: www.isca.in, www.isca.me Received 6th April 2015, revised 28th April 2015, accepted 8th May 2015 Abstract Cadmium (Cd2+) is extremely toxic heavy metal which adversely affects the growth and yield of plants. Therefore, study was conducted to evaluate the effects of various Cd2+ concentrations (0.5mM, 1 mM, 1.5 mM and 2mM) on seed germination and seedling growth of twenty Brassica juncea Cultivars. Various parameters tested in present study includes root and shoot length, seedling size, fresh and dry matter, seed germination percentage, tolerance indices and percent phytotoxicity. Results indicate that the significant inhibitory effect was observed at all the four concentrations of Cd2+ as compared to control. As the Cd2+ concentration increases, all the tested parameters except Tolerance Indices decrease. Present study also reveals that Cultivar RB-50 may show better performance under Cd+2stresses while Cultivar RH-0406 might have poor performance. From these results it can concluded that Cultivar RB-50 might germinate well on Cd2+ contaminated soils, however more studies are needed to signify its effects on growth and yield of Brassica juncea. Keywords: Cadmium, Brassica juncea, seed germination, tolerance indices, percent phytotoxicity.Introduction Indian mustard (Brassica juncea) is an important oil seed crop being cultivated in 53 countries all over the world. Brassica juncea (B. juncea) is a fast growing plant which produces a high biomass even in heavy metal contaminated soils. Yield of B. juncea is negatively affected by presence of heavy metals in the soil. The unrestricted development activities e.g. Urbanization and Industrialization, carried out haphazardly during past few years have caused serious problems of environmental contamination. Agricultural soils are continuously polluted with harmful heavy metals that hamper the growth of plants and cause significant yield reduction and loss in their productivity. Deshveer and Singh2 reported that the yield of Brassica juncea is of low magnitude, despite the use of N and P fertilizers. Foy et al3 and Sheoran et al4 observed that indiscriminate usage of phosphatic fertilizers in agricultural fields builds up cadmium level in the soil which negatively affects plant growth and economic yield. Among various heavy metals Cd+2 is a non essential and extremely deleterious, it is discharged into the agricultural soils worldwide from various sources such as industrial, mining and farming practices and has been ranked Number 7 among the top 20 toxins which has harmful effects on the human health by entering into the food chains6,7 . It is quickly absorbed by roots of plants growing on Cd+2 polluted soils and transported to leaves in the same way as the essential micronutrient metal ions8,9. Plants growing on Cd+2 polluted soil accumulate it, in all plant parts, resulting in growth retardation , affects nutrient uptake, cause alteration in the chloroplast structure, inactivates enzymes, reduce photosynthesis and activates enzymatic and non enzymatic antioxidant machinery10,11. Phytotoxicity of Cd varies with its concentration in the soil as well as with the plant species and cultivars. Some plant species or cultivars have developed tolerance to Cd. Plant varieties show difference in their Cd detoxification capacity in between and within the plant species, which has a beneficial role in the expression of high tolerance in crop plants to Cd toxicity. Therefore screening of plants varieties with the ability to detoxify Cd in the agricultural soils could be the best strategy to overcome the harmful and deleterious consequences of Cd in crop plants. The aims of present study were: In vitro analysis for the impact of Cd toxicity in twenty Brassica junceaCultivars. Screening of Brassica juncea Cultivars for Cd tolerance using germination markers.Material and Methods Approximately 20 Cultivars of Brassica juncea were procured from SKN University Jobner and CCSHAU Hisar. Uniformly selected seeds were surface sterilized with 5% NaOCl for 5 minutes and then washed repeatedly for two to three times with distilled water to prevent fungal/bacterial contamination. Filter papers were also sterilized in autoclave to reduce any chances of microbial growth. Heavy metal test solutions were prepared using four different concentrations of Cadmium (Cd+2) viz. 0.5mM, 1mM, 1.5mM and 2 mM. The chemicals used were of analytical reagent grade. Distilled water was used as control. Seeds were germinated in glass Petri dishes of 15 cm diameter lined with filter paper circles moistened with distilled water as control and various concentrations of Cadmium to impose four levels of stress. Nearly 10 seeds were sown in each Petri dish and incubated in growth chamber set at 25 ± 2°C for 7 days and each treatment was replicated thrice. Occurrence of Germination was considered when roots were 2mm long; Germination Research Journal of Biological Sciences ___________________________________________________________ ISSN 2278-3202 Vol. 4(5), 80-86, May (2015) Int. Res. J. Biological Sci. International Science Congress Association 81 percentage was recorded in every 24h, till the end of experiment. The following parameters were analyzed for the study. Germination percentage: A mean of 10 seeds were taken and expressed as percentage. G.P (%) = Total number of seeds germinated × 100 Total number of seeds Root length and Shoot length: The length of root and shoot of seedlings were recorded at 7th day of germination. Mean values were calculated for both root and shoot length and values were expressed in cm. Seedling Height: Heights of seedling were recorded at 7th day of germination. Mean values were calculated and expressed in cm. Fresh Matter: The Fresh Weight was recorded from the 7th day old seedling. The whole seedling was surface dried with the blotting paper and their fresh weight were recordedDry Matter: The same seedlings were used and dried in oven for 24 h at 80°C and weighed again. This represented the dry matter. Tolerance Indices (T.I): Tolerance indices were determined with the formula given by Iqbal and Rahmati12T.I = Mean root length in metal solution × 100 Mean root length in control Percent Phytotoxicity: Percent Phytotoxicity was determined using the formula of Chou et al. PP = Radicle length in control – Radicle length of test × 100 Radicle length of control Results and Discussion Root length, Shoot length and Seedling size: The statistical analysis (ANOVA) for root length, shoot length and seedling size revealed that cultivars and Cd concentration along with their interactions were highly significant. The data presented in table-1 and table-2 indicates the decrease in trend with respect to root length (RL), shoot length (SL) and seedling size (SS) of Cd+2 treated seedlings over control. As the concentration of Cd+2 increases root length, shoot length and seedling size decreases figure-1. Cultivar RB-50 showed minimum decrease in RL, SL and SS as compared to other 19 Cultivars at all four concentrations of Cd+2 (0.5 mM, 1 mM, 1.5 mM, 2mM). Whereas, cultivar RH-0406 showed maximum decrease in RL, SL and SS at all four concentrations of Cd+2The metal imposed high toxicity at 1.5 mM and 2 mM to all the 20 cultivars. In all the cultivars, the roots were found to be more sensitive to Cd+2 than shoots, which correspond to higher content of Cd+2 in roots than that in the shoots. The greater reduction of root length than shoots could be a result of more absorption of Cd+2 in roots14. Figure-1 Effect of different concentration of Cd+2 on the growth of germinated seedlings of Brassica juncea grown for 7 days invitro Control 0.5mM 1mM 1.5mM 2mM Research Journal of Biological Sciences ___________________________________________________________ ISSN 2278-3202 Vol. 4(5), 80-86, May (2015) Int. Res. J. Biological Sci. International Science Congress Association 82 Table-1 Effect of different concentrations of Cd+2 on Root and Shoot length of Brassica juncea cvRoot Length Shoot length Varieties Control Cd 0.5mM Cd 1mM Cd 1.5mM Cd 2mM Variety Mean Control Cd 0.5mM Cd 1mM Cd 1.5mM Cd 2mM Variety Mean RH-0749 11.07 5.78 1.48 1.34 1 4.13 2.47 2.06 1.71 1.45 1.32 1.8 Pusa Bold 8.24 3.14 1.91 1.76 1.45 3.3 2.28 1.84 1.51 1.46 1.11 1.64 GM-3 7.29 5.11 1.01 0.82 0.77 3 2.56 1.97 1.29 1.17 1.23 1.64 RH-0119 10.04 2.41 1.08 0.74 0.5 2.95 1.97 1.76 1.36 1.28 1.16 1.5 RH-9304 7.55 2.11 1.67 1.36 0.65 2.67 2.14 1.95 1.79 1.77 1.68 1.86 RGN-145 9.46 1.54 1.47 0.49 0.48 2.69 2 1.61 1.51 1.5 1.49 1.62 RH-781 6.48 2.78 2.19 0.74 0.47 2.53 2.5 2.39 1.74 1.14 1.06 1.77 I-2 6.93 6.01 2.58 2.28 0.33 3.63 2.37 1.61 1.59 1.46 1.13 1.63 V-7 8.63 5.87 0.67 0.58 0.34 3.22 3.09 2.39 1.53 1.12 0.9 1.81 RB-50 7.7 7.49 2.27 1.42 1.02 3.98 2.96 2.5 2.26 1.64 1.26 2.12 RH-8113 11.63 2.15 1.43 0.84 0.87 3.38 2.23 1.62 1.55 1.54 1.45 1.68 RH-8812 7.61 5.87 2.83 1.74 1.03 3.82 2.01 1.81 1.66 1.39 1.24 1.62 RH-0406 8.51 1.89 1.83 0.81 0.69 2.75 1.73 1.65 1.51 1.11 0.99 1.4 PBR-357 7.64 6.6 1.97 1.14 1.04 3.68 2.27 2.06 1.97 1.67 1.64 1.92 ROHINI 8.77 5.93 1.53 1.16 0.67 3.61 2.55 2.46 1.73 1.71 1.2 1.93 VARUNA 8.57 3.64 1.28 0.49 0.49 2.89 2.85 2.83 2.17 1.66 1.31 2.16 PCR-7 9.7 1.85 0.89 0.58 0.49 2.7 2.92 2.11 1.71 1.32 1.19 1.85 RL-1359 5.99 5.93 0.69 0.63 0.48 2.74 2.42 2.33 1.44 1.44 1.28 1.78 RH-819 8.56 4.09 1.47 1.36 0.85 3.26 2.43 1.98 1.8 1.21 1.11 1.71 RH-30 8.33 5.23 2.16 1.09 0.57 3.48 2.36 2.21 1.69 1.45 1.41 1.82 Conc Mean 8.43 4.27 1.62 1.07 0.71 3.22 2.41 2.06 1.68 1.42 1.26 1.76 - - - - - - - - - - - - - For means of S.Em± C.D.5% - C.V.%= 14.85 - S.Em± C.D.5% - C.V.%= 12.18 - Variety(var) 0.12 0.34 - - - - 0.06 0.15 - - - - Concentration (conc) 0.06 0.17 - - - - 0.03 0.08 - - - - VarXConc 0.28 0.77 - - - - 0.12 0.35 - - - - - - - - - - - - - - Each data is an average of three replicatesSeed Germination Percentage: The statistical analysis (ANOVA) for seed germination percentage reveals that cultivars and Cd+2 concentrations along with their interactions were highly significant. The effect of different concentrations of Cd+2 on the germination percentage on 20 cultivars of B. junceais shown in table-3. The germination was highest when seeds were grown in distilled water as control; however Cd+2treatments reduced seed germination of B.juncea at different concentrations. The inhibition of seed germination increased with increasing concentration of Cd+2. It was observed that maximum germination percentage recorded is (91.33%) for Cultivar RH-0119 and minimum germination percentage recorded is (76%) for Cultivar PCR-7 at 2mM Cd+2. Whereas Cultivar RB-50 showed better performance at all four concentrations of Cd+2 treatments. The results of the present study clearly revealed that metal Cd+2 negatively affect the seed germination in Brassica juncea. This observation is in similarity with the previous findings of Seyed et al15 who reported that germination rate was reduced in Canola (Barassica napus), Wheat (Triticum aestivum) and Safflower (Carthamus tinctorious ) with increase in concentration of Cd+2. Reduction in seed germination is may be due to the interference and alterations in the cell membrane permeability properties by Cd+2, which resulted in reduction of water absorption and transport16. Seedling Fresh and Dry Matter: The statistical analysis (ANOVA) for seedling fresh and dry matter reveals that cultivars and Cd+2 concentrations along with their interactions were highly significant. Effect of different concentrations of metal Cd+2 on the fresh and dry weight of germinated seedlings of B.juncea is shown in table-4. Seedling fresh weight and dry weight were highest when seeds were treated and grown in distilled water as control; however Cd+2 treatments reduced fresh weight and dry weight of B.juncea at different concentrations. The reduction was increased with increasing concentration of Cd+2.Cultivar RB-50 showed minimum reduction in fresh and dry weight at all four concentrations of Cd+2 treatments as compared to rest of the 19 Cultivars and Cultivar RH-819 showed maximum reduction in fresh weight Research Journal of Biological Sciences ___________________________________________________________ ISSN 2278-3202 Vol. 4(5), 80-86, May (2015) Int. Res. J. Biological Sci. International Science Congress Association 83 and Cultivar RH-781 showed maximum reduction in dry weight at all four concentrations of Cd+2 treatments. The total dry matter accumulation was comparatively less affected by Cd+2 in all 20 cultivars of B.juncea. It has been reported that with an increasing concentration of Cd+2 biomass accumulations reduces and it may be the result of inhibition in chlorophyll synthesis and photosynthesis17 Tolerance Indices and Percent Phytotoxicity: The Tolerance indices and phytotoxicity, both are the parameters which depend on the root length of the seedlings. The phytotoxicity was found to be increased in all the 20 cultivars with increased in Cd+2supply figure-2. The present work showed that there exist a perfect negative correlation between Tolerance indices and Percent Phytotoxicity. The Cultivar RB-50 showed least phytotoxicity (86.8%) and high metal tolerance (13.2%) at 2mM concentration of Cd+2 as compared to other 19 cultivars where as Cultivar RH-0119 showed highest phytotoxicity (95.02%) and least metal tolerance (4.98%) at 2mM concentration of Cd+2as compared to other 19 cultivars. The decrease in metal tolerance index and increase in percent phytotoxicity with increase in Cd+2 concentrations in the root have been observed earlier in Cicer arientinum and Phaseolus mungo18. Conclusion The results of present investigation reveals that Cd+2 treatments played an inhibitory role for the seed germination and seedling growth of Brassica juncea Cultivars. Cd+2 treatments was found to be responsible for decrease in root- shoot length, seedling size, germination percentage, fresh and dry matter, and percentage of tolerance indices in all the Brassica junceacultivars. The intensity of inhibition was found to be directly proportional to the concentration of Cd+2 solutions employed. Results presented shows the existence of genotypical variations among Brassica juncea Cultivars with respect to tolerance in Cd+2 toxicity. Results of present study also indicates that Cultivar RB-50 showed least reduction in Root Length, Shoot Length, Seedling Size, Fresh matter and Dry matter; It also showed high metal tolerance and least phytotoxicity as compared to other cultivars. Whereas, Cultivar RH-0406 showed maximum reduction in Root Length, Shoot Length and Seedling Size. These findings reveals that Cultivar RB-50 might be the most tolerant cultivar and may be successfully grown on Cd+2 contaminated soils. 20406080100120020406080100120Tolerance Indices v/s Percent Phytotoxicity Tolerance Indices v/s Percent Phytotoxicity Figure-2 Effect of different concentrations of Cd+2 on Tolerance indices and Percent Phytotoxicity of Brassica juncea cv Research Journal of Biological Sciences ___________________________________________________________ ISSN 2278-3202 Vol. 4(5), 80-86, May (2015) Int. Res. J. Biological Sci. International Science Congress Association 84 Table-2 Effect of different concentrations of Cd+2 on Seedling size of Brassica juncea cv Varieties Control Cd 0.5mM Cd 1mM Cd 1.5mM Cd 2mM Variety Mean RH - 0749 13.54 7.84 3.17 2.79 2.33 5.93 Pusa Bold 10.52 4.98 3.42 3.22 2.89 5.01 GM - 3 9.85 7.05 2.3 2.02 1.98 4.64 RH - 0119 12.01 4.17 2.45 2.02 1.66 4.46 RH - 9304 9.7 4.05 3.46 3.14 2.33 4.54 RGN - 145 11.33 3.15 2.98 1.99 1.98 4.29 RH - 781 8.99 5.18 3.93 1.88 1.53 4.3 I - 2 9.31 7.62 4.17 3.73 1.46 5.26 V - 7 11.75 8.27 2.2 1.7 1.25 5.03 RB - 50 10.67 9.99 4.37 3.06 2.27 6.07 RH - 8113 13.87 3.77 2.75 2.38 2.32 5.02 RH - 8812 9.61 7.68 4.49 3.13 2.27 5.44 RH - 0406 10.24 3.54 3.34 1.93 1.68 4.14 PBR - 357 9.91 8.66 3.94 2.8 2.69 5.6 ROHINI 11.32 8.3 9 3.25 2.87 1.87 5.54 VARUNA 11.42 6.47 3.45 2.15 1.8 5.06 PCR - 7 12.62 3.96 2.6 1.9 1.67 4.55 RL - 1359 8.41 8.26 2.13 2.07 1.76 4.53 RH - 819 10.99 6.07 3.27 2.58 1.96 4.97 RH - 30 10.69 7.44 3.86 2.49 1.98 5.29 Conc Mean 10.84 6.33 3.28 2.49 1.98 4.98 F or means of S.Em± C.D.5% - C.V.%= 10.26 - Variety(var) 0.13 0.37 - - - - Concentration (conc) 0.07 0.18 - - - - VarXConc 0.3 0.82 - - - - Each data is an average of three replicates. Table-3 Effect of different concentrations of Cd+2 on Germination percentage (-value) of Brassica juncea cv. Varieties Control Cd 0.5mM Cd 1mM Cd 1.5mM Cd 2mM Variety Mean RH - 0749 77.71 71.57 68.86 66.14 66.14 70.09 Pusa Bold 75.00 68.86 68.86 66.14 63.43 68.46 GM - 3 77.71 68.86 66.14 63.43 61.22 67.47 RH - 0119 90.00 77.71 71.57 68.86 68.86 75.40 RH - 9304 77.71 68.86 66.14 61.22 61.22 67.03 RGN - 145 68.86 68.86 66.14 63.43 63.43 66.14 RH - 781 90.00 77.71 68.86 66.14 63.43 73.23 I - 2 90.00 68.86 66.14 66.14 63.43 70.91 V - 7 77.71 68.86 63.43 63.43 63.43 67.37 RB - 50 68. 86 66.14 63.43 61.22 59.00 63.73 RH - 8113 90.00 77.71 68.86 66.14 63.43 73.23 RH - 8812 68.86 68.86 66.14 63.43 63.43 66.14 RH - 0406 68.86 68.86 63.43 63.43 61.22 65.16 PBR - 357 83.86 77.71 66.14 63.43 61.22 70.47 ROHINI 71.57 68.86 68.86 59.00 56.79 65.02 VARUNA 68.86 66.14 63.43 63.43 61.22 64.62 PCR - 7 66.14 63.43 61.22 59.00 54.78 60.92 RL - 1359 68.86 68.86 68.86 63.43 61.22 66.24 RH - 819 71.57 66.14 63.43 61.22 61.22 64.72 RH - 30 68.86 66.14 66.14 66.14 56.79 64.82 Conc Mean 76.05 69.95 66.30 63.74 6 1.74 67.56 For means of S.Em± C.D.5% - C.V.%= 7.24 - Variety (var) 1.26 3.52 - - - - Concentration(conc) 0.63 1.76 - - - - VarXConc 2.82 7.88 - - - - Each data is an average of three replicates. Research Journal of Biological Sciences ___________________________________________________________ ISSN 2278-3202 Vol. 4(5), 80-86, May (2015) Int. Res. J. Biological Sci. International Science Congress Association 85 Table-4 Effect of different concentrations of Cd+2 on Fresh and Dry matter on Brassica juncea cvFresh matter Varieties Dry matter Control Cd 0.5mM Cd 1mM Cd 1.5mM Cd 2mM Variety Mean Control Cd 0.5mM Cd 1mM Cd 1.5mM Cd 2mM Variety Mean RH-0749 0.313 0.292 0.281 0.202 0.196 0.257 0.063 0.062 0.059 0.055 0.049 0.058 Pusa Bold 0.27 0.198 0.161 0.159 0.13 0.184 0.053 0.05 0.045 0.043 0.039 0.046 GM-3 0.327 0.303 0.224 0.192 0.189 0.247 0.062 0.062 0.061 0.058 0.053 0.059 RH-0119 0.59 0.117 0.115 0.086 0.082 0.198 0.046 0.046 0.045 0.045 0.044 0.045 RH-9304 0.176 0.213 0.206 0.204 0.194 0.199 0.056 0.05 0.049 0.048 0.048 0.05 RGN-145 0.279 0.267 0.213 0.239 0.202 0.24 0.043 0.038 0.037 0.036 0.034 0.037 RH-781 0.373 0.261 0.219 0.191 0.171 0.243 0.046 0.041 0.034 0.021 0.02 0.032 I-2 0.265 0.229 0.213 0.196 0.189 0.218 0.056 0.049 0.045 0.044 0.043 0.047 V-7 0.255 0.233 0.215 0.159 0.144 0.201 0.051 0.046 0.045 0.045 0.043 0.046 RB-50 0.182 0.211 0.199 0.184 0.182 0.191 0.049 0.045 0.046 0.044 0.042 0.045 RH-8113 0.244 0.218 0.193 0.184 0.165 0.201 0.064 0.063 0.063 0.063 0.058 0.062 RH-8812 0.263 0.22 0.197 0.184 0.165 0.206 0.052 0.05 0.045 0.043 0.042 0.046 RH-0406 0.286 0.23 0.213 0.207 0.178 0.223 0.061 0.055 0.053 0.051 0.05 0.054 PBR-357 0.319 0.317 0.302 0.232 0.225 0.279 0.054 0.053 0.049 0.046 0.045 0.049 ROHINI 0.279 0.26 0.219 0.212 0.209 0.236 0.058 0.054 0.046 0.044 0.039 0.048 VARUNA 0.333 0.307 0.241 0.23 0.226 0.268 0.045 0.04 0.036 0.038 0.038 0.039 PCR-7 0.492 0.364 0.271 0.27 0.191 0.318 0.062 0.058 0.048 0.047 0.042 0.051 RL-1359 0.274 0.272 0.199 0.194 0.173 0.222 0.055 0.05 0.049 0.046 0.044 0.049 RH-819 0.177 0.176 0.165 0.101 0.055 0.135 0.051 0.048 0.044 0.044 0.042 0.046 RH-30 0.315 0.253 0.251 0.228 0.222 0.254 0.055 0.054 0.054 0.051 0.049 0.053 Conc Mean 0.301 0.247 0.215 0.193 0.174 0.226 0.054 0.051 0.048 0.046 0.043 0.048 - - - - - - - - - - - - - For means of S.Em± C.D.5% - C.V.%= 15.609 S.Em± C.D.5% - C.V.%= 8.712 - Variety(var) 0.009 0.025 - - - - 0.001 0.003 - - - - Concentrations (conc) 0.005 0.013 - - - - 0.001 0.002 - - - - VarXConc 0.02 0.057 - - - - 0.002 0.007 - - - - Each data is an average of three replicates. 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